DDRUGS OF ABUSE ANALYSIS RUGS OF ABUSE ANALYSIS

48 Pages·2007·3.11 MB·English

by  Melissa Clark

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Waters System Solutions for Drugs of Abuse Analysis
Waters Corporation has over 40 years history of developing innovative HPLC, mass spectrometry, software,
chemistry and support services. Waters can now provide forensic toxicology laboratories with complete solutions
that will improve the accuracy and precision of assays while increasing productivity. Waters MassTrak™ Systems
bring the power of LC/MS to your laboratory in a robust, easy-to-use and cost-effective package. These systems
offer new levels of sample throughput, sensitivity, specifi city and fl exibility for both screening and confi rmation
applications. They are supported by a dedicated toxicology applications development group that has extensive
experience in developing validated methods for drugs of abuse analysis.
The MassTrak™ System for routine drugs of abuse confirmation
analysis, consisting of an Alliance® HT HPLC system and the
Quattro micro™ API incorporating TargetLynx™ Application
Manager, sets new standards for sample throughput, sensitivity
and ease of use.
The Quattro micro™ API can also be used for toxicology
screening applications using the unique ChromaLynx™
chromatographic data processing software and in-source
CID libraries.
The MassTrak™ system incorporating the LCT Premier™ XE represents
a new powerful solution for forensic toxicology screening
applications. The LCT Premier™ XE is based on time of flight
(TOF) technology, which combined with the ACQUITY UPLC™
system provides fast, reliable exact mass measurement. The
combination of high full scan sensitivity and routine exact mass
measurement (< 5 ppm) enables identification of unknown
compounds with the highest degree of confidence.
The LCT Premier™ XE, shown here with the ACQUITY UPLC™
System, represents a major advance for screening applications
and provides the ultimate sensitivity and speed of analysis.
The MassTrak™ System incorporating the Quattro Premier™ XE
raises the performance bar for drugs of abuse analysis. The
Quattro Premier™ XE is a high-performance benchtop tandem
quadrupole instrument, featuring advanced Travelling Wave
(T-Wave™) technology. The ultra fast scanning speed enabled by
T-Wave™ technology enables the Quattro Premier™ XE to take full
advantage of the exceptionally high chromatographic resolution
of the ACQUITY. The combination of the ACQUITY UPLC™ and
Quattro Premier™ XE delivers unmatched sensitivity and analysis
speed for drugs of abuse confirmation analysis.
Chemistries for the Drugs of Abuse Laboratory
Selecting an HPLC column can be a daunting task with hundreds of stationary phases to choose from. Waters makes it easy
by providing innovative stationary phases that provide superior peak shapes, long column lifetimes and complementary
selectivities. Whether you’re working with simple or complex matrices, biological or non-biological samples, acidic, basic
or neutral molecules. No one offers you more proven sample preparation tools for LC/MS/MS and GC/MS challenges.
Table of Contents
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-6
ChromaLynx™ Application Manager for Systematic Toxicological Analysis . . . . . . . . . . . . . . . . . . . . . . . . 7-8
TargetLynx™ Application Manager for Confirmation Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-10
General Unknown Screening for Drugs in Biological Samples by LC/MS . . . . . . . . . . . . . . . . . . . . . . . 11-14
A rapid and Sensitive Method for the Quantitation of
Amphetamines in Human Saliva . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15-18
Opiates: Use or Abuse? Quantification of Opiates in Human Urine . . . . . . . . . . . . . . . . . . . . . . . . . . . 19-20
Quantification of M orphine, M orphine-3-Glucuronide and
Morphine-6-Glucuronide in Biological Samples by LC/MS/MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21-22
Development of a Rapid and Sensitive Method for the Quantification
of Benzodiazepines in Plasma and Larvae by LC/MS/MS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23-25
Detection of N ordiazepam and O xazepam in
Calliphora Vicina Larvae using LC/MS/MS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26-28
Quantitative Analysis of Δ 9-Tetrahydrocannabinol in Preserved
Oral Fluid by LC/MS/MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29-33
Simultaneous Analysis of G HB and its Precursors in Urine
Using LC/MS/MS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34-37
Determination of Aconitine in Body Fluids by LC/MS/MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38-40
Published References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41-43
Compound Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Cautionary Statement:
The MassTrak™ systems are CE marked and declared as in vitro diagnostic devices in the European
Union under EU directive 98/79/EC, however the application notes described in this document are
for Forensic use only, they are not to be used for any medical device diagnostic application.
©2007 Waters Corporation.
