chemosensors

Review
New Applications of Gas Chromatography and Gas
Chromatography-Mass Spectrometry for Novel Sample
Matrices in the Forensic Sciences: A Literature Review
Oliver Gould , Ngoc Nguyen and Kevin C. Honeychurch *

School of Applied Sciences, University of the West of England, Frenchay Campus, Coldharbour Lane,
Bristol BS16 1QY, UK
* Correspondence: kevin.honeychurch@uwe.ac.uk

Abstract: The investigation of novel sample matrices in the forensic sciences offers several possible
advantages, such as allowing for results to be obtained in cases where common sample types are
absent. This review focuses on the application of gas chromatography and gas chromatography–mass
spectrometry (GC-MS) for the determination of drugs in alternative sample matrices, including hair,
sweat, meconium, breast milk, and vitreous humour. Less common sample types are also reported
including air, cerumen, insects, and their larvae and pupae. The application of pyrolysis GC-MS
(Py GC-MS) is also reviewed, showing the possibility of determining high molecular weight drugs
which would commonly be unattainable by GC-MS. The application of Py GC-MS for the simulation
and investigation of the underlying chemistry and the products formed in the smoking of drugs is
also reported.

Keywords: gas chromatography; gas chromatography–mass spectrometry; pyrolysis GC-MS; forensic;

hair; sweat; meconium; breast milk; vitreous humour

Citation: Gould, O.; Nguyen, N.;

Honeychurch, K.C. New
Applications of Gas Chromatography 1. Introduction
and Gas Chromatography-Mass
Proven as powerful analytical techniques, both gas chromatography (GC) and gas
Spectrometry for Novel Sample
chromatography–mass spectrometry (GC-MS) have a long application history. As is the
Matrices in the Forensic Sciences: A
case for all chromatographic techniques, GC separates mixtures by exploiting differences
Literature Review. Chemosensors 2023,
11, 527. https://doi.org/10.3390/
in component distribution between two phases; the stationary phase and the moving or
chemosensors11100527
more commonly called, carrier gas moving phase. An aliquot of the sample mixture to be
separated is introduced to the moving gas phase, just before it encounters the stationary
Academic Editors: María José phase. The sample component separation is dictated by differences in their equilibria
Aliaño-González, Irene Domínguez
between the two phases. Those with low affinity for the stationary phase spend more
Pérez and Roberto Romero-González
time in the moving carrier gas phase and exit the column more quickly (i.e., have shorter
Received: 24 August 2023 retention times). As there is little interaction between molecules and the carrier gas phase,
Revised: 25 September 2023 this phase plays only a small role in separation, acting as only a way to carry the sample
Accepted: 3 October 2023 components through the column. The main separation mechanism is governed by the
Published: 7 October 2023 sample components’ vapor pressures and their sorption to the stationary phase. As a result,
chromatographic separation needs to be carried out at temperatures high enough for the
components’ vapor pressures to be high enough to allow them to exit the column (retention
time) in a realistically short analysis time. However, this temperature needs to be at a value
Copyright: © 2023 by the authors.
such that there are still large enough differences between the individual sample compound’s
Licensee MDPI, Basel, Switzerland.
vapor pressure and stationary phase interactions to allow for chromatographic separation.
This article is an open access article
Too high a temperature can result in poor resolution and the risk of column degradation, too
distributed under the terms and
conditions of the Creative Commons
low a temperature results in long retention times and poor chromatographic performance.
Attribution (CC BY) license (https://
Early applications of GC utilized a single temperature (isothermal) to elute the sample
creativecommons.org/licenses/by/ components; an approach which gives satisfactory results for a few sample components,
4.0/). but not for the majority. Consequently, nowadays most GC separations are undertaken

Chemosensors 2023, 11, 527. https://doi.org/10.3390/chemosensors11100527 https://www.mdpi.com/journal/chemosensors

Chemosensors 2023, 11, 527 2 of 34

using temperature programming. The analysis is started at a low temperature, suitable

for the more volatile sample components. The temperature is then increased in a linear
manner as the analysis proceeds, until it reaches a temperature appropriate for the least
volatile sample components. Using such an approach, each compound starts to migrate
rapidly as the temperature reaches the appropriate level for it. This has been shown to be
highly successful, obtaining some of the highest chromatographic efficiencies presently
available. Using modern capillary columns, theorical plate values exceeding 100,000 can be
obtained [1] compared to only 8000 to 12,000 for high performance liquid chromatography
(HPLC) [2]. Notably, the heating of the sample required for GC analys means that the
analyte needs to be both sufficiently volatile and thermally stable. This can limit the
compounds that can be successfully determined, and derivatization steps are often required
to overcome these problems, an issue generally not affecting techniques such as HPLC.
The volatility of a number of commonly encountered analytes is governed by inter-
molecular hydrogen bonding. The presence of such bonds requires more energy (heat)
when changing from a liquid to a vapor. Consequently, the removal of hydrogen bonding
effects can markedly increase volatility. Such an approach has made the GC determination
of high-boiling point alcohols, phenols, carboxylic acids, and amines a possibility. Removal
of hydrogen bonding can also offer improvements in chromatographic performance as
bonding between the molecule and polar groups present in the stationary phase is less-
ened. As a result, poor chromatographic behaviour, such as peak tailing is alleviated.
The ‘removal’ of these offending hydrogen bonding groups is commonly undertaken by
forming a suitable derivative of these prior to introduction of the analyte to the GC. Many
alcohols, phenols, and amines undergo hydrogen bonding but can be determined by GC as
their acetyl derivatives. Such derivatives can be readily formed by reaction with reagents
such as ethanoic anhydride in the presence of a catalyst, such as hydrochloric acid, before
introduction to the GC. However, probably, one of the most commonly employed deriva-
tization methods is the formation of trimethylsilyl derivatives employing derivatizing
agents such as trifluoro-ethanoic anhydride or N-trifluoroacetyl imidazole can be used
to give their corresponding trifluoro acetyl derivatives. Many alternative methods now
exist for their preparation, such as the reaction of trimethylchlorosilane (TMS) in pyridine
or hexamethyldisilazane (HMDS) and N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA).
Notably, the GC analysis of carboxylic acids is also commonly undertaken by converting
these compounds to their corresponding methyl esters. There are a number of methods
commonly used to obtain this, such as; diazomethane, or methanolic solutions of boron
trifluoride. This is an attractive approach for their GC-MS analysis as select ion mode (SIM)
can readily be used for the monitoring of the indicative, but the uncommonly encountered
m/z 74 ion resulting from the McLafferty rearrangement of the methyl ester.
Helium has proven to be a particularly suitable mobile phase carrier gas for GC
applications. It is both inert in the chromatographic system itself and in the ionization
sources of mass spectrometric detectors. However, it is not a sustainable, renewable
source and as a result there is a growing demand to develop methods that are based on
alternative carrier gases, such as; nitrogen or hydrogen. Nevertheless, the elution of sample
components from the GC in a gas phase has been an attractive feature for GC, as a wide
range of different detector types can be used to give both qualitative and quantitative
outputs. A large number of different detectors are commonly utilized, such as flame
ionization [3], electron capture [4], and nitrogen–phosphorus [5] detectors, to mention but
a few.
One principle draw back with GC is its inability to identify unknown sample com-
ponents. Identification with such detector systems can only be made by running known
standards and then comparing relative retention times. However, one now commonly
employed approached, which to large extent overcomes these issues, is the coupling of GC
with mass spectrometry (MS). MS is extremely good for allowing for the identification of
unknown compounds, be it from user interpretation of mass spectra or via computer-based
matching with various libraries of MS spectra. However, MS alone is poor when faced
Chemosensors 2023, 11, 527 3 of 34

with mixtures; an issue alleviated when coupled with techniques such as GC, which is
particularly good at separating pure compounds out of complex mixtures. Such a coupling
makes GC-MS an extremely powerful technique for a number of different disciplines, such
as forensic science, where quantification and identification of unknown sample components
in complex samples is required.
The first on-line coupling of gas chromatography to a mass spectrometer was reported
in the late 1950s [6,7]. The availability of smaller, more powerful computers allowed for the
further development of the technique and nowadays GC-MS is commonly seen in most
laboratories throughout the world. The ability to handle large volumes of data has allowed
for more complex systems such as two-dimensional gas chromatography (GC × GC) [8] to
be more routinely applied.
MS as a detector system for GC offers a number of advantages. The detector can
be applied in the full scan mode, for near universal detection or in a more selective and
sensitive SIM mode. The full scan mode involves scanning the mass range recorded over a
predefined range. This is typically from m/z 50 to m/z 500 but is dependent on the mass
range anticipated for the sample components of interest. This provides a qualitative and
quantitative picture of the sample, allowing for the detection of any compound eluting
from the GC that is ionizable and produces fragments with m/z values in the investigated
range. The application of lower mass settings can result in interference from nitrogen
(m/z 28), carbon dioxide (m/z 44), or Argon (m/z 40) present in relatively large concen-
trations in air. The approach is less sensitive as, if a very large mass range is selected,
sensitivity is adversely affected, as the number of scans per unit time will decrease. Quanti-
tively, the full scan mode offers the advantage of allowing the mass spectrum of individual
eluting compounds to be obtained and matched against commercially available library
spectra for the identification of unknows. Greater sensitivity and selectivity can be obtained
using the SIM approach. In the selected ion monitoring mode, only the m/z of the selected
fragments are collected. This is a more targeted approach, used for the determination of
known components in the sample. The approach offers greater sensitivity as, if a small
mass range is selected, sensitivity is improved, as the number of scans per unit time is
increased compared to the full scan mode. This feature also helps eliminate interferences
arising due to other ions that can be present in complex sample matrices. Signal-to-noise
ratios are also improved, allowing for improvements in detection limits.
Modern tandem mass spectrometry (MS/MS) instruments utilize the technique of
multiple reaction monitoring (MRM). Ions are first formed, and a compound indicative
predefined selection is made by the first quadrupole. These ions are referred to as precursor
or parent ions and are then fragmented in the collision cell to give further fragment ions
known as product or daughter ions. A second quadrupole then selects a predefined sample
of these. Unlike SIM or full scan mass spectrometry, it is not the actual m/z of the ion
that is used as the analytical result. In MRM, it is the value of the ion transition that is
utilized. Multiple reaction monitoring allows for increased selectivity, due to the specific
collision-induced fragmentations of precursor ions and improved signal-to-noise ratios.
Technology such as pyrolysis GC-MS (Py-GC-MS) has also helped expand the range
of samples that can be successfully investigated. Its application for the determination of
low-volatility compounds was first described in the early 1960s [9–12] and later in 1967 for
Py-GC-MS [13,14]. The addition of mass spectrometry, as a detection system, improves
the utility of the approach, as pyrolysis products formed can firstly be separated by GC,
then identified from their mass spectra. Early recognition of the power of this approach
was demonstrated by its application on the Viking Lander in the search for life on the
planet Mars in 1976 [15], and is now common on a number of missions [16]. Present
day applications of Py-GC-MS range from research, quality control, and characterization
of materials [17] and environmental investigations, including microplastics [18,19], geol-
ogy [16,20], fuels, conservation [21], as well as medicines [22], and for the identification of
bacteria [23]. In forensic science, the technique is commonly employed in the investigation
Chemosensors 2023, 11, 527 4 of 34

of condom materials and lubricants [24], photocopier toner [25,26], packaging tapes [27,28],
and paint samples [29].
Urine and blood remain the most commonly employed biological samples used
for the determination and monitoring of drugs and other substances. However, issues
can be associated with these samples. Urine offers a small detection window of usually
2–4 days and is usually favoured for investigating drug use in sport or in drug management
programs. The risks of tampering or adulteration during transportation and storage are
possible, and issues of privacy in sample collection can also be a problem [30,31]. Blood
has a smaller detection window of 2–24 h and is commonly applied to determine current
intoxication levels [32]. Collection of the sample can be invasive and expensive, needing
trained personnel. Problems can also be associated with locating veins for sampling in
certain groups, such as intravenous drug users.
Hair has also been investigated as an alternative less invasive sample allowing for a
long detection window, being able to show historic patterns of drug use [33]. Nevertheless,
limitations to this as a sample include complex methods of extraction and relatively high
analysis costs [34]. Questions regarding external contamination, particularly for smoked
substances and the low concentrations they represent, coupled with the often-limited
quantities of sample available, can also be an issue [35]. Studies have also shown that
when hair is heated concentrations and the nature of the drugs present can be affected.
In 2016, Ettlinger and Yeagles [36] reported that levels of ∆9-tetrahydrocannabinol (THC)
and cocaine decreased following heat treatment of hair, being converted to cannabinol and
benzoylecgonine, respectively.
In this present review we have focused on the discussion of alternative sample ma-
trixes and their analysis by GC and GC-MS for applications in the forensic sciences. The
review covers recent advances in both GC and GC-MS applications applicable to the
forensic discipline. The applications discussed include sweat and skin [37–52], ceru-
men [53–56], meconium [57–59], breast milk [60,61], larvae, pupae and insects [62–65],
Vitreous humour [66–71], drug paraphernalia [72] cosmetics and fragrances [73–78], and
air, gases and vapours [79–84]. A further section is dedicated to the application of pyrolysis
GC-MS [85–98].

