Methods in

Molecular Biology 1872

Loralie J. Langman
Christine L.H. Snozek
Editors

LC-MS in Drug
Analysis
Methods and Protocols
Second Edition
METHODS IN MOLECULAR BIOLOGY

Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes:

http://www.springer.com/series/7651
 LC-MS in Drug Analysis

Methods and Protocols

Second Edition

Edited by

Loralie J. Langman
Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA

Christine L.H. Snozek

Department of Laboratory Medicine and Pathology, Mayo Clinic in Arizona, Scottsdale, AZ, USA
Editors
Loralie J. Langman Christine L.H. Snozek
Department of Laboratory Department of Laboratory
Medicine and Pathology Medicine and Pathology
Mayo Clinic Mayo Clinic in Arizona
Rochester, MN, USA Scottsdale, AZ, USA

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-4939-8822-8 ISBN 978-1-4939-8823-5 (eBook)
https://doi.org/10.1007/978-1-4939-8823-5
Library of Congress Control Number: 2018957456

© Springer Science+Business Media, LLC, part of Springer Nature 2012, 2019

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is
concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction
on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation,
computer software, or by similar or dissimilar methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply,
even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations
and therefore free for general use.
The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to
be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty,
express or implied, with respect to the material contained herein or for any errors or omissions that may have been made.
The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

This Humana Press imprint is published by the registered company Springer Science+Business Media, LLC, part of
Springer Nature.
The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A.
Preface

The second edition of this book is again intended to provide detailed LC-MS(/MS)
procedures for the analysis of compounds of clinical significance. The main focus points
for this edition were new developments including novel drugs (both therapeutic and
recreational) and updated methodologies, as well as discussing alternate matrices not
addressed in the first edition.
We thank our colleagues who contributed to the contents of the book for the countless
hours of work that these chapters represent. We hope that you, the reader, find this book
useful.

Rochester, MN, USA Loralie J. Langman

Scottsdale, AZ, USA Christine L.H. Snozek

v
Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 An Introduction to Drug Testing: The Expanding Role
of Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Christine L.H. Snozek, Loralie J. Langman, and Steven W. Cotten
2 Quantification of Eight Cannabinoids Including Cannabidiol
in Human Urine Via Liquid Chromatography Tandem Mass Spectrometry . . . . 11
Karl B. Scheidweiler and Allan J. Barnes
3 Analysis of Benzodiazepines for Drug-Facilitated Assaults
and Abuse Settings (Urine) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Olaf H. Drummer, Matthew Di Rago, and Dimitri Gerostamoulos
4 Targeted Opioid Screening Assay for Pain Management Using
High-Resolution Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Darlington Danso, Loralie J. Langman, and Paul J. Jannetto
5 Measurement of Buprenorphine and Norbuprenorphine in Urine. . . . . . . . . . . . . 51
Andrea R. Terrell, Vipin Adhlakha, and Poluru Reddy
6 Quantitation of Tapentadol by Liquid Chromatography: Tandem
Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Graham R. Jones and Russell P. Handy
7 Therapeutic Drug Monitoring of Lacosamide by LC-MS/MS . . . . . . . . . . . . . . . . 67
He Sarina Yang and Leslie Edinboro
8 LC-MS/MS Method for the Quantification of the Leflunomide
Metabolite, Teriflunomide, in Human Serum/Plasma . . . . . . . . . . . . . . . . . . . . . . . 75
Geoffrey S. Rule, Alan L. Rockwood, and Kamisha L. Johnson-Davis
9 Analysis of Tryptic Peptides from Therapeutic Monoclonal Antibodies
Using LC-MS/MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Maria Alice V. Willrich
10 Quantification of Methotrexate in Human Serum and Plasma
by Liquid Chromatography Tandem Mass Spectrometry. . . . . . . . . . . . . . . . . . . . . 101
Gabrielle N. Winston-McPherson, Michael Schmeling,
and Andrew N. Hoofnagle
11 Simultaneous Determination of Tacrolimus and Cyclosporine A
in Whole Blood by Ultrafast LC-MS/MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Matthew W. Bjergum, Paul J. Jannetto, and Loralie J. Langman
12 Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)
Method to Quantify Gabapentin and Pregabalin in Urine. . . . . . . . . . . . . . . . . . . . 119
Stephen Merrigan and Kamisha L. Johnson-Davis
13 The Evolving Landscape of Designer Drugs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
Sherri L. Kacinko and Donna M. Papsun

vii
viii Contents

14 Analysis of Synthetic Cannabinoid Metabolites by Liquid

Chromatography-Tandem Mass Spectrometry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
Gregory C. Janis
15 Quantification of Designer Opioids by Liquid Chromatography–Tandem
Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
Sherri L. Kacinko and Joseph W. Homan
16 Screening Analysis for Designer Stimulants by LC-MS/MS . . . . . . . . . . . . . . . . . . 165
Piotr Adamowicz and Bogdan Tokarczyk
17 Drug Screening Using Liquid Chromatography Quadrupole
Time-of-Flight (LC-QqTOF) Mass Spectrometry. . . . . . . . . . . . . . . . . . . . . . . . . . . 181
Jennifer M. Colby and Kara L. Lynch
18 Alternate Matrices: Meconium, Cord Tissue, Hair, and Oral Fluid . . . . . . . . . . . . 191
Kendra L. Palmer and Matthew D. Krasowski
19 Salt-Assisted Liquid-Liquid Extraction of Meconium for Analysis
of Cocaine and Amphetamines by Liquid Chromatography-Tandem
Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
Melissa M. Goggin and Gregory C. Janis
20 Detection of In Utero Cannabis Exposure in Umbilical Cord Tissue
by a Sensitive Liquid Chromatography-Tandem Mass
Spectrometry Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
Fang Wu, Triniti L. Jensen, and Gwendolyn A. McMillin
21 Quantitation of Ethyl-β-D-Glucuronide in Human Umbilical Cord
Tissue by Liquid Chromatography Tandem Mass Spectrometry
(LC-MS/MS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
Simuli L. Wabuyele and Gwendolyn A. McMillin
22 Analysis of Drugs in Oral Fluid Using LC-MS/MS . . . . . . . . . . . . . . . . . . . . . . . . . 237
Cynthia A. Coulter and Christine M. Moore
23 Determination of Cocaine and Metabolites in Dried Blood Spots
by LC-MS/MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
Lars Ambach and Christophe Stove

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
Contributors

PIOTR ADAMOWICZ  Institute of Forensic Research, Krakow, Poland

VIPIN ADHLAKHA  Aria Diagnostics, Indianapolis, IN, USA
LARS AMBACH  Faculty of Pharmaceutical Sciences, Laboratory of Toxicology, Department of
Bioanalysis, Ghent University, Ghent, Belgium
ALLAN J. BARNES  Quest Diagnostics, Chantilly, VA, USA
MATTHEW W. BJERGUM  Clinical Mass Spectrometry Lab, Superior Drive Support Center,
Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA
JENNIFER M. COLBY  Department of Pathology, Microbiology, and Immunology, Vanderbilt
University Medical Center, Nashville, TN, USA
STEVEN W. COTTEN  Department of Pathology and Laboratory Medicine, School of Medicine,
University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
CYNTHIA A. COULTER  Toxicology Analytical Services, Immunalysis Corporation, Pomona,
CA, USA
DARLINGTON DANSO  Department of Laboratory Medicine and Pathology, Mayo Clinic,
Rochester, MN, USA
MATTHEW DI RAGO  Faculty of Medicine, Nursing and Health Sciences, Department of
Forensic Medicine, School of Public Health and Preventive Medicine, Monash University,
Southbank, VIC, Australia; Victorian Institute of Forensic Medicine, Southbank, VIC,
Australia
OLAF H. DRUMMER  Faculty of Medicine, Nursing and Health Sciences, Department of
Forensic Medicine, School of Public Health and Preventive Medicine, Monash University,
Southbank, VIC, Australia
LESLIE EDINBORO  Quest Diagnostics Nichols Institute of Chantilly, Chantilly, VA, USA
DIMITRI GEROSTAMOULOS  Faculty of Medicine, Nursing and Health Sciences, Department
of Forensic Medicine, School of Public Health and Preventive Medicine, Monash University,
Southbank, VIC, Australia; Victorian Institute of Forensic Medicine, Southbank, VIC,
Australia
MELISSA M. GOGGIN  MedTox Laboratories, Laboratory Corporation of America Holdings,
St. Paul, MN, USA
RUSSELL P. HANDY  Alberta Office of the Chief Medical Examiner, Edmonton, AB, Canada
JOSEPH W. HOMAN  NMS Laboratories, Willow Grove, PA, USA
ANDREW N. HOOFNAGLE  Department of Laboratory Medicine, University of Washington,
Seattle, WA, USA
GREGORY C. JANIS  MedTox Laboratories, Laboratory Corporation of America Holdings,
St. Paul, MN, USA
PAUL J. JANNETTO  Department of Laboratory Medicine and Pathology, Mayo Clinic,
Rochester, MN, USA; Clinical Mass Spectrometry Lab, Superior Drive Support Center,
Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA
TRINITI L. JENSEN  ARUP Laboratories, Salt Lake City, UT, USA
KAMISHA L. JOHNSON-DAVIS  ARUP Institute for Clinical and Experimental Pathology,
Salt Lake City, UT, USA; Department of Pathology, University of Utah Health Sciences
Center, Salt Lake City, UT, USA
GRAHAM R. JONES  Alberta Office of the Chief Medical Examiner, Edmonton, AB, Canada

ix
x Contributors

SHERRI L. KACINKO  NMS Laboratories, Willow Grove, PA, USA

MATTHEW D. KRASOWSKI  Department of Pathology, University of Iowa Hospitals and
Clinics, Iowa City, IA, USA
LORALIE J. LANGMAN  Department of Laboratory Medicine and Pathology, Mayo Clinic
College of Medicine, Rochester, MN, USA; Clinical Mass Spectrometry Lab, Superior Drive
Support Center, Department of Laboratory Medicine and Pathology, Mayo Clinic,
Rochester, MN, USA
KARA L. LYNCH  Department of Laboratory Medicine, University of California San
Francisco, San Francisco, CA, USA
GWENDOLYN A. MCMILLIN  Department of Pathology, University of Utah, Salt Lake City,
UT, USA; ARUP Laboratories, Salt Lake City, UT, USA; ARUP Institute for Clinical
and Experimental Pathology, Salt Lake City, UT, USA; Department of Pathology,
University of Utah School of Medicine, Salt Lake City, UT, USA
STEPHEN MERRIGAN  ARUP Institute for Clinical and Experimental Pathology, Salt Lake
City, UT, USA; ARUP Laboratories, Salt Lake City, UT, USA
CHRISTINE M. MOORE  Toxicology Analytical Services, Immunalysis Corporation, Pomona,
CA, USA
KENDRA L. PALMER  Department of Pathology, University of Iowa Hospitals and Clinics,
Iowa City, IA, USA
DONNA M. PAPSUN  NMS Laboratories, Willow Grove, PA, USA
POLURU REDDY  Aria Diagnostics, Indianapolis, IN, USA
ALAN L. ROCKWOOD  Florida State University, Tallahassee, FL, USA; Rockwood Scientific
Consulting, Salt Lake City, UT, USA
GEOFFREY S. RULE  ARUP Institute for Clinical and Experimental Pathology, Salt Lake
City, UT, USA; ARUP Laboratories, Salt Lake City, UT, USA
KARL B. SCHEIDWEILER  Quest Diagnostics, Chantilly, VA, USA
MICHAEL SCHMELING  Department of Laboratory Medicine, University of Washington,
Seattle, WA, USA
CHRISTINE L.H. SNOZEK  Department of Laboratory Medicine and Pathology, Mayo Clinic
in Arizona, Scottsdale, AZ, USA
CHRISTOPHE STOVE  Faculty of Pharmaceutical Sciences, Laboratory of Toxicology,
Department of Bioanalysis, Ghent University, Ghent, Belgium
ANDREA R. TERRELL  Phoenix Laboratories, Indianapolis, IN, USA
BOGDAN TOKARCZYK  Institute of Forensic Research, Krakow, Poland
SIMULI L. WABUYELE  ARUP Institute for Clinical and Experimental Pathology, Salt Lake
City, UT, USA; ARUP Laboratories, Salt Lake City, UT, USA
MARIA ALICE V. WILLRICH  Division of Clinical Biochemistry and Immunology, Department
of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA
GABRIELLE N. WINSTON-MCPHERSON  Department of Laboratory Medicine, University of
Washington, Seattle, WA, USA; Henry Ford Hospital, Detroit, MI, USA
FANG WU  Department of Pathology, University of Utah, Salt Lake City, UT, USA
HE SARINA YANG  Quest Diagnostics Nichols Institute of Valencia, Valencia, CA, USA
 Chapter 1

An Introduction to Drug Testing: The Expanding Role of Mass

Spectrometry
Christine L.H. Snozek, Loralie J. Langman, and Steven W. Cotten

Abstract
Measurement of drugs and their metabolites in biological fluids is the foundation of both therapeutic drug
monitoring (TDM) and toxicology. The introduction of methods based on mass spectrometry (MS),
coupled with gas or liquid chromatography, has revolutionized these areas. This chapter will introduce
the reader to the application of MS to TDM and toxicology, the steps that should be considered during
implementation and the processes that should be implemented to assure continued quality. Points of
emphasis include advances and recent trends since the publication of the first edition of this book, such
as high-resolution mass spectrometry and increased interest in alternate matrices.

Key words Mass spectrometry, Gas chromatography, Liquid chromatography, Therapeutic drug
monitoring, Toxicology, Drug testing

1 Introduction

Measurement of drugs and their metabolites in biological fluids is

the foundation of both therapeutic drug monitoring (TDM) and
toxicology. Though different in their application, each of these
disciplines depends upon accurate identification and quantification
if drug measurements are to be useful. Thousands of methods are
described for drug analysis, but until recently most have relied upon
analytical tools that suffer from lack of specificity and sensitivity,
namely, spectrophotometry and immunoassays. It must be
acknowledged that the methods utilizing each of these allowed
TDM and toxicology to grow and mature, but it is mass spectrom-
etry that is taking the analysis of drugs and drug metabolites into
new directions.
In recent years, mass spectrometry whether single stage, tan-
dem (MS/MS), or high resolution (HRMS), coupled with liquid
chromatography (LC) or gas chromatography (GC), has emerged
as a powerful tool for clinical and toxicology laboratories.

Loralie J. Langman and Christine L.H. Snozek (eds.), LC-MS in Drug Analysis: Methods and Protocols,
Methods in Molecular Biology, vol. 1872, https://doi.org/10.1007/978-1-4939-8823-5_1,
© Springer Science+Business Media, LLC, part of Springer Nature 2019

1
2 Christine L.H. Snozek et al.

Gas Chromatography
Liquid Chromatography

Mass Library
Sample Preparation Separation Ionization Detection
Analyzer(s) Scanning/Identification

Solid-Phase Extraction Chemical Ionization (CI)

Liquid-Liquid Extraction Electron Impact (EI)
Derivatization Electrospary Ionization (ESI)
Matrix Assisted Laser Desorption Ionization (MALDI)

Fig. 1 Current applications of mass spectrometry in the clinical laboratory

Improvements in user interfaces, computing power, and column

characteristics have expanded the potential of MS-based systems
from rigid and cumbersome techniques to limitless, adaptable
open-source platforms capable of identifying numerous analytes
in a single sample. The sensitivity and accuracy achievable have
naturally found applications in the areas of TDM and toxicological
analysis and beyond (Fig. 1). Evidence of the transition to the
clinical setting is seen not only by the number of publications but
also in the development of quality management guidelines and
standards [1, 2].
This volume will provide the reader with a number of applica-
tions to both disciplines which will be introduced in the following
pages. This chapter will also discuss some of the issues one should
consider when migrating to MS-based methods. The emphasis of
this second edition is on newer drugs (e.g., designer drugs of
abuse) and technologies that have emerged since the first edition,
as well as a focus on alternate matrices beyond traditional blood and
urine specimens.

2 Therapeutic Drug Monitoring

Therapeutic drug monitoring is an integral part of personalized

medicine. By providing accurate quantification of drug concentra-
tions in the circulation using blood, serum, or plasma, TDM is used
to maximize the effect of certain prescribed drugs by achieving a
therapeutic concentration as quickly as possible while simulta-
neously minimizing unwanted or toxic side effects. The drugs
typically monitored are those with narrow therapeutic indices and
for which there are established relationships between the concen-
tration found within the circulation and the observed effects of the
drug. For these drugs, the delicate balance between efficacy (ED50)
 Introduction to Drug Testing 3

and toxicity (LD50) dictates the need for accurate quantification.

TDM also provides a means of assessing compliance, ensuring
correct dosing, and identifying drug-drug interactions. Ironically
TDM initially developed in parallel with the introduction of
chromatography-based methods into the clinical laboratory in the
early 1970s.
Unfortunately, the methods were time-consuming and in the
1980s were replaced with immunoassay-based methods which
remain in use in many laboratories today. While these methods
offer advantages of ease of use and availability on many analytical
platforms, they suffer from issues of sensitivity and specificity. Posi-
tive and negative interferences are well documented and if not
recognized can lead to inappropriate patient care. In addition,
these methods usually cross-react with structurally related metabo-
lites which may or may not contribute to the pharmacological
activity of the parent drug. Other compounds which also share
structural features with the drug being measured are also likely to
cross-react with the antibody, and so the presence of such com-
pounds also poses problems. Finally, the limit of detection of many
immunoassays is insufficient for current TDM applications. For
example, evaluation of methotrexate clearance uses low micromolar
cutoffs, at a concentration range where immunoassay cross-
reactivity with metabolites becomes a significant concern. Clinical
laboratories have thus replaced many of the TDM analyses using
immunoassay with methods using LC-MS/MS [3–7].
Monitoring of immunosuppressant therapy for solid organ
transplant patients using LC-MS/MS was perhaps one of the first
applications that adopted MS analysis. Subtherapeutic doses can
result in transplant rejection, while overdosing can cause serious
toxicity or death. It is therefore imperative to closely monitor
individual patient drug levels for proper treatment. LC-MS/MS
permits simultaneous quantification of immunosuppressant drugs
from one whole blood sample [8–10]. Whole blood samples are
lysed and precipitated (either offline or online) followed by mass
analysis with internal standards. Comparisons of LC-MS/MS with
immunoassays for immunosuppressants often demonstrate lower
drug concentrations in LC-MS/MS assays [9], with the difference
being attributed to the continued issue of cross-reactivity of immu-
noassay antibodies with drug metabolites.
A relatively new arena for TDM opened with the introduction
of monoclonal antibody (mAb) therapies, used to treat a variety of
conditions including Crohn’s disease and ulcerative colitis. These
biologic agents provide the ability to target specific aspects of the
immune system, to modulate immune response against self- and
foreign antigens. Measurement of mAb concentrations gives some
indication of likelihood of successful therapy and can aid in distin-
guishing subtherapeutic concentrations from nonresponsiveness to
treatment. However, many of the mAb therapies are themselves
4 Christine L.H. Snozek et al.

immunogenic, necessitating simultaneous measurement of the

mAb and neutralizing antibodies that block the activity of the
drug [11].
The aforementioned drugs or classes represent but a few of
those for which LC-MS/MS methods have been described. Meth-
ods for antiepileptic, antidepressant, antimicrobial, and chemother-
apeutic agents are readily found, with some being the only methods
described. Clearly another advantage in the adoption of MS tech-
niques is the ability to develop assays in-house rather than having to
wait for immunoassay manufacturers.

3 Toxicology

Originally the “study of poisons,” toxicology has evolved into a

broader, highly diverse field. Today we recognize that poisons are
readily found in the home, work, and environment, can be
man-made or naturally occurring, and may be therapeutic in
other circumstances. Unfortunately, toxicity may not become
apparent until after the offending agent is metabolized or even
cleared by the body. This poses an interesting challenge. As with
TDM, the use of mass spectrometry-based methods has extended
the testing range and may, in the near future, facilitate discovery
long after a toxin is gone. Still in the early stages and not quite ready
for clinical or forensic application, proteomic and metabolomic
profiling using mass spectrometry have revealed patterns that one
day will likely be used to identify toxins months or years after an
exposure.
In the clinical setting, broad screens seeking offending agents
were largely abandoned in the 1990s, again primarily due to the
lengthy times required to complete the analyses and the change in
focus to drugs of abuse. Broad screens are not high-volume tests,
but there are clinical situations in which such a test is useful in
excluding a toxin as a cause of the patient’s symptoms. One such
example is highlighted by the emergence over the last decade of
designer drugs, e.g., synthetic cannabinoids, cathinones, and fenta-
nyl analogs. The rapid evolution of new psychoactive substances
(NPS), in efforts to evade detection and legislation, has created
intense interest in technologies such as high-resolution mass spec-
trometry (HRMS) which can tentatively identify novel compounds
without a spectral library and before standard reference material is
available. HRMS complements existing technologies but may even-
tually supplant GC-MS-based screening as the preferred choice for
small molecule identification. Confirmation against a spectral
library or reference standard can be done for known compounds
as with conventional screens; for emerging compounds, HRMS
data can be acquired in an untargeted manner and reanalyzed at a
later date [12].
 Introduction to Drug Testing 5

For many years, GC-MS was considered the gold standard for
confirmation of the presence of abused drugs in urine, but as
methods and libraries have developed, many laboratories have
turned to LC-MS/MS for these analyses. In the instance of pain
management, screening and confirmatory drug testing using
LC-MS/MS allow for detection of both morphine-based and syn-
thetic opioids. The technique provides superior sensitivity and
specificity compared to immunoassays which are usually targeted
to morphine and may thus fail to detect synthetic opioids and
oxycodone. The ability to detect multiple parent drugs and meta-
bolites in a relatively short run makes LC-MS/MS a powerful tool
in support of pain management testing. The use of this technique
also provides an extended analytical measuring range of approxi-
mately 105 ng/mL that is most useful in this setting [13].
Matrices other than blood and urine are an increasingly impor-
tant component of clinical and forensic toxicology. Meconium and
umbilical cord tissue can reveal in utero exposure to illicit drugs.
Oral fluid, hair, and dried blood spots all provide certain advantages
over traditional analytical matrices. For example, oral fluid testing
can allow for observed collection of a specimen during a roadside
stop from a seemingly intoxicated driver, while dried blood spots
can facilitate testing collected in remote locations far from a refer-
ence laboratory.

4 Evaluation of Methods

A detailed discussion of chromatographic methods is beyond the

scope of this book, though some features will be discussed in this
section. Generally, these techniques are used to separate the drugs
of interest from other compounds present in the sample. Afterward
the analyte is introduced into the mass spectrometer which serves as
the detector. Which chromatographic technique is used depends
upon the sample and the volatility and solubility of the target
analyte.
Depending upon the biological matrix and target compounds
being analyzed, pre-analytical treatment of the sample is generally
necessary as seen in Fig. 2. Removal of interfering components such
as proteins and lipids improves sensitivity by decreasing the com-
plexity of the mixture analyzed. Liquid-liquid or solid-liquid
extraction may be necessary prior to introduction on the chroma-
tography system to enrich for target compounds. For compounds
present in low abundance, extraction followed by evaporation of
the solvent to dryness effectively concentrates the sample, improv-
ing sensitivity. For relatively abundant compounds, a simple depro-
teinization step (i.e., the dilute and shoot method) may suffice. In
this, the sample is mixed with an organic compound such as aceto-
nitrile (spiked with the appropriate internal standard) and
6 Christine L.H. Snozek et al.

Exogenous Endogenous
immunosuppressants hormones
neurological agents vitamin D
antiviral agents organic acids
Therapeutic acylcarnitines
Drug
Monitoring

Mass
Spectrometry
D.O.A Forensics poisons
opiates
Confirmatory and toxins
steroids Drug Testing Toxicological D.O.A
Analysis

Fig. 2 Pipeline of steps involved in mass spectrometry analyses

centrifuged, and the supernatant is injected. These methods work

fairly well for many LC-MS- or LC-MS-/MS-based methods.
Internal standards should be added at the beginning of analysis
as both a quality control measure and to facilitate quantification.
Where possible, it is recommended that analogs labeled with a
stable isotope, e.g., deuterium or 13C, of the primary analyte of
interest be used as the internal standard. If such is not available, it is
acceptable to use a compound that is structurally related. The
internal standard must undergo all steps of the procedure (extrac-
tion, derivatization, evaporation, etc.) in order to serve the purpose
of identifying problems that could arise during the sample prepara-
tion. Since small amounts of the unlabeled compound may con-
taminate the internal standard, it should be checked by analyzing a
blank sample to which the internal standard is added.
It is usually necessary to separate the analytes of interest from
each other and from unrelated compounds by the use of a column.
In liquid chromatography, gradient solvent systems of methanol,
water, or acetonitrile (or mixtures thereof) are frequently used to
sequentially elute compounds based on polarity and affinity for the
column. As laboratorians face increasing work demands, further
simplification of methods by direct introduction of the sample
into the mass spectrometer has been explored and continues to
gain in popularity. Online sample extraction, for example, by tur-
bulent flow chromatography (e.g., Thermo Cohesive), or high-
throughput solid phase extraction (e.g., Agilent RapidFire), can
greatly facilitate assay throughput by reducing analytical run time
and/or allowing multiplexed analysis.
 Introduction to Drug Testing 7

After separation, the compounds are ionized prior to mass

analysis. Charged compounds are shuttled into the mass analyzer
which, depending upon the method, selects ions based on prede-
termined mass-to-charge ratio (m/z) criteria or scans within
defined m/z ranges. If tandem mass spectrometry (MS/MS) is
used, the selected ionized compounds are further fragmented fol-
lowed by an additional m/z detection to obtain spectra for both
precursor (formerly known as “parent”) and product (formerly
known as “daughter”) ions. The data are transformed into a recog-
nizable mass spectrum which, in conventional mass spectrometry, is
subsequently compared to expected values for the target com-
pounds or internal standards for quantification or to a library of
chemical spectra to obtain the identity of the compounds analyzed.
Untargeted HRMS permits tentative identification even in the
absence of a known spectral library.

5 Quality Assurance

Each laboratory should develop a quality assurance program based

upon their respective regulatory guidelines and needs. Such pro-
grams provide guidance to the analyst regarding method validation
and maintenance with sufficient checks to assure that the results
reported are as accurate as possible. Table 1 provides a list of
documents that may be useful in developing a robust quality assur-
ance program.
Method validation should include assessment of the limit of
detection (LOD), limit of quantification (LOQ), linearity, selectiv-
ity, accuracy, precision, carryover, matrix effects, and reference
intervals. For those drugs and analytes regulated under the Clinical
Laboratory Improvement Amendments (CLIA) of 1988, the labo-
ratory should use the mandatory precision limits to targeted day-
to-day precision necessary for successful proficiency testing
[14]. Alternatively, one should consider the application of the

Table 1
CLSI documents relevant to mass spectrometry analysis

Useful resources and documents for quality assurance programs

CLSI C43-A2 Gas chromatography-mass spectrometry confirmation of drugs
CLSI C50 Mass spectrometry in the clinical laboratory
CLSI EP 05 Evaluation of precision performance of quantitative measurement methods
CLSI EP 06 Evaluation of the linearity of quantitative measurement methods
CLSI EP09 Method comparison and bias estimation using patient samples
CLSI C24 Assessment of laboratory tests when proficiency testing is not available
8 Christine L.H. Snozek et al.

analysis when setting precision and accuracy goals. If TDM is the

application, the actual precision and accuracy needs may exceed
those of CLIA.
Each analytical run should include an adequate number of
quality control samples containing the targeted analytes or drugs
considered representative of those expected. Concentrations
should target decision points and span the analytical measuring
range. For example, a method used for TDM of a drug should
include control samples below, within, and above the therapeutic
range. Assays frequently used to monitor clearance, e.g., lefluno-
mide and methotrexate, should include a quality control near the
clinical decision limit or the LOQ, as appropriate. A method used
for confirmation of the presence of an abused drug should include
control samples in which the drug is absent and present near and
above the defined cutoff. Blind quality control samples may be
included to assess the nonanalytical portions of the entire process.
Proficiency testing is also an important part of quality assurance.
Clinical laboratories operating under CLIA must have control
compounds for both quantitative and qualitative confirmatory
drug testing and control compounds for each drug class surveyed
in broad-spectrum screening using GC-MS and must participate in
proficiency testing for each analyte reported [14]. In these chal-
lenges, the proficiency samples are tested as ordinary samples. The
development of new methods for various drugs is often ahead of
the availability of commercial sources of such samples. In these
cases, it is reasonable for several laboratories performing the analysis
to exchange samples on a regular basis (at a minimum of twice
per year).
Forensic laboratories in the United States still face some issues
related to lack of standardization and accreditation [15, 16]. Some
of the outlined challenges regarding standardization and profi-
ciency related to analyte identification should be addressed through
the development and adoption of standards of drugs and drug
metabolites suitable for mass spectrometry. For example, national
and international authorities have published guidelines for drug
testing that include mass spectrometry as the premier analytical
method for unambiguous analyte identification [17, 18]. However,
further work remains to be done in this arena.

6 Conclusions

Recent years have seen expansion of the importance of mass spec-

trometry to TDM and toxicology. TDM has expanded from its
historical arena of small-molecule agents to embrace proteolysis-
aided quantitation of mAb therapies. HRMS and LC-MS/MS have
rapidly risen to the forefront of efforts to detect and identify a
growing and ever-changing list of NPS. The ability to
 Introduction to Drug Testing 9

retrospectively identify compounds seen during untargeted acqui-

sition on HRMS has allowed better epidemiological tracking of
issues related to NPS such as outbreaks of toxicity after exposure
to a new agent or contaminated batch of drug.
As separation techniques continue to improve and hardware
and software platforms advance, the role of mass spectrometry in
the clinical lab will continue to grow. When evaluating a new mass
spectrometry method, the concepts of linearity, sensitivity, specific-
ity, accuracy, and precision should be at the forefront. Proper
validation will ensure that the quality of the diagnostic data
provided remains high with the improved fidelity afforded by
mass spectrometry.

References
1. CLSI (2010) Gas chromatography/mass spec- 8. Salm P, Taylor PJ, Rooney F (2008) A high-
trometry confirmation of drugs; approved performance liquid chromatography-mass
guideline, 2nd edn. CLSI Document C43-A2. spectrometry method using a novel atmo-
Clinical and Laboratory Standards Institute, spheric pressure chemical ionization approach
Wayne, PA for the rapid simultaneous measurement of
2. CLSI (2007) Mass spectrometry in the clinical tacrolimus and cyclosporin in whole blood.
laboratory: general principles and guidance; Ther Drug Monit 30:292–300
approved guideline. CLSI Document C50-A. 9. Koster RA, Dijkers ECF, Uges DRA (2009)
Clinical and Laboratory Standards Institute, Robust, high-throughput LC-MS/MS
Wayne, PA method for therapeutic drug monitoring of
3. Kirchherr H, Kühn-Velten WN (2006) Quan- cyclosporine, tacrolimus, everolimus, and siro-
titative determination of forty-eight antide- limus in whole blood. Ther Drug Monit
pressants and antipsychotics in human serum 31:116–125
by HPLC tandem mass spectrometry: a multi- 10. Bogusz MJ et al (2007) Simultaneous LC-MS-
level, single-sample approach. J Chromatogr B MS determination of cyclosporine a, tacroli-
Analyt Technol Biomed Life Sci 843:100–113 mus, and sirolimus in whole blood as well as
4. Zhang Y, Mehrotra N, Budha NR, Christensen mycophenolic acid in plasma using common
ML, Meibohm B (2008) A tandem mass spec- pretreatment procedure. J Chromatogr B Ana-
trometry assay for the simultaneous determina- lyt Technol Biomed Life Sci 850:471–480
tion of acetaminophen, caffeine, phenytoin, 11. Vande Casteele N et al (2014) Therapeutic
ranitidine, and theophylline in small volume drug monitoring in inflammatory bowel dis-
pediatric plasma specimens. Clin Chim Acta ease: current state and future perspectives.
398:105–112 Curr Gastroenterol Rep 16:378
5. Sørensen LK (2010) Determination of acidic 12. Wu AH, Gerona R, Armenian P, French D,
and neutral therapeutic drugs in human blood Petrie M, Lynch KL (2012) Role of liquid
by liquid chromatography-electrospray tandem chromatography-high-resolution mass spec-
mass spectrometry. Forensic Sci Int. https:// trometry (LC-HR/MS) in clinical toxicology.
doi.org/10.1016/j.forsciint.2010.07.016 Clin Toxicol (Phila) 50:733–742
6. Subramanian M, Birnbaum AK, Remmel RP 13. Mikel C, Almazan P, West R, Crews B et al
(2008) High-speed simultaneous determina- (2009) LC-MS/MS extends the range of
tion of nine antiepileptic drugs using liquid drug analysis in pain patients. Ther Drug
chromatography-mass spectrometry. Ther Monit 31:746–748
Drug Monit 30:347–356 14. Centers for Medicare & Medicaid Services
7. Nair H, Lawrence L, Hoofnagle AN (2012) (2003) Interpretive guidelines for laboratories.
Liquid chromatography-tandem mass spec- http://www.cms.gov/CLIA/03_Interpretive_
trometry work flow for parallel quantification Guidelines_for_Laboratories.asp. Accessed
of methotrexate and other immunosuppres- 20 Nov 2017
sants. Clin Chem 58:943–945
10 Christine L.H. Snozek et al.

15. Committee on Identifying the Needs of the quality management system in drug testing
Forensic Sciences Community, National laboratories. http://www.unodc.org/
Research Council (2009) Strengthening foren- documents/scientific/QMS_Ebook.pdf.
sic science in the United States: a path forward. Accessed 20 Nov 2017
The National Academies Press, Washington, 18. United States Department of Justice Drug
D.C. Enforcement Administration (2010) Scientific
16. Kaye DH (2010) The good, the bad, the ugly: working group for the analysis of seized drugs
the NAS report on strengthening forensic sci- recommendations. http://www.swgdrug.org/
ence in America. Sci Justice 50:8–11 Documents/SWGDRUG%
17. United Nations Office on Drugs and Crime 20Recommendations%20Version%207-1.pdf.
(2009) Guidance for the implementation of a Accessed 20 Nov 2017
 Chapter 2

Quantification of Eight Cannabinoids Including Cannabidiol

in Human Urine Via Liquid Chromatography Tandem Mass
Spectrometry
Karl B. Scheidweiler and Allan J. Barnes

Abstract
Medical and recreational cannabis legalization has highlighted the importance of being able to identify
recent cannabis use and impairment. Monitoring minor plant cannabinoids has been proposed to assist in
identifying recent cannabis use. Additionally, cannabidiol (CBD) has been proposed for epilepsy, pain,
inflammatory disorder, anxiety, and addiction treatment; therefore, monitoring CBD is of increasing
clinical importance. However, few methods exist capable of monitoring extensive panels of traditional
cannabinoid analytes and minor cannabinoids (including CBD). This chapter details a liquid chromatogra-
phy tandem mass spectrometry method capable of measuring Δ9-tetrahydrocannabinol (THC),
11-hydroxy-THC, 11-nor-9-carboxy-THC, cannabinol, cannabigerol, tetrahydrocannabivarin (THCV),
and its metabolite, 11-nor-9-carboxy-THCV, in urine.

Key words Cannabinoids, Cannabidiol, Urine, Liquid chromatography, Mass spectrometry, Pipette
tip extraction

1 Introduction

Interest in monitoring cannabis use and abuse has increased in light

of the recent and ongoing legislation regarding medical and recrea-
tional cannabis [1, 2]. Δ9-Tetrahydrocannabinol (THC), the psy-
choactive component of cannabis, is highly lipophilic yielding
prolonged excretion into blood and urine after cessation of cannabis
use, confounding distinction between recent use and residual excre-
tion post-cessation [3]. Therefore monitoring minor cannabinoids
present in cannabis and their metabolites has been proposed for
possibly assisting in distinguishing recent use from prolonged
excretion. Several minor cannabinoids, such as cannabidiol
(CBD), cannabinol (CBN), cannabigerol (CBG), tetrahydrocanna-
bivarin (THCV), and its metabolite, 11-nor-9-carboxy-THCV

Loralie J. Langman and Christine L.H. Snozek (eds.), LC-MS in Drug Analysis: Methods and Protocols,
Methods in Molecular Biology, vol. 1872, https://doi.org/10.1007/978-1-4939-8823-5_2,
© Springer Science+Business Media, LLC, part of Springer Nature 2019

11
12 Karl B. Scheidweiler and Allan J. Barnes

(THCVCOOH), were proposed as possible markers of recent can-

nabis intake in different biological matrices [4–8].
CBD is a plant cannabinoid present in cannabis that binds CB2
cannabinoid receptors and has been proposed to be clinically useful
for treating many clinical syndromes including epilepsy, pain,
inflammatory disorders, and anxiety [9, 10]. CBD also has been
studied for treating cannabis and opioid addiction [11]. Therefore,
methods capable of monitoring CBD are of increasing interest.
THC is rapidly metabolized to its equipotent metabolite
11-hydroxy-THC (11-OH-THC) and further to its main inactive
metabolite, 11-nor-9-carboxy-THC (THCCOOH). All three
undergo glucuronidation, are excreted into urine, and are tradi-
tional cannabis testing analytical target analytes. Urine cannabinoid
testing for multiple cannabinoid compounds and their metabolites
presents several challenges, mainly that cannabinoids are extensively
glucuronidated requiring synthesis of glucuronide reference stan-
dards and/or hydrolysis [12]. Glucuronide reference standards are
currently available only for THC and THCCOOH; thus, hydrolysis
is required to be able to effectively monitor additional cannabinoids
in urine. It previously has been reported that only the
THCCOOH-glucuronide ester linkage is easily cleaved via alkaline
hydrolysis, while the ether-linked THC-glucuronide bond is not
[12]. Therefore previous methods targeting both THC and
THCCOOH in urine required tandem hydrolysis via enzyme glu-
curonidase followed by alkaline hydrolysis [12] or directly moni-
toring both the free and glucuronidated forms of THC and
THCCOOH [13, 14]. Similar research found that CBD was also
extensively glucuronidated in urine, requiring enzymatic hydrolysis
before analysis, since CBD-glucuronide reference standards are not
commercially available [5]. This chapter details a liquid chromatog-
raphy tandem mass spectrometry (LC-MS/MS) method following
an effective overnight enzyme hydrolysis, employing pipette tip
solid phase extraction, targeting THC, 11-OH-THC,
THCCOOH, CBD, CBN, CBG, THCV, and THCVCOOH.
Development and validation of the enzymatic hydrolysis and
LC-MS/MS method were recently reported [15].

2 Materials

Prepare all solutions with ultrapure water (resistivity of 18 MΩ cm

with total organic content <10 ppb) and LCMS-grade solvents.

2.1 Supplies and 1. β-Glucuronidase from overexpressing recombinant E. coli

Equipment (EBG™; 50,000 U/mL).
2. WAX-S tips (1 mL/20 mg resin with 40 mg salt), DPX Labs, or
similar.
 Cannabinoids in Urine 13

3. Volumetric glassware: 5–1000 mL volumetric flasks, 0.5–5 mL

class A volumetric pipettes.
4. 12 mL glass screw-top bottles with Teflon-lined screw caps.
5. Adjustable pipettes: 10–100, 100–1000, and 500–5000 μL.
6. 17  60 mm glass vials with Teflon-lined screw caps.
7. 1 mL 96-well plates.
8. Plate kit with 500 μL glass inserts and mat cover.
9. 1.5 mL polypropylene microcentrifuge tubes.
10. Magnetic stir plate.
11. pH meter.
12. Shaking water bath.
13. Automated liquid handling system, e.g., Tecan Freedom
EVO® 100.
14. Microcentrifuge for 17  60 mm tubes and 1.5 mL microcen-
trifuge tubes.
15. Centrifuge for 96-well plates.
16. Vortexer.
17. LC-MS/MS, e.g., Sciex 5500 QTRAP® triple quadrupole/
linear ion trap mass spectrometer with a Turbo V ion spray
source coupled with a high-performance liquid chromatogra-
phy (HPLC) system consisting of a DGU-20A3 degasser,
LC-20ADxr pumps, SIL-20ACxr autosampler, and a
CTO-20 column oven.
18. Analytical column: UCT Selectra DA 100  2.1 mm, 3 μm.
19. In-line filter: Phenomenex KrudKatcher Ultra HPLC.

2.2 Cannabinoid 1. Calibrator intermediate individual analyte stock solutions:

Calibrator and Quality 100 mg/L of THC, 11-OH-THC, THCCOOH, CBD,
Control Solutions CBN, CBG, and THCV. Transfer 0.5 mL of each 1.0 mg/
mL stock solution of THC, 11-OH-THC, THCCOOH,
CBD, CBN, CBG, and THCV to individual 5 mL volumetric
flasks. Fill to 5 mL with methanol. Cap, mix well, and transfer
to 12 mL glass screw-top bottle.
2. Calibrator 9 10 working standard: 5000 μg/L THCCOOH.
Add 0.5 mL of 100 mg/L THCCOOH stock solution to a
10 mL volumetric flask and fill to 10 mL with methanol. Cap,
mix well, and transfer to a 12 mL screw-top glass bottle.
Calibrator concentrations are shown in Table 1 (see Note 1).
3. Calibrator 8 10 working standard: 2500 μg/L of all analytes.
Add 250 μL of each 100 mg/L stock solution of THC,
11-OH-THC, THCCOOH, CBD, CBN, CBG, THCV, and
THCVCOOH into a 10 mL volumetric flask and fill to 10 mL
14 Karl B. Scheidweiler and Allan J. Barnes

Table 1
Final concentrations (μg/L) for calibrators in urine

Calibrator 1 2 3 4 5 6 7 8 9
THC 1 2 5 10 20 50 100 250 –
CBG 1 2 5 10 20 50 100 250 –
11-OH-THC – 2 5 10 20 50 100 250 –
CBD – 2 5 10 20 50 100 250 –
CBN – 2 5 10 20 50 100 250 –
THCV – 2 5 10 20 50 100 250 –
THCVCOOH – 2 5 10 20 50 100 250 –
THCCOOH 1 2 5 10 20 50 100 250 500

with methanol. Cap, mix well, and transfer to a 12 mL screw-

top glass bottle.
4. Calibrator 7 10 working standard: 1000 μg/L of all analytes.
Add 4 mL of calibrator 8 working standard stock solution to a
10 mL volumetric flask and fill to 10 mL with methanol. Cap,
mix well, and transfer to a 12 mL screw-top glass bottle.
5. Calibrator 6 10 working standard: 500 μg/L of all analytes.
Add 5 mL of calibrator 7 working standard stock solution to a
10 mL volumetric flask and fill to 10 mL with methanol. Cap,
mix well, and transfer to a 12 mL screw-top glass bottle.
6. Calibrator 5 10 working standard: 200 μg/L of all analytes.
Add 4 mL of calibrator 6 working standard stock solution to a
10 mL volumetric flask and fill to 10 mL with methanol. Cap,
mix well, and transfer to a 12 mL screw-top glass bottle.
7. Calibrator 4 10 working standard: 100 μg/L of all analytes.
Add 5 mL of calibrator 5 working standard stock solution to a
10 mL volumetric flask and fill to 10 mL with methanol. Cap,
mix well, and transfer to a 12 mL screw-top glass bottle.
8. Calibrator 3 10 working standard: 50 μg/L of all analytes.
Add 5 mL of calibrator 4 working standard stock solution to a
10 mL volumetric flask and fill to 10 mL with methanol. Cap,
mix well, and transfer to a 12 mL screw-top glass bottle.
9. Calibrator 2 10 working standard: 20 μg/L of all analytes.
Add 4 mL of calibrator 3 working standard stock solution to a
10 mL volumetric flask and fill to 10 mL with methanol. Cap,
mix well, and transfer to a 12 mL screw-top glass bottle.
10. Calibrator 1 10 working standard: 10 μg/L of all analytes.
Add 5 mL of calibrator 2 working standard stock solution to a
10 mL volumetric flask and fill to 10 mL with methanol. Cap,
 Cannabinoids in Urine 15

mix well, and transfer to a 12 mL screw-top glass bottle (see

Note 2).
11. Quality control (QC) intermediate individual analyte stock
solutions: 100 mg/L of THC, 11-OH-THC, THCCOOH,
CBD, CBN, CBG, and THCV. Transfer 0.5 mL of each
1.0 mg/mL stock solution of THC, 11-OH-THC,
THCCOOH, CBD, CBN, CBG, and THCV to individual
5 mL volumetric flasks. Use different 1.0 mg/mL ampules
than were used for the calibrators. Fill to 5 mL with methanol.
Cap, mix well, and transfer to 12 mL glass screw-top bottle.
12. High QC 10 working standard: 4000 μg/L THCCOOH
and 2000 μg/L all other analytes. Add 200 μL of each
100 mg/L stock solution of THC, 11-OH-THC, CBD,
CBN, CBG, THCV, and THCVCOOH into a 10 mL volu-
metric flask. Add 400 μL of 100 mg/L THCCOOH stock
solution to the same flask. Fill to volume with methanol.
Cap, mix well, and transfer to a 12 mL screw-top glass bottle
(see Note 3).
13. Mid QC 10 working standard: 300 μg/L for all analytes. Add
30 μL of each 100 mg/L stock solution of THC, 11-OH-
THC, THCCOOH, CBD, CBN, CBG, THCV, and
THCVCOOH to a 10 mL volumetric flask and fill to 10 mL
with methanol. Cap, mix well, and transfer to a 12 mL screw-
top glass bottle.
14. Low QC 10 working standard: 30 μg/L for all analytes. Add
1 mL of mid QC working standard stock solution to a 10 mL
volumetric flask and fill to 10 mL with methanol. Cap, mix
well, and transfer to a 12 mL screw-top glass bottle.
15. Hydrolysis control: 6045 μg/L THCCOOH-glucuronide.
Add 604.5 μL of 100 μg/mL THCCOOH-glucuronide stan-
dard to a 10 mL volumetric flask and fill to 10 mL with
methanol. Cap, mix well, and transfer to a 12 mL screw-top
glass bottle. Yields 4000 μg/L THCCOOH if hydrolysis effi-
ciency is 100%.

2.3 Solutions and 1. Internal standard intermediate stock solution: 10 mg/L of

Buffers each deuterated compound. Add 0.5 mL of each 100 mg/L
stock solution of d3-THC, d3-11-OH-THC, d9-THCCOOH,
d3-CBD, and d3-CBN to a 5 mL volumetric flask. Fill flask to
5 mL with methanol, cap, and mix well. Store solution in
capped, screw-top glass 17  60 mm vial at 20  C.
2. 50 μg/L internal standard working solution: pipette 250 μL of
10 mg/L internal standard intermediate stock solution into a
50 mL volumetric flask. Fill to 50 mL with methanol, cap, and
mix well. Transfer working internal standard into a 50 mL
16 Karl B. Scheidweiler and Allan J. Barnes

screw-top glass bottle, cap tightly, and store at 20  C (see

Note 4).
3. 2 M sodium phosphate monobasic solution. Weigh out 60 g of
sodium phosphate monobasic, and transfer to a 250 mL volu-
metric flask. Fill with water to 250 mL, cap, and mix until
powder is completely dissolved.
4. 2 M sodium phosphate dibasic solution. Weigh out 71 g of
sodium phosphate dibasic and transfer to a 250 mL volumetric
flask, fill with water to 250 mL, cap, and mix until powder is
completely dissolved.
5. 2 M sodium phosphate buffer, pH 6.8. Transfer 100 mL of 2 M
sodium phosphate dibasic solution to a 1 L beaker. Add mag-
netic stir bar, place on magnetic stir plate at slow stir speed, and
insert calibrated pH electrode. While stirring, slowly add 2 M
sodium phosphate monobasic solution until pH 6.8  0.1 is
achieved. Transfer solution to glass reagent bottle, and store
capped at room temperature. Solution expires after 1 month or
sooner if precipitants are observed.
6. 5% aqueous formic acid: fill 1000 mL volumetric flask to
approximately 50% full with deionized water, add 1000 μL of
concentrated formic acid, and fill volumetric flask to 1000 mL
with deionized water. Store in screw-top glass reagent bottle at
room temperature; stable for 2 weeks.
7. Mobile phase A: 0.15% formic acid in water. Fill a 1000 mL
volumetric flask with approximately 500 mL of water, slowly
add 1.5 mL of formic acid, fill with water to 1000 mL, cap, and
mix well. Transfer solution to 1000 mL glass reagent bottle and
place on LC-MS/MS instrument.
8. Mobile phase B: 0.15% formic acid in acetonitrile. Fill a
1000 mL volumetric flask with approximately 500 mL of ace-
tonitrile, slowly add 1.5 mL of formic acid, fill with acetonitrile
to 1000 mL, cap, and mix well. Transfer solution to 1000 mL
glass reagent bottle and place on LC-MS/MS instrument.

3 Methods

3.1 Sample 1. Add 200 μL of negative urine to a labeled glass 17  60 mm

Preparation screw-top vial for each calibrator, QC, and hydrolysis control.
2. Add 20 μL of the appropriate calibrator or QC working stan-
dard solution to each vial (see Note 5).
3. Prepare the negative control by pipetting 200 μL of negative
urine and 20 μL of methanol into a glass 17  60 mm screw-
top vial.
 Cannabinoids in Urine 17

4. Pipette 200 μL of each patient urine and 20 μL of methanol

into labeled glass 17  60 mm screw-top vials.
5. Add 20 μL of working internal standard (containing d3-THC,
d3-11-OH-THC, d9-THCCOOH, d3-CBD, and d3-CBN) to
all calibrators, controls, and patient specimens.
6. Add 50 μL of 2 M sodium phosphate buffer, pH 6.8 to all
calibrators, controls, and patient specimens.
7. Add 40 μL of EBG recombinant E. coli β-glucuronidase
(2000 U/sample) to each vial and mix gently.
8. Cap samples and incubate for 16 h at 37  C in a shaking
water bath.
9. After incubation, transfer samples into 1.5 mL polypropylene
microcentrifuge tubes.
10. Add 620 μL of acetonitrile to each tube.
11. Centrifuge at 15,000g, 4  C for 10 min.
12. Transfer 550 μL of supernatant to a 1 mL, 96-deep-well plate
and transfer the plate to an automated liquid handling system.
13. Program the liquid handling system to add 200 μL of 5%
aqueous formic acid, and then perform four aspiration/dis-
pense cycles through 1 mL WAX-S tips.
14. Transfer 60 μL from the upper, organic layer into clean 0.5 mL
glass inserts residing in a 1 mL 96-well plate containing 140 μL
of mobile phase A (see Notes 6 and 7).
15. Cap inserts, vortex, and centrifuge at 700g, 4  C for 5 min.
16. Transfer the plate containing inserts to the autosampler.

3.2 Liquid 1. Analyze specimens on a Sciex 5500 QTRAP with a Selectra DA

Chromatography 100  2.1 mm, 3 μm column combined with a KrudKatcher
Tandem Mass Ultra HPLC in-line filter.
Spectrometry Analysis 2. Injection volume: 45 μL.
3. Column temperature: 40  C.
4. Flow rate: 0.5 mL/min.
5. Gradient program (see Note 8):
(a) 30% mobile phase B (MPB) for 0.5 min.
(b) 48% MPB at 1.0 min.
(c) 75% MPB at 10.0 min.
(d) 95% MPB at 10.5 min held for 2.0 min.
(e) Re-equilibrate to 30% MPB over 0.1 min, and hold for
2.0 min.
(f) Divert flow to waste from 0 to 2.5 min and for the last
6.1 min.
(g) Total run time of 14.6 min.
18 Karl B. Scheidweiler and Allan J. Barnes

6. Mass spectrometer mode: electrospray ionization (ESI) with

multiple reaction monitoring (MRM) in positive mode (see
Note 9).
7. Method parameters (see Note 10):
(h) Curtain gas flow 45 L/min.
(i) Medium collision gas.
(j) Ion spray voltage 5500 V.
(k) Source temperature 600  C.
Ion source gases 1 and 2 were optimized for each MRM period:
(l) Period I 50 L/min (gas 1) and 60 L/min (gas 2).
(m) Period II 45 L/min and 70 L/min.
(n) Period III 50 L/min and 70 L/min.
(o) Period IV 55 L/min and 70 L/min.
(p) MS/MS settings are displayed in Table 2.
8. Construct linear calibration curves daily from peak area ratios
of analytes to their respective internal standard, with 1/x2
weighting.
9. Construct calibration curves from 1 to 250 μg/L for THC and
CBG, 2–250 μg/L for 11-OH-THC, CBD, CBN, THCV, and
THCVCOOH and 1–500 μg/L for THCCOOH. Figure 1
shows extracted ion chromatograms of representative blank
human urine fortified to contain analytes at their low QC
concentrations.

4 Notes

1. Store all working calibrator, QC, hydrolysis control, and inter-

nal standard solutions at 20  C. Solutions expire after
6 months.
2. Note that calibrator 1 contains all compounds; however,
11-OH-THC, CBD, CBN, THCV, and THCVCOOH are
not quantitated below calibrator 2.
3. QC working solutions at low, medium, and high concentra-
tions are prepared across the linear dynamic range using differ-
ent ampoules than for calibrators. Store all working QC
solutions at 20  C. Solutions expire after 6 months.
4. Internal standard working and 10 mg/L stock solutions are
stored at 20  C and expire after 6 months.
5. Calibrator, QC, and hydrolysis control 10 methanolic work-
ing standard solutions are fortified into urine daily to avoid
storage stability issues for cannabinoids in urine [16].
 Cannabinoids in Urine 19

Table 2
LC-MS/MS parameters for cannabinoids, their metabolites, and internal standards in human
hydrolyzed urine

Analyte Q1 (amu) Q3 (amu) DP (V) CE (eV) CXP (V) MRM period Dwell time (msec)
THC 315.0 193.1 91 31 20 4 58
315.0 123.1 91 43 14 4 58
11-OH-THC 331.0 201.0 31 33 18 2 58
331.0 313.2 31 19 30 2 58
THCCOOH 345.0 193.0 61 35 14 2 58
345.0 327.2 61 21 28 2 58
CBD 315.0 193.0 51 29 18 3 58
315.0 123.0 51 41 14 3 58
CBN 311.0 223.0 51 29 18 4 58
311.0 241.1 51 25 24 4 58
CBG 317.0 193.2 36 21 14 3 58
317.0 122.8 36 41 18 3 58
THCV 287.1 165.0 26 29 14 3 58
287.1 123.0 26 41 10 3 58
THCVCOOH 317.0 165.1 31 35 14 1 120
317.0 271.1 31 25 20 1 120
d3-THC 318.1 196.1 76 31 20 4 58
318.1 123.0 76 43 14 4 58
d3-11-OH-THC 334.1 201.0 26 35 16 2 58
334.1 316.0 26 19 30 2 58
d9-THCCOOH 354.1 196.1 56 35 18 2 58
354.1 335.8 56 23 28 2 58
d3-CBD 318.1 195.9 26 27 16 3 58
318.1 135.2 26 25 10 3 58
d3-CBN 314.0 223.1 51 29 18 4 58
314.0 241.0 51 25 22 4 58
THC delta9-tetrahydrocannabinol, 11-OH-THC 11-hydroxy-THC, THCCOOH 11-nor-9-carboxy-THC, CBD canna-
bidiol, CBN cannabinol, CBG cannabigerol, THCV delta9-tetrahydrocannabivarin, THCVCOOH 11-nor-9-carboxy-
THCV
Bold masses depicted quantifier transitions. DP declustering potential, CE collision energy, CXP collision cell exit
potential, MRM scheduled multiple reaction monitoring
20 Karl B. Scheidweiler and Allan J. Barnes

Fig. 1 Extracted ion chromatogram showing quantifier MRM transitions after injecting an extract prepared
from 200 μL fortified urine containing each analyte at 3 μg/L. Δ9-Tetrahydrocannabinol (THC: m/z 315–193),
11-hydroxy-THC (11-OH-THC: m/z 331–201), 11-nor-9-carboxy-THC (THCCOOH: m/z 345–193), cannabinol
(CBN: m/z 311–223), cannabigerol (CBG: m/z 317–193), tetrahydrocannabivarin (THCV: m/z 287–165), and its
metabolite, 11-nor-9-carboxy-THCV (THCVCOOH: m/z 317–165)

6. The 500 μL glass inserts are used since cannabinoids were

found to bind polypropylene plates, while stability was main-
tained using the glass inserts. More than 50% loss was observed
for 2 h. when polypropylene plates (lacking glass inserts) were
used in the 4  C autosampler.
7. Glass inserts are small targets for the automated liquid handling
system and autosampler requiring careful adjustment of instru-
ment settings to ensure proper pipetting/injection.
8. If it is desired to target fewer analytes than THC, 11-OH-
THC, THCCOOH, CBD, CBN, CBG, THCV, and
THCVCOOH, a more simplified (faster) gradient could be
implemented; however, full revalidation of the method would
be required.
 Cannabinoids in Urine 21

9. Monitor two transitions for each analyte and internal standard.

Optimize MS/MS parameters via infusion of individual ana-
lytes at 10 μL/min (20 μg/L).
10. Mass spectrometer settings may differ for individual
instruments.

Acknowledgments

This research was funded by the Intramural Research Program of

the National Institute on Drug Abuse, National Institutes of
Health. Recombinant EBG™ β-glucuronidase from E. coli was
provided via a Materials Transfer Agreement between the National
Institutes of Health and KURA Biotec. The authors thank Yves-
Vincent Duperron (KURA Biotec) for his technical assistance.

References
1. Hall W, Kozlowski LT (2017) The diverging 7. Hidvegi E, Somogyi GP (2010) Detection of
trajectories of cannabis and tobacco policies in cannabigerol and its presumptive metabolite in
the United States: reasons and possible impli- human urine after Cannabis consumption.
cations. Addiction. https://doi.org/10.1111/ Pharmazie 65(6):408–411
add.13845 8. Kemp PM, Abukhalaf IK, Manno JE, Manno
2. Smith A (2017) 10 things to know about legal BR, Alford DD, Abusada GA (1995) Cannabi-
pot. http://money.cnn.com/2017/04/19/ noids in humans. I. Analysis of delta
news/legal-marijuana-420/index.html. 9-tetrahydrocannabinol and six metabolites in
Accessed 1 Aug 2017 plasma and urine using GC-MS. J Anal Toxicol
3. Bergamaschi MM, Karschner EL, Goodwin 19(5):285–291
RS, Scheidweiler KB, Hirvonen J, Queiroz 9. Campbell CT, Phillips MS, Manasco K (2017)
RH, Huestis MA (2013) Impact of prolonged Cannabinoids in pediatrics. J Pediatr Pharma-
cannabinoid excretion in chronic daily cannabis col Ther 22(3):176–185. https://doi.org/10.
smokers’ blood on per se drugged driving laws. 5863/1551-6776-22.3.176
Clin Chem 59(3):519–526. https://doi.org/ 10. Blessing EM, Steenkamp MM, Manzanares J,
10.1373/clinchem.2012.195503 Marmar CR (2015) Cannabidiol as a potential
4. Aizpurua-Olaizola O, Zarandona I, Ortiz L, treatment for anxiety disorders. Neurothera-
Navarro P, Etxebarria N, Usobiaga A (2017) peutics 12(4):825–836. https://doi.org/10.
Simultaneous quantification of major cannabi- 1007/s13311-015-0387-1
noids and metabolites in human urine and 11. Sloan ME, Gowin JL, Ramchandani VA, Hurd
plasma by HPLC-MS/MS and enzyme-alkaline YL, Le Foll B (2017) The endocannabinoid
hydrolysis. Drug Test Anal 9(4):626–633. system as a target for addiction treatment: trials
https://doi.org/10.1002/dta.1998 and tribulations. Neuropharmacology doi:
5. Bergamaschi MM, Barnes A, Queiroz RH, https://doi.org/10.1016/j.neuropharm.
Hurd YL, Huestis MA (2013) Impact of enzy- 2017.05.031
matic and alkaline hydrolysis on CBD concen- 12. Abraham TT, Lowe RH, Pirnay SO, Darwin
tration in urine. Anal Bioanal Chem 405 WD, Huestis MA (2007) Simultaneous GC-
(14):4679–4689. https://doi.org/10.1007/ EI-MS determination of Delta9-
s00216-013-6837-x tetrahydrocannabinol, 11-hydroxy-Delta9-
6. ElSohly MA, deWit H, Wachtel SR, Feng S, tetrahydrocannabinol, and 11-nor-9-carboxy-
Murphy TP (2001) Delta9- Delta9-tetrahydrocannabinol in human urine
tetrahydrocannabivarin as a marker for the following tandem enzyme-alkaline hydrolysis.
ingestion of marijuana versus Marinol: results J Anal Toxicol 31(8):477–485
of a clinical study. J Anal Toxicol 25 13. Andersson M, Scheidweiler KB, Sempio C,
(7):565–571 Barnes AJ, Huestis MA (2016) Simultaneous
22 Karl B. Scheidweiler and Allan J. Barnes

quantification of 11 cannabinoids and metabo- β-glucuronidase hydrolysis and quantification

lites in human urine by liquid chromatography of eight urinary cannabinoids and metabolites
tandem mass spectrometry using WAX-S tips. by liquid chromatography tandem mass spec-
Anal Bioanal Chem 408(23):6461–6471. trometry. Drug Test Anal 10(3):518–529.
https://doi.org/10.1007/s00216-016-9765-8 https://doi.org/10.1002/dta.2230
14. Skopp G, Potsch L (2002) Stability of 11-nor-- 16. Desrosiers NA, Lee D, Scheidweiler KB,
delta(9)-carboxy-tetrahydrocannabinol glucu- Concheiro-Guisan M, Gorelick DA, Huestis
ronide in plasma and urine assessed by liquid MA (2014) In vitro stability of free and glucur-
chromatography-tandem mass spectrometry. onidated cannabinoids in urine following con-
Clin Chem 48(2):301–306 trolled smoked cannabis. Anal Bioanal Chem
15. Sempio C, Scheidweiler KB, Barnes AJ, Huestis 406(3):785–792. https://doi.org/10.1007/
MA (2018) Optimization of recombinant s00216-013-7524-7
 Chapter 3

Analysis of Benzodiazepines for Drug-Facilitated Assaults

and Abuse Settings (Urine)
Olaf H. Drummer, Matthew Di Rago, and Dimitri Gerostamoulos

Abstract
An overview of the detection of benzodiazepines and their respective metabolites and target analytes in
urine by LC-MS/MS is described. This overview shows substantial differences in the approach to detection
using this technique including optional use of β-glucuronidase to hydrolyze conjugates present in urine.
There are also significant variations in the extraction method employed from the use of direct injection,
liquid-liquid extraction to solid-phase extraction options, with little apparent difference in limits of
detection. Chromatography was largely based on the use of C18-bonded columns; however both C8-
and phenyl-bonded columns were used to affect separation. Modern-day tandem mass spectrometers are
capable of exceptional sensitivity enabling detection of sub-nanogram per milliliter amounts in urine, which
provide for longer detection times in the urine of suspected drug-facilitated assaults. A method employed in
the laboratory of the authors is provided by way of an example for readers wishing to establish a method in
their own laboratory.

Key words Tandem mass spectrometry, Liquid chromatography, Benzodiazepines, Metabolites,

β-Glucuronidase, Solid-phase extraction, Liquid-liquid extraction, Novel psychoactive drugs

1 Introduction

Benzodiazepines are a class of drugs that interact with the GABAA

receptor and comprise well over 50 substances that range from
short-acting hypnotics to long-acting anxiolytics. Examples of
short-acting benzodiazepines include midazolam, oxazepam, and
temazepam, while diazepam is a common example of a longer-
acting drug used largely to relieve anxiety and act as minor tran-
quilizers. Increasingly, novel, but illicit, benzodiazepines are
becoming available as novel psychoactive drugs that have been
detected in many countries [1].
Benzodiazepines are one of the more common drug types to be
used by assailants to sedate victims and render them unable or less
able to resist an assault (sexual and/or to engage in theft). This
crime is often termed drug-facilitated (sexual) assault (DFSA).

Loralie J. Langman and Christine L.H. Snozek (eds.), LC-MS in Drug Analysis: Methods and Protocols,
Methods in Molecular Biology, vol. 1872, https://doi.org/10.1007/978-1-4939-8823-5_3,
© Springer Science+Business Media, LLC, part of Springer Nature 2019

23
24 Olaf H. Drummer et al.

Importantly, most members of this class of drug also cause amnesia,

reducing the ability of the victim to remember details of the assault.
In a recent review, the incidence of benzodiazepines in victims of
DFSA ranged from 3.5% to 38.7% with a median of 11% [2],
although they were often in combination with other drugs, includ-
ing alcohol, illicit, and prescription drugs.
This chapter provides an overview of published techniques for
benzodiazepines and their relevant metabolites and close analogs in
urine using liquid chromatography tandem mass spectrometry
(LC-MS/MS) and provides a method for their determination in
urine using this technique.

1.1 Benzodiazepines Once consumed benzodiazepines are mostly metabolized and

and Their Analogs excreted into urine. Metabolites range from simple glucuronide
conjugates such as with oxazepam to hydroxylation and subsequent
conjugation and/or dealkylation. The dominant form in urine is
often a metabolite rather than the parent drug. A few drugs that are
structurally unrelated to benzodiazepines bind to a neighboring
site of the GABAA receptor and function largely as hypnotics. These
include zolpidem and zopiclone (Z-drugs) and are included in this
chapter.
Table 1 lists the individual members of the benzodiazepine
class and the related Z-drugs and identifies key metabolites that
need to be targeted in a urine analysis. The substances that possess a
hydroxy group will usually also exist as a glucuronide metabolite.
Hence to achieve maximum sensitivity for detection of the
non-conjugated metabolite in urine, a de-conjugation step is
advised prior to an extraction or preparation step and introduction
into the chromatograph.

1.2 Overview There have been numerous methods published using LC-MS/MS
of Published Methods to determine the presence of benzodiazepines in urine, particularly
over the last 10 years. A selection of those specializing in drugs used
in assault cases, and in particular benzodiazepines and/or their
Z-drug analogs, and relevant metabolite analytes (n ¼ 20) are
summarized in Table 2 [3–22]. The list is restricted to references
published over the last 10 years.
The methods vary substantially in their approach; most (15 of
20) use some form of hydrolysis to liberate conjugated benzodia-
zepines from their respective glucuronides, although there is sig-
nificant variation in the source of enzyme used and the hydrolysis
conditions. The optimization of these hydrolysis conditions is
rarely reported, let alone for all of the known conjugates.
LC-MS/MS used in these publications represented all of the
major instrument manufacturers and will vary substantially in sen-
sitivity due to the configuration and design of the mass analyzers.
Many of the more recent analyzers show much higher sensitivity
than the older LC-MS/MS instruments. Almost all LC-MS/MS
 Benzodiazepines in Urine 25

Table 1
Selection of benzodiazepines and related drugs, their key metabolites, and target analytes in urine

Benzodiazepine Key metabolites Key analyte for urinalysis

Alprazolam 1-Hydroxy alprazolam 1-Hydroxy alprazolam
(hydrolysis)
Bromazepam 3-Hydroxy bromazepam and glucuronides 3-Hydroxy bromazepam
(hydrolysis)
Clobazam Norclobazam (desmethylclobazam) Clobazam and norclobazam
Clonazepam 7-Acetamido-, 7-amino-, and 3-hydroxy- 7-Aminoclonazepam
clonazepam
Clonazolam 7-Amino-, 7-acetamido-clonazolam 7-Aminoclonazolam
Chlordiazepoxide Demoxepam, oxazepam, and glucuronide Oxazepam (hydrolysis)
Diazepam Nordiazepam, oxazepam, temazepam, and their Oxazepam, temazepam
glucuronides (hydrolysis)
Etizolam 1- and 8-hydroxy etizolam 1-Hydroxyetizolam
Estazolam 4-Hydroxy- and 1-oxo-estazolam 4-Hydroxyestazolam
(hydrolysis)
Flunitrazepam 7-Acetamido-, 7-aminoflunitrazepam 7-Aminoflunitrazepam
Flurazepam N-1-Desalkyl-, hydroxyethylflurazepam N-1-Desalkylflurazepam
(hydrolysis)
Lorazepam Lorazepam glucuronides Lorazepam (hydrolysis)
Lormetazepam Lorazepam and glucuronides Lorazepam (hydrolysis)
Midazolam 1-Hydroxy midazolam 1-Hydroxy midazolam
Nordiazepam Temazepam and oxazepam glucuronides Temazepam and oxazepam
(hydrolysis)
Oxazepam Oxazepam glucuronides Oxazepam (hydrolysis)
Phenazepam 3-Hydroxy-phenazepam and glucuronides 3-Hydroxy-phenazepam
(hydrolysis)
Temazepam Oxazepam, temazepam, and glucuronides Oxazepam and temazepam
(hydrolysis)
Nitrazepam 7-Acetamido-, 7-aminonitrazepam 7-Aminonitrazepam
Tetrazepama 3-Hydroxyl and glucuronides Tetrazepam
Triazolam 1-Hydroxy triazolam 1-Hydroxy triazolam
(hydrolysis)
Zopiclone N-Desmethyl-zopiclone, zopiclone-N-oxide Zopiclone, N-desmethyl-
zopiclone
Zolpidem Carboxy metabolite of 4-methyl Zolpidem and carboxy
metabolite
Note: Hydrolysis refers to hydrolysis of urine liberating conjugated drug or metabolite
a
Also metabolized to diazepam
 26

Table 2
Analysis parameters for selected publications describing tandem LC-MS detection of benzodiazepines in urine

Mass spectrometer, number of

benzodiazepine (BZ) analytes,
Reference Pre-treatment of urine Extraction Chromatography conditions LOD
Olaf H. Drummer et al.

