Drugs of Abuse Analysis WATERS LAB

DRUGS OF ABUSE ANALYSIS
APPL IC ATION NOTEBOOK
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, specicity and exibility for both screening and conrmation
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 youre 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 Morphine, Morphine-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 Nordiazepam and Oxazepam in
Calliphora Vicina Larvae using LC/MS/MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26-28
Quantitative Analysis of 9-Tetrahydrocannabinol in Preserved
Oral Fluid by LC/MS/MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29-33
Simultaneous Analysis of GHB 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
With LC/MS/MS, amphetamines, opiates,

benzodiazepines, GHB and many other drugs can
be analyzed without extensive sample pre-treatment
and without derivatization. The sensitivity of
LC/MS/MS allows the use of small sample volumes.
Thus, volume-limited samples from alternate matrices,
e.g. hair, sweat and oral fluid can be used in
addition to blood, plasma or urine. MassTrak
Systems equipped with the TargetLynx application
manager enable the quantification and verification
of drugs of abuse in a single chromatographic run
with a high degree of confidence.
The utilization of LC/MS (and particularly

LC/MS/MS) in forensic toxicology laboratories has
increased significantly in recent years. The sensitivity,
rapid analysis, selectivity and simple sample pretreatment requirements have led to LC/MS/MS
methods being adopted as the first choice for many
important drugs of abuse analysis applications.
In addition to the common illicit drugs such as
amphetamines, opiates, cannabis, LSD and cocaine,
many prescribed legal drugs have a high potential
for abuse and are knowingly abused or accidentally
misused e.g. benzodiazepines and the prescribed
opiates, methadone and buprenorphine.
The Waters toxicology application group has gained

extensive expertise in developing and refining
LC/MS/MS methods for the quantification of a
wide range of drugs of abuse in their application
laboratories in Manchester (UK), Paris (France) and
Milford (USA) and in collaboration with many forensic
laboratories in Europe. Some of these methods are
documented in this application notebook.
Drugs of abuse analysis is typically a two-part

process - an initial screening test is usually followed
by a confirmatory analysis of putative positive results
screens. The most widely used screening technique
is immunoassay while GC/MS is the most utilized
technique for confirmation analysis. LC/MS/MS is
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 chromatographytandem mass spectrometry

(LC/MS/MS) is now a widely accepted technique
in forensic toxicology laboratories for quantitative
and confirmatory analysis. It is particularly useful for
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.
Opiates from plasma and urine

Benzodiazepines from plasma, urine and fly larvae
GHB from urine
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.
5
Systematic Toxicological Analysis or General

Unknown Screening (GUS)
A comprehensive LC/MS library is now available

for use on Waters LC/MS (single quadrupole) and
LC/MS/MS (tandem quadrupole) systems. It is based
on a generic chromatographic run using electrospray
ionization (ESI) and mass spectra recorded at multiple
cone voltages. Controlled, reproducible fragmentation
is caused by in-source fragmentation providing spectra
of structurally-related fragments ions; the higher the
cone voltage, the more fragmentation is observed.
Identification of compounds from this type of experiment
is based on matching the spectra from multiple cone
voltage spectra at a single retention time.
LC/MS is also a powerful tool for systematic

toxicological analysis (STA). Waters has developed
the ChromaLynx Application Manager, a unique
data processing tool that can search LC/MS libraries
based on cone voltage fragmentation. ChromaLynx
Application Manager provides automatic deconvolution
and exhaustive examination of complex chromatograms
to identify individual components including minor and
closely-eluting peaks. Individual components are then
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
tested for use with the Waters ZQ single quadrupole
and Quattro micro tandem mass spectrometer systems.
LC/MS is now increasingly used in screening

applications. Until recently, the widespread
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
reduced making the technique more widely accessible.
GC/MS/MS
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.
6
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
laboratories. Due to the polar, and non volatile nature
of many toxicologically relevant compounds, LC/MS
methods are now increasingly utilised in toxicological
screening applications. In these applications there is
usually a need to detect and identify toxic compounds
from complex chromatograms resulting from the
analysis of biological fluids such as blood and urine.
Chromalynx addresses many of the requirements of

the toxicology laboratory for screening applications.
It is designed for automated processing of LC/MS and
GC/MS data and some of the key features are:
Detection of all component peaks in a
sample, including peaks not seen in total
ion chromatogram (TIC) traces
Spectral deconvolution and peak identification
Manually reviewing complex chromatograms to

detect and identify potential toxic compounds can
be a laborious, time-consuming and subsequently
expensive task. In a manual process, closely eluting
or low intensity components can easily be missed.
ChromaLynx automates this manual task, enabling
rapid detection and identification of compounds
from complex mixtures.
Automated library searching at multiple

cone voltages
Automatic scoring of the library search
Combination of retention time and
mass spectra in the library search.
Results displayed in user-friendly browser
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 Morphine.
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.
7
Flexibility and Ease of Use
Spectral Deconvolution
ChromaLynx
Following acquisition of full scan spectra recorded at

multiple cone voltages, ChromaLynx will analyse
each chromatogram and extract full scan spectra
and extract specific ion chromatograms to detect
the presence of components.
has been designed to be easy to use

while offering flexibility. It consists of a method editor,
to set up chromatographic data processing and library
search parameters. The method editor can be viewed
spreadsheet-style for ease of review and allows editing
of parameters for peak location and detection, and
subsequent identification using LC/MS and GC/MS
libraries. The processed data is displayed in the
ChromaLynx browser for ease of review.
The key to the exceptional performance of ChromaLynx

is a new, proprietary chromatography deconvolution
algorithm. This algorithm efficiently locates peaks in
a chromatogram and extracts clean mass spectra
of eluting components. The extracted mass spectra
can then be searched against LC/MS and GC/MS
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.
ChromaLynx can be used with both LC/MS and

GC/MS data and can accommodate both exact mass
and nominal mass data.
Summary
ChromaLynx Application Manager sets new standards
for the analysis of complex chromatograms resulting from
LC/MS or GC/MS analysis of physiological samples
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.
Figure 2: ChromaLynx method editor displaying

chromatogram data processing parameter set up. In this
example 7 chromatograms will be processed, five recorded
in positive Ion mode and two in negative ion mode.
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

library searching of all peaks and filtering of results for
retention time and cone voltage used.
Figure 4: ChromaLynx Browser displaying identified compounds,

candidate compounds, results of a library search and total ion
chromatograms recorded at different cone voltages.
8
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,
it is essential that extensive analytical information is
provided to confirm the presence of a suspected drug.
TargetLynx is ideal for this application, where the
presence of a suspected drug can be confirmed by
the presence of a number of different diagnostic ions
and MRM transitions. Confirmation analysis using
TargetLynx is enhanced as ion ratio measurements
and chromatographic retention time information is also
incorporated in the analytical procedure.
Quantitation using LC/MS/MS is now well established

in many forensic, environmental, clinical and veterinary
applications. There are often legal, environmental,
human health and financial implications arising from
the results of quantitative MS analyses. This has led
to an increased demand by regulatory and legal
authorities for extra confirmatory and quality control
checks. Regulatory or statutory methods often require,
for example, the monitoring of multiple structurally
specific fragment ions, maximum chromatographic
peak width and/or retention time. To calculate and
check these manually is a labour intensive, timeconsuming and subsequently costly task.
168
100
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
quantitative results.
Quantitation
Confirmation 1
105
TargetLynx is able to rapidly identify and flag

samples where, for example:
Confirmation 2
82
93
Analytes are above a specified concentration

0
50
Analyte confirmatory ion ratios are outside

specified limits
One or more analyte signal-to-noise ratios are
below a defined value
119
150
122
100
290
150
200
250
m/z
300
Figure 1: Mass Spectrum of the cocaine metabolite

Benzoylecgonine.Transition 290/168 is used for quantitation.
Two further MRM transitions 290/105 and 290/82 are
monitored for additional confirmation by TargetLynx.
An analyte retention time or relative retention time

is outside limits
The coefficient of determination (r2) of the
calibration curve exceeds a defined value
Flexibility and Ease of Use

TargetLynx can be used with LC/MS/MS and
GC/MS/MS data. This data can be SIM (Selected
Ion Monitoring), MRM (Multiple Reaction Monitoring)
or full scan/full spectrum. In the case of full scan/full
spectrum data, extracted ion chromatograms are used
for quantitation.
Drugs of Abuse and Forensic Toxicology

LC/MS/MS is now increasingly used in forensic
toxicology laboratories for drugs of abuse confirmation
and quantitation applications. LC/MS/MS is typically
used for confirmation analysis, following a positive
immunoassay analysis that indicates the presence of
a class of drugs or specific drug, or when there is
evidence present that a drug is likely to be present in
a sample.
TargetLynx consists of a method editor, to set up

processing parameters, and a browser, to view
processed data. The method editor can be viewed
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.
9
Processed data is displayed in the TargetLynx browser

for ease of review. A variety of sample flags allows
easy location and interrogation of samples falling out
with the defined confirmatory and QC criteria.
The Compound Summary Report, Sample Summary

Report, Totals Report and Samples Report allow the
user to display calibration information per compound,
report one compound/sample/totals group per page
or split and print summary reports per sample.
Data can be exported from the TargetLynx browser
as a XML or comma separated text file into a LIMS
system.
Method Setup
Data Acquisition
Summary
Data Processing
TargetLynx Application Manager 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
quantitative results.
Review Results
Reporting
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,
calibration curve and chromatograms.
TargetLynx features various reporting options, with

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.
10
General Unknown Screening for Drugs in Biological

Samples by LC/MS
Luc Humbert1, Michel Lhermitte1, Frederic Grisel2
1Laboratoire de Toxicologie & Gnopathologie, CHRU Lille, France
2Waters Corporation, Guyancourt, France
Introduction
Electrospray is a soft ionization technique that mainly

leads to protonated molecular ions in positive ion
mode and to deprotonated molecular ions in negative
ion mode. In order to get more specific structural
information, it is possible to induce fragmentation of
these molecular ions in the source region of a mass
spectrometer. This can be achieved by increasing
the voltage applied to the sampling cone area where
ions transit from a high pressure region to a low
pressure region. Molecular ions then collide with
neutral molecules in the source region and fragment
into characteristic ions. This is referred to as insource
collision induced dissociation (CID). Using this process
reproducible LC/MS mass spectra can be used to
produce a library of mass spectra .
Identification of drugs of abuse and toxicants in

biological fluids is currently performed by a variety
of analytical techniques including immunoassays
and chromatographic techniques such as GC/MS
and LC with UV detection. Although these techniques
are well established and widely used, they suffer
from limitations for many toxicologically important
compounds. For example, sensitivity is often a
limitation with LC/UV techniques as newer drugs
are used at lower therapeutic concentrations. In
addition, LC/UV methods can require extensive
sample preparation. GC/MS is often referred to as
the gold standard in toxicology laboratories, but
even GC/MS has significant limitations for toxicology
screening applications where rapid sample analysis
is a requirement. Many substances encountered in
toxicology laboratories are non-volatile, polar or
thermally labile and cannot be directly analyzed
by GC/MS. These compounds usually require time
consuming derivatization prior to analysis.
H3O+
analyte
[analyte +H]+
LC/MS, using electrospray ionization (ESI), is ideally

suited to polar, non volatile and, thermally unstable
compounds and potentially provides a powerful means
of identifying many toxicologically relevant compounds
rapidly without the need for sample derivatization.
ESI
CID
H2O
[analyte +H]+
ion+
neutral
Figure 1. Atmospheric Pressure Ionisation (API) process - This

soft ionisation process leads to cations in positive ion mode and
anions in negative ion mode which are generally stable. These
molecular ions can be fragmented in the source region of
LC/MS instruments.
Historically, the lack of availability of LC/MS libraries

and reliable LC/MS chromatographic deconvolution
software has limited the widespread use of this
technique for screening applications. However, with
the recent development of a unique LC/MS library
and ChromaLynx chromatographic deconvolution
software, LC/MS can now be considered a powerful
and practical alternative to traditional screening
methods.
Extractor
Sample Cone
& Cone Gas
LC/MS Library Concept

The electrospray ionization process, used in
LC/MS systems, is very different from the electron
impact (EI) Ionization used in GC/MS systems,
thereby preventing the use of commercial EI mass
spectra libraries such as NIST, Wiley, and
Pfleger- Maurer-Weber.
Figure 2. InSource-CID An example showing fragmentation

of the moleculer ion (m/z 195) of caffeine at 60V in the
Quattro micro API ion source.
11
In the current version of the library, this approach

has been used for over 500 compounds, which
corresponds to approximately 2600 mass
spectra. These compounds represent 90% of the
intoxication cases encountered in Europe. In addition
chromatographic retention times are also stored for
each compound in the library. The library
is easy to maintain and user appendable.
LC Separation Method
An identical generic LC method is used both to
generate the library mass spectra and for sample
analysis. The generic gradient method has been
developed based on water and acetonitrile buffered
with 5 mM ammonium formate at pH 3. The total run
time including system and column re-equilibration is
26 minutes.
Figure 3. Extensive structural information is stored for each

component in the library as mass spectra can be stored at every
significant cone voltage in both positive and negative ion mode.
Positive ESI @ 90 V
ChromaLynx Application Manager

Chromatogram examination is at least as important
as the content and structure of the library. The
chromatogram from a typical toxicological analysis
will usually be complex and exhibit dozens of peaks.
Compounds of interest can be difficult to identify
especially at low concentrations when they can be
hidden in the base line or when they closely elute.
ChromaLynx application manager includes a unique
algorithm to specifically process multifunctional LC/MS
data. The process can be ultimately as exhaustive
as analyzing each scan for each cone voltage; this
enables the detection of the maximum number of
components in a chromatogram.
Positive ESI @ 75 V
Positive ESI @ 60 V
Positive ESI @ 45 V
Positive ESI @ 30 V
Positive ESI @ 15 V
Unlike other LC/MS/MS screening techniques,

