PC-POD-CA-001-v04

PERFORM Operating Document

Use and Maintenance of QQQ LC-MS

PC-POD-CA-001-v04

Revision History

Version Reason for Revision Date

04 Condense version 06-Aug-2020

1. Introduction
The content of this PERFORM Operating Document (POD) provides guidelines for the
use and maintenance of the QQQ LC-MS.

The PERFORM Centre Clinical Analysis suite has an Agilent 6460 Triple Quad Mass Spectrometer
(LC-MS) which is a bench top QQQ providing atmospheric pressure ionization of liquid
samples. A wide variety of compound classes may be analyzed using either ESI or APCI.
Molecular weight information as well as structural information through collision induced
dissociation provides useful qualitative sample information. This is well suited for both routine
and complex analyses such as metabolism, toxicology, environmental, food safety, pharmaceutical,
and drugs of abuse testing. This system provides analysis with greater sensitivity and productivity
for quantifying pharmaceutical candidates, measuring trace-level environmental or food
contaminants, or confirming biomarkers. Mass Hunter Quantitative Analysis software provides
quantitative results with minimal manual intervention.

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2. Definition of Terms and abbreviations

LC-MS Liquid Chromatography-Mass Spectrometry

MS Mass Spectrometer
QQQ Triple Quadrupole
UPLC Ultra Performance Liquid Chromatography
HPLC High Performance Liquid Chromatography
N2 Nitrogen
ESI Electrospray Ionization
APCI Atmospheric Pressure Chemical Ionization
BinPump Binary Pump
TCC Thermostat Column Compartment
TFA Trifluoroacetic acid
MP Mobile Phase
EMV Electron multiplier voltage
MW Molecular weight
L Liter
Min Minute
mL Milliliter
µA Micro Ampere

3. Overview of LC-MS
• The 1290 Infinity II LC System (UPLC 1290) is equipped with a binary pump
for two pairs of solvents (A1,A2 and B1, B2) and is capable to withstand 1200
Bars of operational pressure. Column and sample temperature is controlled
by using column and auto-sampler thermostats.
The chromatography system is coupled to mass spectrometer via ESI or APCI
interfaces (sources) which are interchangeable.
• Two supplies of nitrogen are required to run MS analysis; One, from nitrogen
tank (ultrapure grade) to operate the collision cell and another from N2
generator to operate ESI or APCI source, The first source is equipped with
filters which should be replaced at least once a year or more frequently if

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required by workload. The second source of N2 is supplied by a passive N2

generator connected to a stationary source from Agilent. The pressure should
be maintained sufficiently high to provide 80-100 psi pressure of nitrogen at
the generator output. Diffusion/selection membrane in passive generator
should be replaced at least once a year.
• There are 3 pumps (1 mechanical and 2 turbo) to create the vacuum in LC-
MS system.
• There are 2 ion sources APCI and high efficiency Jet stream ion source ESI
which are readily interchangeable.
• Built-in system logbook keeps log of errors for the sample running. Logbook
deletes old entries each time a new worklist is run so it should be either
saved or printed if required to report any malfunction of the instrument.
• Mass Hunter software suit is used for data analysis and is suitable for research
and development (R&D) purposes. For compliance, additional software can be
purchased.

3.1 Training requirements

Prior to using the LC-MS System individuals should:
• Read and sign this POD.
• Undergo appropriate LC-MS System training and/or provide a proof of an
external training to the Supervisor, Clinical Analysis prior to use.

3.2 General precautions

• Use only water and high purity solvents (LC-MS grade): do not use water
generated by Millipore system in the lab. Avoid contamination by using clean
pipets, bottles and glassware.
• Make sure the level of oil in the mist filter from the mechanical pump is
cleared regularly by opening the ballast valve for a few minutes. MS must be
on standby before the ballast valve is opened. The valve should be closed for
the routine operation. Make sure that there is no leak of oil. Check the oil
level in the rough pump.
• If the rough pump overheats, it will beep. Let it cool by opening the front cover.
• The oil in the rough pump should be replaced and MS ion transfer capillary
cleaned every 6 months. About 1.5 L of oil (Agilent 6040-0834) is required for
replacement. To change the oil, turn off the LC-MS and shutdown the MS: In
order and from the software, UPLC is on standby mode, right click on QQQ
pane in the Data Acquisition software and press Vent. Wait 40 minutes. Then
close the valve from the compress air (on the wall 90° clockwise). Turn off
the computer, switch off the MS, UPLC, and the oil pump via their power
buttons. The oil pump and capillary can be cleaned. To turn on the MS, open
the compress air slowly and then manually turn on UPLC, MS and oil pump.
Wait until UPLC injector finish the initialization. Turn on the computer, open
Data acquisition software, right-click on Pump Down in the MS pane. Wait