ElSohly et al. β-Glucuronidase/sulfatase LLE ChCl3/isopropanol (9:1) Luna C8 (4.6  100 mm, 3 μm), Agilent 1100 SL TOF-MS ESI
[3] (4 h, 37  C) 0.1% acetic acid/ACN 22 BZ/metabolites
LOD 0.5–3 ng/mL
Glover and β-Glucuronidase LLE DCM/DCE/heptane/ HyPURITY C8 (3  150 mm), 1% API 3000 TQ API
Allen [4] (PV) (0.5 h, 56  C) isopropanol HCOOH, 1% isopropanol ACN 6 BZ
gradient LOD 2.5–5 ng/mL
Guale et al. None SPE Strata-X-Drug B Eclipse Plus C18 (3  100 mm, Agilent 6230 TOF-MS,
[5] 1.8 μm), 5 mM NH4COO, 5–60% 15 BZ/metabolites,
MeOH gradient LOD not given
Hegsted β-Glucuronidase (HP-2) Direct injection ACQUITY HSS T3 (2.1  100 mm, Waters Xevo TQ-S ESI
et al. [22] (1 h, 65  C) and without 1.8 μm), 0.1% HCOOH/MeOH, 5 BZ
50 C LOD 2–6 ng/mL
Jagerdeo and β-Glucuronidase SPE Isolute SLE, Cortex C18 (2.1  50 mm, 1.6 μm), Waters Orbitrap
Schaff [6] (RA) (0.5 h, 68  C) DCM/isopropanol (95:5) 0.1% HCOOH/ACN 22 BZ/metabolites,
LOD 1 ng/mL
Jeong et al. None Direct injection Zorbax SB-C18 (2.1  100 mm, API 3200 TQ ESI
[7] 3.5 μm), 2 mM trifluoroacetate/ 18 BZ
0.2% acetic acid ACN gradient LOD 0.3–3 ng/mL
Karampela None Direct injection XTerra MS C8 (2.1  250 mm, Shimadzu 2010EV ESI LC-MS,
et al. [8] 5 μm), 0.05% HCOOH/ACN 10 BZ
gradient LOD >10 ng/mL
Lee et al. [9] None Ethyl acetate LLE ACE5 C18 (4.6  250 mm, 5 μm), API 4000 Q-Trap ESI
0.1% HCOOH/ACN gradient 21 BZ and others
LOD >0.5 ng/mL
Mata et al. β-Glucuronidase (HP-2) DPX WAX tips ACQUITY BEH C18 Waters Xevo TQ-S, ESI
[18] (3 h, 55  C) (2.1  100 mm, 1.7 μm), water 17 BZ,
0.1% HCOOH/ACN 0.1% LOD 0.5–12.5 ng/mL
HCOOH
Marin et al. β-Glucuronidase SPE Trace-B, ethyl acetate: NH3 XTerra MS C18 (2.1  150 mm, Waters, Micromass, Alliance,
[10] (BL) (2 h, 60  C) (98:2) 3.5 μm), 100 mM HCOOH/ Quattro ESI
ACN gradient 13 BZ/metabolites
LOD <10 ng/mL
Ming and β-Glucuronidase Direct injection ACQUITY BEH C18 Waters TQD ESI,
Heathcote (EC) (1.5 h 60  C) (2.1  50 mm, 1.7 μm), 0.2% 13 BZ/metabolites LOD
[11] HCOOH/MeOH gradient, 45 C 0.5–2 ng/mL
Perez et al. β-Glucuronidase (HP-2) SPE UCT Clean Screen XCEL I, ACQUITY BEH C18 (1.7 μm, Waters Quattro micro, ESI
[19] (1 h, 55  C) ethyl acetate: NH4OH (100:2) 2.1  50 mm), (A) 0.1% HCOOH 5 BZ, LOD 2–16 ng/mL
and (B) acetonitrile
Petterson β-Glucuronidase (10 min, Direct injection ACQUITY BEH phenyl column Waters Xevo TQ ESI
Bergstrand 25  C) (1.0  50 mm, 1.7 μm), 11 designer BZ
et al. [12] ACN/0.1% HCOOH gradient LOD 1–10 ng/mL
Remane et al. β-Glucuronidase Direct injection Zorbax Eclipse XDB C18 API 4000 Q-Trap ESI
[13] (EC) (0.5 h, 55  C) and (4.6  100 mm, 5 μm), 50 mM 24 BZ/metabolites plus others
without HCOOH LOD meet SOFT criteria
Rosano et al. β-Glucuronidase (1 h, Direct injection ACQUITY BEH Phenyl Waters Xevo TQD, ESI
[20] 55  C) (2.1  50 mm, 1.7 μm), 2 mM 16 BZ,
NH4COO/2 mM NH4COO, LOD 25 ng/mL
0.1% HCOOH in ACN
Salomone β-Glucuronidase LLE pH 7.5, DCM/propan-2-ol Eclipse XDB C18 (4.6  50 mm, API 3200 triple Q-Trap,
Benzodiazepines in Urine

et al. [14] (HP) (1 h, 55  C) (85:15) 1.8 μm), MeOH/water gradient 23 BZ/analogs, LOD
0.5–30 ng/mL

(continued)
27
 28

Table 2
(continued)

Mass spectrometer, number of

benzodiazepine (BZ) analytes,
Reference Pre-treatment of urine Extraction Chromatography conditions LOD
Schaefer et al. β-Glucuronidase (1.5 h, Direct injection – column switching Cyclone MAX (2.1  50 mm, 5 μm), Thermo Fisher TSQ ESI
[15] 40  C) 10 mM NH4HCO3/0.1% 10 BZ/metabolites
HCOOH/ACN LOD mostly <10 ng/mL
Olaf H. Drummer et al.

Tang et al. β-Glucuronidase (A) (1 h, SPE Waters Oasis MCX (combined Eclipse C8 (3  100 mm, 1.8 μm), Agilent TQ ESI
[21] 55  C) acidic, basic) 5 mM NH4COO, 0.1% HCOOH, 14 BZ
MeOH LOD 2–100 ng/mL
Verplaetse β-Glucuronidase Bond Elut Plexa PCX SPE, elution ACQUITY C18 (2.1  50 mm, API 3200 Q-Trap, ESI,
et al. [16] (PV) (3 h, 65  C) acetone/CHCl3 (1:1), and 1.7 μm), 10 mM NH4HCO3 29 BZ/metabolites
ammoniated ethyl acetate MeOH gradient LOD >0.02 ng/mL
Xiong et al. None Online SPE (Oasis HLB) with XTerra MS C18 (2.1  150 mm, Waters Quattro Premier XE ESI,
[17] formic acid 3.5 μm), ACN/MeOH/water 8 BZ and glucuronidated
metabolites
LOD from 0.2 ng/mL
 Benzodiazepines in Urine 29

methods listed in Table 2 use electrospray ionization techniques

(ESI). However, ESI is more likely to exhibit ion suppression or ion
enhancement compared to atmospheric pressure chemical ioniza-
tion (APCI).
Recently, a series of designer benzodiazepines have appeared
throughout much of the world, presumably to overcome laws
surrounding availability of benzodiazepines in various countries
[1, 23]. These include clonazolam, deschloroetizolam, diclazepam,
etizolam, flubromazepam, flubromazolam, flutazolam, meclonaze-
pam, nifoxipam, phenazepam, and pyrazolam [12], although some
of these compounds have previously been available in other parts of
the world, e.g., phenazepam in Russia. These analytes have been
shown to be easily analyzed by LC-MS/MS using a rapid
de-conjugation step with β-glucuronidase at 25  C over 10 min
and direct injection of the supernatant into an ultrahigh-pressure
liquid chromatograph (UPLC) tandem mass spectrometer under
positive ESI. This technique was an adaptation of a previously
published method for a wider range of psychoactive drugs in
urine that did not utilize a hydrolysis step [24].
One limitation with most methods utilizing LC-MS/MS is
when multiple-reaction mode (MRM) is used and preselected tran-
sitions are monitored, the resulting method will be limited to
identification of known substances, rather than screening for any
unknown substances, such as other benzodiazepines. The use of
data-dependent acquisition (DDA) modality for ions that exceed a
certain threshold is fragmented (and collected) allowing for both
screening and identification on a single chromatographic run
[25, 26].
The following describes a method used in the authors’ labora-
tory for the detection of a range of psychotropic drugs, including
benzodiazepines, in urine. This method is typically used in cases of
suspected drug-facilitated assault (see Notes 1, 2, and 3) and gen-
erally follows a general unknown screen using one or more meth-
ods, such as high-resolution LC-MS, targeted tandem LC-MS,
GC-MS, or a benzodiazepine immunoassay screen.

2 Materials

Prepare all buffers in deionized water using chemicals that are at

least analytical reagent grade. Use solvents that are of liquid chro-
matography grade.
Some of the newer substances may be sourced from Toronto
Research Chemicals (Canada), Cayman Chemical (USA), or
Chiron (Norway).
Deuterated benzodiazepine internal standards are available
from AptoChem, Cerilliant, LGC, or Lipomed (see Note 4).
30 Olaf H. Drummer et al.

2.1 Solutions 1. Borate buffer, pH 9.8: Dissolve 0.775 g of boric acid and 4.1 g
and Reagents of sodium tetraborate in 250 mL of water. Adjust to pH 9.8
(See Note 5) using 1 M sodium hydroxide.
2. 1.1 M sodium acetate buffer, pH 4.5: Dissolve 22.56 g of
anhydrous sodium acetate in 250 mL of water. Adjust to pH
4.5 with glacial acetic acid.
3. 1 M sodium hydroxide (NaOH): Add 4 g of sodium hydroxide
to 100 mL of water. Mix well.
4. β-Glucuronidase: Commercial enzyme sourced from red aba-
lone (Haliotis rufescens) 7000 units (U) per mL or 14,000 U
per sample.
5. Extraction solvent: Dichloromethane/isopropanol/ethyl ace-
tate, 1:1:3 v/v/v. Add 100 mL each of dichloromethane and
isopropanol to 300 mL of ethyl acetate, and mix gently.
6. Stock standard: 1 mg/mL each drug or metabolite. Dilute
2.00 mg of each benzodiazepine or metabolite in 2.00 mL
methanol. Stock standards can be kept for at least 3 months
stored at 20  C.
7. Working standard 1 (WS1): Dilute 0.1 mL of each stock stan-
dard to 1 mL with methanol (see Note 6).
8. Working standard 2 (WS2): Dilute 0.05 mL of each stock
standard to 5 mL with water.
9. Working standard 3 (WS3): Dilute 0.05 mL of each stock
standard to 50 mL with water.
10. Mobile phase A: 50 mM ammonium formate pH 3.5. Add
3.15 g of ammonium formate to 1 L of water. Adjust pH to
3.5 with concentrated formic acid.
11. Mobile phase B: 0.1% formic acid in acetonitrile. Add 1 mL of
formic acid to 1 L of acetonitrile.

2.2 Supplies 1. pH meter.

and Equipment 2. Reciprocating shaker or vortex mixer.
3. Benchtop centrifuge.
4. Nitrogen evaporator.
5. LC-MS/MS such as SCIEX 3200 quadrupole mass spectrom-
eter operated in multiple reaction monitoring modes.
6. Agilent Zorbax Eclipse XDB-C18 (4.6 mm  150 mm, 5 μm
particle size) or similar.
 Benzodiazepines in Urine 31

3 Methods

3.1 Sample Analysis 1. Obtain drug-free urine (with consent) from a volunteer, and
use this to prepare a series of calibration standards for one or
more of the benzodiazepine and/or metabolites (see Note 7).
Typically a range of concentrations are prepared at or above the
limit of detection to a concentration within the range of the
spectrometer and often seen in samples. The typical calibration
ranges for some of the more common analytes are listed in
Table 3 (see Note 8).

Table 3
Multiple-reaction mode transitions and typical calibration range for selected benzodiazepines,
metabolites, and related analogs

Calibration Parent First Second Other measured

Analyte range (mg/L) mass transition transition transitions
Alprazolam 0.01–0.25 309 281 205 274
1-Hydroxyalprazolam 0.01–0.25 325 216 189 297, 197
Flunitrazepam 0.005–0.25 314 268 239 183
7-Aminoflunitrazepam 0.005–0.25 284 135 93 226, 227
7-Aminodesmethylflunitrazepam 0.005–0.05 270 121 222
Hydroxyflunitrazepam 0.005–0.25 330 284 237
Desmethylflunitrazepam 0.005–0.25 300 254 198
Clonazepam 0.005–0.25 316 270 214 241
7-Aminoclonazepam 0.005–0.25 286 222 121 250
Nitrazepam 0.0050–0.25 282 236 180 207
7-Aminonitrazepam 0.005–0.25 252 121 208 94, 77
Bromazepam 0.01–0.25 316 182 209 181
Clobazam 0.01–0.25 301 193 224 259, 105, 153
Desmethylclobazam 0.01–0.25 287 245 210
Chlordiazepoxide 0.01–0.25 300 282 227
Diazepam 0.01–1 285 154 193 222
Estazolam 0.01–0.25 296 268 206
Etizolam 0.01–0.25 343 314 259
Flurazepam 0.01–0.1 388 315 317 134
Desalkylflurazepam 0.01–0.25 289 140 226
Lorazepam 0.01–0.25 321 275 303 229

(continued)
32 Olaf H. Drummer et al.

Table 3
(continued)

Calibration Parent First Second Other measured

Analyte range (mg/L) mass transition transition transitions
Lormetazepam 337 291 75 289, 317
Midazolam 0.01–0.25 326 291 249 223
Nordiazepam 0.01–1 271 140 165 208
Oxazepam 0.01–1 287 241 269 104
Prazepam 0.005–0.25 325 271 140 272
Phenazepam 0.05–0.50 349 206 179 242
Triazolam 0.005–0.25 343 308 239
1-Hydroxytriazolam 0.005–0.05 359 331 239
Temazepam 0.01–1 301 235 177 193, 255, 283
Zopiclone 0.01–0.25 389 245 217 112
Zolpidem 0.01–0.10 308 263 235 236

2. For example, a calibration range of 5–3000 ng/mL is shown

below using the prepared working standards (WS1–WS3) for
2 mL urine. These will usually contain several benzodiazepines
and/or relevant metabolites.

Concentration Volume Volume Volume

(ng per mL of urine) WS1 (μL) WS2 (μL) WS3 (μL)
5 10
10 20
30 60
100 20
300 60
1000 20
3000 60

3. Obtain at least two control specimens containing a known

amount of the benzodiazepine and/or metabolite (see Note 9).
4. Add 0.5 mL of 1.1 M sodium acetate buffer (pH 4.5) and
β-glucuronidase enzyme (14,000 U total) to 2 mL urine speci-
 Benzodiazepines in Urine 33

Shake on
2mL Urine
reciprocating Centrifuge 3000
Sample, QC or
shaker 5 min, rpm, 5 min
Calibrator
5000 rpm

Add 14000 IU
Abalone Add 6mL Transfer solvent
β-glucuronidase extraction solvent layer to glass vial
enzyme

Adjust sample
Add 0.5 mL 1.1M Evaporate to Inject 3uL into
pH to 8.5 with
sodium acetate dryness under N2 LC-MS/MS
1M sodium
buffer gas @ 40˚C system
hydroxide

Add 1mL 1M
Incubate Reconstitute with Transfer to
borate buffer pH
overnight @ 55˚C 0.05mL methanol autosampler vial
9.8

Fig. 1 Flow chart for extraction of benzodiazepines from urine

mens, prepared standards, and control samples. Incubate over-

night at 55  C (see Note 10).
5. To each hydrolyzed urine sample, add 1 mL of borate buffer
(pH 9.8) and 0.3 mL of 1 M sodium hydroxide to adjust pH to
8.5.
6. Add 6 mL of extraction solvent to each sample and gently
agitate on a reciprocating shaker or vortex mixer for 5 min
(see Fig. 1).
7. Centrifuge tubes briefly to separate layers. Separation should
occur quickly.
8. Transfer solvent (top layer) to clean glass tubes, and evaporate
to dryness under a stream of nitrogen gas at 40  C.
9. Reconstitute residue in 0.05 mL of methanol and transfer to
autosampler vials for analysis.

3.2 Analysis 1. Analytical column: Agilent Zorbax Eclipse XDB-C18

(4.6 mm  150 mm, 5 μm particle size) (see Note 11).
2. Column temperature: 60  C.
3. Gradient (can be optimized for separation of similar
compounds):
34 Olaf H. Drummer et al.

Mobile phase B (%) Time (min) start Flow rate (mL/min)

10 1.4
10 1 2.2
100 18 2.2
100 20 2.2
10 20.1 1.4
10 24 1.4

4. Injection volume: 3 μL. Operate the autosampler at room

temperature, and rinse the needle with methanol before and
after every injection.
5. Analyze by mass spectrometry (see Notes 12 and 13). Example
chromatograms of a standard extract and a DFSA case are
shown in Fig. 2.
6. Evaluate MRM for the presence of one of more of the target
analytes (see Note 14). If MRM are present and meet accep-
tance criteria and substance is above the reporting limit, then
the identification can be accepted.
7. An approximate concentration can be calculated based on the
calibration responses of the calibrator(s) (see Note 15).
8. Depending on the type of case and laboratory policy, another
test may be required including the presence of identified sub-
stance(s) in other specimens (see Notes 16 and 17).

4 Notes

1. A reference aimed at providing detailed guidance over optimi-

zation of LC-MS methods can be found on the web under the
Royal Society of Chemistry and the National Measurement
System (UK) [27].
2. Analytical methods should be validated to internationally
recognized guidelines. An example of validation procedures
and performance levels has been published by the Scientific
Working Group in Toxicology (SWGTOX, 2013) [28]. Any
good-quality HPLC instrument can be used. Window detec-
tion threshold of 5% is usually acceptable for retention time
monitoring.
3. The procedure is also useful for the detection of other drugs of
interest including drugs of abuse and a range of prescribed and
over-the-counter drugs.
4. Deuterated internal standards should be included and paired
with the target analytes to correct for any changes in recovery
 Benzodiazepines in Urine 35

Fig. 2 (a) Calibration standard in urine containing benzodiazepines and some metabolites at 0.25 mg/L, except
for flurazepam, nordiazepam, and temazepam which are at 0.1 mg/L. (b) Chromatogram of a urine from a
DFSA case containing alprazolam (0.01 mg/L) and its 1-hydroxy metabolite (0.03 mg/L) and diazepam with
three of its metabolites, nordiazepam (0.2 mg/L), oxazepam (0.02 mg/L), and temazepam (0.2 mg/L)
36 Olaf H. Drummer et al.

and MS response. A number of these are available for commer-

cial suppliers and include d5-alprazolam, d5-diazepam,
d5-nordiazepam, d5-oxazepam, d5-temazepam, and the
d5-amino-benzodiazepine metabolites. More than one deuter-
ated internal standard is recommended for longer or more
complex runs.
5. Reagents can be stored at ambient temperature for up to
3 months.
6. Calibration standards can be made fresh for each batch or larger
batch (e.g., 10 mL) aliquoted into small volumes sufficient for
each assay and stored frozen for up to 6 months at 20  C.
7. When performing a chromatography-based screen prior to this
assay, the type of benzodiazepine and its approximate concen-
tration will be suspected. This will give the analyst information
on what drugs to include in this method and the corresponding
calibration concentrations. It is highly recommended to also
include with each batch at least one and preferably several
matrix-matched calibrators at various concentrations. These
calibrators allow analysts to ensure that both the chromatogra-
phy and MS conditions are satisfactory and that substances in
unknown samples can be confirmed and nominated an approx-
imate concentration. This allows some interpretation to be
made with respect to known pharmacokinetics of the drug in
question.
8. Samples containing large concentrations may require dilution if
a more accurate estimate of concentration is required as the ion
source or detector will show saturation effects at higher con-
centrations, leading to substantial nonlinearity. Due to signifi-
cant sensitivity of LC-MS/MS techniques, detection limits can
easily reach below 1 ng/mL.
9. Quality controls are also highly recommended to include in
each batch to provide an indicator of accuracy of quantifica-
tions from each analytical run. Commercial controls are avail-
able for the more common benzodiazepines. Acceptance limits
are generally around 20% of the mean but can be set to two
standard deviations obtained from several analytical batches.
When dealing with low detection limits, such as often seen in
DFSA cases, benzodiazepine/metabolite concentrations may
be closer to LOD than at the middle or high end of the
calibration range.
10. A hydrolysis marker is recommended, such as oxazepam glucu-
ronide, or any other available glucuronide metabolite to pro-
vide a measure of the extent of hydrolysis in each batch.
Addition of deuterated glucuronide analyte to each sample
can also allow for monitoring of hydrolysis performance in
each sample.
 Benzodiazepines in Urine 37

11. Many alternative column dimensions, particle sizes, and sta-

tionary phases may be used, in which case optimization of
instrument conditions is required including flow rate (see
Table 2).
12. Each LC-MS/MS will require specialized setup and optimiza-
tion. Parameters such as ion spray voltage; source temperature;
curtain, heater, and nebulizing gas flows; detector dwell times,
and MRM detection windows will need to be optimized for the
compounds to be monitored. Analytes to be detected will
require instrument-specific optimization of parent mass and
product transitions to be monitored. At least two transitions
are required for detection and confirmation, in combination
with transition ratio and retention time matching with certified
standards. Table 3 summarizes the transitions for most of the
more common benzodiazepines and metabolites using the ABI
3200 Q-Trap.
13. Other LC-MS/MS systems can be used, provided that MS
parameters have been optimized for all the analytes. Instru-
ment parameters such as source, quadrupole, and detector
settings can yield different analyte responses, resulting in vary-
ing limits of detection and quantification. These will need to be
optimized for each laboratory.
14. Acceptance criteria for transition ratios should be established.
This is often 20% (absolute) of a reference response from a
known positive result included in the analytical run; however,
criteria need to be established from validation data. If these
criteria are not met, then identification has not occurred.
15. Calibration curves are established by calculating peak area
ratios for the various calibration standards over the peak area
of the respective internal standard and obtaining a regression fit
from the lowest to highest calibrator. These regression fits are
typically >0.99 (r2) unless a clear outlier can be determined.
The peak area to internal standard ratio for any identified
benzodiazepine or metabolite in the specimens is then used
in the regression fit to obtain the concentration in the sample.
This regression calculation and computation is typically done
in software that comes with the instruments, and this may
require nonlinear treatment with a quadratic fit most common.
Quality controls run with each batch must meet the laboratory
acceptance criteria; otherwise an assay would need to be
repeated if quantitative data is reported.
16. It is highly recommended that an experienced toxicologist
reviews results in case a metabolite can be sourced from inges-
tion of other benzodiazepines.
38 Olaf H. Drummer et al.

17. The Society of Forensic Toxicologists (SOFT) has recom-

mended detection limits and cutoffs for drugs and metabolites
of relevance to drug-facilitated assaults; see http://www.soft-
tox.org/files/MinPerfLimits_DCF2017.pdf.

Acknowledgments

The authors gratefully acknowledge the staff of the toxicology

section of the Victorian Institute of Forensic Medicine, many of
whom have contributed to the validation and use of the procedure
outlined in this chapter.

References

1. Sundstrom M, Pelander A, Angerer V, 8. Karampela S, Vardakou I, Papoutsis I et al

Hutter M, Kneisel S, Ojanpera I (2013) A (2012) Direct urine analysis for the identifica-
high-sensitivity ultra-high performance liquid tion and quantification of selected benzodiaze-
chromatography/high-resolution time-of- pines for toxicology screening. J Chromatogr B
flight mass spectrometry (UHPLC-HR- 902:42–46
TOFMS) method for screening synthetic can- 9. Lee H-H, Lee J-F, Lin S-Y, Chen B-H (2016)
nabinoids and other drugs of abuse in urine. Simultaneous identification of abused drugs,
Anal Bioanal Chem 405(26):8463–8474 benzodiazepines, and new psychoactive sub-
2. Anderson LJ, Flynn A, Pilgrim JL (2017) A stances in urine by liquid chromatography tan-
global epidemiological perspective on the toxi- dem mass spectrometry. Kaohsiung J Med Sci
cology of drug-facilitated sexual assault: a sys- 32(3):118–127
tematic review. J Forensic Legal Med 47:46–54 10. Marin SJ, Coles R, Merrell M, McMillin GA
3. ElSohly MA, Gul W, ElSohly KM, Avula B, (2008) Quantitation of benzodiazepines in
Khan IA (2006) LC-MS-(TOF) analysis urine, serum, plasma, and meconium by
method for benzodiazepines in urine samples LC-MS-MS. J Anal Toxicol 32(7):491–498
from alleged drug-facilitated sexual assault vic- 11. Ming DS, Heathcote J (2011) A rapid and
tims. J Anal Toxicol 30(8):524–538 accurate UPLC/MS/MS method for the
4. Glover SJ, Allen KR Measurement of benzo- determination of benzodiazepines in human
diazepines in urine by liquid chromatography- urine. J Chromatogr B 879(5):421–428
tandem mass spectrometry: confirmation of 12. Pettersson Bergstrand M, Helander A, Beck O
samples screened by immunoassay, pp (2016) Development and application of a
1758–1001 Electronic multi-component LC-MS/MS method for
5. Guale F, Shahreza S, Walterscheid JP et al determination of designer benzodiazepines in
(2013) Validation of LC-TOF-MS screening urine. J Chromatogr B Analyt Technol Biomed
for drugs, metabolites, and collateral com- Life Sci 1035:104–110
pounds in forensic toxicology specimens. J 13. Remane D, Wetzel D, Peters FT (2014) Devel-
Anal Toxicol 37(1):17–24 opment and validation of a liquid
6. Jagerdeo E, Schaff JE (2016) Rapid screening chromatography-tandem mass spectrometry
for drugs of abuse in biological fluids by ultra (LC-MS/MS) procedure for screening of
high performance liquid chromatography/ urine specimens for 100 analytes relevant in
Orbitrap mass spectrometry. J Chromatogr B drug-facilitated crime (DFC). Anal Bioanal
1027:11–18 Chem 406(18):4411–4424
7. Jeong Y-D, Kim MK, Suh SI, In MK, Kim JY, 14. Salomone A, Gerace E, Brizio P, Gennaro MC,
Paeng K-J (2015) Rapid determination of ben- Vincenti M (2011) A fast liquid
zodiazepines, zolpidem and their metabolites chromatography-tandem mass spectrometry
in urine using direct injection liquid chromato- method for determining benzodiazepines and
graphy–tandem mass spectrometry. Forensic analogues in urine. Validation and application
Sci Int 257:84–92 to real cases of forensic interest. J Pharm
Biomed Anal 56(3):582–591
 Benzodiazepines in Urine 39

15. Schaefer N, Peters B, Schmidt P, Ewald AH 93 conventional and emerging drugs of abuse
Development and validation of two LC-MS/ and their metabolites in urine by UHPLC-
MS methods for the detection and quantifica- MS/MS. J Chromatogr B Analyt Technol
tion of amphetamines, designer amphetamines, Biomed Life Sci 969:272–284
benzoylecgonine, benzodiazepines, opiates, 22. Hegstad S, Hermansson S, Betner I, Spigset O,
and opioids in urine using turbulent flow chro- Falch BM (2014) Screening and quantitative
matography. Anal Bioanal Chem 405 determination of drugs of abuse in diluted
(1):247–258 urine by UPLC-MS/MS. J Chromatogr B
16. Verplaetse R, Cuypers E, Tytgat J (2012) The Analyt Technol Biomed Life Sci
evaluation of the applicability of a high pH 947-948:83–95
mobile phase in ultrahigh performance liquid 23. Hoiseth G, Tuv SS, Karinen R (2016) Blood
chromatography tandem mass spectrometry concentrations of new designer benzodiaze-
analysis of benzodiazepines and pines in forensic cases. Forensic Sci Int
benzodiazepine-like hypnotics in urine and 268:35–38
blood. J Chromatogr A 1249:147–154 24. Al-Saffar Y, Stephanson NN, Beck O (2013)
17. Xiong L, Wang R, Liang C et al (2015) Deter- Multicomponent LC-MS/MS screening
mination of co-administrated opioids and ben- method for detection of new psychoactive
zodiazepines in urine using column-switching drugs, legal highs, in urine-experience from
solid-phase extraction and liquid chromatogra- the Swedish population. J Chromatogr B Ana-
phy–tandem mass spectrometry. J Chromatogr lyt Technol Biomed Life Sci 930:112–120
A 1395:99–108 25. Sturm S, Hammann F, Drewe J, Maurer HH,
18. Mata DC, Davis JF, Figueroa AK, Stanford MJ Scholer A (2010) An automated screening
(2016) Ultra performance liquid chromatogra- method for drugs and toxic compounds in
phy with tandem mass spectrometry for the human serum and urine using liquid
quantitation of seventeen sedative hypnotics chromatography-tandem mass spectrometry. J
in six common toxicological matrices. J Anal Chromatogr B Analyt Technol Biomed Life Sci
Toxicol 40(1):58–63 878(28):2726–2732
19. Perez ER, Knapp JA, Horn CK, Stillman SL, 26. Sundström M, Pelander A, Ojanpera I (2017)
Evans JE, Arfsten DP (2016) Comparison of Comparison of post-targeted and pre-targeted
LC-MS-MS and GC-MS analysis of benzodiaz- urine drug screening by UHPLC-HR-
epine compounds included in the drug demand QTOFMS. J Anal Toxicol
reduction urinalysis program. J Anal Toxicol 40 27. Sargent M (ed) (2013) Guide to achieving
(3):201–207 relaible quantitative LC-MS measurements,
20. Rosano TG, Ohouo PY, Wood M (2017) 1st edn. RSC Analytical Standards Committee,
Screening with quantification for 64 drugs Cambridge
and metabolites in human urine using UPLC- 28. SWGTOX 2013 Scientific working group for
MS-MS analysis and a threshold accurate cali- forensic toxicology (SWGTOX) standard prac-
bration. J Anal Toxicol:1–11 tices for method validation in forensic toxicol-
21. Tang MH, Ching CK, Lee CY, Lam YH, Mak ogy 20 May
TW (2014) Simultaneous detection of
 Chapter 4

Targeted Opioid Screening Assay for Pain Management

Using High-Resolution Mass Spectrometry
Darlington Danso, Loralie J. Langman, and Paul J. Jannetto

Abstract
The use and adherence monitoring of opioids in pain management is recommended by numerous clinical
practice guidelines. Many physicians use urine immunoassay screening tests, which suffer from a lack of
sensitivity and specificity, to verify compliance to pain medications. However, several immunoassay tests are
required to comprehensively detect the synthetic, semisynthetic, and natural opioids due to the limited
cross-reactivity of each assay. Superior testing strategies are required to specifically identify low concentra-
tions of opioids found in adherent pain management patients. Therefore we present a method for the
qualitative identification of 33 opioids and metabolites (codeine, codeine-6-β-glucuronide, morphine,
morphine-6-β-glucuronide, 6-acetylmorphine, hydrocodone, norhydrocodone, dihydrocodeine, hydro-
morphone, hydromorphone-3-β-glucuronide, oxycodone, noroxycodone, oxymorphone, oxymorphone-
3-β-glucuronide, noroxymorphone, meperidine, normeperidine, methadone, EDDP, propoxyphene, nor-
propoxyphene, tramadol, O-desmethyltramadol, tapentadol, tapentadol-β-glucuronide, N-desmethylta-
pentadol, buprenorphine, norbuprenorphine, norbuprenorphine glucuronide, naloxone, naloxone
glucuronide, fentanyl, and norfentanyl) in unhydrolyzed urine using a liquid chromatography tandem
mass spectrometry (LC-MS/MS) with high-resolution, accurate-mass Orbitrap detection.

Key words Opioids, High-resolution mass spectrometry, Pain management, Accurate-mass

1 Introduction

Opioids are a large class of medications commonly used to relieve

acute and chronic pain or help manage opioid abuse and depen-
dence [1, 2]. Medications that fall into this class include buprenor-
phine, codeine, fentanyl, hydrocodone, hydromorphone,
methadone, morphine, oxycodone, oxymorphone, tapentadol, tra-
madol, and others. Opioids work by binding to the opioid recep-
tors that are found in the brain, spinal cord, gastrointestinal tract,
and other organs [3]. Common side effects depend on the dose and
include drowsiness, confusion, nausea, constipation, and in severe
cases respiratory depression [3]. These medications can also pro-
duce physical and psychological dependence and have a high risk

Loralie J. Langman and Christine L.H. Snozek (eds.), LC-MS in Drug Analysis: Methods and Protocols,
Methods in Molecular Biology, vol. 1872, https://doi.org/10.1007/978-1-4939-8823-5_4,
© Springer Science+Business Media, LLC, part of Springer Nature 2019

41
42 Darlington Danso et al.

for abuse and diversion which is one of the main reasons many
professional practice guidelines recommend compliance testing in
patients prescribed these medications.
Opioids are readily absorbed from the gastrointestinal tract,
nasal mucosa, and lungs and after subcutaneous or intramuscular
injection. Opioids are primarily excreted from the kidney in both
free and conjugated forms. This assay does not hydrolyze the urine
sample and therefore allows detection of both parent drugs and
metabolites (including glucuronide forms). The detection window
for most opioids in urine is approximately 1–3 days with longer
detection times for some compounds (e.g., methadone). We
describe a liquid chromatography tandem mass spectrometry
(LC-MS/MS) method with high-resolution, accurate-mass Orbi-
trap detection to qualitatively (present vs. not detected) identify
33 opioid compounds (parent drugs and metabolites) in urine to
help determine compliance and/or identify illicit opioid drug use.
The method is a simple dilute-and-shoot in which urine samples are
diluted (1:10) with internal standard (IS) in clinical laboratory
reagent water (CLRW) and then subjected to chromatographic
separation on a Restek Ultra Biphenyl column and analyzed on
the Orbitrap with heated electrospray ionization in positive mode.
The mass spectrometry method is full MS followed by data-
dependent MS2. This data-dependent experiment collects full
scan data and MS/MS spectra for all compounds defined in the
inclusion list. A step gradient elution of the Ultra Biphenyl column
achieves a complete chromatographic separation of isobaric com-
pounds with a total run time of 8 min. Compound identification
combines exact mass (<5 ppm), delta retention time, and MS2
spectral library match.

2 Materials

2.1 Samples and 1. Collect urine samples under proper supervision to ensure spec-
Reagents imen integrity and to avoid specimen manipulation that might
cause false negative results (see Note 1).
2. 0.5% formic acid in CLRW. Add 5 mL of formic acid to a 1 L
reagent bottle. Bring to volume with CLRW and mix thor-
oughly. Stable for 1 month at 20–27
C.
3. Negative (drug-free) urine. Obtain drug-free urine commer-
cially (e.g., UTAK) or from volunteers. Verify as drug-free by
assaying prior to use in this method.
4. Wash solution # 1: 2% acetonitrile/1% formic acid in water.
Add 20 mL of acetonitrile and 10 mL of formic acid to a 1 L
reagent bottle. Bring to volume with CLRW and mix thor-
oughly. Stable for 1 month at 20–27
C.
 Opioids by HRMS 43

5. Wash solution # 2: 45% acetonitrile/45% isopropanol/10%

acetone. Add 450 mL of isopropanol, 450 mL of acetonitrile,
and 100 mL of acetone to a 1 L reagent bottle, then mix
thoroughly. Stable for 1 month at 20–27
C.
6. Mobile phase 1: 10 mM ammonium formate/0.1% formic acid
in CLRW. Add 1.26 g of ammonium formate, 2 L of CLRW,
and 2 mL of formic acid to a 2 L reagent bottle, then mix
thoroughly. Stable for 1 month at 20–27
C.
7. Mobile phase 2: 0.1% formic acid in acetonitrile. Add 2 L of
acetonitrile and 2 mL of formic acid to a 2 L reagent bottle,
then mix well. Stable for 2 months at 20–27
C.
8. 50:50 methanol/CLRW. Add 5 mL of methanol and 5 mL of
CLRW in a flask and mix well. Make fresh when needed.

2.2 Standards and Targeted drugs and stock concentrations are shown in Table 1.
Controls
1. Intermediate stock internal standards: 10 μg/mL of each deut-
erated compound. To separate 10 mL volumetric flasks, add
1 mL of each 100 μg/mL stock solution (dihydrocodeine-d6,
buprenorphine-d4, norbuprenorphine-d3, fentanyl-d5,
norfentanyl-d5) or 100 μL of each 1 mg/mL stock solution
(oxycodone-d6 or methadone-d9). Bring to volume with
methanol. Stable 10
C in amber glass vials with Teflon
caps for 2 years.
2. Working internal standard: 7.5 ng/mL of oxycodone-d6, dihy-
drocodeine-d6, and methadone-d9, 1.0 ng/mL of fentanyl-d5
and norfentanyl-d5, and 2.5 ng/mL of buprenorphine-d4 and
norbuprenorphine-d5. Add 75 μL of the oxycodone-d6 inter-
mediate, 75 μL of the dihydrocodeine-d6 intermediate, 75 μL
of the methadone-d9 intermediate, 25 μL of the
buprenoprhine-d4 intermediate, 25 μL of the
norbuprenorphine-d5 intermediate, 10 μL of the fentanyl-d5
intermediate, and 10 μL of the norfentanyl-d4 intermediate
stock solution to a 100 mL volumetric flask. Bring to volume
with 0.5% formic acid in CLRW. Mix well for 20–30 min.
Stable for 2 months at 2–8
C.
3. Intermediate opioid standard: 10 μg/mL of each drug. Add
100 μL of each 1.0 mg/mL stock of methadone, EDDP,
meperidine, cis-tramadol, tapentadol, o-desmethyltramadol,
dihydrocodeine, n-desmethyltramadol, norhydrocodone, oxy-
codone, noroxycodone, hydromorphone, oxymorphone, nor-
meperidine, morphine, noroxymorphone, hydrocodone,
codeine, and naloxone to a 10 mL volumetric flask. Bring to
volume with methanol, and mix well. Stable at or below
10
C in amber glass vials with Teflon caps for 2 years or
until manufacturer expiration of stock I, whichever comes first.
44 Darlington Danso et al.

Table 1
Standard concentrations (stock and intermediate calibration solution), negative control, cutoff, and
positive control values for the targeted opioid screen

Positive
Stock Intermediate Negative Cutoff control
concentration concentration control value value value
Drug (mg/mL) (μg/mL) (ng/mL) (ng/mL) (ng/mL)
Codeine 1.0 10.0 10.0 25.0 50.0
Codeine-6-β-glucuronide 0.1 10.0 50.0 100.0 200.0
Morphine 1.0 10.0 10.0 25.0 50.0
Morphine- 1.0 10.0 50.0 100.0 200.0
6-β-glucuronide
6-Acetylmorphine 1.0 10.0 10.0 25.0 50.0
Hydrocodone 1.0 10.0 10.0 25.0 50.0
Norhydrocodone 1.0 10.0 10.0 25.0 50.0
Dihydrocodeine 1.0 10.0 10.0 25.0 50.0
Hydromorphone 1.0 10.0 10.0 25.0 50.0
Hydromorphone- 0.1 10.0 50.0 100.0 200.0
3-β-glucuronide
Oxycodone 1.0 10.0 10.0 25.0 50.0
Noroxycodone 1.0 10.0 10.0 25.0 50.0
Oxymorphone 1.0 10.0 10.0 25.0 50.0
Oxymorphone- 0.1 10.0 50.0 100.0 100.0
3-β-glucuronide
Noroxymorphone 1.0 10.0 10.0 25.0 50.0
Fentanyl 1.0 1.0 1.0 2.0 5.0
Norfentanyl 1.0 1.0 1.0 2.0 5.0
Meperidine 1.0 10.0 10.0 25.0 50.0
Normeperidine 1.0 10.0 10.0 25.0 50.0
Naloxone 1.0 10.0 10.0 25.0 50.0
Naloxone- 1.0 10.0 50.0 100.0 200.0
3-β-glucuronide
Methadone 1.0 10.0 10.0 25.0 50.0
EDDP 1.0 10.0 10.0 25.0 50.0
Propoxyphene 1.0 10.0 10.0 25.0 50.0
Norpropoxyphene 1.0 10.0 10.0 25.0 50.0
Tramadol 1.0 10.0 10.0 25.0 50.0

(continued)
 Opioids by HRMS 45

Table 1
(continued)

Positive
Stock Intermediate Negative Cutoff control
concentration concentration control value value value
Drug (mg/mL) (μg/mL) (ng/mL) (ng/mL) (ng/mL)
O-Desmethyltramadol 1.0 10.0 10.0 25.0 50.0
Tapentadol 1.0 10.0 10.0 25.0 50.0
N-Desmethyltapentadol 1.0 20.0 20.0 50.0 100.0
Tapentadol-β-glucuronide 0.1 10.0 50.0 100.0 200.0
Buprenorphine 1.0 2.0 2.0 5.0 10.0
Norbuprenorphine 1.0 2.0 2.0 5.0 10.0
Norbuprenorphine 1.0 10.0 10.0 20.0 50.0
glucuronide

4. Intermediate glucuronide standard: 10 μg/mL of each conju-

gated metabolite. Add 1.0 mL of each 100 μg/mL stock of
morphine-6β-D-glucuronide, naloxone-3β-D-glucuronide,
tapentadol-β-D-glucuronide, codeine-6β-D-glucuronide, oxy-
morphone-3β-D-glucuronide, and hydromorphone-3β-D-glu-
curonide to a 10 mL volumetric flask. Bring to volume with
50:50 methanol/CLRW and mix well. Stable at or below
10
C in amber glass vials with Teflon caps for 2 years or
until manufacturer expiration of stock I, whichever comes first.
5. 10 μg/mL norbuprenorphine glucuronide intermediate stan-
dard (see Note 2). Add 100 μL of the 1.0 mg/mL norbupre-
norphine glucuronide stock to a 10 mL volumetric flask. Bring
to volume with methanol, and mix well. Stable at or below
10
C in amber glass vials with Teflon caps for 2 years or until
manufacturer expiration of stock I, whichever comes first.
6. 1.0 μg/mL fentanyl and norfentanyl intermediate standard.
Add 10 μL of each 1.0 mg/mL stock of fentanyl and norfenta-
nyl to a 10 mL volumetric flask and bring to volume with
methanol. Stable at or below 10
C in amber glass vials with
Teflon caps for 2 years or until manufacturer expiration of
stock I, whichever comes first.
7. 2.0 μg/mL buprenorphine and norbuprenorphine intermedi-
ate standard. Add 20 μL of each 1.0 mg/mL stock of bupre-
norphine and norbuprenorphine to a 10 mL volumetric flask
and bring to volume with methanol. Stable at or below 10
C
in amber glass vials with Teflon caps for 2 years or until manu-
facturer expiration of stock I, whichever comes.
46 Darlington Danso et al.

8. 20 μg/mL N-desmethyltapentadol intermediate standard. Add

200 μL of 1.0 mg/mL N-desmethyltapentadol stock to a
10 mL volumetric flask and bring to volume with methanol.
Stable at or below 10
C in amber glass vials with Teflon caps
for 2 years or until manufacturer expiration of stock I, which-
ever comes.
9. Cutoff calibrator. To a 50 mL volumetric flask, add 100 μL of
the fentanyl and norfentanyl intermediate. Add 125 μL of the
buprenorphine and norbuprenorphine intermediate. Add
125 μL of the N-desmethyltapentadol intermediate. Add
100 μL of the norbuprenorphine glucuronide intermediate.
Add 500 μL of the intermediate glucuronide standard. Add
125 μL of the intermediate opioid standard. Bring to volume
with negative urine, then mix well for 30 min. Stable for 1 year
at 10
to 35
C.
10. Negative control: 50% of the cutoff concentration. To a
50 mL volumetric flask, add 50 μL of the fentanyl and norfen-
tanyl intermediate. Add 50 μL of the buprenorphine and nor-
buprenorphine intermediate. Add 50 μL of the N-
desmethyltapentadol intermediate. Add 50 μL of the norbu-
prenorphine glucuronide intermediate. Add 250 μL of the
intermediate glucuronide standard. Add 50 μL of the interme-
diate opioid standard. Bring to volume with negative urine,
and then mix well for 30 min. Stable for 1 year at 10
to
35
C.
11. Positive control: 200% of the cutoff concentration. To a
50 mL volumetric flask, add 250 μL of the fentanyl and nor-
fentanyl intermediate. Add 250 μL of the buprenorphine and
norbuprenorphine intermediate. Add 250 μL of the N-des-
methyltapentadol intermediate. Add 250 μL of the norbupre-
norphine glucuronide intermediate. Add 1000 μL of the
intermediate glucuronide standard. Add 250 μL of the inter-
mediate opioid standard. Bring to volume with negative urine,
and then mix well for 30 min. Stable for 1 year at 10
to
35
C.

2.3 Supplies and 1. 12  75 mm disposable borosilicate glass test tubes (manual

Equipment method/daily prime).
2. Microplate 96 deep-well PP square well, 2 mL/well, DNase/
RNase free.
3. Restek Ultra BiPh analytical column, 50  3.0 mm, 3 μm.
4. Centrifuge with rotors for 12  75 mm test tubes.
5. Adhesive seal.
6. Vortexer.
7. TraceFinder 4.1 clinical research software.
 Opioids by HRMS 47

8. Thermo Scientific Q Exactive Plus, Dionex UltiMate degasser

3000, Leap Ctc Pal auto-injector, or equivalent, and Dionex
UltiMate 3000 RS Pump.

3 Methods

3.1 Extraction 1. Mix all patient samples briefly, aliquot into labelled
Procedure (see Note 3) 12  75 mm disposable borosilicate glass test tubes, and cen-
trifuge for 10 min at 2850 g.
2. Aliquot 100 μL of each blank, standard, control, and patient
sample into its own sample well of a 96-well plate. This assay
uses a one point calibration (the cutoff calibrator), a negative
control, a positive control, and a blank (drug-free urine) with
each batch (see Note 4).
3. Add 900 μL of working internal standard to each well, and then
mix by vortexing.
4. Cover the plate with an adhesive seal for analysis.

3.2 Analysis 1. Injection volume: 20 μL.

2. Flow rate: 0.50 mL/min.
3. Total run time: 8 min.
4. Mobile-phase gradient conditions are shown in Table 2 (see
Notes 5 and 6). Figure 1 shows chromatograms demonstrat-
ing separation of isobaric compounds.
5. Mass spectrometer source conditions are shown in Table 3.
6. Analyze data: Compound identification is by retention time,
exact masses (m/z) at 5 ppm (Table 4), and spectral library
match (see Note 7).

Table 2
Gradient conditions

Start Time (s) % Mobile phase B

0.00 30 0
0.50 250 20
4.67 50 100
5.50 35 100
6.08 130 0
48 Darlington Danso et al.

Fig. 1 Chromatograms showing the separation of isobaric compounds

Table 3
Mass spec parameters (source conditions)

HESI source Actual

Sheath gas flow rate 55
Aux gas flow rate 15
Sweep gas flow rate 2
Spray voltage (kv) 3.50
Spray current (μA) 0.0


Capillary temp ( C) 320
S-lens RF level
Aux gas heater temp (
C) 350

4 Notes

1. Common adulterants known to affect results are water, soap,

bleach, vinegar, and salt. Preferred specimen volume size is
5 mL and a minimum of 1.0 mL. From the results of 28 days’
stability studies conducted, samples stored refrigerated were
stable for 14 days, those stored frozen were stable for
28 days, and those stored ambient were stable for 72 h. Icteric
and hemolyzed samples are rejected.
2. With a lower cutoff value (10 μg/mL) than the rest of the
glucuronides, the norbuprenorphine glucuronide intermediate
standard is made by itself. Unique intermediates of fentanyl,
norfentanyl, buprenorphine, norbuprenorphine, and
 Opioids by HRMS 49

Table 4
Transitions and retention times for drug identification

Rt Rt
Drug m/z (min) Drug m/z (min)
Codeine 300.15918 3.10 Meperidine 248.16428 3.86
Codeine-6-β-glucuronide 476.19151 2.62 Normeperidine 234.14871 3.85
Morphine 286.14337 1.97 Naloxone 328.15433 2.90
Morphine-6-β-glucuronide 462.17586 1.77 Naloxone-3-β-glucuronide 504.18642 1.82
6-Acetylmorphine 328.15359 3.37 Methadone 310.21628 4.10
Hydrocodone 300.15912 3.71 EDDP 278.19012 4.11
Norhydrocodone 286.14328 3.50 Propoxyphene 340.22687 4.02
Dihydrocodeine 302.17435 2.83 Norpropoxyphene 308.20056 4.11
Hydromorphone 286.14346 2.42 Tramadol 264.19547 3.83
Hydromorphone-3-β- 462.17586 1.43 O-Desmethyltramadol 250.17993 3.12
glucuronide
Oxycodone 316.15387 3.48 Tapentadol 222.18501 3.83
Noroxycodone 302.13846 3.28 N-desmethyltapentadol 208.16959 3.83
Oxymorphone 302.13837 2.12 Tapentadol-β-glucuronide 398.21713 3.29
Oxymorphone-3-β- 478.17080 1.08 Buprenorphine 468.31061 3.97
glucuronide
Noroxymorphone 288.12303 1.63 Norbuprenorphine 414.26343 3.85
Fentanyl 337.22711 3.97 Norbuprenorphine glucuronide 590.29597 3.51
Norfentanyl 233.16461 3.76
Rt retention time

n-desmethyltapentadol are made for similar reasons. This

makes the preparation of the working standard and controls
in the matrix easier.
3. All procedures are carried out at room temperature. Standard,
controls, and reagents are allowed to equilibrate to room tem-
perature before using. All necessary precautions should be
taken when it comes to handling of samples to avoid potential
infections.
4. Sample preparation could be readily automated. Carryover
studies should always be investigated during method develop-
ment to rule out the possibility of false positives from spiked
patient samples and also from analytes with high concentration.
5. Interference studies for the common prescribed drugs, over-
the-counter drugs, therapeutic drugs, and common drugs of
50 Darlington Danso et al.

abuse should be conducted during method development.

Matrix effects should also be investigated during method
development.
6. The gradient elution shown off of Ultra Biphenyl Restek col-
umn achieved complete chromatographic separation of isobaric
compounds.
7. Since this is a qualitative assay, results are reported “present”
when analyte concentrations are above the cutoff or “not
detected” when concentrations are below the cutoff. Accep-
tance criteria are expected retention time and actual retention
time of each analyte within 0.10 min, better than 5 ppm mass
accuracy; and spectral library score >80%.

References

1. Owen GT, Burton AW, Schade CM, Passik S http://www.cdc.gov/mmwr/volumes/65/rr/

(2012) Urine drug testing: current recommen- pdfs/rr6501e1.pdf. Accessed 15 Jun 2017
dations and best practices. Pain Physician 15: 3. Gutstein HB, Akil H (2006) Opioid analgesics.
ES119–ES133 In: Brunton LL, Lazo JS, Parker KL (eds) The
2. Centers For Disease Control And Prevention pharmacological basis of therapeutics, 11th edn.
Public Health Service U S Department Of McGraw-Hill Companies Inc., Goodman & Gil-
Health And Human Services (2016) Centers man’s http://www.accessmedicine.com/con
for disease control guideline for prescribing tent.aspx?aID¼940653
opioids for chronic pain—United States.
 Chapter 5

Measurement of Buprenorphine and Norbuprenorphine

in Urine
Andrea R. Terrell, Vipin Adhlakha, and Poluru Reddy

Abstract
Buprenorphine, a synthetic opioid possessing both analgesic and opioid receptor antagonist properties, has
proven to be an effective therapeutic aid for opioid dependency and chronic pain management. The
downside, as with all opioids, natural or synthetic, is its potential for misuse and abuse. The euphoria
induced by buprenorphine leads to abuse. Additionally, individuals with an active addiction to short-acting
opioids such as heroin may use buprenorphine between doses of their drug of choice to stave off withdrawal
symptoms. As such, buprenorphine monitoring is utilized in medication-assisted therapy programs for
opioid dependency, as well as chronic pain management settings. Buprenorphine may also be included in
drug testing programs for law enforcement purposes. The assay described here was designed to detect and
quantify both buprenorphine and its metabolite norbuprenorphine.

Key words Buprenorphine, Norbuprenorphine, LC-MS/MS, Opioid

1 Introduction

Buprenorphine is a versatile synthetic opioid. In the 1970s bupre-

norphine was used as a low-dose analgesic for postoperative and
cancer patients. Reports of abuse began soon after its introduction.
In 1985 the United States classified buprenorphine as a schedule V
narcotic analgesic [1]. In 2002 the DEA placed all formulations
containing buprenorphine onto schedule III and approved it for
medication-assisted treatment of opioid dependency [2].
According to statistics provided by the Drug Abuse Warning
Network (DAWN), buprenorphine-related ED visits involving
nonmedical use did not reach a measurable level until 2006, when
there were a reported 4440 visits to emergency departments. The
number of these visits then increased 384% to 21,483 visits in
2011. More recently, visits increased 51% from 14,266 visits in
2009 to 21,483 visits in 2011 [3].

Loralie J. Langman and Christine L.H. Snozek (eds.), LC-MS in Drug Analysis: Methods and Protocols,
Methods in Molecular Biology, vol. 1872, https://doi.org/10.1007/978-1-4939-8823-5_5,
© Springer Science+Business Media, LLC, part of Springer Nature 2019

51
52 Andrea R. Terrell et al.

It has 25–40 times the analgesic efficacy of morphine [4]. The

primary receptors that mediate opioid effects are the μ and the ĸ
receptors. Buprenorphine’s versatility lies in its dual mechanisms of
action, having both μ (agonist) and ĸ (antagonist) mediating prop-
erties. This facilitates the drug’s effectiveness in opioid dependency
treatment (antagonist activity) and in chronic pain management
(agonist activity). The drug may also be slowly withdrawn without
the discomfort that often accompanies methadone withdrawal.
Buprenorphine’s bioavailability is maximized with sublingual
administration. Sublingual formulations of buprenorphine, alone
or in combination with naloxone, are available. When used as
directed, the buprenorphine/naloxone combination delivers the
analgesic and antagonist effects of the buprenorphine, without
the full antagonist effects of the naloxone. If the combination
drug is injected, the bioavailability of the naloxone is increased
which precipitates withdrawal and blocks the euphoric and analge-
sic properties in opioid-dependent individuals [5]. An extended
release transdermal formulation designed for relief of moderate to
severe chronic pain was approved in 2010. In a study comparing
sublingual vs. transdermal buprenorphine in osteoarthritis patients,
both forms were similarly effective, with transdermal buprenor-
phine demonstrating better tolerability.
Buprenorphine is metabolized in the liver via the CYP3A4
pathway to norbuprenorphine, its primary metabolite. Both bupre-
norphine and norbuprenorphine are pharmacologically active.
Accordingly, both compounds should be monitored. Additionally,
both parent and metabolite are extensively metabolized to their
glucuronide conjugates. Drug monitoring in urine is performed for
purposes of assessing compliance or detecting illicit use; thus, it is
desirable to measure total drug in the sample. Free buprenorphine
and norbuprenorphine generally represent just 1% of a dose, so
hydrolysis is performed prior to measurement in urine.
Commercially available immunoassays are often used to screen
for buprenorphine in urine samples. Liquid chromatography with
mass spectrometric detection is now commonly used for confirma-
tion, or as the presumptive and definitive test, if immunoassay is not
used. Enzymatic hydrolysis has proven to be more effective than
acid or base hydrolysis for releasing the glucuronide conjugate from
buprenorphine and norbuprenorphine.
This chapter describes a method for quantitative determination
of total buprenorphine and norbuprenorphine in urine utilizing
liquid chromatography with tandem mass spectrometry (LC-MS/
MS). The method, as described here, was extracted from a larger
analytical procedure for the quantitative analysis of 48 drugs and
metabolites in urine. The chromatography conditions described in
this chapter have been optimized for the 48-drug panel. Adjust-
ments should be made to optimize for a method that contains only
buprenorphine, norbuprenorphine, and corresponding internal
standards.
 Buprenorphine and Norbuprenorphine 53

2 Materials

2.1 Prepared 1. Mobile phase A: 0.1% formic acid in water: Break 1 ampule of
Reagents (See Note 1) high-grade formic acid and transfer the entire contents (1 mL)
using a glass pipette (see Note 2) into 1 L of HPLC grade
water. Store at room temperature for up to 1 month (see
Note 3).
2. Mobile phase B: 0.1% formic acid in methanol: Break 1 ampule
of high-grade formic acid and transfer the entire contents
(1 mL) using a glass pipette into 1 L of HPLC grade methanol.
Store at room temperature for up to 1 month.
3. Needle rinse: 60/20/20 isopropanol/methanol/acetonitrile:
Combine 200 mL of methanol, 200 mL of acetonitrile, and
600 mL of isopropanol. Store at room temperature for up to
1 month.
4. 200 mM phosphate buffer, pH 6.8: Weigh out 8.0 g of
sodium phosphate monobasic dihydrate and 8.7 g of sodium
phosphate dibasic dihydrate and mix to dissolve in 500 mL of
water. Using pH paper or a pH meter, verify pH is 6.8 (0.2).
Store at room temperature for up to 2 months.
5. 20 mM ammonium formate buffer in 80/20 water/meth-
anol, pH 3.7: Weigh 250 mg of ammonium formate and
dissolve in 160 mL of mobile phase A. Add 40 mL of mobile
phase B. Using pH paper or a pH meter, verify pH is 3.7
(0.2). Store at room temperature for up to 1 month.
6. Internal Standard (IS) spiking solution: 50 ng/mL each
IS. Combine 10 μL of 100 μg/mL buprenorphine-d4 stock
solution, 10 μL of 100 μg/mL norbuprenorphine-d3 stock
solution, 5 mL of acetonitrile, and 15 mL of water in a glass
or polypropylene tube. Mix well. Store refrigerated for up to
1 month.
7. Composite mix solution (CMS BUP): 10,000 ng/mL
buprenorphine and norbuprenorphine. Combine 100 μL
of 100 μg/mL buprenorphine standard, 100 μL of 100 μg/
mL norbuprenorphine standard, and 800 μL of acetonitrile in a
glass screw cap vial. Mix well.
8. Composite spiking solution (CSS) 1: 2000 ng/mL bupre-
norphine and norbuprenorphine. Combine 200 μL of CMS
BUP with 800 μL of acetonitrile in a glass screw cap vial. Store
at 20  C for up to 2 months.
9. CSS 2: 400 ng/mL buprenorphine and norbuprenor-
phine. Mix 200 μL of CSS 1 with 800 μL of acetonitrile in a
glass screw cap vial. Store at 20  C for up to 2 months.
54 Andrea R. Terrell et al.

10. CSS 3: 100 ng/mL buprenorphine and norbuprenor-

phine. Mix 200 μL of CSS 2 with 600 μL of acetonitrile in a
glass screw cap vial. Store at 20  C for up to 2 months.
11. CSS 4: 20 ng/mL buprenorphine and norbuprenorphine.
Mix 100 μL of CSS 3 with 400 μL of acetonitrile in a glass
screw cap vial. Store at 20  C for up to 2 months.
12. System suitability solution: Combine 4 mL of water and
1 mL of methanol in a glass or polypropylene vial. Add 10 μL
of CSS 1, mix, and store refrigerated for up to 1 month.
13. Quality control (QC) mix solution (QMS BUP): Using a
different aliquot of stock standard material as the calibrators,
combine 100 μL of 100 μg/mL buprenorphine standard,
100 μL of 100 μg/mL norbuprenorphine standard, and
800 μL of acetonitrile in a glass screw cap vial. Mix well.
Store at 20  C for up to 2 months.
14. QC composite spiking solution 1 (QCC 1): 2000 ng/mL
buprenorphine and norbuprenorphine. Combine 200 μL of
QMS BUP with 800 μL of acetonitrile in a glass screw cap vial.
Mix well. Store at 20  C for up to 2 months.
15. QCC 2: 500 ng/mL buprenorphine and norbuprenor-
phine. Combine 100 μL of QCC1 with 300 μL of acetonitrile
in a glass screw cap vial. Mix well. Store at 20  C for up to
2 months.

2.2 Preparation of 1. Cal 1: 2 ng/mL both analytes. Combine 100 μL of CSS 4 and
Urine Calibrators and 900 μL of urine in a glass screw cap vial. Store at 20  C for up
Controls to 2 months (see Note 4).
2. Cal 2: 5 ng/mL both analytes. Combine 50 μL of CSS 3 and
950 μL of urine in a glass screw cap vial. Store at 20  C for up
to 2 months.
3. Cal 3: 10 ng/mL both analytes. Combine 100 μL of CSS 3 and
900 μL of urine in a glass screw cap vial. Store at 20  C for up
to 2 months.
4. Cal 4: 20 ng/mL both analytes. Combine 50 μL of CSS 2 and
950 μL of urine in a glass screw cap vial. Store at 20  C for up
to 2 months.
5. Cal 5: 50 ng/mL both analytes. Combine 125 μL of CSS 2 and
875 μL of urine in a glass screw cap vial. Store at 20  C for up
to 2 months.
6. Cal 6: 100 ng/mL both analytes. Combine 50 μL of CSS 1 and
950 μL of urine in a glass screw cap vial. Store at 20  C for up
to 2 months.
 Buprenorphine and Norbuprenorphine 55

7. Cal 7: 200 ng/mL both analytes. Combine 100 μL of CSS

1 and 900 μL of urine in a glass screw cap vial. Store at 20  C
for up to 2 months.
8. QC: low – 6 ng/mL both analytes. Add 4.6 mL of blank urine
to tube, followed by 340 μL of acetonitrile, and then 60 μL of
QCC 2. Vortex well. Store at 20  C for up to 2 months.
9. QC: mid – 18 ng/mL both analytes. Add 4.6 mL of blank
urine to tube, followed by 355 μL of acetonitrile, and then
45 μL of QCC 1. Vortex well. Store at 20  C for up to
2 months.
10. QC: high – 72 ng/mL both analytes. Add 4.6 mL of blank
urine to tube, followed by 220 μL of acetonitrile, and then
180 μL of QCC 1. Vortex well. Store at 20  C for up to
2 months.

2.3 Supplies and 1. Sciex 4500 system with Turbo V Source and diverter valve.
Analytical Equipment 2. MultiQuant software v3.0 or later.
3. Shimadzu Nexera XR HPLC System.
4. LC-20AD XR binary pumps.
5. SIL-20AC Autosampler.
6. CTO-20AC column oven.
7. CBM-20A System Controller.
7. Phenomenex HPLC column, 2.6 μm, Kinetex Biphenyl,
50  3.0 mm.
8. Phenomenex SecurityGuard ULTRA UHPLC Biphenyl Car-
tridges, 3.0 mm.
9. Phenomenex SecurityGuard ULTRA Holder.
10. Screw cap glass vials capable of holding 2 mL.
11. Glass autosampler vials with inserts.
12. β-glucuronidase (IMCSzyme [recombinant], >50,000 U/
mL, or similar).
13. 1.5 mL or 2.0 mL microcentrifuge tubes.
14. Polypropylene tubes capable of holding >5 mL.
15. pH meter or pH paper.

3 Method

3.1 Sample 1. Aliquot 50 μL of urine from each calibrator, QC, and sample
Preparation into a microcentrifuge tube.
2. For each double blank and negative control, aliquot 50 μL of
blank urine.
56 Andrea R. Terrell et al.

3. Add 50 μL of the composite IS spiking solution to each of the

calibrators, QCs, negative controls, and samples and then mix.
Do not add IS to the double blanks.
4. Add 50 μL of 200 mM phosphate buffer pH 6.8 and vortex.
5. Add 20 μL IMCS enzyme (50,000 U/mL) and mix gently.
6. Incubate at 55  C for 30 min.
7. Add 200 μL of 20 mM ammonium formate buffer pH 3.7 and
vortex.
8. Centrifuge at 30,000g for 10 min.
9. Transfer approximately 200 μL of supernatant to autosampler
vials with inserts and cap. Store refrigerated until analysis.

3.2 Analysis 1. Inject 100 μL of the system suitability standard before each
batch.
2. Injection volume: 5 μL.
3. Needle rinse mode: before and after aspiration.
4. Autosampler temperature: 8  C.
5. Flow rate: 0.7 mL/min.
6. Column oven temperature: 40  C.
7. Gradient conditions are shown in Table 1.
8. Divert flow to waste for the first 1 min.
9. Mass spectrometer conditions are shown in Table 2.
10. Analyte-specific parameters are shown in Table 3.
11. Process the data using the first transition for each analyte as the
quantifier, and the second transition as the ion ratio qualifier.
12. Use linear regression model with 1/x2 weighting for all
analytes.

Table 1
Chromatography gradient

Time (min) % Mobile phase B

1.5 20
3.0 42
4.6 82
4.7 95
5.2 95
5.21 5
6.0 (Stop)
 Buprenorphine and Norbuprenorphine 57

Table 2
Mass spectrometer parameters

Parameter Setting
Polarity ESI positive/negative switching
Curtain gas 30 psi
CAD gas 10
Ionspray voltage (Pos) 2500 V
Ionspray voltage (Neg) 4500 V
Temperature 550  C
Ion source gas 1 60 psi
Ion source gas 2 60 psi
Acquisition time 5.9 min
Q1 resolution Unit
Q3 resolution Unit
MRM pause time 3 ms
Settling time 50 ms
MRM window (Pos) 40 s
Target scan time (Pos) 0.3 s
MRM window (Neg) 20 s
Target scan time (Neg) 0.05 s
EP 10 V

Table 3
Analyte-specific parameters

Q1 mass Q3 mass RT (min) Analyte DP CE CXP

468.2 414.2 4.66 Buprenorphine 1 100 50 16
468.2 396.1 4.66 Buprenorphine 2 100 54 14
468.2 55.1 4.66 Buprenorphine 3 100 92 14
472.2 59.1 4.66 Buprenorphine-d4 100 92 14
414.2 152.0 4.34 Norbuprenorphine 1 120 130 12
414.2 115.0 4.34 Norbuprenorphine 2 120 124 8
414.2 165.0 4.34 Norbuprenorphine 3 120 98 12
417.2 83.1 4.34 Norbuprenorphine-d3 120 74 8
58 Andrea R. Terrell et al.

Fig. 1 Positive sample from patient using Suboxone

Fig. 2 A sample that has been adulterated by “pill scraping” where a small amount of pill or film is directly
added to the urine sample. In this scenario, samples will have an elevated concentration of parent drug and
little to no metabolite

Fig. 3 Negative sample

13. Confirm that batch meets acceptance criteria (see Note 5).
14. Report patient samples using a cutoff of 10 ng/mL. Results
below this are reported as negative or not detected (see Note
6). Figures 1, 2, and 3 show examples of patient results using
this method.

4 Notes

1. All reagents should be HPLC grade or higher.

2. Do not use a plastic pipette to transfer the formic acid. The
concentrated acid will leach plasticizers out of the plastic and
cause a large late eluting peak.
3. Never wash mobile phase bottles with soap or detergent. This
causes high background and can never be rinsed out
completely. If a bottle is accidentally washed with soap or
detergent, discard it or use it for non-LC/MS assays.
 Buprenorphine and Norbuprenorphine 59

4. The concentration of the lowest calibrator is assigned as the

lower limit of quantitation (LLOQ), and the highest calibrator
concentration is the upper limit of quantitation (ULOQ). The
analytical range for buprenorphine and norbuprenorphine
using this method is 2–200 ng/mL.
5. Batch acceptance criteria: The back-calculated concentrations
of each calibrator and QC must be within 20% (for the
LLOQ, 25%) and 25% of target, respectively. The analyte
retention times of QCs must match within 0.05 min of the
mean retention time in calibrators. The analyte ion ratio in QCs
must match within 20% of the mean ion ratio in calibrators.
The analyte response in blank urine may not exceed 30% of the
response at the LLOQ.
6. Analyte identification criteria: The retention time for transi-
tions 1 and 2 must be within 0.05 min of the mean retention
time in the calibrators and within 0.01 min of each other. The
ion ratio must be within 20% of the mean ion ratio in
calibrators.

References
1. Samhsa.gov/medication-assisted-treatment/ 4. Baselt RC (2017) Disposition of toxic drugs and
buprenorphine chemicals in man, 11th edn. Biomedical Publi-
2. Drug Enforcement Administration Office of cations, Seal Beach, CA
Diversion Control, Drug and Chemical Evaluation 5. Yokell MA et al (2011) Buprenorphine and
Section, July 2013 publication “Buprenorphine” buprenorphine/naloxone diversion, misuse,
3. Crane EH (2015) Emergency department visits and illicit use: an international review. Curr
involving narcotic pain relievers. The CBHSQ Drug Abuse Rev 4(1):28–41
report, November 5
 Chapter 6

Quantitation of Tapentadol by Liquid Chromatography:

Tandem Mass Spectrometry
Graham R. Jones and Russell P. Handy

Abstract
Tapentadol is an orally active analgesic with a similar structure to tramadol. Its primary mechanism of action
is agonist action on the mu-opioid receptor. The method described here quantitates tapentadol in the
whole blood using a matching deuterated internal standard, extraction via a protein “crash” with acetoni-
trile, followed by analysis using liquid chromatography-tandem mass spectrometry.

Key words Tapentadol, Deuterated, Mu-opioid, LC-MS/MS

1 Introduction

Tapentadol is a synthetic mu-opioid agonist with a similar structure

to tramadol and is used to treat moderate to severe acute pain in
patients 18 years of age or older. It is available in the USA as
50–100 mg normal-release tablets and 50–250 mg extended-
release tablets. Recommended doses for adults are 50–100 mg
every 4–6 h or 50–250 mg twice daily for the extended release
form, up to a daily maximum recommended dose of 600 mg.
Tapentadol has an estimated half-life of 3–7 h, a volume of distri-
bution of 6–9 L/kg, and relatively low protein binding (Fb 0.20)
[1, 2].
Tapentadol undergoes extensive first-pass metabolism by phase
2 glucuronidation to form the O-glucuronide. After oral adminis-
tration 70% of the dose is excreted in the urine (55% O-glucuronide
and 15% sulfate). Only about 3% of tapentadol is excreted
unchanged in the urine. Phase 1 oxidative metabolism via
CYP2C9 and CYP2C19 is a minor metabolic route, forming pri-
marily N-desmethyltapentadol. None of the metabolites of tapen-
tadol appear to have analgesic activity [2]. Therefore this analytical
procedure only measures the active parent drug.

Loralie J. Langman and Christine L.H. Snozek (eds.), LC-MS in Drug Analysis: Methods and Protocols,
Methods in Molecular Biology, vol. 1872, https://doi.org/10.1007/978-1-4939-8823-5_6,
© Springer Science+Business Media, LLC, part of Springer Nature 2019

61
62 Graham R. Jones and Russell P. Handy

2 Materials

2.1 Solvents 1. 50:50 methanol/water (v/v). Add 100 mL methanol to a

(See Note 1) graduated cylinder. Fill to 200 mL with deionized water and
mix. Store at room temperature.
2. Mobile phase A: 0.1% v/v formic acid in deionized water. Add
1 mL of formic acid to a 1 L graduated cylinder. Fill to volume
with deionized water and mix.
3. Mobile phase B: acetonitrile.
4. Working standard A: 25 μg/mL tapentadol. Add 0.125 mL of
1 mg/mL tapentadol methanolic stock to a 5 mL volumetric
flask. Fill to volume with 50:50 methanol:water. Mix well and
store at 4
C.
5. Working standard B: 2.5 μg/mL tapentadol. Add 0.5 mL of
working standard A to a 5 mL volumetric flask. Fill to volume
with 50:50 methanol:water. Mix well and store at 4
C.
6. Working internal standard: 2.5 μg/mL tapentadol-D3. Add
0.125 mL of 0.1 mg/mL tapentadol-D3 methanolic stock to
a 5 mL volumetric flask. Fill to volume with 50:50 methanol:
water. Mix well and store at 4
C.
7. Quality control: 0.5 μg/mL tapentadol. Add 12.5 μL of 1 mg/
mL tapentadol methanolic stock to a 25 mL volumetric flask.
Fill to volume with drug-free whole blood and mix well. Ali-
quot into 1 mL vials and freeze at   20
C (see Note 2).