2. Methods
To compile this paper our primary search engine was Google Scholar; however, we
also used Web of Science, and the University of the West of England library. Only papers
with full access and English as the published language were included. Since the nature of
the paper is to discuss the role of GC/GC-MS in the analysis of novel, niche, or underused
sample matrices, we did not set an exclusion date range to enable us to provide a broad
overview of the matrices.
The search terms began with GC or GC-MS with the AND Boolean operator followed
by one or more of: drugs, illicit substances, forensic, volatile compounds, sweat, fragrance,
cosmetics, skin, earwax, meconium, breast milk, larvae, pupae, insects, vitreous humour,
air, and/or vapor.

3. Alternative Sample Matrixes

3.1. Sweat and Skin
Sweat is a fluid secreted from the apocrine and eccrine glands distributed over the
body. It is used to maintain and regulate a constant body temperature [37]. The application
of sweat allows for a possible alternative, non-invasive sample with a large detection
window of one to four weeks, offering cumulative information on drug use and an eco-
nomic alternative to blood sampling. Sweat is readily accessible compared to other bodily
fluids [38] and can be obtained via relatively tamper-proof collection methods and avoids
problems with privacy and the transport of biohazardous fluids. The large detection win-
dow makes sweat an effective approach to determine drug use and is reported to be useful
Chemosensors 2023, 11, 527 5 of 34

in drug testing programs. This includes incidences where sweat on clothing has been used
to detect drugs [39].
The quantity of sweat produced varies from individual to individual, and depends
on the volume of sweat they secrete per gland, and is also affected by changes in their
activity, emotions, and heat [40]. Sweat has low tonicity and is more acidic than blood.
Basic drugs will therefore preferably accumulate in sweat rather than in the blood [41,42].
The concentration gradient that is set up between blood and sweat allows for diffusion
of the free fraction of the drug through the lipid bilayer and its excretion onto the skin
in sweat. Although this passive diffusion mechanism is the main route, excretion also
occurs via sebum and intracellular diffusion [43]. A number of chemical and biochemical
processes can also affect excretions, including possible dissociation constants, molecular
mass, lipophilicity, and protein binding [44].
Cone et al. [44] have shown that a dose as low as 1–5 mg of cocaine was sufficient
to give detectable levels of cocaine and its metabolites in sweat. Published methods
for the detection of substituted amphetamine class drugs such as methamphetamines
and MDMA [45,46], nicotine, cocaine [38,47] opiates and heroin [44], THC and other
cannabinoids [48] have been given. Barnes et al. [45] have adapted a method to specifically
target one or two drug classes to examine sweat following daily doses of methamphetamine
using GC-MS and reported that detection of cumulative doses was successful. Others have
investigated methods for simultaneous drug detection. Kintz et al. [49] have developed a
method for the determination for some of the most commonly abused drug classes; opiates,
amphetamine, cocaine, and cannabinoids using both GC-MS or LC-MS in sweat; whilst
Cone et al. [44] have adapted a GC-MS technique previously applied to oral fluid and
blood, for the detection of heroin, cocaine, and their metabolites in sweat.
One common approach for the collection of sweat is from patches worn for a known
time period, such as weeks or as short as just a few hours. Patches can be hidden and
are generally non-invasive, making them acceptable to participants in trials or testing.
However, due to differences in the density of sweat glands across the body, levels of
detection can differ at different sampling sites; a factor that can have a bearing on the
results obtained [50]. In one such study [50], an eightfold difference in the concentrations
recorded from patches from the lower back and from the upper shoulder were recorded.
Investigations on the effectiveness of sweat patches, compared with urine analysis,
for drug screening of cocaine and opiates have previously been conducted by several
researchers. Preston et al. [47] determined that the greater quantity of cocaine in sweat
patches compared to urine tests was due to different excretion mechanisms, whilst Huestis
et al. [51] detected less opiates. Both, however, obtained results indicating that sweat
patches generated more favourable and reliable results than urine.
A solid phase extraction and GC-MS method for 3,4-methylenedioxymethamphetamine
(MDMA), 3,4-methylenedioxyamphetamine (MDA), 3-hydroxy-4-methoxymethamphetamine
(HMMA), 3-hydroxy-4-methoxyamphetamine (HMA), 3,4-methylenedioxyethylamphetamine
(MDEA), methamphetamine (MAMP), and amphetamine (AMP) in sweat was reported
by De Martinis et al. [46] Drugs were eluted from PharmChekTM sweat patches with a
pH 5.0 acetate buffer, extracted by solid phase extraction and determined using GC-MS
with selected ion monitoring. Limits of quantification (LOQ) for MDMA, MDEA, MAMP,
and AMP were 2.5 ng/patch and 5 ng/patch for MDA and HMA and HMMA.
In addition to the detection of elicit substances from sweat, there is also a slowly
emerging field which attempts to identify an individual based on their produced scent
profile. In 2014, Choi, and Oh [52] used two dimensional GC-MS to determine the charac-
teristics of human sweat profiles, 574 compounds were identified including compounds
associated with age [52]. While the 2014 Choi and Oh study shows promise and significant
potential for forensic utility, it is difficult to find other examples of this type of research;
this might be due in part to a general lack of forensic research funding and the need for
high-resolution mass spectrometers, which generally are not used in forensic laboratories.
Chemosensors 2023, 11, 527 6 of 34

3.2. Cerumen
Recently, a number of reports have investigated other possible samples, including
cerumen (earwax) [53–56]; bone [99,100]; adipocere, also known as corpse wax, grave wax
or mortuary wax; brain tissue; flies; and pupae [58]. Meier et al. reported the detection time
window of cerumen, commonly referred as earwax, to be reportedly in excess of that of
urine but shorter than that reported for hair [55]. Their study showed that in all cases of the
recent use of drugs such as opiates, amphetamine and derivatives, cocaine, methadone and
diazepam, investigations of the corresponding cerumen samples were found to be positive.
In cases where drugs could only be detected in urine, cerumen samples were also found to
be positive. However, where only the hair was positive, drug levels in cerumen were found
in only 52.5% of the cases investigated. However, cannabis use was only detected in 31.6%
of cerumen samples of deceased cannabis users. Unexpectedly, THC was not detected but
its oxidized form, cannabinol, was recorded. Sampling of the cerumen was undertaken
using a cotton swab from both ears prior to autopsy, to avoid contamination with blood.
These were then dried at room temperature for 24 h and extracted by sonicating for
1 h in 1 mL of ethyl acetate containing internal standard. Samples were centrifuged
and evaporated to dryness and reconstituted in the mobile phase for LC-TOF MS and
LC–MS/MS analysis.
Gonçalves Barbosa et al. [56] investigated poisoning in cattle by the pyrethroid insecticide,
bifenthrin. Via examination of the animal’s earwax by headspace/gas chromatography—mass
spectrometry (HS/GC–MS) they were able to detect the presence of bifenthrin in the earwax
of the exposed animals. Samples of earwax were collected directly using a metallic curette.
These were then weighed into 20 mL GC headspace vials, and internal standard was added.
The vials were sealed and examined by HS/GC–MS. This was confirmed by comparison of
the sample’s MS spectrum with a bifenthrin standard, and from its retention time relative
to the internal standard, 3-methylcyclohexanone.
Nicotine has been determined in human cerumen by HS/GC–MS [54]. Cerumen
samples were collected from three different groups based on their smoking habits (non-
smoker, passive, and active smokers). Nicotine cerumen levels were reported to be much
lower than its metabolite, cotinine, even for active smokers, which contrasted with nicotine
levels reported in scalp hair, where the cotinine levels were much lower than nicotine. The
authors concluded that cerumen was potentially a better sample for the measurement of
biological levels as it is better protected from external contamination than hair. Nicotine
concentration profiles, and the related compounds, o-nicotine, cotinine, and anabasine in
the cerumen samples investigated were studied further by data mining and it was shown
to be possible to discriminate between the three different groups. These applications are
summarized in Table 1.
Chemosensors 2023, 11, 527 7 of 34

Table 1. Determination of drugs in sweat, earwax, bone, meconium, breast milk, insects and larvae, and vitreous humour.

Analytes Matrix Derivatization Sample Pre-Treatment Type of GC LOD Comments Ref

Cocaine and heroin,
morphine, 6-acetylmorphine,
Sweat patch extracted with
ecgonine methyl ester,
BSTFA with 1% internal standard by shaking, Cocaine, heroin, and Sweat patch worn for a
ecgonine ethyl ester,
Sweat TMCS, N-Methyl-bis- centrifuged and filtered, and GC-MS/EI metabolites period of several days to [44]
cocaethylene,
trifluoroacetamide then purified by SPE, 1.0 ng/patch. several weeks at a time.
benzoylecgonine,
evaporated, and derivatized.
norcocaine, norcocaethylene,
and benzoylnorecgonine
Patches (PharmChek™)
were folded and placed in
acetate buffer (pH 4.0) for Weekly sweat patches were
MTBSTFA with 1% 30 min at room temperature. applied to participants, one
Limit of quantification of
MAMP and AMP Sweat TBDMCS and BSTFA The solution was then GC-MS/EI on the back and one on the [45]
2.5 ng/patch.
with 1% TMCS. purified by SPE. Analytes abdomen, and removed after
eluted and evaporated to a week.
dryness under N2 and
derivatized.
Limits of quantification A sweat patch was applied
Drugs were eluted from for MDMA, MDEA, for various periods prior to,
MDMA, MDA, HMMA,
Triethylamine in PharmChek™ sweat patches MAMP and AMP were during, and after MDMA
HMA, MDEA, Sweat GC-MS/EI [46]
heptane and HFAA. with sodium acetate buffer, 2.5 ng/patch, and administration. A 1.0 mg/kg
methamphetamine and AMP
extracted with disk SPE. 5 ng/patch for MDA, of MDMA was administered
HMA and HMMA. orally to a participant
3 ng/mL for cocaine and
The absorbent sweat pad
2 ng/mL for
was extracted with 75%
benzoylecgonine and Sweat and urine specimens
methanol/25% 0.2 M
ecgonine methyl ester. collected from
Cocaine, benzoylecgonine, Sweat and sodium acetate, pH 5.0 by GC-MS/EI in
-- Limits of quantitation 44 methadone-maintenance [47]
and ecgonine methyl ester. urine shaking for 30 min; eluent SIM
were 4 ng/mL for patients. ELISA
was then analysed by ELISA
cocaine and 2 ng/mL for immunoassay compared.
and confirmed by GC-MS by
benzoylecgonine and
SIM.
ecgonine methyl ester.
Chemosensors 2023, 11, 527 8 of 34

Table 1. Cont.