ChromaLynx application manager enables a
complete and systematic chromatogram examination.
This type of data processing is essential for systematic
toxicological screening or general unknown screening.
ChromaLynx application manager selects a single
mass spectrum at a given scan and extracts up to 8 of
the most intense ions and reconstructs corresponding
ion chromatograms. These ion chromatograms
are then examined and components are detected
according to user defined parameters. Detected
components are then searched against
library spectra.
Figure 4. Loxapine, a tranquilizer agent. Mass spectra recorded

at 6 different CV values using in-source CID.The degree of
fragmentation increases with the cone voltage.
Using in-source CID, it is possible to generate mass

spectra exhibiting different fragmentation patterns
according to the value of the cone voltage applied
in the source. This can be done in both positive and
negative ion modes. These spectra can then be used
to build a library.
12
1911
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TIC
5.59e9
8.00
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5: Scan ES+
TIC
4.47e9
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4: Scan ES+
TIC
2.72e9
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3: Scan ES+
TIC
3.13e9
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2: Scan ES+
TIC
3.05e9
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1: Scan ESTIC
4.51e7
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Positive ESI @ 75 V
2.00
4.00
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Positive ESI @ 60 V
2.00
4.00
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Positive ESI @ 45 V
2.00
4.00
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Positive ESI @ 30 V
2
2.00
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Positive ESI @ 15 V
2
2.00
4.00
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Negative ESI @ 30 V
1911
62
2.00
4.00
This area represents only 4 minutes out of the 26

minutes of the whole chromatogram for function 3
recorded in positive ion mode at 30V. ChromaLynx
will process all chromatograms to achieve a detailed
and efficient screening. ChromaLynx application
manager automatically processes data in minutes that
would take hours manually.
7: Scan ES+
TIC
5.39e9
Positive ESI @ 90 V
6.00
Mass Spectrum Extraction and Library

Search Process
Once chromatographic components have been detected,
ChromaLynx automatically extracts mass spectra of
the individual components. This is performed taking into
account possible interferences due to closely eluting
peaks. It is possible to customize parameters in order
to get precise background subtraction depending on
the peak width and tolerance on apex determination.
Extracted mass spectra of detected components are then
compared to library mass spectra.
Time
26.00
Figure 5. Urine extract - one single sample analysis leads to 7

chromatograms. Manual examination of each chromatogram
would be time consuming and not feasible for a routine
toxicology laboratory. Analysis of the area circled in the
chromatogram above by ChromaLynx (figure 6b) illustrates
the presence of several peaks that would be missed on
examination of the total ion chromatogram.
13.08
706433728
1911
6a
In order to improve the specificity of the screening

technique, additional filters have been developed to
enhance the quality of the screening and to get more
relevant results. Retention time filters as well as cone
voltage filters are available and user defined tolerance
parameters can be implemented.
3: Scan ES+
TIC
2.95e9
Area
12.49
501322304
11.67
166387152
14.60
115287184
13.84
46390212
14.07
20840152
Time
11.50
12.00
12.50
13.00
13.50
14.00
14.50
15.00
1911
2 3
100
Figure 6a. Close-up view of the 11-15 minutes section of

chromatogram area for function 3 acquired in positive ESI @ 30
V. Here the total ion chromatogram (TIC) indicates that only one
component elutes at 13.8 minutes.
11.50
12.50
13.00
13.50
14.00
147
130
Time
14.50
318
RT 14.13 min
12.20
12.00
Peak #5
100
279
161 205 233
319
293
357 381 390
11.73
6b
14.77
11.15
RT 14.01 min
%
0
327
Peak #4
100
13.60
11.61
%
117
161
175
205
240
265 282
316
328
367 383
14.19
11.96
12.73
14.01
13.37
14.31
11.50
11.75
12.00
12.25
12.50
12.75
13.00
13.25
13.50
13.75
14.00
14.25
14.50
14.75
Figure 6b. Close-up view of the 11 - 15 minutes section of

chromatogram area for function 3 acquired in positive ESI @ 30
V. Using extracted ion chromatograms shows that at least three
components elute between 13.5 and 13.8 minutes.
RT 13.84 min
101 130
263
208 224
266
306
339 367 383
251
RT 13.72 min
101
145
177
208
227
252
301 329
Peak #1
100
%
175
Peak #2
100
%
265
Peak #3
100
Time
3
11.25
1911
100
14.13
361
371 398
329
RT 13.66 min
100
0
100
142
150
177
190 242 251

200
250
284
295
300
361
386 393
m/z
350
Figure 7. Chromatogram acquired in positive ESI @ 30 V and

corresponding mass spectra of 5 components detected by
ChromaLynx. Automated spectral deconvolution allows extraction
of clean mass spectra that can be used for library searching.
13
Application Example - Polyintoxication
Tramadol was the only expected compound that was

not detected. In addition, three unexpected compounds
were also Detected - Meprobamate, Acepromazine
and Bromazepam. It was highly likely that these three
compounds were the cause of the intoxication.
A urine sample was taken from a suspected

intoxication. It was known that the person was
taking a number of prescribed drugs. The urine
samples were analyzed by LC/MS to identify the
cause of intoxication. Toxicologists were looking
for both expected compounds due to the regular
treatment and unexpected active substances that
may have been taken accidently or deliberately.
Candidate Average Fit (%)
Method and Instrumentation

Analytical Equipment and Instrumentation
Waters Toxicology Screening LC/MS System
comprising of:
ZQ Single Quadrupole Mass Spectrometer
Alliance 2695 Separations Module
Analyte Name
Status
Origin
Nicotine
Unexpected molecule
Smoker / Contamination
6 Functions
56.1
Trimetazidine
Expected molecule
Medication
63.3
Acetaminophen
Expected molecule
Medication
62.3
Caffeine
Expected molecule
Medication
74.0
Quinine
Expected molecule
Medication
70.3
Zolpidem
Expected molecule
Medication
94.7
Meprobamate
Unexpected molecule
Unknown
55.3
Mianserin
Expected molecule
Medication
67.1
Acepromazine
Unexpected molecule
Unknown
57.6
10
Bromazepam
Unexpected molecule
Unknown
53.1
11
Hydroxyzine
Expected molecule
Medication
88.2
12
Propoxyphene
Expected molecule
Medication
62.1
13
Tramadol
Expected molecule
Medication
Not found
MassLynx 4.0 Data Station

ChromaLynx 4.0 Application Manager
Green Triangles indicate a
component Identified with
a high degree of confidence
Sample Preparation
Liquid/liquid extraction at 2 pH (4.5 & 9.0) using
dichloromethane/ether/hexane [30:50:20] + 0.5%
isoamylic alcohol.
LC Separation Method
Waters XTerra MS Column & Precolumn: C18,
3.5 m, 2.1 mm id x 150 mm (10 mm for precolumn)
Top three candidates are displayed

for each component
Compounds confidently Identified
Column Oven Temperature: 30 C
Comparison with Library spectra
Mobile Phase based on Water/Acetonitrile with

Ammonium Formate 5 mM @ pH 3
Gradient: 5% organic to 90% organic from 2 min.
to 16 minutes
Figure 8: ChromaLynx browser showing a list of candidate

compounds, chromatogram recorded at different cone voltages
and comparison of a unknown spectra against library spectra.
MS Operating Conditions
Capillary 3.5 kV in both positive and negative ion
modes
Conclusion
Source Temperature @ 120 C & Desolvation

Temperature @ 250 C
Using the combination of in-source CID at multiple

cone voltages and retention time data results in a
library containing detailed information for each
compound. With the development of ChromaLynx
data chromatographic deconvolution software, LC/MS
can now be considered a powerful tool for toxicology
screening applications.
Desolvation Gas Flow Rate @ 350 l/h &

Cone Gas Flow Rate @ 100 l/h
Function 1: Full Scan - Negative ESI from 100 to 650
amu in 250 ms @ 30 Volts
Functions 2 to 7: Full Scan - Positive ESI from 100 to
650 amu in 250 ms @ from15 Volts to 90 Volts
The unique ChromaLynx deconvolution algorithm ensures

that the maximum number of components are detected.
The unique algorithm enables low intensity and closely
eluting peaks to be detected and identified. The accuracy
of the library search process is enhanced by utilizing
multiple mass spectra per component and retention time.
Results
From the resultant analysis, 8 out of 9 expected
components were successfully identified by the
ChromaLynx data processing library search process.
14
A Rapid and Sensitive Method for the

Quantitation of Amphetamines in Human Saliva
1Michelle
1Waters,
Wood*, 2 Gert De Boeck, 2Nele Samyn, 1Don Cooper and 1Michael Morris
Manchester, UK. 2National Institute of Criminalistics and Criminology (NICC), Belgium.
Introduction
MS conditions
Ecstasy (MDMA), EVE (MDEA) and MDA are

amongst the most frequently used recreational
drugs. Target analysis of these drugs and other
amphetamines in biological samples is of great
importance for clinical and forensic toxicologists
alike. Plasma and urine are currently the most
common matrices investigated. However, due to the
invasive nature of such samples (and the associated
inconvenience of sample collection) there is an
increasing interest in the use of saliva as an alternative
marker for drug abuse.
Mass spectrometer: Quattro Ultima tandem

mass spectrometer.
Ionisation mode:
ES positive ion
Capillary voltage: 1.5 kV

MS/MS:
Collision gas: Argon at

2.5 x 10-3 mbar
Precursor
(m/z)
Product
(m/z)
Cone
Voltage
(V)
Collision
Energy
(eV)
MDEA
208
163
50
12
MDEA D5
213
163
50
12
Methamphetamine
150
91
50
15
Methamphetamine D14
164
98
50
18
Compound
Due to the limited volume of sample (usually <100 L)

the traditional methods for amphetamine analysis
(i.e. GC/MS) may not be sufficiently sensitive to
allow quantitation. In addition, the high viscosity
of saliva can lead to problems during solid-phase
extraction. Therefore, we have developed an
alternative method. Amphetamines were isolated from
saliva using a simple methanol clean-up procedure
and subsequently analysed using LC/MS/MS.
The developed method has a total analysis time
(including sample preparation) of less than 15 minutes
and allows the simultaneous analysis of several
amphetamines in saliva. Limits of detection of 1 ng/mL
saliva (or better) were achieved.
Amphetamine
136
91
60
17
Amphetamine D11
147
98
60
16
Ephedrine
166
148
30
12
Ephedrine D3
169
151
40
12
MDA
180
105
30
22
MDA D5
185
168
50
10
MDMA
194
163
60
12
MDMA D5
199
165
60
13
Table 1. MRM transitions and conditions for the measurement of

several amphetamines and their internal standards.
Methods and Instrumentation

LC conditions
LC System:
Waters Alliance 2690
Results and Discussion
Column:
Conventional C18
(100 x 2.1 mm, 3.5 m)
Mobile phase:
(A) =10 mM ammonium acetate
MRM transitions were determined for six commonly

abused amphetamines and their deuterated analogues.
The resultant transitions and conditions used are given
in Table 1. Examples of MS and product ion spectra
are given in Figure 1.
(B) = 95% acetonitrile: 5% 10 mM

ammonium acetate
Isocratic elution
(85:15)
Flow rate:
0.3 mL/min
Injection volume:
10 L
Standard curves were prepared by dilution of a

mixture of amphetamines in mobile phase followed
by LC/MS/MS analysis. Figure 2 shows the MRM
chromatograms acquired simultaneously during a
single injection (6 out of 12 shown). The typical
linearity of response, in the absence of biological
matrix, is demonstrated in Figure 3a.
15
148.2
108.1
In order to extend the experiment for determination

of amphetamines in oral fluid, a series of calibrators
(0.1-500 ng/mL) were prepared by adding
amphetamines to blank saliva. Following isolation
from the matrix using a simple methanol extraction
procedure (Figure 4), samples were analysed using
LC/MS/MS. The amphetamines were quantified
by reference to their respective deuterated internal
standards. Once again, responses were linear over
the range investigated (Figure 3b).
166.2
100
100
110
129.1
143.2
130
140
120
167.2
176.2 179.2
149.2
150
160
170
180
190
m/z
210
200
148.4
100
In order to assess the feasibility of using oral fluid as

an alternative specimen for drug abuse, saliva samples
collected from current amphetamine users were analysed
using the developed method. Figure 5 shows the
resultant MRM chromatograms for one of the oral fluid
samples found to be positive for the presence of the
amphetamines MDEA, MDMA and MDA. It should be
noted that the designer drug MDA is also a metabolite
of MDMA and MDEA. The results from 10 individuals
are summarised in Figure 6 and demonstrate that,
within this particular group, MDMA (Ecstasy) was the
most commonly used amphetamine (with concentrations
ranging from 3.1 to > 3000 ng/mL). The results also
demonstrate the trend for multiple, rather than single,
drug use.
166.3
0
100
110
120
130
140
150
160
170
180
190
200
m/z
210
100
208.4
163.2
135.1
105.1
129.1
209.3
176.2 179.2
0
90
100
110
120
130 140 150
160
170
180 190 200
210
m/z
163.1
100
208.3
90 100
110
120
130 140 150
160
170
180 190 200
210
m/z
100
213.4
163.2
105.2 108.1
124.2
214.3
176.1
212.4
0
100
110
120
130
140
150
100
160 170 180 190 200
217.4
m/z
210 220
163.2
100
%
0
MRM of 12 channels ES+

194>163
100
%
0

180>105
100
%
0

166>148
100
%
0

136>91
100
%
0

150>91
100
%
0
0.00

208>163
0.50
1.00
1.50
2.50
2.00
Time
3.00
Figure 2. MRM chromatograms obtained for a single injection

of a mixture of amphetamines(100 ng/mL) in mobile phase
(respective internal standards not shown).
133.3 135.3
Compound name: MDEA

Coefficient of Determination: 0.998354
Calibration curve: 23664.8* x + 1238.54
Response type: External Std. Area
Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None
213.4
m/z
100
110
120
130
140
150
160 170
180 190 200
210
220
12437615
Figure 1. MS (top trace) and product ion spectra (lower trace)

for (a) ephedrine, (b) MDEA and (c) MDEA-D5.
Response
50
100
150
200
250
300
350
400
450
pg/l
500
Figure 3a. Typical linearity of response for MDEA in the absence

of biological matrix.
16
Concentration (ng/mL)
Compound name: MDEA

Response type: External Std. Area
12437615
Response
50
100
150
200
250
300
350
400
450
pg/l
500
350
300
250
200
150
100
50
0
1
Individual #
Figure 3b. Typical linearity of response for saliva containing

MDEA.
10
Methamphetamine
Amphetamine
Ephedrine
MDA
MDEA
MDMA
Figure 6. Summary of results obtained from LC/MRM analysis

of 10 saliva samples collected from current amphetamine users.
N.B. MDMA concentrations ranged from 3 to over 3000 ng/mL.
50 l saliva
+
200 L methanol
(containing internal standards)
Centrifuge 13,000 rpm

(to collect supernatant)
HPLC/MRM analysis
Total analysis time: 15 mins
Figure 4. Schematic overview of the developed LC/MRM technique.
93918
100
%
0

180>105
100
%
0

166>148
100
%
0

136>91
100
%
0

150>91
100
%
0
0.00
8421488
0.50
1.00
1.50
Saliva spiked with amphetamines were firstly

extracted using methanol. Following centrifugation,
supernatants were analysed using LC/MRM analysis.
Amphetamines were quantified by reference to their
internal standards.