2.2 Supplies and 1. 10 mL glass tubes

Equipment 2. 1 mL autosampler vials
3. Vortexer
4. Centrifuge with adaptor for 10 mL tubes
5. Triple quadrupole mass spectrometer using electrospray ioni-
zation source, e.g., Agilent model 6410 with 12,000 series LC
system, or similar
6. Analytical column: Agilent Poroshell SB-C18, 2.1  100 mm,
2.7 μm

3 Methods

3.1 Preparation of 1. Appropriately label a set of 10 mL glass tubes and a set of 1 mL

Working Standards autosampler vials. Label for one tube for each standard, blank
and Unknown Samples and control, and two tubes for each specimen (see Note 3).
2. Add 0.5 mL drug-free whole blood to each 10 mL tube for the
blank and standards. Add 0.5 mL of the control to the
 Tapentadol by LC-MS/MS 63

Table 1
Spiking scheme for calibration standards

Final conc. tapentadol (mg/L) 0.025 0.05 0.1 0.25 0.5 1 2.5 5
Working standard A (μL) – – – – 10 20 50 100
Working standard B (μL) 5 10 20 50 – – – –

appropriate tube. Add 0.5 mL of specimen, in duplicate, to the

appropriate specimen tubes.
3. To the standard tubes, add the working standard according to
Table 1.
4. Add 0.050 mL of working internal standard to each tube.
Vortex and allow to stand 10 min.
5. Add 0.5 mL of 50:50 methanol/water to each tube. Vortex
to mix.
6. Add 2 mL acetonitrile to each tube while vortexing, and con-
tinue to vortex for 10–15 s. Allow to sit for 10 min and then
re-vortex briefly. Centrifuge at 2000g for 5 min.
7. Transfer an aliquot of supernatant to a 1.5 mL autosampler
vial. Cap tightly.
8. Inject 0.5 μL on the LC-MS/MS.

3.2 Analysis 1. Place the extracts on the autosampler in the following order:
blank, calibrators in order of lowest to highest concentrations,
blank (can reinject from same vial), QC sample, unknown
samples, and 0.5 mg/L calibrator (reinject from same vial)
(see Note 4).
2. Autosampler parameters cool to constant 4
C.
3. Injection volume 0.5 μL.
4. Column temperature 45
C.
5. Flow rate 0.5 mL/min.
6. Mobile phase program (see Note 5):
(a) 0.0 min: mobile B ratio 10%
(b) 4.0 min: mobile B ratio 50%
(c) 6.0 min: mobile B ratio 50%
(d) 6.01 min: mobile B ratio 95%
(e) 8.5 min: mobile B ratio 95%
(f) 8.51 min: mobile B ratio 10%
(g) 14.0 min: mobile B ratio 10%
7. Ion source parameters: gas temp ¼ 350
C, gas flow 11 L/min;
nebulizer ¼ 35 psi, capillary voltage 4000 V.
64 Graham R. Jones and Russell P. Handy

Table 2
Analytical and detection conditions for tapentadol and the deuterated internal standard

Qualifier
Qualifier Qualifier Qualifier #2
Ret. Quantifier Quantifier #1 #1 #2 collision
time Precursor product collision product collision product energy
Compound (min) ion (m/z) ion (m/z) energy (V) ion (m/z) energy (V) ion (m/z) (V)
Tapentadol 3.6 222.2 107 24 121 18 77 57
Tapentadol- 3.6 225.2 107 24 121 18 77 57
D3

Fig. 1 MRM chromatograms of tapentadol and tapentadol-D3, showing the quantifying ion transitions 222.2 to
107 and 225.2 to 107, respectively, and the corresponding qualifying ions of 121 and 77

8. Mass spectrometer parameters: Compound-dependent para-

meters are listed in Table 2 (see Note 6).
9. Figure 1 shows the chromatography of a 0.025 mg/L calibra-
tor containing tapentadol and the internal standard. One quan-
titation transition and two qualifying transitions are shown for
each compound (see Note 7).

4 Notes

1. Unless otherwise stated, all reagents are of HPLC or analytical

grade. Formic acid is 98–100%.
2. Use a different source of tapentadol solution than that used for
the calibrators.
3. Case blood samples are run in duplicate as a repeatability check
for difficult postmortem blood samples.
4. Sample order is at the discretion of the user. An unextracted
standard may be injected prior to the main run in order to
verify condition, including the retention time window. An
extracted blank sample is injected after the highest calibrator
to verify lack of carryover. If applicable, for long runs, a
 Tapentadol by LC-MS/MS 65

calibrator is reinjected after every ten samples as a calibration

stability check.
5. The original procedure was developed to quantify tramadol
and methadone as well as tapentadol, which is why the LC
gradient is more complex than might be required to quantify
tapentadol alone.
6. Quantifier and qualifier multiple reaction monitoring (MRM)
transitions are based on a single precursor ion for each com-
pound. Mass spectrometry parameters will vary between
instrument and manufacturers and must be optimized for the
specific LC-MS/MS instrument used.
7. Percent accuracy was established in whole blood calibrations
(n ¼ 3) from 0.025 mg/L (112.9%, 1.7% CV) and 5.0 mg/L
(99.8%, 0.3% CV) and independently prepared whole blood
controls (n ¼ 15) 0.5 mg/L (98.8%, 3.4% CV). Recovery
spikes into postmortem case blood (n ¼ 10) averaged 96.2%
(1.9% CV) at 0.075 mg/L and 100.1% (1.65% CV) at 4.0 mg/
L. Ionization efficiency of tapentadol extracted from the whole
blood (n ¼ 5) was 101.4% (1.8% CV) compared with extraction
from DIW (n ¼ 5). QC samples run over a 12-month time
period from a single frozen batch showed excellent stability
(mean 0.48 mg/L, 5.8% CV; n ¼ 14).

References

1. Baselt RC (2017) Disposition of toxic drugs and 2. Ortho-McNeil-Janssen (2010) Nucynta (tapen-
chemicals in man, 11th edn. Biomedical Publi- tadol) prescribing information. PriCara Divi-
cations, Seal Beach, CA sion, Raritan, NJ
 Chapter 7

Therapeutic Drug Monitoring of Lacosamide by LC-MS/MS

He Sarina Yang and Leslie Edinboro

Abstract
High-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) has become a primary
analytical methodology in therapeutic drug monitoring of antiepileptic drugs (AEDs). To demonstrate the
utility of LC-MS/MS in measuring drug concentrations in serum or plasma, analysis of lacosamide
(Vimpat™) is discussed in this chapter. Lacosamide is an example of the newer-generation AEDs. The
drug is extracted by protein precipitation and dilution of the serum specimen. A small volume of the
extracted specimen is injected into a reversed-phase chromatography column, and lacosamide is identified
by positive electrospray ionization (ESI) mass spectrometry in the multiple reaction monitoring (MRM)
mode, which provides selectivity for quantitative analysis. A deuterated internal standard is used to correct
for any loss of analyte during the process of extraction and analysis. A seven-point calibration curve and two
levels of quality controls are included in each batch.

Key words Antiepileptic drugs, Lacosamide, LC-MS/MS, Therapeutic drug monitoring

1 Introduction

In the last 20 years, 14 so-called new generation of AEDs have

entered the market, including eslicarbazepine, felbamate, gabapen-
tin, lacosamide, lamotrigine, levetiracetam, oxcarbazepine, prega-
balin, rufinamide, stiripentol, tiagabine, topiramate, vigabatrin, and
zonisamide [1]. Compared to the first-generation AEDs, the newer
agents generally have wider therapeutic ranges and fewer serious
adverse effects. However, the pharmacokinetics of the new AEDs
show significant interindividual variability due to sex, age, race,
hepatic metabolism, renal function, and concomitant medications
[1]. Therapeutic drug monitoring (TDM) is of particular impor-
tance in the clinical management of AED therapy, as therapeutic
and toxic effects of AEDs have been found to be related to serum
concentration [2]. TDM of new antiepileptic drugs can be of great
benefit in the treatment of seizure disorders and in prevention of
adverse drug effects. TDM can also aid in individualizing therapy,
adjusting for variable or nonlinear pharmacokinetics, and managing

Loralie J. Langman and Christine L.H. Snozek (eds.), LC-MS in Drug Analysis: Methods and Protocols,
Methods in Molecular Biology, vol. 1872, https://doi.org/10.1007/978-1-4939-8823-5_7,
© Springer Science+Business Media, LLC, part of Springer Nature 2019

67
68 He Sarina Yang and Leslie Edinboro

special populations such as pregnant women. In addition, clinicians

rely upon TDM to assess patient compliance and to prevent drug
misuse, particularly in patients with psychiatric disorders [3].
In this chapter, lacosamide high-performance liquid chroma-
tography-mass spectrometry (LC-MS/MS) analysis is used as an
example of a procedure for quantifying new-generation AEDs in
serum or plasma; analysis of most of the other new-generation
AEDs was presented in the first edition of this book. Lacosamide
is a novel functionalized amino acid that is used as an adjunct
therapy for the treatment of partial-onset seizures and focal epilep-
sies [4]. It is also being investigated as a treatment for diabetic
neuropathic pain [5]. Lacosamide is thought to have dual mechan-
isms of action [6]. It selectively enhances slow inactivation of
voltage-gated sodium channels, which stabilizes hyper-excitable
neuronal membrane and inhibits neuronal firing. In addition, it
modulates the collapsing response mediator protein-2 (CRMP-2).
The role of CRMP-2 in seizures has not been fully elucidated;
however, its expression is altered in epilepsy and other neurodegen-
erative disorders. Lacosamide has low plasma protein binding
(<15%) and minimal clinically significant drug-drug interactions.
Overall, lacosamide exhibits predictable pharmacokinetics with no
clinically significant differences in pharmacokinetics between chil-
dren, young adults, and elderly patients [1].
Automated immunoassay methods have been widely used for
TDM of anticonvulsants since the 1980s. However, antibodies
used in these methods may cross-react with metabolites of the
drug in question [7]. Moreover, immunoassays are not available
for many newer-generation AEDs [8]. In recent years, LC-MS/MS
has become the primary analytical methodology in therapeutic
monitoring of newer AEDs. Compared to immunoassays,
LC-MS/MS offers superior sensitivity, specificity, accuracy, and
throughput in measuring the concentration of drugs in biological
samples. Only a small amount of specimen (100–300 μL) is needed
for analysis. Here, we describe a detailed lacosamide LC-MS/MS
method used in a large clinical laboratory.

2 Materials

2.1 Internal 1. Internal standard working solution: 5 μg/mL lacosamide-13C,

Standard, Calibrators, D3. Add 100 μL of 1 mg/mL lacosamide-13C, D3 stock solu-
Controls, and Buffers tion to a 20 mL volumetric flask. Fill to volume with methanol
and mix well. Store at 2–8
C for 1 month or  10 to 30
C
for 6 months.
2. 50% methanol. Add 5 mL of methanol to a 10 mL volumetric
flask. Fill to volume with clinical laboratory reagent water
 TDM of Lacosamide by LC-MS/MS 69

(CLRW) and mix. Prepare fresh prior to preparation of

standards.
3. 200 μg/mL lacosamide standard working solution: Add 1 mL
of 1 mg/mL lacosamide stock solution to a 5 mL volumetric
flask. Fill to volume with 50% methanol and mix well. Prepare
fresh prior to preparation of standards.
4. Calibrator 1: 0.5 μg/mL lacosamide. Add 25 μL of lacosamide
standard working solution to a 10 mL glass volumetric flask
containing approximately 5 mL of drug-free serum. Fill to
volume with drug-free serum. Aliquot 1 mL into 1.5 mL vials
and store frozen. Stable for 6 months (see Note 1).
5. Calibrator 2: 1 μg/mL lacosamide. Add 50 μL of lacosamide
standard working solution to a 10 mL glass volumetric flask
containing approximately 5 mL of drug-free serum. Fill to
volume with drug-free serum. Aliquot 1 mL into 1.5 mL vials
and store frozen. Stable for 6 months.
6. Calibrator 3: 2 μg/mL lacosamide. Add 100 μL of lacosamide
standard working solution to a 10 mL glass volumetric flask
containing approximately 5 mL of drug-free serum. Fill to
volume with drug-free serum. Aliquot 1 mL into 1.5 mL vials
and store frozen. Stable for 6 months.
7. Calibrator 4: 4 μg/mL lacosamide. Add 200 μL of lacosamide
standard working solution to a 10 mL glass volumetric flask
containing approximately 5 mL of drug-free serum. Fill to
volume with drug-free serum. Aliquot 1 mL into 1.5 mL vials
and store frozen. Stable for 6 months.
8. Calibrator 5: 8 μg/mL lacosamide. Add 400 μL of lacosamide
standard working solution to a 10 mL glass volumetric flask
containing approximately 5 mL of drug-free serum. Fill to
volume with drug-free serum. Aliquot 1 mL into 1.5 mL vials
and store frozen. Stable for 6 months.
9. Calibrator 6: 10 μg/mL lacosamide. Add 500 μL of lacosamide
standard working solution to a 10 mL glass volumetric flask
containing approximately 5 mL of drug-free serum. Fill to
volume with drug-free serum. Aliquot 1 mL into 1.5 mL vials
and store frozen. Stable for 6 months.
10. Calibrator 7: 20 μg/mL lacosamide. Add 1000 μL of lacosa-
mide standard working solution to a 10 mL glass volumetric
flask containing approximately 5 mL of drug-free serum. Fill to
volume with drug-free serum. Aliquot 1 mL into 1.5 mL vials
and store frozen. Stable for 6 months.
11. Quality control 1: 5 μg/mL lacosamide. Reconstitute lyophi-
lized serum quality controls (UTAK Laboratories Bi-Level
AED II serum toxicology control or similar), by adding exactly
5 mL of CLRW using a volumetric pipette, and mix well on a
70 He Sarina Yang and Leslie Edinboro

shaker. Reconstituted control material is stored at 2–8
C and is

stable for 25 days after reconstitution (see Note 2).
12. Quality control 2: 20 μg/mL lacosamide. Reconstitute lyophi-
lized serum quality controls (UTAK Laboratories Bi-Level
AED II serum toxicology control or similar), by adding exactly
5 mL of CLRW using a volumetric pipette, and mix well on a
shaker. Reconstituted control material is stored at 2–8
C and is
stable for 25 days after reconstitution.
13. Mobile phase A: 0.1% formic acid in water. Add 1.0 mL of
formic acid to 999 mL of CLRW. Stable at room temperature
for 1 month.
14. Mobile phase B: 0.1% formic acid in acetonitrile. Add 1.0 mL
of formic acid to 999 mL of acetonitrile. Stable at room tem-
perature for 1 month.
15. Autosampler wash solution: 50% methanol in water. Add
500 mL of methanol to 500 mL of water. Stable in room
temperature for a month.

2.2 Supplies 1. Agilent 1200 series pump system operating in laminar flow
and Analytic mode (or equivalent).
Equipment 2. Applied Biosystems API 3200 mass spectrometer
(or equivalent).
3. Sciex Analyst® software.
4. Restek guard column.
5. Restek Pinnacle DB Biphenyl 5 μm 50 x 2.1 mm column.
6. Allegra X-15R benchtop centrifuge, Beckman Coulter.
7. Autosampler Vials-Robo Type 1, Class A (or equivalent).
8. Flat-bottom glass inserts (250 μL).
9. Vials with caps and septa.
10. Vortex mixer.
11. Eppendorf microcentrifuge or equivalent.
12. Tubes, Eppendorf 1.5 mL.
13. 2 mL and 1 mL 96-well collection plates.

3 Methods

3.1 Extraction 1. Pipette 100 μL of each plasma or serum specimen, calibrator,

of Lacosamide from and control to its own 1.5 mL Eppendorf tube or individual well
Plasma and Serum of a 2 mL 96-well plate (see Note 3).
2. Add 20 μL of internal standard to each tube or well.
3. Add 200 μL of methanol to each vial or well to precipitate
proteins. Vortex to mix then centrifuge for 15 min at 4
C.
 TDM of Lacosamide by LC-MS/MS 71

Tubes are centrifuged at 18,538  g; 96-well plates are centri-

fuged at 4000  g.
4. Transfer 150 μL of supernatant off the protein pellet into indi-
vidual autosampler vials with flat-bottom glass inserts or a new
1 mL 96-well collection plate.
5. Place samples into the autosampler rack (see Note 4).

3.2 LC-MS/MS 1. Inject samples in the following order: calibration standards

Analysis (S1–S7), blank, quality control level 1, quality control level
2, unknown samples, additional quality controls.
2. Injection volume: 10 μL.
3. Column temperature: room temperature (see Note 5).
4. Gradient times, flow rates, and solution percentages are listed in
Table 1.
5. Mass spectrometry source: electrospray ionization positive mode.
6. Source parameters: curtain gas ¼ 20 psi, ion spray volt-
age ¼ 5500 V, ion source temperature ¼ 550
C.
7. Two characteristic fragment ions (or transitions) for lacosamide
and one transition for the internal standard are shown in Table 2
(see Note 6).
8. Chromatography of lacosamide and the internal standard are
shown in Fig. 1 (see Note 7).

Table 1
Gradient program

Step Total time (min) Flow rate (mL/min) A (%) [aqueous] B (%) [organic]
0 0.10 0.75 90 10
1 0.20 0.75 30 70
2 1.90 0.75 10 90
3 2.00 0.75 90 10

Table 2
Mass transitions for lacosamide and internal standard

Substance Q1 (m/z) Q3 (m/z) Q3 qualifier (m/z)

Lacosamide 251.4 91.0 108.0
13
Lacosamide- C, D3 255.4 91.0 --------
72 He Sarina Yang and Leslie Edinboro

Fig. 1 Example of chromatography for lacosamide (254.1/91.0 and 254.1/108.0) and internal standard
lacosamide-13C, D3 (255.4/91.0)

3.3 Data Analysis 1. Generate calibration curves using a weighted (1/x) linear
regression curve.
2. Quantitate analyte peaks by normalizing the peak area of the
quantifier ion of each compound to the internal standard.
3. Calculate lacosamide concentration from the calibration curve
(see Note 8).
4. Analytical measure range (AMR) of this assay is 0.5 to 20 μg/mL.

4 Notes

1. Calibrators are stable for 6 months if aliquoted in 1 mL vials and

kept frozen at  20
C.
2. Store reconstituted UTAK controls at 2–8
C. Once reconsti-
tuted they are stable for 25 days.
3. Spiking studies demonstrate that serum, EDTA plasma, and
heparin plasma are all acceptable for analysis. Serum stability is
at least 5 days at ambient, 14 days refrigerated, and 30 days
frozen storage temperature. Hemoglobin, triglycerides, and bil-
irubin do not significantly interfere with measurement of laco-
samide by this method.
4. Post-extraction stability studies indicate that extracted samples
are stable for 24 h.
5. Back pressure of column should be monitored. Sudden increase
of back pressure may indicate clogging of column, and sudden
decrease of pressure may be due to leaking in the HPLC system.
6. To ensure the correct identification of lacosamide, in each batch,
the retention time of lacosamide peak should be within 0.2 s of
the established time. The peak area of internal standard should
be within 30% of the established value and remain stable within
the run. The chromatogram should be symmetrical, well
resolved, and Gaussian distributed regarding the morphology,
free of tailing or fronting peak, or any interference peak.
 TDM of Lacosamide by LC-MS/MS 73

7. If a peak shows a sign of having column overload (peak front-

ing), sample should be repeated at twofold or fourfold dilution
to ensure correct quantitation.
8. Using a calibration curve, the relative abundance of a given ion is
converted to an absolute amount of the original molecule.
Regression coefficient of the calibration curve should be above
0.98. Only one calibrator can be excluded from the curve. If the
lowest calibrator is removed, patient samples having results
lower than the second lowest calibrator need to repeated; if the
highest calibrator is removed, patient samples having results
higher than the second highest calibrator need to be repeated.

References
1. Krasowski MD (2010) Therapeutic drug moni- retigabine and eslicarbazepine acetate. Expert
toring of the newer anti-epilepsy medications. Opin Pharmacother 13(5):699–715
Pharmaceuticals 3:1909–1905 5. Jacob S, Nair AB (2016) An updated overview
2. Anderson GD (2008) Pharmacokinetic, phar- on therapeutic drug monitoring of recent anti-
macodynamics, and pharmacogenetic targeted epileptic drugs. Drug R D 16:303–316
therapy of antiepileptic drugs. Ther Drug 6. Ben-Menachem E (2008) Lacosamide: an inves-
Monit 30(2):173–180 tigational drug for adjunctive treatment of
3. Deeb S, McKeown DA, Torrance HJ, Wylie FM, partial-onset seizures. Drugs Today 44
Logan BK, Scott KS (2014) Simultaneous anal- (1):35–40
ysis of 22 antiepileptic drugs in postmortem 7. Eadie MJ (1998) Therapeutic drug monitor-
blood, serum and plasma using LC-MS-MS ing—antiepileptic drugs. Br J Clin Pharmacol
with a focus on their role in forensic cases. J 46:185–193
Anal Toxicol 38(8):485–494 8. Krasowski MD 2013 Antiepileptic drugs: thera-
4. Patsalo PN, Berry DJ (2012) Pharmacotherapy peutic drug monitoring of the newer generation
of the third-generation AEDs: lacosamide, drugs. Clinical Laboratory News
 Chapter 8

LC-MS/MS Method for the Quantification of the Leflunomide

Metabolite, Teriflunomide, in Human Serum/Plasma
Geoffrey S. Rule, Alan L. Rockwood, and Kamisha L. Johnson-Davis

Abstract
Leflunomide is a prodrug that is metabolized to the active metabolite, teriflunomide (A77 1726), to inhibit
the enzyme dihydroorotate dehydrogenase and decrease the synthesis of pyrimidine nucleotides for DNA
and RNA synthesis. Teriflunomide is primarily used for the treatment of rheumatoid arthritis and multiple
sclerosis.
A liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated
to quantify the drug teriflunomide over a concentration range of 5 ng/mL–200 μg/mL in serum or plasma.
The calibration curve was divided into two separate overlapping regions of the analytical measurement
range, with a high curve and a low curve range. Samples are first analyzed using the high-range calibration
curve after a 100-fold dilution of the sample extract. Samples falling below the upper curve region are
evaluated again without dilution and quantified, if possible, against the low curve calibration standards.
This method can be used to support therapeutic drug monitoring of patients that are administered with
leflunomide therapy.

Key words Leflunomide, Teriflunomide, Immunosuppressant, LC/MS/MS

1 Introduction

Leflunomide is a prodrug approved by the FDA in 1998 and

brought to market by Sanofi-Aventis under the name Arava®. It is
administered largely for the treatment of rheumatoid arthritis and is
classified as a disease-modifying antirheumatic drug (DMARD) and
functions to slow disease progression [1]. Leflunomide contains an
isoxazole ring which is opened nonenzymatically to the active
metabolite, teriflunomide. Another drug formulation, marketed
as Aubagio®, consists of the ring-opened form and is used for the
treatment of active relapsing-remitting multiple sclerosis [2]. Leflu-
nomide has also been prescribed, off-label, for other uses in renal

Loralie J. Langman and Christine L.H. Snozek (eds.), LC-MS in Drug Analysis: Methods and Protocols,
Methods in Molecular Biology, vol. 1872, https://doi.org/10.1007/978-1-4939-8823-5_8,
© Springer Science+Business Media, LLC, part of Springer Nature 2019

75
76 Geoffrey S. Rule et al.

transplant patients, treating cytomegalovirus viremia [3], BK virus-

associated nephropathy [4], for treatment in psoriatic arthritis [5]
and Wegener granulomatosis, systemic lupus erythematosus, sar-
coidosis, and more [6].
Teriflunomide inhibits cell proliferation of activated lympho-
cytes by inhibition of the enzyme, dihydroorotate dehydrogenase,
to decrease the synthesis of pyrimidine nucleotides for DNA and
RNA synthesis [7, 8]. Once the drug is metabolized, circulating
concentrations of leflunomide after oral administration are gener-
ally very low in comparison with levels of teriflunomide [9]. In vivo,
leflunomide is rapidly and, essentially, completely converted to
teriflunomide that has both immunosuppressive and antiviral
effects. Studies have shown a correlation between teriflunomide
blood concentrations of 40 μg/mL and progressive clearance of
BK polyomavirus (BKV) [10, 11]. BKV is a common infection in
immunosuppressed renal transplant patients that can lead to failure
and loss of grafted kidneys. Teriflunomide reaches a maximum
plasma concentration approximately 6–12 h following oral admin-
istration [7]. The drug is cleared both in the urine and in the feces
(approximately 43% and 48%, respectively) [7]. It has a steady-state
volume of distribution (Vd) of just 0.13 L/kg and clearance of
approximately 31 mL/h [7].
Both leflunomide and teriflunomide have the same molecular
weight, and after collision-induced dissociation of the protonated
parent by tandem mass spectrometry, both compounds have the
same two most abundant product ions. Consequently, chro-
matographic separation of leflunomide from teriflunomide is nec-
essary to eliminate the possibility of interference in the
quantification of the parent drug and metabolite.
The therapeutic range for teriflunomide is not well established;
however serum/plasma concentrations >40 μg/mL tend to corre-
late with improved patient outcome. The drug is teratogenic; thus,
clearance (defined as concentrations <20 ng/mL) is monitored as
well. Patient drug concentrations in serum or plasma could range
from several hundred μg/mL down to the medical decision point
of 20 ng/mL [12]. Here we describe a method that covers a
40,000-fold range, from 5 ng/mL to 200 μg/mL, by the imple-
mentation of two separate but overlapping calibration curves. A
single extraction procedure is utilized for both the high and low
curve ranges, with the exception that samples analyzed for the
higher concentration range are diluted 100-fold prior to analysis.
Earlier methods for determination of teriflunomide or leflunomide
have been described in the literature as using HPLC/UV [9, 13,
14] or LC/MS/MS [15].
 Teriflunomide LC-MS/MS Method 77

2 Materials

2.1 Supplies 1. Volumetric glassware and pipettes.

and Equipment 2. Pipette tips.
3. Agilent 1200 series HPLC pump.
4. CTC autosampler.
5. AB Sciex API4000 triple quadrupole mass spectrometer with
Analyst quantitation software (ver. 1.5.1). The instrument
employs an ESI interface, multiple reaction monitoring
(MRM), unit resolution performed in negative
ionization mode.
6. Autosampler vials.
7. Analytical column; 2 mm  10 cm, Luna PFP [2] (pentafluor-
ophenyl) phase on 3 μm particles.
8. Microcentrifuge tubes (1.7 mL).
9. Vortex mixer.
10. Centrifuge (to accommodate microcentrifuge tubes and capa-
ble of achieving 207083 rcf).

2.2 Standards The high calibration curve consists of standards 6–10; the low
and Controls calibration curve consists of standards 1–5. Controls for the high
curve are D, E, and F; controls for the low curve are A, B, and C.
1. Stock solution: 10 mg/mL teriflunomide in DMSO. Add
10 mg of teriflunomide powder to 1 mL of dimethyl sulfoxide
(DMSO). Mix until completely dissolved.
2. Standard 10: 200 μg/mL teriflunomide. Add 5 μL of stock
solution to 245 μL of drug-free serum. Vortex to mix. Make
fresh daily.
3. Standard 9: 50 μg/mL teriflunomide. Add 40 μL of standard
10–120 μL of drug-free serum. Vortex to mix. Make fresh daily.
4. Standard 8: 10 μg/mL teriflunomide. Add 10 μL of standard
10–190 μL of drug-free serum. Vortex to mix. Make fresh daily.
5. Standard 7: 2 μg/mL teriflunomide. Add 10 μL of standard
10–990 μL of drug-free serum. Vortex to mix. Make fresh daily.
6. Standard 6: 0.8 μg/mL teriflunomide. Add 100 μL of standard
7–150 μL of drug-free serum. Vortex to mix. Make fresh daily.
7. Standard 5: 1 μg/mL teriflunomide. Add 200 μL of standard
7–200 μL of drug-free serum. Vortex to mix. Make fresh daily.
8. Standard 4: 0.5 μg/mL teriflunomide. Add 100 μL of standard
5–100 μL of drug-free serum. Vortex to mix. Make fresh daily.
9. Standard 3: 0.1 μg/mL teriflunomide. Add 25 μL of standard
4–100 μL of drug-free serum. Vortex to mix. Make fresh daily.
78 Geoffrey S. Rule et al.

10. Standard 2: 0.02 μg/mL teriflunomide. Add 10 μL of standard

4–240 μL of drug-free serum. Vortex to mix. Make fresh daily.
11. Standard 1: 0.005 μg/mL teriflunomide. Add 5 μL of standard
4–495 μL of drug-free serum. Vortex to mix. Make fresh daily.
12. Control F: 170 μg/mL teriflunomide. Use a separate prepara-
tion of stock solution than for calibrators. Add 170 μL of stock
solution to a 10 mL volumetric flask. Fill to volume with drug-
free serum. Vortex mix and aliquot into microcentrifuge tubes.
Store at 20  C or lower until use.
13. Control E: 80 μg/mL teriflunomide. Use a separate prepara-
tion of stock solution than for calibrators. Add 80 μL of stock
solution to a 10 mL volumetric flask. Fill to volume with drug-
free serum. Vortex mix and aliquot into microcentrifuge tubes.
Store at 20  C or lower until use.
14. Control D: 1 μg/mL teriflunomide. Add 125 μL of control E
to a 10 mL volumetric flask. Fill to volume with drug-free
serum. Vortex mix and aliquot into microcentrifuge tubes.
Store at 20  C or lower until use.
15. Control C: 0.8 μg/mL teriflunomide. Add 100 μL of control E
to a 10 mL volumetric flask. Fill to volume with drug-free
serum. Vortex mix and aliquot into microcentrifuge tubes.
Store at 20  C or lower until use.
16. Control B: 0.1 μg/mL teriflunomide. Add 12.5 μL of control
E to a 10 mL volumetric flask. Fill to volume with drug-free
serum. Vortex mix and aliquot into microcentrifuge tubes.
Store at 20  C or lower until use.
17. Control A: 0.02 μg/mL teriflunomide. Add 200 μL of control
D to a 10 mL volumetric flask. Fill to volume with drug-free
serum. Vortex mix and aliquot into microcentrifuge tubes.
Store at 20  C or lower until use.
18. Internal standard/protein precipitating solution: 333 ng/mL
d4-teriflunomide. Add 30 μL of a 1 mg/mL stock solution of
the deuterated internal standard to 30 mL of 1:1, methanol/
acetonitrile containing 0.1% formic acid. Allow the solution to
mix for 30 min. at room temperature (20–25  C) prior to
storage.

2.3 Solutions 1. Mobile phase a: 0.1% formic acid in deionized water. Add 1 mL
of formic acid to 999 mL of deionized water in a graduated
cylinder. Mix.
2. Mobile phase B: 0.1% formic acid in 1:1:18 water/methanol/
acetonitrile. Add 1 mL of formic acid, 50 mL of deionized
water, and 50 mL of methanol to a 1 L graduated cylinder. Fill
to volume with acetonitrile and mix.
 Teriflunomide LC-MS/MS Method 79

3. Double blank solution: 0.1% formic acid in 1:1 methanol/

acetonitrile. Add 1 mL formic acid to 500 mL of methanol
and 500 mL of acetonitrile to a 1 L graduated cylinder,
and mix.
4. Dilution solution: 0.1% formic acid in 25% water, 75% 1:1
methanol/acetonitrile. Add 1 mL of formic acid, 250 mL of
deionized water, 375 mL of methanol, and 375 mL of acetoni-
trile to a 1 L graduated cylinder, and mix.
5. Autosampler needle wash 1; 4:1 methanol/water containing
0.1% trifluoroacetic acid. Add 1 mL of trifluoroacetic acid and
200 mL of deionized water to a 1 L graduated cylinder. Fill to
volume with methanol and mix.
6. Autosampler needle wash 2; 2:3 methanol/water. Add 400 mL
of methanol to 600 mL of deionized water in a graduated
cylinder and mix.
7. Test injection solution: 100 pg/μL teriflunomide and lefluno-
mide in 1:3 water/acetonitrile. Combine 100 μL each of
10 μg/mL solutions of teriflunomide and leflunomide with
2.45 mL of water in a 10 mL volumetric flask. Fill to volume
with acetonitrile and mix.
8. Heparinized or EDTA plasma, plain (red top) serum: Collect
and process specimens according to standard phlebotomy pro-
cedures. Freeze specimens at 20  C or colder and transport.

3 Methods

3.1 Sample 1. Label a microcentrifuge tube for each standard [1–10], control
Preparation (A–F), patient sample, blank, and double blank (see Note 1).
2. Add 100 μL of each blank serum, standard, control, and patient
serum/plasma to the appropriate microcentrifuge tubes.
3. Add 300 μL of “double blank solution” to the tubes designated
double blanks.
4. Add 300 μL of “working internal standard” to all other blanks,
calibrators, controls, and patient samples (see Note 2).
5. Cap tubes and vortex mix for 2 min.
6. Centrifuge for 10 min at 20783 rcf.
7. Prepare samples for injection:
(a) High curve: Transfer 2 μL of each patient, calibrator 6–10,
control D-F, and blank supernatant into autosampler vials
containing 200 μL of dilution solution. Cap and mix.
(b) Low curve: Transfer 100 μL of each calibrator 1–5, control
A–C, blank, and low patient (see Note 3) supernatant into
autosampler vials and cap.
80 Geoffrey S. Rule et al.

Table 1
Chromatography conditions

Step Total time (min) Flow rate (μL/min) A (%)a B (%)

0 0.0 300 40 60
1 0.2 300 40 60
2 2.0 300 0 100
3 4.0 300 0 100
4 4.1 300 40 60
5 4.9 300 40 60
a
A(%) and B(%) refer to the percentages of mobile phases A and B, respectively

Table 2
MS/MS operating parameters

MS/MS parameter table

Curtain gas (CUR): 25 psi
Temperature (TEM):500  C
Ion source gas 1 (GS1): 35 psi
Ion source gas 2 (GS2): 30 psi
Nitrogen gas is used for both GS1 and GS2
Ihe: ON
IonSpray voltage (IS): 4300 V
Collision gas (CAD): Medium
Declustering potential (DP): 55 V
Entrance potential (EP): 10 V
Detector parameters (negative)
CEM 2500

3.2 Analysis 1. Inject test solution of leflunomide and teriflunomide (see

Note 4).
2. Inject 3 μL of each sample in sequence.
3. Inject high curve samples first. Determine if any patients need
to be run on the low curve, then prepare as described above and
inject if required.
4. Analytical column temperature: 24  C.
5. LC gradient is shown in Table 1.
6. MS parameters are as shown in Tables 2 and 3 (see Note 5).
 Teriflunomide LC-MS/MS Method 81

Table 3
List of precursor and product ions for teriflunomide and internal standard

Collision cell
Precursor ion Product ion Dwell (msec) Collision energy (eV) exit potential
Teriflunomide 269.1 82.0 50 17 5
269.1 160.0 50 26 2
d4-teriflunomide 273.1 82.0 50 27 5
273.1 164.0 50 34 2

XIC of -MRM (4 pairs): 269.000/160.000 Da from Sample 1 (Test inject std) of Run14.w if... Max. 3.1e5 cps.

1.06e6
1.00e6

N
9.00e5
H F F
8.00e5 N
F O
F
7.00e5 O OH
F N
N
F H
Intensity, cps

6.00e5 O
MW = 270
teriflunomide
5.00e5 MW = 270
4.00e5 leflunomide
2.18
3.00e5
2.49

2.00e5

1.00e5

0.00
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
Time, min

Fig. 1 Extracted ion current chromatograms of teriflunomide in standard 1 (5 ng/mL), and internal standard (d4
teriflunomide), showing two transitions for each

7. Collect and analyze data using AB Sciex Analyst® software (ver

1.5.2). Example extracted ion current profiles for the low curve
standard 1 and internal standard are shown in Fig. 1.
8. Integrate peak height and perform linear regression analysis of
the calibration standards with 1/x2 weighted regression of peak
height ratio vs. analyte concentration. The unknown peak
height value divided by the internal standard peak height
value gives the ratio.
82 Geoffrey S. Rule et al.

4 Notes

1. For each curve range, both a blank and double blank sample are
prepared. A blank is analyzed as the first and last injection of
each sequence and consists of a known negative serum sample.
A double blank is analyzed after each high standard. A “double
blank” sample is one prepared in identical fashion to the ordi-
nary blank but in the absence of internal standard. Placing this
sample after the high calibration standard allows one to distin-
guish between autosampler carryover and unlabeled analyte
that may be contributed through the internal standard addition
as an impurity.
2. Prepare the low and high calibration curve standards, all quality
control samples and patient samples in the same batch. Prepare
each patient sample with 100 dilution, as described, and
evaluate against the high curve calibration standards. If within
the high calibration curve region, report the determined value.
Samples above 200 μg/mL must be extracted a second time
with appropriate matrix dilution. Patient samples below
0.8 μg/mL (the lower limit of quantitation of the high curve)
can be prepared according to instructions for the low curve,
then analyzed along with the low curve calibrators and quality
control samples. Report the resulting value as appropriate.
3. The concentration of internal standard utilized is equivalent to
1 μg/mL in the plasma/serum sample. This concentration is
near the low end of the upper curve and at the high end of the
lower curve. It is possible to use this same solution (concentra-
tion) for both curves since there is a lack of a significant analyte
isotope interference with the IS and because only a small
amount of unlabeled analyte is present in the IS. We discuss
this topic elsewhere in detail [16].
4. A test injection is made with a solution of teriflunomide and
leflunomide to verify instrument performance (sensitivity and
retention time) and chromatographic separation (resolution)
prior to each batch of samples. Results are compared with
historical values, and a judgment is made regarding system
suitability for patient sample analysis.
5. Although the parent drug, leflunomide, is generally found only
at very low concentrations, if at all, it is separated chromato-
graphically from the metabolite, teriflunomide, due to the fact
that the two have both the same precursor and product ion
masses.
 Teriflunomide LC-MS/MS Method 83

Acknowledgments

The authors would like to thank the ARUP Institute for Clinical
and Experimental Pathology for making this work possible.

References

1. Schattenkirchner M (2000) The use of lefluno- 11. Huttemann M, Shipkova M, Klett C,

mide in the treatment of rheumatoid arthritis: Hasche G, Wilhelm J, Bolley R,
an experimental and clinical review. Immuno- Olbricht C, Wieland E (2013) Total and
pharmacology 47:291–298 free plasma concentrations of the active
2. Oh J, O’ Connor PW (2013) Teriflunomide metabolite of leflunomide in relation to
for the treatment of multiple sclerosis. Semin therapeutic outcome in kidney transplant
Neurol 33:45–55 recipients with BK-virus nephropathy.
3. Chacko B, John GT (2012) Leflunomide for Transplant Proc 45:1611–1613
cytomegalovirus: bench to bedside. Transplant 12. Brent RL (2001) Teratogen update: repro-
Infect Dis 14:111–120 ductive risks of leflunomide (Arava); a pyrimi-
4. Teschner S, Gerke P, Geyer M, Wilpert J, dine synthesis inhibitor: counseling women
Krumme B, Benzing T, Walz G (2009) Leflu- taking leflunomide before or during preg-
nomide therapy for polyomavirus-induced allo- nancy and men taking leflunomide who are
graft nephropathy: efficient BK virus contemplating fathering a child. Teratology
elimination without increased risk of rejection. 63:106–112
Transplant Proc 41:2533–2538 13. Chan V, Charles BG, Tett SE (2004) Rapid
5. Babic-Naglic D, Anic B, Novak S, Grazio S, determination of the active leflunomide metab-
Martinavic Kaliterna D (2010) Treatment of olite A77 1726 in human plasma by high-
rheumatoid and psoriatic arthritis review of performance liquid chromatography. J Chro-
leflunomide. Reumatizam 57:161–162 matogr B Analyt Technol Biomed Life
803:331–335
6. Pinto P, Dougados M (2006) Leflunomide in
clinical practice. Acta Reumatol Portuguesa 14. van Roon EN, Yska JP, Raemaekers J, Jansen
31:215–224 TL, van Wanrooy M, Brouwers JR (2004) A
rapid and simple determination of A77 1726 in
7. Prakash A, Jarvis B (1999) Leflunomide: a human serum by high-performance liquid
review of its use in active rheumatoid arthritis. chromatography and its application for optimi-
Drugs 58:1137–1164 zation of leflunomide therapy. J Pharm Biomed
8. Breedveld FC, Dayer JM (2000) Leflunomide: Anal 36:17–22
mode of action in the treatment of rheumatoid 15. Parekh JM, Vaghela RN, Sutariya DK,
arthritis. Ann Rheum Dis 59:841–849 Sanyal M, Yadav M, Shrivastav PS (2010)
9. Schmidt A, Schwind B, Gillich M, Brune K, Chromatographic separation and sensitive
Hinz B (2003) Simultaneous determination determination of teriflunomide, an active
of leflunomide and its active metabolite, A77 metabolite of leflunomide in human plasma
1726, in human plasma by high-performance by liquid chromatography-tandem mass spec-
liquid chromatography. Biomed Chromatogr trometry. J Chromatogr B Analyt Technol
17:276–281 Biomed Life Sci 878:2217–2225
10. Mutlu D, Saglik I, Koyun M, Comak E, 16. Rule GS, Clark ZD, Yue B, Rockwood AL
Mutlu E, Uslu Gokceoglu A, Cagla Dogan S, (2013) Correction for isotopic interferences
Dinckan A, Akbas SH, Akkaya B, Akman S, between analyte and internal standard in
Suleymanlar G, Colak D (2013) BK virus infec- quantitative mass spectrometry by a nonlinear
tions in pediatric kidney transplant recipients. calibration function. Anal Chem
Mikrobiyol Bul 47:461–471 85:3879–3885
 Chapter 9

Analysis of Tryptic Peptides from Therapeutic Monoclonal

Antibodies Using LC-MS/MS
Maria Alice V. Willrich

Abstract
Immunotherapies are a hot topic, with the potential to impact our understanding of the immune system
and treat a diverse array of conditions. Therapeutic monoclonal antibodies (mAbs) are part of this revolu-
tion, and clinical chemists are aware of the success of the biologic drugs. Antibodies are not just immuno-
assay reagents anymore but are also present in clinical serum samples from more and more patients each day.
The clinical laboratory will have many roles as mAb therapies expand, including the development of new
assays to differentiate a mAb from an endogenous, disease-causing clone and monitoring therapeutic drugs
for better patient outcomes and assessing for the loss of response to therapy.
Therapeutic mAbs use has expanded significantly in the last 5 years, and depending on their target or
their concentration, they may impact routine clinical testing for patients. Optimizing therapy during the
induction phase to keep the mAb concentrations above certain thresholds has proven to be associated with
improved responses and better outcomes in chronic conditions such as inflammatory bowel disease. This
chapter will describe a LC-MS/MS protocol for analysis of tryptic peptides unique to infliximab (clonotypic
peptides) for quantitation of the mAb. The protocol can be adapted to other mAbs with similar outcomes
and is a useful, relatively simple strategy for measurement of mAbs.

Key words LC-MS/MS, Infliximab, Therapeutic monoclonal antibodies, Trypsin, Tryptic peptides,
Method development

1 Introduction

Therapeutic monoclonal antibodies (mAbs) are a relatively novel,

growing field in the pharmaceutical industry, with over $100 billion
dollars in sales across the world in 2017. Intact therapeutic mAbs
are typically modeled after the human IgG class of immunoglobu-
lins (Igs): homodimers consisting of two identical glycosylated
heavy chains (50–70 kDa) and two identical light chains
(22–24 kDa) linked together by disulfide bonds. The Ig is further
segmented at the hinge region by function, with the Fab (fragment
antigen binding) representing the upper segment of the protein

Loralie J. Langman and Christine L.H. Snozek (eds.), LC-MS in Drug Analysis: Methods and Protocols,
Methods in Molecular Biology, vol. 1872, https://doi.org/10.1007/978-1-4939-8823-5_9,
© Springer Science+Business Media, LLC, part of Springer Nature 2019

85
86 Maria Alice V. Willrich

Antigen binding site

Fragment
antigen
binding (Fab)

Disulfide bonds
hinge

CH2 CH2

Crystallizable
fraction (Fc)

CH3 CH3

Fig. 1 Immunoglobulin structure. Each immunoglobulin (Ig) consists of two

identical glycosylated heavy chains (50–70 kDa) and two identical light chains
(22–24 kDa). The light chains are linked to the heavy chains by a single disulfide
bond, while the heavy chains are linked together by two or more disulfide bonds
depending on the Ig isotype and/or subclass. The Ig is further segmented at the
hinge region by function, with the Fab (fragment antigen binding) representing
the upper segment of the protein and the Fc (crystallizable fraction) portion
representing the lower segment of the protein. VL, variable region of the light
chain; VH, variable region of the heavy chain; CL, constant region of the light
chain; CH1, CH2, and CH3, constant regions 1, 2, and 3 of the heavy chain

and the Fc (crystallizable fraction) portion representing the lower

segment of the protein (Fig. 1).
The two Fab arms contain the N-terminal portion of the heavy
chain and are associated with either a kappa or lambda light chain.
Together the N-terminal portions of the heavy and light chains
form the variable region, which contains the antigen-binding com-
plementary determining region (CDR). The C-terminal portion of
each heavy and light chain contains the constant region. Therapeu-
tic mAbs are usually of the IgG kappa isotype, predominantly of the
IgG1 subclass, with a few being IgG2, IgG4, or hybrids. When
using mass spectrometry to characterize and quantify mAbs, the
variable region is the target, and its uniqueness is compared to the
endogenous polyclonal Ig repertoire [1].
Therapeutic mAbs have unique characteristics when compared
to small molecule drugs. They do not undergo traditional phase I
or phase II metabolism or elimination via cellular transporters.
Instead, the Fc portion is recognized by receptors on the surface
of endothelial cells that internalize the Igs, leading to lysosomal
degradation [2]. The pharmaceutical industry has invested in sev-
eral modifications (Fc engineering) to increase mAb half-lives in
circulation in order to increase efficacy. Most mAbs in clinical use
have half-lives of a week or longer, resulting in long dosing intervals
(2–8 weeks). Intravenous administration allows for application of
larger volumes, lower immunogenicity, and higher bioavailability,
 LC-MS/MS for Therapeutic Monoclonal Antibodies 87

whereas more convenient subcutaneous injections will work well

for smaller volumes [1].
Most of the clinical indications for monitoring mAbs concen-
trations are related to loss of response to therapy and poor thera-
peutic efficacy. Clinical tests for infliximab, adalimumab,
certolizumab, vedolizumab, and eculizumab are available commer-
cially in the United States, and there are many more in the pipeline.
Infliximab and adalimumab are among the top 5 mAb best sellers,
and serum measurement of mAb concentrations at trough is
increasingly becoming part of routine patient management. Espe-
cially considering the high cost of therapy, which can vary from
$15,000 a year for TNF inhibitors to $500,000 a year for comple-
ment inhibitors, both the healthcare system and patients can signif-
icantly benefit from higher rates of success and fewer treatment
failures. Methodologies for measurement of infliximab and adali-
mumab vary widely and include both mAb quantitation and the
measurement of immunogenicity, i.e., the presence of anti-drug
antibodies. Immunogenicity is invariably assessed using immunoas-
says, due to its heterogeneous nature. The mAb quantitation may
be performed using immunoassays, cell-based assays [3, 4], liquid
chromatography [5], or mass spectrometry [6]. Currently mass
spectrometry plays a central role in the routine quality control of
therapeutic mAb production [7–9], and new mass spectrometry-
based methodologies for monitoring are continuously appearing in
the literature [1, 10–13].

1.1 Tryptic Peptide In order to quantify mAbs using mass spectrometry, the mAb of
Methods interest must first be differentiated from the very similar polyclonal
background of over 1 g/dL of endogenous human Igs in serum.
Historically, proteins have been quantitated by LC-MS/MS using
specific tryptic fragments (referred to as “proteotypic” peptides)
[14]. Multiple tryptic peptides can be quantitated at the same time,
as shown for the quantitation of IgG subclasses in serum by
LC-MS/MS [15]. For chimeric mAbs, whose entire variable region
is of animal origin, such as infliximab and rituximab, trypsin diges-
tion is possible as the nonhuman variable region is large (>250
amino acids). This increases the likelihood of finding unique signa-
ture peptides on the light chain and/or heavy chain which is specific
to that mAb and not found in the human polyclonal background.
Not every mAb will be amiable to the digest method especially
as newer mAbs are humanized (only small portions of the variable
region are of animal source, and they are engrafted onto a human
framework, such as eculizumab) or even fully human (genetically
engineered antibodies generated by phage display libraries, such as
adalimumab). Experiments to find a peptide specific for adalimu-
mab, for instance, failed to detect a tryptic peptide not found in the
polyclonal serum background (data not published). For other
mAbs, sensitivity of the method will be the limiting factor. For
88 Maria Alice V. Willrich

example, while rituximab is a chimeric IgG1 mAb similar to inflix-

imab, it was found that the tryptic method did not allow for the
sensitivity needed for a clinical assay, without pre-analytical
immunoenrichment.
One of the challenges with the development of a tryptic
method for large proteins such as mAbs is the standardization of
the digest. Below we describe a method for LC-MS/MS quantita-
tion of tryptic peptides unique to infliximab, a chimeric (70%
human/30% murine) IgG1 kappa monoclonal antibody targeting
tumor necrosis factor (TNF) alpha. Briefly, the protocol utilizes a
simple and relatively inexpensive purification or Ig-enrichment step
using the Ig fraction after saturated ammonium sulfate precipita-
tion. The mixture is then treated with trifluoroethanol (TFE),
dithiothreitol (DTT), and iodoacetamide (IAA) to unfold the pro-
tein, reduce the disulfide bonds between the heavy and light chains,
and prevent their reformation. Trypsin is then added to cut the
chains into specific peptides: two of these peptides being the
infliximab-unique peptides from the light and heavy chain variable
regions of the mAb (Fig. 2). After addition of isotopically labeled

1) Digest mAb to
generate clonotypic
peptides

2) Quantify mAb from C C

mixture containing H
2
H
2

non-specific peptides
using LC-MS/MS C
H
C
H
3 3
Intensity (cps)

LC retention time (min)

Fig. 2 Trypsin digestion will generate unique peptides for quantitation.

Pre-analytical steps will promote denaturation (protein unfolding) and reduction
(cysteine reduction breaks disulfide bond connecting mAb light and heavy
chains), and alkylation of cysteines prevents disulfide bond from reforming.
Subsequently, digestion by trypsin cleaves the intact immunoglobulin into
smaller peptides. The peptide mixture is separated by liquid chromatography
before analysis by tandem mass spectrometry. Peptides specific to the variable
region of the mAb either on the light chain or heavy chain, which do not cross-
react with human sequences, are used to quantitate the mAb
 LC-MS/MS for Therapeutic Monoclonal Antibodies 89

peptide retention time standards, the mixture is subjected to

reverse-phase C8 liquid chromatography and selective reaction
monitoring using a triple quadrupole mass spectrometer. Fragment
ions from the infliximab-specific peptides are monitored and com-
pared to a standard curve for quantitation [6]. This method can be
adapted and translated to other mAbs with similar outcomes.

2 Materials

Prepare all solutions using ultrapure water and analytical grade

reagents. Reagents should be prepared at room temperature, unless
otherwise noted.

2.1 Reagents and 1. 2,2,2-Trifluoroethanol (TFE): When handling it, wear gloves
Mobile Phases and pipette under a hood. Store at room temperature.
2. 50 mM ammonium bicarbonate: Weigh out 791 mg of ammo-
nium bicarbonate and dissolve in 200 mL of water, mixing well
at room temperature. Store at room temperature.
3. Saturated ammonium sulfate solution: Weigh out 150 g of
ammonium sulfate. Add 200 mL of water. Keep solution at
room temperature. Mix periodically over 8 h or more to ensure
solution is saturated and crystals are still visible (see Note 1).
Immediately before use, mix solution for 30 min using a stir
plate. Store at room temperature.
4. 200 mM dithiothreitol (DTT). Weigh out 30.85 mg of DTT.
Add 1 mL of 50 mM ammonium bicarbonate to dissolve. This
solution can be scaled up for larger batches; each sample requires
0.01 mL of DTT. This reagent should be prepared fresh daily;
do not store (see Note 2).
5. 200 mM iodoacetamide (IAA). Weigh out 37 mg of IAA. Add
1 mL of 50 mM ammonium bicarbonate to dissolve the powder.
This solution can be scaled up for larger batches; each sample
requires 0.02 mL of IAA. This reagent should be prepared daily.
Once ready, wrap the solution in aluminum foil (see Note 3) and
keep it at room temperature.
6. 1 mg/mL trypsin from bovine pancreas. Weigh out 5 mg of
trypsin. Dissolve in 5 mL of 50 mM ammonium bicarbonate.
The solution should be prepared fresh daily (see Note 4).
7. 1% formic acid. To a 25 mL volumetric flask, add 25 μL of
formic acid. Bring up to volume with water, mixing well. The
solution should be stored at room temperature.
8. Mobile phase A: 0.1% formic acid in water. In a 2 L reagent
bottle, add 2 L of water. Then, add 2 mL of formic acid, mixing
well. The solution should be stored at room temperature and
discarded after 1 week of use.
90 Maria Alice V. Willrich

9. Mobile phase B: 0.1% formic acid in acetonitrile. In a 2 L reagent

bottle, add 2 L of acetonitrile. Then, add 2 mL of formic acid,
mixing well. Degas the solution. Store at room temperature and
discard after 1 week of use.

2.2 Standards and Calibrators (standards) and controls are prepared by spiking inflix-
Controls imab into normal pooled human serum (see Note 5). Prepare
standards using volumetric flasks and good laboratory pipetting
practices.
1. 10 mg/mL Infliximab stock solution: Obtain the pharmaceu-
tical preparation (trade name Remicade, Janssen Biotech) from
a pharmacy. Keep refrigerated at 4  C until the day of reconsti-
tution. The infliximab vial contains 100 mg lyophilized powder
of infliximab. Add 10 mL of water using a volumetric pipette to
the entire vial contents to obtain a concentration of 10 mg/mL
(see Notes 6 and 7).
2. 100 μg/mL infliximab standard. Add 1 mL of 10 mg/mL
infliximab stock solution to a 100 mL volumetric flask. Bring
to volume with normal (drug-free) human serum. Aliquot
350 μL into microcentrifuge tubes; stable frozen for 2 years
at < 60  C.
3. 50 μg/mL infliximab standard. Add 25 mL of the 100 μg/mL
infliximab standard to a 50 mL volumetric flask. Bring to
volume with normal (drug-free) human serum. Aliquot
350 μL into microcentrifuge tubes; stable frozen for 2 years
at < 60  C.
4. 20 μg/mL infliximab standard. Add 10 mL of the 100 μg/mL
infliximab standard to a 50 mL volumetric flask. Bring to
volume with normal (drug-free) human serum. Aliquot
350 μL into microcentrifuge tubes; stable frozen for 2 years
at < 60  C.
5. 10 μg/mL infliximab standard. Add 5 mL of the 100 μg/mL
infliximab standard to a 50 mL volumetric flask. Bring to
volume with normal (drug-free) human serum. Aliquot
350 μL into microcentrifuge tubes; stable frozen for 2 years
at < 60  C.
6. 5 μg/mL infliximab standard. Add 2.5 mL of the 100 μg/mL
infliximab standard to a 50 mL volumetric flask. Bring to
volume with normal (drug-free) human serum. Aliquot
350 μL into microcentrifuge tubes; stable frozen for 2 years
at < 60  C.
7. 2 μg/mL infliximab standard. Add 1 mL of the 100 μg/mL
infliximab standard to a 50 mL volumetric flask. Bring to
volume with normal (drug-free) human serum. Aliquot
350 μL into microcentrifuge tubes; stable frozen for 2 years
at < 60  C.
 LC-MS/MS for Therapeutic Monoclonal Antibodies 91

8. 1 μg/mL infliximab standard. Add 1 mL of the 100 μg/mL

infliximab standard to a 100 mL volumetric flask. Bring to
volume with normal (drug-free) human serum. Aliquot
350 μL into microcentrifuge tubes; stable frozen for 2 years
at < 60  C.
9. Low quality control (QC): 3 μg/mL infliximab. Add 30 μL of
the 10 mg/mL infliximab stock solution to a 100 mL volumet-
ric flask. Bring to volume with normal (drug-free) human
serum. Aliquot 1 mL into microcentrifuge tubes; stable frozen
for 2 years at < 60  C (see Note 8).
10. Medium QC: 10 μg/mL infliximab. Add 100 μL of the
10 mg/mL infliximab stock solution to a 100 mL volumetric
flask. Bring to volume with normal (drug-free) human serum.
Aliquot 1 mL into microcentrifuge tubes; stable frozen for
2 years at < 60  C.
11. Medium-high QC: 25 μg/mL infliximab. Add 250 μL of the
10 mg/mL infliximab stock solution to a 100 mL volumetric
flask. Bring to volume with normal (drug-free) human serum.
Aliquot 1 mL into microcentrifuge tubes; stable frozen for
2 years at < 60  C.
12. High QC: 80 μg/mL infliximab. Add 800 μL of the 10 mg/
mL infliximab stock solution to a 100 mL volumetric flask.
Bring to volume with normal (drug-free) human serum. Ali-
quot 1 mL into microcentrifuge tubes; stable frozen for 2 years
at < 60  C.
13. 0.2 mg/mL horse IgG digestion standard working solution:
Pipette 1 mL of 10 mg/mL stock horse IgG solution into a
50 mL volumetric flask and bring to volume with water. Trans-
fer contents to a 50 mL Falcon tube and store frozen at -20  C
(see Note 9).
14. Isotopically labeled tryptic peptides (13C, 15N stable isotopes):
Synthesize stock powder of isotopically labeled peptides for the
light chain (YASESMSGIPSR) and heavy chain (GLEW-
VAEIR) of infliximab. A 0.04 nM scale is recommended,
using a combination of 13C and 15N on valine (V), isoleucine
(I), and proline (P) amino acids of the peptides (see Table 1).
Keep stock peptides under vacuum at room temperature inside
a desiccator.
15. 1 mg/mL isotopically labeled tryptic peptides stock solutions:
Weigh out 10 mg of each labeled peptide separately. Add
10 mg of one of the peptides to a 10 mL class A volumetric
flask. Bring to volume with 1% formic acid, mixing well. Repeat
the procedure with the second labeled peptide. Store frozen at
80  C.
92 Maria Alice V. Willrich

Table 1
Tryptic peptides and transitions used to quantify infliximab

Peptides Transitions DP (V) CE (V) CXP (V)

Infliximab heavy chain peptide (1071.57 Da)
GLEWVAEIR
-y4 ion 537.1/488.4 70 23 45
-y5 ion 537.1/587.5 70 25 45
GLEWV(+6)AEI(+7)R
-y4 ion+7 543.4/495.4 40 23 25
-y5 ion+13 543.4/600.5 40 25 25
Infliximab light chain peptide (1283.58 Da)
YASESMSGIPSR
-y6 ion 643.2/616.3 78 37 45
-y10 ion 643.2/1050.5 78 27 45
YASESMSGI(+7)P(+6)SR
-y6 ion+13 649.5/629.6 160 37 45
-y10 ion+13 649.5/1063.7 160 27 45
Horse IgG light chain peptide (1377.74 Da)
VNNQALPQPIER
-y4 ion 689.9/514.3 81.4 35.1 45
-y6 ion 689.9/739.4 81.4 35.1 45
Notes: DP declustering potential, V volts, CE collision energy, CXP collision cell exit potential

16. 5 μg/mL isotopically labeled standard working solutions: Add

50 μL of the 1 mg/mL YASESMSGIPSR light chain peptide
stock solution to a 10 mL volumetric flask. To the same flask,
add 50 μL of the 1 mg/mL GLEWVAEIR heavy chain peptide
stock solution. Bring to volume with 1% formic acid, mixing
well. Transfer contents to a Falcon tube. The working solution
is stable for 1 year after prepared when kept frozen at 20  C.

2.3 Supplies and 1. Disposable culture tubes, 75  12 mm, 4.5 mL polypropylene.

Instrumentation 2. Pipette tips, various sizes.
3. HandiStep or other repeat pipetter.
4. Eppendorf barrels, various sizes.
5. 96 deep-well 1 mL round microtiter plate.
6. A triple quadrupole mass spectrometer (API5000, AbSciex) or
similar is used to monitor multiple ion pairs.
 LC-MS/MS for Therapeutic Monoclonal Antibodies 93

7. A Cohesive LX4 multiplex system (Shimadzu) or similar chro-

matography apparatus is used for sample separation and intro-
duction into the mass spectrometer.
8. Waters X Bridge C8 3  30 mm; 3.5 μm analytical column.
Similar C8 columns can be used.
9. Precolumn filter: C8 4  2.0 mm.
10. Analyst software from AB Sciex is used for data processing.
11. Orbital shaker.
12. Misonix Ultrasonic Liquid Processor.
13. Benchtop Shaking/Rotating Incubator Scientific Industries
Incubator.
14. Beckman Allegra X-30R Tabletop Plate Centrifuge or
equivalent.

3 Methods

Briefly, the method consists of sample preparation for an overnight

trypsin digestion followed by liquid chromatography separation of
peptides and mass spectrometry detection of analytes and
standards.

3.1 Saturated 1. Label a 75  12 mm polypropylene conical tube for each cali-

Ammonium Sulfate bration standard, blank (normal human serum, no infliximab),
Extraction control material, and patient sample.
2. Transfer 100 μL of calibration working standards, blank, con-
trols, and patient samples to appropriately labeled tubes.
3. Add 50 μL of the 0.2 mg/mL horse IgG working solution to all
tubes. Vortex for 30 s (see Note 10).
4. Add 75 μL of saturated ammonium sulfate to each tube, and
vortex for 30 s.
5. Centrifuge all samples at 3000g for 10 min.
6. Pipette and discard the supernatant off of the pellet now formed
in the tubes.
7. Reconstitute the pellet in 100 μL of 50 mM ammonium bicar-
bonate. Alternate vortexing for 30 s and resting for 30 s, for
10 min or until the pellet is completely dissolved.

3.2 Trypsin Digestion 1. Add 10 μL of 200 mM DTT and 100 μL of TFE to every
sample in the analytical run. The reducing agents will break up
the disulfide bonds which hold the light and heavy chains of the
immunoglobulins together. This will expose the cleavage sites
of the light and heavy chains to trypsin.
94 Maria Alice V. Willrich

2. Vortex each tube for 10 s. Cap and incubate at 55  C for 30 min

in a rocking incubator.
3. Cool the mixture down until it reaches room temperature.
4. Add 20 μL of 200 mM IAA for alkylation. Vortex each tube for
10 s. Incubate at room temperature for 1 h in the dark, on an
orbital shaker.
5. Transfer 75 μL from each polypropylene tube to a 96 deep-well
plate.
6. Once sample transfer is complete, to each well, add:
(a) 200 μL of water
(b) 50 μL of 50 mM ammonium bicarbonate
(c) 50 μL of 1 mg/mL trypsin
7. Cover the plate and vortex for 10–15 s.
8. Sonicate the plate for 1 min.
9. Incubate at 37  C for at least 8 h up to 24 h (overnight) in a
rocking incubator.
10. Add 20 μL of pure formic acid to stop trypsin activity after the
overnight incubation.

3.3 LC-MS/MS Peptides (Table 1) are unique to infliximab heavy and light chains,
Set-Up and and two transitions are monitored per peptide for added specificity.
Quantitation of The primary transition for quantitation is the light chain y6 transi-
Infliximab tion (LC-y6), with the y10 monitored as a qualitative ion. The
heavy chain y4 ion (HC-y4) is used as a second quantification ion;
the HC-y5 ion was also added as a qualitative ion.
1. Add 5 μL of the 5 μg/mL isotopically labeled standard working
solutions (retention time standards) to each well. Vortex the
plate for 10 s. These are used to monitor the retention time of
each desired peptide (clonotypic peptides from the light and
heavy chains of infliximab).
2. Place the plate in the Cohesive LX4 autosampler. Make sure
mobile phases A and B are in place, columns are equilibrated,
and the chromatography method is set up on your preferred
software (see Note 11).
3. Inject 20 μL of each digest onto a Phenomenex C8 Security-
Guard column (4  2.0 mm ID) followed by a Waters XBridge
C8 column (3.0  30 mm; 3.5 μm) with a flow of 400 μL/min.
4. Separate peptides using a 5.5 min gradient from 95% aqueous
(A: water + 0.1% formic acid) to 35% organic (B: acetoni-
trile + 0.1% formic acid) (Fig. 3).
5. Set instrument Turbo V ion source conditions as IS, 5500;
TEM, 600; CAD, 12; CUR, 40; GS1, 35; GS2, 30; and EP,
8. The DP, CE, and CXP values for the SRM transitions were
 LC-MS/MS for Therapeutic Monoclonal Antibodies 95

1.8e4
Heavy chain-y4

1.3e5
Heavy chain-y4 (IS)

4000 Light chain-y6

Intensity (cps)

2.6e4 Light chain-y6 (IS)

8.0e4
Horse IgG Light Chain-y4

1 2 3 4 5
Time (min)

Fig. 3 Chromatograms for heavy chain, light chain, horse IgG, and labeled internal standard peptides
transitions. The chromatograms illustrated above show a sample containing 10 μg/mL of infliximab. Horse
IgG was added at 50 μg/mL; isotopically labeled internal standards were added at 5 μg/mL

optimized by infusion experiments using isotope-labeled pep-

tides during the test development stage.
6. Generate a 7-point standard curve (1, 2, 5, 10, 20, 50, and
100 μg/mL) using infliximab spiked into pooled human serum
standards. Normalize peptide peak areas to horse IgG peptide
peak areas (see Note 12).
7. Quantitate both the light chain y6 and the heavy chain y4
transitions. The two transitions should match within 20% (see
Note 13).
8. Calculated concentrations: Results are automatically calculated
by the LC-MS/MS analyst software. Quantitation results are in
μg/mL and are read directly from the calibration curve with no
corrections unless a sample is diluted. If the sample is diluted (see
Note 14), the technologist should multiply the observed value
times the entered dilution factor.
9. Make sure controls used for the assay are within the specified
acceptable ranges for the test. We recommend the following
Westgard Rules for quality control or CLSI guideline
EP05 [16].
96 Maria Alice V. Willrich

4 Notes

1. The saturated ammonium sulfate solution is a critical step in

precipitating the immunoglobulins and leaving albumin and
other smaller molecular weight proteins in solution. Its prepa-
ration is critical for the success of all downstream procedures.
The saturated solution should be kept at room temperature for
at least 8 h prior to use under stirring. If crystals are not visible
after stirring is stopped, make sure to add more ammonium
sulfate to the solution under stirring until the crystals become
visible.
2. DTT is a reducing agent, which in this protocol will reduce the
light chains from the heavy chains of the immunoglobulins and
expose the sites of cleavage to trypsin. The reagent should be
prepared daily, as its reducing potency is decreased with stor-
age. Preparations stored frozen for more than 24 h significantly
decrease the signal intensity of the peptides cleaved by trypsin.
3. IAA serves as an alkylating agent, preventing the reconnection
of the disulfide bonds between the immunoglobulin light and
heavy chains after they have been reduced by DTT. The reagent
is light sensitive and should be kept in the dark until ready to
use. The reagent should be prepared fresh daily.
4. Trypsin is a serine protease which cleaves peptide chains at the
carboxyl side of amino acids, lysine and arginine, except when
either is followed by a proline. The optimal temperature for
trypsin activity is at 37  C, and optimal pH is between 7.8 and
8.7, although there is a degree of hydrolysis at pH closer to 6.5
or 7.0. When adding the trypsin to the prepared sample mix-
ture, monitor the pH in the first few runs to make sure it is
within 6.5 and 8.0. If it is not, modifying the amounts of
reagents you are adding in each step to reach the desired pH
may be helpful for troubleshooting purposes. Trypsin activity is
stopped at acid pH below 3.0.
5. During the protocol development stages, we attempted to
prepare the standards in buffer or a different matrix other
than serum. The results of those experiments were very low
or absence of cleavage by trypsin, and therefore, no peptides
were generated. This suggests trypsin needs a certain protein
ratio to successfully promote cleavage. Therefore, the recom-
mendation is to have standards and controls in serum. The
standards should be prepared weekly. We suggest taking out a
set of working standards at the beginning of each week and
discard any leftover volume at the end of the week. Keep the
working standards refrigerated in-between uses.
6. Only reconstitute the drug in water once you are ready to
proceed with preparations of calibrators and controls. The
 LC-MS/MS for Therapeutic Monoclonal Antibodies 97

drug is only stable in water for 2 h, according to the package

insert.
7. Although not mandatory, to verify the concentration of the
working standards, one can use a nephelometric/turbidimetric
total IgG assay, available in most clinical laboratories. This can
be useful if the working standards prepared do not perform
accordingly.
8. Ideally, best laboratory practices recommend that quality con-
trol preparations be different from the calibrators used to make
the standard curve. However, quality control materials are not
readily available for infliximab. Alternatively, residual waste
leftover serum from patients can be used as quality control
materials if there is enough volume to establish a range of
acceptable results and if the sample stability supports such a
practice.
9. The tryptic method for quantitation of infliximab described
here utilizes a surrogate IS from a different species (horse)
since a stable isotope-labeled version of infliximab was not
available at time of development and the ones available today
are prohibitively expensive. Horse IgG is added to patient
serum before immunoglobulin enrichment by ammonium sul-
fate protein crash to monitor digestion efficiency.
10. The horse IgG works well as an internal standard for this assay
since it undergoes trypsin digestion with the sample and
accounts for the variation in that process. When analyzing
results from quantitation, it is important to observe stability
in the peak intensities of the horse IgG transitions. A minimum
peak area intensity threshold should be defined, and significant
variation (>20%) or trends within an analytical run may be
important clues for troubleshooting.
11. Preventive maintenance and rigorous cleaning are very impor-
tant for instruments used for proteomics assays. The test
method shown here is considered relatively “dirty,” since sam-
ples do not undergo significant extraction or purification.
Therefore, an abundant quantity of peptides and small proteins
is being injected onto the chromatographic column and the
ionization source. It is important to divert non-desired chro-
matographic portions to waste to save the instrument and
minimize interferences. In addition, we suggest a stringent
monthly cleaning schedule to prevent build-up.
12. Acceptable standard values should read within 10% of expected
target value. The correlation coefficient (R2) of the curve
should be greater than 0.99 for acceptance. Outlying standards
may be deleted to improve the observed standard concentra-
tions, R2 value, and controls. No more than two nonconsecu-
tive standards may be deleted from a particular curve. If the
98 Maria Alice V. Willrich

100 μg/mL standard is deleted, all patient samples with results

>50 μg/mL should be repeated. If the 1 μg/mL standard is
deleted, concentrations <2 μg/mL cannot be reported.
13. Quantitation from the LC-y6 and HC-y4 ions should match
within 20%. Good agreement between quantitation from dif-
ferent transitions is additional evidence of complete digestion
by trypsin. Due to the lack of a stable isotopically labeled
internal standard for the entire protein, our experience has
shown that when the quantitation from the two peptides
(HC-y4 and LC-y6) differs by more than 20%, the LC-y6 is
the more robust transition to be used for quantitation, since it
elutes closer to the retention time of the horse IgG internal
standard. When troubleshooting, the HC-y4 transition may
show discrepant results in the presence of polyclonal hyper-
gammaglobulinemia in patient samples. In addition, disagree-
ment between HC-y4 and LC-y6 may indicate contamination
of the instrument, usually occurring at the ionization source or
first quadrupole. Rigorous maintenance is required when that
happens.
14. If sample dilutions are required, the dilution should be
prepared with normal (infliximab-free) human serum.