Introduction
The utilization of LC/MS (and particularly With LC/MS/MS, amphetamines, opiates,
LC/MS/MS) in forensic toxicology laboratories has benzodiazepines, G HB and many other drugs can
increased significantly in recent years. The sensitivity, be analyzed without extensive sample pre-treatment
rapid analysis, selectivity and simple sample pre- and without derivatization. The sensitivity of
treatment requirements have led to LC/MS/MS LC/MS/MS allows the use of small sample volumes.
methods being adopted as the first choice for many Thus, volume-limited samples from alternate matrices,
important drugs of abuse analysis applications. e.g. hair, sweat and oral fluid can be used in
addition to blood, plasma or urine. MassTrak™
In addition to the common illicit drugs such as
Systems equipped with the TargetLynx™ application
amphetamines, opiates, cannabis, LSD and cocaine,
manager enable the quantification and verification
many prescribed ‘legal’ drugs have a high potential
of drugs of abuse in a single chromatographic run
for abuse and are knowingly abused or accidentally
with a high degree of confidence.
misused e.g. benzodiazepines and the prescribed
opiates, methadone and buprenorphine. The Waters toxicology application group has gained
extensive expertise in developing and refining
Drugs of abuse analysis is typically a two-part
LC/MS/MS methods for the quantification of a
process - an initial screening test is usually followed
wide range of drugs of abuse in their application
by a confirmatory analysis of putative positive results
laboratories in Manchester (UK), Paris (France) and
screens. The most widely used screening technique
Milford (USA) and in collaboration with many forensic
is immunoassay while GC/MS is the most utilized
laboratories in Europe. Some of these methods are
technique for confirmation analysis. LC/MS/MS is
documented in this application notebook.
now an established technique for confirmation analysis
and is increasingly used for screening applications. Areas of focus have been on:
Amphetamines from plasma, urine and saliva
Basic drugs in saliva
Targeted and Confirmatory Analysis
THC in saliva
Liquid chromatography—tandem mass spectrometry
(LC/MS/MS) is now a widely accepted technique Opiates from plasma and urine
in forensic toxicology laboratories for quantitative
Benzodiazepines from plasma, urine and fly larvae
and confirmatory analysis. It is particularly useful for
GHB from urine
polar, non-volatile and thermally labile compounds
that are difficult to analyze by gas chromatography
(GC). In addition, the reduced sample pre-treatment
requirements and short run times of LC/MS/MS,
compared to GC/MS, make this technique attractive
for high-throughput laboratories and for laboratories
tasked with providing rapid results.
Waters latest mass spectrometer systems, the ACQUITY™ SQD and
ACQUITY™ TQD combine ease of use and robustness with the speed
and sensitivity of UPLC® technology.
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©2007 Waters Corporation.
Systematic Toxicological Analysis or General A comprehensive LC/MS library is now available
Unknown Screening (GUS) for use on Waters LC/MS (single quadrupole) and
LC/MS/MS (tandem quadrupole) systems. It is based
LC/MS is also a powerful tool for systematic
on a generic chromatographic run using electrospray
toxicological analysis (STA). Waters has developed
ionization (ESI) and mass spectra recorded at multiple
the ChromaLynx™ Application Manager, a unique
cone voltages. Controlled, reproducible fragmentation
data processing tool that can search LC/MS libraries
is caused by in-source fragmentation providing spectra
based on cone voltage fragmentation. ChromaLynx™
of structurally-related fragments ions; the higher the
Application Manager provides automatic deconvolution
cone voltage, the more fragmentation is observed.
and exhaustive examination of complex chromatograms
Identification of compounds from this type of experiment
to identify individual components including minor and
is based on matching the spectra from multiple cone
closely-eluting peaks. Individual components are then
voltage spectra at a single retention time.
searched against library spectra and the results are
displayed in an easy-to-use browser format. The current version of the Waters toxicology library
contains spectra from > 500 compounds, and has been
LC/MS is now increasingly used in screening
tested for use with the Waters ZQ™ single quadrupole
applications. Until recently, the widespread
and Quattro micro™ tandem mass spectrometer systems.
implementation of LC/MS has primarily been limited
by the lack of commercially available LC/MS libraries,
and the perceived high capital costs of LC/MS
systems. In recent years the capital cost of LC/MS has GC/MS/MS
reduced making the technique more widely accessible. Waters Corporation also offers GC/MS/MS systems
for forensic toxicology. The Quattro micro™ GC is
the most sensitive GC-tandem quadrupole mass
spectrometer on the market today. Waters offers
GC/MS/MS systems for analytes which have
traditionally been submitted for GC/MS analysis.
GC/MS/MS offers enhanced sensitivity and specificity
over GC/MS and allows for reduced sample clean-up.
Waters Quattro micro™ GC - tandem mass spectrometer system for the most
demanding GC applications.
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©2007 Waters Corporation.