Analytes Matrix Derivatization Sample Pre-Treatment Type of GC LOD Comments Ref

In total, 11 daily cannabis
Patches were extracted with users after cessation of drug
Derivatized with
methanol/sodium acetate GC/MS Limit of quantification, use. PharmChek® Sweat
∆9-tetrahydrocannabinol Sweat trifluoroacetic [48]
buffer pH 5, by shaking and negative ion CI. 0.4 ng/patch. patches worn for 7 days.
anhydride
isolated by SPE. Percent recovery from
patches was 44–46%.
Sweat pads extracted in
Heroin, methanol containing
6-monoacetylmorphine, deuterated internal
morphine, codeine, cocaine, standards by shaking.
benzoylecgonine, ecgonine Methanol solution was
AMP and related
methyl ester, divided into 3 for
compounds with
∆9-tetrahydrocannabinol, buprenorphine testing,
heptafluorobutyric
nordiazepam, oxazepam, amphetamines testing, and Buprenorphine was
anhydride. Other From 0.01 to
AMP, methamphetamine, Sweat the remainder for the other GC-MS/EI identified and quantitated [49]
drugs by silylation 2.0 ng/patch
methylenedioxyam- compounds. Evaporated to by LC/MS.
with BSTFA and
phetamine, dryness. Amphetamine and
trimethylchlorosi-
metbylenedioxymetham- related compounds were
lane.
phetamine, identified after
metbylenedioxyethylam- derivatization with
phetamine and heptafluorobutyric
buprenorphine. anhydride; other drugs were
derivatized by sialylation.
The percentage
The absorbent pad was cross-reactivity at 10 ng/mL
removed and extracted with for each analyte was 100%
buffer, 75% methanol/25% for morphine, 28% for
Heroin, 6-acetylmorphine, 0.2 M sodium acetate ELISA and heroin, 30% for
Sweat --- GC-MS, 3 ng/mL [51]
morphine, and codeine (pH 5.0), by shaking. The GC-MS 6-acetylmorphine, 588% for
eluent was then analysed codeine, 143% for
according to package hydrocodone, 16% for
directions by ELISA. hydromorphone, and 30%
for oxymorphone
Chemosensors 2023, 11, 527 9 of 34

Table 1. Cont.

Analytes Matrix Derivatization Sample Pre-Treatment Type of GC LOD Comments Ref

The monitored ions for
Earwax samples (20 mg) quantification were m/z
Cotinine, a major metabolite
transferred to GC vials, and HS/GC–MS by 84 for nicotine, o-nicotine
of nicotine, o-nicotine, and Earwax --- --- [54]
3-methyl cyclohexanone was SIM. and anabasine, m/z 98 for
anabasine.
added as internal standard. cotinine, and at m/z 69 for
3-methyl cyclohexanone (IS).
Samples were weighed into m/z ratios of 181 (base peak
Earwax in GC headspace vials, and IS, HS/GC–MS by of bifenthrin) and 112 and
Bifenthrin --- --- [56]
cattle 3-methylcyclohexanone SIM. 69 (molecular ion and base
added. peak of IS).
Soft tissues were removed
from the bone. Samples were
cut in small fragments, dried
and pulverized using a ball
mill. An aliquot of bone
powder and internal
standard solution and
methanol were vortexed and
incubated and sonicated and
Pregabalin, m/z;
centrifuged. The
41-43-55-69-84-141.
supernatants were recovered
Quetiapine and pregabalin Human bone GC–MS by SIM 0.1 ng/mg for both. Quetiapine m/z [99]
and evaporated. PBS; 0.1 M,
144-210-239-321. Sertraline
pH 6 was added and
(IS) m/z; 159-262-274-304
samples were subjected to a
SPE and eluted with
dichloromethane:isopropanol:
ammonia and then
evaporated. Samples were
reconstituted with 100 µL of
ethyl acetate, vortexed
before introduction to the
GC-MS.
Chemosensors 2023, 11, 527 10 of 34

Table 1. Cont.

Analytes Matrix Derivatization Sample Pre-Treatment Type of GC LOD Comments Ref

Fatty acid ethyl esters. 0.3 g Meconium added to a 2.5 ng/g cocaine, 6 ng/g
MeconiumGC-FIDCocaine, 10 mL glass tube spiked with GC-MS in SIM anhydroecgonine methyl
60 µL acetonitrile,
benzoylecgonine, ISTD 200 ng/g and mode, ester, 8 ng/g Meconium samples were
20µL MSTFA,
anhydroecgonine methyl Meconium methanol added for multi-step cocaethylene, 10 ng/g spiked, there were no [58]
vortexed then heated
ester (metabolite of crack extraction. An amount of temperature benzoylecgonine, non-spiked samples tested.
60 ◦ C for 20 min.
cocaine), nicotine, and 0.1 M HCL was added prior ramp 15 ng/g nicotine, 5 ng/g
cotinine to the pipette SPE process. cotinine
Organophosphates:
chlorpyrifos, dichlorvos,
dimethoate, Liquid extraction with
demeton-smethyl, ethion, acetonitrile and internal GC-MS, SIM Range from 0.21 to
Meconium [59]
malathion, omethoate, standards. Dry residue mode 320.43 µg/g
pirimiphos-methyl, reconstituted with toluene.
pyrazophos, and
tolclofos-methyl
A very simple extraction
20 target analytes, only DMP, Liquid/liquid extraction method was used, which has
Range from 0.5 to
DEP, DIBP, DBP, and DEHP Breast Milk with acetonitrile saturated in GC-MS/MS the benefit of being easily [61]
211.2 µg/kg
detected. n-hexane. reproducible, no dangerous
levels were found.
Organochlorine Pesticides
(aldrin, dieldrin, endrin,
alpha-endosulfan,
beta-endosulfan, endosulfan
A 60 m column with a
sulphate, heptachlor, All organopesticides
Breast Milk Soxhlet extraction GC-MS 54.9 min sample [101]
heptachlor epoxide isomer B, LOD 0.01 ng/mL
analysis time.
methoxychlor, endrin ketone,
endrin, pp’-DDE, pp’-DDD,
pp’-DDT, α−HCH, β−HCH,
γ−HCH and δ−HCH)
Heroin and associated Flies and Alkali–acid base extraction
GC-MS [63]
metabolites larvae method
Chemosensors 2023, 11, 527 11 of 34

Table 1. Cont.

Analytes Matrix Derivatization Sample Pre-Treatment Type of GC LOD Comments Ref

Larvae,
Washed with DCM,
pupae, spent
crystalized with liquid
pupae and Trifluoroacetic GC-MS SIM Negative in adult Calliphora
Methamphetamine nitrogen, homogenised, and 5 ng/mg [64]
adult anhydride. mode vomitoria L.
extracted with methanol
Calliphora
before derivatisation.
vomitoria L.
Blowfly Centrifuged homogenate of
BSTFA at 70 ◦ C for
larvae reared the larvae and or liver has
15 min for the
on liver the supernatant removed
Opiates, cocaine, barbituates, detection of GC-MS SIM Methods are difficult to
samples prior to derivatisation. SPE Not reported [65]
and antidepressents morphine-2TMS and mode follow.
which tested or liquid-liquid extraction
benzoylecgonine-
positive for flash-alkylation with
TMS
the drugs MethElute.
Internal standards and
ascorbic acid are mixed and
precipitated with zinc
sulphate, then centrifuged.
GC-MS Full
Drug and metabolite Vitreous The supernatant is treated
scan m/z Not reported [66]
screening Humour with sodium acetate. This is
43–550
then filtered through an SPE
column. Sample extracts are
reconstituted with ethyl
acetate.
THC-COOH-d3 was added
to the biological sample.
Hydrolysis was performed
11-Nor-9-carboxy-∆9 Vitreous MSTFA heated at with sodium hydroxide and
GC-MS 1 ng/mL [68]
tetrahydrocannabinol Humour 90 ◦ C for 30 min hexane, the aqueous layer
was transferred, acidified
with HCL and extracted
with hexane/ethyl acetate.
Chemosensors 2023, 11, 527 12 of 34

Table 1. Cont.

Analytes Matrix Derivatization Sample Pre-Treatment Type of GC LOD Comments Ref

Samples mixed with
phosphate buffer 0.25 M
pH 8.4 along with
deuterated analogues of
Meprobamate, morphine, Bile, Vitreous BSTFA/TMCS
each analyte.
cyamemazine, caffeine, Humour, morphine analysed GC-MS/MS Not reported [69]
For morphine, meprobamate,
diazepam, and citalopram Whole blood as TMS derivitive
cyamemazine, and caffeine
an automated SPE method
was used before phosphate
buffer was added.
Antidepressant drugs;
amitriptyline, nortriptyline, SPE using a non-polar C8
citalopram, clomipramine, sorbent and phosphate
fluoxetine, maprotiline, Dry residues were buffer 0.1 M pH 6.0, columns
mirtazapine, sertraline and Vitreous derivatized with are then acidified with acetic Dynamic range 5-500 ng/mL
GC-MS SIM 1.5 ng/mL for all
venlafaxine, and 4 of their Humour, HFBA in ethyl acid and washed with r2 = 0.99. %RSD less than [71]
mode analytes
metabolites, Whole Blood acetate at 50 ◦ C methanol. Then, derivatized, 10.9% for all analytes.
desmethylmaprotiline, 30 min. and evaporated, dry samples
desmethylmirtazapine, were reconstituted with
desmethylsertraline, ethyl acetate.
O-desmethylvenlafaxine,
AMP amphetamine; BSTFA N,O-bis(trimethylsilyl)trifluoroacetamide; CI chemical ionization; ELISA enzyme-linked immunosorbent assay; GC-MS/EI gas chromatography–mass
spectrometry electron impact ionization; HMA 3-hydroxy-4-methoxyamphetamine, HMMA 3-hydroxy-4-methoxymethamphetamine; HS/GC-MS headspace gas chromatography–mass
spectrometry; HFAA heptafluorobutyric acid anhydride; IS internal standard; LOD limit of detection; MAMP methamphetamine; MDA 3,4-methylenedioxyamphetamine; MDEA
3,4-methylenedioxyethylamphetamine; MTBSTFA N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide; MDMA 3,4-methylenedioxymethamphetamine; PBS Phosphate buffered saline;
SIM select ion mode; SPE solid phase extraction; TBDMCS N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide; TMCS trimethylchlorosilane; DMP dimethyl phthalate; DEP diethyl
phthalate; DIEP diisobutyl phthalate; DBP di-n-butyl phthalate; DEHP bis(2-ethylhexyl) phthalate.
Chemosensors 2023, 11, 527 13 of 34

3.3. Meconium
Meconium is the earliest stool of a mammalian infant resulting from defecation. Unlike
later faeces, meconium is composed of materials ingested during the time the infant spends
in the uterus. Consequently, it can be utilized as a sample to investigate in utero exposure.
Recently Mozaner-Bordin et al. used GC-MS [57] to analyse 50 meconium samples,
and were able to detect and quantify cocaine, benzoylecgonine, anhydroecgonine methyl
ester (metabolite of crack cocaine), nicotine, and cotinine. A solid-phase extraction method
was used to extract the analytes, based on the modification of a disposable pipette tip with
solid-phase material. The resulting residue was derivatized with acetonitrile and MSTFA.
A splitless injection was used with a 30 m × 0.25 mm capillary column (0.25 µm film
thickness), these column dimensions are probably the most commonly used in most labs.
The temperature ramp was carried out in different stages starting at 70 ◦ C and working up
to 320 ◦ C, with the total run taking around 20 min. The MS was set to SIM mode with EI,
following method development three ions were selected for each analyte to allow for both
identification and quantification. This process can help reduce noise and increase sensitivity,
which is beneficial for targeted analysis. This group calculated with standards the linearity,
LLOD, and LLOQ, which are common in GC-MS studies. However, this group also took
the step of measuring the precision and accuracy over several days using a QC, which
would have to be performed in any forensic lab carrying out this type of testing; while
many labs carry out this type of procedure it is not always reported in the written article.
Cocaine was detected at the 2.5 ng/g level, however, the LOQ was 10 ng/g. Nicotine was
the most inconsistent, with recovery percentages ranging from 50.72 to 95.33% [58].
Analysis of meconium is not only limited to the detection of illicit substances. A study
of new-borns from Thailand used meconium to determine organophosphate exposure.
Eight different organophosphates were found across 98% of the 68 babies analysed. This
study showed that there were no statistical differences in exposure to babies whose mothers
worked in agriculture and those who lived within 1 km of agricultural sites [58]. Again,
this group used an Agilent GC-MS with a 30 m × 0.25 mm capillary column (0.25 µm film
thickness), and stepped up the temperature ramp starting at 50 ◦ C and going up to 290 ◦ C.
The MS used EI mode and SIM. The total analysis time was 33 min. This paper provides
fewer details in terms of the methods and, specifically, the testing of the methods for
consistency. There was no derivatization step here, but there was a liquid extraction process.
An amount of 0.15 g meconium was added to a phosphate buffer/methanol solution and
vortexed prior to internal standard addition. Acetonitrile was added prior to centrifugation,
and this process was repeated twice. The evaporated residue was reconstituted with
toluene prior to GC-MS injection (using a pulsed splitless injection) [59]. The benefit of
standard methods being used by multiple groups and labs around the world is that they are
tried and tested and thus are easier to present as forensically valid; as this paper suggests,
the future is not necessarily new instruments or modifications but new applications and
sample preparations.