Acknowledgments

The author would like to thank her colleagues Dr. Melissa

R. Snyder and Dr. David L. Murray, who were mentors of this
test development project, and Dr. David Barnidge and Paula Lad-
wig who were instrumental in the analytical development experi-
ments and assay performance validation. The author reports an
intellectual property/royalty income interest in an LC-MS/MS-
based method for measurement of therapeutic monoclonal
antibodies.

References
1. Ladwig PM, Barnidge DR, Willrich MAV 3. Lazar-Molnar E, Delgado JC (2016) Immuno-
(2017) Mass spectrometry approaches for genicity assessment of tumor necrosis factor
identification and quantitation of therapeutic antagonists in the clinical laboratory. Clin
monoclonal antibodies in the clinical labora- Chem 62(9):1186–1198. https://doi.org/10.
tory. Clin Vaccine Immunol 24(5). https:// 1373/clinchem.2015.242875
doi.org/10.1128/CVI.00545-16 4. Pavlov IY, Carper J, Lazar-Molnar E, Delgado
2. Ordas I, Mould DR, Feagan BG, Sandborn WJ JC (2016) Clinical laboratory application of a
(2012) Anti-TNF monoclonal antibodies in reporter-gene assay for measurement of func-
inflammatory bowel disease: tional activity and neutralizing antibody
pharmacokinetics-based dosing paradigms. response to infliximab. Clin Chim Acta
Clin Pharmacol Ther 91(4):635–646. 453:147–153. https://doi.org/10.1016/j.
https://doi.org/10.1038/clpt.2011.328 cca.2015.12.015
 LC-MS/MS for Therapeutic Monoclonal Antibodies 99

5. Wang SL, Ohrmund L, Hauenstein S, in serum using nanobody enrichment coupled

Salbato J, Reddy R, Monk P, Lockton S, to MALDI-TOF mass spectrometry. Clin
Ling N, Singh S (2012) Development and val- Chem. https://doi.org/10.1373/clinchem.
idation of a homogeneous mobility shift assay 2015.253781
for the measurement of infliximab and 11. Ladwig PM, Barnidge DR, Willrich MA
antibodies-to-infliximab levels in patient (2016) Quantification of the IgG2/4 kappa
serum. J Immunol Methods 382 monoclonal therapeutic Eculizumab from
(1–2):177–188. https://doi.org/10.1016/j. serum using Isotype specific affinity purifica-
jim.2012.06.002 tion and microflow LC-ESI-Q-TOF mass spec-
6. Willrich MA, Murray DL, Barnidge DR, Lad- trometry. J Am Soc Mass Spectrom. https://
wig PM, Snyder MR (2015) Quantitation of doi.org/10.1007/s13361-016-1566-y
infliximab using clonotypic peptides and selec- 12. Mills JR, Barnidge DR, Murray DL (2015)
tive reaction monitoring by LC-MS/MS. Int Detecting monoclonal immunoglobulins in
Immunopharmacol 28(1):513–520. https:// human serum using mass spectrometry. Meth-
doi.org/10.1016/j.intimp.2015.07.007 ods 81:56–65. https://doi.org/10.1016/j.
7. Jawa V, Joubert MK, Zhang Q, Deshpande M, ymeth.2015.04.020
Hapuarachchi S, Hall MP, Flynn GC (2016) 13. Mills JR, Kohlhagen MC, Dasari S, Vander-
Evaluating immunogenicity risk due to host boom PM, Kyle RA, Katzmann JA, Willrich
cell protein impurities in antibody-based MA, Barnidge DR, Dispenzieri A, Murray DL
biotherapeutics. AAPS J 18(6):1439–1452. (2016) Comprehensive assessment of
https://doi.org/10.1208/s12248-016-9948- M-proteins using nanobody enrichment cou-
4 pled to MALDI-TOF mass spectrometry. Clin
8. Wang D, Wynne C, Gu F, Becker C, Zhao J, Chem 62(10):1334–1344. https://doi.org/
Mueller HM, Li H, Shameem M, Liu YH 10.1373/clinchem.2015.253740
(2015) Characterization of drug-product- 14. Anderson L, Hunter CL (2006) Quantitative
related impurities and variants of a therapeutic mass spectrometric multiple reaction monitor-
monoclonal antibody by higher energy C-trap ing assays for major plasma proteins. Mol Cell
dissociation mass spectrometry. Anal Chem 87 Proteomics 5(4):573–588. https://doi.org/
(2):914–921. https://doi.org/10.1021/ 10.1074/mcp.M500331-MCP200
ac503158g 15. Ladwig PM, Barnidge DR, Snyder MR, Katz-
9. Zhang B, Jeong J, Burgess B, Jazayri M, mann JA, Murray DL (2014) Quantification of
Tang Y, Taylor Zhang Y (2016) Development serum IgG subclasses by use of subclass-specific
of a rapid RP-UHPLC-MS method for analysis tryptic peptides and liquid chromatography—
of modifications in therapeutic monoclonal tandem mass spectrometry. Clin Chem 60
antibodies. J Chromatogr B Analyt Technol (8):1080–1088. https://doi.org/10.1373/
Biomed Life Sci 1032:172–181. https://doi. clinchem.2014.222208
org/10.1016/j.jchromb.2016.05.017 16. CLSI (2004) Evaluation of precision perfor-
10. Kohlhagen MC, Barnidge DR, Mills JR, mance of quantitative measurement methods:
Stoner J, Gurtner KM, Liptac AM, Lofgren approved guideline, 2nd edn. CLSI document
DI, Vanderboom PM, Dispenzieri A, Katz- EP05-A2 edn. Clinical and Laboratory Stan-
mann JA, Willrich MA, Snyder MR, Murray dards Institute, Wayne, PA
DL (2016) Screening method for M-proteins
 Chapter 10

Quantification of Methotrexate in Human Serum and Plasma

by Liquid Chromatography Tandem Mass Spectrometry
Gabrielle N. Winston-McPherson, Michael Schmeling,
and Andrew N. Hoofnagle

Abstract
Mass spectrometry (MS) is a highly specific and sensitive technique that is used for the detection of many
different analytes with diverse chemical characteristics. It has been adopted by clinical laboratories for the
quantification of small molecules and, by extension, has been widely used for therapeutic drug monitoring.
It is an attractive alternative to immunoassay methods, because it is not subject to the same interferences. A
limitation of MS (relative to immunoassays) is the turnaround time. However, this can be addressed by
workflow parallelization with other assays. Herein we describe a tandem LC-MS/MS method for the
detection and quantification of methotrexate in human plasma with a lower limit of quantification of
0.01 μM and within-assay and between-assay coefficients of variation of less than 15%. This method lacks
interference from high-abundance metabolites and utilizes kindred chromatography to improve turn-
around time in the therapeutic drug monitoring laboratory.

Key words Methotrexate, Therapeutic drug monitoring, LC-MS/MS

1 Introduction

Mass spectrometry has gained prominence in the clinical laboratory

over the last 30 years and is now being used in all areas of clinical
diagnostics [1, 2]. The technique lends itself well to the detection
and quantification of small molecules that have been traditionally
measured using immunoassays. An advantage of mass spectrome-
try, over immunoassay methods, is the improved specificity
[3]. This characteristic is important for assays used in therapeutic
drug monitoring, as inaccurate measurements of concentration can
be deleterious [4]. In the case of methotrexate, immunoassays
suffer from cross-reactivity with methotrexate metabolites,
7-hydroxymethotrexate (7-OHMTX), and 2,4-diamino-N10-
methylpteroic acid (DAMPA), leading to falsely elevated measure-
ments [5]. Moreover, the availability of methotrexate

Loralie J. Langman and Christine L.H. Snozek (eds.), LC-MS in Drug Analysis: Methods and Protocols,
Methods in Molecular Biology, vol. 1872, https://doi.org/10.1007/978-1-4939-8823-5_10,
© Springer Science+Business Media, LLC, part of Springer Nature 2019

101
102 Gabrielle N. Winston-McPherson et al.

Fig. 1 The structure of folate, methotrexate, and methotrexate metabolites. Key structural differences are
highlighted

immunoassays is limited as a widely used platform, the Abbott

DX/FLX, was discontinued.
Methotrexate is a toxic folic acid analog that is used clinically as
both an immunosuppressant and a chemotherapeutic agent
(Fig. 1). It functions by inhibiting essential cellular mechanisms
including DNA synthesis and DNA repair [6]. As an immunosup-
pressant, methotrexate is given at relatively low doses over long
periods of time as a treatment for disorders like psoriasis and
Crohn’s disease. It is given in higher doses, when used as a chemo-
therapeutic agent, requiring close monitoring and possible rescue
with leucovorin [7]. Glucarpidase is another agent used for metho-
trexate rescue that functions by aiding in the metabolism of meth-
otrexate to DAMPA [8, 9].
Therapeutic drug monitoring of methotrexate is necessary for
patients due to possible hepatic toxicity and nephrotoxicity
[10]. Additionally, therapeutic drug monitoring is important as
there is large pharmacokinetic variability between patients, making
proper dosing highly individualized [11, 12]. Mass spectrometry
methods for measuring methotrexate have been reported and
shown improved performance characteristics over immunoassays
[13–16]. It is an attractive alternative to immunoassay methods
because it is not subject to the same interferences [17]. One limita-
tion of LC-MS/MS can be delayed turnaround time. However, this
can be addressed by utilizing the same chromatographic mobile
 Quantification of Methotrexate in Human Serum and Plasma by Liquid. . . 103

phase and solid phase as other assays, with different gradients as

needed. This kindred chromatographic design can be applied to
different analytes and allows multiple assay workflows to be run in
parallel with one another and in series on the LC-MS/MS system,
as we have demonstrated previously [18]. For example, the chro-
matographic column and mobile phases that are used in this proce-
dure are now also used in our laboratory to quantify other
therapeutic drugs and metabolites, including sirolimus, cyclospor-
ine, tacrolimus, voriconazole, posaconazole, and teriflunomide,
which saves the time it normally takes to change and purge mobile
phases and to change and equilibrate columns.
We have developed an LC-MS/MS method for the quantifica-
tion of methotrexate, after extraction from plasma [18]. The lower
limit of quantification is 0.01 μM. This method is not susceptible to
analytical interference from methotrexate metabolite 7-OHMTX
(MW 470 g/mol) which has a molecular weight difference of only
16 g/mol from methotrexate (Fig. 1). Theoretically, 7-OHMTX
could fragment to give a m/z ratio the same as methotrexate leading
to a positive interference in MS/MS methods. Moreover, this assay
is not susceptible to interference by DAMPA (MW 325 g/mol),
which has been shown to cause false elevations of methotrexate in
immunoassays methods particularly after glucarpidase treatment
[4]. In addition, kindred chromatography across therapeutic drug
monitoring assays improves turnaround time.

2 Materials

2.1 Equipment 1. Waters 2795 XE Alliance HT HPLC system

and Supplies 2. Micromass Quattro Micro™ API tandem mass spectrometer
3. Ascentis Express C18 column, 2.0 cm
2.1 mm, 2.7 μm
4. Nitrogen generator
5. Multi-tube vortexer
6. Microcentrifuge
7. Adjustable pipettes and appropriate tips
8. Glass vials w/ screw neck
9. Screw cap 12
32 PTFE/silicon septa
10. 96-well collection plates
11. Pall adapter collar for centrifugation
12. Nova-Pak C-18 2.1
10 mm cartridge, waters
13. Sentry 2.1 mm guard holder, waters
14. Sealing tape pad (3 M)
15. Cap Mat for AcroPrep
16. Defibrinated plasma, UTAK
104 Gabrielle N. Winston-McPherson et al.

2.2 Reagents 1. 1 M ammonium acetate: Weigh 38.54 g of crystalline ammo-

nium acetate and transfer to a 1 L graduated cylinder contain-
ing 200 mL of water. Add water (~300 mL) to bring the final
volume to 500 mL (see Note 1).
2. 0.1 M NaOH: To a volumetric 100 mL flask, add approxi-
mately 70 mL of water. Add 0.4 g of NaOH pellets. Swirl
gently to dissolve. Fill to volume with water and mix (see
Note 2).
3. 1 mg/mL methotrexate stock standard: Weigh 5.0 mg of
methotrexate and transfer to a bottle containing 5 mL of
0.1 M NaOH. Swirl to mix and aliquot 1 mL into 1.6 mL
tubes (see Note 3).
4. 0.1 mg/mL methotrexate-d3 stock standard: Weigh 0.5 mg of
methotrexate-d3 and transfer to a bottle containing 5 mL of
0.1 M NaOH. Swirl to mix and aliquot 1 mL into 1.6 mL tubes
(see Note 3).
5. Methotrexate and methotrexate-d3 tuning solution: Into a
20 mL volumetric flask, add 0.1 mL of 1.0 mg/mL methotrex-
ate stock standard. To the same flask, add 1.0 mL of metho-
trexate-d3 stock standard (0.1 mg/mL). Add methanol to a
final volume of 20 mL. Swirl to mix (see Note 3).
6. Precipitation reagent containing internal standard: 0.05 μM
methotrexate-d3 in 1:1 methanol/acetonitrile. In a 25 mL
vial, mix 10 mL of HPLC grade acetonitrile and 10 mL of
methanol. Add 6 μL of 0.1 mg/mL methotrexate-d3 standard
solution (see Note 4).
7. Mobile phase A: 2 mM ammonium acetate/0.1% formic acid in
water. Measure 1 L of water using a graduated cylinder and add
to a 1 L Erlenmeyer flask. Add 2 mL of 1 M ammonium acetate
to flask. Add 1.14 mL of formic acid (88%) to flask and swirl to
mix (see Note 5).
8. Mobile phase B: 2 mM ammonium acetate/0.1% formic acid in
methanol. Measure 1 L of methanol using a graduated cylinder
and add to a 1 L Erlenmeyer flask. Add 2 mL of 1 M ammo-
nium acetate to flask. Add 1.14 mL of formic acid (88%) to
flask and swirl to mix (see Note 5).

2.3 Standards 1. Working standard 1: 10 μM methotrexate. Measure 200 mL of

defibrinated plasma using a graduated cylinder and transfer to a
bottle. Add 0.91 mL of 1.0 mg/mL methotrexate stock stan-
dard. Mix well.
2. Working standard 2: 5 μM methotrexate. Measure 100 mL of
defibrinated plasma using a graduated cylinder and transfer to a
bottle. Add 100 mL of working standard 1 (10 μM methotrex-
ate). Mix well.
 Quantification of Methotrexate in Human Serum and Plasma by Liquid. . . 105

3. Working standard 3: 1 μM methotrexate. Measure 180 mL of

defibrinated plasma using a graduated cylinder and transfer to a
bottle. Add 20 mL of working standard 1 (10 μM methotrex-
ate). Mix well.
4. Working standard 4: 0.5 μM methotrexate. Measure 100 mL of
defibrinated plasma using a graduated cylinder and transfer to a
bottle. Add 100 mL of working standard 3 (1.0 μM metho-
trexate). Mix well.
5. Working standard 5: 0.05 μM methotrexate. Measure 180 mL
of defibrinated plasma using a graduated cylinder and transfer
to a bottle. Add 20 mL of working standard 4 (0.5 μM metho-
trexate). Mix well.
6. Working standard 6: 0.01 μM methotrexate. Measure 160 mL
of defibrinated plasma using a graduated cylinder and transfer
to a bottle. Add 40 mL of working standard 5 (0.01 μM meth-
otrexate). Mix well.
7. Methotrexate low control: UTAK level 2, target value
0.075 μmol/L methotrexate. Reconstitute each vial with
5.0 mL of reagent grade water, using a volumetric pipet.
Recap and let it sit for 10–15 min. Swirl gently until a homoge-
neous mixture is attained. Aliquot 1.0 mL into 1.5 mL polypro-
pylene tubes and store at 4  C. Stable for 25 days at 4  C.
8. Methotrexate high control: UTAK level 4, target value
0.75 μmol/L methotrexate. Reconstitute each vial with
5.0 mL of reagent grade water, using a volumetric pipet.
Recap and let it sit for 10–15 min. Swirl gently until a homoge-
neous mixture is attained. Aliquot 1.0 mL into 1.5 mL polypro-
pylene tubes and store at 4  C. Stable for 25 days at 4  C.

3 Methods

3.1 Extraction 1. Bring samples and supplies to room temp. Mix all standards,
controls, blanks, and samples well before pipetting.
2. Label a 1.6 mL microcentrifuge tube for each blank, standard,
control, and sample. The suggested sample order is given
below (see Note 6):
(a) Blank
(b) Standard 6
(c) Standard 5
(d) Standard 4
(e) Standard 3
(f) Standard 2
(g) Standard 1
(h) Blank
106 Gabrielle N. Winston-McPherson et al.

Fig. 2 Protein precipitation with organic solvent. The reaction mixture is shown
prior to addition of sample (a), after addition of sample and mixing (b), and after
centrifugation (c)

(i) Control low

(j) Control medium
(k) Control high
(l) Experimental/patient specimens
3. Add 400 μL of methotrexate-d3 precipitation solution to each
labeled tube (Fig. 2a).
4. Add 100 μL of blanks, standards, controls, and samples, to the
appropriately labeled tube making sure to vortex each sample
before addition. After addition, a copious amount of white
precipitate should be visible in each sample (Fig. 2b).
5. Place the tubes into the micro-tube vortexer.
6. Cap the tubes, switch the mixing speed knob to the maximum
setting, and mix the samples for 5 min (see Notes 7 and 8).
7. Centrifuge for 10 min at 15,600
g (Fig. 2c).
8. Pipette transfer at least 250 μL of each supernatant into a
corresponding 96-well collection plate (see Notes 9 and 10).
9. Apply 3 M tape (plate seals) on the top of the collection plate
to seal.

3.2 LC-MS/MS 1. Injection volume: 20 μL.

Analysis 2. Analytical column: Ascentis Express C18 column,
2.0 cm
2.1 mm, 2.7 μm.
3. Column temperature: 55 C.
Quantification of Methotrexate in Human Serum and Plasma by Liquid. . . 107

Table 1
Chromatographic program for the assay

Time (min) Flow (min/mL) Mobile phase A (%) Mobile phase B (%)
0 0.6 90 10
0.6 0.6 0 100
1.2 0.6 90 10
1.8 End N/A N/A

Fig. 3 Liquid chromatography tandem mass spectrometry of the method

4. Solvent elution program: see Table 1.

5. Expected retention times: Methotrexate and methotrexate-d3:
0.95–1.25 min (Fig. 3) (see Notes 11 and 12).
108 Gabrielle N. Winston-McPherson et al.

6. Transitions monitored: Methotrexate m/z 455 !308; metho-

trexate-d3 m/z 458 ! 311 (see Note 13).
7. Instrument parameters are capillary voltage (1 kV), desolvation
temperature (400  C), desolvation gas (nitrogen), collision gas
(argon), collision gas pressure (9 psi), cone voltage (30 V),
collision energy (20 V), and dwell times (200 ms per channel)
(see Note 14).

4 Notes

1. Once prepared the ammonium acetate reagent is stable for

6 months when stored refrigerated (4–8  C).
2. The sodium hydroxide reagent is stable at ambient temperature
for 1 year after preparation.
3. Working standards made from methotrexate and
d3-methotrexate are stable for 1 year when stored frozen
(20  C). The tuning solution is stable frozen (20  C) for
2 years.
4. The precipitation reagent must be made fresh daily. Observa-
tions have shown that the internal standard is unstable in
solution. The mixture of methanol and acetonitrile may be
prepared several minutes before use, but the internal standard
should be added right before the precipitation reagent is to be
used (within 30 s to 1 min). Mix the solution well prior to
pipetting to ensure thorough incorporation of the internal
standard.
5. Mobile phase can be stored at ambient temperature and is
stable for 1 month.
6. It is very important to prepare the plate in such a fashion as to
minimize potential carryover. This assay has demonstrated car-
ryover at a level of >26 μM methotrexate. Carryover can be
minimized by pipetting lower concentration standard prior to
high concentration standards. Because the first samples from
patients are generally much higher in concentration than
subsequent samples from the same patients, they can be
pipetted at the end of the batch to avoid carryover in a similar
fashion.
7. It is important to secure the tubes in the micro-tube vortexer.
Our laboratory uses a cut piece of cardboard that is secured
over the tops of all tubes and fixed in place using scotch tape.
8. After mixing, the precipitate may be stuck in the bottom of the
tube or on the walls. It is important to dislodge any precipitate
that has been collected in the tube and resuspend the mixture
prior to centrifugation. This can be achieved by scraping each
Quantification of Methotrexate in Human Serum and Plasma by Liquid. . . 109

tube one time on a tube rack to fully suspend all precipitate,

flicking the samples 1–2 times or briefly vortexing.
9. The supernatant can also be poured carefully into a 96-well
collection plate. While pipetting is more accurate, pouring the
sample is quicker and not susceptible to dripping due to the
low surface tension of the supernatant.
10. Place the 96-well collection plate on a dark background prior
to transfer of the supernatant. Our laboratory uses a dark
colored, laminated, piece of cardstock. Visualization of transfer
is much easier when the plate is rested on a dark background.
11. Retention times vary from column to column. Expected reten-
tion times are generated as the mean  SD during develop-
ment, but observed retention times for patient samples are
generally within 0.02 min of the standards for that batch.
12. To evaluate susceptibility to interference from DAMPA or
7-OHMTX, 2 μM 7-OHMTX and DAMPA were added to a
residual patient plasma specimen that contained 0.77 μM
methotrexate. The sample was extracted per the standard pro-
tocol and analyzed using the same chromatographic program.
For this experiment, transitions for DAMPA (m/z 326 !176)
and 7-OHMTX (m/z 471 !491) were added. Unique reten-
tion times were observed for DAMPA (1.09 min) and
7-OHMTX (1.11 min) as compared to methotrexate and
methotrexate-d3 (Fig. 3). The measured concentration of
endogenous methotrexate was unchanged from the amount
measured prior to addition of 7-OHMTX and DAMPA.
13. Several accreditors and CLSI recommend the use of confirma-
tory ion transitions in LC-MS/MS assays as best practice in
demonstrating the specificity of the assay in each sample.
Older, less sensitive instruments, such as the instrument used
in this assay, require longer dwell times to achieve the sensitiv-
ity that is needed for useful clinical assays. The inclusion of
confirmatory ion transitions on these less sensitive instruments
decreases the number of peaks across the chromatographic
curve, which leads to higher imprecision of the assay. As a
result, for therapeutic drug monitoring assays in which the
analyte is expected to be present, we rely on the peak shape
and retention time of the unlabeled analyte and its labeled
internal standard to confirm the specificity of the assay. On
newer more sensitive instruments, confirmatory transitions
could be added.
14. Please note that these instrument parameters will need to be
optimized for different systems.
110 Gabrielle N. Winston-McPherson et al.

References
1. Jannetto PJ, Fitzgerald RL (2016) Effective 12. Aumente D, Buelga DS, Lukas JC et al (2006)
use of mass spectrometry in the clinical labora- Population pharmacokinetics of high-dose
tory. Clin Chem 62:92–98. https://doi.org/ methotrexate in children with acute lympho-
10.1373/clinchem.2015.248146 blastic leukaemia. Clin Pharmacokinet
2. Strathmann FG, Hoofnagle AN (2011) Cur- 45:1227–1238. https://doi.org/10.2165/
rent and future applications of mass spectrom- 00003088-200645120-00007
etry to the clinical laboratory. Am J Clin Pathol 13. Przybylski M, Preiss J, Dennebaum R, Fischer J
136:609–616. https://doi.org/10.1309/ (1982) Identification and quantitation of
AJCPW0TA8OBBNGCK methotrexate and methotrexate metabolites in
3. Garg U, Zhang YV (2016) Mass spectrometry clinical high-dose therapy by high pressure liq-
in clinical laboratory: applications in therapeu- uid chromatography and field desorption mass
tic drug monitoring and toxicology. In: Clini- spectrometry. Biomed Mass Spectrom
cal applications of mass spectrometry in drug 9:22–32. https://doi.org/10.1002/bms.
analysis. Springer, New York, pp 1–10 1200090106
4. Al-Turkmani MR, Law T, Narla A, Kellogg 14. Wu D, Wang Y, Sun Y et al (2015) A simple,
MD (2010) Difficulty measuring methotrexate rapid and reliable liquid chromatography-mass
in a patient with high-dose methotrexate- spectrometry method for determination of
induced nephrotoxicity. Clin Chem 56:1792. methotrexate in human plasma and its applica-
https://doi.org/10.1373/clinchem.2010. tion to therapeutic drug monitoring. Biomed
144824 Chromatogr BMC 29:1197–1202. https://
5. Albertioni F, Rask C, Eksborg S et al (1996) doi.org/10.1002/bmc.3408
Evaluation of clinical assays for measuring 15. Schofield RC, Ramanathan LV, Murata K et al
high-dose methotrexate in plasma. Clin Chem (2015) Development and validation of a turbu-
42(39):39–44 lent flow chromatography and tandem mass
6. Bath RK, Brar NK, Forouhar FA, Wu GY spectrometry method for the quantitation of
(2014) A review of methotrexate-associated methotrexate and its metabolites. J Chroma-
hepatotoxicity. J Dig Dis 15:517–524. togr B Analyt Technol Biomed Life Sci
https://doi.org/10.1111/1751-2980.12184 1002:169–175. https://doi.org/10.1016/j.
jchromb.2015.08.025
7. Widemann BC, Adamson PC (2006) Under-
standing and managing methotrexate nephro- 16. Gunther V, Mueller D, von Eckardstein A,
toxicity. Oncologist 11:694–703. https://doi. Saleh L (2016) Head to head evaluation of
org/10.1634/theoncologist.11-6-694 the analytical performance of two commercial
methotrexate immunoassays and comparison
8. Cavone JL, Yang D, Wang A (2014) Glucarpi- with liquid chromatography-mass spectrome-
dase intervention for delayed methotrexate try and the former fluorescence polarization
clearance. Ann Pharmacother 48:897–907. immunoassay. Clin Chem Lab Med
https://doi.org/10.1177/ 54:823–831. https://doi.org/10.1515/cclm-
1060028014526159 2015-0578
9. Green JM (2012) Glucarpidase to combat 17. Steinborner S, Henion J (1999) Liquid-liquid
toxic levels of methotrexate in patients. Ther extraction in the 96-well plate format with
Clin Risk Manag 8:403–413. https://doi.org/ SRM LC/MS quantitative determination of
10.2147/TCRM.S30135 methotrexate and its major metabolite in
10. Ahmed YAAR, Hasan Y (2013) Prevention and human plasma. Anal Chem 71:2340–2345
management of high dose methotrexate toxic- 18. Nair H, Lawrence L, Hoofnagle AN (2012)
ity. J Cancer Sci Ther 5:106–112. https://doi. Liquid chromatography–tandem mass spec-
org/10.4172/1948-5956.1000193 trometry work flow for parallel quantification
11. Evans WE, Crom WR, Abromowitch M et al of methotrexate and other immunosuppres-
(1986) Clinical pharmacodynamics of high- sants. Clin Chem 58:943. https://doi.org/
dose methotrexate in acute lymphocytic leuke- 10.1373/clinchem.2011.175067
mia. N Engl J Med 314:471–477. https://doi.
org/10.1056/NEJM198602203140803
 Chapter 11

Simultaneous Determination of Tacrolimus and

Cyclosporine A in Whole Blood by Ultrafast LC-MS/MS
Matthew W. Bjergum, Paul J. Jannetto, and Loralie J. Langman

Abstract
Numerous methods for the measurement of tacrolimus and cyclosporine A involving traditional liquid
chromatography tandem mass spectrometry (LC-MS/MS) have previously been described. The majority of
these methods use solid-phase extraction, liquid-liquid extraction, or protein precipitation extraction with
instrument run times greater than 15 s per sample. Continued demands in clinical labs for greater efficiency
and throughput have put increased stress on traditional technologies such as high-performance liquid
chromatography-ultraviolet detection (HPLC-UV) and traditional LC-MS/MS. As an improvement to the
existing methods, we describe a sensitive ultrafast LC-MS/MS with run times of less than 15 s per sample.

Key words Prograf, FK506, Neoral, CSA, Gengraf, Mass spectrometry, Rapid fire

1 Introduction

The use of cyclosporine and tacrolimus has led to major advances in

the field of transplantation, with excellent short-term outcome
concerning prevention of rejection. Patient and graft survival rates
have improved secondary to lower incidence of acute rejection
episodes and severe infectious complications [1]. However, the
toxicity of these drugs is the Achilles’ heel of current immunosup-
pressive regimens. Therapeutic drug monitoring of immunosup-
pressant drugs that have a narrow therapeutic index is an
increasingly useful tool for minimizing toxicity while maximizing
prevention of graft loss and organ rejection. Patients treated with
calcineurin inhibitors (either cyclosporine or tacrolimus) are at high
risk of developing renal injury [2]. Renal effects include tubular
dysfunction and rarely hemolytic uremic syndrome that can lead to
acute graft loss [3]. Attention must be paid to drug dose, other side
effects, and drug interactions to minimize toxicity and maximize
efficacy of these drugs. Tacrolimus is a macrolide antibiotic derived
from the fungus Streptomyces tsukubaensis. Like cyclosporine,

Loralie J. Langman and Christine L.H. Snozek (eds.), LC-MS in Drug Analysis: Methods and Protocols,
Methods in Molecular Biology, vol. 1872, https://doi.org/10.1007/978-1-4939-8823-5_11,
© Springer Science+Business Media, LLC, part of Springer Nature 2019

111
112 Matthew W. Bjergum et al.

tacrolimus inhibits calcineurin to suppress T cells. Tacrolimus is

metabolized by cytochrome P450 (CYP) 3A4; thus, its concentra-
tions are affected by drugs which inhibit (calcium channel blockers,
antifungal agents, some antibiotics, grapefruit juice) or induce
(anticonvulsants, rifampin) this enzyme [4].
Since 90% of tacrolimus is in the cellular components of the
blood, especially erythrocytes, the whole blood is the preferred
specimen for analysis of trough concentrations. Target steady-
state concentrations vary depending on clinical protocol, the pres-
ence or risk of rejection, time from transplant, type of allograft,
concomitant immunosuppression, and side effects (mainly nephro-
toxicity). Optimal trough blood concentrations are generally
between 5.0 and 15.0 ng/mL. Higher levels are often sought
immediately after transplant, but as organ function stabilizes at
about 4 weeks from transplant, doses are generally reduced in stable
patients for most solid organs [5].
Cyclosporine is a lipophilic polypeptide used to prevent rejec-
tion after solid organ transplantation; it suppresses T cell activation
by inhibiting calcineurin to decrease IL-2 production. There is
substantial inter-patient variability in absorption, half-life, and
other pharmacokinetic parameters. Cyclosporine is extensively
metabolized by CYP3A4 to at least 30 less active metabolites,
many of which are detected on immunoassays [6].
With 80% of cyclosporine sequestered in erythrocytes, the
whole blood is the preferred specimen for analysis. Dose is adjusted
initially (up to 2 months posttransplant) to maintain concentrations
generally between 150 and 400 ng/mL. After the first two postop-
erative months, the target range is generally lower, between 75 and
300 ng/mL [7].
Cyclosporine A and tacrolimus are each extracted from the
whole blood using osmotic shock lysis followed by protein precipi-
tation using methanol and zinc sulfate heptahydrate. The precipita-
tion solution also contains cyclosporine A-13C2,d4 and ascomycin
as internal standards. Samples are vortexed and centrifuged, and the
supernatant is analyzed by liquid chromatography tandem mass
spectrometry (LC-MS/MS).

2 Materials

All reagents are of HPLC or analytical grade.

2.1 Prepared 1. Mobile phase 1: 10 mM ammonium acetate, 0.1% formic acid,

Reagents and 0.01% trifluroacetic acid (TFA) prepared in water. To a 2 L
volumetric flask, add 2000 mL of type I water. While stirring,
add 1.54 g of ammonium acetate, 2 mL of formic acid, and
180 μL of TFA.
 Tacrolimus and Cyclosporine by Ultra-fast LC-MS/MS 113

2. Mobile phase 2: 50:50 methanol/water. Add 1 L of methanol

to a 2 L volumetric flask. Fill to volume with water and
mix well.
3. Mobile phase 3: 10 mM ammonium acetate, 0.1% formic acid,
0.01% TFA, and 10% acetonitrile prepared in methanol. To a
2 L volumetric flask, add 200 mL of acetonitrile. While stirring,
add 1.54 g of ammonium acetate, 2 mL of formic acid, 180 μL
of TFA, and 1800 mL of methanol.
4. Tacrolimus stock II standard, 10 μg/mL tacrolimus: Add
100 μL of 1.0 mg/mL tacrolimus reference standard to a
10 mL volumetric flask, bring to volume with methanol, and
mix well. Stable up to 2 years stored at
20  C in screw-cap
amber vials with rubber/Teflon septa.
5. Cyclosporine A-13C2,d4 stock II standard, 100 μg/mL cyclo-
sporine A-13C2,d4: Add 5.0 mg of cyclosporine A-13C2,d4 to a
50 mL volumetric flask, bring to volume with methanol, and
mix well. Stable up to 2 years stored at
20  C in screw-cap
amber vials with rubber/Teflon septa.
6. Ascomycin stock II standard, 100 μg/mL ascomycin: Add
5.0 mg of ascomycin to a 50 mL volumetric flask, bring to
volume with methanol, and mix well. Stable up to 2 years
stored at
20  C in screw-cap amber vials with rubber/Teflon
septa.
7. Working Standard 1: 1.0 ng/mL tacrolimus/25 ng/mL cyclo-
sporine A. Add 20 mL of water to a 1000 mL volumetric flask,
then add 100 μL of tacrolimus stock II standard and 25 μL of
1.0 mg/mL cyclosporine A reference standard. Bring to vol-
ume with bovine whole blood and mix well (see Note 1). Stable
up to 6 months at or below at
60  C.
8. Working Standard 2: 5.0 ng/mL tacrolimus/150 ng/mL
cyclosporine A. Add 20 mL of water to a 1000 mL volumetric
flask, then add 500 μL of tacrolimus Stock II standard and
150 μL of 1.0 mg/mL cyclosporine A reference standard.
Bring to volume with bovine whole blood and mix well. Stable
up to 6 months at or below at
60  C.
9. Working Standard 3: 10.0 ng/mL tacrolimus/400 ng/mL
cyclosporine A. Add 20 mL of water to a 1000 mL volumetric
flask, then add 1000 μL of tacrolimus Stock II standard, and
400 μL of 1.0 mg/mL cyclosporine A reference standard.
Bring to volume with bovine whole blood and mix well. Stable
up to 6 months at or below at
60  C.
10. Working Standard 4: 20.0 ng/mL tacrolimus/700 ng/mL
cyclosporine A. Add 20 mL of water to a 1000 mL volumetric
flask, then add 2000 μL of tacrolimus Stock II standard and
700 μL of 1.0 mg/mL cyclosporine A reference standard.
114 Matthew W. Bjergum et al.

Bring to volume with bovine whole blood and mix well. Stable
up to 6 months at or below at
60  C.
11. Working Standard 5: 40.0 ng/mL tacrolimus/1000 ng/mL
cyclosporine A. Add 20 mL of water to a 1000 mL volumetric
flask, then add 4000 μL of tacrolimus Stock II standard and
1000 μL of 1.0 mg/mL cyclosporine A reference standard.
Bring to volume with bovine whole blood and mix well. Stable
up to 6 months at or below at
60  C.
12. Working Internal Standard: 2.0 ng/mL ascomycin/50 ng/mL
cyclosporine A-13C2,d4. Add 20 mL of water to a 2000 mL
volumetric flask, add 56 g of zinc sulfate heptahydrate, and mix
until completely dissolved. Add 1800 μL of methanol, 1000 μL
of 100 μg/mL Cyclosporine A-13C2,d4, and 50 μL of 100 μg/
mL ascomycin; mix well. Stable up to 14 days at or below at

10  C.
13. Quality control 1: 5.0 ng/mL tacrolimus/100 ng/mL cyclos-
porine A, in drug-free whole blood. Obtained commercially.
14. Quality control 2: 15.0 ng/mL tacrolimus/300 ng/mL
cyclosporine A, in drug-free whole blood. Obtained
commercially.
15. Quality control 3: 30.0 ng/mL tacrolimus/500 ng/mL
cyclosporine A, in drug-free whole blood. Obtained
commercially.

2.2 Supplies and 1. Agilent RapidFire 365.

Analytical Equipment 2. Triple quadrupole mass spectrometer using electrospray ioniza-
tion source. e.g., Agilent Model 6495 or similar.
3. Centrifuge with deep well plate holders.
4. SPE cartridge: Agilent RapidFire-C18.
5. 96 deep-well plates. 1.5 mL.
6. 96 square-well plates. 2.0 mL.

3 Methods

3.1 Preparation of 1. Prepare working calibration standards, quality controls, and

Working Standards, unknown samples for the run: Mix samples for 5 min. Add
Controls, and 200 μL of each working calibration standard, quality control,
Unknown Samples and unknown sample to its own well of a 96 square-well plate,
(Each Run) 2.0 mL.
2. Add 200 μL of water to each sample well (see Note 2).
3. Vortex plate for 15 s.
4. Add 300 μL of working internal standard to each well (see Note
3).
 Tacrolimus and Cyclosporine by Ultra-fast LC-MS/MS 115

5. Vortex and centrifuge for 10 min at 2845 g.

6. Transfer 400 μL of each standard, control, and sample superna-
tant to a 96 deep-well plate, 1.5 mL.

3.2 Analysis 1. Place the sample extracts in the 96-well plate in the following
order (see Note 4):

Calibration standards, in order of lowest to highest concentration

(see Note 5).
Blank whole blood (carryover) control immediately after highest-
concentration calibration standard (see Note 6).
Quality controls, in order of lowest to highest concentration (see
Note 7).
Unknown samples and additional quality controls.

2. Set RapidFire 365 method to the following parameters:

(a) Pump flow rates:
Pump 1, mobile phase 1: 1.5 mL/min.
Pump 2, mobile phase 2: 1.5 mL/min.
Pump 3, mobile phase 3: 1.25 mL/min.
(b) Cycle durations in milliseconds:
Aspirate state/volume: 600 (see Note 8).
Load/wash state: 3000.
Extra wash state: 2000.
Elute state: 3500.
Re-equilibrate state: 500.
(c) Ion source parameters: gas temp ¼ 150  C, gas
flow ¼ 14 L/min, nebulizer ¼ 20 psi, sheath gas
temp ¼ 300  C, sheath gas flow ¼ 11 L/min, nozzle
voltage ¼ 300 V, and capillary voltage ¼ 5000 V.
(d) iFunnel parameters: high pressure ¼ 120 V and low
pressure ¼ 80 V.
(e) Mass spectrometer parameters: Compound-dependent
parameters are detailed in Table 1 (see Note 9).
3. Figure 1 shows the chromatography of a working standard con-
taining tacrolimus and internal standard (ascomycin).
4. Figure 2 shows the chromatography of a working standard con-
taining cyclosporine A and internal standard.
116 Matthew W. Bjergum et al.

Table 1
Analytical and detection conditions for tacrolimus, cyclosporine A, and internal standards

Mean
Retention Quantifier Quant. Qualifier Qual. #1 ratio
time Precursor product collision product ion collision qualifier
Drug (Min.) ion (m/z) ion (m/z) energy (V) #1 (m/z) energy (V) MRM #1
Tacrolimus 0.145 821.8 768.5 17 786.5 13 35
Ascomycin 0.146 809.6 756.6 17 774.6 14 41
Cyclosporine 0.144 1219.8 1202.6 14 1184.5 30 9
A
Cyclosporine 0.144 1225.8 1208.6 15 – – –
A- 13C2,d4

Fig. 1 Chromatography of a working standard containing tacrolimus and internal standard (ascomycin)

4 Notes

1. Use freshly thawed and strained bovine whole blood. Add

appropriate volume of stock II standard, mix, and allow to
equilibrate for 24 h.
2. Tacrolimus is extensively bound to the FK-binding protein
within the red blood cells (RBCs). Osmotic lysis is used to
burst the RBCs and thus releasing tacrolimus protein complexes.
 Tacrolimus and Cyclosporine by Ultra-fast LC-MS/MS 117

Fig. 2 Chromatography of a working standard containing cyclosporine A and internal standard

3. Zinc sulfate/methanol denatures and dissociates the protein

which causes the release of tacrolimus.
4. Sample order is at the discretion of the user; the rationale for this
order is as follows. The highest concentration standard is placed
last in the calibration curve and is followed by a blank sample to
assess any carryover. Quality control samples are interspersed
with unknown samples to monitor the success of analysis
throughout the run. To ensure at least 10% of each clinical run
is comprised of quality controls and calibrators, we run one
control after every nine patient samples.
5. Ensure that the curve is drawn correctly and there are no points
that are excluded from a good linear or quadratic fit. The
recommended R2 value is greater than 0.990. Cyclosporine
standards were calibrated based on a quadratic 1/x2 curve fit.
Tacrolimus standards were calibrated based on a linear 1/x2
curve fit.
6. A peak with the same retention time as tacrolimus or cyclospor-
ine should have a calculated concentration of less than 50% of
working standard.
7. Calculated results should fall within two standard deviations of
the expected value, exhibit good chromatography, and stable
retention times.
8. The instrument aspirates for a specified time rather than speci-
fied volume.
118 Matthew W. Bjergum et al.

9. Quantifier and qualifier MRM (multiple reaction monitoring)

transitions are based on a single precursor ion for each drug.
Cyclosporine internal standard produces minimal fragment ions
that are reliable and reproducible. Mass spectrometry para-
meters will vary between instruments and must be optimized
for the specific LC-MS/MS used.

References

1. Gabardi S, Halloran PF, Friedewald J (2011) 5. Scott LJ, McKeage K, Keam SJ et al (2003)
Managing risk in developing transplant Drugs 63:1247–1297. https://doi.org/10.
immunosuppressive agents: the new regulatory 2165/00003495-200363120-00006
environment. Am J Transplant 11:1803–1809. 6. Dunn CJ, Wagstaff AJ, Perry CM, Plosker GL,
https://doi.org/10.1111/j.1600-6143.2011. Goa KL (2001) Cyclosporin: an updated review
03653.x of the pharmacokinetic properties, clinical effi-
2. Burdmann EA, Andoh TF, Yu L, Bennett WM cacy and tolerability of a microemulsion-based
(2003) Cyclosporine nephrotoxicity. Semin formulation (neoral)1 in organ transplantation.
Nephrol 23(5):465–476 Drugs 61:1957–2016
3. Naesens M, Kuypers DR, Sarwal M (2009) Cal- 7. Moyer TP, Post GR, Sterioff S et al (1988)
cineurin inhibitor nephrotoxicity. Clin J Am Soc Cyclosporine nephrotoxicity is minimized by
Nephrol 4:481–508 adjusting dosage on the basis of drug concen-
4. Kahan BD, Keown P, Levy GA, Johnston A tration in blood. Mayo Clin Proc 63
(2002) Therapeutic drug monitoring of immu- (3):241–247
nosuppressant drugs in clinical practice. Clin
Ther 24:330–350
 Chapter 12

Liquid Chromatography-Tandem Mass Spectrometry

(LC-MS/MS) Method to Quantify Gabapentin and Pregabalin
in Urine
Stephen Merrigan and Kamisha L. Johnson-Davis

Abstract
Gabapentin and pregabalin are anticonvulsant drugs that are also utilized for pain management. A mass
spectrometry method was developed and validated to quantify gabapentin and pregabalin in urine to
support testing for adherence.

Key words Anticonvulsants, Mass spectrometry, LC-MS/MS, Urine, Pain management

1 Introduction

Gabapentin (Neurontin) is an anticonvulsant drug that was

approved for use as an adjunctive treatment of partial seizures in
both children and adults [1]. Gabapentin is also indicated for the
management of postherpetic neuralgia in adults [2, 3]. It is also
used as treatment for neuropathic pain following spinal cord injury,
post-traumatic stress disorder, poststroke pain syndrome, alcohol
withdrawal, migraine therapy, hot flashes associated with prostate
cancer treatment, and postoperative pain after cancer surgery
[3, 4]. Although gabapentin is structurally similar to gamma-
amino butyric acid (GABA), gabapentin does not interact with
GABA receptors, nor is it converted to GABA or a GABA agonist
[5, 6]. The general mechanism by which gabapentin exerts its
anticonvulsant action is unknown; however, the drug binds to the
alpha 2-delta subunit of the voltage-gated calcium channels in the
CNS to decrease calcium influx, which will reduce neurotransmitter
release [4]. Gabapentin is not metabolized in the liver, nor does it
induce liver enzymes. The drug circulates relatively unbound in
serum, with a protein-bound fraction of about 3% and has a volume
of distribution of approximately 58 L [6, 7]. Gabapentin is

Loralie J. Langman and Christine L.H. Snozek (eds.), LC-MS in Drug Analysis: Methods and Protocols,
Methods in Molecular Biology, vol. 1872, https://doi.org/10.1007/978-1-4939-8823-5_12,
© Springer Science+Business Media, LLC, part of Springer Nature 2019

119
120 Stephen Merrigan and Kamisha L. Johnson-Davis

eliminated via the kidneys with an elimination half-life of approxi-

mately 5–7 h [5–7]. Impaired renal function can substantially
decrease the clearance of gabapentin. The major side effects of the
drug include somnolence, dizziness, ataxia, fatigue, and nystagmus.
Administration of gabapentin does not influence the pharmacoki-
netics of conventional anticonvulsant drugs nor are the pharmaco-
kinetics of gabapentin modified by the presence of other
anticonvulsant drugs [5].
Pregabalin (Lyrica) is an analog of the inhibitory neurotrans-
mitter GABA, as well as gabapentin. The mechanism of action is
not fully elucidated; however, it is known that the drug binds to the
alpha2-delta subunit of the voltage-gated calcium channels in the
CNS to decrease neurotransmitter release [8]. The drug is utilized
for the management of neuropathic pain associated with diabetic
peripheral neuropathy, postherpetic neuralgia, fibromyalgia,
generalized anxiety disorder, spinal cord injury, and others
[2, 9–13]. Pregabalin is also indicated for adjunctive therapy for
adult patients with partial-onset seizures [3, 14]. Pregabalin is
rapidly absorbed after oral administration; the drug is not highly
protein bound; it is not metabolized by the liver but is excreted in
the urine, unchanged [15, 16]. The elimination half-life is around
5–7 h., and the volume of distribution is about 0.5 L/kg. Prega-
balin side effects include asthenia, dry mouth, constipation, periph-
eral edema, dizziness, somnolence, ataxia, confusion, and blurred
vision [8].
Therapeutic drug monitoring (TDM) of gabapentin and preg-
abalin for seizure management is not widely employed but is clini-
cally beneficial, especially in patients with poor renal function, since
both drugs are excreted via the kidneys. TDM is typically per-
formed on serum/plasma samples in order to assist clinicians to
manage drug therapy within the therapeutic range. In the field of
pain management, pregabalin and gabapentin can be used in com-
bination with other drugs to treat pain, such as opiates and benzo-
diazepines [17]. Urine drug testing is widely utilized to assist
clinicians to assess patient adherence/compliance to prescribed
medications. A mass spectrometry method to quantify gabapentin
and pregabalin in urine can be used to support testing for medica-
tion adherence/compliance.

2 Materials

2.1 Supplies 1. Cellulose acetate filter for plasma preparation, 0.45 μm.
and Equipment 2. Volumetric glassware and pipettes.
3. 10 L container with a spigot or tap.
4. Transfer pipettes with tips.
5. 96 deep-well plate.
 Gabapentin and Pregabalin in Urine 121

6. 96 deep-well plate mat cover.

7. Centrifuges with rotors for 96-well plates and for 250 mL
bottles.
8. CTC Pal autosampler with cooled sample tray, Agilent series
1200 HPLC pump, and an AB SCIEX API 4000 mass spec-
trometer with analyst software 1.5.1. or similar.
9. Phenomenex Kinetex, 2.1 mm  50 mm 2.6 μm, Biphenyl
HPLC column, or similar.
10. AB SCIEX Quantitation Wizard Software.

2.2 Solutions 1. Synthetic urine: Add 100 g of urea, 35 g of sodium chloride,

14 g of potassium phosphate, 5.0 g of creatinine, and 2.75 g of
sodium phosphate to a 5 L beaker. Fill to volume with 18 MΩ
water. Stir to mix. Store at < 60  C, stable for 1 year.
2. Blank plasma, e.g., expired plasma from a blood bank: Centri-
fuge plasma at 7826 rcf for 30 min at 4  C, filter through
0.45 μm cellulose acetate filter to remove particulates. Test
plasma to ensure it is negative for gabapentin and pregabalin.
Store at < 20  C, stable 6 months (see Note 1).
3. Calibrator 1: 0.25 μg/mL gabapentin and pregabalin. Add
12.5 μL each of 1.0 mg/mL gabapentin and pregabalin to a
labeled 50 mL volumetric flask . Fill to volume with blank
plasma. Add a stir bar and insert the stopper. Stir at room
temperature for 1 h or until all reference material is in solution
(see Note 2).
4. Calibrator 2: 0.5 μg/mL gabapentin and pregabalin. Add
25 μL each of 1.0 mg/mL gabapentin and pregabalin to a
labeled 50 mL volumetric flask. Fill to volume with blank
plasma. Add a stir bar and insert the stopper. Stir at room
temperature for 1 h or until all reference material is in solution.
5. Calibrator 3: 2.5 μg/mL gabapentin and pregabalin. Add
125 μL each of 1.0 mg/mL gabapentin and pregabalin to a
labeled 50 mL volumetric flask. Fill to volume with blank
plasma. Add a stir bar and insert the stopper. Stir at room
temperature for 1 h or until all reference material is in solution.
6. Calibrator 4: 5.0 μg/mL gabapentin and pregabalin. Add
250 μL each of 1.0 mg/mL gabapentin and pregabalin to a
labeled 50 mL volumetric flask. Fill to volume with blank
plasma. Add a stir bar and insert the stopper. Stir at room
temperature for 1 h or until all reference material is in solution.
7. Calibrator 5: 25 μg/mL gabapentin and pregabalin. Add
1250 μL each of 1.0 mg/mL gabapentin and pregabalin to a
labeled 50 mL volumetric flask. Fill to volume with blank
plasma. Add a stir bar and insert the stopper. Stir at room
temperature for 1 h or until all reference material is in solution.
122 Stephen Merrigan and Kamisha L. Johnson-Davis

8. Calibrator 6: 50 μg/mL gabapentin and pregabalin. Add

2500 μL each of 1.0 mg/mL gabapentin and pregabalin to a
labeled 50 mL volumetric flask. Fill to volume with blank
plasma. Add a stir bar and insert the stopper. Stir at room
temperature for 1 h or until all reference material is in solution.
9. Calibrator 7: Blank. Process 50 mL blank plasma as described
above, but do not add gabapentin or pregabalin stock
solutions.
10. Quality control 1: 5 μg/mL gabapentin and pregabalin. Add
15 mL synthetic urine to a labeled 25 mL volumetric flask. Add
125 μL each of 1.0 mg/mL gabapentin and pregabalin. Fill to
volume with synthetic urine. Add a stir bar and insert a stopper
into the flask. Stir the solutions for 1 h at room temperature
(see Note 3).
11. Quality control 2: 15 μg/mL gabapentin and pregabalin. Add
15 mL synthetic urine to a labeled 25 mL volumetric flask. Add
375 μL each of 1.0 mg/mL gabapentin and pregabalin. Fill to
volume with synthetic urine. Add a stir bar and insert a stopper
into the flask. Stir the solutions for 1 h at room temperature.
12. Internal standard/precipitation solution: Obtain a 10 L con-
tainer with a spigot or tap. Add 4.0 L of methanol and 4.0 L of
acetonitrile to the container. Add 4.8 mL of 0.1 mg/mL
gabapentin-d10 internal standard and 4.8 mL of 0.1 mg/mL
pregabalin-d6 internal standard using an accurate syringe or
pipette. Add a stir bar and mix at room temperature for 1 hour.
Aliquot into appropriately labeled 1.0 L bottles (see Note 4).
13. Mobile phase A/postcrash diluent: 0.1% formic acid in clinical
laboratory reagent water. Add 1 mL of formic acid to a 1 L
beaker; fill to 1 L with 18 MΩ water. Stir to mix. Store at
23  C, stable 1 month (see Note 5).
14. Mobile phase B: 0.1% formic acid in acetonitrile. Add 1 mL
formic acid to a 1 L beaker; fill to 1 L with mass spectrometry
grade acetonitrile. Store at 23  C, stable for 3 months.
15. 1:1:1 methanol:acetonitrile:water (v/v). Add 10 mL each of
HPLC grade methanol, acetonitrile, and water to a 50 mL
glass bottle and cap. Make fresh before use.
16. Prime intermediate solution: Add 10 μL each of 1.0 mg/mL
gabapentin and pregabalin stocks to a 5 mL volumetric flask
and bring to volume with HPLC grade methanol. Mix well.
Store at 4  C, stable for 3 months.
17. Prime solution: Add 325 μL of the prime intermediate solution
to a 25 mL volumetric flask and bring to volume with 1:1:1
methanol:acetonitrile: water. Transfer 1.6 mL of prime solu-
tion to autosampler vials and cap. Stable for 1 year at < 60  C.
 Gabapentin and Pregabalin in Urine 123

3 Methods

3.1 Sample 1. Pipette 20 μL of each calibrator, quality control, and patient

Preparation sample into its own well of a 96-well plate (see Note 6).
2. Add 380 μL internal standard precipitation solution to each
well and seal with a plate mat.
3. Mix by a 30 s vortex at 1006 rcf.
4. Centrifuge for 5 min at 3500 rcf.
5. Pipette 380 μL of postcrash diluent (mobile phase A) into
each well of a second 96-well plate.
6. Using the 8-channel pipette, pipette 20 μL supernatant from
precipitated sample plate into the second plate containing the
postcrash diluent with pipette tips immersed in liquid. Rinse
tips by drawing diluent into expelled sample tips and
re-expelling.
7. Seal the plate with a plate mat cover.

3.2 Analysis This assay employs electrospray ionization in positive ion mode and
multiple reaction monitoring (MRM) of mass transitions.
1. Set autosampler parameters according to Table 1. The injec-
tion volume is 10 μL.
2. Set mobile phase gradient according to Table 2. The flow rate
is 0.4 mL/min (see Note 7).
3. Set integrated switching valve parameters according to
Table 3.
4. Set mass spectrometer conditions according to Table 4.
5. Run samples. Retention times and MRM transitions are
shown in Tables 5 and 6, respectively.
6. Analyze data. The calibration curve is linear with 1/x weight-
ing (see Note 8).

4 Notes

1. The calibrators were made in plasma because this assay was

designed to run both urine and plasma matrices. Sample
preparation for the two matrices is the same.
2. Calibrators and blank can be transferred in 400 μL aliquots to
labeled tubes and capped tightly. Placed upright in a < 60  C
freezer; these solutions are stable for 1 year.
3. Controls can be transferred in 150 μL aliquots to appropri-
ately labeled tubes. Cap tightly. Store at < 60  C and the
solution is stable for 1 year.
124 Stephen Merrigan and Kamisha L. Johnson-Davis

Table 1
Autosampler parameters

Loop volume 1 (μL) 20

Injection volume (μL) 10
Airgap volume (μL) 3
Front volume (μL) 5
Rear volume (μL) 5
Filling speed (μL/s) 5
Pull-up delay (ms) 3000
Inject to LC Vlv1
Injection speed (μL/s) 5
Pre inject delay (ms) 500
Post inject delay (ms) 500
Needle gap valve clean (mm) 3
Valve clean time solvent 1 (s) 3
Valve clean time solvent 2 (s) 3
Post clean time solvent 1 (s) 2

Table 2
Mobile phase gradient

Total time Flow rate Mobile phase Mobile phase

Step (min) (μL/min) aqueous (%) organic (%)
0 0.00 400 95.0 5.0
1 1.50 400 87.0 13.0
2 2.00 400 83.0 17.0
3 2.10 400 5.0 95.0
4 2.90 400 5.0 95.0
5 3.00 400 95.0 5.0
6 3.50 400 95.0 5.0

4. The internal standard precipitation solution will be run with

every batch of samples using prepared aliquots. Store at
2–8  C and the solution is stable for 1 year. Validate the
solution prior to placing into use by confirming performance
of a calibration curve and accuracy of control values.
 Gabapentin and Pregabalin in Urine 125

Table 3
Integrated valve parameters

Valco valve Diverter

Position Total time (min) Position
Default position 0 Left
1 1.0 Right
2 2.0 Left

Table 4
Mass spectrometer parameters

Curtain gas (CUR) 50 psi

Ion spray voltage (IS) 2000 V
Temperature (TEM) 500  c
Ion source gas 1 (GS1) 55 psi
Ion source gas 2 (GS2) 60 psi
Collision gas (CAD) 7
Declustering potential (DP) 50 V
Entrance potential (EP) 10 V

Table 5
Approximate retention times

Drug Retention time (minutes)

Gabapentin 1.51
Gabapentin-d10 1.46
Pregabalin 1.23
Pregabalin-d6 1.2

5. Prepare the mobile phases as accurately as possible. Variations

may affect retention times.
6. Urine samples in plastic containers are acceptable, refrigerated
(4  C) up to 1 month or frozen ( 20  C) for up to 1 month.
Assay procedure is performed at room temperature. If patient
samples are frozen, allow samples to thaw before analysis.
7. Allow the column to equilibrate for 20 min before use.
8. Acceptable calibration requires an R2 value greater than 0.995
and control values within two standard deviations of the
126 Stephen Merrigan and Kamisha L. Johnson-Davis

Table 6
MRM transitions

Q1 Q3 Collision Collision exit Dwell

Analyte (m/z) (m/z) energy (V) potential (V) (m s)
Gabapentin (quantifier ion) 172.1 137.1 21 27 50
Gabapentin (qualifier ion) 172.1 95.1 31 15 50
Pregabalin (quantifier ion) 160.1 125.2 18 25 50
Pregabalin (qualifier ion) 160.1 83.2 22 15 50
Gabapentin-d10 (quantifier ion) 182.1 147.2 21 27 50
Gabapentin-d10 (qualifier ion) 182.1 103.5 31 15 50
Pregabalin-d6 (quantifier ion) 166.1 131.1 18 25 50
Pregabalin-d6 (qualifier ion) 166.1 89.2 22 15 50

mean. Quantitation is based on peak area ratio of analyte to

deuterated internal standard in the sample compared to the
calibration curve. Specificity of analysis is confirmed by the
respective qualitative mass transition ratio in the sample com-
pared to average transition ratios in the calibration curve
30%.

Acknowledgments

The authors would like to thank the ARUP Institute for Clinical
and Experimental Pathology for support.

References

1. Dougherty JA, Rhoney DH (2001) Gabapen- 6. McLean MJ (1995) Gabapentin. Epilepsia 36

tin: a unique anti-epileptic agent. Neurol Res (Suppl 2):S73–S86
23:821–829 7. Beydoun A, Uthman BM, Sackellares JC
2. Ettinger AB, Argoff CE (2007) Use of antiepi- (1995) Gabapentin: pharmacokinetics, efficacy,
leptic drugs for nonepileptic conditions: psy- and safety. Clin Neuropharmacol 18:469–481
chiatric disorders and chronic pain. 8. Taylor CP, Angelotti T, Fauman E (2007)
Neurotherapeutics 4:75–83 Pharmacology and mechanism of action of
3. Krasowski MD, McMillin GA (2014) Advances pregabalin: the calcium channel alpha2-delta
in anti-epileptic drug testing. Clin Chim Acta (alpha2-delta) subunit as a target for antiepi-
436:224–236 leptic drug discovery. Epilepsy Res
4. Chang CY, Challa CK, Shah J, Eloy JD (2014) 73:137–150
Gabapentin in acute postoperative pain man- 9. Shneker BF, McAuley JW (2005) Pregabalin:
agement. Biomed Res Int 2014:631756 a new neuromodulator with broad thera-
5. Goa KL, Sorkin EM (1993) Gabapentin. A peutic indications. Ann Pharmacother
review of its pharmacological properties and 39:2029–2037
clinical potential in epilepsy. Drugs 10. Tassone DM, Boyce E, Guyer J, Nuzum D
46:409–427 (2007) Pregabalin: a novel gamma-
 Gabapentin and Pregabalin in Urine 127

aminobutyric acid analogue in the treatment of 14. Hamandi K, Sander JW (2006) Pregabalin: a
neuropathic pain, partial-onset seizures, and new antiepileptic drug for refractory epilepsy.
anxiety disorders. Clin Ther 29:26–48 Seizure 15:73–78
11. Zareba G (2005) Pregabalin: a new agent for 15. Blommel ML, Blommel AL (2007) Pregabalin:
the treatment of neuropathic pain. Drugs an antiepileptic agent useful for neuropathic
Today 41:509–516 pain. Am J Health Syst Pharm 64:1475–1482
12. Guglielmo R, Martinotti G, Clerici M, Janiri L 16. Schulze-Bonhage A (2013) Pharmacokinetic
(2012) Pregabalin for alcohol dependence: a and pharmacodynamic profile of pregabalin
critical review of the literature. Adv Ther and its role in the treatment of epilepsy. Expert
29:947–957 Opin Drug Metab Toxicol 9:105–115
13. Buoli M, Caldiroli A, Serati M (2017) Pharma- 17. Heltsley R, Depriest A, Black DL, Robert T,
cokinetic evaluation of pregabalin for the treat- Caplan YH, Cone EJ (2011) Urine drug test-
ment of generalized anxiety disorder. Expert ing of chronic pain patients. IV. Prevalence of
Opin Drug Metab Toxicol 13:351–359 gabapentin and pregabalin. J Anal Toxicol
35:357–359
 Chapter 13

The Evolving Landscape of Designer Drugs

Sherri L. Kacinko and Donna M. Papsun

Abstract
Since 2008 there has been an onslaught of new drugs in the illicit marketplace. Often referred to as
“research chemicals,” “designer drugs,” or “novel psychoactive substances” (NPS), these substances are
used for their pharmacological effects which are often similar to more widely known drugs such as ecstasy or
heroin. In some cases users specifically seek out these new chemicals, in other cases they are simply
purchasing what they believe to be their normal drug of choice from a dealer, but the product is not
what it is purported to be. Implementation of national and international systems to monitor the appearance
of new compounds enables laboratories to be prepared with validated tests to detect them in biological
specimens. The most common classes of NPS are synthetic cannabinoids, novel opioids, novel benzodia-
zepines, stimulants, and hallucinogens. Within these groups the compounds may be drugs that were
originally synthesized for research purposes during the pursuit of new therapeutic agents such as the
synthetic cannabinoid JWH-018 and the designer opioid U47700. Others like etizolam are compounds
used in other countries but not commonly seen in the USA. Some are drugs synthesized specifically to
circumvent legal controls. In all cases, these compounds present a unique challenge to forensic toxicology
laboratories which must quickly develop and validate analytical methods for the identification and quantifi-
cation in biological matrices.
This chapter is a condensed and updated version of an article originally published in Clinical and Forensic
Toxicology News.

Key words Research chemicals, Novel psychoactive substances, Synthetic drugs, Synthetic cannabi-
noids, Analogs

1 History of Novel Psychoactive Substances [1]

Designer drugs, research chemicals, novel psychoactive substances

(NPS), and “legal highs” are terms used to describe drugs designed
specifically to skirt legal controls. Although this movement picked
up steam in the new millennium, the search for new recreational
substances, either through repurposing pharmaceutical research or
modifying existing drugs of abuse, is not new.
The second International Opium Convention, effective in
1928, specifically banned morphine and heroin [2]. In response,
chemists began synthesizing esters of morphine with similar

Loralie J. Langman and Christine L.H. Snozek (eds.), LC-MS in Drug Analysis: Methods and Protocols,
Methods in Molecular Biology, vol. 1872, https://doi.org/10.1007/978-1-4939-8823-5_13,
© Springer Science+Business Media, LLC, part of Springer Nature 2019

129
130 Sherri L. Kacinko and Donna M. Papsun

structures and effects to heroin such as benzoylmorphine and

acetylpropionylmorphine, which were considered legal alternatives
to morphine and heroin.
Dr. Albert Hofmann, a chemist at Sandoz Laboratories in
Basel, Switzerland, was investigating potential medical uses of
ergot alkaloid derivatives when he synthesized lysergic acid diethy-
lamide (LSD) in 1938. The psychedelic properties of LSD were
discovered when Dr. Hofmann accidently ingested the drug, and in
1947 Sandoz Laboratories introduced it as a medication for the
treatment of psychiatric disorders [3]. LSD continued to be a
popular drug for psychiatric research and recreational use and was
eventually controlled by the DEA in 1968. Shortly thereafter the
synthesis of ALD-25 or 1-acetyl-LSD led to the first prosecution of
a drug analogue.
In 1965, Dr. Alexander Shulgin obtained a drug enforcement
agency (DEA) license and began synthesizing chemicals in a back-
yard laboratory, thus launching the modern era of designer drugs.
Dr. Shulgin developed a protocol for evaluating new drugs which
included initial testing in animals before human experiments began.
The protocol included details on determining an effective dose by
administration of increasing doses and use of a rating scale. The
subjects of these experiments were friends and acquaintances of
Dr. Shulgin and his wife. Comprehensive narratives on these experi-
ences were published in two books, Phenethylamines I have Known
and Loved (PIHKAL, 1991) and Tryptamines I have Known and
Loved (TIHKAL, 1997). These two books are often considered to
be “cookbooks” used by manufacturers looking to introduce these
drugs to a new generation of users.
While Dr. Shulgin was evaluating stimulant and hallucinogenic
drugs in California, Dr. John W. Huffman was at Clemson Univer-
sity studying the cannabinoid receptor system to gain more under-
standing of their role in diseases such as multiple sclerosis. As part
of his research, Dr. Huffman and his team synthesized more than
400 compounds which have varying degrees of binding affinity and
activity at cannabinoid receptor 1 (CB1) and cannabinoid receptor
2 (CB2). CB1 is the receptor which is responsible for the desired
effects of delta-9-tetrahydrocannabinol (THC), the primary active
component of marijuana, and CB2 is primarily a peripheral receptor
involved in immune system modulation. Dr. Huffman was search-
ing for compounds with affinity for and activity at the CB2 recep-
tor, but over two decades later, experimental drug users
“rediscovered” Dr. Huffman’s work and the potential in CB1
receptor agonists.
 Designer Drugs 131

2 Monitoring NPS

In 2007, the European Monitoring Centre for Drugs and Drug

Addiction (EMCDDA) updated its guidelines for the early warning
system (EWS) [4]. The purpose of this update was to expand the
scope from new synthetic drugs to all new psychoactive substances.
Under these guidelines a steady increase in the number and scope
of new compounds has been reported every year since 2008. Not
only has the number of monitored compounds increased; the types
are also changing. In addition to synthetic cannabinoids, opioids
and benzodiazepines have gained popularity in recent years.
In the USA, the National Drug Early Warning System
(NDEWS) was created in 2014 as a cooperative agreement between
the National Institute on Drug Abuse and the University of Mary-
land [5]. NDEWS collects information from a variety of sources
including localized early warning systems to provide a national
warehouse for information on drug trends and emerging novel
psychoactive substances.

3 Classes of NPS

3.1 Synthetic The first incidence of synthetic cannabinoids being detected in the
Cannabinoids USA occurred in late 2008 when US Customs and Border Patrol
verified the presence of HU-210 in packets of an herbal incense
product called “spice.” HU-210 was already a controlled sub-
stance, but shortly thereafter another uncontrolled drug,
JWH-018, was detected in similar products. This compound was
one of the 400 substances synthesized by Dr. Huffman. Since early
2009 the components of these herbal incenses have changed rap-
idly. As soon as the DEA adds a substance to the list of scheduled
compounds, chemists make slight modifications to the structure to
create a compound that is not listed.
A thorough review on the pharmacology and toxicology of
synthetic cannabinoids was published in 2014 and updated, along
with other classes of synthetic cannabinoids, in 2017 [6, 7]. Recrea-
tional synthetic cannabinoids target the CB1 receptor though many
also bind to and have an effect at the CB2 receptor. It is possible
that CB2 receptor activity modulates some of the undesirable
effects seen with the pure CB1 receptor agonists. The most com-
mon in vivo test for cannabinoids is the mouse tetrad test which
evaluates the effect of a compound on several different mouse
behaviors. This series of tests determines the dose of drug required
to change specific behaviors by 50% (ED50). By comparing the
ED50 of one drug with the ED50 of another drug, the potency can
be estimated. Unfortunately, this information is only available for a
handful of synthetic cannabinoids being used; the mouse tetrad has
not been performed on the vast majority of substances.
132 Sherri L. Kacinko and Donna M. Papsun

General adverse effects associated with synthetic cannabinoid

use include tachycardia, hypertension, agitation, hallucinations,
nausea, vomiting, tremors, seizures, anxiety, and paranoia. The
type and degree of effect vary significantly between different com-
pounds, and some compounds such as XLR-11 have been asso-
ciated with specific health effects. Following a cluster of exposures
to XLR-11, researchers noted that there appeared to be an associa-
tion between exposure to this substance and acute kidney injury
[8]. Furthermore, synthetic cannabinoids have been indicated by
pathologists as a cause of death [7].