194>163
12912
100
%
0
2.00

208>163
2.50
In a separate (controlled) study, blood and saliva were

collected from experienced MDMA-users (n=12) at
various times following oral administration of
75 mg MDMA. Samples were analysed using GC/MS
(blood) and LC/MRM (oral fluid). Blood concentrations
of MDMA ranged from 21 to 295 ng/mL. The
corresponding saliva concentrations were usually
higher and ranged from 47 to > 6000 ng/mL. In both
matrices peak MDMA levels were generally observed
between 2 and 4 hours after administration. Figure 7
shows the mean MDMA levels in blood and oral fluid
and also demonstates the clear relationship between
the two matrices.
Time
3.00
Figure 5. MRM chromatogram for an oral fluid sample found to

be positive for the presence of MDEA, MDMA and MDA.
17
Mean MDMA in plasma

(ng/mL)
The use of oral fluid as a non-invasive alternative

to blood or urine as a marker for drug use, is an
attractive possibility. Collection of this biological
sample requires no special equipment or facilities and
can be supervised, thus removing the opportunity for
sample adulteration.
To this end we have developed a simple, rapid
method that allows the simultaneous quantitation
of several amphetamines in saliva during a single
chromatographic run. The procedure involves the
extraction of amphetamines from saliva followed by
LC/MRM analysis and is less time-consuming and
labour-intensive than the existing GC/MS method.
1400
200
180
160
140
120
100
80
60
40
20
0
1200
1000
800
600
400
200
Mean MDMA in plasma

(ng/mL)
Conclusions
0
0
60
120
180
240
300
Time after admininistration (minutes)
Figure 7. Mean MDMA levels (n=12) in plasma and oral fluid

following a single administration of 75 mg MDMA.
The developed method has been successfully applied

to the analysis of saliva samples collected from current
amphetamine users in an on-going study to assess the
feasibility of oral fluid as a convenient, non-invasive
specimen for monitoring drug abuse.
18
Opiates: Use or Abuse?

Quantification of Opiates in Human Urine
1Michelle Wood 1, Kevin Rush 2, Michael Morris1 and Allan Traynor 2
Waters Corporation, Manchester, UK
2Medscreen Ltd., London, UK
Introduction
Methodology
Heroin is a highly addictive drug. It is processed

from morphine, a naturally occurring substance
extracted from the seedpod of the Asian opium
poppy (Figure 1). Abuse of heroin is associated
with serious health conditions, including fatal
overdose, collapsed veins and an increased risk
of infectious diseases such as hepatitis, HIV/AIDS
and tuberculosis. Once inside the body it is rapidly
metabolised to morphine (Figure 2), which is then
excreted in the urine.
Sample preparation
Urine samples were prepared for LC/MS/MS analysis

by means of a simple, generic solid-phase extraction
(SPE) procedure. A Waters Oasis HLB Extraction
Cartridge (1 cc/30 mg) was firstly conditioned with
methanol (1 mL) followed by water (1 mL). Urine
samples (spiked with deuterated internal standards) for
SPE were diluted into water (300 L urine into 700 L
water before applying to the pre-conditioned cartridge).
The cartridge was washed with 5% methanol before
elution of the sample using 1 mL 100% methanol. Ten
microlitres (10 L) of the eluant were analysed using LC
in conjunction with multiple reaction monitoring (MRM).
The presence of morphine in urine cannot alone

be used as a marker for illicit heroin abuse since
morphine and codeine (which is also metabolised
to morphine) can be found in prescriptive medicines
and foods. For example, such medicines are valuable
treatments for pain, coughs and diarrhea. Ingestion of
pastries containing poppy seeds has also been shown
to lead to the presence of morphine and codeine
in the urine (Hayes et al., 1987). However, the
intermediate metabolite of heroin,
6-monoacetylmorphine (6-MAM) can be used as a
specific marker for heroin as it does not result from the
metabolism of either morphine or codeine. In addition,
acetylcodeine is a known impurity of illicit heroin
synthesis and may be used to distinguish between the
pharmacologically pure heroin that is used in heroin
maintenance programs and illicit street heroin.
LC/MS/MS
A Quattro micro triple quadrupole mass spectrometer
fitted with ZSpray ion interface was used for all
analyses. Ionization was achieved using electrospray
in the positive ionization mode (ES+). Details of the
MRM conditions are given in Table 1.
LC analyses were performed using a Waters LC
2790 separations module. Chromatography was
achieved using a Waters Nova-Pak CN HP column
(3.9 x 75 mm) eluted isocratically with 2 mM
ammonium acetate:methanol (50:50) containing 0.5%
formic acid at a flow rate of 0.3 mL/min. The column
temperature was maintained at 30 C. All aspects of
system operation and data acquisition were controlled
using MassLynx software with automated data
processing using the QuanLynx program.
We have developed an LC/MS/MS method that

allows the simultaneous quantification of several
opiates in urine. The method can also be used to
establish whether morphine present in the urine has
originated from illicit heroin use.
Precursor
ion
(m/z)
Product
ion
(m/z)
Morphine
286
Morphine - d3
289
Codeine
Dihydrocodeine (DHC)
Compound
Cone
Voltage
(V)
Collision
energy
(eV)
165
42
38
165
45
40
300
165
45
40
302
199
45
32
38
6-Monoacetyl morphine (6-MAM)
328
165
50
6-Monoacetyl morphine (6-MAM)-d6
334
165
45
38
6-acetylcodeine
342
165
50
38
Table 1: MRM transitions and conditions for the measurement

of several opiates. The conditions for deuterated morphine and
6-MAM (d3 and d6 respectively) were also included for the
purpose of internal standardisation.
Figure 1: The Asian

opium poppy,
Papaver somniferum.
19
6-MAM
Acetylcodeine
CH3
H3C
CH3
CH3
CH3
Heroin
CH3
O
HO
CH3
H3C
CH3
CH3
HO
OH
H3C
Morphine
OH
Codeine
Results
342>165
A series of calibrators (0.5-250 ng/mL) were prepared

by adding opiate standards to blank urine. Urine
samples (either calibrators or unknown samples) were
then extracted using the SPE method described above
prior to LC/MRM analysis.
328>165
302>199
300>165
Following LC/MRM analysis, the areas under the

specific MRM chromatograms were integrated.
Figure 3 shows the MRM chromatograms of various
opiates obtained with a 10 L injection of the
5 ng/mL urine calibrator. The opiates were quantified
by reference to the integrated area of the deuterated
internal standards. Responses were linear for all
compounds (Figure 4 shows a typical standard curve
for 6-MAM in urine).
286>165
Figure 3. MRM chromatograms for: (top to bottom) acetylcodeine,

6-MAM, DHC, codeine and morphine. Responses were obtained
with a 10 L injection of the 5 ng/mL urine calibrator.
Compound name: 6-MAM

Response type: Internal Std. (Ref 4), Area* (IS Conc./IS Area)
41.9
Summary
We describe a sensitive method for the simultaneous
analysis of several opiates in urine. The method
involves a simple SPE purification step prior to analysis
using LC/MRM and may be used to identify cases of
heroin abuse.
Response
ng/mL
0
0
20
40
60
80 100 120 140 160 180 200 220 240
Figure 4. Standard curve for 6-MAM. Responses were calculated

in reference to the integrated area of the deuterated internal
standards.
References
Hayes LW, Krasselt WG and Mueggler PA. Concentrations
of morphine and codeine in serum and urine after ingestion of
poppy seeds. Clinical. Chemistry. 1987; 33: 806-808.
20
Quantification of Morphine, Morphine-3-Glucuronide

and Morphine-6-Glucuronide in Biological Samples by LC/MS/MS
1Michelle Wood and Michael Morris.
Waters Corporation, Manchester, UK.
Introduction
Morphine is a potent analgesic isolated from the
opium poppy papaver somniferum and traditionally
used for the treatment of moderate to severe pain.
Analgesia results from the action of morphine at
the opioid receptors of the spinal cord and brain
(Figure 1), where it attenuates both the speed of the
impulse and the perception of pain.
In human subjects, morphine is extensively metabolised
(primarily by conjugation with glucuronic acid) to
form morphine-3-glucuronide (M3G) and morphine-6glucuronide (M6G). Whilst, the principal metabolite
i.e. M3G, has little or no analgesic effect, M6G has
been shown to be highly effective and is believed
likely to contribute significantly to the overall
effectiveness of morphine1. Hence, quantification of
both the parent drug and metabolites is desirable for
pharmacokinetic studies.
Previously we have described a LC/MS/MS method
that allows the quantification of morphine and several
other opiates in urine2. Here we present a simple
method that enables the quantification of morphine
in plasma, whole blood and urine. Furthermore this
procedure allows differentiation between two isobaric
glucuronide metabolites.
Cartridge (1 cc/30 mg) was firstly conditioned with

1 mL volumes of each of the following: methanol,
water and ammonium carbonate (10 mM, pH 8.8).
Samples (100 L, spiked with deuterated internal standards) were made up to a final volume of 1 mL with
ammonium carbonate before applying to the
pre-conditioned cartridge. The cartridge was then
washed with 1 mL ammonium carbonate before elution
of the sample using 100% methanol (0.5 mL). Eluents
were dried using a Savant Speedvac Plus evaporator and then redissolved in 100 L of mobile phase.
Reconstituted samples were briefly vortex mixed before
the analysis of 10 L using LC in conjunction with multiple reaction monitoring (MRM).
LC/MS/MS
A Waters Quattro micro triple quadrupole mass
spectrometer fitted with ZSpray ion interface was
used for all analyses. Ionisation was achieved using
electrospray in the positive ionisation mode (ES+).
Details of the MRM conditions are given in Table 1.
Compound
Figure 1. Image of
guinea-pig brain.
The red areas
represent the highest
density of opioid
receptors; yellow areas
represent moderate
density; whilst blue,
purple and white
represent low density.
Precursor
ion
(m/z)
Product
ion
(m/z)
Cone
Voltage
(V)
Collision
energy
(eV)
Morphine
286
165
45
38
Morphined3
289
165
45
40
MorphineM3Gglucuronide
462
286
45
28
MorphineM3Gd3-glucuronide
465
289
45
30
MorphineM6Gglucuronide
462
286
45
28
Table 1: MRM transitions and conditions for the measurement

of morphine and its metabolites. The deuterated analogues of
morphine and morphine-3-glucuronide were also included for the
purpose of internal standardisation.
LC analyses were performed using a Waters

2795 separations module. Chromatography was
achieved using a C18 column (3.9 x 150 mm) eluted
isocratically with 0.1% formic acid:acetonitrile (97:3)
at a flow rate of 0.3 mL/min. Column temperature was
maintained at 30 C. All aspects of system operation
and data acquisition were controlled using MassLynx
4.0 software with automated data processing using
the QuanLynx program.
Methodology
Sample preparation
Biological samples were prepared for LC/MS/MS

analysis by means of a simple, solid-phase extraction
(SPE) procedure. A Waters Oasis HLB extraction
21
Results
A series of calibrators (0.5-500 g/L) were prepared
in duplicate by adding standards to blank plasma,
whole blood or urine. Samples were then extracted
using the SPE method described above prior to
LC/MRM analysis.
100
462>286
M6G
%
0
MOR
100
286>165
Following LC/MRM analysis, the areas under the

specific MRM chromatograms were integrated.
Figure 2 shows the extracted MRM chromatogram

of morphine, M3G and M6G obtained with a 10 L
injection of the 5 g/L plasma calibrator. Opiates
were quantified by reference to the integrated area
of the deuterated internal standards. Responses were
linear (r = >0.999) over the range investigated for all
3 compounds and in each matrix (Figure 3 shows a
typical standard curve for M3G in urine).
1.00
2.00
3.00
4.00
5.00
6.00
7.00
Time
Figure 2. MRM chromatogram for morphine (MOR), M3G and

M6G. The above responses were obtained with a 10 L injection
of the 5 g/L plasma calibrator. Due to the isobaric nature of
M3G and M6G chromatographic resolution is required to enable
identification.
Compound name: Morphine-3-glucuronide

Correlation coefficient: r= 0.999883, r 2 = 0.999766
Calibration curve: 0.09404 * x + 0.100968
Response type: Intermal Std ( Ref 4 ), Area * ( IS Conc. / IS Area )
Curve type: Linear, Origin: Exclude, Weighting: 1/x Axis trans: None
Summary
We present a sensitive method for the quantification of
morphine and its glucuronide metabolites. The method
involves a simple SPE purification prior to analysis
using LC/MRM and is suitable for plasma, whole
blood or urine samples.
47.1
1.06
Response
Response
References
0.00
0.0
0.0
0
1. The Analgesic Effect of Morphine-6-Glucuronide. R Osborne,

P Thomson, S Joel, D Trew, N Patel and M Slevin.
Br J Clin. Pharmacol. 1992. 34 (2) 130-8.
100
200
300
2.0
400
g/L
4.0 6.0 8.0 10.0
g/L
500
Figure 3. Standard curve for M3G in urine. Responses

(duplicates) were calculated in reference to the integrated area of
the deuterated internal standards. The inserted figure shows the
response for the range 0-10 g/L.
2. Opiates: Use or Abuse? Quantification of Opiates in

Human Urine. (Waters Application Brief WAB45).
M Wood, K Rush*, M Morris and A Traynor*.
Clinical Applications Development Group,
UK Limited, Manchester, UK.
*Medscreen Ltd., 1A Harbour Quay, 100 Prestons Rd, London.
22
Development of a Rapid and Sensitive Method

for the Quantification of Benzodiazepines in
Plasma and Larvae by LC/MS/MS
Gert De Boeck1, Nele Samyn 1, Karen Pien2, Patrick Grootaert 3 and Michelle Wood 4,
National Institute of Criminalistics and Criminology (NICC), Brussels, Belgium. 2 Free University of Brussels, Belgium.
3 Royal Belgian Institute of Natural Sciences, Brussels, Belgium. 4 Waters Corporation, Manchester, UK.
1
Introduction
Benzodiazepines are the most widely prescribed
psychoactive drugs in the world for the symptomatic
treatment of anxiety and sleep disorders. However,
misuse of these compounds has been reported and
they are frequently encountered in postmortem blood
analysis (suicide or accidental death).
Here we describe the development of a rapid and
sensitive LC/MS/MS method for the quantification of
10 benzodiazepines. Limits of detection of 0.2 g/L
or better were achieved when just 25 L plasma
was used.
Conventional Phenyl Column

(2.1 x 150 mm, 5 m)
Mobile phase :
A =10:10:80 acetonitrile:
methanol: 20 mM ammonium acetate
Curve number
0
25
60
60
0
0
1
1
7 (concave)
6 (linear)
1
1
A series of calibrators (1, 10, 40, 100, 200, 400

and 800 g/L) were prepared by adding the
benzodiazepines to drug-free plasma. Plasma
samples were isolated from the matrix using a
simple acetonitrile clean-up procedure (which also
incorporates the addition of the internal standards).
Figure 2 shows the MRM chromatograms of the
benzodiazepines obtained with a 10 L injection of
the 10 g/L plasma calibrator. Quantification was
performed by integration of the area under the specific
MRM chromatograms. Figure 3 shows a typical
standard curve for diazepam in plasma.
LC/MS/MS conditions
Column:
B (%)
100
75
40
40
100
100
Figure 1 shows the MS and MS/MS spectra for

alprazolam. Table 1 summarizes the MRM transitions
and conditions used for this and several other
benzodiazepines (and their respective deuterated
analogues). The latter were used as internal standards
for quantification purposes.
Experimental Conditions
A (%)
0
0.5
8
11
12
15
In addition, we present the application of this method

to the analysis of benzodiazepines in Calliphora
vicina larvae. Insects and their larvae are commonly
used in the estimation of postmortem interval.
Furthermore, they may serve as a reliable alternate
source for toxicological analysis in the absence of
suitable tissues and fluids that are normally taken for
this purpose.
LC System:
Time (min)
B = 95:5 acetonitrile:
20 mM ammonium acetate
Flow rate:
0.25 mL/min
Injection volume:
10 L
MS conditions:
Mass spectrometer: Quattro Ultima

Ionisation Mode:
ES positive ion
Capillary voltage : 3kV

MS/MS:
MRM analysis (Table 1).

