DDRUGS OF ABUSE ANALYSIS RUGS OF ABUSE ... in developing validated methods for drugs of abuse...

DRUGS OF ABUSE ANALYSIS DRUGS OF ABUSE ANALYSIS

APPLICATION NOTEBOOKAPPLICATION NOTEBOOK

Waters Corporation has over 40 years history of developing innovative HPLC, mass spectrometry, software, chemistry and support services. Waters can now provide forensic toxicology laboratories with complete solutions that will improve the accuracy and precision of assays while increasing productivity. Waters MassTrak™ Systems bring the power of LC/MS to your laboratory in a robust, easy-to-use and cost-effective package. These systems offer new levels of sample throughput, sensitivity, specifi city and fl exibility for both screening and confi rmation applications. They are supported by a dedicated toxicology applications development group that has extensive experience in developing validated methods for drugs of abuse analysis.

Waters System Solutions 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 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.

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.

Selecting an HPLC column can be a daunting task with hundreds of stationary phases to choose from. Waters makes it easy by providing innovative stationary phases that provide superior peak shapes, long column lifetimes and complementary selectivities. Whether you’re working with simple or complex matrices, biological or non-biological samples, acidic, basic or neutral molecules. No one offers you more proven sample preparation tools for LC/MS/MS and GC/MS challenges.

Chemistries for the Drugs of Abuse Laboratory

©2007 Waters Corporation.

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 inCalliphora Vicina Larvae using LC/MS/MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26-28

Quantitative Analysis of Δ9-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

Table of Contents

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.


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 pre-treatment 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.

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.

Targeted and Confirmatory AnalysisLiquid chromatography—tandem 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.

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 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.

Areas of focus have been on:

Amphetamines from plasma, urine and saliva

Basic drugs in saliva

THC in saliva

Opiates from plasma and urine

Benzodiazepines from plasma, urine and fly larvae

GHB from urine

5

Introduction

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.


Systematic Toxicological Analysis or General Unknown Screening (GUS)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.

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.

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.

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.

GC/MS/MSWaters 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.

6

Waters Quattro micro™ GC - tandem mass spectrometer system for the most demanding GC applications.

ChromaLynx™ Application Manager for Systematic Toxicological Analysis

IntroductionThe 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.

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.

When combined with a powerful multi-function LC/MS library, ChromaLynx™ offers the most comprehensive LC/MS solution for screening applications.

ChromaLynx™ for the Forensic Toxicology LaboratoryChromalynx™ 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

• Automated library searching at multiplecone 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


7

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.

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.

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

Figure 1:


Flexibility and Ease of UseChromaLynx™ 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.

ChromaLynx™ can be used with both LC/MS and GC/MS data and can accommodate both exact mass and nominal mass data.

Spectral Deconvolution 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.

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.

SummaryChromaLynx™ 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.

8

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.

Figure 3: Library search method editor enabling automatic library searching of all peaks and filtering of results for retention time and cone voltage used.

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.

Visual comparison of component mass spectrum against proposed library match

Library search displays top three Candidates identified at specific point in the chromatogram

Figure 4: ChromaLynx™ Browser – displaying identified compounds, candidate compounds, results of a library search and total ion chromatograms recorded at different cone voltages.


9

IntroductionQuantitation 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, time-consuming and subsequently costly task.

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.

TargetLynx™ is able to rapidly identify and flag samples where, for example:• Analytes are above a specified concentration• Analyte confirmatory ion ratios are outside

specified limits• One or more analyte signal-to-noise ratios are

below a defined value• An analyte retention time or relative retention time

is outside limits• The coefficient of determination (r2) of the

calibration curve exceeds a defined value

Drugs of Abuse and Forensic ToxicologyLC/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.

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.

Flexibility and Ease of UseTargetLynx™ 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.

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.

m/z50 100 150 200 250 300

%

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100 168

105

82

93119 150

122290

Quantitation

Confirmation 1

Confirmation 2

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™.

TargetLynx™ Application Manager for Confirmation Analysis


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.

ReportingCustomisation and Export of Data

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’.

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.

SummaryTargetLynx™ 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.

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.

10

Method Setup

Data Acquisition

Data Processing

Reporting

Review Results

Figure 2: TargetLynx™ Method Editor – showing customisable selection of relevant displayed parameters.

Figure 3: TargetLynx™ browser – showing results summary with flags, calibration curve and chromatograms.

Moving the cursor over the sample of interest, displays tool tips with explanations of why the sample has been flagged.

IntroductionIdentification 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.

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.

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.

LC/MS Library ConceptThe 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.

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 in–source collision induced dissociation (CID). Using this process reproducible LC/MS mass spectra can be used to produce a library of mass spectra .

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.


11

ion+

ESICID

analyte

neutral

[analyte+H]+

H3O+ H2O

[analyte+H]+ +

Figure 2. InSource-CID – An example showing fragmentationof the moleculer ion (m/z 195) of caffeine at 60V in theQuattro micro™ API ion source.

General Unknown Screening for Drugs in Biological Samples by LC/MS

Luc Humbert1, Michel Lhermitte1, Frederic Grisel21Laboratoire de Toxicologie & Génopathologie, CHRU Lille, France2Waters Corporation, Guyancourt, France

Extractor

Sample Cone& Cone Gas


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.

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 MethodAn 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.

ChromaLynx™ Application ManagerChromatogram 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.