3.2 Opioids Novel opioid compounds have a longer history than synthetic
cannabinoids, but after a long period of inactivity with respect to
new compounds, acetyl fentanyl burst onto the recreational drug
scene in 2013, and then in late 2015, numerous other opioid-
related compounds began to appear. These primarily consist of
substances synthesized by pharmaceutical companies looking for
new analgesic drugs for pain treatment which were abandoned at
some point in the development process. These represent a chemi-
cally diverse class of substances, most of which do not have struc-
tural similarity to opiates.
There is evidence that these drugs are both sought after by
drug users for recreational use and by drug manufacturers as adul-
terants to more commonly abused opioids such as heroin. While
novel opioids may be structurally dissimilar, in general, these com-
pounds target the μ-opioid receptor. Activation of this receptor
causes analgesia, sedation, and euphoria which are the desired
effects for users. Adverse effects include itching, nausea, constipa-
tion, and decreased respiration. Users develop tolerance to both the
desired and undesired effects of opioid compounds, thus requiring
larger and larger doses. The development of tolerance along with
unknown identity of street drugs is a dangerous combination. The
potency of these compounds varies greatly. Individuals who believe
they are using heroin may use a dose consistent with previous doses
that resulted in the desired effect, but the presence of another,
more potent, opioid could make this dose deadly. Also of concern
is the increased toxicity of opioids when combined with benzodia-
zepines. Taken alone prescription benzodiazepines rarely result in
death, but the combination of benzodiazepines and opioids can
have a synergistic effect on respiratory depression.
Overdose with novel opioids is assumed to be similar to that
which is observed with any other μ-opioid receptor agonist and, like
overdoses with heroin or prescription opiates such as oxycodone or
oxymorphone, can be treated with naloxone. Naloxone acts as
antagonist at the μ-opioid receptor. Since naloxone has a must
stronger binding affinity for the receptor than opioid agonists, it
can replace them at the receptor and block additional receptors,
quickly and effectively reversing the central nervous system (CNS)
 Designer Drugs 133

depression caused by opioids. Since naloxone is extremely safe,

several states have made it available over-the-counter, and first
responders are usually equipped with the drug as a mechanism of
reducing opioid overdose-related deaths. This antidote is very
much needed as novel opioids are being increasingly found in
postmortem cases.

3.3 Benzodiazepines Benzodiazepines are one of the most commonly prescribed psycho-
tropic drug classes in the USA. There are 14 benzodiazepines
available by prescription used to treat a variety of physical and
psychological conditions. Compounds such as midazolam and tria-
zolam have half-lives of 2–3 h and are often used to induce anes-
thesia or as sleep medication. Alprazolam, lorazepam, and
clonazepam are common medications used to treat anxiety, panic
disorders, and seizures with effects lasting for 6–10 h. Chlordiaz-
epoxide which is used as a muscle relaxant and diazepam which is an
anti-anxiolytic are two examples of long-acting benzodiazepines.
These compounds typically have half-lives exceeding 24 h. The
gamma-aminobutyric acid A receptor is the target receptor for
benzodiazepines. This receptor has multiple binding sites which
may explain the differences in degree and type of reactions observed
from different benzodiazepines. All benzodiazepines have sedating
effects making them a target for abuse. In general, the drugs are
safe, but when combined with other CNS depressants, such as
alcohol or opioids, the danger increases.
Novel benzodiazepines are often referred to as “research ben-
zos” by drug users. Medications available in other countries but not
legal prescription medications in the USA, and compounds which
are not used in any country comprise this group. Phenazepam and
etizolam were the first two benzodiazepines to appear in the illicit
drug market in the USA. The DEA reports that between 2008 and
2013, there were 284 reports of phenazepam, and the drug was
found in 31 states [9]. Etizolam appeared in 2012 with a total of
140 reports between its first appearance and June 2014 according
to the DEA (14). Other compounds that have been reported
include bromazepam, flubromazepam, flubromazelam deloraze-
pam, diclazepam, and clonazelam.
Some signs and symptoms of benzodiazepine overdose include
anxiety, agitation, dizziness, confusion, nystagmus, slurred speech,
altered mental state, amnesia, hypotension, and impaired cogni-
tion. Respiratory depression may occur with benzodiazepine use,
the degree of which will be significantly increased if used in combi-
nation with other central nervous system depressants. Flumazenil is
a specific antidote for benzodiazepine overdose, but the risk of
flumazenil may outweigh potential benefits, so it is ideally only
used to treat acute overdose in the benzodiazepine naı̈ve individual.
Designer benzodiazepines are increasingly being detected in hospi-
talizations, impaired driving cases, and overdose deaths.
134 Sherri L. Kacinko and Donna M. Papsun

3.4 Other Classes At the same time synthetic cannabinoids were making an appear-
ance in the USA, synthetic cathinones and sympathomimetic
amines – derivatives of cathinone and methamphetamine – were
gaining in popularity. Commonly called “bath salts” or “plant
food,” the first generation of these products contained compounds
such as MDPV, mephedrone, and methylone. Although the pro-
ducts were all classified under the same street name of “bath salts”
and later “party pills” or “party powders,” the types and activities of
the compounds or combinations of compounds they contained
varied greatly including stimulants, hallucinogens, entactogens,
and dissociative anesthetics.
These substances are chemically similar to cathinone, a natural
stimulant found in the khat plant, as well as amphetamine and
methamphetamine, which are two stimulants that are routinely
found as drugs of abuse. Small chemical changes to the structure
of cathinone and/or amphetamine result in several new drugs that
pharmacologically behave very similarly, producing the same spec-
trum of stimulant effects such as increased energy, tachycardia, and
hypertension.
Like synthetic cannabinoids, trends in the popularity of these
compounds can be traced to legislation controlling them. Follow-
ing passage of the Synthetic Drug Abuse Prevention Act of 2012,
the stimulant MDPV was replaced by alpha-PVP; reported “flakka”
use soared between 2014 and 2015; however, after China was
pressured by the USA to ban sale of number of chemicals, including
alpha-PVP, the chemical has disappeared. Methylone was a popular
entactogen among attendees at electronic music festivals but over
time was replaced by ethylone followed by butylone and
dibutylone.
2C-B and 5-MeO-DiPT are just two of the many psychedelic
substances described by Alexander Shulgin in his drug tomes. Of
the novel psychoactive substances that would be considered syn-
thetic hallucinogens, most only have appeared in a handful of cases,
with no one particular substance gaining significant popularity
[10, 11].
Dissociative anesthetics such as methoxetamine and 3-meth-
oxy-phencyclidine are structurally related to ketamine and phency-
clidine and are reported to have similar effects. Though the
mechanism of action is not completely understood, these com-
pounds are antagonists at the NMDA receptor. Antagonism of
this receptor results in hallucinations and dissociation.

4 Challenges of NPS

NPS present many challenges to clinical and forensic toxicologists

primarily due to the rapid change in compounds being used. From
an analytical perspective, the wide range of chemical structures and
 Designer Drugs 135

classes comprising NPS means multiple tests must be run to deter-

mine what drug might be present. In addition, it is nearly impossi-
ble for a laboratory to maintain an up-to-date test due to lack of
resources including analytical standards. Also, labs must weigh the
high cost of developing a test for a new drug with the realization
that the window of use might be narrow. By the time the test is
ready and meets industry standards, users may have moved on to
another substance. Labs must also be aware that new isomers of
existing compounds may interfere with their existing test. For
example, many of the fentanyl analogs have the same molecular
weight and fragmentation patterns, so assays which employ short
run times may not be able to resolve these isomers.
While the novel benzodiazepines and fentanyl-related substances
might cross-react with routine urine drug screens, other types of
NPS may not be detected in a patient arriving at the emergency
department. Therefore, clinical toxicologists must treat patients
suffering from toxic effects of drugs without knowing the specific
compound. Additionally, most of these drugs have never been tested
on animals, and there is limited information on their pharmacology.
Therefore it is difficult to assess the role a designer drug has in a case
because little to no information is available correlating presence or
quantitation in biological fluids to effects on the human body.

References
1. Kacinko SL, Papsun D (June 2016) Designer 6. Gurney S (2014) Pharmacology, toxicology,
drugs keep evolving: novel psychoactive sub- and adverse effects of synthetic cannabinoid
stances challenge laboratories and laws. Clinical drugs. Forensic Sci Rev 26(1):53–78
and Forensic Toxicology News (Quarterly, 7. Logan BK, Mohr ALA, Friscia M et al (2017)
AACC/CAP), 1-8. Available from: https:// Reports of adverse events associated with use of
www.aacc.org/publications/clinical-and-foren novel psychoactive substances, 2013–2016: a
sic-toxicology-news review. J Anal Toxicol:1–38
2. Cambridge University Press (1913) The sec- 8. Murphy TD, Weidenbach KN, Van Houten C
ond international opium conference. Am J Int (2013) Acute kidney injury associated with
Law 7:838–847 synthetic cannabinoid use—multiple states,
3. Hoffmann, A. (1996). LSD: Completely Per- 2012. MMWR Morb Mortal Wkly Rep
sonal. Presentation, 1996 Worlds of Con- 62:93–98
sciousness Conference. Available at http:// 9. Drug Enforcement Agency, Office of Diversion
www.maps.org/news-letters/v06n3/ Control, Drug & Chemical Identifcation Sec-
06346hof.html. tion. (2014). Phenazepam. Retrieved from
4. EMCDDA–Europol (2009) Annual Report on http://www.deadiversion.usdoj.gov/drug_
the implementation of Council Decision chem_info/phenazepam.pdf.
2005/387/JHA, European Monitoring Cen- 10. Araújo AM, Carvalho F, Bastos M de L et al
tre for Drugs and Drug Addiction and Euro- (2015) The hallucinogenic world of trypta-
pol, Publications Office of the European mines: an updated review. Arch Toxicol
Union, Luxembourg 89:1151–1173
5. National Drug Early Warning System 11. Dean BV, Stellpflug SJ, Burnett AM et al
(NDEWS) https://www.drugabuse.gov/ (2013) 2C or not 2C: phenethylamine
related-topics/trends-statistics/national-drug- designer drug review. J Med Toxicol
early-warning-system-ndews 9:172–178
 Chapter 14

Analysis of Synthetic Cannabinoid Metabolites by Liquid

Chromatography-Tandem Mass Spectrometry
Gregory C. Janis

Abstract
The analysis of synthetic cannabinoid compounds in a urine sample is currently one of the more complex
tasks facing toxicologists. The list of prevalent compounds in circulation at any given time is constantly in
flux, changing at a rapid rate to avoid legal control and to a lesser extent to avoid detection. Even with
knowledge of the chemical entities, their detection in urine is complicated by the fact that they are present
exclusively as both phase I metabolites and phase II conjugates. With proper knowledge of the correct
analytical targets, relatively simple procedures are capable of extracting and analyzing synthetic cannabi-
noids. Following enzymatic hydrolysis, compounds can be extracted through liquid partitioning proce-
dures, and the extracts are analyzed via LC-MS/MS.

Key words Synthetic cannabinoids, Spice, K2, Urine drugs of abuse

1 Introduction

Few things have challenged analytical toxicology to the degree of

synthetic cannabinoids. The inherent complexity of detecting
xenobiotics is significantly more challenging when targeting any
designer drugs, and targeting synthetic cannabinoids has raised the
challenge several steps higher. The rapid proliferation of synthetic
cannabinoids in combination with significant phase I metabolic
transformations results in the field of analytical toxicology perpetu-
ally one step behind drug trends.
Synthetic cannabinoids first appeared as drugs of abuse in
England in approximately 2004. At that time, the active com-
pounds consisted primarily of JWH-018, a research tool originally
developed by John Huffman of Clemson University [1] and later
clandestinely produced as a street drug. JWH-018 is just one of a
large family of structural derivatives possessing activity at one or
more of the cannabinoid receptor subtypes. In addition to the
chemical species related to JWH-018, multiple other families of

Loralie J. Langman and Christine L.H. Snozek (eds.), LC-MS in Drug Analysis: Methods and Protocols,
Methods in Molecular Biology, vol. 1872, https://doi.org/10.1007/978-1-4939-8823-5_14,
© Springer Science+Business Media, LLC, part of Springer Nature 2019

137
138 Gregory C. Janis

Fig. 1 Structures of representative synthetic cannabinoids

cannabinoid receptor agonists were developed as research tools or

as potential drug candidates. However, since the original introduc-
tion of clandestinely produced JWH-018, clandestine chemistry
has taken over developing new derivatives of synthetic cannabinoids
for use as drugs of abuse with nearly wavelike consistency. The
evolution of clandestine synthetic cannabinoids follows a relatively
logical path of structural permutations. Minor permutations such as
the addition of fluorine to known active structures are common. So
are minor changes in aliphatic regions of active molecules including
changing the length of carbon chains or cyclizing aliphatic chains.
Some examples of the dominant synthetic cannabinoids over the
decade are shown in Fig. 1. These drugs are typically applied to
plant-based substrates with the intention of smoking the material
similar to marijuana.
As neither animal nor human clinical trials were ever initiated
on these compounds, there was no knowledge of the safety or
metabolism of these drugs nor was there any knowledge of the
metabolism or excretion of synthetic cannabinoids. Knowing the
metabolism of these drugs is a prerequisite to urine sample analysis.
Through forensic and in vitro testing, it was determined that
JWH-018 and its most similar analogs were primarily metabolized
via terminal oxidation of the aliphatic chain and subsequently fur-
ther oxidized to a terminal carboxylic acid. Phase I metabolites are
then subjected to phase II glucuronide conjugation [2, 3].
Un-metabolized parent synthetic cannabinoids do not generally
 Synthetic Cannabinoids 139

appear in urine samples; thus urine-based assays must target the

proper metabolites for each targeted species.
The requirement for metabolic knowledge and corresponding
reference standards is just one complication of synthetic cannabi-
noid analysis. Analysis is additionally challenging due to the con-
stant evolution of these compounds. Historically, the typical
lifespan of any specific synthetic cannabinoid as a drug of abuse is
less than a year. In the period following a specific synthetic canna-
binoid entering the drug scene, legal restrictions are applied and
enforced. In response, clandestine manufacturing has historically
switched to alternative analogs lacking legal restrictions. Testing
laboratories must then identify the new chemical entity and, once
again, predict and determine the most appropriate metabolites to
analytically target.
The typical method of self-administering synthetic cannabi-
noids adds yet another layer of complexity for toxicological study.
Many of the drug entities are structurally modified by pyrolysis
[4, 5]. Thus, the chemical species a user is exposed to may not be
the same species as found in the dosing product.
Despite the plethora of synthetic cannabinoids, some analytical
strategies have proven to be applicable to the analysis of most
compounds. Liquid chromatographic analysis is straightforward.
Both C18 and C8 columns with typical gradients can produce
adequate separations, although isobaric isomers exist within the
family of drugs. One must also be aware that while the parent
drugs are amines, predominant metabolites are often zwitterions
(see Fig. 2). The existence of zwitterions does require the use of an
acidic mobile phase to obtain sufficient ionization for positive ion
mass spectrometry. Tandem mass spectrometry methods are set up
using compound-specific fragmentation. Quantitation is per-
formed against a concurrently analyzed calibration curve of the
analytes of interest.

2 Materials

2.1 Solutions 1. 50:50 methanol/water (v/v): Add 100 mL of methanol and

and Standards 100 mL of deionized water to a glass container, cap tightly, and
mix. May be stored at room temperature for up to 6 months.
2. Analytical stock standards: 500 μg/mL of each drug. Prepare
stock solutions of the analytical targets by accurately weighing
5 mg of each of the following reference standards into individ-
ual 10 mL volumetric flasks and diluting to volume with aceto-
nitrile, AB-CHMINACA butanoic acid, AB-FUBINACA
oxobutanoic acid, AB-PINACA pentanoic acid, ADBICA
N-(4-hydroxypentyl), ADBICA pentanoic acid,
ADB-PINACA pentanoic acid, AKB-48 N-pentanoic acid,
140 Gregory C. Janis

JWH-018
JWH-018 Petanoic acid

MAB-CHMINACA MAB-CHMINACA Acid

Fig. 2 Predominant metabolism of JHW-018 and MAB-CHIMACA

JWH-018 N-pentanoic acid, JWH-073 N-butanoic acid,

MAB-CHMINACA butanoic acid, MAM2201 N-pentanoic
acid, and UR-144 N-pentanoic acid.
3. 1.0 μg/mL combined working standard: Combine 20 μL of each
of the 500 μg/mL stocks in a single 10 mL volumetric flask.
Dilute to volume with 50:50 methanol/water (see Note 1).
4. 50 ng/mL combined working standard: Pipette 50 μL of the
1 μg/mL combined working standard into a 10 mL volumetric
flask. Dilute to volume with 50:50 methanol/water.
5. Internal standard stock solutions: Weigh approximately 1 mg
of each of the following isotopically labeled analogs into a
10 mL volumetric flask and dilute to volume with acetonitrile,
JWH 073 N-butanoic acid D5, JWH 018 N-pentanoic acid
D4, AM2201 N-(4-hydroxypentyl) D5, UR-144 N-pentanoic
acid D5.
6. Internal standard spiking solution: 100 ng/mL of each internal
standard. Aliquot 100 μL of each 100 μg/mL internal standard
 Synthetic Cannabinoids 141

stock in a 10 mL volumetric flask and dilute to volume with

50:50 methanol/water.
7. 50:50 hexane/ethyl acetate (v/v): Add 500 mL of hexane and
500 mL of ethyl acetate to a glass container, cap tightly, and
mix. May be stored at room temperature for up to 6 months.
8. 0.1 N hydrochloric acid (HCl): Add approximately 40 mL of
type I water to a 50 mL volumetric flask. Pipette 431 μL of
concentrated HCl (35%, 11.6 M) to the flask. Bring to volume
with reagent grade water. Transfer to a sealable glass bottle.
Cap tightly and store at room temperature and is stable 1 year
at ambient temperature.
9. 10 M potassium hydroxide: Weigh 560 g of solid potassium
hydroxide pellets into a 1 L reagent bottle. Dissolve pellets in
500 mL of reagent grade water. Dilute with an additional
500 mL of reagent grade water. Cap tightly and store at
room temperature and is stable 1 year at ambient temperature.
10. 50 mM potassium phosphate hydrolysis buffer, pH6.0: Add
approximately 3600 mL of type 1 reagent grade water to a 4 L
beaker. Add 13.6 mL of concentrated phosphoric acid (85%,
14.75 M) to the beaker with a graduated cylinder and mix.
Adjust the pH to 5.0 with 10 M potassium hydroxide. Dilute
to volume with type 1 water. Transfer to an amber reagent
bottle and label with appropriate information. Cap tightly and
store at room temperature and is stable 1 year at ambient
temperature.
11. Mobile phase A, 10 mM ammonium acetate, 0.1% formic acid:
Triple rinse a clean 1 L volumetric flask with type 1 laboratory
grade water. Add approximately 500 mL of type I laboratory
water to the volumetric flask. Weigh 0.77 g of high purity
ammonium acetate (>99.99%) into a new reagent boat. Trans-
fer the contents to the volumetric flask. Add 1.0 mL of high
purity concentrated formic acid (>99%, 30 M) to the volumet-
ric flask, dilute to volume with water, and mix. Stable when
stored in a glass bottle at room temperature for up to 6 weeks.
12. Mobile phase B: Methanol, HPLC grade.
13. 50:50 methanol/ammonium acetate with formic acid: In a
glass container, combine 250 mL of HPLC grade methanol
and 250 mL of mobile phase A. Stable when stored at room
temperature for up to 6 weeks.

2.2 Supplies 1. β-Glucuronidase enzyme (E. coli with approximately 140 U/mg
and Equipment of activity at 37
C).
2. Multi-tube vortex (VWR VX-2500 or similar).
3. Heat block for extraction tubes.
4. Nitrogen evaporator, e.g., TurboVap or similar.
142 Gregory C. Janis

5. Centrifuge (Beckman Coulter Allegra 25R, or similar).

6. LC-MS vials and caps.
7. Waters HSS T3, 50  2.1 mm, 1.8 μm column.
8. Thermo BetaBasic C18 guard column, 10  2.1 mm, 5 μm.
9. LC-MS/MS system (Waters classic UPLC and Sciex 5500
triple quadrupole).
10. 15 mL disposable glass round bottom tubes.
11. 10 mL disposable glass round bottom tubes.

3 Methods

3.1 Calibrator 1. Pipette 0.500 mL of negative urine into labeled glass 15 mL

and Control extraction tubes for calibrators and controls.
Preparation 2. Pipette 10 μL of the 0.05 μg/mL combined working standard
into the tube labeled standard 1. Final concentration: 1.0 ng/
mL.
3. Pipette 50 μL of the 0.05 μg/mL combined working standard
into the tube labeled standard 2. Final concentration: 5.0 ng/
mL.
4. Pipette 25 μL of the 1.0 μg/mL combined working standard
into the tube labeled standard 3. Final concentration: 50.0 ng/
mL.
5. Use standard solutions prepared from an independently
prepared lineage of stock standards to prepare quality controls.
Fortify 500 μL of negative urine with 30 μL of the 0.05 μg/mL
combined standard for the low quality control. Spike 500 μL of
negative urine with 20 μL of the 1.0 μg/mL combined stan-
dard for the high quality control.

3.2 Extraction 1. Pipette 0.5 mL of each sample into labeled 15 mL glass tubes.
2. Add 20 μL of the internal standard spiking solution into each
calibrator, control, and sample tube.
3. Add 500 μL of 50 mM potassium phosphate buffer, pH 6 to
each tube.
4. Add 20 μL of β-glucuronidase enzyme to each tube (see Note 2).
5. Vortex tubes at medium speed for 1 min.
6. Incubate samples at approximately 55
C for 20 min.
7. Remove samples promptly and place at room temperature for
approximately 10 min.
8. Add 1.0 mL of 0.1 N HCl to each tube and vortex mix samples
for 1 min.
 Synthetic Cannabinoids 143

9. Add 5.0 mL of 50% hexane/50% ethyl acetate to each tube (see

Note 3).
10. Cap and vortex mix tubes at low speed for 5 min while main-
taining sample mixing.
11. Centrifuge tubes at 1300 g for 5 min.
12. Using disposable pipettes, transfer the upper, organic layer into
properly labeled 15 mL disposable centrifuge tubes.
13. Dry the organic extracts under a stream of N2 while heating at
approximately 45
C for approximately 15 min, and remove
promptly when dry.
14. Add 200 μL of 50:50 methanol and 10 mM ammonium ace-
tate þ0.1% formic acid to each tube.
15. Vortex tubes at high speed for 5 min.
16. Transfer extract into a properly labeled disposable glass auto-
sampler vial with flat insert and cap tightly.

3.3 LC-MS/MS 1. Column temperature: 50
C.

Parameters 2. Flow rate: 0.7 mL/min.
3. Injection volume: 10 μL.
4. Autosampler temperature: 10
C.
5. Gradient elution profile: Shown in Table 1.
6. Monitored transitions and analyte to internal standard pairs are
listed in Table 2 (see Note 4 and Fig. 3).

Table 1
UPLC gradient

Time (minute) A% B% Flow (mL/min)

0.00 72 28 0.700
0.50 52 48 0.700
5.50 52 48 0.700
6.00 32 68 0.700
7.80 32 68 0.700
8.40 28 72 0.700
8.50 72 28 0.700
9.00 70 28 0.700
Table 2
144

Monitored tandem mass spectrometer transitions

Quantitative
Precursor fragment Collision
Analyte/IS Parent species (m/z) (m/z) RT (min) energy Paired internal standard
UR-144 N-pentanoic acid UR-144/XLR-11 342.2 144.1 6.42 48 UR-144 N-pentanoic acid-D5
Gregory C. Janis

JWH-073 N-butanoic acid JWH-073 358.0 155.0 4.06 30 JWH 073 N-butanoic acid-D5
AB-CHMINACA butanoic acid AB-CHMINACA 358.3 241.1 7.05 25 UR-144 N-pentanoic acid-D5
ADBICA N-(4-hydroxypentyl) ADBICA 360.1 156.1 4.06 30 AM2201 N-(4-hydroxypentyl)-
D5
AB-PINACA pentanoic acid AB-PINACA and 361.1 217.0 1.37 43 RCS-4 N-pentanoic acid-D5
F-AB-PINACA
JWH-018 N-pentanoic acid JWH-018 and AM2201 372.0 155.0 5.39 27 JWH 018 N-pentanoic acid-D4
ADBICA pentanoic acid ADBICA 374.2 244.1 2.01 30 RCS-4 N-pentanoic acid-D5
ADB-PINACA pentanoic acid ADB-PINACA 375.3 245.2 2.01 32 RCS-4 N-pentanoic acid-D5
MAM-2201 N-pentanoic acid MAM-2201 386.2 141.1 6.36 54 JWH 398 N-pentanoic acid-D5
AKB-48 N-pentanoic acid AKB-48 and 5F-AKB-48 396.3 107.1 7.11 62 UR-144 N-pentanoic acid-D5
AB-FUBINACA oxobutanoic acid AB-FUBINACA 399.3 253.3 1.87 28 RCS-4 N-pentanoic acid-D5
MAB-CHMINACA butanoic acid MAB-CHMINACA 372.6 145.0 7.70 52 UR-144 N-pentanoic acid-D5
JWH 073 N-butanoic acid-D5 – 363.2 155.1 3.96 33
RCS-4 N-pentanoic acid-D5 – 357.2 135.1 2.85 45
UR-144 N-pentanoic acid-D5 – 347.3 125.0 6.41 30
AM2201 N-(4-hydroxypentyl)-D5 – 381.3 155.1 4.57 45
JWH 018 N-pentanoic acid-D4 – 376.3 155.1 5.35 30
 Intensity, cps
0.00
5.00e5
1.00e6
1.50e6
2.00e6
2.50e6
3.00e6
3.50e6
4.00e6
4.50e6
5.00e6
5.50e6
6.00e6
6.50e6
7.00e6
7.50e6
8.00e6
8.50e6
9.00e6
9.50e6
1.00e7
1.05e7
1.10e7
1.15e7

0.0

Fig. 3 Representative chromatogram

0.5

AB-PINACA Pentanoic Acid

1.0

AB-FUBINACA Oxobutanoic Acid

1.5

ADB-PINACA Pentanoic Acid

2.0
2.5
2.46

ADBICA Pentanoic Acid

3.0
3.5

ADBICA N-(4-hydroxypentyl)
4.0
4.5
Time, min
JWH-073 N-Butanoic Acid

5.0
JWH-018 N-Pentanoic Acid

5.5
MAM-2201 N-Pentanoic Acid

6.0
6.25

6.5
6.49
UR-144 N-Pentanoic Acid AB-CHMINACA Butanoic Acid

7.0
7.5
AKB-48 N-Pentanoic Acid

8.0
AB-CHMINACA Butanoic Acid

8.5
9.0
145 Synthetic Cannabinoids
146 Gregory C. Janis

4 Notes

1. When preparing calibrators and quality control samples, one

must keep in mind the poor water solubility of the free drugs
and metabolites. Insolubility can result in analyte losses during
storage. Calibrators and quality controls should be kept frozen
when not in use. This is less of a concern for actual samples as
phase II conjugation significantly improves water solubility.
2. In urine, phase II conjugates must be hydrolyzed releasing free
drug phase II metabolites. Attempts to analyze intact conju-
gates in urine are not practical as few reference standards are
available for the conjugated drugs. Both chemical and enzy-
matic hydrolysis methods have been employed. However, some
more recent compounds contain ester and amide moieties;
these compounds may degrade at elevated pH. Thus, enzy-
matic hydrolysis is typically preferred. Beta glucuronidase
from abalone and E. coli can both be used successfully; how-
ever, recombinant E. coli has been shown to be particularly
efficient. With the addition of E. coli derived beta glucuroni-
dase, phase II glucuronide conjugates can be efficiently hydro-
lyzed in as little as 30 min with mild incubation.
3. Like tetrahydrocannabinol (THC) and its primary urine
metabolite carboxy-THC, unconjugated synthetic cannabi-
noids are significantly nonpolar. Thus, liquid-liquid extraction
is capable of easily extracting the drugs and unconjugated
metabolites. Mixtures of hexane and ethyl acetate have proven
to extract metabolites of synthetic cannabinoid from urine with
a high degree of efficiency while avoiding significant analytical
interference. The organic extracts only need to be dried and
reconstituted prior to LC-MS/MS analysis. Solid-phase tech-
niques, again exploiting the nonpolar nature of the drugs, have
also been successfully employed for extraction of synthetic
cannabinoid metabolites.
4. Quantitation of donor samples is achieved by comparing the
analyte to internal standard response of detected peaks to the
generated calibration curve for the corresponding analyte. Uti-
lize a 1/x weighted linear regression for all analytes.

References

1. Huffman JW, Dai D, Martin BR, Compton DR the cannabimimetic JWH-018 using chemically
(1994) Design, synthesis and pharmacology of synthesised reference material for the support of
cannabimimetic indoles. Bioorg Med Chem Lett LC-MS/MS-based drug testing. Anal Bioanal
4(4):563–566 Chem 401(2):493–505
2. Beuck S, Möller I, Thomas A, Klose A, 3. Chimalakonda KC, Bratton SM, Le VH, Yiew
Schlörer N, Sch€anzer W, Thevis M (2011) Struc- KH, Dineva A, Moran CL, James LP, Moran JH,
ture characterisation of urinary metabolites of Radominska-Pandya A (2011) Conjugation of
 Synthetic Cannabinoids 147

synthetic cannabinoids JWH-018 and cannabimimetic effects in mice. J Toxicol Sci

JWH-073, metabolites by human 42(3):335–341
UDP-glucuronosyltransferases. Drug Metab 5. Daw RC, Grabenauer M, Pande PG, Cox A,
Dispos 39(10):1967–1976 Kovach AL, Davis KH, Wiley JL, Stout PR,
4. Kaizaki-Mitsumoto HK, Funada M, Odanaka Y, Thomas BF (2014) Pyrolysis studies of synthetic
Kumamoto H, Numazawa S (2017) Pyrolysis of cannabinoids in herbal products. Drug Alcohol
UR-144, a synthetic cannabinoid, augments an Depend 140:e44
affinity to human CB1 receptor and
 Chapter 15

Quantification of Designer Opioids by Liquid

Chromatography–Tandem Mass Spectrometry
Sherri L. Kacinko and Joseph W. Homan

Abstract
Opioids including heroin and commonly prescribed drugs such as oxycodone and fentanyl are among the
most commonly abused drugs. In recent years, the abuse of opioids has spread beyond these commonly
encountered analytes and now includes novel psychoactive drugs such as AH-7921 and U47700 and a
variety of fentanyl-related compounds such as acetyl fentanyl and furanyl fentanyl. The assay described is for
the quantitative determination of 19 designer opioids in serum, plasma, and whole blood. Also included is a
discussion on the challenges of keeping an analytical method current as new analytes appear on the illicit
drug market.

Key words Designer opioids, Novel opioids, Research opioids, LC-MS/MS

1 Introduction

Chapter 13 provides a brief history of designer opioids. Analysis of

biological matrices for designer opioids by liquid chromatogra-
phy–tandem mass spectrometry (LC-MS/MS) generally does not
present an analytical challenge for laboratories with experience in
analyzing these types of specimens for classical opioids. Rather, the
challenge is the availability of certified reference materials and
deuterated internal standards and the variety of chemical structures
that comprise designer opioids. It is also important to be forward
thinking during method development so that the final method can
be quickly updated to include new analytes. While there are a few
published gas chromatography-mass spectrometry (GCMS) meth-
ods for designer opioids including acetyl fentanyl and butyryl fen-
tanyl [1–4], the preferred method for quantification of designer
opioids appears to be LC-MS/MS [5–14]. In general, analytes of
interest are extracted from biological specimens using either liquid-
liquid or solid-phase extraction before being introduced in a
LC-MS/MS system.

Loralie J. Langman and Christine L.H. Snozek (eds.), LC-MS in Drug Analysis: Methods and Protocols,
Methods in Molecular Biology, vol. 1872, https://doi.org/10.1007/978-1-4939-8823-5_15,
© Springer Science+Business Media, LLC, part of Springer Nature 2019

149
150 Sherri L. Kacinko and Joseph W. Homan

The presented method is a modification of a validated method

for MT-45, butyryl fentanyl, AH-7921, and acetyl fentanyl in
serum, plasma, and whole blood. The original method had a run
time of only 5.00 min (min) during which all four analytes were
completely resolved. However, after approximately 5 months of
routine use in a large reference laboratory, a large interfering peak
appeared in the window for AH-7921. After identifying the inter-
fering substance as U47700, minor method modifications allowed
for the separation of AH-7921 and U47700. At the same time, new
fentanyl analogs were added to the analytical method. An extended
run time of 6.00 min allows for the resolution of the majority of
analytes; two pairs of isomers are resolved. Calibrators are prepared
with a mixed analyte spiking solution prepared in serum, while bulk
controls are prepared in serum, frozen and diluted with whole
blood at the time of analysis. Solid-phase extraction is used to
clean up and concentrate the samples. Separation is achieved
using a Zorbax Rx-SIL normal phase analytical column with gradi-
ent elution by Waters Acquity ultra performance liquid chromatog-
raphy system. A Waters TQD tandem mass spectrometer with a
positive-ion electrospray source is used to identify and quantify
analytes of interest. Table 1 lists the analytical ranges for all com-
pounds included in the panel.

2 Materials

2.1 Prepared All reagents are reagent grade or better.

Reagents
1. 5% ammonium hydroxide in acetonitrile (ACN): In the fume
hood, add 5.0 mL of ammonium hydroxide and 95 mL of
ACN to a 100 mL bottle; mix thoroughly. Prepare fresh daily.
2. 0.1% formic acid in ACN: Transfer 999 mL of ACN to a 1.0 L
glass bottle. Add 1.0 mL of formic acid. Cap and mix by
inversion.
3. 0.1% formic acid in deionized (DI) water: Transfer 999 mL of
DI water to a 1.0 L glass bottle. Add 1.0 mL of formic acid.
Cap and mix by inversion.
4. 0.1 N hydrochloric acid (HCl): Transfer 8.4 mL of HCl to a
1.0 L volumetric flask containing approximately 400 mL of DI
water. Dilute to volume with DI water; mix thoroughly.
5. 0.1 M sodium phosphate buffer, pH 6.0: Add 3.4 g of sodium
phosphate dibasic and 21.1 g of sodium phosphate monobasic
to approximately 200 mL of DI water in a 2.0 L volumetric
flask. Swirl to dissolve and dilute to volume with DI water.
Adjust pH to 6.0 with 0.5 M sodium phosphate dibasic. Mix
thoroughly.
 Designer Opioids in Blood 151

Table 1
Designer opioid analytes

Analyte Analytical range (ng/mL) Stock concentration Group

4-ANPP 0.1–10 Powder A
4-Methoxybutyryl fentanyl 0.1–10 Powder A
4-Methylphenethyl acetyl fentanyl 0.1–10 Powder A
Acryl fentanyl 0.1–10 Powder A
Alpha-methyl fentanyl 0.1–10 Powder A
a
Butyryl fentanyl 0.1–10 Powder A
Carfentanil 0.1–10 1000 ng/mcL A
Furanyl fentanyl 0.1–10 Powder A
MT-45 0.1–10 1000 ng/mcL A
Ortho-fluorofentanyl 0.1–10 Powder A
Para-fluorobutyryl fentanyla 0.1–10 Powder A
Para-fluorofentanyl 0.1–10 Powder A
Valeryl fentanyl 0.1–10 Powder A
AH-7921 0.2–20 1000 ng/mcL B
U-47700 0.2–20 Powder B
U-50488 0.2–20 Powder B
Beta-hydroxythiofentanyl 0.5–50 Powder C
D5-Beta-hydroxythiofentanyl IS Powder
D5-Para-fluorofentanyl IS Powder
D3-Alpha-methyl fentanyl IS Powder
D5-Methylphenethyl acetyl fentanyl IS Powder
13C6-Acetyl fentanyl IS 50 ng/mcL
D6-U-47700 IS Powder
D3-AH-7921 IS 100 ng/mcL
a
Some analytes are not resolved by this method and are reported as indicated: butyryl/isobutyryl fentanyl and para-
fluorobutyryl fentanyl/para-fluoroisobutyryl fentanyl (FIBF). IS internal standard

6. 0.5 M sodium phosphate dibasic: Transfer 35.5 g of sodium

phosphate dibasic to a 500 mL volumetric flask. Dissolve and
dilute to volume with DI water. Mix thoroughly.
7. Mobile phase A, ammonium formate, pH 4.0: Transfer
900 mL of DI water to a 1.0 L beaker. Add 2 mL of formic
acid, and adjust to pH 4.0 with approximately 2 mL of
152 Sherri L. Kacinko and Joseph W. Homan

ammonium hydroxide while mixing. Dilute to volume with DI

water, and mix thoroughly.
8. Mobile phase B: HPLC-grade ACN.

2.2 Calibrators See Table 1 for information on certified reference materials (CRM)
used to prepare calibrators, internal standard (IS), and controls.
1. 100 ng/μL individual stock standards: Transfer 1.0 mg of
4-ANPP and U-47700, 1.2 mg of U-50488, and 1.1 mg of
the remaining powdered CRM drugs, each to their own 10 mL
volumetric flasks (see Note 1). Dilute to volume with methanol
(MeOH). Store in amber glass vials with Teflon-lined caps.
Unless otherwise noted, all stock solutions are stable for
1 year when stored in appropriate container at < 10  C.
2. 100 ng/μL MT-45 and carfentanil substock standards: Com-
bine 100 μL of each 1000 ng/μL stock of MT-45 or carfentanil
with 900 μL of MeOH. Discard immediately after use.
3. Mixed bulk standard 1: 100 ng/mL Group A drugs, 200 ng/
mL Group B drugs, and 500 ng/mL Group C drug. Transfer
50 μL of each 100 ng/μL Group A drug stock, 10 μL of the
1000 ng/mL AH-7921 stock, 100 μL of each remaining
100 ng/μL Group B drug stock, and 250 μL of the 100 ng/
μL beta-hydroxythiofentanyl stock to a plastic 50 mL volumet-
ric flask, and dilute to volume with drug-free human serum.
Transfer 0.3 mL aliquots to labeled 2.0 mL plastic snap-cap
conical tubes for storage in freezer (see Note 2).
4. Intermediate 10 calibrator 1: Thaw one tube of mixed bulk
standard 1, and vortex briefly to mix. The undiluted mixed
bulk standard is used as 10 calibrator 1.
5. Intermediate 10 calibrator 2: Transfer 200 μL of intermediate
10 calibrator 1 and 200 μL of drug-free serum to a
12  75 mm test tube. Mix well. Discard immediately after
use. Concentrations of the 10 calibrators are shown in
Table 2.
6. Intermediate 10 calibrator 3: Transfer 200 μL of intermediate
10 calibrator 2 and 300 μL of drug-free serum to a
12  75 mm test tube. Mix well. Discard immediately after use.
7. Intermediate 10 calibrator 4: Transfer 200 μL of intermediate
10 calibrator 3 and 600 μL of drug-free serum to a
12  75 mm test tube. Mix well. Discard immediately after use.
8. Intermediate 10 calibrator 5: Transfer 200 μL of intermediate
10 calibrator 4 and 300 μL of drug-free serum to a
12  75 mm test tube. Mix well. Discard immediately after use.
9. Intermediate 10 calibrator 6: Transfer 200 μL of intermediate
10 calibrator 5 and 200 μL of drug-free serum to a
12  75 mm test tube. Mix well. Discard immediately after use.
 Designer Opioids in Blood 153

Table 2
Intermediate 10 and working calibrators

Prepared concentration (ng/mL)

Calibrator Group Aa Group B Group C

10 cal 1 100 200 500
10 cal 2 50 100 250
10 cal 3 20 40 100
10 cal 4 5.0 10 25
10 cal 5 2.0 4.0 10
10 cal 6 1.0 2.0 5.0
Working cal 1 10 20 50
Working cal 2 5 10 25
Working cal 3 2 4 10
Working cal 4 0.5 1.0 2.5
Working cal 5 0.2 0.4 1.0
Working cal 6 0.1 0.2 0.5
a
Drugs are categorized as Groups A, B, and C as noted in Table 1

10. Working calibrators: Transfer 50 μL of each intermediate 10

calibrator to a labeled 13  100 mm test tube. Add 450 μL of
drug-free human whole blood preserved with potassium oxa-
late/sodium fluoride (KOx/NaF) to each tube, and mix well.
Table 2 lists the final concentration of the working calibrators.

2.3 Controls 1. 100 ng/μL individual IS stock standards: Transfer 1.0 mg of

and Internal Standards d6-U-47700 and 1.1 mg of the remaining powdered IS CRMs
(IS) (d5-beta-hydroxythiofentanyl, d5-para-fluorofentanyl, d3-alpha-
methyl fentanyl, d5-methylphenethyl acetyl fentanyl), each to its
own 10 mL volumetric flask. Dilute to volume with MeOH and
mix well. Store in amber glass vials with Teflon-lined caps.
2. Working IS: Add 40 μL each of d3-alpha-methyl fentanyl,
d5-para-fluorofentanyl, and d5-4-methylphenethyl acetyl fenta-
nyl, 80 μL each of 13C6-acetyl fentanyl and d6-U-47700, and
200 μL each of d3-AH-7921 and d5-beta-hydroxythiofentanyl
to a 200 mL volumetric flask. Dilute to volume with MeOH.
Store in amber glass bottle with Teflon-lined cap. Preparation
instructions and final concentrations are shown in Table 3.
3. Carryover check control stock solution: 5.0 ng/μL furanyl
fentanyl and 4-ANPP and 10 ng/μL U-47700. Transfer
0.5 mL of 100 ng/μL furanyl fentanyl stock solution, 0.5 mL
154 Sherri L. Kacinko and Joseph W. Homan

Table 3
Preparation of working internal standard solution

Stock concentration Amount to add Final concentration

Analyte (ng/μL) (μL) (ng/μL)
13C6-Acetyl fentanyl 50 80 0.02
D3-AH-7921 100 200 0.1
D3-Alpha-methyl fentanyl 100 40 0.02
D5-Para-fluorofentanyl 100 40 0.02
D5-Beta-hydroxythiofentanyl 100 200 0.1
D5-4-Methylphenethyl acetyl fentanyl 100 40 0.02
D6-U-47700 100 80 0.04

of 100 ng/μL 4-ANPP stock solution, and 1.0 mL of 100 ng/

μL U-47700 stock solution to a 10 mL volumetric flask. Dilute
to volume with MeOH. Store in an amber bottle with Teflon-
lined cap.
4. Working carryover check control: 150 ng/mL furanyl fentanyl
and 4-ANPP, 300 ng/mL U-47700. Combine 15 μL of carry-
over check control stock solution and 0.5 mL of drug-free
blood in a 13  100 mL test tube. Mix well. Sample is now
ready for extraction.
5. 1 mg/mL individual stock quality control (QC) solutions:
Transfer 10 mg of 4-ANPP and U-47700, 12 mg of
U-50488, and 11 mg of the remaining powdered CRM
drugs, each to their own 10 mL volumetric flasks. Dilute to
volume with MeOH. Store in amber glass vial with Teflon-
lined cap.
6. Mixed substock QC solution: 10 ng/μL Group A drugs,
20 ng/μL Group B drugs, and 50 ng/μL Group C drug.
Transfer 100 μL of each individual Group A QC stock solution,
200 μL of each individual Group B QC stock solution, and
500 μL of beta-hydroxythiofentanyl QC stock solution to a
10 mL volumetric flask. Dilute to volume with MeOH. Store in
amber glass vial with Teflon-lined cap.
7. Bulk high control: Transfer 175 μL of mixed substock QC
solution to a plastic 25 mL volumetric flask, and dilute to
volume with drug-free serum. Transfer 0.2 mL aliquots to
labeled 2.0 mL plastic snap-cap conical tubes for storage in
freezer (stable for 6 months). Table 4 lists the concentration of
each analyte in the bulk low, bulk high, low working, and high
working controls.
 Designer Opioids in Blood 155

Table 4
Concentration of bulk 10 and working low and high controls

Concentration (ng/mL)
Analyte Bulk 10 high Bulk 10 low Working low Working high
4-ANPP 100 3.5 0.35 7.0
4-Methoxybutyryl fentanyl 100 3.5 0.35 7.0
4-Methylphenethyl acetyl fentanyl 100 3.5 0.35 7.0
Acryl fentanyl 100 3.5 0.35 7.0
Alpha-methyl fentanyl 100 3.5 0.35 7.0
Butyryl fentanyl/isobutyryl fentanyl 100 3.5 0.35 7.0
Carfentanil 100 3.5 0.35 7.0
Furanyl fentanyl 100 3.5 0.35 7.0
MT-45 100 3.5 0.35 7.0
Ortho-fluorofentanyl 100 3.5 0.35 7.0
Para-fluorobutyryl fentanyl/FIBF 100 3.5 0.35 7.0
Para-fluorofentanyl 100 3.5 0.35 7.0
Valeryl fentanyl 100 3.5 0.35 7.0
AH-7921 200 7.0 0.7 14
U-47700 200 7.0 0.7 14
U-50488 200 7.0 0.7 14
Beta-hydroxythiofentanyl 500 17.5 1.75 35

8. Bulk low control: Transfer 1250 μL of bulk high control to a

plastic 25 mL volumetric flask, and dilute to volume with drug-
free serum. Transfer 0.2 mL aliquots to labeled 2.0 mL plastic
snap-cap conical tubes for storage in freezer (stable for
6 months).
9. Low and high working controls (prepared at time of analysis):
Thaw one bulk high control and one bulk low control. Transfer
50 μL of each bulk control to its own individually labeled
13  100 mm tube. Add 450 μL drug-free blood and mix well.

2.4 Supplies 1. Agilent Plexa PCX 3.0 mL/60 mg solid-phase extraction

and Instrumentation (SPE) columns.
2. Waters TQD tandem mass spectrometer with Waters Acquity
ultra performance liquid chromatography system.
3. Zorbax Rx-SIL 3  100 mm, 1.8 μm analytical column with
Optimize EXP filter, 0.2 μm.
156 Sherri L. Kacinko and Joseph W. Homan

4. Vacuum manifold for SPE.

5. 12  75 mm and 13  100 mm test tubes.
6. Amber bottles with Teflon-lined caps.
7. Amber glass vials with Teflon-lined caps.
8. 2.0 mL plastic snap-cap conical tubes.
9. Volumetric glassware.
10. Pipettes and tips.
11. Vortexer.
12. Centrifuge with adaptors for 13  100 mm tubes.
13. Nitrogen evaporator.

3 Methods

3.1 Sample 1. Prepare working calibrators and controls as described above

Preparation (see Note 3).
2. Transfer 0.5 mL of blank blood (for matrix blank), DI water
(reagent blank), and each patient specimen to their own appro-
priately labeled 13  100 mm test tubes (see Note 4 for samples
with expected concentrations above the analytical range).
3. Add 25 μL working IS solution to each tube; vortex briefly
to mix.
4. Add 1.0 mL of HPLC-grade ACN to each tube. Vortex for
approximately 10 s to mix.
5. Centrifuge all test tubes at 2100g for approximately 10 min.
6. Transfer supernatant to new, appropriately labeled
13  100 mm test tubes.
7. Add 1.0 mL of 0.1 M sodium phosphate buffer, pH 6.0, to
each test tube; vortex briefly to mix.

3.2 Solid-Phase 1. Place one SPE column for each sample to be extracted in a
Extraction vacuum manifold rack.
2. Condition columns with 2.0 mL of HPLC-grade ACN; aspi-
rate slowly through column.
3. Equilibrate columns with 2.0 mL of DI water; aspirate through
column. Do not allow columns to dry before application of
samples.
4. Transfer prepared samples to columns; aspirate slowly.
5. Rinse columns with 2.0 mL of 0.1 N HCl. Follow by rinsing
with 2.0 mL of ACN; aspirate slowly.
6. Place new, labeled glass tubes beneath each SPE column. Add
2.0 mL of 5% ammonium hydroxide in ACN to elute by
gravitational flow.
 Designer Opioids in Blood 157

7. Evaporate to dryness at 55  5  C under nitrogen flow.

8. Reconstitute in 500 μL of 0.1% formic acid in ACN; vortex
thoroughly for approximately 30 s.
9. Transfer to appropriately labeled 0.5 mL plastic autosampler
vials, and cap with Teflon-lined snap caps.

3.3 LC-MS/MS 1. Place samples in autosampler in the following order:

Analysis (a) Reagent blank.
(b) Working carryover check control.
(c) Matrix blank.
(d) Calibrators from highest to lowest (see Note 5).
(e) High control.
(f) Patient samples (bracketed by controls).
(g) Low control.
2. Inject 20 μL in full loop mode.
3. Column temperature: 40  C.
4. Flow rate: constant 0.6 mL/min.
5. Mobile phase ratio: 10% A, 90% B. No gradient.
6. MS parameters (should be optimized for instrument used):
(h) Polarity: positive mode.
(i) Capillary voltage: 0.50 kV.
(j) Cone voltage: 35.00 V.
(k) Extractor voltage: 3.00 V.
(l) RF voltage: 0.10 V.
(m) Source temperature: 120  C.
(n) Desolvation temperature: 450  C.
(o) Cone gas flow: 35 L/h.
(p) Desolvation gas flow: 1000 L/h.
(q) Collision gas flow: 0.15 mL/min.
(r) MSMS mode entrance: 5.00.
(s) MSMS mode collision energy: 16.00.
(t) MSMS mode exit: 1.00.
(u) Gain: 1.00.
7. Data acquisition parameters are outlined in Table 5 (see
Note 6).
8. Figure 1 portrays a calibrator containing all analytes and IS,
with an expanded view in Fig. 2. Two transitions are monitored
for each analyte (Table 6).
158 Sherri L. Kacinko and Joseph W. Homan

Table 5
Data acquisition parameters

Time (min) Channel Reaction Dwell (secs) Cone volt. Col. energy
1.400–2.400 1 359.20 > 111.00 0.007 35 34
2 359.20 > 192.10 0.007 35 22
3 364.20 > 111.00 0.007 35 34
4 364.20 > 192.10 0.007 35 22
1.500–2.500 1 395.30 > 113.00 0.007 40 34
2 395.30 > 335.20 0.007 40 18
1.700–2.700 1 365.20 > 105.00 0.006 45 36
2 365.20 > 188.20 0.006 45 24
1 369.30 > 105.10 0.006 50 38
2 369.30 > 188.10 0.006 50 24
1 375.10 > 105.00 0.006 45 40
2 375.10 > 188.10 0.006 45 24
1.800–2.800 1 355.30 > 105.10 0.006 48 38
2 355.30 > 188.10 0.006 48 24
3 360.30 > 105.10 0.006 48 38
4 360.30 > 188.10 0.006 48 24
1 281.20 > 105.00 0.006 35 30
2 281.20 > 188.20 0.006 35 16
1.900–2.900 1 335.20 > 105.00 0.006 45 32
2 335.20 > 188.20 0.006 45 22
1 351.10 > 105.00 0.006 45 36
2 351.10 > 188.00 0.006 45 22
2.000–3.000 1 381.20 > 105.00 0.006 45 38
2 381.20 > 188.20 0.006 45 24
2.100–3.100 1 349.10 > 169.00 0.006 45 18
2 349.10 > 181.00 0.006 45 22
2.200–3.200 1 351.20 > 91.10 0.006 45 38
2 351.20 > 202.10 0.006 45 22
3 354.20 > 91.10 0.006 45 38
4 354.20 > 202.10 0.006 45 22

(continued)
 Designer Opioids in Blood 159

Table 5
(continued)

Time (min) Channel Reaction Dwell (secs) Cone volt. Col. energy
2.500–3.500 1 337.20 > 119.00 0.006 45 34
2 337.20 > 202.20 0.006 45 22
3 342.20 > 119.00 0.006 45 34
4 342.20 > 202.20 0.006 45 22
1 323.10 > 105.00 0.006 45 36
2 323.10 > 188.00 0.006 45 22
3 329.10 > 105.00 0.006 45 36
4 329.10 > 188.00 0.006 45 22
3.200–5.400 1 329.20 > 204.10 0.03 45 22
2 329.20 > 284.10 0.03 45 18
3 335.20 > 173.00 0.03 45 28
4 335.20 > 284.10 0.03 45 18
3.600–5.400 1 329.10 > 173.00 0.03 45 26
2 329.10 > 284.00 0.03 45 16
3 334.10 > 178.00 0.03 45 26
4 334.10 > 289.00 0.03 45 16
4.100–5.400 1 369.20 > 112.00 0.03 45 30
2 369.20 > 298.10 0.03 45 20

4 Notes

1. Many of the powdered CRM drugs are sold as hydrochloride

salts. The calculations are adjusted to ensure that the stock
standard concentrations reflect the molecular weight of the
free base rather than the hydrochloride salt. Similar adjustment
is done for the QC and internal standard measurements.
2. The described calibrator and control preparation scheme allows
for preparation of bulk material that can be frozen and used for
at least 6 months. For bulk preparation, serum demonstrated
better reproducibility than preparing the bulk calibrators and
controls in whole blood. Intermediate 10 and working cali-
brators are all prepared at the time of analysis. Controls can be
prepared using different sources or lot numbers than the stan-
dards; however, with novel drugs, multiple sources or lots
might not be available. If different sources or lot numbers are
160 Sherri L. Kacinko and Joseph W. Homan

Figure 2

100

e
%

a c
b

g
f
h i

0
1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 Time

Fig. 1 Multiple reaction monitoring (MRM) chromatogram of extracted whole blood fortified with 10–50 ng/mL
of each compound (a) beta-hydroxythiofentanyl, (b) carfentanil, (c) MT-45, (d) alpha-methyl fentanyl, (e)
4-methylphenethyl acetyl fentanyl, (f) acetyl fentanyl, (g) U-47700, (h) AH-7921, (i) U-50488

100

c f

i
% g
a e
b
h

0
2.20 2.25 2.30 2.35 2.40 2.45 2.50 2.55 2.60 2.65 Time

Fig. 2 Expanded view of Fig. 2 across 2.20–2.70 min (a) valeryl fentanyl, (b)para-fluorobutyryl fentanyl/FIBF,
(c) 2-furanyl fentanyl, (d) ortho-fluorofentanyl, (e) 4-ANPP, (f) butyryl fentanyl/isobutyryl fentanyl, (g) acryl
fentanyl, (h) 4-methoxybutyryl fentanyl, (i) para-fluorofentanyl
 Designer Opioids in Blood 161

Table 6
Analyte retention times and monitored MRM transitionsa

Quantifier Qualifier
Analyte Retention time (min) transition transition
Beta-hydroxythiofentanyl 1.92 359.2 > 192.1 359.2 > 111
D5-Beta-hydroxythiofentanyl 1.93 364.2 > 192.1 364.2 > 111.0
Carfentanil 1.94 395.3 > 335.2 395.2 > 113.0
Valeryl fentanyl 2.17 365.2 > 188.2 365.2 > 105.0
Para-fluorobutyryl fentanyl 2.19 369.3 > 188.1 369.3 > 105.1
Furanyl fentanyl 2.22 375.1 > 188.1 375.1 > 105.0
Ortho-fluorofentanyl 2.23 355.3 > 105.1 355.3 > 188.1
4-ANPP 2.28 281.2 > 105.0 281.2 > 188.2
Butyryl fentanyl/isobutyryl fentanyl 2.33 351.1 > 105.0 351.1 > 188.0
4-Methoxybutyryl fentanyl 2.36 381.2 > 105.0 381.2 > 188.2
Para-fluorofentanyl 2.37 355.3 > 105.1 355.3 > 188.1
Acryl fentanyl 2.37 335.2 > 105.0 335.2 > 188.2
D5-Para-fluorofentanyl 2.39 360.3 > 105.1 360.3 > 188.1
MT-45 2.55 349.1 > 181.0 349.1 > 169.0
Alpha-methyl fentanyl 2.62 351.2 > 91.1 351.2 > 202.1
D3-Alpha-methyl fentanyl 2.63 354.2 > 91.1 354.2 > 202.1
4-Methylphenethyl acetyl fentanyl 2.88 337.2 > 119.0 337.2 > 202.2
D5-4-Methylphenethyl acetyl fentanyl 2.90 342.2 > 119.0 342.2 > 202.2
13C6-Acetyl fentanyl 2.96 329.1 > 188.0 329.1 > 105.0
U-47700 3.61 329.2 > 284.1 329.2 > 204.1
D6-U-47700 3.95 335.2 > 284.1 335.2 > 173.0
AH-7921 3.95 329.1 > 284.0 329.1 > 173.0
D3-AH-7921 3.96 334.1 > 289.0 334.1 > 178.0
U-50488 4.63 369.2 > 298.1 369.2 > 112.0
a
Analytes listed below IS used for quantification

not available controls should be prepared by a different indi-

vidual or at a different time from the same materials as calibra-
tion standards.
3. Enough low and high controls should be prepared to bracket
patient samples as required by laboratory standard operating
procedures. A general rule of thumb is that fortified controls
162 Sherri L. Kacinko and Joseph W. Homan

should comprise at least 5% of the samples being analyzed and

they should be interspersed throughout the run with no more
than ten patient specimens between each control sample.
4. Samples that are expected to be above the analytical range for
one or more analytes can be diluted with blank whole blood. A
dilution control is not necessary because the working controls
are prepared by diluting the 10 control. If more than 10
dilution of a patient specimen is required, an appropriate dilu-
tion control should be prepared by applying the sample dilu-
tion factor to the 10 control.
5. If no carryover check control is used, inject the matrix blank
after the highest standard to check for carryover.
6. Acquisition parameters should be optimized for the instrumen-
tation employed. The method, as written, has the following
limitations:
(a) Butyryl fentanyl and isobutyryl fentanyl are co-eluting
isobaric compounds and cannot be distinguished based
on this methodology. Results are reported as the pair.
(b) Para-fluorobutyryl fentanyl and para-fluoroisobutyryl fen-
tanyl (FIBF) are co-eluting isobaric compounds and can-
not be distinguished based on this methodology. Results
are reported as the pair. Relative retention time (RRT)
shifts may occur, report cases that are within 2% of
average standard RRT as positive.
(c) 3-methyl fentanyl may interfere with the quantitation of
butyryl fentanyl/isobutyryl fentanyl by LC-MS/MS.
(d) Meta-fluorofentanyl may interfere with the quantitation
of para-fluorofentanyl and ortho-fluorofentanyl by
LC-MS/MS.

References
1. McIntyre IM, Trochta A, Gary RD et al (2016) 5. Kronstrand R, Thelander G, Lindstedt D et al
An acute Butyr-fentanyl fatality: a case report (2014) Fatal intoxications associated with the
with postmortem concentrations. J Anal Tox- designer opioid AH-7921. J Anal Toxicol
icol 40:162–166 38:599–604
2. McIntyre IM, Trochta A, Gary RD et al (2015) 6. Fels H, Krueger J, Sachs H et al (2017) Two
An acute acetyl fentanyl fatality: a case report fatalities associated with synthetic opioids:
with postmortem concentrations. J Anal Tox- AH-7921 and MT-45. Forensic Sci Int 277:
icol 39:490–494 e30–e35
3. McIntyre IM, Gary RD, Joseph S et al (2017) 7. Vorce SP, Knittel JL, Holler JM et al (2014) A
A fatality related to the synthetic opioid fatality involving AH-7921. J Anal Toxicol
U-47700: postmortem concentration distribu- 38:226–230
tion. J Anal Toxicol 41(2):158–160 8. Poklis J, Poklis A, Wolf C et al (2016) Two fatal
4. Fort C, Curtis B, Nichols C et al (2016) Acetyl intoxications involving Butyryl fentanyl. J Anal
fentanyl toxicity: two case reports. J Anal Tox- Toxicol 40:703–708
icol 40:754–757
 Designer Opioids in Blood 163

9. Mohr ALA, Friscia M, Papsun D et al (2016) 12. Sofalvi S, Schueler HE, Lavins ES et al (2017)
Analysis of novel synthetic opioids U-47700, An LC-MS-MS method for the analysis of Car-
U-50488 and Furanyl fentanyl by LC–MS/MS fentanil, 3-Methyl fentanyl, 2-Furanyl fentanyl,
in postmortem casework. J Anal Toxicol acetyl fentanyl, fentanyl and Norfentanyl in
40:709–717 postmortem and impaired-driving cases. J
10. Fleming SW, Cooley JC, Johnson L et al Anal Toxicol 41:473–483
(2017) Analysis of U-47700, a novel synthetic 13. Shanks KG, Behonick GS (2017) Detection of
opioid, in human urine by LC-MS-MS and Carfentanil by LC-MS-MS and reports of asso-
LC-QToF. J Anal Toxicol 41:173–180 ciated fatalities in the USA. J Anal Toxicol
11. Papsun D, Krywanczyk A, Vose JC et al (2016) 41:466–472
Analysis of MT-45, a novel synthetic opioid, in 14. Seither J, Reidy L (2017) Confirmation of Car-
human whole blood by LC–MS-MS and its fentanil, U-47700 and other synthetic opioids
identification in a drug-related death. J Anal in a human performance case by LC-MS-MS. J
Toxicol 40:313–317 Anal Toxicol 41:493–497
 Chapter 16

Screening Analysis for Designer Stimulants by LC-MS/MS

Piotr Adamowicz and Bogdan Tokarczyk

Abstract
The increase in the number of new substances appearing on the drug market has been observed in the
1980s and 1990s of the last century, when many phenethylamine and tryptamine derivatives entered the
market. However, the phenomenon of mass marketing of new designer stimulants (being the components
of so-called “legal highs”) began to develop since 2006 in Europe, and it was something new. Since then,
the number of stimulants introduced on the drug market is growing regularly, rapidly, and intensively. Such
a situation creates a need for comprehensive screening methods for detection of these drugs in biological
specimens. The fast and simple liquid chromatography-tandem mass spectrometry qualitative screening
procedure presented here is designed to detect and identify a wide range of designer stimulants in the
blood. The assay has wide applicability for rapid screening of new stimulants in forensic or clinical samples.
The procedure can be easily modified for additional novel psychoactive substances.

Key words Designer stimulants, New psychoactive substances (NPS), Legal highs, Drug screening,
Blood analysis, LC-MS/MS

1 Introduction

In recent years, many designer stimulants have appeared on the

drug market, and currently still the upward trend in the number of
new drugs is continuing in many countries worldwide [1]. These
substances are also known as “legal highs” or “research chemicals”
and are sold mainly on the Internet. Officially, they are not intended
for human consumption and are sold under trivial names, as col-
lectibles, plant fertilizers, bath salts, room fresheners, or herbal
incenses, in order to avoid criminal responsibility. Designer drugs
are characterized by similar chemical structures to controlled sub-
stances. This is due to the fact that these substances are synthesized
in order to circumvent the existing drug laws, usually by changing
the structure of known illegal drugs. New designer stimulants are
an important challenge for toxicologists. Screening of biological
material for the presence of designer stimulants should be a part of
routine procedures used in clinical and forensic laboratories.

Loralie J. Langman and Christine L.H. Snozek (eds.), LC-MS in Drug Analysis: Methods and Protocols,
Methods in Molecular Biology, vol. 1872, https://doi.org/10.1007/978-1-4939-8823-5_16,
© Springer Science+Business Media, LLC, part of Springer Nature 2019

165
166 Piotr Adamowicz and Bogdan Tokarczyk

However, complex methods enabling the detection of such a wide

spectrum of these compounds in biological materials are lacking.
Constantly emerging drugs require that the methodology of their
determination in biological material must be constantly updated.
Unfortunately, immunochemical tests are not effective for diverse
and variable new drugs. Analytical methods used in many labora-
tories such as gas chromatography coupled with mass spectrometry
(GC-MS) or high-performance liquid chromatography with diode
array detection (HPLC-DAD) are often insufficient. Due to insuf-
ficient sensitivity, the application of these methods is limited to
acute or fatal poisonings.
Only modern coupled techniques, especially liquid chromatog-
raphy with tandem mass spectrometry (LC-MS/MS), have the
features that enable a comprehensive analysis of a wide range of
designer stimulants in biological material [2–4]. In this context, we
present a fast and simple LC-MS/MS screening procedure for the
detection and identification of many popular designer stimulants in
the blood in a single run. The method allows the screening of
80 designer stimulants using only 0.2 mL of the whole blood.
The most important advantage of this method is that the procedure
can be easily modified for more drugs. A similar procedure was used
for the screening for 143 new psychoactive substances [5].

2 Materials

2.1 Laboratory 1. Liquid chromatograph coupled with mass detector (e.g., Agi-
Equipment lent Technologies 1200 Series liquid chromatograph with
6460 Triple Quad mass spectrometer).
2. Agilent Technologies SB-C18 chromatography column
(2.1
50 mm, 1.8 μm).
3. Thermoblock with evaporator.
4. Minicentrifuge for vials.
5. Vortexer.
6. Single-channel automatic pipettes.
7. Freezer for temperatures below 15  C.
8. Refrigerator providing a temperature below 4  C.

2.2 Reagents All solvents should be gradient grade or higher (see Note 1).
and Solutions
1. Mobile phase A: 0.1% formic acid in water (v/v). Add 1 mL of
formic acid to 1 L of distilled water (or proportionately smaller
volumes) (see Note 2).
2. Mobile phase B: 0.1% formic acid in acetonitrile (v/v). Add
1 mL of formic acid to 1 L of acetonitrile (or proportionately
smaller volumes).
 Designer Stimulants 167

3. Working internal standard solution: 1 μg/mL mephedrone-D3

in methanol. Add 10 μL of 0.1 mg/mL mephedrone-D3 drug
standard to 990 μL of methanol. Internal standard solutions
must be stored below 15  C, but not longer than 7 days (see
Note 3).
4. Spiking controls solutions: 1 μg/mL selected stimulant(s) in
methanol. Add 10 μL of each 1 mg/mL standard for the drug
(s) of interest to 990 μL of methanol to obtain the concentra-
tion of 10 μg/mL. Dilute the obtained solution 10
, for
example, add 100 μL to 900 μL of distilled water to obtain
the final concentration of 1 μg/mL. These solutions must be
stored below 15  C, but not longer than 7 days (see Note 3).
5. Precipitation solution: iced acetonitrile (< 15  C).

2.3 Supplies 1. 2 mL Eppendorf vials.

2. 2 mL glass vials with screw caps.
3. Polypropylene inserts (for autosampler vials).
4. Autosampler vials.
5. Graduated cylinder (0.5 L or 1 L).
6. Pipette tips.

3 Methods

3.1 Sample 1. Defrost unknown blood samples for approximately 30 min at

Preparation room temperature.
2. Defrost drug-free blood for controls for 30 min at room
temperature.
3. Pipette 0.2 mL of each unknown blood sample into a 2 mL
Eppendorf vial. Pipette 0.2 mL of drug-free blood into a 2 mL
vial for each control to be used.
4. Add 2 μL (final concentration 10 ng/mL) or 20 μL (final
concentration 100 ng/mL) of each spiking control solution
to the appropriate vial of drug-free blood, and vortex to mix
(see Note 4).
5. Add 20 μL of working internal standard solution to all vials to
obtain a final concentration of 100 ng/mL of mephedrone-D3.
Wait 10 min.
6. Precipitate the samples with iced acetonitrile (see Note 5). Add
600 μL of acetonitrile in 50 μL portions, vortexing the samples
for 10 s after each addition. After adding the last portion of
acetonitrile, mix the samples for 5 min, and centrifuge at
13,000 rpm (15,682
g) for 5 min (see Note 6).
7. Transfer the organic solvent to a 2 mL glass vial.
168 Piotr Adamowicz and Bogdan Tokarczyk

8. Evaporate the acetonitrile to dryness under air or nitrogen at

30  C (see Note 7).
9. Dissolve the dry residues in 100 μL of mobile phase A, and
transfer to inserts for autosampler vials.

3.2 Chromatographic 1. Column temperature: 20  C. It is suggested to equip the

and Spectrometric column with an inline filter (4.6 mm, 0.2 μm) (see Note 8).
Conditions 2. Mobile phase flow rate: 0.3 mL/min. Gradient conditions are
shown in relation to mobile phase B content:
0 min—10%
6 min—100%
7 min—10%
14 min—10%
3. Injection volume: 10 μL.
4. Set the mass detector mode to positive ionization (ESIþ) and
dynamic multiple reaction monitoring (dMRM) (see Note 9).
5. Set the monitored MRM transitions, fragmentor voltages, and
collision energies for individual compounds according to data
presented in Table 1 (see Note 10). Example chromatograms
are shown in Fig. 1.
6. Set the remaining mass detector parameters as follows: capillary
voltage, 3000 V; gas flow (nitrogen), 11 L/min; and gas
temperature, 325  C; sheath gas flow, 11 L/min; sheath gas
temperature, 325  C; nebulizer pressure, 40 psi; and retention
time window for all compounds, 1 min (see Note 11).