ChromaLynx™ Application Manager for Systematic
Toxicological Analysis
Introduction ChromaLynx™ for the Forensic
Toxicology Laboratory
The need to qualitatively analyze complex mixtures
is frequently encountered in forensic toxicology Chromalynx™ addresses many of the requirements of
laboratories. Due to the polar, and non volatile nature the toxicology laboratory for screening applications.
of many toxicologically relevant compounds, LC/MS It is designed for automated processing of LC/MS and
methods are now increasingly utilised in toxicological GC/MS data and some of the key features are:
screening applications. In these applications there is
• Detection of all component peaks in a
usually a need to detect and identify toxic compounds
sample, including peaks not seen in total
from complex chromatograms resulting from the
ion chromatogram (TIC) traces
analysis of biological fluids such as blood and urine.
• Spectral deconvolution and peak identification
Manually reviewing complex chromatograms to
• Automated library searching at multiple
detect and identify potential toxic compounds can
cone voltages
be a laborious, time-consuming and subsequently
expensive task. In a manual process, closely eluting • Automatic scoring of the library search
or low intensity components can easily be missed. • Combination of retention time and
ChromaLynx™ automates this manual task, enabling mass spectra in the library search.
rapid detection and identification of compounds
• Results displayed in user-friendly browser
from complex mixtures.
with report generator option
When combined with a powerful multi-function LC/MS
library, ChromaLynx™ offers the most comprehensive
LC/MS solution for screening applications.
Figure 1:
ChromaLynx™ is able to confidently detect and locate closely eluting peaks. Here
Cocaine is identified with a high degree of confidence in a very complex area of
the Chromatogram.
Total Ion Chromatogram (TIC) traces from 7 functions
from a LC/MS analysis of a urine sample. Several
components were identified by ChromaLynx™
including, Ecgoninemethyl ester, Morphine,
Benzolyecognine, Cocaine and Noscapine
ChromaLynx™ is able to detect and locate low intensity peaks. Peak eluting at 3.14 mins was subsequently identified as M orphine.
On visual inspection, there is no conclusive evidence that a significant component elutes at 3.14 minutes. The unique ChromaLynx™
deconvolution algorithm clearly indicates that a component is present and has been confidently identified.
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©2007 Waters Corporation.
Flexibility and Ease of Use Spectral Deconvolution
ChromaLynx™ has been designed to be easy to use Following acquisition of full scan spectra recorded at
while offering flexibility. It consists of a method editor, multiple cone voltages, ChromaLynx™ will analyse
to set up chromatographic data processing and library each chromatogram and extract full scan spectra
search parameters. The method editor can be viewed and extract specific ion chromatograms to detect
spreadsheet-style for ease of review and allows editing the presence of components.
of parameters for peak location and detection, and
The key to the exceptional performance of ChromaLynx™
subsequent identification using LC/MS and GC/MS
is a new, proprietary chromatography deconvolution
libraries. The processed data is displayed in the
algorithm. This algorithm efficiently locates peaks in
ChromaLynx™ browser for ease of review.
a chromatogram and extracts ‘’clean’’ mass spectra
ChromaLynx™ can be used with both LC/MS and of eluting components. The extracted mass spectra
GC/MS data and can accommodate both exact mass can then be searched against LC/MS and GC/MS
and nominal mass data. libraries. ChromaLynx™ has been designed to exploit a
unique multi-function electrospray LC/MS library based
on in-source collision induced (CID) mass spectra. By
recording mass spectra at multiple cone voltages in both
positive and negative ion mode extensive information
is acquired on samples. To further enhance the library
search process, ChromaLynx™ also uses retention time
information as a search parameter.
Summary
ChromaLynx™ Application Manager sets new standards
Figure 2: ChromaLynx™ method editor displaying
chromatogram data processing parameter set up. In this for the analysis of complex chromatograms resulting from
example 7 chromatograms will be processed, five recorded LC/MS or GC/MS analysis of physiological samples
in positive Ion mode and two in negative ion mode. such as blood and urine. A unique algorithm enables
ChromaLynx™ to locate peaks in a chromatogram and
then automatically compare the mass spectra against
library mass spectra. When using LC/MS mass spectra
recorded at multiple cone voltages (using in-source
CID) combined with retention time information further
enhances the component identification processes.
Green peaks indicate a component identified
with a high degree of confidence
List of possible components present. Green
indicates confident identification, yellow tentative
identifications, red indicates a poor library match
Library search results for
component in-source at
three different cone voltages.
Library search displays top three
Candidates identified at specific
point in the chromatogram
Visual comparison of component mass
spectrum against proposed library match
Figure 3: Library search method editor enabling automatic
Figure 4: ChromaLynx™ Browser – displaying identified compounds,
library searching of all peaks and filtering of results for
candidate compounds, results of a library search and total ion
retention time and cone voltage used.
chromatograms recorded at different cone voltages.
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©2007 Waters Corporation.