Collision gas Argon
at 2.5 x 10-3 mbar
23
Responses were linear, in all cases, over the range

investigated (Coefficient of Determination > 0.99).
Compound
Precursor ion
(m/z)
Product ion
(m/z)
308.8
313.8
315.8
319.9
284.9
289.8
313.9
320.8
320.8
326.8
270.9
275.9
287.0
291.7
324.9
330.0
300.9
305.8
342.9
349.0
280.9
285.8
269.8
273.8
154.0
153.7
267.9
274.8
274.7
280.8
139.8
139.8
240.8
245.8
270.9
276.0
255.0
259.8
307.7
313.9
Alprazolam
Alprazolam-d5
Clonazepam
Clonazepam-d4
Diazepam
Diazepam-d5
Flunitrazepam
Flunitrazepam-d7
Lorazepam
Lorazepam-d4
Nordiazepam
Nordiazepam-d5
Oxazepam
Oxazepam-d5
Prazepam
Prazepam-d5
Temazepam
Temazepam-d5
Triazolam
Triazolam-d4
Cone Voltage Collision energy

(V)
(eV)
70
100
80
100
60
80
80
80
60
80
80
80
60
80
80
80
60
60
60
60
25
25
25
25
25
25
25
25
23
23
25
25
26
26
25
25
25
25
25
25
Table 1. MRM transitions and conditions for the measurement of

10 benzodiazepines.
Note that due to the isobaric nature between these benzodiazepines and
Figure 1. MRM chromatograms for (top to bottom): lorazepam,

temazepam, triazolam, prazepam, oxazepam, diazepam,
alprazolam, flunitrazepam, nordiazepam and clonazepam.
Responses were obtained with a 10 L injection of the 10 g/L
plasma calibrator.
their deuterated analogues alternative precursor ions were utilised.
308.9
100
280.9
100
B
311.0
180.8
280.9
166.9
182.8
212.9
226.8 254.8 273.9
140.8 152.9
274.0
282.9
312.0
164.9
301.1
m/z
120
140
160
180
200
220
240
260
280
300
240.8
205.9
138.0
0
100
309.0
250.9
226.9
m/z
100
320
120
140
160
180
200
220
240
260
280
300
320
Figure 2. MS and MS/MS spectra of alprazolam.
For all compounds, LODs of 0.2 g/L (or better) and LOQs of 1 g/L (or better) were achieved. The precision
of the assay was assessed by performing replicate (n=5) extractions of plasma samples containing low, medium
and high concentrations of the benzodiazepines (i.e. 2, 40 and 200 g/L respectively). Coefficients of variation
(%CVs) were found to be highly satisfactory (<15%).
24
Compound name: Diazepam

Response type: Internal Std (Ref 10), Area* (IS Conc./IS Area)
100
276>140
979
%
0
100
Response
271>140
%
0
100
100
200
300
400
500
600
g/L
800
700
Figure 3.Typical response for plasma containing diazepam.

Diazepam spiked plasma was firstly extracted using acetonitrile
prior to analysis using LC/MRM. Benzodiazepines were
quantified by reference to their deuterated internal standards.
1mL ACN and I.S.

(nordiazepam d5 & oxazepam d5)
vortex thoroughty
6.00
8.00
Conclusion
We have developed a simple, rapid method that
allows the simultaneous quantification of 10
benzodiazepines in plasma a single chromatographic
run. LODs were better than 0.2 g/L when only
25 L plasma was used. The method involves a simple
protein precipitation step with acetonitrile followed by
LC/MS/MS analysis.
Larvae were reared on artificial foodstuff (beefheart)

spiked with a range of concentrations of nordiazepam
(0, 0.5, 1 and 2 g/g). Post-feeding larvae were
harvested (after 7 days) for analysis of drug content
by LC/MS/MS. Figure 4 outlines the initial sample
preparation method used for these specimens. All
control larvae reared on spiked foodstuff were positive
for nordiazepam and the metabolite oxazepam. All
control samples were negative. Figure 5 shows the
MRM chromatograms obtained following LC/MS/MS
analysis of a control larva and a larva positive for
nordiazepam. The method was sufficiently sensitive to
measure benzodiazepines in single larvae whereas
previous analytical techniques e.g. GC/MS, RIA, TLC
have required pools i.e. typically 20 larvae.
500L H2O
mix throughly
271>140
Time
10.00
Figure 5. MRM chromatograms obtained with the analysis

of larvae that were reared on artificial foodstuff spiked with
Nordiazepam at 0 and 1 g/g (A and B respectively). Figure
C shows the MRM chromatogram for the internal standard i.e.
Nordiazepam-d5.
The developed LC/MS/MS was subsequently applied

to the analysis of Calliphora vicina larvae in a study to
assess the feasibility of using insects and their larvae
as alternate specimens in the absence of any suitable
human specimens for toxicological analysis.
After 7 days
The method was subsequently applied to the analysis of

Calliphora vicina larvae in a study designed to assess
the feasibility of using insects as alternate specimens in
the absence of any suitable human tissues.
The sensitivity was such that it was possible to detect
benzodiazepines in single larvae whereas previous
methods have required pools.
Dry to 100L
LC/MS/MS
analysis
(10L aliquot)
Filter
Figure 4. Preparation of larvae for LC/MS/MS analysis.
25
Detection of Nordiazepam and Oxazepam in

Calliphora Vicina Larvae using LC/MS/MS
Karen Pien1; Patrick Grootaert2; Gert De Boeck3; Nele Samyn3; Tom Boonen 4; Kathy Vits 4; Michelle Wood 5; Michael Morris5.
University of Brussels, Belgium; 2Royal Institute of Natural Sciences, Brussels, Belgium;
3National Institute of Criminalistics and Criminology (NICC), Section Toxicology, Brussels, Belgium;
4National Institute of Criminalistics and Criminology, Brussels, Belgium; 5Waters, Manchester, UK.
1Free
Introduction
Sample preparation
In addition to their use in the estimation of postmortem

interval, insects may serve as reliable alternate source
for toxicological analyses in the absence of tissues
and fluids normally taken for such purpose. To date,
a variety of compounds have been measured in fly
larvae and pupae using different analytical procedures
i.e. (Radio-Immunoassay (RIA), Gas Chromatography
(GC) and Thin-Layer Chromatography (TLC)). In these
studies a minimum of 1g (approximately 20 larvae)
was needed to detect the toxic compound.
Individual larvae and pupae samples were prepared

for LC/MS/MS as follows; the sample was transferred
to a vial containing 500 L water and vortex-mixed
thoroughly. One millilitre of acetonitrile (containing
deuterated internal standards) was then added and
the samples mixed for a further minute. The mixture
was evaporated to ~100 L and then filtered. A 10 L
aliquot was analysed using LC/MS/MS.
In this study we used LC/MS/MS (Liquid

Chromatography-Tandem Mass Spectrometry)
to detect the benzodiazepine Nordiazepam
and its metabolite Oxazepam, in single larvae
of the Calliphora vicina. Benzodiazepines are
prescribed for the symptomatic treatment of
anxiety and sleep disorders. They are frequently
encountered in postmortem blood analysis
(suicide or accidental deaths).
Sample
Target Concentration
(g/g)*
Control
Nor I (human therapeutic dose)
0.5
Nor II (human lethal dose)
Nor III (2x human lethal dose)
Table 1: Target concentration of Nordiazepam in larval food.

*Concentrations expressed in g/g food.
In addition, we compared the development of

postfeeding larvae and pupae fed on different
concentrations of Nordiazepam.
LC/MS/MS
LC Conditions
Experimental Conditions
LC System:
Column:
Conventional Phenyl Column

(2.5 x 150 mm, 5 m)
Mobile Phase:
A=10:10:80
acetonitrile:methanol:
20 mM ammonium acetate
Study design
Flies and larvae were from a stock colony of

Calliphora vicina maintained in an environmental
chamber at 18-24 C and 60-70 % humidity with
cyclical artificial lighting simulating 16 h daylight
and 8 h darkness.
B=95:5 acetonitrile: 20 mM
ammonium acetate
Larvae were reared on artificial food (beef heart)

spiked with a range of concentrations of Nordazepam
(Table 1). Post-feeding larvae were harvested from day
4 till day 8. Thirty larvae were boiled and conserved
(in a mixture of ethanol and acetic acid) prior to
measurement of length. Another 30 were used for
toxicological analysis. These were weighed and then
killed, by freezing to -20 C. The larvae were stored
at -20 C until analysis.
Time (min)
A (%)
B (%)
Curve number
0
0.5
8
11
12
15
100
75
40
40
100
100
0
25
60
60
0
0
1
1
7 (concave)
6 (linear)
1
1
Flow Rate: 0.25 mL/min

Injection Volume: 10 L
26
MS Conditions
Compound
Mass Spectrometer: Quattro Ultima

triple quadrupole
Nordiazepam
Nordiazepam-d5
Oxazepam
315
Oxazepam-d5
327
Ionisation Mode:
ES+
Capillary Voltage:
3kV
Precursor Ion
(m/z)
Product Ion
(m/z)
Cone Voltage
(V)
Collision Energy
(eV)
278
91
30
30
325
109
38
25
86
28
18
270
35
25
Table 2: MRM transitions and conditions for them LC/MS/MS analysis

of Nordiazepam and Oxazepam. Deuterated analogues were also
included as internal standards.
Results
All larvae, pupae and food spiked with Nordiazepam
were positive for the drug, whereas all control
samples were negative.
Peak concentrations of Nordiazepam were measured

on day 4 for NOR I, II and III, followed by a
precipitous fall of larval Nordiazepam concentrations.
From day 7, Nordiazepam was not detectable in a
single larva.
Figures 1 and 2 show the larvae Nordiazepam and

Oxazepam concentrations from days 4 - 8. Figure 3
shows the MRM chromatograms obtained following
the LC/MS/MS analysis of a control larva and a
Nordiazepam positive larva.
Peak concentrations of Oxazepam were measured

on day 5 for NOR II and III and at day 6 for NOR
I. Low concentrations of Oxazepam were still
measured at day 8. In this study, two patterns of
development were observed; the post-feeding larvae
fed on Control, NOR I and NOR III food regime
developed at approximately the same rate and each
demonstrated wandering-phase behaviour at day
6, pupation at day 8 and emerging of adult flies at
day 18.
Figure 1: Concentration of Nordiazepam in larva reared

(for 4-8 days) on foodstuff spiked with Nordiazepam. Mean
concentrations are plotted ( 1SD).
276>140
271>140
271>140
Figure 2: Concentration of Oxazepam in larva reared (for

4-8 days) on foodstuff spiked with Nordiazepam. Mean
concentrations are plotted ( 1SD).
Figure 3: MRM chromatograms obtained with the analysis
of larvae that were reared on artificial foodstuff spiked with
Nordiazepam at 0 and 1 g/g (A and B respectively). Figure
C shows the MRM chromatogram for the internal standard i.e.
Nordiazepam d5.
27
Discussion and Conclusions
In contrast, the development of larvae fed with the

NOR II regime was 1 day later in all stages.
We have developed a method that allows the

detection of Nordiazepam and its metabolite
Oxazepam in single larvae. Larval drug
concentrations showed a stepwise increase with
increasing drug concentrations in the foodstuff.
It was clear that Nordiazepam was metabolized
to Oxazepam, which was still detectable at day 8.
Nordiazepam was detectable until day 6.
Control maggots were negative.
Post-feeding larval length is shown in Table 3;

no significant differences were observed.
Day 4
Day 5
Day 6
Day 7
Day 8
Control
16.7
17.8
15.2
15.8
15.9
Nor I
16.9
17.7
16.3
15.6
15.2
Nor II
17.2
17.3
16.7
15.5
15.8
Nor III
16.9
17.1
16.9
15.8
15.1
No differences were seen on the post-feeding larval

length, but differences in post- feeding larval weight
and development were seen in the NOR II larvae.
The reason of this disturbance is not yet understood,
but is presumably because larval physiology is
disturbed to a greater extent by this drug level. This
study indicates that an estimation of the postmortem
interval based on the length of the post-feeding
larvae of Calliphora vicina, which have fed on
tissues containing Nordiazepam, will have no error.
However an error, of up to 24 hours, can be made
if the estimation is based on duration of larvaland
puparial stages.
Table 3: Mean post-feeding larval length (mm).
Control
Day 4
Day 5
Day 6
Day 7
Day 8
69.5
84.5
78.2
86.2
71.4
Nor I
73.4
87.5
80.5
Nor II
110.8
105.8
89
101.7
78.5
96
92.9
Nor III
82.5
83.5
83.5
84
83.1
Table 4: Mean post-feeding larval weight (mg).
Post-feeding larval weight is shown in Table 4:

although no significant differences were seen in
larvae reared on Control, NOR I and NOR III food
regimes, the mean weight of larvae fed on NOR II
was significantly higher. This observation was also
confirmed in a second rearing experiment.
28
Quantitative Analysis of 9-Tetrahydrocannabinol

in Preserved Oral Fluid
1Marleen
Laloup 1, Maria del Mar Ramirez Fernandez1, Michelle Wood 2, Gert De Boeck1, Cecile Henquet 3, Viviane Maes 4, Nele Samyn1
Institute of Criminalistics and Criminology (N.I.C.C), Brussels, Belgium; 2Waters Corp., Manchester, UK;.
3Maastricht University, The Netherlands 4, Free University of Brussels, Brussels, Belgium
1National
The purpose of this study was to develop and validate

a rapid and sensitive LC/MS/MS method that would
be suitable for the analysis of THC in oral fluid
samples collected with the Intercept.