4 Notes

1. Store acetonitrile used for precipitation at temperatures below

15  C. Store distilled water at temperatures below 4  C. Store
commercially purchased stock drug solutions at temperatures
below 15  C. Until analysis, keep blood samples at tempera-
tures below 15  C.
2. Keep mobile phase A in dark glass bottle to retard bacteria and
algae growth.
3. Changes of standard concentration may occur in working solu-
tions maintained over a prolonged period of time.
4. In order to control the sensitivity of the method as well as if the
results are positive, the selected stimulants should be spiked
into drug-free blood samples to prepare controls at concentra-
tions of 10 and 100 ng/mL. Samples are initially run without
drug-specific positive controls. The internal standard is used in
the initial run as an indicator of successful extraction and
Table 1
List of designer stimulants covered by presented method with their names, mass spectrometer parameters, and retention times

Retention Relative
Precursor Product Fragmentor Collision time retention
Name ion ion voltage [V] energy [V] RT [min] time RRT
2-AI (2-aminoindane) 134.1 117 87 12 1.2 0.48
115 24
91.1 32
2-DPMP (desoxypipradrol) 252.2 167 120 16 5.4 2.16
91.1 24
65.1 60
2-FA/4-FA (2/4-fluoroamphetamine) 154.1 137 85 4 1.6 0.64
109 16
83 44
2,3-DMEC (2,3-dimethylethcathinone) 206.2 188.2 56 8 4.8 1.92
159.2 16
158.2 32
2,4-DMEC (2,4-dimethylethcathinone) 206.2 115.1 68 60 5.1 2.04
91.2 60
72.2 12
3,4-DMMC (3,4-dimethylmethcathinone) 192.1 174.1 85 8 5.03 2.01
159.1 20
3-CMC (3-chloromethcathinone) 198.1 180.1 25 8 2.54 1.02
145.1 16
144.1 32
4-CMC (4-chloromethcathinone) 198.1 180 83 8 2.54 1.02
Designer Stimulants

145.1 16
144.1 36
(continued)
169
 170

Table 1
(continued)

Retention Relative
Precursor Product Fragmentor Collision time retention
Name ion ion voltage [V] energy [V] RT [min] time RRT
3-EMC (3-ethylmethcathinone) 192.1 91.2 58 40 4.8 1.92
77.1 52
65.2 60
3-FMC (3-fluoromethcathinone) 182.1 164.1 93 8 1.3 0.52
149 20
148 36
3-FPM (3-fluorophenmetrazine) 196.1 135.1 25 20 2.2 0.88
115.1 32
Piotr Adamowicz and Bogdan Tokarczyk

109.1 24
3-MEC (3-methylethcathinone) 192.1 174.2 62 8 3.4 1.36
145.2 16
144.2 28
4-BMC (brephedrone, 4-bromomethcathinone) 242 145.1 85 12 3.6 1.44
144 36
77.1 60
4-CEC (4-chloroethcathinone) 212.1 194.1 86 8 4.23 1.69
144.1 28
77.1 60
4Cl-α-PVP (4-chloro-α-pyrrolidinovalerophenone) 266.1 125 102 24 5.47 2.19
111 48
74.1 132
4-EEC (4-ethylethcathinone) 206.2 188.2 25 8 4.98 1.99
159.1 16
144.1 28
4-EMC (4-ethylmethcathinone) 192.1 174.1 68 8 4.68 1.87
145.1 20
144.1 36
4-FMA (4-fluoromethamphetamine) 168.1 137.1 62 8 1.85 0.74
109 20
83.1 44
4-FMC (flephedrone, 4-fluoromethcathinone) 182.1 164.1 83 8 1.29 0.52
149 20
148 36
4-MBC (benzedrone) 254.2 236.1 87 8 5.55 2.22
91.1 24
65.1 60
4-MDMC (4-methyldimethcathinone) 192.1 91.2 54 40 2.9 1.16
77.2 60
72.2 28
4-MEAP (NEMNP, 220.2 202.1 78 8 5.25 2.10
4-methyl-α-ethylaminopentiophenone) 144.1 32
91.1 48
4-MEC (4-methylethcathinone) 192.1 174.1 143 8 3.34 1.34
145.1 16
91.1 36
4-MeMABP (4-methylbuphedrone) 192.1 174.2 56 8 4.4 1.76
145.2 20
144.2 32
4-MPD (4-methylpentedrone) 206.2 188.1 80 8 5.11 2.04
144.1 36
77.1 60
4-MPHP (PV-4, 40 -methyl-α-pyrrolidinohexiophenone) 260.2 140.1 104 24 5.72 2.29
Designer Stimulants

105.1 20
91.1 48
171

(continued)
 172

Table 1
(continued)

Retention Relative
Precursor Product Fragmentor Collision time retention
Name ion ion voltage [V] energy [V] RT [min] time RRT
4-MTA (4-methylthioamphetamine) 182.1 165 62 4 4.46 1.78
137 20
117 16
5-APB (5-(2-aminopropyl)benzofuran) 176.1 159.1 70 4 3.17 1.27
131.1 16
77 44
5-MAPB (5-(2-methylaminopropyl)benzofuran) 190.1 159.1 68 8 4.0 1.60
131 20
Piotr Adamowicz and Bogdan Tokarczyk

91.1 36
6-APB (6-(2-aminopropyl)benzofuran) 176.1 159.1 64 4 4.04 1.62
131 20
91.1 36
6-EAPB (1-(benzofuran-6-yl)-N-ethylpropan-2-amine) 204.1 159.2 25 8 4.7 1.88
131.1 20
91.1 36
6-IT (6-(2-aminopropyl)indole) 175.1 158.1 60 4 2.53 1.01
130 20
117 24
6-MAPB (6-(2-methylaminopropyl)benzofuran) 190.1 159.2 25 8 4.2 1.68
131.1 16
91.1 36
α-MT (α-methyltryptamine) 175.1 158.1 58 4 2.35 0.94
130 24
117 28
α-PVP (α-pyrrolidinopentiophenone) 232.2 126.1 85 24 4.56 1.82
91 24
77 48
α-PBP (α-pyrrolidinobutiophenone) 218.2 112.1 83 24 2.57 1.03
91.1 20
77.1 48
α-PPP (α-pyrrolidinopropiophenone) 204.1 133 120 16 1.65 0.66
105.1 24
98.1 28
α-PVT (α-pyrrolidinopentiothiophenone) 238.1 126.1 41 20 3.18 1.27
111 36
97 20
BDB (1,3-benzodioxolylbutanamine) 194.1 179.1 143 12 5.1 2.04
164.1 20
77.1 52
bk-DMBDB (dibutylone) 236.1 161 145 16 2.38 0.95
86.1 24
65.1 56
bk-MDDMA (dimethylone) 222.1 147 83 16 1.5 0.60
91.1 36
72.1 16
BMDP (4-methylenedioxy-N-benzylcathinone) 284.1 266.1 87 8 5.18 2.07
91 24
65.1 60
Buphedrone 178.1 160.1 85 4 1.83 0.73
91.1 16
77.1 48
Bupropion 240.1 184 93 4 5.05 2.02
Designer Stimulants

166 12
131.1 24
173

(continued)
 174

Table 1
(continued)

Retention Relative
Precursor Product Fragmentor Collision time retention
Name ion ion voltage [V] energy [V] RT [min] time RRT
Butylone 222.1 204.1 91 4 2.14 0.86
174.1 12
131 32
BZP (benzylpiperazine) 177.1 91.1 101 20 0.5 0.20
85.1 12
65.1 52
D2PM (diphenylprolinol) 254.2 236.1 83 8 4.91 1.96
165 56
Piotr Adamowicz and Bogdan Tokarczyk

130 28
Diethylpropion 206.2 105.1 112 16 1.88 0.75
100.1 20
77.1 48
Ephedrone 164.1 146 85 8 1.05 0.42
131 16
77 52
Ethylone 222.1 204.1 89 4 1.72 0.69
174.1 12
146.1 24
Ethylphenidate 248.2 84.1 85 20 5.1 2.04
56.1 56
55.1 56
Ethcathinone 178.1 160.1 89 8 1.31 0.52
131 16
117 28
Eutylone 236.1 218.1 89 8 2.97 1.19
188.1 16
174 32
MDPBP (30 ,40 -methylenedioxy- 262.2 161.1 118 16 3.6 1.44
α-pyrrolidinobutyrophenone) 112.1 24
65.1 60
MDPPP (30 ,40 -methylenedioxy- 248.1 147 91 20 2.08 0.83
α-pyrrolidinopropiophenone) 98.1 24
91.1 48
MDPV (methylenedioxypyrovalerone) 276.2 175.1 124 16 4.9 1.96
135 20
126.1 24
MeBP (methylbuphedrone) 191.2 105.1 99 20 0.74 0.30
79.1 40
77.1 52
MeOPP (para-methoxyphenylpiperazine) 193.1 150 116 16 2.1 0.84
120 36
65.1 60
Mephedrone/2-MMC/3-MMC 178.1 160.1 87 8 2.5 1.00
145.1 20
77.1 56
MePPP (40 -methyl-α-pyrrolidinopropiophenone) 218.2 119.2 54 24 4.1 1.64
98.2 28
91.2 40
Metamfepramone (dimethylcathinone) 178.1 133 85 12 1.2 0.48
105.1 20
72.1 24
Methedrone (4-methoxymethcathinone) 194.1 176.1 85 8 1.8 0.72
Designer Stimulants

161 16
146 28
175

(continued)
 176

Table 1
(continued)

Retention Relative
Precursor Product Fragmentor Collision time retention
Name ion ion voltage [V] energy [V] RT [min] time RRT
Mexedrone 208.1 158.2 56 8 3.3 1.32
119.1 20
91.2 40
MDMC (methylone) 208.1 190 91 4 1.56 0.62
160 12
132 24
MPA (methiopropamine) 156.1 125 58 8 1.0 0.40
97 20
Piotr Adamowicz and Bogdan Tokarczyk

58.2 4
MPBP (40 -methyl-α-pyrrolidinobutiophenone) 232.2 161.1 85 12 4.79 1.92
105.1 24
91.1 48
MPHP (40 -methyl-α-pyrrolidinohexiophenone) 260.2 140.2 25 28 5.9 2.36
105.1 24
91.1 52
MPPP (desmethylprodine) 248.2 174.1 97 8 4.61 1.84
44.2 20
42.2 60
Naphyrone 282.2 211.1 126 12 5.76 2.30
155 24
141 20
NEB (N-ethylbuphedrone) 192.1 174.1 68 8 2.25 0.90
130 32
91.1 28
NEH (N-ethylhexedrone) 220.2 130.1 84 36 5.2 2.08
91.1 32
77.1 60
N-ethylpentylone (ephylone) 250.1 232.1 84 8 4.7 1.88
202.2 16
174.1 32
Pentedrone (α-methylaminovalerophenone) 192.1 174.1 89 8 3.91 1.56
132.1 16
91.1 24
Pentylone (methylenedioxypentedrone) 236.1 218.1 62 8 4.4 1.76
188.1 16
175.1 20
PV-7 (α-PHP, α-pyrrolidinohexiophenone) 246.2 140.2 54 24 5.3 2.12
91.2 24
77.2 60
PV-8 (α-PHPP) 260.2 154.2 5 28 5.62 2.25
91.1 24
77.1 60
TFMPP (trifluoromethylphenylpiperazine) 231.1 188 122 16 5.08 2.03
145 44
44.1 20
Mephedrone-D3 (internal standard) 181.1 163.1 87 8 2.5 1.00
Designer Stimulants
177
178 Piotr Adamowicz and Bogdan Tokarczyk

Fig. 1 Example of chromatograms obtained for blood from real forensic cases in which 3-methylethcathinone
(a), 4-chloroethcathinone (b), and N-ethylpentylone (c) were revealed. Subsequent analysis by other quanti-
tative methods determined drug concentrations to be, respectively, 3-MEC, 69 ng/mL; 4-CEC, 49 ng/mL; and
N-ethylpentylone, 196 ng/mL
 Designer Stimulants 179

analysis, based on acceptable intensity and retention time.

Samples that meet internal standard criteria and demonstrate
the presence of another compound(s) in Table 1 are repeated,
alongside drug-free blood spiked with standards of the appro-
priate drug or drugs.
5. We find that the use of iced acetonitrile (stored at temperatures
below 15  C) provides better isolation efficiency.
6. Never add the entire volume of acetonitrile at once because it
causes immediate protein precipitation, which significantly
reduces the yield of the process.
7. In this form, the samples may be frozen at temperatures below
15  C and stored up to 1 month. Do not evaporate at higher
temperatures. Some compounds (especially cathinones) might
be lost.
8. The column used tends to clog up during biological material
analysis. The use of interchangeable filters significantly
increases the life of the column.
9. The application of dynamic MRM provides an enhanced sensi-
tivity (due to increased number points per peak) by utilizing
the retention time window of each analyte [6].
10. Monitor three MRM pairs for each compound. This ensures
the specificity of the method. The procedure is open, meaning
that the procedure can be easily extended for additional new
designer drugs. The most intense MRM transitions for sub-
stances not listed here can be easily obtained with the use of the
Agilent Mass Hunter Optimizer. This software automatically
selects four of the most abundant fragment ions for defined
precursor ion and optimizes fragmentor voltage and the
corresponding collision energy for each transition.
11. Interpretation of results: Concentrations are estimated using
the spiked controls. Samples for which the estimated concen-
tration (based on comparison with the controls) is higher than
the cutoff of 5 ng/mL are considered as presumptive positive
and subjected to subsequent targeted quantitative analyses
(not part of this procedure).

References
1. European Monitoring Centre for Drugs and chromatographic and spectrometric methods.
Drug Addiction (2017) European Drug Report Aust J Forensic Sci 49:637. https://doi.org/
2017: Trends and Developments, Publications 10.1080/00450618.2016.1167240
Office of the European Union, Luxembourg. 3. Ellefsen KN, Concheiro M, Huestis MA (2016)
http://www.emcdda.europa.eu/system/files/ Synthetic cathinone pharmacokinetics, analytical
publications/4541/TDAT17001ENN.pdf. methods, and toxicological findings from
Accessed 11 July 2017 human performance and postmortem cases.
2. Zuba D, Adamowicz P (2016) Distinction of Drug Metab Rev 48(2):237–265. https://doi.
constitutional isomers of mephedrone by org/10.1080/03602532.2016.1188937
180 Piotr Adamowicz and Bogdan Tokarczyk

4. Namera A, Kawamura M, Nakamoto A, Saito T, tandem mass spectrometry. Drug Test Anal 8
Nagao M (2015) Comprehensive review of the (7):652–667
detection methods for synthetic cannabinoids 6. Stone P, Glauner T, Kuhlmann F, Schlabach T,
and cathinones. Forensic Toxicol 33 Miller K (2009) New dynamic MRM mode
(2):175–194 improves data quality and triple quad quantifica-
5. Adamowicz P, Tokarczyk B (2016) Simple and tion in complex analyses. Technical overview.
rapid screening procedure for 143 new psycho- Agilent technologies. https://www.agilent.
active substances by liquid chromatography- com/cs/library/technicaloverviews/Public/
5990-3595en_lo%20CMS.pdf.
 Chapter 17

Drug Screening Using Liquid Chromatography Quadrupole

Time-of-Flight (LC-QqTOF) Mass Spectrometry
Jennifer M. Colby and Kara L. Lynch

Abstract
Drug screening using high-resolution mass spectrometers, including quadrupole time-of-flight mass ana-
lyzers (QqTOFs), is becoming increasingly popular due to the additional flexibility that these instruments
offer laboratories. Liquid chromatography (LC) coupled to TOF, as in an LC-QqTOF, offers comparable
sensitivity and a shortened method development time relative to triple quadrupole-based mass spectrome-
try. In addition, LC-QqTOF data that are collected in untargeted mode can be analyzed retrospectively to
detect additional compounds that were not predefined targets. Much of the power of LC-QqTOF lies in
data processing, and the data analysis workflow that a lab uses must be adequately validated.

Key words High-resolution mass spectrometry, Time of flight, Drug screen, Toxicology

1 Introduction

High-resolution mass spectrometry (HRMS) using quadrupole

time-of-flight (QqTOF) or Orbitrap technology has gained recog-
nition as a valuable tool for broad-spectrum drug screening in a
variety of biological matrices. In contrast to triple quadrupole-
based liquid chromatography tandem mass spectrometry
(LC-MS/MS) methods, which collect nominal mass data primarily
in a targeted manner, HRMS instruments collect untargeted, accu-
rate mass data for precursor and product ions. Similar to LC-MS/
MS methods, compounds can be identified by targeted data analysis
(i.e., library searching) in which the precursor mass, retention time,
and product ion spectrum are compared to that of a reference
standard. It has been demonstrated that the detection capabilities
of LC-QqTOF for drug screening using targeted data analysis are
comparable to LC-MS/MS [1]. In addition to targeted analysis,
other data analysis strategies are possible (e.g., suspect and untar-
geted screening) [2–4]. These additional strategies are particularly
attractive in the setting of drug screening, as they allow for the

Loralie J. Langman and Christine L.H. Snozek (eds.), LC-MS in Drug Analysis: Methods and Protocols,
Methods in Molecular Biology, vol. 1872, https://doi.org/10.1007/978-1-4939-8823-5_17,
© Springer Science+Business Media, LLC, part of Springer Nature 2019

181
182 Jennifer M. Colby and Kara L. Lynch

identification of compounds that are not included in the library or

are unexpected. As a result, HRMS permits identification of both
expected and unexpected compounds in a single analytical run.
Tentative identification of unknown compounds can be made in
the absence of a reference standard or a library spectrum. Once a
compound is tentatively identified, an analytical standard can be
purchased and tested to confirm that the analytical retention time
and fragmentation pattern match that observed in the test sample.
The illicit drug market is constantly evolving, making it difficult
for analytical toxicology laboratories to maintain methods capable
of detecting all potential toxins and emerging drugs. Since data are
acquired in an untargeted manner with HRMS, they can be retro-
spectively analyzed for new and emerging novel psychoactive sub-
stances (NPS) and synthetic designer drugs, adding to the appeal of
this technology in the clinical and forensic setting. The addition of
emerging drugs to HRMS methods using targeted data analysis
only requires the purchase of an analytical standard to establish the
retention time and acquire a high-resolution mass spectrum for
addition to the spectral library used by the laboratory. Subsequently
the limit of detection, matrix effects, and other validation para-
meters can be evaluated following each laboratory’s protocol.
HRMS has clearly emerged as the predominant method for the
detection of NPS; however, this technology is not readily available
in most routine clinical and forensic laboratories. This chapter will
describe an HRMS method using an LC-QqTOF instrument for
drug screening. It will primarily focus on the use of targeted data
analysis for drug screening. This method can be adapted to include
any pharmaceutical or illicit drug with the purchase of an analytical
standard. Validation of the analytical method is further described in
Thoren et al. (2016), and validation of the data analysis parameters
is described in Colby et al. [5].

2 Materials

2.1 Standards 1. 50:50 methanol/acetonitrile: Mix 15 mL of methanol with

and Reagents 15 mL of acetonitrile (see Note 1). Solution is stable at
15–30
C for 1 year when stored in glass bottles (e.g., Pyrex).
2. Specimen preparation buffer: Mix 25 mL of 50:50 methanol/
acetonitrile with 175 mL of water to prepare 200 mL of
preparation buffer. Solution is stable at 15–30
C for 1 year
when stored in glass bottles (e.g., Pyrex).
3. 1 M ammonium formate: Measure 6.305 g of ammonium
formate using an analytical balance. Add the ammonium for-
mate to a 100 mL volumetric flask, and fill to 100 mL with
water. Solution is stable at 15–30
C for 1 year when stored in
glass bottles (e.g., Pyrex).
Drug Screening Using Liquid Chromatography Quadrupole Time-of-Flight. . . 183

4. Mobile phase A: 5 mM ammonium formate and 0.05% formic

acid in water. Add 10 mL of 1 M ammonium formate, 1 L of
water, and 1 mL of formic acid to a graduated cylinder, and then
add water to bring the volume to 2 L. Solution is stable at
15–30
C for 1 month when stored in glass bottles (e.g., Pyrex).
5. Mobile phase B: 0.05% formic acid in 50:50 methanol/aceto-
nitrile. Mix 1 L of acetonitrile with 1 L of methanol in a 2 L
graduated cylinder. Using a volumetric pipette, remove 1 mL
of the mix, and then add 1 mL of formic acid. Solution is stable
at 15–30
C for 1 month when stored in glass bottles (e.g.,
Pyrex).
6. Pump wash solution: 50:50 water/methanol. Mix 1 L of water
with 1 L of methanol in a 2 L graduated cylinder. Solution is
stable at 15–30
C for 6 months when stored in glass bottles
(e.g., Pyrex).
7. Quality control mix: 1 μg/mL each of 20–25 drugs in 50:50
methanol/acetonitrile (see Notes 2 and 3). Add 5 mL of 50:50
methanol/acetonitrile to a 10 mL volumetric flask. Add 10 μL
of each 1 mg/mL drug reference standard to be included. Fill
to volume with 50:50 methanol/acetonitrile, and mix well.
Prepare 1 mL aliquots in glass vials with PTFE-lined caps.
Example mixes are shown in Table 1. Mixes are stable at
20
C for 1 year, or until the stock standard solutions expire,
whichever comes first.
8. Fentanyl-D5 internal standard working solution: 1 μg/mL
fentanyl-D5 in specimen preparation buffer. Add 100 μL of
fentanyl-D5 stock standard (100 μg/mL) to 9.9 mL of speci-
men preparation buffer. Prepare 1 mL aliquots in glass vials
with PTFE-lined caps. Stable for 1 year at 20
C, or until the
stock solution expires, whichever comes first.
9. Serum/plasma protein dump solution: 200 ng/mL fentanyl-
D5 in acetonitrile. Add 500 μL of fentanyl-D5 stock standard
(100 μg/mL) to a 250 mL volumetric flask, and fill with
acetonitrile. Prepare 10 mL aliquots in glass vials or bottles.
Stable for 1 year at 20
C, or until the stock solution expires,
whichever comes first.
10. Negative control: Drug-free human urine or serum. Stable at
4
C until expiration date on label (see Note 4).
11. Positive controls: 100 ng/mL of each quality control mix. Add
1 mL of each quality control mix into its own 10 mL volumet-
ric flask. Fill to volume with drug-free human urine or serum
(match the matrix of the samples to be tested). Depending on
the drugs that it contains, the positive control mix may be
stable at 4
C for up to 30 days or up to 1 year at 80
C.
Prior to freezing, prepare aliquots with a volume that can be
used within the refrigerated stability window of the mix (see
Note 5).
184 Jennifer M. Colby and Kara L. Lynch

Table 1
Example of drug mixes

Mix A Mix B Mix C

2C-T2 6-APB/5-APB Delta9-THC
4-MePPP 7-Aminoclonazepam 2-Hydroxyethylflurazepam
4-MTA 7-Aminonitrazepam 6-monoacetylmorphine
Amoxapine Aripiprazole 9-hydroxyrisperidone
Atropine Brompheniramine maleate Acetaminophen
Cannabidiol Buprenorphine glucuronide Alpha-hydroxytriazolam
Chlorpheniramine Cannabinol Diazepam
Clomipramine Cathinone Droperidol
Clonazepam Codeine Gabapentin
Desipramine Desalkylflurazepam Hydrocodone
Dihydrocodeine Diphenoxylate Levorphanol
Diphenhydramine Flunitrazepam Loxapine
DOM Hydroxyzine Methadone
Doxepin Meperidine Methylphenidate
Ecgonine methyl ester Meprobamate Mianserin HCl
Fluoroamphetamine Olanzapine N-Desmethylclomipramine
Lorazepam Oxcarbazepine Nicotine
MBDB Phentermine Nordoxepin
Methocarbamol Quetiapine Norketamine
Oxycodone Zaleplon Venlafaxine
0
4-MePPP 4 -methyl-α-pyrrolidinopropiophenone, 4-MTA 4-methylthioamphetamine, DOM 2,5-dimethoxy-4-methy-
lamphetamine, MDBD methylbenzodioxolylbutanamine, APB 2-aminopropylbenzofuran, THC tetrahydrocannabinol,
HCl hydrochloride

2.2 Supplies 1. 12  75 mm plastic test tubes.

2. 13  100 mm glass tubes (see Note 6).
3. 2 mL autosampler vials and caps with polytetrafluoroethylene
(PTFE)-lined septa.
4. Amber vials with PTFE-lined caps for drug and internal stan-
dard solutions.
5. Volumetric glassware (pipettes and flasks, Class A).
6. Graduated cylinder (2 L).
7. Glass bottles to store reagents.
 Drug Screening Using Liquid Chromatography Quadrupole Time-of-Flight. . . 185

8. Plastic transfer pipettes (see Note 7).

9. UPLC columns (Kinetex C18, 2.6 μm, 3  50 mm) and guard
columns (SecurityGuard ULTRA cartridges, C18) or similar.
10. APCI calibration solution for 5600 TripleTOF®.

2.3 Equipment 1. Benchtop centrifuge (Eppendorf Microcentrifuge 5430, or

similar).
2. Sample concentrator/evaporator (TurboVap LV evaporator, or
similar).
3. Air displacement pipettes (Eppendorf Reference 2 Pipettes, or
similar).
4. Vortexer.
5. Centrifuge (Eppendorf 5810 or similar).
6. An HPLC system compatible with ABSciex Analyst software
(e.g., Shimadzu LC-20ADXR Prominence) with degasser,
binary pump, solvent-switching valve, temperature-controlled
autosampler, and temperature-controlled column
compartment.
7. Nitrogen generator or liquid N2 dewar capable of supplying
curtain, collision, and source gases.
8. 5600 TripleTOF® quadrupole time-of-flight mass spectrome-
ter with a DuoSpray™ source and automatic calibrant delivery
system.
9. Analyst® 1.5, PeakView® 2.0, and MasterView™ 1.0 software.

3 Methods

3.1 Analysis of Urine 1. Aliquot approximately 1 mL of each patient urine specimen

into a 12  75 mm plastic test tube using a disposable, plastic
transfer pipet. Cap the tube.
2. Centrifuge all patient urine specimens for 10 min at 450  g in
the centrifuge.
3. Label an amber autosampler vial for the negative control,
positive control, and each patient urine. Due to the possibility
of carryover, a double blank (no drug, no internal standard)
sample must be injected between each patient sample. Each
double blank can be injected up to five times before being
replaced with a fresh vial.
4. Pipet 1000 μL of sample preparation buffer into each double
blank vial.
5. Pipet 700 μL of sample preparation buffer into each control
and patient vial.
186 Jennifer M. Colby and Kara L. Lynch

6. Pipet 100 μL of internal standard into each vial, except for the
double blank.
7. Pipet 200 μL of negative control, positive control, and patient
urine to the correspondingly labeled vials.
8. Cap the vials and vortex to mix.
9. Place the vials in the autosampler tray for testing.

3.2 Analysis 1. Aliquot 250 μL of each serum or sodium heparin plasma speci-
of Serum/Plasma men or control sample using an air displacement pipette with
disposable tips into a 1.5 mL microcentrifuge tube. Add
750 μL of protein dump solution. Pipette up and down to
mix. Cap the tube. Vortex for at least 30 s.
2. Centrifuge specimens for 10 min at 8500  g in a benchtop
centrifuge.
3. Carefully remove samples from the centrifuge, ensuring that
the pellet at the bottom of the tube is not disturbed.
4. Open the tube, and transfer 750 μL of supernatant into a
13  100 mm glass tube. Some supernatant should remain in
the tube.
5. Dry supernatant under nitrogen flow at 37
C for 20 min or
until dry.
6. Label an amber autosampler vial for the negative control,
positive control, each patient specimen, and double blank sam-
ples. Due to the possibility of carryover, a double blank
(no drug, no internal standard) sample must be injected
between each patient sample.
7. Transfer 1000 μL of sample preparation buffer to the double
blank vial. Cap and place in the autosampler tray.
8. Pipet 100 μL of sample preparation buffer into the tubes con-
taining dried control and patient samples, ensuring that the
dried sample is resuspended.
9. Pipet the resuspended patient and control samples into the
appropriate autosampler vials.
10. Cap the vials and place in the autosampler tray for testing.

3.3 Instrument Liquid chromatography system:

Operating Conditions
1. Injection volume: 10 μL
2. Flow rate: 400 μL/min
3. LC parameters:
0 min: 2% mobile phase B (MPB)
10 min: 98% MPB
Wash 2 min at 100% MPB
 Drug Screening Using Liquid Chromatography Quadrupole Time-of-Flight. . . 187

Re-equilibrate 2 min at 2% MPB

5600 QTOF mass spectrometer:
4. Ion source: positive electrospray, 500
C, ion spray voltage
floating 5500 V, declustering potential 100 V
5. Gas settings: source gas 1–30 PSI, source gas 2–30 PSI, curtain
gas 25 PSI
6. Ion release delay: 67
7. Ion release width: 25
8. Full-scan TOF-MS from 50 to 700 Da
9. Information-dependent acquisition of product ion spectra for
20 candidate ions per cycle
10. Automatic calibration verification of TOF and MS/MS mass
accuracy every five injections

3.4 Data Analysis 1. Load the data file(s) and premade targeted analysis extraction
ion chromatogram (XIC, Fig. 1) list into MasterView (see
Notes 8 and 9).

5.0e5

4.5e5

4.0e5

3.5e5

3.0e5
Intensity

2.5e5

2.0e5

1.5e5

1.0e5

5.0e4

0.0e0
1 2 3 4 5 6 7 8 9
Time, min

Fig. 1 Extracted-ion chromatogram of a mix of 40 drugs and metabolites. Drug (retention time in minutes):
morphine (2.59), oxymorphone (2.81), hydromorphone (3.01), codeine (3.49), amphetamine (3.69), oxycodone
(3.73), 6-monoacetylmorphine (3.88), hydrocodone (3.88), MDA (3.89), methamphetamine (3.90), desmethyl-
tramadol (3.94), MDMA (4.05), benzoylecgonine (4.5), norfentanyl (4.56), tramadol (4.85),
7-aminoclonazepam (4.96), cocaine (5.11), norbuprenorphine (5.71), fentanyl (6.12), fentanyl-D5 (6.12),
flurazepam (6.33), midazolam (6.38), EDDP (6.49), buprenorphine (6.51), clonazepam (6.73), alpha-
hydroxymidazolam (6.80), alpha-hydroxytriazolam (7.12), nitrazepam (7.15), methadone (7.21), alpha-
hydroxyalprazolam (7.22), oxazepam (7.33), lorazepam (7.43), flunitrazepam (7.44),
2-hydroxyethylflurazepam (7.45), alprazolam (7.57), triazolam (7.58), desalkylflurazepam (7.66), temazepam
(7.77), nordiazepam (7.88), diazepam (8.33)
188 Jennifer M. Colby and Kara L. Lynch

2. Ensure that ion extraction settings display precursor mass

search of 30 ppm, a retention time window of 15 s, and a
minimum peak intensity of 1 count per second.
3. Verify that a combined score threshold of >70 and combined
scoring algorithm using 10% mass error, 10% retention time
error, 10% isotope error, and 70% library match have been
programmed as positivity criteria.
4. Process data.
5. Review presumptive positive extracted ion chromatograms
for visual assessment of peak shape and library match (see
Note 10).

4 Notes

1. All reagents, including water and solvents, must be analytical

grade or better. Glassware used to prepare reagents should be
free of detergents or other residues. Dedicated glassware used
only for LC-MS reagent preparation is ideal. For measurement
purposes, Class A volumetric glassware should be used when-
ever possible. Though they are not critical, positive displace-
ment pipettes with disposable tips may be useful in measuring
small volumes of solvent (e.g., for measuring drug standards).
2. Once opened, drug standards should be transferred to amber
colored vapor-tight vials (PTFE-lined caps) for storage at
20
C or 80
C. Wrapping the top of the sealed vial with a
narrow strip of Parafilm can help preserve standards for future
use. Using amber vials helps protect drugs that degrade in
light.
3. Each mix should contain different drugs. Drugs should not be
grouped into mixes by class; instead, each mix should contain
drugs with varying retention times and physical/chemical
properties. The mixes of drug standards may be used in
method development and validation, as well as to spike positive
QC samples. Spiked samples can be used to establish retention
time, product ion spectrum, and to verify lower limit of
detection.
4. Each new lot of drug-free human serum or urine should be
tested in-house to verify that it is truly free of all dugs. Most
commercial laboratories that prepare human-derived materials
for sale do not test for every drug and, in particular, do not test
for a range of novel psychoactive substances. It is far less costly
to determine up front whether a lot is suitable for use than to
have to discard contaminated quality control materials.
5. The goal of quality control is to verify instrument performance
across all drug categories and to confirm the chromatographic
Drug Screening Using Liquid Chromatography Quadrupole Time-of-Flight. . . 189

separation meets expectations. The method is tested by injec-

tion of negative and positive control samples. To prevent wast-
age of reagents and analytical time, the positive control sample
contains only a subset of the 100+ compounds in the method.
The drugs are chosen based on prevalence in the population,
chromatographic retention time, and presence of isomers. The
presence/absence of each compound, peak area, and retention
time are recorded for the drugs present in the positive control.
These parameters are tracked over time to demonstrate stability
of the method.
6. The wider the diameter of the glass tube that is used for
evaporating samples, the faster they will dry. The choice of
what tube to use depends on what tubes will fit in the drying
apparatus that is in use in the laboratory.
7. The use of plastic transfer pipettes should be undertaken with
caution. Compounds may be more susceptible to adsorb to
some brands of pipettes than others. Once a suitable pipette is
identified, introduction of a new supplier of pipettes should be
avoided. If this is unavoidable, adsorption can be tested using
spiked samples.
8. One of the rationales for collecting untargeted, full scan,
HRMS data is that different data analysis techniques can be
used to maximize the information gleaned from each sample.
The data analysis workflow that is of greatest utility in routine
analysis is known as targeted analysis. To perform targeted
analysis, the analyst must know the accurate mass, chro-
matographic retention time, isotope pattern, and product ion
spectrum of the targeted compounds. Accurate mass and iso-
tope pattern can be predicted based on the empirical formula of
the compound of interest. Retention times must be established
on the LC-MS system where the method will be performed. It
is ideal to collect product ion spectra on the LC-MS where the
method will be run, using a dedicated product ion scan. The
spectrum can be added to the in-house compound library.
Retention times are typically established by averaging the
observed retention time from 3 injections on 2 individual
columns. Targeted data analysis methods may include any
number of compounds. Including additional compounds is
simple, compared to a traditional targeted acquisition, because
compound-dependent parameters do not need to be devel-
oped. If the retention time is established and the product ion
spectrum is included in the library, the compound can be
detected using the targeted compound list. Additional valida-
tion experiments (e.g., recovery, lower limit of detection, inter-
ference testing, etc.) are necessary when adding a new
compound to the method, but these are not typically too
labor intensive.
190 Jennifer M. Colby and Kara L. Lynch

9. After the parameters of interest have been established, a tar-

geted compound list is built in MasterView. Positivity is
assessed using combined scores. Additional details on how to
establish and validate data analysis parameters can be found in
Colby, Thoren, and Lynch, Journal of Analytical Toxicology,
2017 Jan; 41(1): 1–5.
10. Compounds identified by the targeted data analysis approach
may be reported as positive or as presumptive positive, depend-
ing on the laboratory’s reporting criteria.

References
1. Thoren KL, Colby JM, Shugarts SB, Wu AHB, Screening new psychoactive substances in urban
Lynch KL (2016) Comparison of information- wastewater using high resolution mass spec-
dependent acquisition on a tandem quadrupole trometry. Anal Bioanal Chem 408:4297
TOF vs a triple quadrupole linear ion trap mass 4. Moschet C, Piazzoli A, Singer H, Hollender J
spectrometer for broad-spectrum drug screen- (2013) Alleviating the Reference standard
ing. Clin Chem 62:170–178 dilemma using a systematic exact mass suspect
2. Bade R, Rousis NI, Bijlsma L, Gracia-Lor E, screening approach with liquid
Castiglioni S, Sancho JV et al (2015) Screening chromatography-high resolution mass spec-
of pharmaceuticals and illicit drugs in wastewater trometry. Anal Chem 85:10312
and surface waters of Spain and Italy by high 5. Colby JM, Thoren KL, Lynch KL (2016) Opti-
resolution mass spectrometry using UHPLC- mization and validation of high-resolution mass
QTOF MS and LC-LTQ-Orbitrap MS. Anal spectrometry data analysis parameters. J Anal
Bioanal Chem 407:8979 Toxicol 1:1–5
3. González-Mariño I, Gracia-Lor E, Bagnati R,
Martins CPB, Zuccato E, Castiglioni S (2016)
 Chapter 18

Alternate Matrices: Meconium, Cord Tissue, Hair,

and Oral Fluid
Kendra L. Palmer and Matthew D. Krasowski

Abstract
Drug testing commonly involves serum, blood, or urine. More recently, alternative specimens for drug
testing have been increasingly used for clinical and forensic toxicology. Examples include oral fluid (saliva),
hair, meconium, and umbilical cord tissue. Each of these matrices has unique properties that provide
advantages for certain applications. Oral fluid has easier and less invasive collection requirements than
urine, the most common specimen for drug screening. Oral fluid drug testing is common in Europe and
steadily gaining popularity in the United States. Hair accumulates drugs and drug metabolites and provides
a much longer window of detection than blood or urine. Meconium and umbilical cord tissue each allow for
assessment of prenatal drug exposure over the course of months. Limitations of these alternative matrices
include need for laboratory-developed tests (exception being some oral fluid immunoassays), challenges
with the specimen matrix, and incomplete understanding of drug incorporation and kinetics. This chapter
briefly describes each of the above alternative specimens in terms of their utility, advantages, and limitations.

Key words Drug screening, Neonatal testing, In utero exposure, Saliva testing, Detection window

1 Introduction

Historically, blood and urine were the mainstay specimens for drug
testing; however, the use of alternative specimens in drug testing
has become increasingly important in clinical and forensic toxicol-
ogy. Some commonly used alternative specimens include oral fluid,
hair, meconium, and umbilical cord tissue. Each of these matrices
has unique qualities which provide advantages in various settings.
This chapter will briefly describe each of the above alternative
specimens in terms of their utility, advantages, and limitations.
Detailed testing methods will be presented in subsequent chapters.

Loralie J. Langman and Christine L.H. Snozek (eds.), LC-MS in Drug Analysis: Methods and Protocols,
Methods in Molecular Biology, vol. 1872, https://doi.org/10.1007/978-1-4939-8823-5_18,
© Springer Science+Business Media, LLC, part of Springer Nature 2019

191
192 Kendra L. Palmer and Matthew D. Krasowski

2 Oral Fluid

Oral fluid is the liquid present in the oral cavity and consists
primarily of saliva which is an aqueous solution produced by the
salivary glands. Other minor constituents include oral mucosal
transudate, bacteria, and epithelial cells. Oral fluid, as an alternative
to blood and/or urine, is becoming a more widely used matrix.
While oral fluid is commonly used in Europe, its use in the United
States is still limited, and oral fluid testing is not widely available on
routine chemistry analyzers. In areas where it is utilized, oral fluid is
predominantly used in workplace testing and drug monitoring in
substance abuse programs [1]. Oral fluid has some advantages over
urine, which is another specimen often used in these settings. The
primary advantage to oral fluid testing is that collection is simple
and noninvasive, which eliminates the need for restroom facilities
and makes it possible to collect in remote settings. Oral fluid
collection can be more closely observed than urine collection,
which limits the opportunity for specimen adulteration and also
the need for same-sex collectors. An interesting feature of oral fluid
as a toxicology specimen is that the parent drug is frequently the
predominant substance [2]. For example, following use of heroin,
oral fluid may contain the parent drug itself in addition to the
metabolite 6-monoacetylmorphine (6-MAM). In contrast, heroin
itself is rarely detected in blood or urine; the metabolites 6-MAM
and morphine are much more common in these specimens.
There are some limitations to oral fluid testing. Drug concen-
trations are typically lower than those found in urine. Also, collect-
ing sufficient sample volume to allow for confirmatory testing,
should it be necessary, can be difficult. This is especially problematic
as some drugs, including anti-adrenergic and anticholinergic drugs,
will result in reduced salivation [3]. The oral fluid can be contami-
nated from ingested food or beverages, and drug concentrations
will be higher for drugs that are smoked, inhaled, insufflated, or
taken orally. When testing in a quantitative manner, this may be a
limitation as concentrations will not correlate well with blood con-
centrations. However, when performing qualitative testing, this
may present as an advantage.
Due to the relative newness of this specimen type, analyte
stability has not been well characterized. Similarly, collection device
designs are still being perfected. Some early sample collection
devices caused adsorption of the drug to the device, resulting in
false-negative test results.
 Alternate Matrices 193

3 Hair

Toxicology testing on hair, in various forms, has occurred since the

mid-1800s. Although limited to heavy metal detection for the first
century, advances in detection methods now allow for detection of
a wide array of drugs and their metabolites. Hair testing is often
used in conjunction with criminal and forensic investigations,
including in drug-related deaths, child protection, and drug-
facilitated crime. Hair testing has also become a component of
drug monitoring in rehabilitation programs, in workplace drug
testing, and for regranting of drivers’ licenses [4].
Hair is a unique matrix for toxicology testing, in that it is able
to provide historical data of drug use and can provide a very large
detection window [5–8]. Once incorporated into the hair, drugs
remain relatively stable and can theoretically be detected for up to
several years, although detection windows of several months are
more likely in practical terms [9]. This provides a detection window
that is substantially longer than other matrices such as blood or
urine, which can only detect use from hours to weeks, depending
on the substance. The longer window of detection can be especially
useful in assessing long-term abstinence. Hair toxicology testing
may also provide some utility in assessing cases of possible child
abuse or any other case when there is a delay in medical presenta-
tion [10]. In settings where a remote but narrow time frame is of
interest, great caution must be used in result interpretation as the
long window of detection can lead to misinterpretation.
Depending on the processing protocol, hair toxicology testing
may also be able to provide information on environmental contam-
ination. In some settings, such as child abuse assessment, the
environmental exposure to illicit substances may be of interest.
However, the potential of environmental contamination can intro-
duce some uncertainty.
One distinct advantage of hair toxicology testing is that samples
may be stored for long periods of time without the need for
refrigeration. As with any specimen type, hair as a matrix for toxi-
cology testing also has some limitations. Hair is a difficult matrix to
analyze, and outside of reference laboratories, few clinical labora-
tories have the capability to perform this type of testing. An addi-
tional limitation of hair testing is that some individuals lack a
sufficient amount of hair to facilitate testing—this is especially
problematic with young children and infants and limits utility in
newborn drug screening. Additionally, some individuals may
remove their hair in an attempt to avoid testing.
Finally, the mechanisms of drug incorporation into hair are not
completely understood, and evidence suggests that there may be
some variability in drug binding which may be based on molecular
size and structure, pH, and lipid solubility [11]. It has also been
194 Kendra L. Palmer and Matthew D. Krasowski

suggested that melanin content of hair may influence the incor-

poration process, which may result in bias depending on hair color.
Although drugs incorporated into hair are fairly stable under nor-
mal conditions, they can undergo decomposition when exposed to
excessive ultraviolet radiation or bleaching. Once incorporated into
the growing hair, it takes 7–10 days before the hair containing drug
reaches the surface and then some additional time before the hair is
long enough to sample. While this delay is not necessarily a limita-
tion, it is a characteristic of hair toxicology testing which must be
understood. There are a sufficient number of complexities to hair
toxicology testing that an understanding of hair anatomy and phys-
iology is required for proper interpretation of results.

4 Meconium

Meconium is the earliest stool which is formed by the fetus. For-

mation begins around the 12th week of gestation and continues to
accumulate until it is passed shortly after delivery. The vast majority
of healthy, full-term newborns pass their meconium within the first
48 h after birth [12]. The meconium is composed of intestinal
epithelial cells, mucus, and bile, as well as substances ingested by
the fetus in utero, including amniotic fluid and lanugo.
For some time, meconium was the gold standard specimen for
detection of fetal drug exposure, and it is still widely used at many
institutions [13, 14]. It also remains an important specimen when
other sample types, such as umbilical cord tissue, are not available.
One characteristic which has made meconium a popular specimen
for newborn drug screening is its large window of detection.
Because meconium accumulates during the entire second and
third trimester, it should theoretically contain any drug that the
fetus was exposed to during that time. In practice, detection of
third trimester drug exposure may be more sensitive than isolated
second trimester exposure, due to dilution and drug degradation
over time [15]. Even with the possibility of false negatives early in
the second trimester, meconium testing provides a much larger
window of detection than other specimens, such as urine, which
may only provide evidence of exposure during the days preceding
delivery [13, 14].
The use of meconium for newborn drug screening also presents
some challenges [13, 14, 16, 17]. There are several pre-analytic
issues which can complicate meconium testing. Infants that are
post-term or that experience stress in utero may pass their meco-
nium prior to delivery, which makes it unavailable for collection
after birth. Similarly, infants born prematurely may have delayed
passage of meconium (on average, 7.8 days for very premature
infants) due to ongoing maturation of intestinal function and
resulting intestinal hypomotility [18]. Meconium passage may be
 Alternate Matrices 195

even further delayed in infants receiving morphine therapy. In

practice, this may lead to missed collections due to expired or
forgotten collection orders. Additionally, the risk factors used to
determine if drug screening is indicated may not be immediately
apparent and may not be discovered until after the meconium has
been discarded.
The stability of drugs and drug metabolites is also variable in
meconium, with particularly low stability of 6-MAM (heroin
metabolite) in meconium at a variety of temperatures (including
refrigerated, room temperature, and body temperature) [19]. Low
stability was also observed for 7-aminoclonazepam (major metabo-
lite of the benzodiazepine clonazepam) and chlordiazepoxide [19].
Many infants share a hospital room with their mother and can
have several family members involved in their care. As a result,
meconium samples can be accidentally or intentionally disposed
of by family members. Meconium can also present some analytic
challenges as well. Meconium is a sticky and heterogeneous matrix
which makes it a challenging specimen. As a result, meconium
testing is performed by very few laboratories. Lastly, there are
some challenges in result interpretation. Any medication given to
the newborn, prior to meconium passage, will also be detected.
This is especially problematic with therapeutic opioids which will be
indistinguishable from maternal opioid abuse during pregnancy.

5 Umbilical Cord Tissue

A newer, alternative matrix which can also be used for newborn

drug testing is umbilical cord tissue. Studies have shown a high rate
of concordance between drug detection from meconium and cord
tissue samples. Testing methods for cord tissue were developed in
2006, and since that time, it has been gaining popularity due to
some distinct advantages over meconium testing [20–24]. Similar
to meconium testing, cord tissue has a large window of detection
and should contain drugs that the fetus was exposed to during the
third and possibly second trimesters [14, 21]. Drugs incorporated
into umbilical cord tissue early in pregnancy are subject to degrada-
tion over time, as with drugs in meconium. Unlike meconium, cord
tissue can be collected at the time of delivery. This solves many of
the sample collection issues that complicate meconium testing,
including premature and delayed passage, discarded samples, and
potential sample tampering. One approach taken by hospitals
which have instituted cord tissue testing involves collecting umbili-
cal cord samples from every delivery and storing the samples for
several weeks until clinical decisions regarding newborn drug test-
ing have been made [25]. Cord tissue specimens can be stored for
several weeks or more at refrigerator temperature. Collecting sam-
ples at the time of delivery also avoids detection of medications
196 Kendra L. Palmer and Matthew D. Krasowski

given to the newborn after delivery, which greatly simplifies result

interpretation.
Umbilical cord tissue testing is not without its own limitations.
Cord tissue may not be available for infants who are transferred
from an outside institution after delivery. Similar to other alterna-
tive matrices, umbilical cord tissue is a difficult matrix to test, and as
a result, few laboratories perform testing. It has been found that
cord tissue testing will detect some medications administered to the
mother during delivery. This is most likely due to contamination
from cord blood. There is not a clear understanding of whether
passive exposure to marijuana or other drugs could result in a
positive result. This combined with various factors affecting drug
detection can make result interpretation difficult. Lastly, detailed
studies of analyte stability in umbilical cord tissue, as have been
done in meconium [19], have yet to be reported.

6 Summary

While each of these alternative specimens cannot be used as a

perfect replacement for blood or urine, each alternative matrix has
unique characteristics which can provide advantages when utilized
in the appropriate settings. Oral fluid can be useful where a nonin-
vasive method to detect recent substance use is needed. Hair testing
is an ideal specimen when historical information of drug use is of
interest. Meconium and cord tissue have similar utility in newborn
drug screening where a large window of detection is desired.

References
1. Bosker WM, Huestis MA (2009) Oral fluid (SPME) with GC/MS. Forensic Sci Int 218
testing for drugs of abuse. Clin Chem 55 (1–3):31–36. https://doi.org/10.1016/j.
(11):1910–1931. https://doi.org/10.1373/ forsciint.2011.10.002
clinchem.2008.108670 6. Bar-Oz B, Klein J, Karaskov T, Koren G (2003)
2. Drummer OH (2005) Review: Pharmacoki- Comparison of meconium and neonatal hair
netics of illicit drugs in oral fluid. Forensic Sci analysis for detection of gestational exposure
Int 150(2–3):133–142. https://doi.org/10. to drugs of abuse. Arch Dis Child Fetal Neo-
1016/j.forsciint.2004.11.022 natal Ed 88(2):F98–F100
3. Aps JK, Martens LC (2005) Review: the physi- 7. Cooper GA (2011) Hair testing is taking root.
ology of saliva and transfer of drugs into saliva. Ann Clin Biochem 48(Pt 6):516–530. https://
Forensic Sci Int 150(2–3):119–131. https:// doi.org/10.1258/acb.2011.011112
doi.org/10.1016/j.forsciint.2004.10.026 8. Nakahara Y (1999) Hair analysis for abused
4. Cooper GA, Kronstrand R, Kintz P, Society of and therapeutic drugs. J Chromatogr B
Hair Testing (2012) Society of Hair Testing Biomed Sci Appl 733(1–2):161–180
guidelines for drug testing in hair. Forensic 9. Henderson GL (1993) Mechanisms of drug
Sci Int 218(1–3):20–24. https://doi.org/10. incorporation into hair. Forensic Sci Int 63
1016/j.forsciint.2011.10.024 (1–3):19–29
5. Aleksa K, Walasek P, Fulga N, Kapur B, 10. Stauffer SL, Wood SM, Krasowski MD (2015)
Gareri J, Koren G (2012) Simultaneous detec- Diagnostic yield of hair and urine toxicology
tion of seventeen drugs of abuse and metabo- testing in potential child abuse cases. J Forensic
lites in hair using solid phase micro extraction
 Alternate Matrices 197

Legal Med 33:61–67. https://doi.org/10. temperatures. J Anal Toxicol 41(3):196–204.

1016/j.jflm.2015.04.010 https://doi.org/10.1093/jat/bkw113
11. Wennig R (2000) Potential problems with the 20. Chittamma A, Marin SJ, Williams JA, Clark C,
interpretation of hair analysis results. Forensic McMillin GA (2013) Detection of in utero
Sci Int 107(1–3):5–12 marijuana exposure by GC-MS, ultra-sensitive
12. Clark DA (1977) Times of first void and first ELISA and LC-TOF-MS using umbilical cord
stool in 500 newborns. Pediatrics 60 tissue. J Anal Toxicol 37(7):391–394. https://
(4):457–459 doi.org/10.1093/jat/bkt052
13. Cotten SW (2012) Drug testing in the neo- 21. Concheiro M, Gonzalez-Colmenero E,
nate. Clin Lab Med 32(3):449–466. https:// Lendoiro E, Concheiro-Guisan A, de
doi.org/10.1016/j.cll.2012.06.008 Castro A, Cruz-Landeira A, Lopez-Rivadulla
14. Marin SJ, Keith L, Merrell M, McMillin GA M (2013) Alternative matrices for cocaine, her-
(2009) Comparison of drugs of abuse detec- oin, and methadone in utero drug exposure
tion in meconium by EMIT II and ELISA. J detection. Ther Drug Monit 35(4):502–509.
Anal Toxicol 33(3):148–154 https://doi.org/10.1097/FTD.
0b013e31828a6148
15. McMillin GA, Wood KE, Strathmann FG, Kra-
sowski MD (2015) Patterns of drugs and drug 22. Marin SJ, Metcalf A, Krasowski MD, Linert BS,
metabolites observed in meconium: what do Clark CJ, Strathmann FG, McMillin GA
they mean? Ther Drug Monit 37 (2014) Detection of neonatal drug exposure
(5):568–580. https://doi.org/10.1097/ using umbilical cord tissue and liquid chroma-
FTD.0000000000000181 tography time-of-flight mass spectrometry.
Ther Drug Monit 36(1):119–124. https://
16. Wingert WE, Feldman MS, Kim MH, Noble L, doi.org/10.1097/FTD.0b013e3182a0d18c
Hand I, Yoon JJ (1994) A comparison of
meconium, maternal urine and neonatal urine 23. Montgomery D, Plate C, Alder SC, Jones M,
for detection of maternal drug use during preg- Jones J, Christensen RD (2006) Testing for fetal
nancy. J Forensic Sci 39(1):150–158 exposure to illicit drugs using umbilical cord
tissue vs meconium. J Perinatol 26(1):11–14.
17. Wood KE, Sinclair LL, Rysgaard CD, Strath- https://doi.org/10.1038/sj.jp.7211416
mann FG, McMillin GA, Krasowski MD
(2014) Retrospective analysis of the diagnostic 24. Montgomery DP, Plate CA, Jones M, Jones J,
yield of newborn drug testing. BMC Preg- Rios R, Lambert DK, Schumtz N, Wiedmeier
nancy Childbirth 14:250. https://doi.org/10. SE, Burnett J, Ail S, Brandel D, Maichuck G,
1186/1471-2393-14-250 Durham CA, Henry E, Christensen RD (2008)
Using umbilical cord tissue to detect fetal expo-
18. Bekkali N, Hamers SL, Schipperus MR, sure to illicit drugs: a multicentered study in Utah
Reitsma JB, Valerio PG, Van Toledo L, Ben- and New Jersey. J Perinatol 28(11):750–753.
ninga MA (2008) Duration of meconium pas- https://doi.org/10.1038/jp.2008.97
sage in preterm and term infants. Arch Dis
Child Fetal Neonatal Ed 93(5):F376–F379. 25. Palmer KL, Wood KE, Krasowski MD (2017)
https://doi.org/10.1136/adc.2008.138024 Evaluating a switch from meconium to umbili-
cal cord tissue for newborn drug testing: A
19. Wu F, Marin SJ, McMillin GA (2017) Stability retrospective study at an academic medical cen-
of 21 cocaine, opioid and benzodiazepine drug ter. Clin Biochem 50(6):255–261. https://doi.
analytes in spiked meconium at three org/10.1016/j.clinbiochem.2016.11.026
 Chapter 19

Salt-Assisted Liquid-Liquid Extraction of Meconium

for Analysis of Cocaine and Amphetamines by Liquid
Chromatography-Tandem Mass Spectrometry
Melissa M. Goggin and Gregory C. Janis

Abstract
Meconium, the first stool of a newborn, can be analyzed to identify prenatal exposure to drugs of abuse.
Meconium accumulates in a fetus during the second and third trimesters of pregnancy providing a wide
window of exposure. Identification of in utero drug exposure is essential for the diagnosis and treatment of
infants for dependency/withdrawal caused from the exposure. However, testing of meconium samples is
often cumbersome and time-consuming. Unlike liquid samples, meconium is a viscous, semisolid, tar-like
substance that needs to be individually weighed prior to extraction. Additionally, the meconium matrix is
not homogeneous and not easily mixed or extracted. A method for analyzing cocaine and metabolites as
well as amphetamines in meconium utilizing ceramic homogenizers prior to salt-assisted liquid-liquid
extraction and liquid chromatography tandem-mass spectrometry (LC-MS/MS) is presented.

Key words Meconium, Ceramic homogenizers, SALLE, Cocaine, Amphetamine

1 Introduction

In a recent survey, 4.7% of pregnant women aged 15–44 reported

using illicit drugs during the previous month [1]. In utero drug
exposure can cause several developmental and behavioral issues for
a developing fetus, newborn, and child. In fact, more than 75% of
newborns exposed to illicit drugs have major medical problems
[2]. For example, neonatal abstinence syndrome, respiratory dis-
tress, low birth weight, and sudden infant death syndrome are all
toxidromes associated with prenatal drug exposure. Furthermore, a
mother with an illicit drug addiction may not be able to provide a
safe and nurturing environment for the infant [3, 4]. Older chil-
dren exposed to illicit drugs in utero also exhibit poor social adjust-
ment, learning disabilities, and attention-focusing issues [4].
Therefore, the detection of illicit drug exposure in utero is crucial

Loralie J. Langman and Christine L.H. Snozek (eds.), LC-MS in Drug Analysis: Methods and Protocols,
Methods in Molecular Biology, vol. 1872, https://doi.org/10.1007/978-1-4939-8823-5_19,
© Springer Science+Business Media, LLC, part of Springer Nature 2019

199
200 Melissa M. Goggin and Gregory C. Janis

to identify and treat developmental difficulties, both medical and

social, which the drug-exposed newborn and child may encounter.
Meconium is the first fecal matter passed by a newborn. It is a
heterogeneous material composed of amniotic fluid, bile, lipids,
and other waste materials that accumulate in the developing fetus
starting around 12 weeks of gestation and continues to form until
birth [5]. Thus, meconium analysis can yield information detailing
a nearly 6-month window of possible drug exposure; meconium is
also easily and noninvasively collected from soiled diapers. How-
ever, meconium analysis presents unique challenges; these are
mostly associated with the matrix’s lack of heterogeneity and its
semisolid composition. The sticky, tar-like consistency of meco-
nium necessitates the individual weighing of samples for analysis.
Additionally, meconium requires large volumes of solvent and long
extraction times with high-energy procedures in order to ensure
the sample is completely extracted in contrast to a superficial extrac-
tion of the surface layer of the sample.
Meconium samples may be initially analyzed with an immuno-
assay kit-based technique to quickly screen a large number of
samples; however, these kits often have high false-positive rates
from similar molecules binding the antibodies. Additionally, the
variation of meconium color can interfere with the immunoassay
measurement also causing false-positives in intensely colored speci-
mens. Therefore, samples that screen positive by immunoassay
need to be confirmed via more specific methods, typically gas
chromatography-mass spectrometry (GC-MS)- or liquid
chromatography-mass spectrometry (LC-MS/MS)-based techni-
ques. Yet another challenge of monitoring drugs of abuse in meco-
nium is whether the confirmation methods target the correct
metabolites as the metabolism of a fetus may create different meta-
bolites than what are observed in adult urine. For example, m-
hydroxybenzoylecgonine and p-hydroxymethamphetamine appear
to be important metabolites of cocaine and methamphetamine in
meconium, while they are not typically monitored in urine [6, 7].
Several extraction techniques have been reported in the litera-
ture for meconium drug testing by mass spectrometry-based meth-
ods. For example, ultrasonic-assisted extraction (UAE),
microwave-assisted extraction (MAE), accelerated solvent extrac-
tion (ASE), and solid-phase extraction (SPE) have been utilized
alone or in combination for meconium extraction prior to GC-MS
or LC-MS/MS confirmation analysis [6, 8, 9]. The majority of
extraction methods combine a method of heating or disrupting
the meconium in a solvent to extract the analytes, followed by
SPE to specifically isolate the analytes of interest. UAE and SPE
methods were evaluated at MedTox Laboratories; however,
depending on the solvent utilized for UAE, the extract was dark
in color and not very clean (methanol), or only the surface of the
meconium appeared to be extracted (acetonitrile). While SPE
 Meconium SALLE 201

cleaned the meconium samples significantly better than UAE alone,

it is relatively expensive and time-consuming. Therefore, a method
utilizing salting-out assisted liquid-liquid extraction (SALLE) aided
by ceramic homogenizer shearing of the meconium was developed
and is presented here.
Several salts can be utilized for SALLE to separate water misci-
ble organic solvents from aqueous samples to directly analyze the
extract by LC-MS/MS without the need for evaporation nor
reconstitution [10]. To simultaneously extract cocaine, ampheta-
mines, and their respective metabolites, ammonium acetate and
acetonitrile were utilized as SALLE reagents. Cocaine (COC) and
cocaethylene (CE) were quantified over a range of 1–500 ng/g,
while benzoylecgonine (BE), m-hydroxybenzoylecgonine
(m-OHBE), amphetamine (AMP), methamphetamine (MAMP),
3,4-methylenedioxyamphetamine (MDA), and
3,4-methylenedioxymethamphetamine (MDMA) were quantified
over respective ranges of 5–1000 ng/g. Extraction recoveries
ranged from 19% for the most polar analyte (m-hydroxybenzoylec-
gonine) to 102% for cocaine.

2 Materials

2.1 Solutions and 1. 50:50 methanol/water (v/v): Add 100 mL of methanol and
Standards 100 mL of water to a glass container, cap tightly, and mix. Store
at room temperature for up to 1 year.
2. 1 μg/mL COC-d3 and CE-d3, 3 μg/mL AMP-d8, MAMP-
d14, MDA-d5, and MDMA-d5 internal standard spiking solu-
tion: Combine 100 μL of 1 mg/mL COC-d3 stock and 1 mL
of 0.1 mg/mL CE-d3 stock, plus 300 μL of each 1 mg/mL
stock of AMP-d8, MAMP-d14, MDA-d5, and MDMA-d5 in a
100 mL volumetric flask, and bring to volume with 50:50
methanol/water. Stable up to 1 year stored at 4
C in glass
screw-cap tubes with Teflon septa.
3. 30 μg/mL standard substock and control substock.
(a) Standard substock: Combine 0.3 mL of each 1 mg/mL
stock of AMP, MAMP, MDA, MDMA, BE, and m-OHBE
in a 10 mL volumetric flask. Fill to volume with 50:50
methanol/water. Stable up to 1 year stored at 4
C in glass
screw-cap tubes with Teflon septa.
(b) Control substock: Repeat step 3a with independent pre-
parations of the standard solution stocks.
4. 25 μg/mL AMP, MAMP, MDA, MDMA, BE, and m-OHBE;
12.5 μg/mL COC and CE spiking standard and spiking
control.
202 Melissa M. Goggin and Gregory C. Janis

(a) Spiking standard: Combine 250 μL each of the 1 mg/mL

AMP, MAMP, MDA, MDMA, BE, and m-OHBE stock
standards, plus 125 μL of the 1 mg/mL COC and CE
stock standards in a 10 mL volumetric flask. Fill to volume
with 50:50 methanol/water. Stable up to 1 year stored at
4
C in glass screw-cap tubes with Teflon septa.
(b) Spiking control: Repeat Step 4a with stock standards
from an independent preparation of the standard solution
stocks.
5. 10 μg/mL AMP, MAMP, MDA, MDMA, BE, and m-OHBE;
2.0 μg/mL COC and CE spiking standard: Combine 2 mL of
the 30 μg/mL standard substock and 1.6 mL of the 25 μg/
mL/12.5 μg/mL spiking standard in a 10 mL volumetric flask.
Fill to volume with 50:50 methanol/water. Stable up to 1 year
stored at 4
C in glass screw-cap tubes with Teflon septa.
6. 2.5 μg/mL AMP, MAMP, MDA, MDMA, BE, and m-OHBE;
0.5 μg/mL COC and CE spiking control: Add 0.5 mL of the
30 μg/mL control substock and 0.4 mL of the 25 μg/mL/
12.5 μg/mL spiking control to a 10 mL volumetric flask. Fill to
volume with 50:50 methanol/water. Stable up to 1 year stored
at 4
C in glass screw-cap tubes with Teflon septa.
7. 0.25 μg/mL AMP, MAMP, MDA, MDMA, BE, and m-
OHBE; 0.05 μg/mL COC and CE spiking standard and spik-
ing control.
(a) Spiking standard: Add 0.25 mL of the 10 μg/mL/
2.0 μg/mL spiking standard to a 10 mL volumetric
flask. Fill to volume with 50:50 methanol/water. Stable
up to 1 year stored at 4
C in glass screw-cap tubes with
Teflon septa.
(b) Spiking control: Add 1 mL of the 2.5 μg/mL/0.5 μg/mL
spiking control to a 10 mL volumetric flask. Fill to volume
with 50:50 methanol/water. Stable up to 1 year stored at
4
C in glass screw-cap tubes with Teflon septa.
8. 3M ammonium acetate: Add approximately 150 mL of deio-
nized water to a 500 mL volumetric flask. Weigh 115.6 g of
ammonium acetate, and add to the volumetric flask. Dilute to
volume with water, and mix. Store in amber bottle at room
temperature for up to 3 months.
9. Mobile phase A, 0.1% formic acid: Add approximately 500 mL
of deionized water to a 1 L volumetric flask. Add 1.0 mL of
formic acid to the volumetric flask, dilute to volume with water,
and mix. Stable when stored in a glass bottle at room tempera-
ture for up to 6 months.
10. Mobile phase B: Methanol, HPLC grade.
 Meconium SALLE 203

11. Autosampler wash #1, 0.1% formic acid in methanol: Using a

graduated cylinder, add 1 L of methanol to a 1 L bottle. Add
1.0 mL of formic acid to the bottle, cover, and mix.
12. Autosampler wash #2, 90% water/10% methanol with 0.1%
formic acid: Using a graduated cylinder, add 900 mL of deio-
nized water to a 1 L glass bottle. Add 100 mL of methanol and
1 mL of formic acid to the bottle, cover, and mix.

2.2 Supplies and 1. Negative meconium (purchased or screened in-house from

Equipment donor specimens) for the preparation of calibrators and
controls.
2. Wooden applicators.
3. Ceramic homogenizers (Agilent or similar).
4. 15 mL polypropylene tubes, capped.
5. Multi-tube vortex (VWR VX-2500 or similar).
6. Centrifuge (Beckman Coulter Allegra 25R, or similar).
7. LC-MS vials and caps.
8. Waters HSS T3, 50  2.1 mm, 1.8 μm column.
9. Thermo BetaBasic C18 guard column, 10  2.1 mm, 5 μm.
10. LC-MS/MS system (Waters classic UPLC and Sciex 5500
triple quadrupole).

3 Methods

3.1 Calibrator and 1. Weigh 0.5 g of negative meconium into a labeled 15 mL tube
Control Preparation for each calibrator and control (see Note 1).
2. Spike the tube labeled Standard 1 with 10 μL of the 0.25/
0.05 μg/mL spiking standard (see Note 2). Final concentration
is 5 ng/g AMP, MAMP, BDA, MDMA, BE, m-OHBE, and
1 ng/g COC, CE.
3. Spike the tube labeled Standard 2 with 20 μL of the 0.25/
0.05 μg/mL spiking standard. Final concentration is 10 ng/g
AMP, MAMP, BDA, MDMA, BE, m-OHBE, and 2 ng/g
COC, CE.
4. Spike the tube labeled Standard 3 with 10 μL of the 10/2 μg/
mL spiking standard. Final concentration is 200 ng/g AMP,
MAMP, BDA, MDMA, BE, m-OHBE, and 40 ng/g
COC, CE.
5. Spike the tube labeled Standard 4 with 20 μL of the 10/2 μg/
mL spiking standard. Final concentration is 400 ng/g AMP,
MAMP, BDA, MDMA, BE, m-OHBE, and 80 ng/g
COC, CE.
204 Melissa M. Goggin and Gregory C. Janis

6. Spike the tube labeled Standard 5 with 20 μL of the

25/12.5 μg/mL spiking standard. Final concentration is
1000 ng/g AMP, MAMP, BDA, MDMA, BE, m-OHBE, and
500 ng/g COC, CE.
7. Spike the tube labeled Low QC with 30 μL of the 0.25/
0.05 μg/mL spiking control (see Note 3). Final concentration
is 15 ng/g AMP, MAMP, BDA, MDMA, BE, m-OHBE, and
3 ng/g COC, CE.
8. Spike the tube labeled Medium QC with 20 μL of the 2.5/
0.5 μg/mL spiking control. Final concentration is 100 ng/g
AMP, MAMP, BDA, MDMA, BE, m-OHBE, and 20 ng/g
COC, CE.
9. Spike the tube labeled High QC with 16 μL of the
25/12.5 μg/mL spiking control. Final concentration is
800 ng/g AMP, MAMP, BDA, MDMA, BE, m-OHBE, and
400 ng/g COC, CE.
10. Reserve one tube of meconium labeled as Standard 0 to con-
tain no added analytes; process this sample in an identical
manner to all calibrators and samples.
11. Reserve one tube of meconium labeled as Blank to contain no
added analytes; do not add internal standard to this sample.

3.2 Extraction 1. Weigh 0.5 g of each meconium sample into a labeled 15 mL

tubes (see Note 4).
2. Add 20 μL of the internal standard spiking solution into each
calibrator, control, and sample tube.
3. Add 1.5 mL of 3M ammonium acetate into each tube (see
Note 5).
4. Add 2 ceramic homogenizers to each tube (see Note 6).
5. Cap each tube, and vortex mix samples utilizing multi-tube
vortex for 10 min at medium speed or until meconium samples
appear to be liquefied.
6. Add 1.25 mL of acetonitrile to each tube, and recap (see
Note 7).
7. Vortex mix for 15 min to extract (see Note 8).
8. Centrifuge tubes for 10 minutes at 1100  g.
9. Transfer 200 μL of the supernatant to labeled LC-MS vials (see
Note 9).
10. Dilute each vial with 400 μL of 0.1% formic acid.
11. Cap each vial and vortex mix prior to LC-MS/MS analysis.

3.3 LC-MS/MS 1. Autosampler temperature: 10
C.

Analysis 2. Injection volume: 8 μL.
3. Column temperature: 50
C.
 Meconium SALLE 205

Table 1
Chromatographic gradient parameters

0.1% Formic acid

Time (min) in water (A), % Methanol (B), % Flow (mL/min)
0.00 70.0 30.0 0.500
0.25 70.0 30.0 0.500
0.70 63.0 37.0 0.500
1.00 50.0 50.0 0.500
1.40 50.0 50.0 0.500
1.50 10.0 90.0 0.500
1.70 70.0 30.0 0.500
2.00 70.0 30.0 0.500

4. Gradient is shown in Table 1 (see Note 10).

5. Ion source parameters: Gas temp ¼ 550
C, curtain gas ¼ 35 psi,
ion spray voltage ¼ 5000 V, ion source gases ¼ 55, declustering
potential ¼ 90 V, entrance potential ¼ 10 V, and exit
potential ¼ 12 V.
6. Mass spectrometer parameters: Compound-dependent para-
meters are listed in Table 2 (see Note 11).
7. Figure 1 presents a representative chromatogram of a standard
containing all analytes and internal standards.

4 Notes

1. The meconium may stick to walls of the tube; therefore, it may

be necessary to centrifuge tubes briefly to get the meconium to
the bottom of tube where it is available for solvent extraction.
2. The SALLE may not separate the acetonitrile as efficiently if
too much methanol is added by utilizing larger spiking volumes
of the calibrator stock solutions.
3. Due to the difficulty in preparing a homogeneous mixture of
meconium, calibrators and controls must be prepared individ-
ually by first weighing out the appropriate amount of meco-
nium (0.5 g for this assay) and spiking with standards to the
desired concentration. Low, mid, and high controls are typi-
cally prepared at approximately 3 the lowest calibrator for the
low and at approximately 80% of the highest calibrator for the
high control. The mid control generally approximates the geo-
metric mean or logarithmic mean of the dynamic range of the
assay.
206 Melissa M. Goggin and Gregory C. Janis

Table 2
Monitored transitions

Quantitative product ion Qualitative product ion

Precursor Collision Collision Retention

Analyte ion (m/z) m/z energy (V) m/z energy (V) time (min)
AMP 136.1 119.1 17 91.1 47 0.60
MAMP 150.1 119.1 15 91.2 40 0.60
MAMP_isotopologuea 152.1 121.1 15 93.2 40 0.60
MDA 180.1 105.1 33 133.2 25 0.60
MDMA 194.5 163.1 22 133.1 25 0.60
COC 304.5 182.1 41 150.1 35 1.00
BE 290.1 168.1 28 105.1 40 0.90
m-OHBE b
306.1 168.1 27 121.1 38 0.60
CE 318.5 196.2 41 150.1 32 1.30
AMP-D8 (IS) 144.1 127.1 12 – – –
MAMP-D14 (IS) 164.1 98.1 35 – – –
MDA-D5 (IS) 185.1 138.1 25 – – –
MDMA-D5 (IS) 199.1 165.1 18 – – –
COC-D3 (IS) 307.1 185.1 25 – – –
BE-D8 (IS) 298.1 171.1 27 – – –
CE-D8 (IS) 326.4 204.2 28 – – –
See Note 11
a

AMP-d8 internal standard is utilized for m-OHBE quantitation

b

4. Meconium is a viscous heterogeneous substance; therefore, it is

important to mix well prior to removing a sample aliquot. This
can be done by kneading the meconium with the wooden
applicator.
5. The 3M ammonium acetate is utilized to solvate the meco-
nium, and the high salt concentration allows acetonitrile to be
utilized for liquid-liquid extraction. Ammonium acetate is also
a LC-MS compatible buffer in case of aqueous phase contami-
nation into the organic extract.
6. The ceramic homogenizers are utilized to break up and liquefy
the meconium prior to liquid extraction. Ceramic beads
(2.8 mm, e.g., ChromTech) have also successfully been utilized
for extraction of smaller meconium sample sizes (0.1 g) by our
laboratory. The use of ceramic homogenizers increased
 Meconium SALLE 207

Fig. 1 A representative chromatogram of a standard containing all analytes and

internal standards
208 Melissa M. Goggin and Gregory C. Janis

Fig. 1 (continued)

extraction efficiency of drugs from authentic meconium sam-

ples by an average of ~43% compared to sonication/mixing
with organic solvent alone.
7. For more polar analytes, a small amount of methanol can be
added to the acetonitrile to improve recoveries from the aque-
ous layer. However, if the methanol concentration is greater
than ~5%, the aqueous and organic layers will not separate.
8. Following vortex mixing, two layers should form in the tube.
The bottom layer will consist of the aqueous buffer, which
should contain most of the dark green color from the meco-
nium. The upper layer will be the acetonitrile containing ana-
lytes and will likely have a yellow color of varying intensities
based on the meconium specimen.
9. Following centrifugation, three layers may be visible, the same
as in Note 8, and an additional layer of meconium debris at the
bottom of the tube.
10. Injection volume may vary based on instrument sensitivity.
 Meconium SALLE 209

11. Methamphetamine levels in excess of 10 the upper limit of

quantitation (1000 ng/g) have frequently been observed by
our laboratory in clinical specimens, at which point the quanti-
tative accuracy, peak shape, retention time, and transition ratios
begin to fail. Samples can be re-extracted with dilution to fall
within the linear range; however, meconium samples are often
limited, and results are time-sensitive as a newborn will likely
only be in the hospital for a couple days. An isotopologue
method for the quantitation of methamphetamine and
amphetamine in urine has recently been validated [11], in
which additional transitions are monitored simultaneously for
methamphetamine and transitions shifted 2 amu to monitor
the isotopologues containing two carbon-13 atoms. The rela-
tive abundance of the isotopologues is much lower than the
base compound, thereby decreasing the sensitivity for high
concentration samples. The same isotopologue methodology
applied for meconium methamphetamine has increased the
linear range 100-fold.