TargetLynx™ Application Manager for Confirmation Analysis
Introduction If the analytical data is required to be used as part of
a police investigation or presented in a court of law,
Quantitation using LC/MS/MS is now well established
it is essential that extensive analytical information is
in many forensic, environmental, clinical and veterinary
provided to confirm the presence of a suspected drug.
applications. There are often legal, environmental,
TargetLynx™ is ideal for this application, where the
human health and financial implications arising from
presence of a suspected drug can be confirmed by
the results of quantitative MS analyses. This has led
the presence of a number of different diagnostic ions
to an increased demand by regulatory and legal
and MRM transitions. Confirmation analysis using
authorities for extra confirmatory and quality control
TargetLynx™ is enhanced as ion ratio measurements
checks. Regulatory or statutory methods often require,
and chromatographic retention time information is also
for example, the monitoring of multiple structurally
incorporated in the analytical procedure.
specific fragment ions, maximum chromatographic
peak width and/or retention time. To calculate and
check these manually is a labour intensive, time-
consuming and subsequently costly task. 168
100
Quantitation
TargetLynx™ automates data acquisition, processing
and reporting incorporating a wide range of
confirmatory checks allowing samples falling
outside user-specified or regulatory thresholds to be
easily identified, giving confidence when reporting 105 Confirmation 1
quantitative results. %
Confirmation 2
TargetLynx™ is able to rapidly identify and flag 82
samples where, for example: 119
150
• Analytes are above a specified concentration 93 122
290
• Analyte confirmatory ion ratios are outside 0 m/z
50 100 150 200 250 300
specified limits
• One or more analyte signal-to-noise ratios are
Figure 1: Mass Spectrum of the cocaine metabolite
below a defined value Benzoylecgonine.Transition 290/168 is used for quantitation.
• An analyte retention time or relative retention time Two further MRM transitions 290/105 and 290/82 are
monitored for additional confirmation by TargetLynx™.
is outside limits
• The coefficient of determination (r2) of the
Flexibility and Ease of Use
calibration curve exceeds a defined value
TargetLynx™ can be used with LC/MS/MS and
GC/MS/MS data. This data can be SIM (Selected
Drugs of Abuse and Forensic Toxicology
Ion Monitoring), MRM (Multiple Reaction Monitoring)
LC/MS/MS is now increasingly used in forensic
or full scan/full spectrum. In the case of full scan/full
toxicology laboratories for drugs of abuse confirmation
spectrum data, extracted ion chromatograms are used
and quantitation applications. LC/MS/MS is typically
for quantitation.
used for confirmation analysis, following a positive
immunoassay analysis that indicates the presence of TargetLynx™ consists of a method editor, to set up
a class of drugs or specific drug, or when there is processing parameters, and a browser, to view
evidence present that a drug is likely to be present in processed data. The method editor can be viewed
a sample. spreadsheet-style for ease of review and allows
all quantitation parameters (for peak location and
detection, calibration curves etc.) and user-defined
criteria for confirmatory and QC checks to be set up.
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©2007 Waters Corporation.
Processed data is displayed in the TargetLynx™ browser The Compound Summary Report, Sample Summary
for ease of review. A variety of sample flags allows Report, Totals Report and Samples Report allow the
easy location and interrogation of samples falling out user to display calibration information per compound,
with the defined confirmatory and QC criteria. report one compound/sample/totals group per page
or split and print summary reports per sample.
Data can be exported from the TargetLynx™ browser
Method Setup
as a XML or comma separated text file into a LIMS
system.
Data Acquisition
Summary
Data Processing
TargetLynx™ Application Manager automates data
acquisition, processing and reporting incorporating a
Review Results wide range of confirmatory checks allowing samples
falling outside user-specified or regulatory thresholds to
be easily identified, giving confidence when reporting
Reporting quantitative results.
TargetLynx™ provides an easy to use and flexible
solution to increase laboratory productivity and
improve the quality of quantitative LC/MS/MS or
GC/MS/MS data.
Moving the cursor over the sample of interest,
displays tool tips with explanations of why the
sample has been flagged.
Figure 2: TargetLynx™ Method Editor – showing customisable
selection of relevant displayed parameters.
Reporting
Customisation and Export of Data
Figure 3: TargetLynx™ browser – showing results summary with flags,
TargetLynx™ features various reporting options, with
calibration curve and chromatograms.
reports being printed directly from the Browser file by
sample or by compound.
Report formats can be customised and can consist of
all or some of the following; the Calibration page,
Compound Summary Report, Sample Summary Report,
Totals Support, Samples Report and Audit Report.
In the Calibration page the user can select how the
data is displayed, for example ‘Show Residuals’,
‘Show Response Curve’ and/or ‘Show QC Points’.
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©2007 Waters Corporation.