3.4. Breast Milk

Similar to meconium, breast milk can serve as an ideal matrix to monitor infants’
exposure to medications, drugs of abuse, pesticides, and metals. In a literature review
by Drabinska et al., (2021) [59] cataloguing volatile compounds identified from healthy
human individuals, the authors cite 290 compounds that have been identified in breast milk.
A number of these compounds came from papers investigating exogenous compounds
such as: organochlorine pesticides, brominated diphenyl ethers, dioxins, polychlorinated
biphenyls, parabens, and flavonoids (along with numerous others). Compounds from
garlic-family food stuffs were also identified in breast milk.
Fan et al., 2020 [60] presented the GC-MS/MS analysis of breast milk to detect con-
tamination with phthalates from breast milk storage bags. Through GC-MS/MS analysis,
six different phthalates were detected from 40 samples, and while some contamination of
breast milk was discovered, the reported intake concentrations were determined to be of no
Chemosensors 2023, 11, 527 14 of 34

risk to the infants. Fan, et al., 2020 [60] used an Agilent GC-MS/MS in EI mode. The column
was again a 30 m × 0.25 mm with a 0.25 µm stationary phase layer. The temperature ramp
used multiple steps starting at 60 ◦ C and moving up to 280 ◦ C. Splitless injection was used;
this technique is common for trace level analysis as all the sample enters the column, as
opposed to the split method, which has the benefit of removing the solvent but also sweeps
out some of the sample (not ideal for trace-level analysis). The samples were extracted
with a liquid–liquid extraction, the solvent used was acetonitrile, saturated with n-hexane;
the samples were reconstituted using n-hexane. The concentration range of the detected
phthalate esters ranged from 0.5 to 211.2 µg/kg [61].
Similarly, Witczak et al., 2020 [61] use GC-MS to assess the presence of organochlorine
pesticides in breast milk. Concentrations of beta-endosulfan showed a positive correlation
to maternal fish and poultry consumption during pregnancy. One of the primary limitations
of GC-MS is the inability to directly analyse aqueous samples due to column degradation,
thus biological samples will, in most cases, require some form of pre-concentration, or
solvent extraction, which in a forensic laboratory can be costly in both time and resources.
In the case of the Witczak study, Soxhlet extraction was carried out but the authors present
limited details as to this process. This group used a 60 m column with a 0.25 mm ID
and 2.25 µm film. Using a 60 m column over the more common 30 m can result in better
resolution and efficiency, as determined by the calculation of theoretical analytical plates.
However, doubling the length of the column will not necessarily double the resolution, but
will result in a longer analysis time, which is evidenced by the 54.9 min per sample analysis
time. There are no details about the MS detector setting provided in this paper. As a result
of the GC being unsuitable to aqueous samples, very often the preferred analysis method
for breast milk involves liquid chromatography, usually coupled to MS or a variant thereof.

3.5. Larvae, Pupae and Insects

Different insects are associated with each of the stages of human and animal decompo-
sition. Different populations of flies, and then beetles, followed by moths, are attracted to
the body as the process of decomposition progresses. Flies from the family Calliphoridae,
especially those from the subfamilies Chrysomyinae, Calliphorinae, and Lucillinae are the
first to be attracted to the body for the process of egg laying. Their offspring consume
dead tissue and, in the process, chemicals such as drugs that may be contained in the
tissue. Consequently, they can be used as an indirect way of assessing the drug levels in
the deceased animal or human. However, in the pupal stage, a significant reduction in the
concentration of drug is seen, as digestion and metamorphosis occur [101].
The possibility of detecting heroin and its metabolites in flies and their larvae feeding
on heroin-fortified meat samples was investigated by Ishak et al. [62]. Flies were collected
from a local wet market. Out of this sample, L. cuprina were selected and then reared till
their third generation. Their eggs were then placed onto separate minced buffalo meat
samples fortified with saline solutions of heroin (0.5 to 10 µg/µL). Every eight hours, ten
individual first-instar larvae, ten individual second-instar larvae, and ten migrating third-
instar larvae were collected. These were then extracted and examined by GC-MS. Using
the toxicology mass spectrum library available with the instrument, the heroin metabolites
tryptophan, hydromorphone, and morphine were determined in the analysis of heroin fed
L. cuprina in the second- and third-instar larvae, but not in the first instar or pupa.
Magni et al. [63] have shown the possibility of determining methamphetamine in the
larvae, pupae, spent pupae, and adults of Calliphora vomitoria L. (Diptera: Calliphoridae)
feeding on liver with doses of methamphetamine matched to human lethal dosages of
5 ng/mg and 10 ng/mg. Larvae, pupae, spent pupae, and adult samples were washed with
dichloromethane. These were then crystallized with liquid nitrogen, homogenized, and a
100 mg aliquot extracted with methanol containing the internal standard, diphenylamine,
by heating at 55 ◦ C for 15 h. After cooling, the organic phase was acidified with trifluoracetic
acid and dried at 70 ◦ C under nitrogen. The resulting sample extract was then derivatized
with trifluoroacetic anhydride, dried at 80 ◦ C, and reconstituted in tert-butyl methyl
Chemosensors 2023, 11, 527 15 of 34

ether. Methamphetamine was determined by GC–MS in the SIM mode using m/z 154,
118, 110, and 91. Methamphetamine was determined in the immature stages and spent
pupae of C. vomitoria that had been previously feeding on liver containing 5 ng/mg and
10 ng/mg methamphetamine at 23 ◦ C. Notably no methamphetamine was recorded in in
C. vomitoria adults as reportedly upon emergence as an adult, the flies rapidly eliminate
methamphetamine.
Campobasso et al. [64] have investigated the relationship between drug concentrations
present in human liver samples obtained from 18 necropsies previously shown positive for
drugs and Blowfly larvae (Diptera: Calliphoridae) reared upon them. Subsequently GC-MS
analysis showed that all drugs recorded in the samples of human tissue were also detected
in insect specimens. Opiates, cocaine and barbiturates, and antidepressants (clomipramine,
amitryptiline, nortryptiline, levomepromezine, and tioridazine) were reported. The larvae
of Lucilia sericata (Diptera: Calliphoridae) were also reared on fortified liver samples and
active feeding mature maggots and post-feeding maggots were collected. Fresh greenbottle
(Lucilia sericata) eggs were transferred onto fortified liver samples and reared in an incubator.
Mature larvae were collected at their peak feeding period, four days after laying, while post-
feeding larvae were collected after six days. Once removed from the food source, maggots
were washed with deionized water and prior to analysis samples were homogenized in
deionized water.
Gas chromatographic mass spectrometry investigations were performed in the SIM
mode following solid-phase extraction and derivatization with BSTF, using the ions: m/z
236, 287, 429 for morphine-2TMS and m/z 82, 240, 361 for benzoylecgonine-TMS. Phenobar-
bital quantification, the internal standard (butalbital) was added followed by liquid–liquid
extraction. Extracts were analysed by GC-MS in the SIM mode following flash-alkylation
with MethElute using the ions; m/z 232, 260 for methylated phenobarbital and m/z 195,
196 for the methylated-internal standard. Thioridazine (m/z 98, 370), clomipramine,
amitriptyline (m/z 58, 202), nortriptyline, and levomepromazine (m/z 58, 328) were deter-
mined again, using GC-MS in the SIM mode following liquid–liquid extraction with SKF
525-A as the internal standard.

3.6. Vitreous Humour

The vitreous humour (VH) is a gel-like liquid that fills the eye; as individuals age
the VH becomes less viscous. Due to the invasive nature of testing, VH analysis is al-
most solely reserved for post-mortem sampling. The VH offers long sample stability and
a reduced need for pre-treatment versus blood, for example. However, there is a limit
to the concentration as any target analyte must cross the blood–brain barrier. In 2016,
Metushi et al. [65] compared the analysis of post-mortem whole blood (WB) to VH analysis
to assess the effectiveness of the VH in forensic toxicology, using GC-MS. VH samples
were extracted from the eye with a syringe (ca. 5 mL), 20 mL of WB was added to a glass
vial with sodium fluoride and potassium oxalate. SPE extraction was used to extract the
analytes from the matrix. In this instance, a 15 m × 0.25 mm column was used with
0.25 µm film thickness. A starting temp of 85 ◦ C and a 40 ◦ C/min ramp was used to 170 ◦ C
before a 4 min hold then another 40 ◦ C/min ramp to 190 ◦ C (held for 5 min) and then the
final stage of 10 ◦ C/min until an end temperature of 300 ◦ C was held for 7 min. The MS
was set to full scan with m/z 43–550. Seventy drugs were identified in this study, all of
which are used in medical or clinical treatments but can also be subject to abuse, e.g., di-
azepam. The group found that while there were more identified analytes from whole blood
(209 HB vs. 169 VH) the two matrices were very similar in terms of concentrations, with the
exceptions of trazodone and diazepam, which were both significantly higher in WB [65].
In a 2008 brief communication, a gas chromatography–flame ionisation detector (GC-
FID) was used to determine the presence of phencyclidine (PCP) and GC-MS to analyse
for cannabinoids both in VH [66]. The results of the PCP analysis were promising with
60% of the samples testing positive for the presence of PCP in concentrations ranging from
30–290 ng/mL, however, the group noted that there was no correlation between VH and
Chemosensors 2023, 11, 527 16 of 34

WB PCP concentrations. The results with regard to cannabinoids were less encouraging
as all screened VH samples tested negative. As this is a brief communication there is only
limited information on the GC-MS methods.
Similar findings were also suggested from an earlier study in 2005 in which GC-MS
was used to determine cannabinoids levels in VH. A 12 m × 0.2 mm (0.33 µm film thickness)
fused silica capillary was used, a simple temperature ramp starting at 160 ◦ C and ending at
280 ◦ C at a rate of 25 ◦ C/min was used. SIM mode detection was performed using m/z 371,
473, and 488 for THC-COOH and m/z 374, 476, and 491 for THC-COOH-d3 . Derivatization
was conducted with MSTFA heated at 90 ◦ C for 30 min. In this study, 11-nor-9-carboxy-
∆9 tetrahtdrocannabinol-d3 was used as an internal standard. However, in the few instances
that cannabinoids were detected, they were found to be below 10 ng/mL. It was suggested
that this is due to the hydrophobic nature of 11-nor-9-carboxy-∆9 tetrahtdrocannabinol [67].
In 2015, Bévalot et al. [68] compared WB and VH from humans and rabbits for the
detection of meprobamate, morphine, cyamemazine, caffeine, diazepam, and citalopram.
The group report that in rabbits all six drugs were detected and showed correlation with WB,
but the same was not true for cyamemazine and diazepam in the human subject. Even so,
all six drugs were detected in humans. Interestingly this group used gas chromatography–
tandem mass spectrometry (GC-MS/MS), which differs from conventional GC-MS by
adding an additional quadrupole inline separated by a collision cell. The ions from the
first quadrupole enter the collision cell where they are fragmented further and then moved
onto the second quadrupole for detection to occur. This process eliminates almost all
background noise resulting in a superior signal-to-noise ratio versus traditional GC-MS.
The net result of this is low limits of detection; thus, for targeted analysis, such as the work
by Bévalot et al., 2015, GC-MS/MS has benefits [68]. However, GC-MS/MS is limited by
lower mass accuracy and resolution; thus, single quad GC-MS remains the preferred option
for untargeted sample analysis [69]. Moreover, GC-MS/MS is not routinely deployed in
forensic laboratories and given the substantial outlay, both financial and in terms of space
and time, there would need to be multiple routine use cases before we see an uptake by
forensic providers.
A new analyte target has been proposed recently: Ntoupa et al., 2020 [70] suggested
that antidepressants would be an ideal analyte target for VH analysis. In this instance SPE-
GC-MS was employed for analysis. The extraction method involves several steps, including
extraction, centrifuging, drying, and derivatization with heptafluorobutyric anhydride
(HFBA) and ethyl acetate for 30 min, which of course is all in addition to the GC-MS
analysis time itself. This leads to a long analysis time, which can be a significant factor in
laboratories with limited resources. The column was a 30 m × 0.25 mm column with a
temperature ramp starting at 100 ◦ C and extending up to 300 ◦ C at a rate of 40 ◦ C/min. A
splitless injector method was used with an injector temp of 240 ◦ C. However, despite the
long sampling times, this method of analysis was able to detect nine antidepressants and
four metabolites with a LOD and LOQ of 1.5 and 5.0 ng/mL. The VH analysis matched
whole blood analysis for the number of times antidepressants were detected, and in 14 cases
yielded a higher concentration in VH compared to whole blood.