Calibrators and quality control (QC) samples
Oral fluid samples used for the preparation of blanks,
calibrators and QC samples were obtained from
healthy volunteers and collected with the Intercept
collection device (OraSure Technologies, Bethlehem,
PA) according to the manufacturers instructions.
Briefly, after gently wiping the collector pad between
gum and cheek for approximately 2 minutes the device
is placed in the supplied vial and sealed. Following
centrifugation, the recovered fluid was spiked with
THC to yield a series of calibrators ranging from
0.1 to 100 ng/mL. QC samples were also prepared
by spiking control oral fluid with THC.
Introduction
Cannabis is the collective term for the psychoactive
substances of the Cannabis sativa plant (Figure 1)
and one of the most frequently used illicit drugs in
the western world. 9-Tetrahydrocannabinol (THC),
the main psychoactive constituent of cannabis, is
deposited in the oral cavity during cannabis smoking.
Over the last few years there has been an increasing
interest in the use of oral fluid to document drug
use. The advantage of this specimen over the more
traditional matrices e.g. urine and blood, is that
collection is almost non-invasive, relatively easy
to perform, and may be achieved under close
supervision to prevent adulteration or substitution of
the sample.
Authentic samples
Oral fluid samples were collected by the police at

roadblocks, the purpose of which, was to intercept
drivers who were driving under the influence of drugs.
The samples were collected at the roadside using the
same procedure as described for the blank samples.
An additional series of authentic samples were
obtained from volunteers with a history of cannabis
use. Once a week, and over 2 consecutive weeks,
subjects received either a placebo cigarette (where
the THC had been extracted) or a marijuana cigarette
which contained 300 g cannabis per kg). Samples
were collected 0.5 hour prior to drug administration
and at various times following drug administration
(0.25, 0.5, 1, 1.25, 1.5 hour).
LC/MS/MS is a technique that lends itself well to the

high-throughput determination of multiple analytes in
oral fluid samples due to its high specificity, sensitivity
and short analysis times1,2.
The Intercept is a FDA cleared oral fluid collection
device that is used on a large scale in the U.S. for
workplace testing3. It is also the device of choice to
collect the samples in a current joint roadside study
between the European Union and the U.S. to detect
driving under the influence of drugs4.
The study protocol was approved by the ethics

committee of the University Hospital of Maastricht in
the Netherlands.
The Intercept collection system utilises a variety of

ingredients to ensure stability and to maintain the
integrity of the sample. However, these ingredients
can also cause interferences e.g. ion suppression
during LC/MS/MS analysis in the absence of a
suitable clean-up method5.
Internal standard solution
An internal standard (IS) working solution of THC-d3

at a concentration of 10 ng/mL was prepared in
methanol.
29
Sample preparation
Extraction was performed using either 100 or 500 L

of the collected specimen. When using 500 L, 50 L
of the IS working solution and 4 mL of hexane were
added; when only 100 L of oral fluid was used, an
additional 400 L of deionised water was added.
After mechanical shaking (30 min) and centrifugation
(10 min at 3000 g), the organic phase was collected
and then evaporated to dryness at 40 C under
nitrogen. The extract was reconstituted in 100 L of
mobile phase.
Figure 2 shows the MRM chromatograms obtained

following the analysis of a sample enriched with THC
and the internal standard i.e. THC-d3.
The usefulness of the liquid/liquid extraction step
was assessed by a comparison of the effect of the
matrix both before and after sample clean-up. Matrix
effects were monitored throughout the whole of the
chromatographic run by performing post-column infusion
experiments6. The effect on THC response obtained
following the injection of a sample prior to extraction
and the same sample after extraction of 100 L and
500 L of oral fluid are given in Figure 3. The results
clearly demonstrate the usefulness of the liquid-liquid
extraction step prior to LC/MS/MS analysis.
LC conditions
LC system:
Waters Alliance System
Column:
Waters XTerra MS C18 column

(2.1 x 150 mm, 3.5 m) at 40 C
Mobile phases:
(A): 1 mM ammonium formate

(B): methanol
Isocratic elution 10:90 (A:B)
Flow rate:
0.2 mL/min
Injection volume:
20 L
Mass spectrometry conditions
Mass spectrometer:
Waters
Quattro Premier tandem
mass spectrometer
Ionisation mode:
ES+
Capillary voltage:
2 kV
Figure 2. MRM chromatograms obtained with a single injection of
a 100 L extracted oral fluid sample enriched with 5 ng/mL THC
and 5 ng/mL THC-d3. The figure shows the response for THCd3 (top trace) and for the two transitions of THC (quantifier and
qualifier middle and bottom trace respectively). Peak intensity is
shown in the top right-hand corner of each chromatogram.
Source temperature: 120 C

Desolvation gas:
Nitrogen at 700 L/Hr, 280 C
MS/MS:
THC m/z 315.2>193.1

(quantification ion) m/z
315.2>259.3 (qualifier ion)
THC-d3 m/z 318.2>196.1
Cannabinol m/z 311.2>223.1
Cannabidiol m/z 315.2>193.1
Collision gas:
Argon at 3.5 x 10-3 mbar
Figure 3. Evaluation of the matrix effect on THC response of an

injection of a mobile phase control (A), a blank sample prior
extraction (B), the reconstituted extract after extraction of 100 L
(C) and the reconstituted extract after 500 L of oral fluid (D). The
shaded area indicates the elution position of THC. Peak intensity
for THC is shown in the bottom right-hand corner.
30
over a period of 15 hours. No instability was noted

over the course of this experiment.
To assess method linearity, limit of quantitation (LOQ),

precision, accuracy and analytical recovery a series
of oral fluid calibrators were prepared and a 100 or
500 L aliquot extracted with hexane prior to analysis
using LC/MS/MS. Quantification was achieved
by integration of the area under the specific MRM
chromatogram. For THC, the response was calculated
in reference to the integrated area of THC-d3.
Cannabidiol and cannabinol are two components that

are also naturally-occuring in the Cannabis sativa plant.
Since the m/z for the precursor mass of cannabinol
is different to that of THC, it does not interfere in
its quantitation. On the other hand, the protonated
molecular species of cannabidiol i.e. m/z 315.2 is
the same as that of THC. Furthermore it shows the
same product ions after collision induced dissociation.
Thus chromatographic separation is essential to
distiguish between these 2 isobaric compounds.
Analysis of standards showed cannabidiol to be
chromatographically resolved from THC (Figure 4).
Linear responses (r = >0.999, 1/x weighting) were

obtained up to 100 ng/mL when 100 L of sample
was extracted and up to 10 ng/mL when 500 L
sample was extracted. Linearity and sensitivity data are
summarised in Table 1. The limit of quantification was
defined as the concentration of the lowest calibrator
which was calculated to be within 20% of the
nominal value and with a % CV less than 20%. This
criteria was met by the lowest calibrator i.e. 0.5 and
0.1 ng/mL when either 100 or 500 L respectively of
the collected sample was extracted.
The utility of the LC/MS/MS method was demonstrated

by the analysis of 102 authentic samples collected
from volunteers who smoked a placebo or marijuana
cigarette. Figure 5 shows the values for THC in
oral fluid collected after smoking the marijuana
cigarette; mean values are plotted as a function of
time. All specimens collected prior to smoking were
negative, with the exception of 3 samples where
concentrations were very low (maximum 2.2 ng/mL).
Peak concentrations occurred 0.5 hour after smoking.
Thereafter concentrations decreased steadily. There
was considerable inter-individual variation in the
observed concentrations; this has also been reported
by other authors7 and may also be as a result of the
lack of exact volume measurement in the device.
Intra-assay and interassay variation (as % CV) were

all found to be highly satisfactory at <6% (Table 2).
Analytical recovery was estimated by comparing the
responses of a 5 ng/mL calibrator (using 100 L of
oral fluid) when the non-deuterated compounds were
added before the extraction step (n= 3) with those
obtained when the non-deuterated analytes were
added after sample preparation (n= 3). The recovery
was found to be satisfactory at 85.6 0.5%
The stability of THC in oral fluid collected by the
Intercept device was assessed by spiking oral fluid
with THC at 3 different concentrations (1, 10 and
100 ng/mL) and then monitoring the stability at 4 C
and at room temperature over a period of 48 hours.
No statistical significant differences could be observed
for the three different concentrations in both conditions.
Forty eight samples were also collected from drivers

intercepted at Belgian roadblocks. Table 3 summarises
the quantitative results for all positive samples and
Figure 6 shows a MRM chromatogram for one such
marijuana user; the presence of cannabidiol was also
noted (at 3.28 min) in this specimen.
The stability of the samples post extraction was

assessed by repeated injections of extracted samples
Linearity Data
volume oral fluid
100L
500L
slope*
intercept*
CV of slope (% over 5
consecutive days)
r 2 (range of 5
consecutive days)
Sensitivity Data
LOQ (ng/mL)
1.0635
5.3976
0.0209
-0.0009
2.9
4.1
0.9993-0.9999
0.9992-0.9999
0.5
0.1
Table 1. Linearity and sensitivity data for THC in oral fluid.Samples were prepared by the liquid-liquid extraction method as described
in the text. *Reported values are the mean of five determinations over 5 consecutive days.
31
Volume Oral
Fluid
100 L
500 L
Concentration
of QC
(ng/mL)
Intra-assay Precision
Mean Concentration
Found
%CV
Accuracy (%)
(ng/mL)
Interassay Precision
Mean Concentration
Found
%CV
Accuracy (%)
(ng/mL)
2.5
2.5
3.6
-1.0
2.4
2.9
25.0
24.8
5.4
-0.7
24.0
5.4
-2.5
-4.1
0.5
0.5
2.5
-2.4
0.5
4.1
-5.5
5.0
4.9
0.4
-2.0
4.7
3.8
-6.8
Table 2. Precision and accuracy data for THC for the extraction of 100 L and 500 L of spiked oral fluid samples.
Intra-assay precision was evaluated by the preparation and analysis of four replicates of a low and a high in a single assay for both
volumes of oral fluid used. Interassay precision was evaluated by the preparation and analysis of each QC over 8 consecutive days
Sample
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Figure 4. LC/MS/MS analysis of THC-d3 (top trace), THC,

cannabidiol (middle trace) and cannabinol (bottom trace). Peak
intensity is shown in the top right-hand corner of each trace.
THC (ng/mL)
5.7
7.0
4.6
18.5
2.5
95.8
0.3
84.7
0.3
0.5
4.5
3.9
31.9
50.8
34.6
56.0
81.1
11.9
107.4
92.1
10.0
17.6
94.8
37.2
Sample
THC (ng/mL)
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
60.2
3.9
52.2
25.4
193.5
111.2
7.3
14.6
1.9
4.7
100.0
23.0
57.1
88.6
3.9
375.8
3.7
4.4
4.2
4.2
4.2
4.1
4.0
4.4
Table 3. Results obtained applying the method to 48 oral fluid

samples collected by the police at the roadside.
THC
(ng/mL)
Figure 5. Box- and whisker plots of THC levels in oral fluid

samples following smoking of a single marijuana cigarette.
Oral fluid samples were taken prior to administration i.e. at
0.5 h, 0.25 h, 0.5 h, 1 h, 1.25 h and 1.5 h after smoking.
Concentrations plotted on the Y-axis are expressed as ng/mL.
The central box represents the values from the lower to upper
quartile (25 to 75 percentile). The middle line represents the
median. The horizontal line extends from the minimum to the
maximum value, excluding outside (not present) and far out
values (cross marker) which are displayed as separate points.
Figure 6. Typical MRM chromatograms obtained following the

analysis of an authentic oral fluid specimen obtained from a
driver in a roadside setting. The calculated concentrations was
5.7 ng/mL. The figure shows the response for THC-d3 (top trace)
and for the two transitions of THC (quantifier and qualifier middle
and bottom trace respectively). Peak intensity is shown in the top
right-hand corner of each trace.
32
Conclusions
References
To the very best of our knowledge, the method

presented here is the first demonstration of the use
of LC/MS/MS for the analysis of THC in oral fluid
samples collected with the Intercept device. The
method is simple and comprises simple liquid/liquid
extraction followed by LC/MS/MS. The method
demonstrates high recovery, excellent precision and
accuracy when using either 100 or 500 L sample.
1. K.A. Mortier, K.E. Maudens, W.E. Lambert, K.M. Clauwaert,

J.F. Van Boxlaer, D.L. Deforce, C.H. Van Peteghem and
A.P. De Leenheer, J. Chromatogr. B 779 (2002) 321330.
2. R. Dams, C.M. Murphy, R.E. Choo, W.E. Lambert,
A.P. De Leenheer and M.A. Huestis, Anal. Chem. 75
(2003) 798804.
3. E.J. Cone, L. Presley, M. Lehrer, W. Seiter, M. Smith,
K.W. Kardos, D. Fritch, S. Salamone, S. Niedbala,
J. Anal. Toxicol. 26 (2002) 541.
4. EU Project ROSITA Roadside Testing Assessment.
http://www.rosita.org.
The LOQ is sufficiently low to meet the requirements

of SAMHSA (2 ng/mL) for oral fluid testing.
Pharmacokinetic studies may require lower LOQs;
these requirements can be met by using larger
volumes of oral fluid.
5. M. Wood, M. Laloup, M. Ramirez Fernandez, K.M. Jenkins,

M.S. Young, J.G. Ramaekers, G. De Boeck, N. Samyn,
Forensic Sci. Int, in press.
6. R. Bonfiglio, R.C. King, T.V. Olah, K. Merkle, Rapid Commun.
Mass Spectrom. 13 (1999) 1175.
The method was successfully applied to the analysis

of samples collected in a controlled cannabis smoking
study and to samples collected at the roadside by
Belgian police.
7. R.S Niedbala, K.W. Kardos, D.F. Fritch, S. Kardos,

T. Fries, J. Waga, J. Robb, E.J. Cone,
J. Anal. Toxicol. 25 (2001) 289.
33
Simultaneous Analysis of GHB and its Precursors in Urine

Using LC/MS/MS
Michelle Wood 1, Marleen Laloup 2, Nele Samyn2, Michael Morris1, Peter Batjoens3 and Gert De Boeck2
Waters Corp., Manchester, UK; 2National Institute of Criminalistics and Criminology (N.I.C.C), Brussels, Belgium;
3Waters Corp., Brussels, Belgium.
sy
Ecsta
Liqui
Fan
tasy
Eas
concentrations; 0, 1, 2, 5, 10, 20, 50 and 80 mg/L.