References
1. SAMHSA (2016) Results from the 2015 isolation of cocaine/crack biomarkers in meco-
national survey on drug use and health: nium. J Chromatogr B 957:14–23
detailed tables. Rockville, MD: Substance 7. Gray TR, Kelly T, LaGasse LL et al (2009)
Abuse and Mental Health Services Administra- Novel biomarkers of prenatal methamphet-
tion. https://www.samhsa.gov/data/sites/ amine exposure in human meconium. Ther
default/files/NSDUH-DetTabs-2015/ Drug Monit 31:70–75
NSDUH-DetTabs-2015/NSDUH-DetTabs- 8. Bordin D, Alves M, Cabices O et al (2014) A
2015.pdf. Accessed 21 June 2017 rapid assay for the simultaneous determination
2. Huestis MA, Choo RE (2002) Drug abuse’s of nicotine, cocaine and metabolites in meco-
smallest victims: in utero drug exposure. nium using disposable pipette extraction and
Forensic Sci Int 128:20–30 gas chromatography-mass spectrometry. J Anal
3. Płotka J, Narkowicz S, Polkowska Ż, Biziuk M, Toxicol 38:31–38
Namieśnik J (2014) Effects of addictive sub- 9. Cabarcos P, Tabernero J Alvarez I et al (2012)
stances during pregnancy and infancy and their A new method for quantifying prenatal expo-
analysis in biological materials. In: Whitacre D sure to ethanol by microwave-assisted extrac-
(ed) Reviews of environmental contamination tion (MAE) of meconium followed by gas
and toxicology, vol 227. Springer International chromatography—mass spectrometry
Publishing, Basel, Switzerland (GC-MS). Anal Bioanal Chem 404:147–155
4. Narkowicz S, Płotka J, Polkowska Ż, Biziuk M, 10. Valente IM, Gonçalves LM, Rodrigues JA
Namieśnik J (2013) Prenatal exposure to sub- (2013) Another glimpse over the salting—out
stance of abuse: a worldwide problem. Environ assisted liquid—liquid extraction in acetoni-
Int 54:141–163 trile/water mixtures. J Chromatogr A
5. Lozano J, Garcia-Algar O, Vall O, Torre R, 1308:58–62
Scaravelli G, Pichini S (2007) Biological matri- 11. Miller AM, Goggin MM, Nguyen A, Gozum
ces for the evaluation of in utero exposure to SD, Janis GC (2017) Profiting from probabil-
drugs of abuse. Ther Drug Monit 29:711–734 ity; combining low and high probability iso-
6. Mantovani Cde C, Lima MB, Oliveira CD, topes as a tool extending the dynamic range
Menck Rde A, Diniz EM, Yonamine M of an assay measuring AMPHETAMINE and
(2014) Development and practical application methamphetamine in urine. J Anal Toxicol
of accelerated solvent extraction for the 41:355–359
 Chapter 20

Detection of In Utero Cannabis Exposure in Umbilical

Cord Tissue by a Sensitive Liquid Chromatography-Tandem
Mass Spectrometry Method
Fang Wu, Triniti L. Jensen, and Gwendolyn A. McMillin

Abstract
In utero exposure to cannabis may cause various short- and long-term health problems in newborns, such as
low birth weight and neonatal withdrawal syndrome. Drug testing with umbilical cord tissue can be used to
identify in utero exposure to cannabis. Here, we described a liquid chromatography-tandem mass spec-
trometry (LC-MS/MS) method that simultaneously quantifies four cannabinoids in umbilical cord tissue,
including tetrahydrocannabinol (THC), 11-nor-Δ9-carboxy-THC (THC-COOH), cannabinol (CBN),
and 11-hydroxy-THC (11-OH-THC). Umbilical cord specimens are weighed and homogenized, and
cannabinoids are extracted using anion exchange solid-phase extraction columns (AX-SPE). Liquid chro-
matography separation is performed, and quantitative results are obtained using LC-MS/MS.

Key words Cannabis exposure, THC, THC-COOH, 11-OH-THC, CBN, Umbilical cord,
LC-MS/MS

1 Introduction

Cannabis is the most commonly used illicit drug in the United

States [1]. According to the results of a 2015 survey, its use is
widespread among young Americans. Among pregnant women,
4.7% admitted to using cannabis in the past month [2]. Adverse
effects of in utero exposure to cannabis on growth, length of
gestation, intelligence, and cognitive functions have been reported
by a large number of studies [3–8]. Moreover, impulsivity and
impaired executive function have been noted in children exposed
to cannabis during gestation [1, 9]. Drug testing has become a
useful tool for timely detection of in utero exposure to cannabis,
and testing results can be used to guide social management and
treatment.
Urine [1], hair [10], meconium [11, 12], and umbilical cord
[13–15] have been used for detection of drug exposure in

Loralie J. Langman and Christine L.H. Snozek (eds.), LC-MS in Drug Analysis: Methods and Protocols,
Methods in Molecular Biology, vol. 1872, https://doi.org/10.1007/978-1-4939-8823-5_20,
© Springer Science+Business Media, LLC, part of Springer Nature 2019

211
212 Fang Wu et al.

newborns. Urine results typically reflect drug exposure in the pre-

ceding 2–3 days. Neonatal hair can detect cumulative exposure to
various drugs during the third trimester [16]. However, hair test-
ing is not always feasible for newborns due to limited hair volume.
Meconium, the first stool from newborns, is able to detect in utero
drug exposure up to 20 weeks after maternal use. It has become a
well-established specimen to assess in utero exposure to drugs
including cannabinoids [12, 17, 18]. However, meconium is not
available in some cases (10–20%) [11] because of early passing prior
to birth; this is particularly seen in the setting of fetal stress caused
by maternal drug use during gestation. Therefore, alternative spec-
imen types are necessary to determine in utero exposure to cannabis
as well as other licit or illicit drugs. Umbilical cord tissue is a
relatively new specimen type, serving as an alternative to meconium
for in utero drug exposure testing. Advantages of umbilical cord
tissue include the amount available for testing [14] as well as easy
specimen collection for all newborns at birth. If prior suspicion of
drug use is indicated, drug testing can be performed immediately
after birth with umbilical cord tissue, making results available faster
than meconium testing results, in most cases. However, the timing
and drug deposition patterns in umbilical cord tissue are not well
studied [19, 20].
Available literature currently provides little published data on
testing for cannabinoids in umbilical cord tissue [15]. This chapter
outlines an LC-MS/MS method for identification and quantifica-
tion of four cannabinoids, including tetrahydrocannabinol (THC),
11-nor-Δ9-carboxy-THC (THC-COOH), cannabinol (CBN), and
11-hydroxy-THC (11-OH-THC), in umbilical cord tissue. Since
metabolic patterns of cannabinoids in umbilical cord tissue are not
well known, results from this assay could be useful to detect and
characterize in utero cannabis exposure. In this chapter, the authors
describe umbilical cord tissue homogenization, solid-phase extrac-
tion (SPE), liquid chromatographic (LC) separation, as well as mass
spectrometric (MS/MS) detection with the focus on extraction
procedure since extraction of cannabinoids from umbilical cord
tissue is a bottleneck in the procedure. The extraction method
described in this chapter could recover 60–86% of the four canna-
binoids from umbilical cord tissue.

2 Materials

Label all reagents, calibrators, and controls with contents, date

prepared, and expiration date. All organic solvents are HPLC
grade or greater. Water should be clinical laboratory reagent
grade or higher.
 Detection of In Utero Exposure to Cannabis 213

2.1 Instrumentation 1. Class A grade glassware.

and Supplies (See 2. Pipettes.
Note 1)
3. Mini food chopper (e.g., Joint Legend Group, model#
JE3049).
4. Stainless steel beads (5.6 mm or similar size).
5. Tissue homogenizer (e.g., Bead Ruptor).
6. Eppendorf microcentrifuge.
7. VWR Advanced Multi-tube Vortexer.
8. Biotage Evolute® AX-SPE columns (60 mg/3 mL).
9. Positive pressure manifold.
10. LC vials (maximum recovery).
11. Nitrogen evaporator/concentrator.
12. Phenomenex Kinetex Evo C18 column (2.1
50 mm, 2.6 μm
particle size).
13. CTC PAL HTC-xt-DLW autosampler.
14. Agilent 1260 infinity series binary pump, degasser, and
column oven.
15. AB SCIEX Triple Quad™ 5500 mass spectrometer.
16. AB SCIEX Analyst and MultiQuant software.

2.2 Stock Four liquid standards containing 1 mg/mL THC, THC-COOH,

Non-deuterated 11-OH-THC, and CBN, respectively, are used to prepare stock
Standard Solutions 1. A tenfold serial dilution is made from stock 1 to obtain stocks
2 and 3. Aliquot and store all stock solutions frozen (65 to
75  C).
1. Stock 1: 1 μg/mL THC, THC-COOH, 11-OH-THC, and
CBN in methanol. Add 5 mL of methanol to a 10 mL glass
volumetric flask. Add 10 μL of each 1 mg/mL liquid standard
into the flask. Fill flask with methanol to the 10 mL line, and
mix well.
2. Stock 2: 100 ng/mL THC, THC-COOH, 11-OH-THC,
and CBN in methanol. Add 1 mL of stock 1 to a 10 mL
glass volumetric flask. Fill flask with methanol to the 10 mL
line, and mix well.
3. Stock 3: 10 ng/mL THC, THC-COOH, 11-OH-THC, and
CBN in methanol. Add 1 mL of stock 2 to a 10 mL glass
volumetric flask. Fill flask with methanol to the 10 mL line,
and mix well.

2.3 Stock Liquid standards containing 1 mg/mL THC, THC-COOH,

Non-deuterated 11-OH-THC, and CBN and 100 μg/mL THC-COOH glucuro-
Control Solutions nide, respectively, are used to prepare stock 4. A tenfold serial
(See Note 2) dilution is made from stock 4 to obtain stocks 5 and 6. Aliquot
and store all stock solutions in freezer (65 to 75  C).
214 Fang Wu et al.

1. Stock 4: 1 μg/mL THC, 11-OH-THC, and CBN and

1.51 μg/mL THC-COOH-glucuronide in methanol. Add
5 mL of methanol to a 10 mL glass volumetric flask. Add
151 μL of THC-COOH glucuronide and 10 μL of THC, 11-
OH-THC, and CBN 1 mg/mL standard into the flask. Fill
flask with methanol to the 10 mL line, and mix well.
2. Stock 5: 100 ng/mL THC, 11-OH-THC, and CBN and
151 ng/mL THC-COOH-glucuronide in methanol. Add
1 mL of stock 4 to a 10 mL glass volumetric flask. Fill flask
with methanol to the 10 mL line and mix well.
3. Stock 6: 10 ng/mL THC, 11-OH-THC, and CBN and
15.1 ng/mL THC-COOH-glucuronide in methanol. Add
1 mL of stock 5 to a 10 mL glass volumetric flask. Fill flask
with methanol to the 10 mL line and mix well.

2.4 Stock Deuterated 1. Stock 7: 100 ng/mL THC-d3, THC-COOH-d3, 11-OH-

Internal Standard THC-d3, and CBN-d3 in methanol. Add 5 mL of methanol
Solution to a 10 mL glass volumetric flask. Add 10 μL of each
100 μg/mL standard of THC-d3, THC-COOH-d3,
11-OH-THC-d3, and CBN-d3, respectively, to the flask.
Fill the flask with methanol to the 10 mL line, and mix well
before use. Aliquot into 2 mL glass vials and store all stock
solutions in freezer (65 to 75  C).

2.5 Buffers and 1. SPE column wash buffer 1: 1% ammonium hydroxide in

Mobile Phases water. Add 80 mL of water to a 100 mL graduated cylinder.
Add 1 mL of ammonium hydroxide into the graduated cylin-
der. Fill to 100 mL with water. Mix before use.
2. SPE column wash buffer 2: methanol.
3. SPE column elution buffer: 2% acetic acid in methanol. Add
80 mL of methanol to a 100 mL graduated cylinder. Add
2 mL of glacial acetic acid into the graduated cylinder. Fill to
100 mL with methanol. Mix before use. SPE column elution
buffer should be prepared fresh daily and stored at room
temperature.
4. Reconstitution buffer: 60:40 methanol/water. Add 60 mL of
methanol to a 100 mL graduated cylinder. Fill to 100 mL with
water. Mix before use.
5. Hydrolysis buffer: 0.5 M NaOH. Add 100 mL of water to a
1 L graduated cylinder. Weigh 20 g of NaOH and transfer to
the cylinder. Fill to 1 L with water. Mix well before use.
6. HPLC mobile phase A: 5 mM ammonium bicarbonate in
water, pH 9.5. Add 100 mL of water to a 1 L graduated
cylinder. Weigh 0.40 g of ammonium bicarbonate and transfer
to the cylinder. Add water to up to the 900 mL mark. Mix and
adjust pH to 9.5 with ammonium hydroxide. Fill to 1 L with
 Detection of In Utero Exposure to Cannabis 215

water. Mobile phase A is stable at room temperature for at

least 10 days.
7. HPLC mobile phase B: methanol. Mobile phase B is stable at
room temperature for at least 14 days.

2.6 Drug-Free 1. Evaluate residual umbilical cord tissue samples using the pre-
Umbilical Cord Pool sented LC-MS/MS method to ensure absence of detectable
cannabinoids. De-identify residual umbilical cord tissue sam-
ples. Pool approximately 80 residual blank umbilical cord
tissue samples. Homogenize drug-free umbilical cord pool
using a mini food chopper (approximately 1/2 cm cubes).
Fill individual 50 mL polypropylene conical centrifuge tubes
(~30 g per tube). Sixty grams of blank umbilical cord tissue is
able to accommodate approximately 1 week of testing if one
run is performed per day. Store in freezer (65 to 75  C);
blank umbilical cord tissue is stable at these temperatures for
at least 1 year and up to two freeze-thaw cycles.

3 Methods

Figure 1 illustrates the schematic workflow of specimen handling.

3.1 Sample, 1. Cut umbilical cord tissue using a clean razor blade for each
Calibrator, and Control sample. Discard used razor blades in the sharp container.
Weigh Out Razors should never be reused.
2. Weigh 1.0  0.1 g of each patient umbilical cord tissue (see
Note 3) into individual 5 mL conical polypropylene tubes
with screw top caps.

Fig. 1 Schematic workflow for processing and analyzing umbilical cord tissue
216 Fang Wu et al.

3. Label eight 5 mL conical polypropylene tubes with screw top

caps from 1 to 8. Tubes 1–5 for calibrator preparation and
tubes 6–8 for controls.
4. Weigh 1.0  0.1 g of pooled blank cord into tubes 1–8 (see
Note 4).
5. Spike internal standard solution: Add 25 μL of stock deuter-
ated internal standard solution (stock 7) to tubes 1–8 and
patient samples.

3.2 Calibrator and A calibration curve is generated with the following calibrator con-
Control Spiking centrations: 0.2, 0.5, 1, 3, and 5 ng/g in drug-free umbilical cord
tissue. Three controls are included at 0, 0.25, and 4 ng/g (i.e.,
negative, low, and high) (see Note 5).
1. Spike tube 1 with 20 μL of stock 3 to create calibrator
1 (0.2 ng/g).
2. Spike tube 2 with 50 μL of stock 3 to create calibrator
2 (0.5 ng/g).
3. Spike tube 3 with 10 μL of stock 2 to create calibrator
3 (1 ng/g).
4. Spike tube 4 with 30 μL of stock 2 to create calibrator
4 (3 ng/g).
5. Spike tube 5 with 50 μL of stock 2 to create calibrator
5 (5 ng/g).
6. Spike tube 6 with 25 μL of stock 6 to create a low QC
(0.25 ng/g).
7. Spike tube 7 with 40 μL of stock 5 to create a high QC
(4 ng/g).
8. Spike tube 8 with stock 7 (internal standard) only. Tube
8 without non-deuterated standard solution is used as a
negative QC.

3.3 Homogenization 1. Add six stainless steel beads into each tube.
and Base Hydrolysis 2. Add 2.5 mL of methanol into each tube.
3. Store samples in freezer (65 to 75  C) for 10 min.
4. Remove samples from freezer and immediately place them in a
homogenizer.
5. Homogenize samples for 4 min at 5 m/s in 30 s intervals with
30 s rest periods to minimize heat exposure to the tissue.
6. Centrifuge homogenates at 20,598
g (14,000 rpm) and
4  C for 10 min in a microcentrifuge.
7. Transfer supernatants to individual pre-labeled 10 mL culture
tubes.
8. Add 2.5 mL of 0.5 M NaOH into each supernatant.
 Detection of In Utero Exposure to Cannabis 217

9. Incubate samples at ambient temperature (23–25  C) on a

multi-tube vortexer at a shaking frequency of 700 rpm for 45
min while shaking to convert THC-COOH glucuronide to
free THC-COOH.

3.4 SPE Extraction 1. Place SPE columns on a positive pressure manifold.

2. Condition individual SPE columns with 1 mL of methanol
followed by 1 mL of water.
3. Load supernatants onto SPE columns (see Note 6).
4. Wash individual SPE columns with 1 mL of 1% ammonium
hydroxide wash buffer.
5. Wash individual SPE columns with 1 mL of methanol.
6. Dry SPE columns at 5 kPa for 1 min to remove residual
methanol.
7. Elute by gravity into individual LC vials with two 0.6 mL
fractions of 2% acetic acid SPE elution buffer.
8. Dry the eluent at 40  C under a gentle nitrogen stream with a
CEREX concentrator 48 or similar evaporator for 20 min (see
Note 7).
9. Reconstitute each sample in 200 μL of reconstitution buffer.
10. Vortex the vials for 5 min at 1200 rpm before LC-MS/MS
analysis.

3.5 Liquid LC-MS/MS analysis is described for an AB SCIEX Triple Quad™

Chromatography- 5500 mass spectrometer interfaced with CTC PAL HTC-xt-DLW
Tandem Mass autosampler and Agilent 1260 Infinity Series binary pump, degas-
Spectrometry (LC-MS/ ser, and column oven.
MS) Analysis (See 1. Column: Kinetex Evo C18 column (2.1
50 mm, 2.6 μm
Note 8) particle size).
2. LC gradient is described in Table 1. Divert the LC eluent to
waste for the first 0.8 min and the final 0.5 min.
3. LC flow rate: of 0.5 mL/min.
4. Injection volume: 40 μL.
5. MS/MS mode: negative electrospray ionization mode.
6. MS/MS parameters are shown in Table 2 (see Note 9).
7. Collect data using scheduled multiple reaction monitoring
(MRM) mode. Use AB SCIEX Analyst software or comparable
for instrument control. Set two transition ions to monitor each
drug analyte, as shown in Table 3.
8. Create a worklist of specimens to be tested.
9. Equilibrate the LC column at the starting conditions (40:60
water/methanol) for 5–10 min before initiating the run. Hold
the column and autosampler at 28  C and 4  C, respectively.
218 Fang Wu et al.

Table 1
Liquid chromatography separation gradienta

Time (min) %A %B
0 40 60
0.5 40 60
1.2 20 80
2.3 20 80
2.7 10 90
3.2 10 90
3.21 5 95
4 5 95
4.01 40 60
4.7 40 60
a
A, 5 mM ammonium bicarbonate in water, pH 9.5; B, methanol; flow rate used is
0.5 mL/min

Table 2
Mass spectrometry parameters

Mass spectrometry parameters

Instrument mode Negative
Curtain gas (psi) 30
Collision gas (psi) 10
Ion spray voltage 4000

Temperature ( C) 550
Nebulizer gas (psi) 25
Heater gas (psi) 30

10. Verify the acquisition worklist for accuracy.

11. Apply the method to the batch. Figure 2 shows representative
LC separation and extracted ion chromatograms of THC,
THC-COOH, 11-OH-THC, and CBN from an authentic
clinical sample.

3.6 Data Analysis 1. Construct calibration curves via linear least-squares regression
with 1/x weighting factor.
2. Review calibrators (see Note 10). Review calibrators for any
missing internal standards. Review retention times (RTs) for
 Detection of In Utero Exposure to Cannabis 219

Table 3
Drug testing components, multiple reaction monitoring transitions, retention time (RT), and internal
standardsa

Non-deuterated analytes/deuterated MRM transitions for MRM transitions for

internal standards RT (min) non-deuterated analytes deuterated analytes
THC/THC-d3 3.2 313.1 ! 245.1 316.0 ! 248.0
313.1 ! 191.1 316.0 ! 194.0
THC-COOH/THC-COOH-d3 1.4 343.1 ! 245.0 346.0 ! 248.2
343.1 ! 191.0 346.0 ! 194.0
11-OH-THC/11-OH-THC-d3 2.1 329.1 ! 268.1 332.1 ! 271.0
329.1 ! 173.0 332.1 ! 173.0
CBN/CBN-d3 3.0 309.3 ! 222.1 312.0 ! 282.0
309.3 ! 171.0 312.0 ! 171.0
a
The LC-MS/MS cutoff used is 0.2 ng/g for each compound

analytes and internal standards for any flags, and review inte-
gration and chromatogram quality.
3. Review QCs. QC values should be within 20% of the target
concentration (see Note 11).
4. Review patient samples (see Note 12).

4 Notes

1. The presented items are in the order of sample preparation, LC,

and MS. Similar instruments can be used.
2. Stock non-deuterated control solution should be prepared
with a different lot number of standard solutions than those
used for stock standard preparation. Quality control samples
contain glucuronidated THC-COOH. Under basic condi-
tions, THC-COOH glucuronide is converted to free drug,
THC-COOH. Hydrolysis efficiency is monitored by conver-
sion yield. Higher concentration per weight of THC-COOH
glucuronide is added to the stock to account for the weight
difference compared to post hydrolysis-free THC-COOH
(molecular weight is 66.2% of the glucuronidated drug).
3. Collect at least 6 in. of umbilical cord. For sample preparation,
1.0  0.1 g of tissue is needed. Drain and discard any blood,
and rinse the exterior of the cord segment with normal saline or
water. Samples received soaking in fluid, as well as samples that
have been excessively scoured or dried have an increased risk of
false-negative results.
4. Use different lot numbers of blank cord to prepare calibrators
and controls.
220 Fang Wu et al.

Fig. 2 Representative LC separation (a) and extracted ion chromatograms with ion transitions noted for (b) THC
(1.3 ng/g), (c) THC-COOH (17.5 ng/g), (d) 11-OH-THC (0.9 ng/g), and (e) CBN (0.3 ng/g) from an authentic
clinical sample

5. Calibrators and QCs should be performed in the same manner

as the patient samples.
6. In order to achieve high recoveries, do not apply too much
pressure to SPE columns and allow supernatants to pass
through SPE columns at 1 drop/4 s.
7. Dry completely but do not overdry! If there is solvent remain-
ing in the vials after 20 min, dry the sample under recom-
mended conditions for a few more minutes (i.e., 2–5 min) to
completely evaporate the solvent.
8. Complete the required instrument maintenance according to
manufacturer’s instruction prior to injecting the controls and
 Detection of In Utero Exposure to Cannabis 221

test specimens. Calibrators and controls must be injected prior

to patient samples.
9. MS parameters were optimized via direct infusion of a mixture
of standards.
10. The calibrators must have acceptable chromatography, reten-
tion time (RT), area counts, and ion ratios. Acceptable criteria
for RT, ion ratios, and chromatography are listed below. If the
ion ratios, RT, and chromatography are outside of the range,
the specimen must be re-injected, re-extracted, or reported as
“not detected.” Manually integrate any compounds not found
automatically.
(a) RT must be 10% of the target of RT for the specimen to
be considered “present.”
(b) Ion ratios must be 30% of expected values.
(c) Ensure the correct peaks are identified in the chromatog-
raphy and no other suspected peaks closer to the target
RT for each compound.
11. Negative control must demonstrate the presence of all deuter-
ated analytes, meet the acceptable criteria, and be negative for
all other non-deuterated analytes (<LOD).
12. All target analytes must meet the acceptable criteria as
described for calibrators.

Acknowledgments

This work was supported by the ARUP Institute for Clinical and
Experimental Pathology.
Disclosure: The authors have no potential conflicts of interest.

References
1. Huestis MA, Choo RE (2002) Drug abuse’s 4. Jutras-Aswad D, DiNieri JA, Harkany T, Hurd
smallest victims: in utero drug exposure. YL (2009) Neurobiological consequences of
Forensic Sci Int 128(1–2):20–30 maternal cannabis on human fetal development
2. Substance AbuseCenter for Behavioral Health and its neuropsychiatric outcome. Eur Arch Psy-
Statistics and Quality. Results from the 2015 chiatry Clin Neurosci 259(7):395–412. https://
National Survey on Drug Use and Health: doi.org/10.1007/s00406-009-0027-z
Detailed Tables SAMHSA. https://www. 5. Calvigioni D, Hurd YL, Harkany T, Keimpema
samhsa.gov/data/sites/default/files/ E (2014) Neuronal substrates and functional
NSDUH-DetTabs-2015/NSDUH-DetTabs- consequences of prenatal cannabis exposure.
2015/NSDUH-DetTabs-2015pdf. Published Eur Child Adolesc Psychiatry 23(10):931–941.
September 8, 2016. Accessed 18 Jan 2017 https://doi.org/10.1007/s00787-014-0550-y
3. Sithisarn T, Granger DT, Bada HS (2012) 6. Behnke M, Smith VC, Committee on
Consequences of prenatal substance use. Int J Substance A, Committee on F, Newborn
Adolesc Med Health 24(2):105–112. https:// (2013) Prenatal substance abuse: short- and
doi.org/10.1515/ijamh.2012.016 long-term effects on the exposed fetus.
222 Fang Wu et al.

Pediatrics 131(3):e1009–e1024. https://doi. method to address the increased utilization of

org/10.1542/peds.2012-3931 umbilical cord in the assessment of in utero
7. Mato S, Del Olmo E, Pazos A (2003) Ontoge- drug exposure. Clin Biochem 49
netic development of cannabinoid receptor (13–14):1092–1095. https://doi.org/10.
expression and signal transduction functional- 1016/j.clinbiochem.2016.04.007
ity in the human brain. Eur J Neurosci 17 15. Chittamma A, Marin SJ, Williams JA, Clark C,
(9):1747–1754 McMillin GA (2013) Detection of in utero
8. Biegon A, Kerman IA (2001) Autoradio- marijuana exposure by GC-MS, ultra-sensitive
graphic study of pre- and postnatal distribution ELISA and LC-TOF-MS using umbilical cord
of cannabinoid receptors in human brain. Neu- tissue. J Anal Toxicol 37(7):391–394. https://
roImage 14(6):1463–1468. https://doi.org/ doi.org/10.1093/jat/bkt052
10.1006/nimg.2001.0939 16. Lozano J, Garcia-Algar O, Vall O, de la
9. Farst KJ, Valentine JL, Hall RW (2011) Drug Torre R, Scaravelli G, Pichini S (2007)
testing for newborn exposure to illicit sub- Biological matrices for the evaluation of in
stances in pregnancy: pitfalls and pearls. Int J utero exposure to drugs of abuse. Ther Drug
Pediatr 2011:951616. https://doi.org/10. Monit 29(6):711–734. https://doi.org/10.
1155/2011/951616 1097/FTD.0b013e31815c14ce
10. Kintz P, Mangin P (1993) Evidence of gesta- 17. Tynon M, Porto M, Logan BK (2015) Simpli-
tional heroin or nicotine exposure by analysis of fied analysis of 11-hydroxy-delta-9-tetrahydro-
fetal hair. Forensic Sci Int 63(1–3):99–104 cannabinol and 11-carboxy-delta-9-
11. Ostrea EM Jr, Brady M, Gause S, Raymundo tetrahydrocannabinol in human meconium:
AL, Stevens M (1992) Drug screening of method development and validation. J Anal
newborns by meconium analysis: a large-scale, Toxicol 39(1):35–40. https://doi.org/10.
prospective, epidemiologic study. Pediatrics 89 1093/jat/bku107
(1):107–113 18. MA ES, Feng S (1998) delta 9-THC metabo-
12. McMillin GA, Wood KE, Strathmann FG, Kra- lites in meconium: identification of 11-OH-
sowski MD (2015) Patterns of drugs and drug delta 9-THC, 8 beta,11-diOH-delta 9-THC,
metabolites observed in meconium: what do and 11-nor-delta 9-THC-9-COOH as major
they mean? Ther Drug Monit 37 metabolites of delta 9-THC. J Anal Toxicol
(5):568–580. https://doi.org/10.1097/ 22(4):329–335
FTD.0000000000000181 19. Gray T, Huestis M (2007) Bioanalytical proce-
13. Concheiro M, Jones HE, Johnson RE, dures for monitoring in utero drug exposure.
Choo R, Shakleya DM, Huestis MA (2010) Anal Bioanal Chem 388(7):1455–1465.
Umbilical cord monitoring of in utero drug https://doi.org/10.1007/s00216-007-1228-
exposure to buprenorphine and correlation 9
with maternal dose and neonatal outcomes. J 20. Colby JM (2017) Comparison of umbilical
Anal Toxicol 34(8):498–505 cord tissue and meconium for the confirmation
14. Haglock-Adler CJ, McMillin GA, Strathmann of in utero drug exposure. Clin Biochem 50
FG (2016) Development of a liquid (13–14):784–790. https://doi.org/10.1016/
chromatography-tandem mass spectrometry j.clinbiochem.2017.03.006
 Chapter 21

Quantitation of Ethyl-β-D-Glucuronide in Human Umbilical

Cord Tissue by Liquid Chromatography Tandem Mass
Spectrometry (LC-MS/MS)
Simuli L. Wabuyele and Gwendolyn A. McMillin

Abstract
In utero exposure to alcohol may adversely affect the development of the embryo or fetus and result in
adverse outcomes known as fetal alcohol spectrum disorders (FASD) which encompass a range of physical,
behavioral, and cognitive impairments in the newborn. Since maternal self-reports are often unreliable,
biomarkers of gestational alcohol consumption are necessary for accurate identification of exposed
newborns at risk. Ethyl-β-D-glucuronide (EtG) is a minor phase II metabolite of ethyl alcohol (ethanol),
formed by enzymatic conjugation of ethanol with glucuronic acid in the liver. As a direct biomarker for
alcohol, detection of EtG in neonatal biological matrices provides accurate identification of maternal
alcohol consumption and fetal alcohol exposure during pregnancy. This chapter describes the quantitation
of EtG in human umbilical cord tissue by liquid chromatography tandem mass spectrometry (LC-MS/MS).

Key words Ethyl glucuronide (EtG), LC-MS/MS, Prenatal alcohol exposure, Umbilical cord, New-
born drug testing

1 Introduction

Alcohol is widely consumed in the United States, and it’s the

second most commonly abused legal psychoactive substance in
pregnancy after nicotine [1]. According to the 2015 Nation Survey
on Drug Use and Health estimates, among pregnant women aged
15–44, 9.3% reported alcohol consumption in the past month,
4.6% reported binge drinking (i.e.,
4 drinks on the same occasion
on at least 1 day in the past 30 days), and 0.8% reported heavy
drinking (i.e., binge drinking on the same occasion on each of 5 or
more days in the past 30 days) [2]. Alcohol consumption during
pregnancy may severely affect the development of the embryo or
fetus and result in adverse outcomes known as fetal alcohol
spectrum disorders (FASD), which encompass a range of physical,
neurobehavioral, and cognitive impairments in the exposed

Loralie J. Langman and Christine L.H. Snozek (eds.), LC-MS in Drug Analysis: Methods and Protocols,
Methods in Molecular Biology, vol. 1872, https://doi.org/10.1007/978-1-4939-8823-5_21,
© Springer Science+Business Media, LLC, part of Springer Nature 2019

223
224 Simuli L. Wabuyele and Gwendolyn A. McMillin

newborn [3]. FASD and the associated secondary disabilities may

have lifelong implications which may result in substantial health
care (medical), economic, and social costs [4]. Early identification
of in utero exposure may aid in early diagnosis of FASD and is
necessary for proper and timely follow-up of exposed newborn
[5, 6]. Given that maternal-self reporting of alcohol use is often
unreliable due to do underreporting, a reliable biomarker of alco-
hol abuse is necessary for accurate detection of in utero alcohol
exposure [6].
Upon ingestion, most of the ethanol (about 90–98%) under-
goes oxidative metabolism in the liver and <1% undergoes
non-oxidative metabolism, whereas a small amount (2–5%) is
excreted unchanged in urine, breath, and sweat (Fig. 1)
[7]. Ethyl-β-D-glucuronide (EtG) is a minor phase II metabolite
of ethyl alcohol (ethanol) formed by enzymatic conjugation of
ethanol with glucuronic acid by UDP-glucuronosyltransferase in
the liver [8, 9]; therefore, EtG is a direct biomarker for alcohol
exposure [10]. Other direct ethanol biomarkers include ethyl sul-
fate (EtS), fatty acid ethyl esters (FAEE), and phosphatidylethanol
(PEth) (Fig. 1) [10].
EtG has been determined to have high sensitivity and specificity
for detection of acute and chronic ethanol exposure in maternal
matrices [11, 12]; however, newborn specimens provide a direct

Fig. 1 Alcohol metabolism (oxidative and non-oxidative pathways) and excretion

 Detection of In Utero Alcohol Exposure 225

measure of fetal alcohol exposure. EtG has been detected in meco-

nium [13, 14], placenta and fetal tissue [15], and umbilical cord
tissue [16] for identification of in utero alcohol exposure
[17]. Umbilical cord specimen testing is a useful alternative to
meconium due to several distinct advantages. Meconium can be
unavailable for testing if it is passed in utero or passage is delayed
and may require multiple collections over several days. However,
umbilical cord is universally available in sufficient amounts immedi-
ately after birth, so that it can be easily and noninvasively collected
in a single step for testing [16, 18, 19].
Currently, the number of analytical methods available for
detection of EtG in umbilical cord specimens is limited. To our
knowledge only one method has been reported in the peer-
reviewed literature [16]. In this chapter, the quantitation of EtG
in human umbilical cord tissue by liquid chromatography
(LC) coupled to tandem mass spectrometry (MS/MS) for assess-
ment of in utero alcohol exposure is described. Umbilical cord
tissue specimens were sliced and weighed. Deuterated internal
standard was added, and samples were homogenized and then
centrifuged. The supernatants were subject to anion exchange
solid-phase extraction (SPE) for cleanup prior to analysis and detec-
tion by LC-MS/MS.

2 Materials

Label all reagents, calibrators, and controls with contents, lot num-
ber, date prepared, expiration dates, and initials of preparer. Use LC
grade or LC-MS grade reagents and follow guidelines/regulations
for proper handling, storage, and disposal of hazardous waste. Note
that similar equipment and supplies to those listed here can be used
as available.

2.1 Supplies 1. Stir sticks, wooden, 5½00 .

2. 50 mL conical sterile polypropylene centrifuge tubes with caps.
3. 7.0 mL polypropylene screw cap tubes with caps (Omni Inter-
national™, Kennesaw, GA).
4. Large plastic antistatic weigh boats.
5. Vials, clear, silanized, Snap-it Cap, 12  32 mm, 1.5 mL.
6. Caps, Snap w/pre-slit, blue PTFE/white silicone.
7. Single edge razor blades.
8. 16  100 mm, 14 mL culture tubes, borosilicate glass.
9. Anion exchange solid-phase extraction (SPE) cartridges,
500-mg bed in a 6 mL reservoir (quaternary amine with chlo-
ride counter ion sorbent).
226 Simuli L. Wabuyele and Gwendolyn A. McMillin

10. Synergi™ Hydro RP, C18 column, 50 mm  3.0 mm, 2.5 μm.
11. Ultra HPLC In-Line Filter, 0.5 μm depth filter  0.00400 ID.
12. 5.6 mm stainless steel UFO beads for Biotage® Bead Ruptor.
13. Mini food chopper/processor, 1.5-cup capacity.
14. Standard glassware, pipette tips, and other supplies.

2.2 Solutions 1. Elution solvent: 2% formic acid in methanol. Add 2 mL of 98%

formic acid to 100 mL of methanol. Elution solvent must be
freshly prepared prior to its use in each batch run.
2. Mobile phase A: 0.1% formic acid in water. Add 1 mL of 98%
formic acid to 1 L of ultrapure water. Stable for 10 days at room
temperature.
3. Mobile phase B: 0.1% formic acid in acetonitrile. Add 1 mL of
98% formic acid to 1 L of acetonitrile. Stable for 14 days at
room temperature.
4. Autosampler wash solvent 1: 0.1% formic acid in 90:10 water/
acetonitrile. Add 1 mL of 98% formic acid and 100 mL of
acetonitrile to 900 mL of ultrapure water. Stable for 14 days
at room temperature (see Note 1).
5. Autosampler wash solvent 2: 60:20:20 isopropanol/acetoni-
trile/methanol. Mix 600 mL of isopropanol (2-propanol),
200 mL acetonitrile, and 200 mL of methanol in a 1 L bottle.
Stable for 14 days at room temperature (see Note 1).

2.3 Samples (see 1. Patient specimen: Collect at least 6 in. of umbilical cord for
Note 2) testing. Excess cord blood should be drained and exterior of
the cord segment rinsed with saline solution or water and
patted dry.
2. Pooled drug-free (blank) umbilical cord: De-identify residual
umbilical cord tissue specimens and screen for the presence of
EtG using the LC-MS/MS method described here. Pool about
80–100 drug-free human umbilical cord samples. Using the
mini food chopper/processor, cut the umbilical cords into
approximately 0.5 cm cubes. Aliquot the pooled umbilical
cord tissue into 50 mL polypropylene tubes (~30 g per tube).
Sixty grams of blank cord is able to accommodate approxi-
mately 1 week of testing if one run is performed per day. Pooled
drug-free (blank) umbilical cord is stable at freezer (65 to
80  C) temperatures for at least 1 year and up to two freeze-
thaw cycles.

2.4 Standards and 1. Standard stock solution: 50 μg/mL EtG in methanol. Add
Calibrators 500 μL of 1.0 mg/mL EtG to 5 mL of methanol in a 10 mL
Class A volumetric flask. Fill to volume with methanol and
mix well.
 Detection of In Utero Alcohol Exposure 227

Table 1
Concentration of calibrators (Cal 1–6) and quality controls (QCs)

Calibrator final conc. (ng/g) QC final conc. (ng/g)

Cal 1 ¼ 5.00 Negative QC ¼ 0.00
Cal 2 ¼ 10.0 Low QC ¼ 6.00
Cal 3 ¼ 25.0 Mid QC ¼ 30.0
Cal 4 ¼ 50.0 High QC ¼ 165
Cal 5 ¼ 100
Cal 6 ¼ 220

2. Calibrator working solution 6: 4.40 μg/mL EtG. Add 880 μL

of standard stock solution to 5 mL of methanol in a 10 mL
Class A volumetric flask then fill to volume with methanol and
mix well (see Note 3 and Table 1).
3. Calibrator working solution 5: 2.00 μg/mL EtG. Add 2.5 mL
of calibrator working solution 6 to 3.0 mL of methanol and
mix well.
4. Calibrator working solution 4: 1.00 μg/mL EtG. Add 2.5 mL
of calibrator working solution 5 to a 5 mL Class A volumetric
flask. Fill to volume with methanol and mix well.
5. Calibrator working solution 3: 0.500 μg/mL EtG. Add
2.5 mL of calibrator working solution 4 to a 5 mL Class A
volumetric flask. Fill to volume with methanol and mix well.
6. Calibrator working solution 2: 0.200 μg/mL EtG. Add
2.0 mL of calibrator working solution 3 to a 5 mL Class A
volumetric flask. Fill to volume with methanol and mix well.
7. Calibrator working solution 1: 0.100 μg/mL EtG. Add
2.5 mL of calibrator working solution 2 to a 5 mL Class A
volumetric flask. Fill to volume with methanol and mix well.

2.5 Quality Controls 1. QC standard stock solution: 50 μg/mL EtG in methanol. Add
(QCs) and Internal 500 μL of 1.0 mg/mL EtG (different lot number) to 5 mL of
Standard methanol in a 10 mL Class A volumetric flask. Fill to volume
with methanol and mix well (see Note 4).
2. QC working solution 4: 3.30 μg/mL EtG. Add 660 μL of QC
standard stock solution to 5 mL of methanol in a 10 mL Class
A volumetric flask. Fill to volume with methanol and mix well
(see Note 5 and Table 1).
3. QC working solution 3: 0.600 μg/mL EtG. Add 1.0 mL of
QC working solution 4 to 4.5 mL of methanol and mix well.
228 Simuli L. Wabuyele and Gwendolyn A. McMillin

4. QC working solution 2: 0.120 μg/mL EtG. Add 1.0 mL of

QC working solution 3 to a 5 mL Class A volumetric flask. Fill
to volume with methanol and mix well.
5. QC working solution 1: 0.000 μg/mL EtG (blank).
Methanol only.
6. Internal standard stock solution: 50 μg/mL EtG-d5 in metha-
nol. Add 500 μL of 1.0 mg/mL Ethyl-β-D-glucuronide-d5 to
5 mL of methanol in a 10 mL Class A volumetric flask. Fill to
volume with methanol and mix well (see Note 6).
7. Internal standard working solution: 0.500 μg/mL EtG-d5 in
methanol. Add 100 μL of internal standard stock solution to
5 mL of methanol in a 10 mL Class A volumetric flask. Fill to
volume with methanol and mix well.

2.6 Equipment and 1. Weighing balance.

Instrumentation 2. Biotage® Bead Ruptor 24.
3. 48-place positive pressure manifold (see Note 7).
4. TurboVap® LV Concentration Evaporator.
5. AB SCIEX Triple Quad™5500 mass spectrometer interfaced
with CTC PAL HTC-xt-DLW autosampler and Agilent 1260
Infinity series binary pump, degasser, and column oven, oper-
ated in negative electrospray ionization mode (see Note 8).
6. AB SCIEX Analyst® and MultiQuant™ software.

3 Methods

3.1 Sample 1. Label a series of 7.0 mL polypropylene screw cap tubes, a series
Preparation of 16  100 mm, 14 mL culture tubes, and a series of auto-
sampler vials with matching identifiers for calibrators, controls,
and patient specimens (i.e., tubes 1–6 for calibrators, tubes
7–10 for the controls, and the other tubes for patient samples).
2. Using a clean razor blade, cut approximately 0.5 cm cubes of
patient cord specimen (see Note 9).
3. Accurately weigh 1  0.025 g of each patient cord specimen
(0.975–1.025 g). Transfer to the appropriate pre-labeled
7.0 mL polypropylene screw cap tubes. Aliquot each patient
cord specimen using a new, clean wooden stir stick for each
specimen.
4. Accurately weigh 1  0.025 g of drug-free umbilical cord
(0.975–1.025 g) to pre-labeled 7.0 mL polypropylene screw
cap tubes 1–10 for calibrators and controls (see Note 10).
5. Add 50 μL of the internal standard working solution to each
tube (see Note 11). It is acceptable to use a repeating pipette.
 Detection of In Utero Alcohol Exposure 229

6. Spike the calibrators.

(a) Add 50 μL of the EtG calibrator working solution 1 to
tube (calibrator) 1.
(b) Add 50 μL of the EtG calibrator working solution 2 to
tube (calibrator) 2.
(c) Add 50 μL of the EtG calibrator working solution 3 to
tube (calibrator) 3.
(d) Add 50 μL of the EtG calibrator working solution 4 to
tube (calibrator) 4.
(e) Add 50 μL of the EtG calibrator working solution 5 to
tube (calibrator) 5.
(f) Add 50 μL of the EtG calibrator working solution 6 to
tube (calibrator) 6.
7. Spike the controls.
(a) Add 50 μL of the EtG QC working solution 1 to tube
7 (negative control) (see Note 12).
(b) Add 50 μL of the EtG QC working solution 2 to tube
8 (low positive control).
(c) Add 50 μL of the EtG QC working solution 3 to tube 9
(mid-positive control).
(d) Add 50 μL of the EtG QC working solution 4 to tube
10 (high positive control).
8. Add 6 large UFO (5.6 mm) stainless steel beads to each tube.
9. Cap the tubes securely and place them in the Bead Ruptor 24.
10. Homogenize at 5.80 m/s for 4 cycles of 30 s, with 30 s pause
in between cycles.
11. Remove samples from the Bead Ruptor 24, and verify that all
specimens have a uniform appearance and no large pieces of
cord are apparent. Samples will feel warm to the touch. Cool
briefly before proceeding.
12. Remove the 6 large UFO (5.6 mm) stainless steel beads from
each sample.
13. Centrifuge samples for 10 min at 0  C and 20,598  g (14,000
rpm). Remove samples from centrifuge carefully without dis-
turbing the pellet.

3.2 Solid-Phase 1. Place the SPE columns on a positive pressure manifold in the
Extraction (SPE) proper sequence in which they are labeled. SPE is by gravity
flow only. Do not apply any pressure to the manifold.
2. Condition the SPE columns with 4 mL of methanol using
gravity flow.
230 Simuli L. Wabuyele and Gwendolyn A. McMillin

3. Equilibrate the SPE columns with 4 mL of ultrapure

de-ionized water using gravity flow.
4. Load sample supernatants in proper sequence onto the SPE
columns using gravity flow.
5. Wash columns (into a waste collection bin) with 3 mL of
ultrapure de-ionized water using gravity flow.
6. Wash columns (into a waste collection bin) with 3 mL of
methanol using gravity flow.
7. Dry the columns at 30–35 psi for 5 min on the positive pressure
manifold.
8. Move SPE columns from the waste position to a collection tube
rack with the pre-labeled 14 mL glass culture tubes.
9. Elute by gravity with 2  1.5 mL of freshly prepared elution
solvent, into corresponding 14 mL glass culture tubes.
10. Place 14 mL glass culture tubes in the concentration evapora-
tor at 40  C and 20 psi for approximately 12 min. Evaporate
the samples to dryness but do not dry for an extended period
of time.
11. Add 1.0 mL of 0.1% formic acid in water (mobile phase A) to
the dried extracts to reconstitute. Vortex mix the tubes briefly.
12. Transfer reconstituted samples from the glass culture tubes
into corresponding pre-labeled autosampler vials, and cap
each vial.
13. Dispose of waste and excess elution solvent into the waste
container.
14. Load the vials on the autosampler for instrumental analysis.

3.3 Instrumental and 1. Flow rate: 0.350 mL/min.

Data Analysis 2. Column temperature ( C): 25  0.5.
3. LC gradient: see Table 2.
4. Autosampler settings: see Table 3.
5. Diverter valve settings: see Table 4.
6. Ion source settings: see Table 5.
7. Multiple reaction monitoring (MRM) transitions and MS para-
meters: see Table 6. The representative mass chromatograms for
a calibrator and a patient sample are shown in Fig. 2.
8. Perform data analysis and quantitation with AB Sciex Multi-
Quant™ software according to the manufacturer’s recommen-
dations (see Note 13).
9. Construct calibration curve (see Note 13) using the six
(non-zero) calibrators. Plot the averaged peak area ratios of
EtG to internal standard (IS) against the nominal EtG
 Detection of In Utero Alcohol Exposure 231

Table 2
LC gradient conditions

Total time (min) A (%) B (%)

0.00 100 0
0.20 99 1
3.00 99 1
3.03 20 80
3.05 2 98
4.05 2 98
4.06 100 0
5.00 100 0

Table 3
Autosampler parameters

Parameter Value
Loop volume 1 (μL) 20
Loop volume 2 (μL) 20
Syringe type 100 μL DLW
Syringe volume (μL) 20
Injection volume (μL) 10.0
Cycle name Analyst LC-Inj DLW Fast_Rev05
Airgap volume (μL) 3
Front volume (μL) 5
Rear volume (μL) 5
Filling speed (μL/s) 5
Pullup delay (ms) 3
Inject to LC Vlv1
Injection speed (μL/s) 5
Pre inject delay (ms) 500
Post inject delay (ms) 500
Needle gap valve clean (mm) 3
Valve clean time solvent 2 (s) 3
Valve clean time solvent 1 (s) 3
Post clean time solvent 1 (s) 2
232 Simuli L. Wabuyele and Gwendolyn A. McMillin

Table 4
Diverter valve parameters

Step Diverter total time (min) Column switching valve positiona

1 0.00 A
2 2.00 B
3 3.20 A
a
valve in position A ¼ sample goes out to waste, valve in position B ¼ samples go to MS

Table 5
MS source parameters

Parameter Value
CUR (Curtain gas, psi) 35
CAD (Collisions gas, psi) 10

TEM (Temperature, C) 600
GS1 (Nebulizer gas, psi) 50
GS2 (Heater gas, psi) 50
IS (IonSpray voltage, V) 3500
Declustering potential (V) 90
Dwell time (ms) 80
Resolution Unit mass (Q1 and Q3)

Table 6
MRM transitions and MS compound-dependent parameters

Entrance Collision Exit potential

Compound Q1 Q3 potential (V) energy (V) (V)
EtG 1 221.2 85.1 10 22 5
EtG 2 221.2 75.1 15 20 10
EtG-d5 1 226.1 85.1 7 22 5
EtG-d5 2 226.1 75.0 13 20 9

concentrations in the calibrators, using linear regression with

1/x2 weighting.
10. Review calibration and QC data (see Note 14). Evaluate cali-
brator and QC peak integration, chromatography, and pres-
ence of internal standard (IS). Review the retention times and
 Detection of In Utero Alcohol Exposure 233

Fig. 2 The representative mass chromatograms for EtG and internal standard in (a) calibrator 1 (5 ng/g) and (b)
internal standard (25 ng/g); (c) positive patient specimen (EtG ¼ 62.9 ng/g) and (d) internal standard (25 ng/g)

ion mass ratios for EtG and IS for any flags. Verify the linear
calibration range for the method (see Note 15).
11. Review patient data. For the patient data to be reported, all
qualitative and quantitative criteria for the analyte and internal
standard established must be met.

4 Notes

1. Potential carryover is dependent on the autosampler used;

therefore, several autosampler wash solvents should be
re-evaluated if a different autosampler is used to ensure there
is no carryover specific to the LC-MS/MS system.
2. Caution should be used when collecting and preparing umbili-
cal cord specimen for testing, to ensure that alcohol containing
products are not used directly on specimen or nearby during
sample processing. Post collection synthesis of EtG may cause
false-positive results [16].
3. Calibrators are prepared by spiking an aliquot of drug-free
human umbilical cord with the calibrator working solution
prepared in methanol.
234 Simuli L. Wabuyele and Gwendolyn A. McMillin

4. Quality control (QC) standards should be prepared from a

second source (i.e., vendor/supplier or different lot number)
than the calibrator standards.
5. Controls (negative, low, mid, and high QCs) are prepared by
spiking an aliquot of drug-free human umbilical cord with the
QC working solutions prepared in methanol.
6. Internal standard is necessary to correct for any variability
throughout the analytical process; therefore, the internal stan-
dard selected should have similar properties as the analyte of
interest. Ideally an isotopically labeled internal standard should
be used as described here.
7. This method was validated using positive pressure for SPE
extraction. The use of other (i.e., vacuum) manifolds was not
evaluated in this work.
8. LC-MS/MS parameters will vary from one instrument or lab-
oratory to another. The parameters described in this method
can be used as starting conditions, but LC and MS parameters
for the analyte and internal standard should be optimized for
the specific instrument utilized.
9. Use a clean razor blade and weigh boat for each test specimen.
Razors should never be reused. Discard razor blades in the
sharps container and any biohazard waste appropriately.
10. Calibrators and controls should have the same matrix as the
patient specimens and should be prepared in the same manner
as the patient samples.
11. Every sample should be analyzed with the internal standard.
12. Negative control is a blank control that does not contain the
analyte of interest, only drug-free umbilical cord tissue and the
internal standard.
13. The MQ4 integration algorithm is used for peak integration.
Integration parameters will need to be determined by the user
based on the quality of the raw data to provide the most
consistent and accurate peak integration. Linear regression
with 1/x2 weighting (where x is the nominal concentration
of a cord calibrator) is performed by the software, which estab-
lishes a linear regression calibration equation, from which the
concentrations of the positive controls and the patient speci-
mens are quantitatively determined and reported.
14. The calibrator and controls must have acceptable chromatog-
raphy (i.e., peak shapes, peak areas) retention times (RT), and
ion mass ratio (IMR) for the target analyte and internal stan-
dard. The concentration of the analyte in the six (non-zero)
calibrators must be within 20% of the target concentration
(accuracy). The correlation coefficient (r) for the calibration
curve generated must be
0.995. The negative control must
 Detection of In Utero Alcohol Exposure 235

demonstrate the presence of the internal standard and should

be negative for the (non-deuterated) analyte and meet all qual-
itative criteria. The positive (low, mid, and high) quality con-
trols (QCs) should contain the reportable compound and the
internal standard at the correct concentrations (i.e., should be
within 20% of the target concentration), and all must meet
the established qualitative criteria for acceptance.
15. The limit of detection (LOD) for EtG in cord is 1 ng/g, the
lower limit of quantitation (LLOQ) is 3.00 ng/g, and the
upper limit of quantitation (ULOQ) is 220 ng/g. The linear
calibration range of the method is 5.00–220 ng/g.

Acknowledgments

This work was supported by the ARUP Institute for Clinical and
Experimental Pathology.
Disclosure: The authors have no potential conflict of interest.

References
1. Forray A (2016) Substance use during preg- 7. Dinis-Oliveira RJ (2016) Oxidative and
nancy. F1000Res 5:F1000 Faculty non-oxidative metabolomics of ethanol. Curr
Rev–F1000 Faculty1887 Drug Metab 17:327–335
2. Center for Behavioral Health Statistics and 8. Walsham NE, Sherwood RA (2012) Ethyl glu-
Quality (2016). 2015 National Survey on drug curonide. Ann Clin Biochem 49:110–117
use and health: detailed tables. Substance Abuse 9. Foti RS, Fisher MB (2005) Assessment of
and Mental Health Services Administration, UDP-glucuronosyltransferase catalyzed forma-
Rockville, MD. https://www.samhsa.gov/ tion of ethyl glucuronide in human liver micro-
data/sites/default/files/NSDUH-DetTabs- somes and recombinant UGTs. Forensic Sci Int
2015/NSDUH-DetTabs-2015/NSDUH- 153:109–116
DetTabs-2015.pdf. Accessed July, 13 10. Bager H, Christensen LP, Husby S et al (2017)
3. Wilhelm CJ, Guizzetti M (2015) Fetal alcohol Biomarkers for the detection of prenatal alco-
Spectrum disorders: an overview from the glia hol exposure: a review. Alcohol Clin Exp Res
perspective. Front Integr Neurosci 9:65 41:251–261
4. Svetlana P, Shannon L, Larry B, et al (2016) 11. Dahl H, Hammarberg A, Franck J et al (2011)
Burden and social cost of fetal alcohol spec- Urinary ethyl glucuronide and ethyl
trum disorders. In: Oxford Handbooks Online sulfate testing for recent drinking in alcohol-
(ed). Oxford, Oxford University Press. http:// dependent outpatients treated with
www.oxfordhandbooks.com/view/10.1093/ acamprosate or placebo. Alcohol Alcohol
oxfordhb/9780199935291.001.0001/ 46:553–557
oxfordhb-9780199935291-e-78. Accessed 12. Morini L, Marchei E, Tarani L et al (2013)
15 Jul 2017 Testing ethylglucuronide in maternal hair and
5. Paley B, O’Connor MJ (2009) Intervention nails for the assessment of fetal exposure to
for individuals with fetal alcohol spectrum dis- alcohol: comparison with meconium testing.
orders: treatment approaches and case manage- Ther Drug Monit 35:402–407
ment. Dev Disabil Res Rev 15:258–267 13. Pichini S, Morini L, Pacifici R et al (2014)
6. Lange S, Shield K, Koren G et al (2014) A Development of a new immunoassay for the
comparison of the prevalence of prenatal alco- detection of ethyl glucuronide (EtG) in meco-
hol exposure obtained via maternal self-reports nium: validation with authentic specimens ana-
versus meconium testing: a systematic litera- lyzed using LC-MS/MS. Preliminary results.
ture review and meta-analysis. BMC Pregnancy Clin Chem Lab Med 52:1179–1185
Childbirth 14:127–127
236 Simuli L. Wabuyele and Gwendolyn A. McMillin

14. Himes SK, Dukes KA, Tripp T et al (2015) 17. Joya X, Friguls B, Ortigosa S et al (2012)
Clinical sensitivity and specificity of meconium Determination of maternal-fetal biomarkers of
fatty acid ethyl ester, ethyl glucuronide, and prenatal exposure to ethanol: a review. J Pharm
ethyl sulfate for detecting maternal drinking Biomed Anal 69:209–222
during pregnancy. Clin Chem 61:523–532 18. Montgomery D, Plate C, Alder SC et al (2006)
15. Morini L, Falcon M, Pichini S et al (2011) Testing for fetal exposure to illicit drugs using
Ethyl-glucuronide and ethyl-sulfate in placen- umbilical cord tissue vs meconium. J Perinatol
tal and fetal tissues by liquid chromatography 26:11–14
coupled with tandem mass spectrometry. Anal 19. Montgomery DP, Plate CA, Jones M et al
Biochem 418:30–36 (2008) Using umbilical cord tissue to detect
16. Jones J, Jones M, Plate C, Lewis D (2012) The fetal exposure to illicit drugs: a multicentered
detection of 1-palmitoyl-2-oleoyl-sn-glycero- study in Utah and New Jersey. J Perinatol
3-phosphoethanol and ethyl glucuronide in 28:750–753
human umbilical cord. Am J Analyt Chem
3:800–810
 Chapter 22

Analysis of Drugs in Oral Fluid Using LC-MS/MS

Cynthia A. Coulter and Christine M. Moore

Abstract
Oral fluid analysis for drugs is increasingly used in a variety of testing areas: pain management and
medication monitoring, parole and probation situations, driving under the influence of drugs (DUID),
therapeutic drug monitoring, and testing for drugs in the workplace. The sample collection itself is
straightforward, rapid, observable, and noninvasive, requiring no special facilities (compared to urine) or
medical personnel (compared to blood). The pH of saliva is slightly acidic relative to blood; therefore, drugs
which are more basic tend to be present in higher concentration in oral fluid than in blood: cocaine,
amphetamines, oxycodone, tramadol, buprenorphine, methadone, and fentanyl. Conversely, acidic drugs
and drugs which are strongly protein bound have lower concentrations in oral fluid than in blood: examples
include benzodiazepines, barbiturates, and carisoprodol. Because of the low volume of specimen available
for analysis and the drug concentrations present (generally much lower than those in urine), efficient
extraction methods and sensitive confirmation procedures are necessary for routine analysis of drugs in oral
fluid. In this chapter, solid-phase extraction methods are described for a variety of drugs with liquid
chromatography—tandem mass spectrometry detection.

Key words Oral fluid, Drugs, Solid-phase extraction, Hydrolysis, LC-MS/MS

1 Introduction

Oral fluid (saliva) consists of gingival crevicular fluid combined with

secretions from the parotid gland (20–25%), the submandibular
gland (70–75%), and other minor salivary glands. It is almost
entirely water, but enzymes, cholesterol, proteins, and electrolytes
are also present. A healthy individual will produce approximately
0.75–1 L per day with a pH value in the range 6.0–7.8. Advantages
of using oral fluid as a test matrix include difficulty of adulteration
(collection can be observed), speed and ease of collection, closer
association with blood concentrations than urine, and potential for
detection of recent intake [1–3]. While saliva can be collected neat
via expectoration (i.e., “spitting”), generally a collection device
consisting of a pad which is then transferred into a transportation
buffer is used. The use of a pad improves the speed of recovery and

Loralie J. Langman and Christine L.H. Snozek (eds.), LC-MS in Drug Analysis: Methods and Protocols,
Methods in Molecular Biology, vol. 1872, https://doi.org/10.1007/978-1-4939-8823-5_22,
© Springer Science+Business Media, LLC, part of Springer Nature 2019

237
238 Cynthia A. Coulter and Christine M. Moore

indicates when adequate sample has been collected (in most

devices) and the buffer assists with drug stability in transportation
and storage; the analytical result can be affected by the mode of
sample collection [4, 5]. In addition, collection devices have differ-
ing volumes of diluent (transportation buffer), so compensating for
the dilution factor before reporting a quantitative result is also a
necessary component of method development and validation.
In contrast to methods used for the analysis of drugs in urine,
oral fluid procedures should include the parent drug (and metabo-
lites), because the compound in highest concentration in saliva
following drug use tends to be the parent drug.
Drug concentrations in oral fluid are also markedly lower than
those detected in urine, so analytical procedures with increased
sensitivity are required. In order to develop laboratory procedures
using liquid chromatography—tandem mass spectrometry
(LC-MS/MS) for drugs in oral fluid, knowledge of the compo-
nents of any transportation buffer in the collected sample is also
essential. Buffers containing non-ionic detergents (e.g., Tween®)
should not be directly injected into LC-MS/MS instruments. In
order to extend the life of chromatographic columns and compo-
nents of the instrument as well as achieve the analytical sensitivity
required for relevant detection of drugs in oral fluid, the analytes
should be efficiently extracted from the matrix prior to injection
and analysis. Methods of sample preparation for large drug panels
include supported liquid extraction [6], liquid-liquid extraction
[7], and solid-phase extraction [8].
In the following procedures, solid-phase extraction methods
are described for a variety of drugs, with LC-MS/MS detection.

2 Materials

All solutions should be prepared with ultrapure water and high

reagent grade salts. All solvents should be of high purity for extrac-
tion, reconstitution, and mobile phase preparation. Drug standards
and deuterated compounds to be used as internal standards should
be of high quality and commercially available (see Note 1). The
volume of each solution can be scaled up as needed depending on
the size of batch to be analyzed.

2.1 Supplies 1. Agilent 1200 Series LC pump, 6430 or 6410 LC-MS/MS, or

similar, operating in electrospray ionization mode.
2. Agilent MassHunter software.
3. Eclipse Plus C18 4.6 mm
50 mm 1.8 μm column or Eclipse
Plus C18 2.1 mm
50 mm 1.8 μm column.
4. Quantisal® oral fluid collection device (see Note 2).
 Drugs in Oral Fluid 239

5. CEREX Clin II extraction columns, 3 mL, 35 mg.

6. Trace-N extraction columns, 3 mL, 15 mg.
7. Positive pressure extraction manifold.
8. Sample concentrator operated under ultrahigh-purity
nitrogen.
9. Heating block capable of temperatures up to 70  C.
10. Vortexer.
11. Borosilicate glassware (see Note 3).
12. Amber borosilicate glass screw top vials.
13. Vacuum oven.
14. Borosilicate glass crimp top high recovery 30 μL reservoir
autosampler vials.
15. Synthetic oral fluid, Immunalysis.
16. Borosilicate glass 900 transfer pipettes.
17. Snap cap 11 mm plastic autosampler vials and caps.

2.2 Prepared 1. Conditioning buffer: 0.1 M sodium phosphate pH 6.0. Add

Reagents for Basic 13.6 g of sodium phosphate to 1 L of deionized (DI) water and
Drugs adjust pH 6.0 by adding ammonium hydroxide. Solution can
be stored up to 6 months.
2. Wash solution: 0.1 M hydrochloric acid. Add 8.4 mL of hydro-
chloric acid to 1 L of DI water. Solution can be stored up to
6 months.
3. Extraction eluant: 78:20:2 methylene chloride/methanol/
ammonium hydroxide. Mix 7.8 mL of methylene chloride,
2 mL of methanol, and 200 μL of ammonium hydroxide.
Prepare fresh daily (see Note 4).
4. Mobile phase A: 20 mM ammonium formate: Add 1.26 g of
ammonium formate to 1 L of HPLC grade water. No adjust-
ment is necessary for the pH. Solution is stored up to
6 months.
5. Mobile phase B: HPLC grade methanol.
6. Reconstitution solution: 50:50 methanol/water. Mix 5 mL of
methanol and 5 mL of water. Prepare fresh daily.
7. Benzoylecgonine (BZE) intermediate solution: 1000 ng/mL
BZE. Add 5 mL of methanol to a 10 mL volumetric flask. Add
10 μL of 1 mg/mL BZE stock solution and bring to volume
with methanol. Mix well. Solution is stable for 6 months when
stored at 20  C.
8. 100 ng/mL BZE methanol spiking solution. Add 5 mL of
methanol to a 10 mL volumetric flask. Add 1 mL of the
1000 ng/mL BZE intermediate solution. Bring to volume
240 Cynthia A. Coulter and Christine M. Moore

with methanol. Mix well. Solution is stable for 6 months when

stored at 20  C.
9. Cocaine (COC) and cocaethylene (CE) intermediate solution:
1000 ng/mL COC and CE. Add 5 mL of acetonitrile to a
10 mL volumetric flask. Add 10 μL of each of the 1 mg/mL
COC and CE stock solutions, and bring to volume with aceto-
nitrile. Solution is stable for 6 months when stored at 20  C.
10. 100 ng/mL COC and CE combined acetonitrile spiking solu-
tion: Add 5 mL of acetonitrile to a 10 mL volumetric flask. Add
1 mL of the 1000 ng/mL COC and CE intermediate solution.
Bring up to volume with acetonitrile. Mix well. Solution is
stable for 6 months when stored at 20  C.
11. 100 ng/mL BZE-d3 methanol internal standard spiking solu-
tion: Add 5 mL of methanol to a 10 mL volumetric flask. Add
10 μL of the 100 μg/mL BZE-d3 stock solution, and bring to
volume with methanol. Mix well. Solution is stable for
6 months when stored at 20  C.
12. 100 ng/mL COC-d3 and CE-d8 acetonitrile internal standard
spiking solution: Add 5 mL of acetonitrile to a 10 mL volu-
metric flask. Add 10 μL each of the 100 μg/mL COC-d3 and
CE-d8 stock solutions, and bring to volume with acetonitrile.
Mix well. Solution is stable for 6 months when stored at
20  C.
13. Quality controls are purchased from an outside vendor and
should be within the linear range of the curve.

2.3 Prepared 1. 8 N sodium hydroxide (NaOH): Add 16 g of NaOH to 50 mL

Reagents of DI water. Mix to dissolve. Solution is stable for 6 months
for Acidic/Neutral when stored at room temperature.
Drugs 2. Sample preparation solution: 0.1 M acetic acid pH 4.0. Add
5.7 mL of glacial acetic acid to 1 L of DI water, and adjust to
pH 4.0 by adding drops of 8 N NaOH. Solution is stable for
6 months when stored at room temperature.
3. Conditioning solution: 0.1 M acetic acid. Add 5.7 mL of glacial
acetic acid to 1 L of DI water. Mix well. Solution is stable for
6 months when stored at room temperature.
4. Step 1 wash solution: 80:20 DI water/glacial acetic acid. Add
8 mL of DI water to a beaker. Add 2 mL of glacial acetic acid,
stirring to mix. Prepare fresh daily.
5. Step 2 wash solution: 40:60 DI water/methanol. Add 4 mL of
DI water and 6 mL of methanol to a beaker and mix. Prepare
fresh daily.
6. Extraction eluant: 98:2 hexane/glacial acidic acid. Add 9.8 mL
of hexane to a beaker. Add 200 μL of glacial acetic acid, stirring
to mix. Prepare fresh daily.
 Drugs in Oral Fluid 241

7. Mobile phase A: 5 mM ammonium formate. Add 0.315 g of

ammonium formate to 1 L of HPLC grade water. Mix to
dissolve. Solution is stored up to 6 months.
8. Mobile phase B: 0.5% formic acid in acetonitrile. Add 5 mL of
formic acid to 1 L of acetonitrile. Mix well. Solution is stored
up to 6 months.
9. Reconstitution solution: 50:50 5 mM ammonium for-
mate:0.5% formic acid in acetonitrile. Add 5 mL of 5 mM
ammonium formate to 5 mL of 0.5% formic acid. Prepare
fresh daily.
10. Delta 9-tetrahydrocannabinol (THC), cannabinol (CBN), and
cannabidiol (CBD) intermediate solution: 1000 ng/mL THC,
CBN, and CBD. Add 5 mL of methanol to a 10 mL volumetric
flask. Add 10 μL of each of the 1 mg/mL THC, CBN, and
CBD stock solutions, and bring to volume with methanol. Mix
well. Solution is stable for 6 months when stored at 20  C.
11. 100 ng/mL THC, CBN, and CBD combined spiking solu-
tion: Add 5 mL of methanol to a 10 mL volumetric flask. Add
1 mL of the 1000 ng/mL THC, CBN, and CBD intermediate
solution. Bring to volume with methanol. Mix well. Solution is
stable for 6 months when stored at 20  C.
12. 10 ng/mL THC, CBN, and CBD methanol spiking solution:
Add 5 mL of methanol to a 10 mL volumetric flask. Add 1 mL
of the 100 ng/mL THC, CBN, and CBD spiking solution.
Bring to volume with methanol. Mix well. Solution is stable for
6 months when stored at 20  C.
13. 100 ng/mL THC-d3 methanol internal standard spiking solu-
tion: Add 5 mL of methanol to a 10 mL volumetric flask. Add
10 μL of the 100 μg/mLTHC-d3 stock solution and bring to
volume with methanol. Mix well. Solution is stable for
6 months when stored at 20  C.
14. Quality controls are purchased from an outside vendor and
should be within the linear range of the curve.