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minimum 4 hours before using the MS and proceed to an autotune before any
run.
• Verify the pressure from the Nitrogen generator, outlet pressure should not
be below 80 psi, if the pressure is too low, the system will indicate an error
and the system will not be able to operate the method.
• Check the calibrant level before tuning. Use different tuning mixes for both
ESI and APCI sources available in the lab.
• Verify that all the cables are properly secured.
• It is important to use appropriate fittings with columns, valves and unions.
Agilent Technologies uses Swagelok fittings but for Waters columns use
Waters fittings.
• Capillary tubing is color coded to identify the internal diameter e.g. Red=
0.12 mm, Green= 0.17 mm and Blue = 0.25 mm. Make sure to use the
right tubing and fittings for the connections.
• The binary pumps should be purged and primed before use.
• Drain any liquid from the LC-MS waste bottle otherwise it will flush back
to LC-MS.
• Set the pressure limits according to your column.
Buffers should be <15 mM and sonicate prior to use. Never leave pure
acetonitrile on the system as it will polymerize if left standing for periods of
time. Always remove the column and store the system with 10% acetonitrile
in HPLC grade water.
• MP bottles should never be covered with Para film. Use Aluminum foil.
• If the sample injection is required using a 96 well plates, configure the
software for 96 well plates. By default it is configured for injection using vials.
• Flush the system with water right after if the buffer has been used to get
rid of the salts. When changing solvents, all channels should be purged at
2 mL/min for 20 minutes/channel.
• If the column is not in use, rinse it with additive free mobile phase and then
with 50-80 % % acetonitrile at 0.1 mL /min for 10-20 column volumes or
as recommended by the column manufacturer.
• If very sticky compounds are used, extra cleaning is required to remove
traces. An Injector Purge Kit is available from Agilent Technologies for
extra cleaning for sticky compounds.
• To change the APCI source put system on standby. Yellow light “should turn
on” on standby mode. Wear gloves while replacing the regular spray shield
for APCI because it may be hot.

3.3 Relevant documents

• Agilent 6460 Series LC-MS QQQ techniques and operation volume 1

Student Manual
• Agilent 6460 Series LC-MS QQQ techniques and operation volume 2
Student Manual
• Nitrogen generator Manual

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4. Procedure
4.1 MS Operation
If the system is not turned on, follow the sequence below to turn it on:
• Check the pressure in N2 generator first for the pressure above 80 psi.
• N2 cylinder is always open for collision energy.
• Turn on the triple Quad.
• Turn on the UPLC and wait for 5 minutes.
• Login with the given user name and password.
• Go to the Mass Hunter Data Acquisition application and turn on the system.

Always perform auto tune once the system is turned “ON”.

If the mass spectrometer is not in use, keep in standby mode. Never turn
the system off except for appropriate maintenance or cleaning.

Follow the instructions from page 195-204 of the student manual LC-
MS QQQ techniques and operation volume 1 for preparing UPLC and
sample preparation procedure. A QQQ routine pre-operation checklist
is included in Appendix I and can be used to verify the LC-MS operation.

If any component of LC-MS system does not work inform the Supervisor, Clinical
Analysis or delegate.

4.2 Data acquisition software layout and instrument controls

Open the data acquisition software. There is a default layout which can be used.
The status of the instrument can be determined by looking at the instrument
status pane. Green means that the instrument is ready for a run. Yellow means
“not ready”, red means that an error has occurred in the module and you can check
the logbook to view the error or hover over the red bar for an error description
(inform the Clinical Analysis Suite supervisor). Blue means that the system is running
and a pink color indicates that the instrument is waiting to inject.

Turn BinPump on and flush the injector. Ensure that lines are clear of bubbles, if not
purge the lines via the BinPump pane. Flush the mobile phase from the binary pump
for 60 sec. In TCC (thermostat column compartment), with direct injection, switch
to column 1 or 2 depending on the choice of the column. Under “Bin Pump” icon
you can set mobile phase composition, gradient steps and times, flow rate. Turn the
pump on.
Save the data in R: drive with your folder. Contact PERFORM IT analyst in order
to obtain access to this drive.

Refer to pages 365-418 of the student manual LC-MS QQQ techniques and

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operation volume 1 to set up all UPLC and QQQ parameters.

Follow the procedure below if you need to perform a Checktune or Autotune

on LC-MS.

4.3 Check Tune and Auto Tune

Refer to page 171 of student manual LC-MS QQQ techniques and
operation volume 1 for Checktune and Autotune procedure.