3.7. Drug Paraphernalia

Recently, Russell et al. [71] have used direct analysis in real-time mass spectrometry
(DART-MS) for the detection of drugs present on swabs taken from drug paraphernalia.
Samples were collected in a four-step procedure. Firstly, the drug paraphernalia sample
was wiped or swabbed. This was then placed into a small paper envelope and mailed to
the laboratory and then extracted and analysed by DART-MS. Samples collected from eight
syringe services programs from November 2021 to August 2022. Out of the 364 items of
drug paraphernalia investigated, which had shown positive for fentanyl or its analogues,
80% also contained xylazine, a drug commonly used as an animal sedative. Interestingly,
heroin was only recorded in 1.9%, and other non-fentanyl opioids, such as tramadol,
methadone, and protonitazene, in only 2.5% of the samples. Among the opioid-positive
Chemosensors 2023, 11, 527 17 of 34

samples, 98.9% contained fentanyl, with smaller contributions from its analogues of 6.3%
fluorofentanyl and 1.6% fentanyl carbamate, with one sample containing fluorofentanyl
only. All other fentanyl analogues, such as fluorofentanyl and fentanyl carbamate, were
also detected with fentanyl.

3.8. Detection of Cosmetics and Fragrances for Forensic Applications

Since the 1970s, there has been a growing body of research involving the detection
of cosmetics for forensic applications. In 2019, a review by Chophi et al. [102] examined
the trends in forensic cosmetic analysis. Amongst the numerous techniques described, the
group suggest attenuated total reflectance–Fourier transform infrared spectrometry (ATR-
FTIR) and Raman spectroscopy as the stand out trends, due to their non-destructive nature.
Despite the suggestions toward spectroscopic methods of analysis, there are still numerous
papers in which GC (coupled to various detectors) was used for cosmetic analysis. Since
there was a 2019 review on the subject and the scope of this paper focuses purely on new
trends in forensic GC/GC-MS analysis, this section will highlight some more recent studies
from the past four years (since 2019, as of the time of writing).
Recently, Ferrari Junior et al., 2022 [73] utilized headspace (HS) GC-MS to analyse
cosmetics seized by the Civil Police of the Federal District in Brazil and, found via deriva-
tization, that formaldehyde concentration levels far exceeded the legal levels (reporting
concentrations of 0.33–4.02%). Direct headspace analysis was used for this analysis; how-
ever, this can suffer from reduced levels of analyte reaching the instrument compared to
preconcentration techniques such as thermal desorption, but it involves far less sample
preparation. This group proposed a novel derivatization method and tested several differ-
ent solutions containing methanol, ethanol, and hydrochloric acid in various ratios. The
group determined that the optimal ratio was 25:25:1 (Methanol, ethanol, hydrochloruic
acid), incubated for 4 h at 60 ◦ C. An injection volume of 250 µL was used at a split ratio of
15:1 on to a 30 m × 0.25 mm column with 1.4 µm film thickness. The GC oven ramp started
at 35 ◦ C and ended at 50 ◦ C with a run time of only 11 min. The cool GC temps and the
thick film column allow for analysis of highly volatile analytes.
With a similar application, Nguyen et al., 2022 [74] used both GC-MS, and GC-FID to
analyse pharmaceutical tablets: over 56 months, 567 tablets were analysed and 119 identi-
fied as being counterfeit. All the pills were analysed in Washington D.C., of the counterfeit
pills, fentanyl was identified as the main psychoactive substance, but other opioids and pre-
cursors were detected. The GC-MS used splitless injection with 30 m × 0.25 mm columns
with 0.25 µm film thickness, and the GC-FID used a 12 m ×0.2 mm column with a 0.33 µm
thick film. This research, much like the work carried out in Brazil, emphasizes the need for
routine testing of even seemingly legitimate products. GC-MS and GC-FID with easy to
automate analytical methods both of samples and backend data are ideally suited to this
type of high-throughput work.
A study on the potential of fragrances to be used as a form of trace evidence was
carried out in 2019 by Gherghel et al. [72] involving experiments on different variables
that may affect the transfer and persistence of fragrances (ageing time of fragrances on the
donor fabric, contact time between the donor fabric and the recipient fabric, and fabric
type). Solid-phase micro-extraction (SPME) GC-MS techniques were used to extract and
analyse the volatile organic compounds (VOCs) in both the primary and secondary fabric.
A 30 m × 0.25 mm column with 0.25 µm film thickness was used, the temperature program
started at 35 ◦ C, increasing to 300 ◦ C, for the first portion a slow ramp of 5 ◦ C/min was
used to 180 ◦ C. There was a 60 min SPME extraction time at 58 ◦ C, with splitless desorption
at 250 ◦ C for 3 min. The results showed that all the VOCs from the donor fabric were
recovered and detected successfully in the secondary fabric despite the short contact time
(10 s) between the two pieces and the up-to-48-hour ageing time of the primary piece.
Moreover, the number of low-volatility compounds recovered from secondary fabric was
higher than that of high-volatility compounds. Therefore, low-volatility compounds were
considered more suitable for forensic analyses than high-volatility compounds. This study
Chemosensors 2023, 11, 527 18 of 34

concluded that VOCs within fragrances that stay on clothing are possibly worth analysing
in cases such as sexual assault or rape, as they could benefit the establishment whether a
contact has occurred between the victim and suspect.
GC-MS was also used in a 2021 study by Raynor et al. [75] to test the persistence
of moisturizer products on human skin, regarding the benefits they might have in in-
vestigating sexual assault cases. The moisturizer components targeted for analysis were
methylparaben, ethylparaben, petrolatum, isopropyl palmitate, glycerol, and cetyl alcohol.
Participants were asked to apply either oil-based or glycerol-based moisturizer, and their
forearm was swabbed within 24 h after the application. Water pre-wetted swabs were found
to be the most effective technique to detect petrolatum compared to those pre-wetted with
ethanol and isopropanol. A GC-MS instrument in full scan mode analysing 30–800 m/z
was used with a 30 m × 0.25 mm × 0.25 µm column. The moisturizer on human skin can be
detected for up to 23.5 h, with the average persistence time of the glycerol-based product of
19.8 ± 1 h and the oil-based product of 13.5 ± 0.7 h. Although this type of evidence holds
potential in forensic applications, its time persistence, sample collection, and sampling
protocols require further investigation and development.
Recently, a proposed analytical method to measure trace levels of nine banned N-
nitrosamines in cosmetic products, which consisted of GC-MS combined with vortex-
assisted dispersive liquid–liquid microextraction (VA-DLLME), was successfully developed
and validated by Schettino, Benedé and Chisvert (2023) [76]. N-nitrosamines are banned
due to their harmful mutagenic, carcinogenic, and teratogenic effects. Therefore, not only
N-nitrosamines are prohibited, but also the substances (secondary alkyl and alkanolamines)
involved in forming N-nitrosamines. Chloroform was used as the extraction solvent as
it allowed the formation of microemulsion by vortexing without the help of a disperser
solvent. Three variables of the VA-DLLME (the vortex time, the extractant volume, and the
ionic strength of the donor phase) were optimized, and an appropriate way to introduce
the sample to the GC was also successfully determined to be a 2 µL injection to a splitless
injector at 230 ◦ C. A 30 m × 0.25 mm × 0.25 µm column with a polyethylene glycol
stationary phase was used. The ramp began at 60 ◦ C and increased to 240 ◦ C. MS acquisition
was carried out in both the SIM and full scan modes. The values achieved from method
LODs and LOQs were recorded to be under the threshold value set by the European
regulation on cosmetic samples. The lowest LOD was 0.2 ng/L for N-nitrosodibutylamine
(NDBA) with a LOQ of 0.6 ng/L.
Hemp oil is a beauty product made from hemp seeds obtained from the Cannabis
Sativa plant. The application of this type of product was claimed to possibly interfere with
the determination of cannabinoid levels in hair samples. To demonstrate the implication,
Paul et al., (2019) [77] conducted research involving applying hemp oil directly to par-
ticipants’ head hair, which was then analysed by GC-MS/MS with the multiple reaction
monitoring (MRM) acquisition mode carried out. The volunteers all declared that they had
neither consumed nor been exposed to cannabis before. They were asked to apply hemp oil
to their hair and still carry out their normal hygiene routine for six weeks. Their hair sam-
ples were collected before and after the hemp oil application for analysis. All samples were
taken through a decontamination process to remove external contamination and sebum.
Then, validated extraction methods were utilised to extract cannabinoids and other drug
compounds from the samples, where methanol, chloroform, and propan-2-ol were used as
solvents. Finally, the extracts underwent an SPE clean-up process using mixed mode car-
tridges to elute the cannabinoids for GC-MS/MS analysis. GC-MS/MS analysis involved a
short 15 m × 0.25 mm column with a 0.25 µm film thickness, the temperature ramp started
at 150 ◦ C with an ending temperature of 320 ◦ C. The total run time was only 8 min, due in
no small part to the short column. The collision gas was argon, but He was used as the car-
rier. The MS was run in multiple reaction monitoring modes. The constituents of cannabis
targeted were THC, the metabolite [11-nor-∆(9)–tetrahydrocannabinol-9-carboxylic acid
(THC-COOH)], and two cannabinoids: cannabinol (CBN) and cannabidiol (CBD). However,
THC and THC-COOH are not likely to be detected in hair since their presence and forma-
Chemosensors 2023, 11, 527 19 of 34

tion are obstructed by the characteristics of hair and the pH environment. The LOD for
the target analytes ranged from 0.01 pg/mg (THC-OH) to 0.91 pg/mg (CBD), and all the
reported RSD% were under 20% with the exception of CBD at 24%. All the volunteers’ hair
samples provided negative results of Cannabis components before hemp oil application
(except for one positive volunteer who was excluded). The post-hemp oil application
outcome showed that 89% of participants’ hair detected one or more cannabis components,
with 33% giving positive results of three major constituents: THC, CBD, and CBN. Despite
the fact that cannabis components were detected in volunteers’ hair after application, only
CBN was detected in the hemp oil, whilst other constituents and metabolites could not be
detected. There was controversy regarding identifying the major constituents of cannabis
in hair. Some suggested it was because of passive exposure, infrequent use of cannabis
or historical exposure, while some opposed it because of chronic and repeated exposure
to cannabis. Therefore, it has been suggested that the hair–cannabis results should be
treated carefully and cautiously, and the consumption of hemp oil should be taken into
consideration prior to interpretation.
GC combined with MS has been successfully applied to analyse fragrance and cream
products for forensic purposes. However, because of its destructive nature, GC-MS some-
times is not as prioritised as other non-destructive techniques in analysing trace evidence
of cosmetics. Nonetheless, it is worth mentioning that in some cases the contribution of
GC-MS is necessary since the information obtained by other techniques is not reliable
enough. In research by Gładysz et al., 2021 [78] involving discriminating lipsticks for
forensic applications, the ATR-FTIR technique was used first as it is non-destructive; yet the
data obtained could not be properly interpreted due to interferences from the substrates.
Therefore, GC-MS was proposed as a confirmatory method since it can provide better data
with less substrate interferences. Table 2 summarises the key analytes, methods, and limits
of detection for the studies relating to the analysis of cosmetic based products.

3.9. Air, Gases and Vapours

The determination of drugs in air has recently been reviewed by Zambas-Adams and
Honeychurch [79] An understanding of the levels of drugs and other forensically important
compounds in air is important for assessing occupational and environmental exposure.
Intelligence on the usage and manufacture of both illicit and licit compounds can also
be gained.
Chemosensors 2023, 11, 527 20 of 34

Table 2. Summary of key information from the detection of cosmetics.