2.4 Prepared 1. 8 N NaOH: Add 16 g of NaOH to 50 mL of DI water. Mix to

Reagents dissolve. Solution is stored at room temperature up to
for Derivatization 6 months.
of Cannabinoids 2. Base hydrolysis solution: 1 N NaOH. Dilute 1 mL of 8 N
NaOH in 7 mL of DI water. Solution is prepared fresh daily.
3. Conditioning solution: 0.1 M acetic acid. Add 5.7 mL of glacial
acetic acid to 1 L of DI water. Solution is stored at room
temperature up to 6 months.
4. Step 1 wash solution: 80:20 DI water/glacial acetic acid. Add
8 mL of DI water to a beaker. Add 2 mL of glacial acetic acid,
stirring to mix. Prepare fresh daily.
242 Cynthia A. Coulter and Christine M. Moore

5. Step 2 wash solution: 40:60 DI water/methanol. Add 4 mL of

DI water and 6 mL of methanol to a beaker and mix. Prepare
fresh daily.
6. Extraction eluant: 98:2 hexane/glacial acidic acid. Add 9.8 mL
of hexane to a beaker. Add 200 μL of glacial acetic acid, stirring
to mix. Prepare fresh daily.
7. Derivatizing reagent catalyst: 10 mM triphenylphosphine
(TPP). Add 0.2 g of TPP to 10 mL of acetonitrile. Mix to
dissolve. Solution is stored at 20  C up to 1 month in an
amber borosilicate glass screw top vial.
8. Derivatizing reagent 1: 10 mM 2,20 -dithiodipyridine (DPDS).
Add 0.2 g of DPDS to 10 mL of acetonitrile. Mix to dissolve.
Solution is stored at 20  C up to 1 month in an amber
borosilicate glass screw top vial.
9. Derivatizing reagent 2: 10 μg 2-picolylamine (2-PA). Add
10 μL of 2-PA to 10 mL of acetonitrile and mix. Solution is
stored at 20  C up to 1 month in an amber borosilicate glass
screw top vial.
10. Reconstitution solution: 50:50 HPLC water/acetonitrile. Add
5 mL of HPLC grade water and 5 mL of acetonitrile to a
beaker. Mix well. Prepare fresh daily.
11. Mobile phase A: 5 mM ammonium formate. Add 0.315 g of
ammonium formate to 1 L of HPLC grade water. Solution is
stored up to 6 months.
12. Mobile phase B: 0.5% formic acid in acetonitrile. Add 5 mL of
formic acid to 1 L of acetonitrile. Solution is stored up to
6 months.
13. THC, CBN, and CBD intermediate solution: 1000 ng/mL
THC, CBN, and CBD. Add 5 mL of methanol to a 10 mL
volumetric flask. Add 10 μL of each of the 1 mg/mL THC,
CBN, and CBD stock solutions, and bring to volume with
methanol. Mix well. Solution is stable for 6 months when
stored at 20  C.
14. 100 ng/mL THC, CBN, and CBD combined spiking solu-
tion: Add 5 mL of methanol to a 10 mL volumetric flask. Add
1 mL of the 1000 ng/mL THC, CBN, and CBD intermediate
solution. Bring to volume with methanol. Mix well. Solution is
stable for 6 months when stored at 20  C.
15. 10 ng/mL THC, CBN, and CBD methanol spiking solution:
Add 5 mL of methanol to a 10 mL volumetric flask. Add 1 mL
of the 100 ng/mL THC, CBN, and CBD spiking solution.
Bring to volume with methanol. Mix well. Solution is stable for
6 months when stored at 20  C.
 Drugs in Oral Fluid 243

16. 100 ng/mL THC-d3 methanol internal standard spiking solu-

tion: Add 5 mL of methanol to a 10 mL volumetric flask. Add
10 μL of the 100 μg/mLTHC-d3 stock solution and bring to
volume with methanol. Mix well. Solution is stable for
6 months when stored at 20  C.
17. 100 ng/mL 11-nor-9-carboxy-delta-9-THC (THC-COOH)
intermediate solution: Add 5 mL of methanol to a 10 mL
volumetric flask. Add 10 μL of the 100 μg/mL THC-COOH
stock solution and bring to volume with methanol. Mix well.
Solution is stable for 6 months when stored at 20  C.
18. 2.5 ng/mL THC-COOH spiking solution: Add 5 mL of
methanol to a 10 mL volumetric flask. Add 25 μL of the
100 ng/mL THC-COOH intermediate solution. Bring to
volume with methanol. Mix well. Solution is stable for
6 months when stored at 20  C.
19. 0.25 ng/mL THC-COOH spiking solution: Add 5 mL of
methanol to a 10 mL volumetric flask. Add 1 mL of the
2.5 ng/mL THC-COOH spiking solution. Bring to volume
with methanol. Mix well. Solution is stable for 6 months when
stored at 20  C.
20. 100 ng/mL THC-COOH-d9 intermediate solution: Add
5 mL of methanol to a 10 mL volumetric flask. Add 10 μL of
the 100 μg/mL THC-COOH-d9 stock solution. Bring to
volume with methanol. Mix well. Solution is stable for
6 months when stored at 20  C.
21. 2.5 ng/mL THC-COOH-d9 internal standard spiking solu-
tion: Add 5 mL of methanol to a 10 mL volumetric flask. Add
25 μL of the 100 ng/mL THC-COOH-d9 intermediate solu-
tion. Bring to volume with methanol. Mix well. Solution is
stable for 6 months when stored at 20  C.
22. Quality controls are purchased from an outside vendor and
should be within the linear range of the curve.

2.5 Prepared 1. Conditioning buffer: 0.1 M sodium phosphate pH 6.0. Add

Reagents for Chiral 13.6 g of sodium phosphate to 1 L of DI water. Mix to dissolve.
Separation While stirring, adjust to pH 6.0 with ammonium hydroxide.
of Amphetamine and Solution is stored up to 6 months.
Methamphetamine 2. Wash solution: 0.1 M hydrochloric acid. Add 8.4 mL of con-
centrated hydrochloric acid to 1 L of DI water. Solution is
stored up to 6 months.
3. Extraction eluant: 98:2 methanol/ammonium hydroxide. Add
9.8 mL of methanol to a beaker. Add 200 μL of ammonium
hydroxide and mix well. Eluant is prepared fresh daily.
244 Cynthia A. Coulter and Christine M. Moore

4. Derivatizing reagent (0.3% Marfey’s reagent): Add 0.001 g of

Marfey’s reagent to 10 mL of acetone. Solution is stable for
3 months.
5. Saturated sodium bicarbonate solution: Add sodium bicarbon-
ate to 10 mL DI water to the point of saturation. Solution is
stable for 3 months.
6. Sodium bicarbonate working solution: Add 1 mL of saturated
sodium bicarbonate solution to 9 mL of water, and vortex to
mix. Make fresh daily.
7. 1 N hydrochloric acid: Dilute 1 mL of hydrochloric acid in
11 mL of DI water. Solution is stable for 3 months.
8. Mobile phase A: 20 mM ammonium formate. Add 1.26 g of
ammonium formate to 1 L of HPLC grade water. No adjust-
ment is necessary for the pH. Solution is stored up to
6 months.
9. Amphetamine (AMP) and methamphetamine (METH) inter-
mediate solution: 2500 ng/mL of each l- and d-isomer. Add
5 mL of methanol to a 10 mL volumetric flask. Add 25 μL of
each 1 mg/mL stock solution of l- and d-AMP and l- and
d-METH. Bring to volume with methanol. Mix well. Solution
is stable for 6 months when stored at 20  C.
10. 250 ng/mL AMP and METH spiking solution: Add 5 mL of
methanol to a 10 mL volumetric flask. Add 1 mL of the
2500 ng/mL AMP and METH intermediate solution. Bring
to volume with methanol. Mix well. Solution is stable for
6 months when stored at 20  C.
11. 250 ng/mL AMP-d5 and METH-d5 internal standard spiking
solution: Add 5 mL of methanol to a 10 mL volumetric flask.
Add 25 μL of each 100 μg/mL AMP-d5 and METH-d5 stock
solution. Bring to volume with methanol. Mix well. Solution is
stable for 6 months when stored at 20  C.
12. Quality controls are purchased from an outside vendor and
should be within the linear range of the curve.

3 Methods

All procedures may be carried out at room temperature in labora-

tory conditions with personnel wearing suitable protective cloth-
ing. Calibrators, controls, and samples are prepared in borosilicate
glass test tubes on the day of extraction, in a 1:3 mixture of
synthetic oral fluid and Quantisal® collection buffer. The volume
of Quantisal® collected sample used in analysis is 1 mL, equivalent
to 0.25 mL of neat oral fluid.
 Drugs in Oral Fluid 245

3.1 Extraction Basic drugs (e.g., amphetamines, cocaine, etc.) diffuse into saliva
and Analysis of Basic from the blood relatively easily. A procedure for the extraction of
Drugs cocaine and its metabolites from oral fluid using a dilution factor of
four is described below. The general method can be applied to
other basic drug classes for extraction.

3.1.1 Preparation 1. Label borosilicate glass test tubes for Calibrators 1–5, each
of Calibrators, Controls, quality control, and specimens.
and Samples 2. Mix 2 mL of synthetic oral fluid with 6 mL of Quantisal®
extraction buffer (or other buffers depending on collection
device).
3. Pipette 1 mL of this mixture into a borosilicate glass test tube
for each calibrator and control (unless using external QC) to
be used.
4. Pipette 1 mL of each sample to be tested into its own borosili-
cate glass test tube.
5. Add 40 μL of each 100 ng/mL internal standard (BZE-d3,
COC-d3, and CE-d8) to each calibrator, control, and
specimen.
6. Do not spike any drug standards into the tube labeled Calibra-
tor 1. This is the negative standard.
7. Spike 10 μL of each 100 ng/mL drug standard (BZE and
COC/CE) into the tube labeled Calibrator 2, to give a final
concentration of 4 ng/mL.
8. Spike 20 μL of each 100 ng/mL drug standard (BZE and
COC/CE) into the tube labeled Calibrator 3, to give a final
concentration of 8 ng/mL.
9. Spike 40 μL of each 100 ng/mL drug standard (BZE and
COC/CE) into the tube labeled Calibrator 4, to give a final
concentration of 16 ng/mL.
10. Spike 80 μL of each 100 ng/mL drug standard (BZE and
COC/CE) into the tube labeled Calibrator 5, to give a final
concentration of 32 ng/mL (see Note 5).
11. Add 1 mL of 0.1 M potassium phosphate pH 6.0 to each tube
as a buffer, and mix well (see Note 6).

3.1.2 Extraction 1. Use one 3 mL, 35 mg CEREX Clin II extraction column per
calibrator, control, and sample. Place each column in the posi-
tive pressure manifold with the pressure setting no higher than
10 psi.
2. Condition the columns by adding 2 mL of methanol to each.
Allow the methanol to flow through the column bed at a rate
no greater than 1 mL/min or one drop at a time. Do not allow
246 Cynthia A. Coulter and Christine M. Moore

the column bed to become dry once the conditioning has

begun (see Note 7).
3. Add 2 mL of 0.1 M sodium phosphate buffer pH 6.0 to each
column. Allow the pH 6.0 buffer to flow through the column
bed at the same rate as the methanol. Do not allow the column
to become dry before loading the sample.
4. Pour each calibrator, control, or sample onto the appropriate
column. Allow the sample to load on the column bed without
the use of positive pressure.
5. Once the samples have completely loaded, wash the columns
with 2 mL of DI water allowing the water to flow at a rate of
1 mL/min. Positive pressure with nitrogen can be used up to a
pressure of 10 psi (see Note 8).
6. Add 2 mL of 0.1 M hydrochloric acid wash solution, allowing
to flow at the same rate as DI water (see Note 9).
7. Dry the columns under nitrogen using positive pressure at
30 psi for 1 min.
8. Remove from pressure and add 1 mL of methanol allowing to
flow at 1 mL/min.
9. Add 1 mL of ethyl acetate, allowing to flow at 1 mL/min.
10. Dry the columns under nitrogen using positive pressure at
30 psi for 7 min.
11. Place clean appropriately labeled borosilicate glass tubes in
extraction collection rack. Dry the tips of the extraction col-
umns and place above the appropriate collection tube.
12. Add 2 mL of extraction eluant to elute basic drugs from
calibrators, controls, and samples.
13. Place tubes in the sample concentrator, and evaporate the
collected eluant under nitrogen (see Notes 10 and 11).
14. Remove dry extraction tubes and add 50 μL reconstitution
solution. Vortex well.
15. Using disposable borosilicate glass transfer pipettes, transfer
each sample to labeled autosampler vials and cap.

3.1.3 Analysis 1. Autosampler temperature: 7  C.

2. Injection volume: 5 μL.
3. Column temperature: 60  C.
4. Binary pump flow rate: 0.2 mL/min (see Note 12).
5. Gradient:
0 min: 80% mobile phase B (MP-B)
4 min: 30% MP-B
5–8 min: hold at 80% MP-B (re-equilibration).
 Drugs in Oral Fluid 247

Table 1
MRM transitions for BZE, COC, CE, and their deuterated analogs

Compound Precursor ion (m/z) Product ion (m/z) Fragmentor voltage (V) Collision energy (V)
BZE-d3 293.3 171.2 120 20
BZE 290.3 168.1 120 15
BZE 290.3 105.1 120 15
COC-d3 307.3 185.3 120 20
COC 304.3 182.3 120 20
COC 304.3 82.3 120 25
CE-d8 326.3 204.4 160 20
CE 318.3 196.4 120 25
CE 318.3 82.2 120 25

6. Total run time: 8 min (see Note 13).

7. MS/MS mode: positive (+) ESI multiple reaction monitoring
(MRM) mode.
8. Nebulizer gas temperature: 350  C.
9. Nebulizer gas flow: 10 L/min.
10. Nebulizer pressure: 40 psi.
11. Positive capillary voltage: 3500 V.
12. The MRM transitions for BZE, COC, and CE are shown in
Table 1 (see Note 14).

3.2 Extraction Acidic/neutral drugs (e.g., barbiturates, THC) do not accumu-

and Analysis late well into oral fluid, but they are easily extracted and analyzed.
of Acid/Neutral Drugs Cannabis is the most widely used illicit drug; detection of the
psychoactive component, THC, in oral fluid is useful for the
identification of recent drug use. A procedure for the extraction
of THC, CBN, and CBD from oral fluid using a dilution factor of
four is described below. The general method can be applied to
other acid/neutral drug classes for extraction; modification of the
LC-MS/MS transitions would be necessary for other drug
classes.

3.2.1 Preparation 1. Label borosilicate glass test tubes for Calibrators 1–5, each
of Calibrators, Controls, quality control, and specimens.
and Samples 2. Mix 2 mL of synthetic oral fluid with 6 mL of Quantisal®
extraction buffer (or other buffers depending on collection
device).
248 Cynthia A. Coulter and Christine M. Moore

3. Pipette 1 mL of this mixture into a borosilicate glass test tube

for each calibrator and control (unless using external QC) to
be used.
4. Pipette 1 mL of each sample to be tested into its own borosili-
cate glass test tube.
5. Add 25 μL of 100 ng/mL internal standard (THC-d3) to each
calibrator, control, and specimen.
6. Do not spike any drug standards into the tube labeled Calibra-
tor 1. This is the negative standard.
7. Spike 25 μL of the 10 ng/mL drug standard (THC, CBN, and
CBD) into the tube labeled Calibrator 2, to give a final con-
centration of 1 ng/mL.
8. Spike 50 μL of the 10 ng/mL drug standard (THC, CBN, and
CBD) into the tube labeled Calibrator 3, to give a final con-
centration of 2 ng/mL.
9. Spike 10 μL of the 100 ng/mL drug standard (THC, CBN,
and CBD) into the tube labeled Calibrator 4, to give a final
concentration of 4 ng/mL.
10. Spike 20 μL of the 100 ng/mL drug standard (THC, CBN,
and CBD) into the tube labeled Calibrator 5, to give a final
concentration of 8 ng/mL (see Note 5).
11. Add 1 mL of 0.1 M acetic acid pH 4.0 to each tube to buffer,
and mix well.

3.2.2 Extraction 1. Use one 3 mL, 15 mg Trace-N extraction column per calibra-
tor, control, and sample. Place each column in the positive
pressure manifold with the pressure setting no higher than
10 psi.
2. Condition the columns by adding 500 μL of methanol to each
one. Allow the methanol to flow through the column bed at a
rate no greater than 1 mL/min or one drop at a time. Do not
allow the column bed to become dry once the conditioning has
begun (see Note 7).
3. Add 100 μL of 0.1 M acetic acid to each column. Allow the
0.1 M acetic acid to flow through the column bed at the same
rate as the methanol. Do not allow the column to become dry
before loading the sample.
4. Pour each calibrator, control, and sample onto the appropriate
column. Allow the sample to load on the column bed without
the use of positive pressure.
5. Once the samples have completely loaded, wash the columns
with 1 mL of Step 1 wash solution. Positive pressure with
nitrogen can be used up to a pressure of 10 psi.
 Drugs in Oral Fluid 249

6. Add 1 mL of Step 2 wash solution (see Note 15).

7. Dry the columns under nitrogen using positive pressure at
30 psi for 7 min.
8. Place clean appropriately labeled borosilicate glass tubes in
extraction collection rack. Dry tips of extraction columns and
place above the appropriate collection tube.
9. Add 1 mL of extraction eluant to elute acidic and neutral drugs
from calibrators, controls, and samples.
10. Place tubes in the sample concentrator and evaporate the col-
lected eluant under nitrogen (see Note 10).
11. Remove dry extraction tubes and reconstitute each tube with
50 μL of reconstitution solution. Vortex well.
12. Using disposable borosilicate glass transfer pipettes, transfer
each sample to labeled plastic autosampler vials and cap.

3.2.3 Analysis 1. Autosampler: 7  C.

2. Injection volume: 10 μL.
3. Column temperature: 60  C.
4. Binary pump flow rate: 0.2 mL/min.
5. Gradient: (see Note 16).
0–2 min: 70% mobile phase B (MP-B)
4–6 min: 90% MP-B
6–8 min: 70% MP-B (re-equilibration)
6. Total run time: 6 min.
7. MS/MS mode: positive (+) ESI multiple reaction monitoring
(MRM) mode.
8. Nebulizer gas temperature: 300  C.
9. Nebulizer gas flow: 10 L/min.
10. Nebulizer pressure: 35 psi.
11. Positive capillary voltage: 3500 V.
12. The MRM transitions for THC, CBN, and CBD are shown in
Table 2 (see Note 14).

3.3 The Use Derivatization is unusual in LC-MS/MS procedures, but the

of Derivatization improvement in sensitivity has allowed these assays to become
in Extraction routine [9]. Derivatization is a helpful tool that can either increase
and Analysis sensitivity for analytes that do not chromatograph well or can be
of Specific Drugs used to separate chiral isomers that don’t separate under normal
and Drug Classes conditions.
250 Cynthia A. Coulter and Christine M. Moore

Table 2
MRM transitions for THC, CBN, and CBD and their deuterated analogs

Compound Precursor ion (m/z) Product ion (m/z) Fragmentor voltage (V) Collision energy (V)
THC-d3 318.3 196.3 150 20
THC 315 193 120 20
THC 315 123 120 30
CBN 311.2 223.1 120 20
CBN 311.2 195.1 120 25
CBD 315 193 120 20
CBD 315 123 120 30

3.3.1 Preparation The identification of THC-COOH, a metabolite of THC, is essen-

of Calibrators, Controls, tial for differentiation of cannabis use from potential passive expo-
and Samples for THC- sure. Unfortunately THC-COOH is present in oral fluid at
COOH concentrations approximately 1000 times lower than those of
THC; in addition THC-COOH has been shown to be glucuroni-
dated in oral fluid; therefore, a hydrolysis procedure was also
incorporated to improve detection. The derivatization of
THC-COOH does not interfere with the analysis of THC, CBN,
and CBD, which do not derivatize.
1. Label borosilicate glass test tubes for Calibrators 1–7, each
quality control, and specimens.
2. Mix 4 mL of synthetic oral fluid with 12 mL of Quantisal®
extraction buffer (or other buffers depending on collection
device).
3. Pipette 2 mL of this mixture into a borosilicate glass test tube
for each calibrator and control (unless using external QC) to
be used.
4. Pipette 2 mL of each sample to be tested into its own borosili-
cate glass test tube.
5. Add 25 μL each of the 100 ng/mL THC-d3 and the 2.5 ng/
mL THC-COOH-d9 internal standard to each calibrator, con-
trol, and specimen.
6. Do not spike any drug standards into the tube labeled Calibra-
tor 1. This is the negative standard.
7. Spike 5 μL of the 100 ng/mL drug standard (THC, CBN, and
CBD) and into the tube labeled Calibrator 2, to give a final
concentration of 1 ng/mL (see Note 5).
8. Spike 25 μL of the 100 ng/mL drug standard (THC, CBN,
and CBD) into the tube labeled Calibrator 3, to give a final
concentration of 5 ng/mL.
 Drugs in Oral Fluid 251

9. Spike 50 μL of the 100 ng/mL drug standard (THC, CBN,

and CBD) and 20 μL of the 0.25 ng/mL THC-COOH drug
standard into the tube labeled Calibrator 4, for final concentra-
tions of 10 ng/mL and 10 pg/mL, respectively.
10. Spike 100 μL of each 100 ng/mL drug standard (THC, CBN,
and CBD) and 40 μL of the 0.25 ng/mL THC-COOH drug
standard into the tube labeled Calibrator 5, for final concen-
trations of 20 ng/mL and 20 pg/mL, respectively.
11. Spike 25 μL of each 1000 ng/mL drug standard (THC, CBN,
and CBD) and 10 μL of the 2.5 ng/mL THC-COOH drug
standard into the tube labeled Calibrator 6, for final concen-
trations of 50 ng/mL and 50 pg/mL, respectively.
12. Spike 50 μL of each 1000 ng/mL drug standard (THC, CBN,
and CBD) and 20 μL of the 2.5 ng/mL THC-COOH drug
standard into the tube labeled Calibrator 7, for final concen-
trations of 100 ng/mL and 100 pg/mL, respectively.
13. Add 0.2 mL of 1 N sodium hydroxide to each calibrator,
control, and sample to hydrolyze prior to solid-phase
extraction.
14. Cap and incubate in the heating block at 60  C for 15 min.
15. Remove from heat and allow to cool to room temperature.
16. Add 0.5 mL of glacial acetic acid to each sample and mix well.

3.3.2 Extraction 1. Use one 3 mL, 15 mg Trace-N extraction column per calibra-
of THC-COOH tor, control, and sample. Place each column in the positive
pressure manifold with the pressure setting no higher than
10 psi.
2. Condition the columns by adding 500 μL of methanol to each.
Allow the methanol to flow through the column bed at a rate
no greater than 1 mL/min or one drop at a time. Do not allow
the column bed to become dry once the conditioning has
begun (see Note 7).
3. Add 100 μL of 0.1 M acetic acid to each column. Allow the
0.1 M acetic acid to flow through the column bed at the same
rate as the methanol. Do not allow the column to become dry
before loading the sample.
4. Pour each calibrator, control, or sample onto the appropriate
column. Allow the sample to load on the column bed without
the use of positive pressure.
5. Once the samples have completely loaded, wash the columns
with 1 mL of Step 1 wash solution, allowing the wash to flow at
a rate of 1 mL/min. Positive pressure with nitrogen can be
used up to a pressure of 10 psi.
252 Cynthia A. Coulter and Christine M. Moore

6. Add 1 mL of Step 2 wash solution, allowing the wash to flow at

a rate of 1 mL/min.
7. Dry the columns under nitrogen using positive pressure at
30 psi for 7 min.
8. Place clean appropriately labeled borosilicate glass autosampler
high recovery reaction vials in extraction collection rack. Dry
tips of extraction columns and place above the appropriate
collection vial (see Note 17).
9. Add 1 mL of extraction eluant to elute THC-COOH, THC,
CBN, and CBD from calibrators, controls, and samples.
10. Evaporate the collected eluant under nitrogen (see Note 10).
11. Remove dry extraction vials and derivatize.
12. To the dry extraction vials, add the following in order:
(a) 20 μL of derivatizing reagent catalyst (TPP)
(b) 20 μL of derivatizing reagent 1 (DPDS)
(c) 20 μL of derivatizing reagent 2 (2-PA)
13. Crimp cap the vials, making sure each is tightly sealed.
14. Heat vials for 10 min at 60  C.
15. Remove vials from heat and allow to cool to room temperature.
16. Remove caps and evaporate to dryness in a vacuum oven.
17. Reconstitute in 50 μL of reconstitution solution. Mix well.

3.3.3 Analysis 1. Autosampler: 7  C.

of THC-COOH 2. Injection volume: 20 μL.
3. Column temperature: 60  C.
4. Binary pump flow rate: 0.2 mL/min.
5. Gradient:
0–2 min: 70% mobile phase B (MP-B)
4–6 min: 90% MP-B
6–8 min: 70% MP-B (re-equilibration)
6. Total run time: 8 min.
7. MS/MS mode: positive (+) ESI multiple reaction monitoring
(MRM) mode.
8. Nebulizer gas temperature: 300  C.
9. Nebulizer gas flow: 10 L/min.
10. Nebulizer pressure: 35 psi.
11. Positive capillary voltage is held at 3500 V.
12. The MRM transitions for THC-COOH, THC, CBN, CBD are
shown in Table 3 (see Note 14).
 Drugs in Oral Fluid 253

Table 3
MRM transitions for THC-COOH, THC, CBN, and CBD and their deuterated analogs

Precursor Product Fragmentor Collision

Compound ion (m/z) ion (m/z) voltage (V) energy (V)
THC-COOH–d9 444 336 140 20
THC-COOH 435 327 130 20
THC-COOH 435 299 130 25
THC-d3 318.3 196.3 150 20
THC 315 193 120 20
THC 315 123 120 30
CBN 311.2 223.1 120 20
CBN 311.2 195.1 120 25
CBD 315 193 120 20
CBD 315 123 120 30

3.3.4 Preparation The legal form of methamphetamine, levo-methamphetamine

of Calibrators, Controls, (l-METH), is present in over-the-counter medications, whereas
and Samples for Chiral the illicit form dextro-methamphetamine (d-METH) is more
Separation of AMP potent and more likely to be abused; in workplace drug testing
and METH programs, there is often a need to differentiate the two. Chiral
separation is possible by treating the sample with Marfey’s reagent.
The resulting chiral derivatives allow chromatographic separation
of the enantiomers of both amphetamine (AMP) and methamphet-
amine (METH) [10].
1. Label borosilicate glass test tubes for Calibrators 1–6, each
quality control, and specimens.
2. Mix 2 mL of synthetic oral fluid with 6 mL of Quantisal®
extraction buffer (or other buffers depending on collection
device).
3. Pipette 1 mL of this mixture into a borosilicate glass test tube
for each calibrator and control (unless using external QC) to
be used.
4. Pipette 1 mL of each sample to be tested into its own borosili-
cate glass test tube.
5. Add 50 μL of each 250 ng/mL internal standard (AMP-d5 and
METH-d5) to each calibrator, control, and specimen.
6. Do not spike any drug standards into the tube labeled Calibra-
tor 1. This is the negative standard.
254 Cynthia A. Coulter and Christine M. Moore

7. Spike 10 μL of 250 ng/mL drug standard (AMP and METH)

into the tube labeled Calibrator 2, to give a final concentration
of 10 ng/mL (see Note 5).
8. Spike 25 μL of each 250 ng/mL drug standard (AMP and
METH) into the tube labeled Calibrator 3, to give a final
concentration of 25 ng/mL.
9. Spike 50 μL of each 250 ng/mL drug standard (AMP and
METH) into the tube labeled Calibrator 4, to give a final
concentration of 50 ng/mL.
10. Spike 100 μL of each 250 ng/mL drug standard (AMP and
METH) into the tube labeled Calibrator 5, to give a final
concentration of 100 ng/mL.
11. Spike 200 μL of each 250 ng/mL drug standard (AMP and
METH) into the tube labeled Calibrator 6, to give a final
concentration of 200 ng/mL.
12. Add 1 mL of 0.1 M potassium phosphate pH 6.0 to each tube
to buffer, and mix well.

3.3.5 Extraction 1. Use one 3 mL, 35 mg CEREX Clin II extraction column per
for Chiral Separation calibrator, control, and sample. Place each column in the posi-
of AMP and METH tive pressure manifold with the pressure setting no higher than
10 psi.
2. Condition the columns by adding 2 mL methanol to each.
Allow the methanol to flow through the column bed at a rate
no greater than 1 mL/min or one drop a time. Do not allow
the column bed to become dry once the conditioning has
begun (see Note 7).
3. Add 2 mL of 0.1 M sodium phosphate buffer pH 6.0 to each
column. Allow the buffer to flow through the column bed at
the same rate as the methanol. Do not allow the column to
become dry before loading the sample.
4. Pour each calibrator, control, or sample onto the appropriate
column. Allow the sample to load on the column bed without
the use of positive pressure.
5. Once the samples have completely loaded, wash the columns
with 2 mL of DI water allowing the water to flow at a rate of
1 mL/min. Positive pressure with nitrogen can be used up to a
pressure of 10 psi.
6. Add 2 mL of 0.1 M hydrochloric acid to each column, allowing
to flow at the same rate as DI water.
7. Dry the columns under nitrogen using positive pressure at
30 psi for 1 min.
 Drugs in Oral Fluid 255

8. Remove from pressure and add 1 mL of methanol allowing to

flow at 1 mL/min.
9. Dry the columns under nitrogen using positive pressure at
30 psi for 7 min.
10. Place clean appropriately labeled borosilicate glass tubes in
extraction collection rack. Dry tips of extraction columns and
place above the appropriate collection tube.
11. Add 2 mL of extraction eluant to elute amphetamines from
calibrators, controls, and samples.
12. Place tubes in the sample concentrator and evaporate the col-
lected eluant under nitrogen (see Note 10).
13. Remove dry extraction tubes and derivatize.
14. Add 50 μL of prepared Marfey’s reagent and 10 μL of a 1:10
dilution of the saturated sodium bicarbonate solution. Cap.
15. Heat tubes 30 min at 50  C.
16. Allow tubes to cool.
17. Add 10 μL of 1 N hydrochloric acid to each tube. Vortex and
transfer to autosampler vials. Cap.

3.3.6 Analysis for Chiral 1. Autosampler: 7  C.

Separation of AMP 2. Injection volume: 5 μL.
and METH
3. Column temperature: 40  C.
4. Binary pump flow: 0.1 mL/min.
5. Gradient:
0 min: 50% MP-B
10 min: 95% MP-B
15–29 min: hold at 50% MP-B.
6. Total run time: 29 min.
7. MS/MS mode: positive (+) ESI multiple reaction monitoring
(MRM) mode.
8. Nebulizer gas: 350  C.
9. Nebulizer gas: flow 10 L/min.
10. Nebulizer pressure: 40 psi.
11. Positive capillary voltage: 4000 V.
12. The MRM transitions for AMP and METH are shown in
Table 4 (see Notes 14 and 18).
256 Cynthia A. Coulter and Christine M. Moore

Table 4
MRM transitions for AMP and METH and their deuterated analogs

Compound Precursor ion (m/z) Product ion (m/z) Fragmentor voltage (V) Collision energy (V)
AMP-d5 393.5 96 80 10
AMP 388.5 119 80 10
AMP 388.5 91 80 35
METH-d5 407.5 92 80 40
METH 402.4 119 80 10
METH 402.4 91 80 30

4 Notes

1. The Agilent Technologies Compendium [11] contains infor-

mation on extraction and LC-MS/MS transitions for the fol-
lowing drugs in oral fluid:
Benzodiazepines: alprazolam, bromazepam, chlordiazepoxide,
clonazepam, diazepam, estazolam, flurazepam, lorazepam,
midazolam, nordiazepam, oxazepam, phenazepam, tema-
zepam, triazolam
Stimulants: Cocaine, benzoylecgonine, cocaethylene, norco-
caine, amphetamine, methamphetamine, MDMA, MDA,
MDEA, methylphenidate, phentermine
Opiates and opioids: Codeine, morphine, 6-acetylcodeine,
6-acetylmorphine, hydrocodone, hydromorphone, oxyco-
done, oxymorphone, buprenorphine, fentanyl, metha-
done, EDDP, meperidine, propoxyphene, tapentadol,
tramadol
Cannabinoids and synthetic cannabinoids: THC, CP 47,497,
CP 47,497-C8, HU-210, JWH-018, JWH-073,
JWH-200, JWH-250, AM-2201
Dissociative drugs: Dextromethorphan, ketamine,
phencyclidine.
Sleep aids: Zaleplon, zolpidem, zopiclone
Antihistamines: Diphenhydramine, doxylamine
Muscle relaxants: Carisoprodol, meprobamate cyclobenzaprine
Antidepressants: Amitriptyline, amoxapine, citalopram, clomip-
ramine, desipramine, dothiepin, doxepin, fluoxetine, flu-
voxamine, imipramine, mianserin, mirtazapine,
nortriptyline, paroxetine, protriptyline, sertraline, trazo-
done, trimipramine, venlafaxine
 Drugs in Oral Fluid 257

Barbiturates: Butalbital, pentobarbital, phenobarbital,

secobarbital
2. Depending upon the method of collection for oral fluid, incor-
poration of a dilution factor may be necessary for quantitation.
Neat oral fluid samples which are collected by expectoration
(“spitting”) into glass vials may need to be centrifuged prior to
extraction to allow any sediment to settle. Most oral fluid
samples are collected using a device which incorporates a pad
which is placed into the mouth; there is likely to be a blue or
yellow dye indication of adequate saliva absorption; then the
pad is placed in a known amount of buffer to assist drug
recovery and stability during transportation. The dilution fac-
tor of 4 used in these procedures is derived from the collection
of oral fluid (1 mL) using the Quantisal® device which is then
placed into transportation buffer (3 mL). Other collection
devices may have different dilution factors which must be
calculated for accurate quantitation.
3. Only borosilicate glassware should be used in the preparation
of THC and THC-COOH specimens and spiking solutions.
4. Laboratory personnel are reducing or eliminating the use of
methylene chloride because of carcinogenic concerns; however
benzoylecgonine recovery is increased when methylene chlo-
ride is used instead of ethyl acetate.
5. The calibration curve consists of 5–7 points including the
negative standard. Additional calibration levels can be
incorporated if high concentrations of drug are suspected.
6. Meperidine (see Subheading 3.1): Most basic drugs can be
loaded onto the extraction columns using a pH 6.0 buffer;
however, meperidine requires a slightly more acidic environ-
ment (pH 4.0) to load efficiently. 0.1 M acetic acid adjusted to
pH 4.0 can be used for both loading and washing purposes.
7. The methanol opens up binding sites in the column packing.
The buffer adjusts the pH of the column to improve binding of
the analytes to the column bed.
8. Carisoprodol (see Subheading 3.1): In order to increase cariso-
prodol recovery from oral fluid, replacing the last wash steps
with methanol and water mixed at a volume ratio of 25:75 wash
followed by hexane (1 mL) has been shown to optimally
recover the drug. Elute carisoprodol and its metabolite, mep-
robamate, with 3 mL ethyl acetate and hexane mixed at a
volume ratio of 50:50 [11].
9. Amphetamine (see Subheading 3.1): When washing the solid-
phase columns, a stronger acid is preferred (hydrochloric .vs.
acetic) to improve recovery; do not use ethyl acetate as part of
the wash. Adding 0.035 N sulfuric acid (10 μL) to the eluant
258 Cynthia A. Coulter and Christine M. Moore

during evaporation assists the volatile amphetamines to stay in

solution. Sulfuric acid cannot be used for chiral separation,
which requires a basic environment.
10. Use a temperature no higher than 40  C and a flow rate that
only flutters the surface of the eluant. This drying flow rate will
differ greatly based on make and model of evaporator and how
many rows of tubes are drying. Do not allow samples to remain
under drying conditions after they have completed
evaporation.
11. Phencyclidine (PCP) (see Subheading 3.1): To increase the
solid-phase extraction recovery of PCP in oral fluid, dry
down samples at ambient temperature; do not over dry samples
while under nitrogen.
12. The flow rate of 0.2 mL/min on the larger bore LC column is
low, but is necessary to separate benzoylecgonine from norco-
caine as they share the same transitions. While norcocaine is not
included in this method, it is possible that its detection is useful
for other biological matrices.
13. Opiates (see Subheading 3.1): Some opioids and metabolites
share transitions in MS/MS mode (e.g., hydrocodone and
codeine, noroxycodone and oxymorphone, etc.). Therefore,
chromatographic separation is critical to correct identification
and quantitation [12].
14. Transition ratios are calculated using the quantifying and qua-
lifying ion transitions at the cutoff concentrations; subse-
quently the established ratio must be met (20%) in order
for a sample to be certified as positive. The use of a second
qualifying transition ion and establishment of a ratio between
quantifying and qualifying transitions intensities gives greater
analyte certainty when identifying a positive sample. Analytes
can have similar transitions and retention times, but the inclu-
sion of the second transition and the transition ratio can be
used to differentiate one analyte from another.
15. Barbiturates (see Subheading 3.2): Reducing the amount of
methanol used in washing the columns to below 25% of the
organic phase increases recovery, e.g., methanol/DI water
volume ratio 25:75.
16. Benzodiazepines (see Subheading 3.2): An isocratic pump pro-
gram of 20 mM ammonium formate pH 8.6: methanol
(50:50) allows for good separation of numerous benzodiaze-
pines. Deuterated clonazepam-d4 interferes with the chroma-
tography of lorazepam; if this is an issue, consider removal or
use a different internal standard.
17. Cannabinoids (see Subheadings 3.2 and 3.3.1): Using high
recovery 1.5 mL reservoir vials greatly increased recovery of
 Drugs in Oral Fluid 259

THC-COOH. The vials are large enough to directly elute the

drugs and have a tapered reservoir that holds the derivatizing
reagents in place. The contents of the vial can then be evapo-
rated and reconstituted in situ, eliminating loss in transfer
steps.
18. Because the l- and d-isomers have the same transitions and
same transition ratios, identification of the l- and d-isomer for
both AMP and METH is based on retention time. The
l-isomer chromatographs first with the d-isomer separated by
approximately 0.3 min downstream.

References

1. Gallardo E, Barroso M, Queiroz JA (2009) zolpidem in oral fluid and its application to
Current technologies and considerations for authentic samples from regular drug users. J
drugs bioanalysis in oral fluid. Bioanalysis 1 Pharm Biomed Anal 74:213–222
(3):637–667 8. Langel K, Gunnar T, Ariniemi K, Rajam€aki O,
2. Vindenes V, Yttredal B, Oiestad EL, Waal H, Lillsunde P (2011) A validated method for the
Bernard JP, Mørland JG, Christophersen AS detection and quantitation of 50 drugs of
(2011) Oral fluid is a viable alternative for abuse and medicinal drugs in oral fluid by gas
monitoring drug abuse: detection of drugs in chromatography-mass spectrometry. J Chro-
oral fluid by liquid chromatography-tandem matogr B Analyt Technol Biomed Life Sci
mass spectrometry and comparison to the 879(13–14):859–870
results from urine samples from patients trea- 9. Coulter C, Garnier M, Moore C (2012) Anal-
ted with methadone or buprenorphine. J Anal ysis of tetrahydrocannabinol and its metabolite,
Toxicol 35(1):32–39 11-nor-d9-tetrahydrocannabinol-9-carboxylic
3. Moore C (2015) Drug testing and adherence acid, in oral fluid using liquid chromatography
monitoring in pain management: Oral fluid with tandem mass spectrometry. J Anal Toxicol
testing. J Opioid Manag 11(1):69–75 36(6):413–417
4. Crouch DJ (2005) Oral fluid collection: the 10. Newmeyer MN, Concheiro M, da Costa JL,
neglected variable in oral fluid testing. Forensic Flegel R, Gorelick DA, Huestis MA (2015)
Sci Int 150(2–3):165–173 Oral fluid with three modes of collection and
5. Coucke LD, De Smet L, Verstraete AG (2016) plasma methamphetamine and amphetamine
Influence of sampling procedure on codeine enantiomer concentrations after controlled
concentrations in oral fluid. J Anal Toxicol 40 intranasal l-methamphetamine administration.
(2):148–152 Drug Test Anal 7(10):877–883
6. Valen A, Leere Øiestad ÅM, Strand DH, 11. Hughes J, Tuyay J, Coulter C, Moore C
Skari R, Berg T (2016) Determination of (2013) Drugs and metabolites in oral fluid:
21 drugs in oral fluid using fully automated Immunoassay screening and LC/MS-MS con-
supported liquid extraction and UHPLC- firmation and quantification. www.agilent.com
MS/MS. Drug Test Anal. https://doi.org/ (Agilent Technologies)
10.1002/dta.2045 12. Tuyay J, Coulter C, Rodrigues W, Moore C
7. Jang M, Chang H, Yang W, Choi H, Kim E, Yu (2012) Disposition of opioids in oral fluid:
BH, Oh Y, Chung H (2013) Development of Importance of chromatography and mass spec-
an LC-MS/MS method for the simultaneous tral transitions in LC-MS/MS. Drug Test Anal
determination of 25 benzodiazepines and 4(6):395–401
 Chapter 23

Determination of Cocaine and Metabolites in Dried Blood

Spots by LC-MS/MS
Lars Ambach and Christophe Stove

Abstract
Cocaine is one of the most common illegal drugs in Europe and other parts of the world. It is mainly
metabolized to benzoylecgonine and ecgonine methyl ester but also to minor metabolites such as norco-
caine and meta-hydroxy-benzoylecgonine. If ethanol is consumed simultaneously, cocaethylene is formed.
Dried blood spots (DBS) are a minimally invasive sampling technique producing easy-to-ship specimens
and have garnered increasing attention in forensic and clinical contexts in recent years. We hereby present a
liquid chromatography/tandem mass spectrometry-based (LC-MS/MS) method for the quantification of
cocaine, benzoylecgonine, ecgonine methyl ester, norcocaine, meta-hydroxy-benzoylecgonine, and
cocaethylene in DBS.

Key words Dried blood spots, Cocaine, Benzoylecgonine, Ecgonine methyl ester, Norcocaine,
Cocaethylene, Meta-hydroxy-benzoylecgonine, HPLC-MS/MS

1 Introduction

Cocaine (COC) is an illicit stimulant of the central nervous system

and the most common illegal drug in Europe after cannabis
[1]. It is the most frequently found stimulant in Southern and
Western Europe with evidence suggesting an increasing use in
recent years [1, 2].
In vivo, COC is rapidly converted into benzoylecgonine
(BE) and ecgonine methyl ester (EME) by cleavage of one of the
respective ester moieties. BE can be formed by carboxylesterases in
the liver or through spontaneous hydrolysis at alkaline or physio-
logical pH. EME can be formed in vivo and in vitro. N-demethyla-
tion to norcocaine (nor-COC) and hydroxylation of BE at the
phenyl ring have been described as minor metabolic steps
[3]. Meta-hydroxy-benzoylecgonine (m-OH-BE) has a longer
half-life than BE [4, 5] and is formed exclusively in vivo [6]. It
can therefore also be used to verify positive COC results if an

Loralie J. Langman and Christine L.H. Snozek (eds.), LC-MS in Drug Analysis: Methods and Protocols,
Methods in Molecular Biology, vol. 1872, https://doi.org/10.1007/978-1-4939-8823-5_23,
© Springer Science+Business Media, LLC, part of Springer Nature 2019

261
262 Lars Ambach and Christophe Stove

O O O
H 3C CH3 H3C H3C CH3
N O N OH N O

O O OH

O O
cocaine benzoylecgonine ecgonine methyl ester

O O O
CH3 H3 C H3 C
CH3
HN O N OH N O
OH
O O O

O O O
norcocaine meta-hydroxy-benzoylecgonine cocaethylene

Fig. 1 Structures of cocaine and the included metabolites

external contamination with COC followed by spontaneous hydro-

lysis to BE is alleged. When ethanol (EtOH) is co-consumed with
COC, cocaethylene (COC-Et) is produced through transesterifica-
tion. An overview of the different structures is given in Fig. 1.
Dried blood spots (DBS) are generally produced by spotting
capillary or venous blood onto filter paper, followed by drying,
extraction, and analysis. DBS provide the opportunity for mini-
mally invasive sampling with only small sample volumes required.
For neonatal metabolic screening, DBS from capillary blood
obtained from heel pricks have been used for decades [7], whereas
for adults, blood obtained from the fingertip by lancet puncture is
commonly used [8, 9]. Venous blood can also be used; in this case,
DBS serve as a means of sample preparation for small sample
volumes [10, 11]. DBS have been used in therapeutic drug moni-
toring [8, 12] as well as for the detection of drugs of abuse,
medications, and their metabolites [13–17].
One of the most prominent challenges associated with DBS is
the influence of the hematocrit (HCT) on analytical results. Due to
higher viscosity at higher HCT, samples with different HCT will
form spots of different sizes. Consequently, sub-punches of these
spots will then represent different volumes of blood, leading to
different quantitative results. Various strategies to overcome this
challenge have been discussed [18].
Several methods for the quantification of COC in dried matrices
have already been published. However, most of these cover a maxi-
mum of 2–3 metabolites [19–23]. We hereby present a more compre-
hensive, sensitive method for the quantification of COC and five of its
metabolites (BE, EME, nor-COC, m-OH-BE, and COC-Et), which
also enables analysts to assess co-consumption of EtOH (through
COC-Et) and verify sample authenticity (through m-OH-BE).
 Cocaine and Metabolites in DBS 263

2 Materials

2.1 Prepared All solvents and reagents are of LC-MS or analytical grade.
Reagents
1. Stock solutions: 1 mg/mL COC, nor-COC, COC-Et, m-OH-
BE; 0.1 mg/mL BE, EME, and internal standards (IS) co-
caine-D3, benzoylecgonine-D8, ecgonine methyl ester-D3,
norcocaine-D3, cocaethylene-D8 reference standards in sealed
glass ampoules. Open, and transfer the contents of the
ampoules into glass vials with a plastic screw cap and rubber/
Teflon septum, and wrap in parafilm. Store vials at
20  C.
2. Blank blood for calibration and quality control (QC) samples:
Obtain blood from healthy volunteers using collection tubes
with sodium fluoride and potassium oxalate (NaF/K2Ox).
Simultaneously collect one ethylenediaminetetraacetic acid
(EDTA) tube of blood and determine the HCT value on a
standard clinical analyzer. Based on the measured HCT, adjust
the HCT of the NaF/K2Ox-stabilized blood to 40% by adding
or removing plasma to or from the blood (see Notes 1 and 2).
3. Extraction solvent: 6 ng/mL of each IS in 2 mM ammonium
acetate. Dissolve 15.42 mg of ammonium acetate in 100 mL of
ultrapure water (resistivity >18 MΩ cm). Add 3 μL of each IS
stock solution to 50 mL of 2 mM ammonium acetate solution.
Store at
20  C (see Note 3).
4. Mobile phase A: 10 mM ammonium formate with 0.1% formic
acid. Dissolve 630.6 mg of ammonium formate in 1 L of
ultrapure water. Add 1000 μL of formic acid. Store at room
temperature. Prepare freshly every week.
5. Mobile phase B: Methanol (MeOH) with 0.1% formic acid.
Add 1000 μL of formic acid to 1 L of MeOH.
6. Stock blood for calibration samples: 1000 ng/mL of each
analyte. Mix 2928 μL of blank blood with 3 μL of each of the
1.0 mg/mL reference solutions of COC, nor-COC, COC-Et,
and m-OH-BE and 30 μL of each of the 0.1 mg/mL reference
solutions of BE and EME. Stock blood also serves as
Calibrator 7.
7. Calibration samples 1–6: Dilute stock blood with blank blood
according to the scheme laid out in Table 1.
8. Stock blood for quality control (QC) samples: 1000 ng/mL of
each analyte. Mix 2928 μL of blank blood with 3 μL of each of
the 1.0 mg/mL reference solutions of COC, nor-COC,
COC-Et, and m-OH-BE and 30 μL of each of the 0.1 mg/
mL reference solutions of BE and EME.
9. QC samples 1–4: Dilute stock blood with blank blood accord-
ing to the scheme laid out in Table 2.
264 Lars Ambach and Christophe Stove

Table 1
Dilution scheme for calibration samples

Calibrator C7 C6 C5 C4 C3 C2 C1
Volume blank blood added (μL) – 300 630 570 630 570 540
Volume stock blood added (μL) 600 300 70 30 – – –
Volume C5 added (μL) – – – – 70 30 –
Volume C3 added (μL) – – – – – – 60
Final concentration (ng/mL) 1000 500 100 50 10 5.0 1.0

Table 2
Dilution scheme for quality control (QC) samples

QC sample QC IV QC III QC II QC I
Volume blank blood added (μL) 150 555 540 580
Volume stock blood added (μL) 450 45 – –
Volume QCIII added (μL) – – 60 20
Final concentration (ng/mL) 750 75 7.5 2.5

2.2 Supplies 1. Blood collection tubes containing NaF/K2Ox or EDTA (see

and Analytical Note 4).
Equipment 2. Whatman® 903 Specimen Collection Paper.
3. Eppendorf 2 mL plastic microcentrifuge tubes or similar.
4. Shaker for 2 mL plastic microcentrifuge tubes, e.g., Biosan
TS-100C Thermo-Shaker.
5. Centrifuge with rotors for microcentrifuge tubes and blood
collection tubes, e.g., Eppendorf Centrifuge 5804R.
6. Sample concentrator, e.g., TurboVap LV or Techne Dri-Block
DB-3D.
7. Standard clinical analyzer capable of measuring hematocrit
(HCT).
8. HPLC system capable of gradient elution and equipped with an
autosampler and column oven, e.g., Shimadzu Prominence
series.
9. Analytical column: Phenomenex Synergi Polar-RP,
100  2.0 mm, 2.5 μm.
10. Triple quadrupole mass spectrometer with electrospray ioniza-
tion source, e.g., SCIEX QTRAP 5500 or similar.
 Cocaine and Metabolites in DBS 265

3 Methods

3.1 Preparation 1. Take up 25 μL of Calibrator 1 with a pipette.

of DBS for Calibrators 2. Transfer the blood in a single drop onto the specimen collec-
and Controls tion paper or collection card. The pipette tip should not touch
the paper (see Notes 5 and 6).
3. Repeat steps 1 and 2 for all calibrators, QCs, and blank blood.
4. Let the DBS dry at room temperature for 2 h (see Notes 7
and 8).

3.2 Extraction 1. Take a 6 mm punch from the center of each DBS (see Note 9).
2. Transfer each punch into its own 2 mL Eppendorf cup.
3. Add 200 μL of extraction solvent to each tube.
4. Cap and shake for 15 min at 1000 rpm/1.12  g at room
temperature.
5. Add 1000 μL of ice-cold acetonitrile (MeCN) (see Note 10).
6. Cap and shake for 10 min at 1000 rpm at room temperature.
7. Centrifuge the samples for 20 min at 14,000  g, 4  C.
8. Transfer the supernatants into labelled glass tubes or vials.
9. Evaporate the supernatants under a stream of nitrogen at 40  C
(see Note 11).
10. Reconstitute in 50 μL of mobile phase A (see Note 12).
11. Transfer to a labelled autosampler vial with a 100 μL insert.

3.3 Analysis 1. Place samples in the autosampler in the following order:

(a) Calibration samples (including blank samples), lowest to
highest concentration.
(b) QC samples, lowest to highest concentration.
(c) Unknown samples.
(d) Solvent blank samples (see Note 13).
2. Set the LC-MS/MS acquisition method to run with the fol-
lowing parameters:
(a) Autosampler parameters: Cool down to 4  C, injection
volume: 10 μL.
(b) HPLC parameters (see Note 14):
l Column temperature: 50  C.
l Flow rate: 0.3 mL/min.
l Gradient program:
0.0–0.5 min: Hold at 0% B.
0.5–0.6 min: Increase linearly to 45% B.
266 Lars Ambach and Christophe Stove

0.6–3.0 min: Increase linearly to 95% B.

3.0–4.0 min: Hold at 95% B.
4.0–4.01 min: Decrease linearly to 0% B.
4.01–6.0 min: Hold at 0% B to re-equilibrate.
(c) Ion source parameters:
l Ion spray voltage: 5500 V.
l Source temperature: 500  C.
l Ion source gas 1: 60 psi.
l Ion source gas 2: 40 psi.
l Curtain gas: 20 psi.
(d) MS parameters:
Compound-dependent parameters are listed in Table 3
(see Notes 15 and 16).
3. A chromatogram of a QC sample at the lower limit of quanti-
tation (LLOQ) of EME (c ¼ 5.0 ng/mL, highest LLOQ of all
analytes) is shown in Fig. 2.

Table 3
Lower limits of quantification (LLOQ) and MRM transitions and parameters

Dwell
LLOQ m/z time DP EP CXP
Analyte (ng/mL) Q1 m/z Q3 (ms) (V) (V) CE (V) (V)
Cocaine 2.5 304.3 82.0/150.0 30 46 10 39/33 12
Benzoylecgonine 1.0 290.1 82.0/168.1/150.1 30 70 10 37/27/32 10
Ecgonine methyl ester 5.0 200.1 82.0/182.1 30 45 10 32/23 9
Norcocaine 2.5 290.2 136.1/168.1/108.0 30 70 10 30/22/44 11
Cocaethylene 2.5 318.2 82.0/150.1 30 70 10 37/33 10
m-OH-benzoylecgonine 2.5 306.3 121.1/82.0 30 70 10 37/36 7
Cocaine-D3 n/a 307.2 185.2 30 70 9 28 10
Benzoylecgonine-D8 n/a 298.3 171.1 30 60 9 27 10
Ecgonine methyl n/a 203.3 185.2 30 70 10 24 10
ester-D3
Norcocaine-D3 n/a 293.2 136.1 30 57 10 30 10
Cocaethylene-D8 n/a 326.3 204.2 30 70 10 28 12
Q1 quadrupole 1, Q3 quadrupole 3, the first transition is used as quantifier, the following transitions as qualifiers, DP
declustering potential, EP entrance potential, CE collision energy, CXP collision cell exit potential
 Cocaine and Metabolites in DBS 267

4 Notes

1. The HCT of the calibration and QC samples should be approx-

imately the (expected) median of the HCT of the unknown
samples to be analyzed in order to minimize the influence of
the HCT effect on the analytical result. The choice of the HCT
of the blood in which calibrators and QC samples are prepared
depends on the sample population. The HCT effect is
compound-dependent. It is thus essential that the HCT effect

Fig. 2 Extracted ion chromatograms for all analytes and internal standards at the lower limit of quantitation of
EME (5.0 ng/mL). Blue ¼ qualifier transition, red/green ¼ quantifier transitions
268 Lars Ambach and Christophe Stove

Fig. 2 (continued)

is evaluated during method validation. Guidelines on how to

do this have been published elsewhere [24].
2. The blood should be kept for a maximum of about 5 days
before hemolysis reaches a critical level. Hemolysis may affect
spreading of the blood during DBS formation and extraction;
therefore, hemolyzed blood may not be used. Freezing likewise
induces hemolysis and is therefore not a suitable means of
preservation of blood intended to produce DBS.
3. Preparation using volumetric glassware is not strictly necessary,
as long as calibrators, QCs, and unknown samples are prepared
with the same batch of extraction solvent. The optimal compo-
sition of the extraction solvent needs to be determined during
method development. Generally, extraction solvents with an
aqueous content between 0% and 20% produce relatively clean
extracts that can usually just be diluted or evaporated and
reconstituted before injection into the LC-MS/MS system.
Extraction with a higher water content will also extract hemo-
globin (and other proteins) from the DBS, and these extracts
therefore require further cleanup before injection. In the pre-
sented method, an entirely aqueous extraction solvent was used
and protein precipitation with acetonitrile was used as a further
purification step to remove hemoglobin from the sample. Liq-
uid-liquid extraction (LLE) or solid-phase extraction (SPE) are
common alternatives for sample purification depending on the
analyte.
4. When analyte stability in the sample matrix is assessed during
method validation, these experiments should be carried out
using EDTA blood in order to avoid interference from a
 Cocaine and Metabolites in DBS 269

stabilizing effect of the NaF/K2Ox. Additionally, incurred

sample stability (without anticoagulant) can also be assessed.
5. For optimal transfer of the full volume, the pipette should best
be held at a ca. 20 angle. Calibrators and controls should
ideally be prepared freshly on the day of analysis. If storage of
DBS is necessary, they should be kept at
20  C or colder.
6. While commercially available card formats are more practical
for the collection of authentic specimens, it is more economical
to use large sheets (A4 or similar) of sample collection paper for
the preparation of calibration and QC samples. Circular pat-
terns can also be printed onto these sheets with a regular laser
printer to aid with the positioning of the blood spots. Dashed
lines should be used for the circles to minimize the interference
of wax contained in the printer toner with the spreading of the
blood on the paper.
7. The drying time may vary according to climate conditions.
Long-term storage of DBS samples should always occur in
sealable plastic bags with desiccant packages.
8. Drying racks are commercially available for card formats. For
drying larger sheets of specimen collection paper, the authors
use a DIY solution in which the paper is horizontally stretched
in the air by attaching binder or foldback clips. The clips can be
freely positioned and attached to the corners of the paper sheet.
9. Punchers of different diameters are commercially available, and
the appropriate punch size needs to be evaluated during
method development. Larger punches lead to increased sensi-
tivity but may lead to problems in samples where patients were
only able to generate small DBS. For 6 mm punches, a regular
(European) office puncher available from stationary stores can
be used.
10. Keeping the MeCN ice-cold (
20  C) instead of at room
temperature and combining it with a sufficient centrifugation
time and speed (20 min, 14,000  g) ensure removal of hemo-
globin from the sample. Use of MeCN at room temperature
and shorter centrifugation led to extracts that were still
colored.
11. When analyzing volatile compounds such as amphetamines or
cathinones, the addition of 20–40 μL of 0.25% hydrochloric
acid (HCl) in MeOH can help to prevent analyte loss through
evaporation by forming nonvolatile hydrochloride salts.
12. The composition of the reconstitution solvent should ideally
reflect the chromatographic starting conditions and should
have similar or lower elution strength than the starting condi-
tions of the LC gradient to ensure optimal peak shape. Lower-
ing the reconstitution volume can provide significant benefits
270 Lars Ambach and Christophe Stove

for method sensitivity. However, it should be large enough to

ensure complete dissolution of the residue. Likewise, larger
injection volumes may result in improved sensitivity.
13. Solvent blank samples (MeOH or mobile phase) should be run
after the highest calibrator and highest QC, in order to assess
potential carry-over, and periodically about every six samples.
Carry-over should be less than 20% of the signal of the calibra-
tor at the lower limit of quantitation.
14. When analyzing BE and nor-COC together, it is important to
choose chromatographic conditions that allow full separation
of the two compounds as they are isomers and share certain
MRM transitions. Transitions that are specific for each analyte
should be used for quantification. If only determination of BE
is desired, interference from nor-COC may not be a significant
problem in practice as nor-COC is only a minor metabolite
occurring at low concentrations in authentic samples. There-
fore, its potential for significant interference with the determi-
nation of BE is limited. However, it should be noted that
nor-COC concentrations can be higher in users with a cholin-
esterase deficiency [25] or when EtOH is co-consumed [26].
15. Commonly, one quantifier transition (usually the most sensi-
tive one) and one qualifier transition are chosen. In the case of
isomeric or isobaric analytes, it is prudent to monitor addi-
tional qualifier transitions as is the case here for BE and
nor-COC. The ratio of the different transitions, referred to as
ion ratio, compared to an analytical standard measured on the
same day (e.g., a calibration sample) should be used as an
additional identification criterion. For LC-MS/MS applica-
tions, ion ratios of unknown samples may not differ more
than 30% from the ion ratios measured in standard
samples [27].
16. The MS parameters presented here are instrument-specific and
may need to be re-optimized when the method is transferred to
another instrument.

References
1. EMCDDA (2017) European drug report 3. Baselt RC (2011) Cocaine. Disposition of toxic
2017: trends and developments. luxembourg: drugs and chemicals in man, 9th edn. Biomed-
european monitoring centre for drugs and ical Publications, Seal Beach, CA, pp 390–394
drug addiction (EMCDDA); June 2017. 4. Moore C, Negrusz A, Lewis D (1998) Deter-
http://www.emcdda.europa.eu/publications/ mination of drugs of abuse in meconium. J
edr/trends-developments/2017 Chromatogr B 713(1):137–146
2. UNODC (2017) World Drug Report 2017. 5. Lester BM, ElSohly M, Wright LL, Smeriglio
Vienna: United Nations Office on Drugs and VL, Verter J, Bauer CR et al (2001) The mater-
Crime (UNODC). Report No.: ISBN 978-92- nal lifestyle study: drug use by meconium toxi-
1-148291-1. http://www.unodc.org/ cology and maternal self-report. Pediatrics 107
wdr2017/ (2):309–317
 Cocaine and Metabolites in DBS 271

6. Klette KL, Poch GK, Czarny R, Lau CO 6-acetylmorphine in blood with use of dried
(2000) Simultaneous GC-MS analysis of blood spots. Ther Drug Monit 30
meta- and para-hydroxybenzoylecgonine and (6):733–739. https://doi.org/10.1097/
norbenzoylecgonine: a secondary method to FTD.0b013e31818d9fdb
corroborate cocaine ingestion using nonhydro- 16. Ingels A-S, De Paepe P, Anseeuw K, Van
lytic metabolites. J Anal Toxicol 24 Sassenbroeck D, Neels H, Lambert WE et al
(7):482–488 (2011) Dried blood spot punches for confir-
7. Guthrie R, Susi A (1963) A simple phenylala- mation of suspected gamma-hydroxybutyric
nine method for detecting phenylketonuria in acid intoxications: validation of an optimized
large populations of newborn infants. Pediat- GC-MS procedure. Bioanalysis 3
rics 32:338–343 (20):2271–2281. https://doi.org/10.4155/
8. Edelbroek PM, van der Heijden J, Stolk LML bio.11.204
(2009) Dried blood spot methods in therapeu- 17. Hernández Redondo A, Schroeck A,
tic drug monitoring: methods, assays, and pit- Kneubuehl B, Weinmann W (2013) Determi-
falls. Ther Drug Monit 31(3):327–336. nation of ethyl glucuronide and ethyl sulfate
https://doi.org/10.1097/FTD. from dried blood spots. Int J Legal Med 127
0b013e31819e91ce (4):769–775. https://doi.org/10.1007/
9. McDade TW, Williams S, Snodgrass JJ (2007) s00414-012-0815-2
What a drop can do: dried blood spots as a 18. De Kesel PMM, Sadones N, Capiau S, Lambert
minimally invasive method for integrating bio- WE, Stove CP (2013) Hemato-critical issues in
markers into population-based research. quantitative analysis of dried blood spots: chal-
Demography 44(4):899–925 lenges and solutions. Bioanalysis 5
10. Ambach L, Hernández Redondo A, König S, (16):2023–2041. https://doi.org/10.4155/
Weinmann W (2014) Rapid and simple bio.13.156
LC-MS/MS screening of 64 novel psychoac- 19. Ellefsen KN, da Costa JL, Concheiro M,
tive substances using dried blood spots. Drug Anizan S, Barnes AJ, Pirard S et al (2015)
Test Anal 6(4):367–375. https://doi.org/10. Cocaine and metabolite concentrations in
1002/dta.1505 DBS and venous blood after controlled intra-
11. Odoardi S, Anzillotti L, Strano-Rossi S (2014) venous cocaine administration. Bioanalysis 7
Simplifying sample pretreatment: application (16):2041–2056. https://doi.org/10.4155/
of dried blood spot (DBS) method to blood bio.15.127
samples, including postmortem, for UHPLC- 20. Saussereau E, Lacroix C, Gaulier JM, Goulle JP
MS/MS analysis of drugs of abuse. Forensic Sci (2012) On-line liquid chromatography/tan-
Int 243:61–67. https://doi.org/10.1016/j. dem mass spectrometry simultaneous determi-
forsciint.2014.04.015 nation of opiates, cocainics and amphetamines
12. den Burger JCG, Wilhelm AJ, Chahbouni A, in dried blood spots. J Chromatogr B
Vos RM, Sinjewel A, Swart EL (2012) Analysis 885–886:1–7. https://doi.org/10.1016/j.
of cyclosporin A, tacrolimus, sirolimus, and jchromb.2011.11.035
everolimus in dried blood spot samples using 21. Mercolini L, Mandrioli R, Gerra G, Raggi MA
liquid chromatography tandem mass spectrom- (2010) Analysis of cocaine and two metabolites
etry. Anal Bioanal Chem 404 in dried blood spots by liquid chromatography
(6–7):1803–1811. https://doi.org/10.1007/ with fluorescence detection: a novel test for
s00216-012-6317-8 cocaine and alcohol intake. J Chromatogr A
13. Sadones N, Capiau S, De Kesel PMM, Lambert 1217(46):7242–7248. https://doi.org/10.
WE, Stove CP (2014) Spot them in the spot: 1016/j.chroma.2010.09.037
analysis of abused substances using dried blood 22. Déglon J, Thomas A, Mangin P, Staub C
spots. Bioanalysis 6(17):2211–2227. https:// (2012) Direct analysis of dried blood spots
doi.org/10.4155/bio.14.156 coupled with mass spectrometry: concepts and
14. Déglon J, Versace F, Lauer E, Widmer C, biomedical applications. Anal Bioanal Chem
Mangin P, Thomas A et al (2012) Rapid 402(8):2485–2498. https://doi.org/10.
LC-MS/MS quantification of the major ben- 1007/s00216-011-5161-6
zodiazepines and their metabolites on dried 23. Antelo-Domı́nguez Á, Cocho JÁ, Tabernero
blood spots using a simple and cost-effective MJ, Bermejo AM, Bermejo-Barrera P, Mor-
sample pretreatment. Bioanalysis 4 eda-Piñeiro A (2013) Simultaneous determina-
(11):1337–1350. https://doi.org/10.4155/ tion of cocaine and opiates in dried blood spots
bio.12.42 by electrospray ionization tandem mass spec-
15. Garcia Boy R, Henseler J, Mattern R, Skopp G trometry. Talanta 117:235–241. https://doi.
(2008) Determination of morphine and org/10.1016/j.talanta.2013.09.010
272 Lars Ambach and Christophe Stove

24. Capiau S, Veenhof H, Alffenaar J-W, Stove CP cocaine interactions in humans. J Pharmacol
(2018) A guideline on bioanalytical method Exp Ther 266(3):1364–1373
validation for dried blood sample analysis. 27. Guidance document on analytical quality con-
Ther Drug Monit (submitted for publication) trol and validation procedures for pesticide
25. Inaba T, Stewart DJ, Kalow W (1978) Metab- residues analysis in food and feed.: European
olism of cocaine in man. Clin Pharmacol Ther Commission, EU Reference Laboratories for
23(5):547–552 Residues of Pesticides; 2014. Report No.:
26. Farré M, de la Torre R, Llorente M, Lamas X, SANCO/12571/2013. http://ec.europa.eu/
Ugena B, Segura J et al (1993) Alcohol and food/plant/plant_protection_products/guid
ance_documents/docs/qualcontrol_en.pdf
 INDEX

A G
Accurate mass .................................................42, 181, 189 Gabapentin ............................................ 67, 119–126, 184
Alcohol biomarkers ....................................................... 224 Glucuronidase ...................................................12, 26, 27,
Anticonvulsants .....................................68, 112, 119, 120 29, 30, 32, 55, 140, 142, 146
Antiepileptic drugs ......................................................4, 67
H
B
High-resolution mass spectrometry (HRMS)................ 1,
Biologics .................................................................. 1, 3, 5, 4, 7, 8, 41–50, 181, 182, 189
12, 68, 135, 149, 165, 166, 179, 181, 258
Buprenorphine ........................................... 41, 43, 45, 46, I
48, 49, 51–59, 187, 256 Immunosuppressant...................................................3, 76,
102, 111, 112
C
Cannabidiol (CBD) .................................................11–21, L
184, 241
Lacosamide ................................................................67–73
Cannabinoids..................................11–20, 130–132, 134, Leflunomide ......................................................... 8, 75–82
137–146, 212, 215, 241–243, 256, 258 Legal highs ........................................................... 129, 165
Cocaine (COC) .......................................... 187, 199–209,
Liquid chromatography (LC)..............................v, 1, 3, 5,
240, 245, 256, 261–270 6, 11–20, 24, 26, 29, 30, 34, 36, 37, 42, 52, 55,
Cyclosporine.................................................103, 111–117 58, 61–65, 67–73, 75–82, 85–98, 101–109,
111–117, 119–126, 137–146, 149–162,
D
165–179, 181–190, 199–209, 211–221,
Designer drugs ........................................... 129–135, 137, 223–235, 237–259, 261–270
165, 179, 182
Designer stimulants ............................................. 165–179 M
Disease-modifying antirheumatic drug Mass spectrometry (MS).............................................. 1–9,
(DMARD) ........................................................... 75
11–20, 24, 28–30, 34, 36, 37, 42, 52, 61–65,
Dried blood spots ............................................ 5, 261–270 67–73, 75–82, 85–98, 101–109, 111–117,
Drug-facilitated assaults............................................23–38 119–126, 137–146, 149–162, 165–179,
181–190, 199–209, 211–221, 223–235,
E
237–259, 261–270
Ethanol biomarkers....................................................... 224 Meconium .......................................................5, 191–196,
Ethyl-glucuronide (EtG) ..................................... 223–235 199–209, 211, 212, 225
Extraction Methotrexate ................................................3, 8, 101–109
liquid-liquid ................................................. 5, 26, 149, Monoclonal antibody (mAb) therapies........... 3, 8, 85–98
199–209, 238, 268
pipette tip .................................................................. 12 N
salt-assisted liquid-liquid ............................... 199–209 Novel benzodiazepines ........................................ 133, 135
solid-phase ..................................................... 6, 12, 26,
Novel opioids ....................................................... 132, 133
28, 113, 146, 149, 150, 154, 156, 200, 212–214, Novel psychoactive drugs .........................................23, 29
217, 220, 225, 229, 230, 238, 251, 258, 268

Loralie J. Langman and Christine L.H. Snozek (eds.), LC-MS in Drug Analysis: Methods and Protocols,
Methods in Molecular Biology, vol. 1872, https://doi.org/10.1007/978-1-4939-8823-5,
© Springer Science+Business Media, LLC, part of Springer Nature 2019

273
 LC-MS IN DRUG ANALYSIS: METHODS AND PROTOCOLS
274 Index
O T
Opioids ..........................................................5, 12, 41–52, Tacrolimus ....................................................103, 111–118
131–133, 149–162, 195, 256, 258 Tapentadol.........................................................41, 43, 45,
Orbitrap ............................................................26, 42, 181 49, 61–65, 256
Teriflunomide ................................................... 75–82, 103
P Therapeutic drug monitoring (TDM)........................ 1–3,
Pain management.................................. 5, 41–50, 52, 120 8, 67–73, 101–103, 109, 111, 120, 262
Time-of-flight (TOF).................................................... 181
Pregabalin .......................................................67, 119–126
Toxicology ..............................................1, 4, 5, 8, 34, 69,
R 70, 131, 137, 182, 190–194

RapidFire ..........................................................6, 113, 115 U

S Ultra-fast mass spectrometry ............................... 111–117
Umbilical cord......................................................... 5, 191,
Synthetic cannabinoids ...................................4, 131–132, 194–196, 211–221, 223–235
134, 137–146, 256