• Wait at least 15 min to stabilize before tuning. There are different low
concentrations tuning mix solutions for ESI and APCI sources from
Agilent Technologies. The flow rate for tuning mix is 0.3 mL /min. Make sure
that there is enough calibrant in the bottle for Checktune or Autotune.
• Check tune once a week when in use or as specified in the study protocol. If
one or two masses do not pass then do Autotune.
• Autotune will automatically change the QQQ parameters and calibrate the
QQQ. Routine use only requires resolution and mass axis verification for
polarity and resolution modes to be used. Select the ions of interest as
Positive or Negative for the Check tune. It will take 15-20 minutes for
every mode. No need to inject anything as it is automatically run by the
system. If there is no peak in the Autotune, a number of issues may be causing
this: the source is dirty, ion transfer capillary is blocked, there’s no calibrant
solution left, and/or calibrant solution is not being delivered to the source. This
is not an extensive list but can help to start troubleshooting. To clean source
flush it with 50:50 methanol and HPLC grade water before repeating.
• After Autotune the EMV is usually 2000 V for old instrument. For the
new instrument the EMV is around 1100 V.
• Check the results if they are acceptable or not. The report will be
automatically saved as PDF under D:\Masshunter\tune\QQQ\G6460A.
• Autotune should be done monthly to optimize ion transmission, update EM
voltage and minimize source exposure to TFA. It takes about 60 minutes to
run the Autotune for both Positive and Negative modes. Flush source with
LC flow after Autotune before running samples in negative ions.

If Checktune fails and abundances drop dramatically, in parallel with a major loss
in assay sensitivity, this suggests a problem with the source and/or mass analyzer. A
major drop in tuning mix mass without a loss in analyte signal suggests a calibrant
problem. The calibrant level may be low or it has been degraded or the diverter
valve may be malfunctioning. Inform Supervisor of Clinical Analysis Suite.

Note: It is useful to monitor the Autotune EMV in the Autotune

report in order to know when the electron multiplier will need to be
replaced. When the sum of the Autotune EMV and the method delta
EMV approach 3000 volts, a new electron multiplier tube should be

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replaced after venting the LC-MS system. Inform Supervisor, Clinical

Analysis who will place a service call.

4.4 Work list

Refer to pages 5-27 of the training manual (volume 2) for the work list preparation.

4.5 Qualitative analysis

Refer to pages 52-104 of the training manual (volume 2) for the Qualitative data
analysis preparation.

4.6 Quantitative Analysis

Refer to pages 178-240 of the training manual (volume 2) for the Qualitative
data analysis preparation.

4.7 Parameters to use in the method optimization

For the method optimization, the parameters for Jet stream ESI and APCI can be
found at: https://perform.concordia.ca/booking/Reference/clinical_analysis/start-
up_parameters_for_qqq_LC-ms.pdf

4.8 Preventive maintenance

Follow Agilent 6400 Maintenance Guide for detailed procedures. In general,
the following is required for the regular maintenance of LC-MS when in use:

Daily:
Flush the nebulizer, clean electrospray spray chamber, clean the APCI
chamber, clean multimode source, check drain bottle.

Weekly:
Clean electrospray spray chamber, clean the APCI chamber, Clean
multimode source, and check the level and color of the roughage pump fluid.
Check the oil mist filter.

Monthly:
Flush the nebulizer. Replace the oil pump every six months. Replace it sooner
if the oil appears dark or cloudy.

Annually:
Replace MS selection wall rotor seal. Multiplier needs replacement every year.
Change Hydrocarbon filters. Wipe the skimmer once a year.

When required:
Clean and adjust electrospray needle as well as the APCI needle. Clean the

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capillary, clean skimmer, change lamp in the source, check for leaks, replace
the LC filter elements, replace electron multiplier horn, replace high energy
dynode. Add rough pump fluid when the pump fluid level is low. Replace
the fuses. Change the seal of the pump when required, needle wash and needle
seat after 60 000 injections.

To adjust the needle, put it in to the magnifier and adjust the needle so that
it is aligned with the tip of ESI or APCI. If it is blocked, dip it in 50:50
methanol and Type 1 water and sonicate it for 3 minutes.

Nitrogen generator:
Refer to Nitrogen generator Manual for the operation, troubleshooting and
maintenance details. Below is the maintenance schedule for N2 generator:

Part Action Frequency

Filters Replace carbon absorber 1x per year
Oxygen sensor Replace oxygen sensor 1x per 3 years
Oxygen sensor Calibrate oxygen sensor 1x per year
Air compressor Replace on failure Expected life > 8000 hrs
Nitrogen compressor Replace on failure Expected life > 8000 hrs
Battery on PCB Replace on failure Expected life > 5 years

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