Analytes Matrix Derivatization Sample Pre-Treatment Type of GC LOD Comments Ref

Methanol, ethanol,
and hydrochloric None. Direct headspace 0.0015% formaldehyde
Formaldehyde Cosmetics Headspace GC-MS RSD less than 12% [74]
acid incubated for 4 h analysis starting concentration
at 60 ◦ C
Screening of pills,
The method used is not GC-MS full scan and
fentanyl main Pharmecutical pills None No quantitative analysis [72]
given. GC-FID
substance
SPME analysis testing
60 min SPME extraction at
Screening of perfume Fabrics None GC-MS full scan Qualitative the persistence of [75]
58 ◦ C.
perfumes on fabrics.
Moisturiser
components:
petrolatum, glycerol, Cetyl alcohol was still
Swabs were extracted into GC-MS direct
cetyl alcohol, Swabs from skin Qualitative detectable from human [76]
400 µL of methanol. injection full scan
isopropyl palmitate, skin after 23.5 h.
ethylparaben, and
methylparaben
Some 0.05 g of cosmetic
samples were spiked with
target analytes. A total of
120 µL of chloroform was GC-MS both in full
N-nitrosamines Costmetic products None 0.2 ng/L N-nitrosodibutylamine. [77]
used for the extraction, the scan and SIM mode.
solution was vortexed and
centrifuged. The collected
extract was directly injected.
Overnight extraction with
methanol dried in a
centrifugal concentrator. 0.12 THC; 0.91 CBD;
∆9 -
Hair was then digested in GC-MS/MS in 0.09 CBN;
tetrahydrocannabiol 20 µL BSTFA (no
Human hair sodium hydroxide and multiple reaction 0.07 THC-COOH; [78]
and associated other details given)
liquid–liquid extraction with monitoring mode 0.01 THC-OH (all
metabolites
chloroform and propan-2-ol. pg/mg)
SPE was used to clean up the
sample.
Chemosensors 2023, 11, 527 21 of 34

3.10. Cannabis Smoke

Cone et al. [80] have reported on a number of studies focused on investigating the
effects of second-hand Marijuana-smoke exposure. In two separate investigations, five drug-
free male volunteers with a history of marijuana use were exposed to the side stream smoke
of 4 and 16 marijuana cigarettes for an hour a day for six successive days. A further third
study was undertaken on two subjects with no previous history of marijuana usage. These
were passively exposed to the smoke of 16 marijuana cigarettes in an unventilated room.
Vacuum elution through a column containing Gas-Chrom Q adsorbent was undertaken to
collect air samples; with exposed tubes eluted with methanol for subsequent determination
by gas chromatography with flame-ionisation detection following derivatization with
N-methyl-N-trimethylsilyl trifluoroacetamide. Levels of THC were reported to be in
the range of 0.9–1.8 µg/L and 3.0–6.7 µg/L, respectively, during the smoking of 4 and
16 marijuana cigarettes. Low-level exposure resulted in urine specimens that tested positive
only infrequently. However, high-level exposure studies showed all subjects exhibited
significant levels of absorbed THC, with detectable levels of cannabinoid metabolites
reported in their urine.

3.11. Synthetic Cannabinoids

The concentrations of synthetic cannabinoid drugs in the air of an English prison
were investigated by Paul et al. [81] using two-dimensional gas chromatography coupled
to time-of-flight mass spectrometry (GC × GC-TOF MS). Sample were obtained using
both fixed sequential samplers and personal air sampling units worn by prison officers.
The air samples were collected onto thermal desorption tubes before examination by
GC × GC-TOF MS. Interestingly, analysis of the air samples taken did not reveal any
synthetic cannabinoids.

3.12. Anaesthetic Gases Exposure

Commonly referred to as laughing gas, nitrous oxide (N2 O), or nitrous is legally
employed as an anaesthetic, especially for surgery, and dentistry. It is also commonly
used for pain relief by health care professionals such as midwifes. Its application has
been reported to possibly lead to concentrations that can be dangerous, especially in
confined spaces. Henderson et al. [82] showed that air levels of N2 O exceeded the legal
occupational exposure standards for N2 O in 76% for the midwives they investigated. A
passive sampling tube was worn by the midwife for the first four hours of their shift. This
contained a steel tube packed with molecular sieve 5A with a diffusive cap on top. This was
then removed and the levels of adsorbed N2 O were determined by thermal desorption–gas
chromatography with electron capture detection.
In 2015, Giuliani et al. used headspace GC-MS to quantify N2 O and apply their
method to a case of N2 O intoxication. To achieve this, two N2 O quantification columns
were sequenced in parallel, one with molecular sieve and the other with Porabond Q;
hydrogen sulphide was used as an internal standard. This group sought to validate their
quantification method over a period of three days, achieving a r2 mean of 0.98, and a LOD
of 5 µM [83]. In the UK in 2019, 9% of 16–24 year-olds reported using N2 O for recreation,
these figures make N2 O the second most used controlled substance with only cannabis
having more users [84]. However, there is still a lot to learn about the abuse of N2 O,
as currently there is limited information on the lethal dosage of N2 O. The Giuliani et al.
2015 [83] paper took samples from the blood and brain of a cadaver, it is hoped that the
presented method of analysis can provide new insights into the lethal dosages of N2 O.
Moreover, it would be further beneficial to forensic science to develop a breath or blood
test that could determine blood concentrations of N2 O in living individuals.

4. Pyrolysis Gas Chromatography for the Investigation of Drugs

As we discussed above, the GC determination of non-volatile compounds that undergo
hydrogen bonding can be achieved by their conversion to volatile derivatives via chemical
Chemosensors 2023, 11, 527 22 of 34

treatment with agents such as TMS and HMDS. However, a number of compounds are
insufficiently volatile due to their relatively high molecular masses, including polymeric
materials such as wood and plastic. The formation of derivatives of these cannot readily
overcome these issues. One solution is to break these large molecules into smaller fragments
which are sufficiently volatile for GC analysis. A number of processes can be used to achieve
this, including hydrolysis, oxidation, and pyrolysis. Hydrolysis and oxidation reactions
require the addition of chemical reagents and off-line reaction steps which result in the
formation of wet reaction products, requiring further processing. However, pyrolysis can
be readily undertaken by the application of heat to the sample in an inert atmosphere
or a vacuum to give reproducible volatile fragments. Small amounts of sample (mg) are
held in contact with a platinum wire or placed in a quartz tube, where a rapid heating to
600–1000 ◦ C can be applied in an inert atmosphere, such as the GC carrier gas. The bonds
of the high-molecular weight sample are cleaved at their weakest points, forming smaller,
more volatile fragments. These are then thermally focused and introduced directly to the
GC, without the need for further clean-up steps. The resulting pyrograms can be used as a
fingerprint to identify the specific polymer or from the mass spectra to identify individual
fragments to obtain structural information.
Commonly, Py-GC-MS is employed in the forensic sciences for investigation of poly-
mers and macromolecules seen in materials such as condom lubricants, paint, and for the
identification of plastics. Alternative applications of Py-GC-MS are summarised in Table 3.
However, studies have shown that it is possible to use Py-GC-MS both quantitatively and
for exploring the reaction processes occurring during the smoking of various drugs. The
first use of Py-GC for the investigation of drugs was described by Janák et al. [9] in 1960,
who described the pyrolysis of the sodium salts of fourteen barbiturates and was able to
show a number of unique fragments were formed. They showed that the fragments from
the drugs were always present and qualitatively and quantitively highly specific. Tsuchi-
hashi, Tatsuno, and Nishikawa [85] have reported the possibility of applying Py-GC-MS for
the determination of eight quaternary ammonium drugs (distigmine bromide, bethanechol
chloride, pancuronium bromide, methylbenactyzium bromide, propantheline bromide,
suxamethonium chloride, neostigmine methylsulphate, and benzethonium chloride) in
urine. As with other quaternary ammonium salts, the drugs are very soluble in water and
cannot be readily extracted by liquid–liquid extraction with organic solvents, making their
extraction from biological fluids difficult. The drugs were first separated by thin-layer
chromatography (TLC) and then pyrolyzed using a Curie-point pyrolizer. The resulting
fragments were then separated by GC and determined by mass spectrometry utilizing SIM.
Detection limits of between 0.01 and 10 µg were reported for each compound. Extraction of
quaternary ammonium compounds by using ODS-cartridge was also examined. Nishikawa
et al. [86] undertook further investigations utilizing TLC and PyGC with flame ionization
detection (FID) for the determination of methylbenactyzium bromide in human urine. In
this investigation they showed it possible to use smaller volumes of urine.
Chemosensors 2023, 11, 527 23 of 34

Table 3. Pyrolysis gas chromatography–mass spectrometry investigations of drugs.

Analyte Pyrolysis Conditions Comments Ref

Platinum crucible using isothermal temperatures of 300 and
400 ◦ C. Pyrolysis analysis was undertaken using isothermal Pyrogram obtained at 300 ◦ C predominated by
Tacrolimus [87]
conditions of 300 ◦ C and 400 ◦ C coupled to GC–MS, in full 2,5-dimethyl-3-hexine-2-5-diol.
scan mode (m/z 25–900).
Investigations made under both anaerobic and aerobic Propionanilide; pyridine, norfentanyl and
Fentanyl conditions coupled to a GC-MS for the modelling of the illicit despropionyl fentanyl formed along with Cl containing [88]
smoking of fentanyl in transdermal patches. compounds, due to the HCl salt of fentanyl.
Pyrolysis of d-methamphetamine made to identify the Amphetamine and dimethylamphetamine formed.
products sealed in a glass tube, wrapped in pyrolysis-foil; Phenylacetone was reported as an oxidative
Methamphetamine [89]
heated at 200–500 ◦ C. Resulting products extracted in degradation product. Above 415 ◦ C toluene, styrene,
methanol. and ethylbenzene were predominated.
Drugs were pyrolyzed as alkaloid salts. The effect of metal
Morphine, codeine phosphate and dihydrocodeine
ions on the pyrolysis reactions were investigated. A mixture
phosphate, 3,4-dimethyl-1,2-dihydroxybenzene. Based
Codeine and morphine of metal powder and Na2 CO3 was added to a piece of [90]
on this compound it was possible to quantify morphine
pyrolysis foil containing the drug and heated (590 ◦ C). The
HCl in urine.
resulting products were identified by GC-MS.
Individual drugs were rapidly heated to 800 ◦ C in quartz
Synthetic cannabinoids (XLR-11, UR-144, capillary tubes under an ambient airflow to approximate the Analysis of thermolysis products was undertaken by
[91]
A-834735, and PB-22) burning end of a cigarette whilst being smoked. Products full-GC-MS (m/z 50–550).
were trapped on a charcoal and passed to the GC-MS.
Synthetic cannabinoids (CUMYL-PICA,
Above 400 ◦ C, toluene, naphthalene, and
5F-CUMYL-PICA, AMB-
Investigations made to simulate the smoking of drugs. 1-naphthalamine were formed. A degradative pathway [92]
FUBINACA, MDMB-
for the liberation of cyanide was shown.
FUBINACA, NNEI, and MN-18)
 5F-CUMYL- Above 400 °C, toluene, naphthalene,
PICA, AMB- Investigations made to simulate the smoking and 1-naphthalamine were formed. A
[92]
FUBINACA, of drugs. degradative pathway for the libera-
MDMB- tion of cyanide was shown.
FUBINACA,
Chemosensors 2023, 11, 527 24 of 34
NNEI, and MN-
18)
Tacrolimus
Tacrolimus(Figure
(Figure1)1)isisa amacrolide
macrolidelactone
lactoneextracted
extractedfrom
fromStreptomyces
Streptomycestsukubaensis
tsukubaensis
with
withreported
reportedpotent
potentimmunosuppressive
immunosuppressive activity. et al.
activity. Böer et al.[87]
[87]have
haveutilized
utilizedPy-GC-MS
Py-GC-
MS and
and thermal
thermal analysis
analysis to to characterize
characterize tacrolimus
tacrolimus raw
raw material.
material. Four
Four samples
samples ofof tacroli-
tacrolimus
from
mus different
from manufacturers
different manufacturers were investigated.
were investigated.

Figure
Figure 1. 1. Structure
Structure ofof Tacrolimus.
Tacrolimus.