Low and high QC samples were prepared by spiking
control urine with the drugs to yield concentrations of
4 and 40 mg/L, respectively.
y La
Blue
nitro
Authentic Samples
Introduction
Gamma-hydroxybutyrate (GHB) is a metabolite
of gamma-aminobutyric acid (GABA) and plays
the role of a central neurotransmitter and
neuromodulator. Since GHB is a normal component of
mammalian metabolism, it is present in all tissues of the
body. Typical urinary GHB concentrations are
< 10 mg/L1,2. In some countries GHB is used clinically
as an intravenous anaesthetic and as a treatment for
narcolepsy, alcoholism and opiate withdrawal. Over
the last few years, GHB has been gaining popularity
amongst club-goers as the recreational drug (Figure 1)
where it is taken for its ability to produce feelings
of euphoria and to enhance sexuality3-5. As a result
of its potent prosexual effects, GHB has also been
increasingly implicated in drug-facilitated sexual
assault 6,7. Ingestion of the chemical precursors of GHB
i.e. gamma-butyrolactone (GBL) and 1,4-butanediol
(1,4-BD) also results in similar physiological effects
since they are rapidly converted to GHB in the body8.
Raised awareness of the effects of these drugs and
their potential for misuse, in addition to their ease of
availability, has resulted in a dramatic increase in
the demand for their analytical determination in both
biological specimens and putative drug preparations.
The purpose of this study was to develop and validate
a sensitive LC/MS/MS procedure that would enable
the simultaneous quantification of GHB, GBL and 1,4BD in urine.
One hundred and eighty two authentic human urine

samples were collected from club-goers attending a
post dance-club chill-out venue and were the result
of 2 separate raids by the Belgian Police Department.
The samples were analysed for GHB and the
precursors using the newly-developed LC/MS/MS
procedure. For comparative purposes, the samples
were also analysed for GHB using a routinely used
GC/MS procedure.
Sample Preparation
Urine samples were diluted (1:20) with an internal

standard solution (GHB-d6 and GBL-d6, at a
concentration of 2 mg/L in deionised water).
LC Conditions
LC system:
Column:
Waters Atlantis dC18 column

(3 x 100 mm, 5 m) at 35 C
Mobile phases:
(A): 0.1% aqueous formic acid

(B): methanol
Isocratic elution:
90:10 (A:B)
Flow rate:
200 L/min.
Inj. volume:
20 L
Mass Spectrometry Conditions
Mass spectrometer: Quattro micro mass spectrometer

Ionisation mode:
ES+
Capillary voltage:
3.5 kV
Source Temperature: 120 C

Desolvation gas:
Nitrogen at 700 L/Hr, 350 C
MS/MS:
Collision gas (argon) at

5 x 10-3 mbar
Samples
Calibrators and quality control (QC) samples

Control urine was spiked with GHB, GBL and 1,4-BD
to yield a series of calibrators at the following
34

1.8 x 10 5
Compound
Precursor
ion
(m/z)
Product
ion
(m/z)
Cone
Voltage
(V)
105
87
10
GHB-d6
111
93
10
GBL
87
45
25
15
GBL-d6
93
49
25
15
1,4-BD
91
73
12
4.2 x 10 5
4.2 x 10 5
1,4,-BD
Collision
energy
(eV)
GHB
1.8 x 10 5
GHB
Multiple reaction monitoring (MRM) transitions were

determined for GHB, the precursors and the internal
standards i.e. GHB-d6 and GBL-d6 (Table 1). Figure 2
shows some examples of product ion spectra.
GBL
4.3 x 10 4
4.3 x 10 4
Figure 3: MRM chromatograms obtained with a single injection

of a control urine sample (left-hand column) prepared by the
dilution method and the same sample enriched with 10 mg/L
of GHB, GBL and 1,4-BD (right-hand column). Peak intensity is
shown in the top right-hand corner of each trace.
Table 1: MRM transitions and conditions for the measurement of

GHB, GBL, 1,4-BD and their deuterated internal standards.
A series of urine calibrators was prepared. Following

preparation i.e. simple dilution, the samples were
analysed using LC/MS/MS. Figure 3 shows the MRM
chromatograms obtained following the analysis of a
control urine sample and the same sample enriched
with GHB, GBL and 1,4-BD.
Quantification was achieved by integration of the area

under the specific MRM chromatogram. For GHB and
GBL, responses were calculated in reference to the
integrated area of their respective deuterated internal
standards. For 1,4-BD the response was calculated
by reference to that of GHB-d6. Linear responses
were obtained for GHB and 1,4-BD over the range
investigated (1-80 mg/L). GBL produced a linear
response over the range 1-50 mg/L.
Precision, intra-assay and interassay variation

(as % CV) were all found to be highly satisfactory at
< 7%. Analytical recoveries ranged from 90-107%
(Table 2).
Low Control
(4 mg/L)
Compound
High Control
(40 mg/L
Mean
(mg/L)
Recovery
(%)
%CV
Mean
(mg/L)
Recovery
(%)
%CV
GHB
4.0
100
3.0
41.0
103
0.5
GBL
3.8
95
4.2
40.2
101
3.9
1,4-BD
3.7
93
2.9
41.3
104
0.7
GHB
3.9
98
3.2
42.7
107
3.5
GBL
3.7
93
3.2
36.1
90
2.9
1,4-BD
4.0
100
2.2
40.0
100
3.1
GHB
4.1
103
5.3
40.0
100
3.4
GBL
4.0
100
6.6
39.8
100
6.3
1,4-BD
3.9
98
3.8
40.5
101
4.7
Precision (n=5)
Intra-assay (n=5)
Figure 2: Product ion spectra for GHB (A), GBL (B) and 1,4BD (C). Pure standards (5 mg/L) were infused into the mass
spectrometer and the cone voltage (CV) optimised for the
precursor ion*. CID was then performed and product ion
spectra acquired under optimum conditions for the most
abundant product ion.
Intrerassay (n=5)
Table 2: Precision and analytical recovery data for GHB and its
precursors in urine.
35
Only two, of these seven samples, were above the

recommended interpretive cut-off concentration of
10 mg/L and were 956 mg/L and 1411 mg/L,
respectively. These two samples were also positive
for GBL. None of the authentic urine samples
contained 1,4-BD.
B
Conclusions
To the very best of our knowledge, the method
presented here is the first demonstration of the use
of LC/MS/MS for the simultaneous analysis of GHB
and its precursors in urine samples. The method is
simple and rapid (total analysis time of <12 mins).
The method offers sufficient sensitivity to enable the
measurement of endogenous levels of GHB and to
identify exogenous ingestion.
Fig. 4: LC/MS analysis of the hydroxybutyric acid isomers.

Ion chromatograms obtained following the analysis of gammahydroxybutyric acid (GHB) only (A) and GHB in the presence of
alpha and beta-hydroxybutyric acid (traces B and C respectively).
Peak intensity is shown in the top right-hand corner of each trace
and is the sum of the responses obtained for both the protonated
and the sodiated species species i.e. m/z 105 + 127.
The LC/MS/MS results obtained following the

analysis of authentic samples, correlated with the
more labour-intensive, time-consuming (~1 hour)
GC/MS method.
The procedure offers several advantages over other
published techniques;
The limit of quantification was defined as the

concentration of the lowest calibrator which was
calculated to be with in 20% of the nominal value
and with a % CV less than 20%. For all of the
analytes of interest, this criteria was met by the 1 mg/L
calibrator and was sufficient to determine endogenous
levels of GHB in the urine.
1. It enables the simultaneous quantification of the

GHB and the precursors in a single analysis;
this can facilitate the identification of the chemical
basis of any seized putative drug preparations or
if present in the biological specimen, can provide
information of the chemical nature of the ingested
drug.
To investigate any potential interference in GHB

quantification by its naturally occuring isomers i.e.
alpha and beta-hydroxybutyric acid, standards
were analysed using the developed LC/MS/MS
method. Both compounds were shown to be
chromatographically resolved from GHB and thus
would not interfere in the quantification of the latter
(Figure 4).
2. It involves fewer manipulations and is less timeconsuming.
The utility of the LC/MS/MS method was demonstrated

by the analysis of one hundred and eighty-two
authentic urine samples. Seven samples contained GHB
at concentrations > 2 mg/L. The same seven samples
were independently identified by the more timeconsuming, labour-intensive GC/MS method.
Although the data presented here indicate that the

actual prevalence of GHB-positives might be quite low,
the hype and publicity surrounding these drugs has led
to a dramatic increase in the number of requests for
their analysis in biological samples (and particularly
in urine). The simplicity and speed of the described
LC/MS/MS technique, should prove a useful means to
meet this current increased demand on laboratories.
36
References
1. Elliott SP. Gamma hydroxybutyric acid (GHB) concentrations in
humans and factors affecting endogenous production.
Forensic Sci. Int 2003;133:9-16.
2. LeBeau MA, Christenson RH, Levine B, Darwin WD and
Huestis MA. Intra- and inter individual variations in urinary
concentrations of endogenous gamma-hydroxybutyrate.
J. Anal. Toxicol 2002;26:340-346.
3. Laborit H. Correlations between protein and serotonin synthesis
during various activities of the central nervous system (slow and
desynchronized sleep, learning and memory, sexual activity,
morphine tolerance, aggressiveness and pharmacological action
of sodium gamma-hydroxybutyrate). Research Communications
in Chemical Pathology and Pharmacology 1972;3:51-81.
4. Ropero-Miller JD and Goldberger BA. Recreational drug current
trends in the 90s. Clin. Lab Med 1998;18:727-746.
5. Bellis MA, Hughes K, Bennett A and Thomson R. The role of an
international nightlife resort in the proliferation of recreational
drugs. Addiction 2003;98:1713-1721.
6. ElSohly MA and Salamone SJ. Prevalence of drugs used in cases
of alleged sexual assault. J. Anal. Toxicol 1999;23:141-146.
7. Ferrara SD, Frison G, Tedeschi L and LeBeau MA. Gammahydroxybutyrate (GHB) and related products. In: LeBeau MA and
Mozayani A, eds. Drug-Facilitated Sexual Assault (DFSA):
A Forensic Handbook. London: Academic Press, 2001:108-126.
8. Palatini P, Tedeschi L, Frison G, Padrini R, Zordan R and
Orlando R et al. Dose-dependent absorption and elimination of
hydroxybutyric acid in healthy volunteers.
Eur. J. Pharmacol 1993;45:353-356.
9. Fieler EL, Coleman DE and Baselt RC. GHB concentrations in
pre and post-mortem blood and urine [Letter].
Clin. Chem 1998;44:692.
37
Determination of Aconitine in Body Fluids by LC/MS/MS

Justus Beike1, Lara Frommherz1, Michelle Wood2, Bernd Brinkmann1 and Helga Khler1
1 Institute of Legal Medicine, University Hospital Mnster, Rntgenstrasse, Mnster, Germany
2 Clinical Applications Group, Waters Corporation, Simonsway, Manchester M22 5PP, UK.
the determination of aconitine in various body fluids8.

The method was fully validated for the determination
of aconitine from whole blood samples and applied
in two cases of fatal poisoning.
Introduction
Plants of the genus Aconitum L (family of
Ranunculaceae) are known to be among the most
toxic plants of the Northern Hemisphere and are
widespread across Europe, Northern Asia and North
America. Two plants from this genus are of particular
importance: the blue-blooded Aconitum napellus L.
(monkshood) which is cultivated as an ornamental
plant in Europe and the yellow-blooded Aconitum
vulparia Reich. (wolfsbane) which is commonly used
in Asian herbal medicine1 (Figure 1).
Many of the traditional Asian medicine preparations
utilise both the aconite tubers and their processed
products for their pharmaceutical properties, which
include anti-inflammatory, analgesic and cardiotonic
effects2-4. These effects can be attributed to the
presence of the alkaloids; the principal alkaloids
are aconitine, mesaconitine, hypaconitine and
jesaconitine.
Figure 1: Aconitum napellus (monkshood) (A)

and Aconitum vulparia (wolfsbane) (B).
The use of the alkaloids as a homicidal agent has

been known for more than 2000 years. Although
intoxications by aconitine are rare in the Western
Hemisphere, in traditional Chinese medicine, the
use of aconite-based preparations is common and
poisoning has been frequently reported. Poisoning
has occurred both during clinical use and also as
consequence of accidental ingestion e.g. by eating
plant material or Aconitum preparations5, 6. The use
of aconite tubers for suicide and homicide purposes
has also been reported 7.
Sample preparation
Biological samples were prepared for LC/MS/MS

by means of a solid-phase extraction (SPE) procedure.
Blood and tissue samples (0.5 g each) were mixed
with 3 mL of 0.15 M phosphate buffer pH 6.0,
homogenised and centrifuged at 5000 g for 10
min. The supernatants were decanted and loaded
on a prepared SPE cartridge. Cartridges were preconditioned with 3 mL methanol, 3 mL water and
1 mL of 0.15 M phosphate buffer pH 6.0. Samples
were allowed to pass through the cartridge under
gravity, before an initial wash step (3 mL water
followed by 1 mL 0.01 M HCl) was performed.
The first symptoms of aconitine poisoning appear