Powder particles of the immunosuppressive tacrolimus were pyrolysised in a plat-

Powder particles of the immunosuppressive tacrolimus were pyrolysised in a plati-
inum crucible using isothermal temperatures of 300 and 400 ◦ C. Pyrolysis analysis was
num crucible using isothermal temperatures of 300 and 400 °C. Pyrolysis analysis was
undertaken using isothermal conditions of 300 0 C and 400 ◦ C coupled to GC–MS, in full
undertaken using isothermal conditions of 300 ⁰C and 400 °C coupled to GC–MS, in full
scan mode (m/z 25–900). The molecular ion of tacrolimus (m/z 804.01) was not recorded
scan mode (m/z 25–900). The molecular ion of tacrolimus (m/z 804.01) was not recorded
and was concluded to indicate that tacrolimus was totally decomposed. The pyrogram
and was concluded to indicate that tacrolimus was totally decomposed. The pyrogram
obtained at 300 ◦ C was predominated by a peak with molecular ion of m/z 142 and was
obtained at 300 ⁰C was predominated by a peak with molecular ion of m/z 142 and was
identified as 2,5-dimethyl-3-hexine-2-5-diol (Figure 2). The pyrogram of tacrolimus ob-
Chemosensors 2023, 11, x FOR PEERidentified
REVIEW as 2,5-dimethyl-3-hexine-2-5-diol (Figure 2). The pyrogram of tacrolimus ob-
tained at 400 ◦ C was found to exhibit four main peaks with abundances >50%, 23 of 34
with a
tained at 400 °C was found to exhibit four main peaks with
smaller fifth peak with a relative abundance inferior to 5%.
abundances >50%, with a
smaller fifth peak with a relative abundance inferior to 5%.

Figure 2. 2,5-dimethyl-3-hexine-2-5-diol.

Fentanyl
Fentanyl is a potent opioid analgesic
analgesic that is increasingly
increasingly abused.
abused. Nishikawa
Nishikawa et et al. [88]
utilized Py-GC-MS
Py-GC-MSto todetermine
determinethe the levels
levels of of fentanyl
fentanyl present
present in transdermal
in transdermal patches.
patches. The
The transdermal
transdermal patches patches are reportedly
are reportedly easy toeasy
obtain to and
obtainare and
abused are by
abused
smokingby smoking
of the drug- of
the drug-containing
containing reservoir reservoir
gel or thegel or thepatch
whole wholeitself.
patchPyrolysis
itself. Pyrolysis investigations
investigations were under-were
undertaken
taken using using both aerobic
both aerobic and anaerobic
and anaerobic conditions
conditions utilizing
utilizing either either air coupled
air of He of He coupled
to the
to the GC-MS. Samples were prepared by sandwiching between
GC-MS. Samples were prepared by sandwiching between 50–100 μg of fentanyl 50–100 µg of fentanyl
HClHCl be-
between
tween two two separatepieces
separate piecesofofquartz
quartzwool
woolinside
insideaaquartz
quartz pyrolysis
pyrolysis tube.
tube. The
The pyrolysis
pyrolysis
tube
tube was
was then
then inserted
inserted into
into the
the platinum
platinum coil
coil filament
filament of of the
the pyroprobe
pyroprobe and and heated
heated in in the
the
presence
presence ofofthethereactant
reactantgas, which
gas, thenthen
which carries the volatile
carries components
the volatile formedformed
components to a sorbent
to a
trap,
sorbentwhere
trap,they
wherearethey
thenare
focused. Figure Figure
then focused. 3 summaries the reactions
3 summaries products
the reactions generated.
products gen-
For the aerobic investigations, during the last minute of flow to the trap,
erated. For the aerobic investigations, during the last minute of flow to the trap, the reac- the reactant gas
was switched from air to the He carrier gas before transfer to the
tant gas was switched from air to the He carrier gas before transfer to the GC, to avoidGC, to avoid damage
to the analytical
damage column.column.
to the analytical Both the anaerobic
Both and aerobic
the anaerobic pyrolysis
and aerobic of fentanyl
pyrolysis and and
of fentanyl the
transdermal patches were reported to give propionanilide as the major
the transdermal patches were reported to give propionanilide as the major pyrolytic prod- pyrolytic product;
pyridine and previously
uct; pyridine and previouslyreported metabolites
reported (norfentanyl
metabolites and despropionyl
(norfentanyl fentanyl)fenta-
and despropionyl were
also reported. Investigations of fentanyl were also reported to give
nyl) were also reported. Investigations of fentanyl were also reported to give chlorine- chlorine-containing
products. These were concluded to result from the HCl salt of fentanyl; a result further
containing products. These were concluded to result from the HCl salt of fentanyl; a result
substantiated by investigations of transdermal patches containing the citrate salt of fentanyl
further substantiated by investigations of transdermal patches containing the citrate salt
not forming chlorine-containing pyrolytic adducts. The authors concluded that it could be
of fentanyl not forming chlorine-containing pyrolytic adducts. The authors concluded that
feasible to identify which salt of the drug had been smoked based on the different pyrolytic
it could be feasible to identify which salt of the drug had been smoked based on the dif-
ferent pyrolytic products formed. The investigation also reported significant polymeric
and hydrocarbon compounds to be formed from the transdermal patch itself.
 the transdermal patches were reported to give propionanilide as the major pyrolytic prod-
uct; pyridine and previously reported metabolites (norfentanyl and despropionyl fenta-
nyl) were also reported. Investigations of fentanyl were also reported to give chlorine-
containing products. These were concluded to result from the HCl salt of fentanyl; a result
Chemosensors 2023, 11, 527 further substantiated by investigations of transdermal patches containing the citrate25 of salt
34
of fentanyl not forming chlorine-containing pyrolytic adducts. The authors concluded that
it could be feasible to identify which salt of the drug had been smoked based on the dif-
ferent pyrolytic
products formed. products formed. also
The investigation The reported
investigation also reported
significant significant
polymeric polymeric
and hydrocarbon
and hydrocarbon
compounds compounds
to be formed to be
from the formed from
transdermal theitself.
patch transdermal patch itself.

Figure 3. Fentanyl pyrolytic product comparison in anaerobic and aerobic trapping conditions, based
onFigure 3. Fentanyl
Nishikawa pyrolytic product comparison in anaerobic and aerobic trapping conditions,
et al. [90].
based on Nishikawa et al. [90].
Illicitly, methamphetamine (MA) is commonly taken by inhalation of the vapor formed
by heating the drug on some suitable substrate, such as aluminium foil. Consequently,
understanding its behaviour upon heating is of interest for both toxicology and forensic
investigations. In light of this, Sato, Hida, and Nagase [89] investigated the mechanism
and the products of the pyrolysis of d-methamphetamine (d-MA). Methamphetamine
-HCI was sealed in a glass tube, wrapped in pyrolysis-foil which was heated to the Curie
point of the foil (200–500 ◦ C). The heated capillary tube was then opened and extracted
with methanol. The resulting solution was diluted 1:10 with dichloromethane, for GC-
MS or 20 mM of ammonium acetate pH 5 buffer for LC/MS analysis. Investigations of
the pyrolysis products of deuterated MA-d3-HCI were also undertaken to explore the
transformation of the methyl group of MA. It was found that at temperatures above 315 ◦ C,
amphetamine (AM) and dimethylamphetamine (DMA) were produced and were concluded
to be formed by demethylation and methylation reactions, respectively. Phenylacetone was
reported as an oxidative degradation product and was detected at the same temperature as
the demethylation and methylation reactions.
It was concluded that these reactions were the main pyrolysis processes occurring at
temperatures below 358 ◦ C. Above 315 ◦ C benzylethyltrimethylammonium (BEMA) was
reported to result from reaction of DMA with a methyl group eliminated from the methy-
lamino group of MA. Consequently, it was concluded that this transformation showed
that both demethylation and methylation reactions occur in the form of the methyl cation.
Above temperatures of 315 ◦ C, thermal degradation of BEMA was reported, occurring via
abstraction of a proton at the β-position and elimination of a trimethylamine, giving allyl-
benzene, cis-β-methylstyrene, and trans-β-methylstyrene. Formation of propylbenzene
at temperatures over 423 ◦ C was reported to show that the elimination of a methylamino
group of MA occurs without the elimination of a hydrogen atom at the 13-position. At tem-
peratures greater than 445 ◦ C the optical isomers, or l-isomers of AM, MA, and DMA, were
reported as the main pyrolysis products formed. Further pyrolysis products of toluene,
styrene, and ethylbenzene were also formed, resulting from cleavage of the C-C bond pf
the α and β-positions at temperatures. A possible mechanism for the formation of these
Chemosensors 2023, 11, 527 26 of 34

products was given and the results were concluded to be important in the field of forensic
science, as erroneous identifications of the abused drug could occur from the formation
of these pyrolysis products. Mitsui et al. [90] investigated the effects of various metals
and inorganic additives on the pyrolysis of codeine and morphine. The drugs were py-
rolyzed as alkaloid salts and the pyrolyzed compounds determined by GC. The authors
investigated the effects of the addition of metal and inorganic compounds to pyrolysis
reactions. A mixture of iron powder and sodium carbonate anhydride (30 mg: 4:1 w/w)
was added to a piece of pyrolysis foil (Curie point of 590 ◦ C). An aqueous solution of the
drug under investigation was then added. The foil was dried on a hot plate for 5 min at
about 100 ◦ C to evaporate the water. After cooling to room temperature, the pyrolysis foil
was inserted into a Curie-point pyrolyzer and the resulting pyrolysis products identified
by GC-MS. The pyrolysis products of morphine hydrochloride, codeine phosphate, and
dihydrocodeine phosphate were identified by their retention times, as was 3,4-dimethyl-1,2-
dihydroxybenzene. Metal powder was added to conduct heat from the pyrolysis foil to the
drug under investigation. Aluminium, iron, chromium, zinc, manganese, nickel, or copper,
were investigated and iron was found to give the highest sensitivity. Similarly, the effect
of the inorganic compounds utilized was also investigated. Sodium carbonate, potassium
iodide, or ammonium sulphate were added with the iron powder and the optimum reaction
mixture was reported as an iron and sodium carbonate ratio of between 5:1 and 2:1. Gas
chromatographic investigations showed a number of peaks, with the largest identified
as 3,4-dimethyl-1,2-dihydroxybenzene from its retention time. Using this peak, it was
Chemosensors 2023, 11, x FOR PEER REVIEW 25 of 34
reported to be possible to construct a calibration curve for morphine hydrochloride with
a correlation coefficient of 0.9998, and a relative standard deviation for the pyrolysis of
2.5% (n = 10). The authors reported on the possibility of extracting drugs from urine with
with dichloromethane
dichloromethane and the and the subsequent
subsequent Py-GCPy-GC
of theirofsalts
theirwith
saltsreportedly
with reportedly high sen-
high sensitivity
sitivity and with good
and with good reproducibility. reproducibility.
Synthetic cannabinoids
cannabinoids are are commonly
commonlysold soldas asherbal
herbal“spice”
“spice”forforsmoking
smokingororby byvap-
va-
ing. Thomas
ping. Thomasetetal.al.[91]
[91]have
haveshown
shownthat thatheating
heatingsynthetic
syntheticcannabinoids
cannabinoidswhichwhichcontain
containa
atetramethylcyclopropyl
tetramethylcyclopropyl ring, as as
ring, illustrated
illustratedforfor
A-834735
A-834735 in Figure 4, can
in Figure result
4, can in the
result in for-
the
mation of thermal
formation degradants
of thermal degradants products which
products can display
which pharmacological
can display pharmacologicalactivity nota-
activity
bly different
notably from from
different the parent compound.
the parent Using Py
compound. UsingGC-MS the authors
Py GC-MS were ablewere
the authors to inves-
able
tigate
to the effects
investigate theofeffects
heating, of similar
heating,tosimilar
that seen
to whilst
that seen smoking
whilstor vaping and
smoking form these
or vaping and
degradants.
form The synthetic
these degradants. Thecannabinoids, XLR-11, UR-144,
synthetic cannabinoids, XLR-11,A-834735, and PB-22,
UR-144, A-834735, andwere in-
PB-22,
were investigated
vestigated via the addition
via the addition of 5 µg of
of 5 μg aliquots aliquots of the individual
the individual drugs toquartz
drugs to separate separate
ca-
quartz ◦ C, and then rapidly heated to 800 ◦ C
pillary capillary tubes.
tubes. These wereThese were equilibrated
equilibrated at 50 °C, at
and50then rapidly heated to 800 °C (20 °C/s)
(20 ◦ C/s)
under an under
ambient anairflow
ambient toairflow to approximate
approximate the burning theend
burning end of a cigarette
of a cigarette being
being smoked.
smoked.
The resultingThe resulting
degradation degradation
productsproducts
were then were then trapped
trapped on a charcoal
on a charcoal desorption
desorption tube at
tube
50 °C.atThe50 ◦probe
C. Thewasprobeheldwas held
at 800 °Catfor
800 ◦ for 20 s, and then airflow was switched to
20 C
s, and then airflow was switched to He while
He
the while the thermolysis/pyrolysis
thermolysis/pyrolysis probe was
probe was returned toreturned 50 ◦ C and equilibrated.
50 °C andtoequilibrated. The charcoalThe de-
charcoal desorption tube was then ◦
sorption tube was then heated to 300heated
°C andtothe300HeCflow anddiverted
the He flow
to thediverted to the GC
GC for separation
for
andseparation
the analysis andofthe analysis of
thermolysis thermolysis
products usingproducts
full-scanusing
mass full-scan massover
spectrometry spectrometry
the mass
over the mass
range, m/z 50–550. range, m/z 50–550.

cannabinoid, A-834735, after Thomas

Figure 4. The thermal degradation of the synthetic cannabinoid, Thomas et
et al.
al. [91].
[91].

In
In aa separate
separatestudy
study[92],
[92],the
the thermal
thermal stability
stability of carboxamide-type
of six six carboxamide-type synthetic
synthetic can-
cannabinoids (CUMYL-PICA, 5F-CUMYL-PICA, AMB-FUBINACA,
nabinoids (CUMYL-PICA, 5F-CUMYL-PICA, AMB-FUBINACA, MDMB-FUBINACA, MDMB-FUBINACA,
NNEI, and MN-18, Figure 5) was undertaken to characterize the possible user exposure
to their thermolysis products formed during their smoking. Smoking of these drugs in-
volves heating them to high temperatures via burning in a cigarette or “joint” and can
result in temperatures of between 700 °C to 900 °C [93]. As a result of the high temperature
that these drugs can be exposed to, their thermal instability can lead to the formation of
 Chemosensors 2023, 11, 527 27 of 34

NNEI, and MN-18, Figure 5) was undertaken to characterize the possible user exposure to
their thermolysis products formed during their smoking. Smoking of these drugs involves
heating them to high temperatures via burning in a cigarette or “joint” and can result
in temperatures of between 700 ◦ C to 900 ◦ C [93]. As a result of the high temperature
11, x FOR PEER REVIEW that these drugs can be exposed to, their thermal instability can lead to the26formation
of 34
of the carcinogenic compound, naphthalene, resulting from the thermal degradation of
the naphthyl group, a common constituent of synthetic cannabinoids. To simulate the
smoking of these drugs, samples were heated sequentially to 200, 400, 600, and 800 ◦ C and
the resultant pyrolysis products separated and determined by GC-MS. Further analysis
quantified the thermolytically generated cyanide and was undertaken by LC–MS/MS.

CUMYL-PICA AMB-FUBINACA MDMB-FUBINACA

NNE1 MN-18
Figure 5. Structures Figure
and designations of carboxamide-type
5. Structures and synthetic cannabinoids.
designations of carboxamide-type synthetic cannabinoids.

Compound-specific pyrolysis products were postulated, and a possible degradative

Compound-specific
pathwaypyrolysis products were
for carboxamide-type postulated,
synthetic andgiven,
cannabinoids a possible degradative
which was postulated to
pathway for carboxamide-type synthetic
result via the formation of ancannabinoids given, which
indole or indazole-amide was postulated
and subsequent to re-
dehydration to give
sult via the formation of anorindole
an indole or indazole-amide
indazole-carbonitrile. andliberation
Thermolytic subsequent dehydration
of cyanide to givewith
was also reported
levels up in levels up to
an indole or indazole-carbonitrile. 27 µg/mg of the
Thermolytic starting drug
liberation material. The
of cyanide wassynthetic cannabinoids
also reported
investigated were reported to also undergo further thermal degradation above 400 ◦ C,
with levels up in levels up to 27 μg/mg of the starting drug material. The synthetic canna-
producing toxic products, including toluene, naphthalene, and 1-naphthalamine. However,
binoids investigated were were
the drugs reported to also
reported to be undergo further
stable at lower thermal
applied degradation
temperatures, with 200above
◦ C being

400 °C, producinggenerally

toxic products, including
insufficient toluene,
to either give naphthalene,
degradants or volatilizeand 1-naphthalamine.
the parent drug. However, at
temperatures between 400–600 ◦ C, CUMYL-PICA, 5F-CUMYL-PICA, AMB-FUBINACA,
However, the drugs were reported to be stable at lower applied temperatures, with 200
and NNEI were reported to undergo extensive degradation while MN-18 and MDMB-
°C being generally insufficient to either give degradants or volatilize the parent drug.
FUBINACA reportedly showed greater stability. The authors postulated that this a was
However, at temperatures between 400–600 °C, CUMYL-PICA, 5F-CUMYL-PICA, AMB-
FUBINACA, and NNEI were reported to undergo extensive degradation while MN-18
and MDMB-FUBINACA reportedly showed greater stability. The authors postulated that
this a was result of the additional nitrogen in the indazole ring of MN-18, compared to
Chemosensors 2023, 11, 527 28 of 34

result of the additional nitrogen in the indazole ring of MN-18, compared to NNEI, which
conferred improved thermal stability to molecule. The effects of pyrolysis for these drugs
was explored by heating sequentially to 200, 400, 600, and 800 ◦ C using methodology
similar to that reported previously [92].
A wide range of different chemical stabilities were found for the drugs investigated,
with 200 ◦ C being generally insufficient to either give degradants or to volatilize the
parent drug. CUMYL-PICA, 5F-CUMYL-PICA, AMB-FUBINACA, and NNEI underwent
extensive degradation at temperatures between 400 and 600 ◦ C, while MN-18 and MDMB-
FUBINACA were reportedly more stable. The authors postulated that, compared to NNEI,
Chemosensors 2023, 11, x FOR PEER REVIEW
the additional nitrogen in the indazole ring of MN-18 confers improved thermal stability. 27 of 34
®
Buscopan (Figure 6a), is commonly used for the relief of abdominal discomfort, pain,
and acute colic. The active ingredient is hyoscine butyl bromide, a quaternary ammonium
compound
ammoniumthat has low lipid
compound solubility
that has andsolubility
low lipid cannot readily pass the
and cannot blood–brain
readily pass thebarrier.
blood–
However, under the thermal conditions commonly seen during
brain barrier. However, under the thermal conditions commonly seen during cigarette smoking, the
cigarette
hyoscine butyl bromide can be converted to scopolamine (Figure 6b). Scopolamine,
smoking, the hyoscine butyl bromide can be converted to scopolamine (Figure 6b). Sco- also
known as hyoscine,
polamine, also knownis a as
naturally occurring
hyoscine, tropaneoccurring
is a naturally alkaloid and anticholinergic
tropane alkaloid anddrug that
anticho-
can be used to treat motion sickness, nausea, vomiting, and can decrease saliva
linergic drug that can be used to treat motion sickness, nausea, vomiting, and can decreaseproduction.
However, it is also smoked
saliva production. However, recreationally for its recreationally
it is also smoked hallucinogenicforproperties. However,prop-
its hallucinogenic this
iserties.
not commonly undertaken, as the experiences are reported to be unpleasant,
However, this is not commonly undertaken, as the experiences are reported to be mentally
and physically, so repeated recreational use is rare [94]. Nevertheless, in situations such
unpleasant, mentally and physically, so repeated recreational use is rare [94]. Neverthe-
as prisons, the smoking of medications such as Buscopan® to give scopolamine has been
less, in situations such as prisons, the smoking of medications such as Buscopan® to give
reported [95,96].
scopolamine has been reported [95,96].

Figure6.6.Structure
Figure Structureofof(a)
(a)hyoscine
hyoscinebutyl
butylbromide
bromideand
and(b)
(b)scopolamine.
scopolamine.

Notably, Deutsch et al. [97] have reported on the problem associated with the deter-
mination of scopolamine by GC-MS. They have shown the possibility of its determination
following derivatization with HFBA. A base ion of m/z 351 was reported for the mass spec-
trum of HFBA derivatized scopolamine corresponding to the molecular ion of the HFB
derivative. However, further analysis of the mass spectrum showed this to be different
Chemosensors 2023, 11, 527 29 of 34

Notably, Deutsch et al. [97] have reported on the problem associated with the determi-
nation of scopolamine by GC-MS. They have shown the possibility of its determination
following derivatization with HFBA. A base ion of m/z 351 was reported for the mass
spectrum of HFBA derivatized scopolamine corresponding to the molecular ion of the HFB
derivative. However, further analysis of the mass spectrum showed this to be different from
that previously reported. This difference was suggested to result from the HFB scopolamine
derivative undergoing a transacylation in the GC-MS inlet system.
Heroin is often inhaled as a vapor via heating on aluminium foil in a process collo-
quially referred to as “chasing the dragon”. Brenneisen and Hasler [98] have studied the
possible products formed by simulating these conditions by heating the heroin samples on
aluminium foil at 250 to 400 ◦ C. The resulting pyrolysis products then being collected in
a condenser trap. Investigation by GC-MS showed 72 pyrolysis products generated from
the heating of diacetylmorphine (street heroin) residues and from the aluminium foil itself.
Reportedly, however, only half of these compounds could be identified. Diacetylmorphine
(base and salt) was found to undergo substitution reactions to complete degradation. Some
typical street heroin constituents, such as, morphine, codeine, acetylcodeine, papaverine,
and caffeine, were found to be heat-stable, with other adulterants such as, noscapine and
paracetamol, being pyrolyzed to a greater extent. The principal chemical reactions leading
to the formation of pyrolysis products were reported to be desacetylation, transacetylation,
N-demethylation, O-methylation, ring cleavage, and oxidation.

5. Conclusions
Gas chromatography is a mature, robust technique and has been considered the
“gold standard” in many analytical forensic investigations. The development of new
technologies with matched capabilities and reliability has not emerged in such a way as to
reduce the relevance of these techniques in forensic science. The work presented herein,
however, demonstrates that rather than looking to new technologies, researchers are finding
increasingly creative and non-traditional targets for analysis. This would seem to be partly
driven by the increased sensitivity advances in modern GC and GC-MS instrumentation,
allowing for smaller sample sizes to be utilized and, hence, opening up the potential for
new sample types. The determination of drugs and potentially other compounds in sweat
can offer an alternative to the more commonly employed urine, blood, and hair analysis.
Similarly, the relatively non-invasive sample cerumen offers advantages, with a detection
window longer than that of urine but shorter than that reported of hair and has been shown
to be possibly less susceptible to contamination. Both meconium and breast milk can be
used to give insights into in utero and infant exposure.
It has been shown to be possible to apply Py-GC-MS beyond its normal application
for the determination of polymeric materials to explore the chemistry and products formed
during the smoking of a number of common drugs. The determination of drugs in insects,
following feeding on meat both fortified and from natural levels found in autopsy-derived
samples, has been shown possible. This is an interesting development and it could be
possible to use this for drug detection in other application areas. Allowing for the natural
abilities of the insect to search out and return with information on the area. Similarly, the
possibility of determining drug and explosive residues in plants and other animals and
could be an area of future study.
Most GC approaches have a relatively low sample throughput, with typical run times
in the order of 15 to 30 min. Additional time is also required for a suitable extraction
and concentration step to be made. There is presently interest in fast chromatography, or
techniques such as DART MS, which do not require a chromatographic step, which could
be applied to overcome this issue. The requirement of the analyte to be both thermally
stable and volatile limits the range of compounds that can be successfully determined
by GC. A factor that has partly led to the increasing popularity of techniques that can be
undertaken at ambient temperatures, such as LC-MS.
Chemosensors 2023, 11, 527 30 of 34

Author Contributions: Conceptualization, K.C.H. and O.G.; methodology, K.C.H. and O.G.; investi-
gation, K.C.H. and O.G.; resources, K.C.H. and O.G.; data curation, K.C.H. and O.G.; writing—original
draft preparation, K.C.H., N.N., and O.G.; writing—review and editing, K.C.H., N.N., and O.G.;
visualization, K.C.H. and O.G.; supervision, K.C.H. and O.G.; project administration, K.C.H. and
O.G.; funding acquisition, K.C.H. All authors have read and agreed to the published version of
the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The authors would like to thank the University of the West of England for
supporting and funding this research with a summer bursary for N.N. We would like to thank the
researchers whose work has been described in this review.
Conflicts of Interest: The authors declare no conflict of interest.

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