~20 min to 2 hours after oral uptake and include
paraesthesia, sweating and nausea. This leads to
severe vomiting, colicky diarrhea, intense pain
and then paralysis of the skeletal muscles. Following
the onset of life-threatening arrhythmia, including
ventricular tachycardia and ventricular fibrillation,
death finally occurs as a result of respiratory
paralysis or cardiac arrest5-7.
Two further washing steps i.e. 2 mL dichloromethane,

followed by 2 mL methanol, were performed before
elution of the aconitine. Cartridges were dried
under vacuum between each of the 3 wash steps.
Aconitine was eluted (2 x 1.5 mL) with a mixture of
dichloromethane:2-propanol:25% aqueous ammonia
(80:20:2). Eluents were pooled and evaporated to
dryness under a stream of nitrogen at 40 C before
reconstitution with 100 L LC mobile phase.
Clearly in the case of suspected aconitine intoxication

there is a need for rapid analytical techniques to
enable prompt diagnosis and treatment. To this end
we have developed a simple LC/MS/MS method for
38
LC/MS/MS
In two forensic cases of suspected aconitine

intoxication, aconitine was detected in the blood
samples and also in the stomach content and urine
of the deceased (Table 1). Figure 3 shows the
chromatogram of the blood sample of aconite victim
no 2. At the time of autopsy the body was already
in an advanced state of putrefaction. Despite these
difficult circumstances, the chromatogram shows a
strong signal for aconitine.
micro
A Quattro
tandem mass spectrometer fitted
with Z-Spray ion interface was used for all analyses.
Ionisation was achieved using electrospray in the
positive ionisation mode (ES+). Detection of aconitine
was performed using multiple reaction monitoring
(MRM). The transition MRM transition m/z 646.4
> m/z 586.5 was used for quantification purposes
and a further two transitions i.e. m/z 646.4 > m/z
526.4 and m/z 646.4 > m/z 368.4 were monitored
for confirmatory purposes.
4.80
LC analyses were performed using an Alliance 2695

separations module (Waters). Chromatography was
achieved using a Waters XTerra RP8 pre-column
(2.1 x 10 mm, 3.5 m) and a XTerra RP8 analytical
column (2.1 x 150 mm, 3.5 m). The column was
maintained at 40 C and eluted isocratically with
0.1 % ammonium acetate (adjusted to pH 6.0
with 1 M acetic acid) and methanol (50:50) at
200 L/min. The injection volume was 10 L and
a total run time of 10 min was used. All aspects of
system operation and data acquisition were controlled
using MassLynx NT 4.0 software with automated
data processing using the QuanLynx program
(Waters).
Case no.
Blood [ng/g]
Aconitine concentration
Stomach content [ng/g]
Urine [ng/mL]
10.0
3.0
Not available
12.1
Not available
180.0
MRM of 3 Channels ES+
100
646.4>586.5
646.4>526.4
646.4>368.4
2.37e3
Time
2.00
4.00
6.00
8.00
10.00
Figure 2: MRM chromatograms for a blood calibrator spiked at

0.1 ng aconitine/g blood. Peak intensity is given in the top righthand corner of the trace.
Summary
We have developed a rapid and sensitive method for
the quantification of aconitine in biological specimens.
The method involves a simple SPE purification prior to
analysis using LC/MRM.
Table 1: Concentrations of aconitine in autopsy samples from two

cases of fatal aconite intoxication.
The utility of the method was demonstrated by its

application to authentic samples in 2 fatal cases of
suspected aconitine poisoning. Blood, urine and
stomach contents were collected during autopsy and
analysed using the developed LC/MS/MS method.
Aconitine could be detected in the blood of both
victims, in the stomach content of one individual and
in the urine of the other.
Results
A series of calibrators (0.1 25 ng/g) were prepared
in duplicate by adding aconitine standards to control
blood. Samples were then extracted, using the
SPE method described above, prior to LC/MS/MS
analysis.
Following analysis, the areas under the specific MRM
chromatograms were integrated. The response was
linear (r2 = 0.999) over the range investigated. The
limit of detection (LOD) of the assay was estimated at
0.1 ng/g blood. Figure 2 shows the responses for the
quantifier and qualifier ions of aconitine obtained with
a calibrator spiked at the LOD.
39
MRM of 3 Channels ES+

646.4>586.5
646.4>526.4
646.4>368.4
1.83e5
4.80
100
2.00
4.00
6.00
8.00
Time
10.00
Figure 3: MRM chromatograms of the blood sample from the

victim in case 2, with 12.1 ng aconitine/g. The chromatograms
show no interferences although the body was in an advanced
state of putrefaction at the time of the autopsy.
References
1. List PH, Hrhammer L (1969). Hagers Handbuch der
Pharmazeutischen Praxis. Vol II, 1066 1082,
Springer Berlin, Heidelberg.
2. Hikino H, Konno C, Takata H, Yamada Y, Yamada C, Ohizumi Y,
Sugio K, Fujimura H (1980). Antinflammatory principles of
Aconitum roots. J Pharmacobiodyn 3: 514 525.
3. Desai HK, Hart BP, Caldwell RW, Jianzhong-Huang JH,
Pelletier SW (1998). Certain norditerpenoid alkaloids and their
cardiovascular action. J Nat Prod 61: 743 748.
4. Ameri A (1998). The effects of Aconitum alkaloids on the central
nervous system. Prog Neurobiol 56: 211 235.
5. Dickens P, Tai YT, But PPH, Tomlinson B, Ng HK, Yan KW (1994).
Fatal accidental aconitine poisoning following ingestion of
Chinese herbal medicine: a report of two cases. Forensic Sci Int
67: 55 58.
6. Chan TY, Tomlinson B, Tse LK, Chan JC, Chan WW, Critchley JA
(1994). Aconitine poisoning due to Chinese herbal medicines: a
review. Vet Hum Toxicol 36: 452-455.
7. Ito K, Tanaka S, Funayama M, Mizugaki M (2000). Distribution
of Aconitum Alkaloids in body fluids and tissues in a suicidal case
of aconite ingestion. J Analytical Toxicol 24: 348 353.
8. Beike J, Frommherz L, Wood M, Brinkmann B, Khler H.
Determination of aconitine in body fluids by LC-MS-MS.
Int. J. Legal Med. 118: 289-293 (2004).
40
Published References
Quantitative Analysis of 9-Tetrahydrocannabinol in

Preserved Oral Fluid by Liquid ChromatographyTandem
Mass Spectrometry
Development of a Rapid and Sensitive Method for the

Quantitation of Amphetamines in Human Plasma and Oral
Fluid by LC/MS/MS
Marleen Laloup a, Maria del Mar Ramirez Fernandez a, Michelle Wood b,

Gert De Boeck a, Ccile Henquet c, Viviane Maesd, Nele Samyna
a National Institute of Criminalistics and Criminology (NICC), Section Toxicology,
Vilvoordsesteenweg 98, 1120 Brussels, Belgium b Waters Corporation,
MS Technologies Centre, Manchester, UK c Department of Psychiatry and
Neuropsychology, South Limburg Mental Health Research and Teaching Network,
EURON, Maastricht University, Maastricht, The Netherlands dDepartment of
Clinical Chemistry-Toxicology, Academic Hospital, Free University of Brussels,
Brussels, Belgium
M. Wood[1], G. De Boeck[2], N. Samyn[2], M. Morris[1], D.P. Cooper[1], R.A.A.

Maes[3], and E.A. De Bruijn[3]
[1]Micromass U.K. Limited, Atlas Park, Simonsway, Wythenshawe, Manchester
M22 5PP, United Kingdom;
[2]National Institute of Criminalistics and Criminology (NICC), Section Toxicology,
Vilvoordsesteenweg 98, 1120 Brussels, Belgium; and
[3]Utrecht Institute of Pharmaceutical Sciences (UIPS), Department of Human
Toxicology, University of Utrecht, Sorbonnelaan 16, 3584 CA Utrecht,
The Netherlands
Abstract
A rapid and sensitive method for the analysis of 9-Tetrahydrocannabinol
(THC) in preserved oral fluid was developed and fully validated. Oral fluid
was collected with the Intercept, a Food and Drug Administration (FDA)
approved sampling device that is used on a large scale in the U.S. for workplace drug testing. The method comprised a simple liquidliquid extraction
with hexane, followed by liquid chromatographytandem mass spectrometry
(LC/MS/MS) analysis. Chromatographic separation was achieved using a
XTerra MS C18 column, eluted isocratically with 1mM ammonium formate
methanol (10:90, v/v). Selectivity of the method was achieved by a combination of retention time, and two precursor-product ion transitions. The use of
the liquidliquid extraction was demonstrated to be highly effective and led
to significant decreases in the interferences present in the matrix. Validation
of the method was performed using both 100 and 500 L of oral fluid. The
method was linear over the range investigated (0.5100 ng/mL and
0.1-10 ng/mL when 100 and 500 L, respectively, of oral fluid were used)
with an excellent intra-assay and inter-assay precision (relative standard
deviations, RSD <6%) for quality control samples spiked at a concentration
of 2.5 and 25 ng/mL and 0.5 and 2.5 ng/mL, respectively. Limits of
quantification were 0.5 and 0.1 ng/mL when using 100 and 500 L,
respectively. In contrast to existing GC/MS methods, no extensive sample
clean-up and time-consuming derivatization steps were needed. The method
was subsequently applied to Intercept samples collected at the roadside and
collected during a controlled study with cannabis.
Abstract
Target analysis of amphetamines in biological samples is of great importance for clinical and forensic toxicologists alike. At present, most laboratories analyze such samples by gas chromatographymass spectrometry.
However, this procedure is labor-intensive and time-consuming, particularly
as a preliminary extraction and derivatization are usually unavoidable.
Here we describe the development of an alternative method. Amphetamines
were isolated from human plasma and oral fluid using a simple methanol
precipitation step and subsequently analyzed using reversed-phase liquid
chromatography tandem mass spectrometry. Quantitation of the drugs
was performed using multiple reaction monitoring. The developed method,
which requires only 50 L of biological sample, has a total analysis time of
less than 20 min (including sample preparation) and enables the simultaneous quantitation of 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxyethylamphetamine, amphetamine,
methamphetamine, and ephedrine in a single chromatographic run. Limits of
detection of 2 g/L or better were obtained. The method has been validated
and subsequently applied to the analysis of plasma and oral fluid samples
collected from current drug users.
Journal of Analytical Toxicology, Volume 27, Number 2, March 2003, pp. 78-87
Development of a Rapid and Sensitive Method for the

Quantitation of Benzodiazepines in Calliphora vicina Larvae
and Puparia by LC/MS/MS
Journal of Chromatography A, 1082 (2005) 1524
M. Wood[1], M. Laloup[2], K. Pien[3], N. Samyn[2], M. Morris[1], R.A.A. Maes[4],

E.A. de Bruijn[4], V. Maes[5], and G. De Boeck[2]
[1]Waters Corporation, MS Technologies Centre, Atlas Park, Manchester, United
Kingdom; [2]National Institute of Criminalistics and Criminology (NICC), Section
Toxicology, Brussels, Belgium; [3]Department of Anatomo-Pathology, Academic
Hospital, Free University of Brussels, Belgium; [4]Utrecht Institute of Pharmaceutical
Sciences (UIPS), Department of Human Toxicology, University of Utrecht, The
Netherlands; and [5]Department of Clinical Chemistry-Toxicology, Academic
Hospital, Free University of Brussels, Belgium
Simultaneous Analysis of Gamma-Hydroxybutyric Acid and

its Precursors in Urine using Liquid ChromatographyTandem
Mass Spectrometry
Michelle Wood a,?, Marleen Laloup b, Nele Samyn b, Michael R. Morris a,
Ernst A. de Bruijn c, Robert A. Maes c, Michael S. Young d, Viviane Maes e,
Gert De Boeck b aWaters Corporation, MS Technologies Centre, Micromass UK
Ltd., Atlas Park, Simonsway, Wythenshawe, Manchester M22 5PP, UK
b National Institute of Criminalistics and Criminology (N.I.C.C.), Section
Toxicology, Vilvoordsesteenweg 98, 1120 Brussels, Belgium c Department of
Human Toxicology, Utrecht Institute of Pharmaceutical Sciences (UIPS),
University of Utrecht, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands
Abstract
We have developed a rapid method that enables the simultaneous analysis of
gamma-hydroxybutyrate (GHB) and its precursors, i.e. gamma-butyrolactone
(GBL) and 1,4-butanediol (1,4-BD) in urine. The method comprised a simple
dilution of the urine sample, followed by liquid chromatographytandem mass
spectrometry (LC/MS/MS) analysis. Chromatographic separation was achieved
using an Atlantis dC18 column, eluted with a mixture of formic acid and methanol. The method was linear from 180 mg/L for GHB and 1,4-BD and from
150 mg/L for GBL. The limit of quantification was 1 mg/L for all analytes.
The procedure, which has a total analysis time (including sample preparation)
of less than 12 min, was fully validated and applied to the analysis of 182
authentic urine samples; the results were correlated with a previously published
GC/MS procedure and revealed a low prevalence of GHB-positive samples.
Since no commercial immunoassay is available for the routine screening of
GHB, this simple and rapid method should prove useful to meet the current
increased demand for the measurement of GHB and its precursors.
Journal of Chromatography A, 1056 (2004) 8390
Abstract
Liquid chromatographytandem mass spectrometry (LC/MS/MS) is emerging as the tool of choice for rapid analysis and the detection of biologically
active compounds in complex mixtures. We describe the development of a
sensitive method for the simultaneous quantitation of 10 benzodiazepines in
Calliphora vicina (Diptera: Calliphoridae) larvae and puparia. The use of
larvae for toxicological analyses offers some technical advantages over putrefied tissue. Four sample pretreatment methods for isolating the benzodiazepines out of larvae were evaluated. A simple homogenization, followed by
acetonitrile precipitation yielded the highest recoveries. Puparia were
pulverized and extracted by ultrasonification in methanol. All extracts were
subsequently analyzed using reversed-phase LC/MS/MS. Larvae and
puparia calibrators containing benzodiazepines at concentrations ranging
from 25 to 750 pg/mg and 50 to 500 pg/mg, respectively, were prepared
and analyzed. The method was demonstrated to be linear over the ranges
investigated. Limits of detection were from 1.88 to 5.13 pg/mg larva and
from 6.28 to 19.03 pg/mg puparium. The developed method was applied
to the determination of nordiazepam and its metabolite oxazepam in larvae
and puparia of the Calliphora vicina fly that had been reared on artificial
foodstuff (beef heart) spiked with 1 g/g nordiazepam. The larvae were
harvested at day 5 for analysis of drug content. The method was sufficiently
41
Furthermore, the processed samples were demonstrated to be stable for 48

h, except for cocaine and benzoylecgonine, where a slight negative trend
was observed, but did not compromise the quantitation. In all cases the
method was linear over the range investigated (2200 g/L) with an excellent intra-assay and inter-assay precision (coefficients of variation <10% in
most cases) for QC samples spiked at a concentration of 4, 12 and 100
g/L. Limits of quantitation were estimated to be at 2 g/L with limits of
detection ranging from 0.2 to 0.5 g/L, which meets the requirements of
SAMHSA for oral fluid testing in the workplace. The method was subsequently applied to the analysis of Intercept samples collected at the roadside by
the police, and to determine MDMA and MDA levels in oral fluid samples
from a controlled study.
sensitive to allow the detection of nordiazepam and oxazepam in a single

larvae.
Journal of Analytical Toxicology, Volume 27, Number 7, October 2003,
pp. 505-512
Determination of Aconitine in Body Fluids by LC/MS/MS

J. Beike1, L. Frommherz1, M. Wood2, B. Brinkmann1 and H. Khler1
Institute of Legal Medicine, University Hospital Mnster, Rntgenstrasse 23,
48149 Mnster, Germany (2) Waters Corporation, MS Technologies Centre,
Atlas Park, Manchester, United Kingdom
(1)
Abstract
Forensic Science International, Volume 150, Issues 2-3 , 10 June 2005,

Pages 227-238
A very sensitive and specific method was developed for the determination
of aconitine, the main toxic alkaloid from plants of the genus Aconitum L.,
in biological samples. The method comprised solid-phase extraction using
mixed-mode C 8 cation exchange columns followed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). Chromatographic separation
was achieved with a RP8 column. Detection of aconitine was achieved using
electrospray in the positive ionisation mode and quantification was performed using multiple reaction monitoring with m/z 646.4 as precursor ion,
i.e. [M+H]+ of aconitine and m/z 586.5, m/z 526.4 and m/z 368.4
as product ions after collision-induced dissociation. The method was fully
validated for the analysis of blood samples: the limit of detection and the
limit of quantitation were 0.1 ng/g and 0.5 ng/g, respectively. Within the
linear calibration range of 0.525 ng/g, analytical recovery was 79.9%.
In two fatal cases with suspected aconite intoxication, aconitine could be
detected in blood samples at concentrations of 10.0 and 12.1 ng/g. In one
case, aconitine could also be detected in the stomach content (3 ng/g) and
in the other in the urine (180 ng/mL).
Recent Applications of LC/MS in Forensic Science

G. De Boeck1, M. Wood 2 and N. Samyn1
Institute of Criminalistics and Criminology, Brussels, Belgium,
2Micromass UK Limited, Wythenshawe, UK.
1National
International Journal of Legal Medicine, Volume 118, Number 5, October

2004, pp. 289-293
Quantitative Analysis of Multiple Illicit Drugs in Preserved Oral

Fluid by Solid-Phase Extraction and Liquid Chromatography
Tandem Mass Spectrometry
Michelle Wood a, Marleen Laloup b, Maria del Mar Ramirez Fernandez b,
Kevin M. Jenkins c, Michael S. Young c, Jan G. Ramaekersd, Gert De Boeck b
and Nele Samyn b,
a Waters Corporation, MS Technologies Centre, Manchester, UK
b Federal Public Service Justice, National Institute of Criminalistics and
Criminology (NICC), Vilvoordsesteenweg 100, 1120 Brussels, Belgium
c Waters Corporation, Milford, MA, USA d Experimental Psychopharmacology
Unit, Brain and Behaviour Institute, Maastricht University, Maastricht, The
Netherlands
Abstract
We present a validated method for the simultaneous analysis of basic drugs
which comprises a sample clean-up step, using mixed-mode solid-phase
extraction (SPE), followed by LC/MS/MS analysis. Deuterated analogues
for all of the analytes of interest were used for quantitation. The applied
LC gradient ensured the elution of all the drugs examined within 14 min
and produced chromatographic peaks of acceptable symmetry. Selectivity
of the method was achieved by a combination of retention time, and two
precursor-product ion transitions for the non-deuterated analogues. Oral fluid
was collected with the Intercept , a FDA approved sampling device that is
used on a large scale in the US for workplace drug testing. However, this
collection system contains some ingredients (stabilizers and preservatives)
that can cause substantial interferences, e.g. ion suppression or enhancement
during LC/MS/MS analysis, in the absence of suitable sample pre-treatment. The use of the SPE was demonstrated to be highly effective and led to
significant decreases in the interferences. Extraction was found to be both
reproducible and efficient with recoveries >76% for all of the analytes.
Introduction
The term forensic science covers those professions that are involved in the
application of the social and physical sciences to the criminal justice system.
Forensic experts are obliged to explain the smallest details of the methods
used, to substantiate the choice of the applied technique and to give their
unbiased conclusions. The final result of the work of the forensic scientist, the
expert evidence, exerts a direct influence on the fate of a given individual.
This burden is a most important stimulus and one that determines the way
of thinking and acting in forensic sciences. Consequently, the methods
applied in forensic laboratories should assure a very high level of reliability
and must be subjected to extensive quality assurance and rigid quality
control programmes.1
Legal systems are based on the belief that the legal process results in justice
a belief that has come under some question in recent years. Of course,
the forensic scientist cannot change scepticism and mistrust single-handedly.
He or she can, however, contribute to restoring faith in the judicial processes
by using science and technology in the search for facts in civil, criminal and
regulatory matters. The ability of mass spectrometry (MS) to extract chemical
fingerprints from microscopic levels of analyte is invaluable in this quest,
enabling the legally defensible identification and quantification of a wide
range of compounds. Recent years have seen the development of powerful
technologies that have provided forensic scientists with new analytical
capabilities, which were unimaginable only a few years ago. Gas
chromatography GC/MS, liquid chromatography LC/MS, isotope ratio MS
and inductively coupled plasma-MS have become routine tools to enable
detection and characterization of minute quantities in what can often be very
complex matrices. In LC/MS, there has been an explosion in the range of
new products available for solving many analytical problems, particularly
those applications in which non-volatile, labile and/or high molecular weight
compounds are being analysed. Many analysts and laboratories have
reached the point at which they are considering the acquisition of LC/MS
instrumentation. According to Willoughby et al. LC/MS has progressed from
the innovators stage through the early adaptors and on to the early
majority stage, and is now open to specialists from a variety of disciplines.
This has been as a direct result of the introduction of robust, user-friendly
atmospheric pressure ionization (API)-MS instruments at an affordable price.
LCGC Europe, Nov 2, 2002
42
Plasma, oral fluid and sweat wipe ecstasy concentrations in

controlled and real life conditions
Nele Samyna, Gert De Boecka, Michelle Woodb, Caroline T. J. Lamersc, Dick De
Waardd, Karel A. Brookhuisd, Alain G. Verstraetee and Wim J. Riedelc
a
Drugs and Toxicology, Section Toxicology, National Institute of Criminalistics

and Criminology, Vilvoordsesteenweg 100, 1120, Brussels, Belgiumb
c
Micromass Ltd., Manchester, UK Experimental Psychopharmacology
Unit, Brain and Behaviour Institute, Maastricht University, Maastricht, The
Netherlands dDepartment of Psychology, University of Groningen, Groningen,
The Netherlands eLaboratory of Clinical BiologyToxicology, Ghent University
Hospital, Ghent, Belgium
Toxicological data and growth characteristics of single postfeeding larvae and puparia of Calliphora vicina (Diptera:
Calliphoridae) obtained from a controlled nordiazepam study
Karen Pien1 , Marleen Laloup2, Miriam Pipeleers-Marichal1, Patrick Grootaert3,
Gert De Boeck2, Nele Samyn2, Tom Boonen4, Kathy Vits4 and Michelle Wood5
(1)
Department of Pathology Academic Hospital, Free University of Brussels,
Brussels, Belgium (2)Section Toxicology, National Institute of Criminalistics and
Criminology (NICC), Brussels, Belgium (3)Department Entomology, Royal Belgian
(4)
Institute of Natural Sciences, Brussels, Belgium Section Micro-traces, National
Institute of Criminalistics and Criminology (NICC), Brussels, Belgium (5)Micromass
UK Limited, Wythenshawe Manchester, UK
Abstract
Abstract
In a double-blind placebo controlled study on psychomotor skills important
for car driving (Study 1), a 75 mg dose of 3,4-methylenedioxymethamphetamine (MDMA) was administered orally to 12 healthy volunteers who were
known to be recreational MDMA-users. Toxicokinetic data were gathered by
analysis of blood, urine, oral fluid and sweat wipes collected during the first
5 hours after administration. Resultant plasma concentrations varied from 21
to 295 ng/mL, with an average peak concentration of 178 ng/mL observed
between 2 and 4 hours after administration. MDA concentrations never
exceeded 20 ng/mL. Corresponding MDMA concentrations in oral fluid, as
measured with a specific LC/MS/MS method (which required only 50 L of
oral fluid), generally exceeded those in plasma and peaked at an average
concentration of 1215 ng/mL. A substantial intra- and inter-subject variability
was observed with this matrix, and values ranged from 50 to 6982 ng/mL
MDMA. Somewhat surprisingly, even 45 hours after ingestion, the MDMA
levels in sweat only averaged 25 ng/wipe.
In addition to this controlled study, data were collected from 19 MDMA-users
who participated in a driving simulator study (Study 2), comparing sober
non-drug conditions with MDMA-only and multiple drug use conditions. In
this particular study, urine samples were used for general drug screening
and oral fluid was collected as an alternative to blood sampling. Analysis
of oral fluid samples by LC/MS/MS revealed an average MDMA/MDEA
concentration of 1121 ng/mL in the MDMA-only condition, with large intersubject variability. This was also the case in the multiple drug condition,
where generally, significantly higher concentrations of MDMA, MDEA and/
or amphetamine were detected in the oral fluid samples. Urine screening
revealed the presence of combinations such as MDMA, MDEA, amph,
cannabis, cocaine, LSD and psilocine in the multiple-drug condition.
Larvae of the Calliphora vicina (Diptera: Calliphoridae) were reared on artificial food spiked with different concentrations of nordiazepam. The dynamics of the accumulation and conversion of nordiazepam to its metabolite
oxazepam in post-feeding larvae and empty puparia were studied. Analysis
was performed using a previously developed liquid chromatography-tandem
mass spectrometry (LC/MS/MS) method. This method enabled the detection
and quantitation of nordiazepam and oxazepam in single larvae and puparia. Both drugs could be detected in post-feeding larvae and empty puparia.
In addition, the influence of nordiazepam on the development and growth
of post-feeding larvae was studied. However, no major differences were
observed for these parameters between the larvae fed on food containing
nordiazepam and the control group. To our knowledge, this is the first report
describing the presence of nordiazepam and its metabolite, oxazepam, in
single Calliphora vicina larvae and puparia.
International Journal of Legal Medicine, Volume 118, Number 4, August
2004, pp. 190-193
To order any of these reprints contact your local Waters office, or go to
www.waters.com/clinical
Forensic Science International, Volume 128, Issues 1-2, 14 August 2002,

Pages 90-97
43
Compound Index
6-MonoacetylMorphine (6-MAM) . . . . . . . . . . . . . 19, 20
Lorazepam . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Alprazolam . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
MDA . . . . . . . . . . . . . . . . . . . . . . . . 15, 16, 17, 42, 43
Amphetamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
MDEA . . . . . . . . . . . . . . . . . . . . . . . . . . 15, 16, 17, 43
Amphetamines . . . . . . . . . . . . . . . . . . . 4, 5, 15, 17, 41
MDMA . . . . . . . . . . . . . . . . . . . 15, 16, 17, 18, 42, 43
Benzodiazepines . . . . . . . . . . . . . . 4, 5, 23, 25, 26, 41
Methamphetamine . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Cannabidiol . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30, 31
Morphine . . . . . . . . . . . . . . . . . . . . . . . 4, 7, 19, 21, 22
Cannabinol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Morphine-3-Glucuronide . . . . . . . . . . . . . . . . . . . . . 4, 21
Clonazepam . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Morphine-6-Glucuronide . . . . . . . . . . . . . . . . . .4, 21, 22
Codeine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Nordiazepam . . . . . . . . . . . . . . . . . . . . . 4, 24, 25, 26
Diazepam . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24, 25
Oxazepam . . . . . . . . . . . . . . . . . . . . 4, 24, 26, 27, 28
Dihydrocodeine (DHC) . . . . . . . . . . . . . . . . . . . . . . . 19
Prazepam . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Ephedrine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Temazepam . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
GHB . . . . . . . . . . . . . . . . . . . 4, 5, 34, 35, 36, 37, 41
Triazolam . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Heroin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
9-Tetrahydrocannabinol . . . . . . . . . . . . . . . . . .4, 29, 41
44
Notes
45
Notes
46
Notes
47
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DRUGS OF ABUSE ANALYSIS

APPL IC ATION NOTEBOOK

Waters System Solutions for Drugs of Abuse Analysis

The MassTrak System for routine drugs of abuse confirmation

The MassTrak system incorporating the LCT Premier XE represents

The MassTrak System incorporating the Quattro Premier XE

Chemistries for the Drugs of Abuse Laboratory

2007 Waters Corporation.

With LC/MS/MS, amphetamines, opiates,

The utilization of LC/MS (and particularly

The Waters toxicology application group has gained

Drugs of abuse analysis is typically a two-part

Areas of focus have been on:

Targeted and Confirmatory Analysis

Liquid chromatographytandem mass spectrometry

Opiates from plasma and urine

Waters latest mass spectrometer systems, the ACQUITY SQD and

Systematic Toxicological Analysis or General

A comprehensive LC/MS library is now available

LC/MS is also a powerful tool for systematic

The current version of the Waters toxicology library

LC/MS is now increasingly used in screening

ChromaLynx Application Manager for Systematic

ChromaLynx for the Forensic

The need to qualitatively analyze complex mixtures

Chromalynx addresses many of the requirements of

Manually reviewing complex chromatograms to

Automated library searching at multiple

When combined with a powerful multi-function LC/MS

Total Ion Chromatogram (TIC) traces from 7 functions

Flexibility and Ease of Use

Following acquisition of full scan spectra recorded at

has been designed to be easy to use

The key to the exceptional performance of ChromaLynx

ChromaLynx can be used with both LC/MS and

Figure 2: ChromaLynx method editor displaying

Green peaks indicate a component identified

List of possible components present. Green

Library search displays top three

Visual comparison of component mass

Figure 3: Library search method editor enabling automatic

Figure 4: ChromaLynx Browser displaying identified compounds,

TargetLynx Application Manager for Confirmation Analysis

If the analytical data is required to be used as part of

Quantitation using LC/MS/MS is now well established

TargetLynx automates data acquisition, processing

TargetLynx is able to rapidly identify and flag

Analytes are above a specified concentration

Analyte confirmatory ion ratios are outside

Figure 1: Mass Spectrum of the cocaine metabolite

An analyte retention time or relative retention time

Flexibility and Ease of Use

Drugs of Abuse and Forensic Toxicology

TargetLynx consists of a method editor, to set up

Processed data is displayed in the TargetLynx browser

The Compound Summary Report, Sample Summary

TargetLynx Application Manager automates data

TargetLynx provides an easy to use and flexible

Moving the cursor over the sample of interest,

Figure 2: TargetLynx Method Editor showing customisable

TargetLynx features various reporting options, with

General Unknown Screening for Drugs in Biological

Electrospray is a soft ionization technique that mainly