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.

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Positive ESI @ 90 V

Positive ESI @ 75 V

Positive ESI @ 60 V

Positive ESI @ 45 V

Positive ESI @ 30 V

Positive ESI @ 15 V

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.

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.


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.

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.

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.

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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.

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.

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1911 1: Scan ES- TIC

4.51e7

Positive ESI @ 90 V

Positive ESI @ 75 V

Positive ESI @ 60 V

Positive ESI @ 45 V

Positive ESI @ 30 V

Positive ESI @ 15 V

Negative ESI @ 30 V

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.

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.

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130 147 293161 233205 279 319381357 390

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101 208145 177 227 252361329301 371 398

329

251242100 142 177 190 284 295 361386 393

Peak #1RT 13.66 min

Peak #2RT 13.72 min

Peak #3RT 13.84 min

Peak #4RT 14.01 min

Peak #5RT 14.13 min

12 3

4 5


Application Example - Polyintoxication

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.

Method and Instrumentation

Analytical Equipment and InstrumentationWaters® Toxicology Screening LC/MS System comprising of: ZQ™ Single Quadrupole Mass Spectrometer Alliance® 2695 Separations Module MassLynx™ 4.0 Data Station ChromaLynx™ 4.0 Application Manager

Sample PreparationLiquid/liquid extraction at 2 pH (4.5 & 9.0) using dichloromethane/ether/hexane [30:50:20] + 0.5% isoamylic alcohol.

LC Separation MethodWaters XTerra® MS Column & Precolumn: C18, 3.5 μm, 2.1 mm id x 150 mm (10 mm for precolumn)Column Oven Temperature: 30 ˚CMobile Phase based on Water/Acetonitrile with Ammonium Formate 5 mM @ pH 3Gradient: 5% organic to 90% organic from 2 min. to 16 minutes

MS Operating ConditionsCapillary 3.5 kV in both positive and negative ion modesSource Temperature @ 120 ˚C & Desolvation Temperature @ 250 ˚CDesolvation Gas Flow Rate @ 350 l/h & Cone Gas Flow Rate @ 100 l/hFunction 1: Full Scan - Negative ESI from 100 to 650 amu in 250 ms @ 30 VoltsFunctions 2 to 7: Full Scan - Positive ESI from 100 to 650 amu in 250 ms @ from15 Volts to 90 Volts

Results

From the resultant analysis, 8 out of 9 expected components were successfully identified by the ChromaLynx™ data processing library search process.

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.

ConclusionUsing 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.

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.

14

Green Triangles indicate a component Identified with a high degree of confidence

Top three candidates are displayed for each component

Compounds confidently Identified

Comparison with Library spectra

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.

Candidate Average Fit (%)

# Analyte Name Status Origin 6 Functions

1 Nicotine Unexpected molecule Smoker / Contamination 56.1

2 Trimetazidine Expected molecule Medication 63.3

3 Acetaminophen Expected molecule Medication 62.3

4 Caffeine Expected molecule Medication 74.0

5 Quinine Expected molecule Medication 70.3

6 Zolpidem Expected molecule Medication 94.7

7 Meprobamate Unexpected molecule Unknown 55.3

8 Mianserin Expected molecule Medication 67.1

9 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


1Michelle Wood*, 2Gert De Boeck, 2Nele Samyn, 1Don Cooper and 1Michael Morris1Waters, Manchester, UK. 2National Institute of Criminalistics and Criminology (NICC), Belgium.

Introduction‘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.

Due to the limited volume of sample (usually <100 μL) 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.

Methods and Instrumentation

LC conditions

LC System: Waters Alliance® 2690

Column: Conventional C18 (100 x 2.1 mm, 3.5 μm)

Mobile phase: (A) =10 mM ammonium acetate

(B) = 95% acetonitrile: 5% 10 mM ammonium acetate

Isocratic elution (85:15)

Flow rate: 0.3 mL/min

Injection volume: 10 μL

MS conditionsMass 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

Results and DiscussionMRM 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.

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.

A Rapid and Sensitive Method for theQuantitation of Amphetamines in Human Saliva

15

Cone Collision Compound Precursor Product Voltage Energy (m/z) (m/z) (V) (eV)

MDEA 208 163 50 12

MDEA D5 213 163 50 12

Methamphetamine 150 91 50 15

Methamphetamine D14 164 98 50 18

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.

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).

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.

©2007 Waters Corporation.16

100%0

100%0

100%0

100

0.00 0.50 1.00 1.50 2.00

MRM of 12 channels ES+ 208>163






2.50 3.00Time

%0

100%0

100%0

00

50 100 150 200 250 300 350 400 450 500pg/µl

12437615

Compound name: MDEACoefficient of Determination: 0.998354Calibration curve: 23664.8* x + 1238.54Response type: External Std. AreaCurve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

Response

100 110 120 130 140 150 160 170 180 190 200 210m/z

100 110 120 130 140 150 160 170 180 190 200 210m/z

0

100

%

166.2

148.2

108.1 143.2129.1 149.2167.2

179.2176.2

0

100

%

148.4

166.3

100 110 120 130 140 150 160 170 180 190 200 210m/z0

100

%

0

100

%

105.1135.1

129.1 176.2 179.2

209.3

90

100 110 120 130 140 150 160 170 180 190 200 210m/z

90

208.4

163.1

208.3

163.2

m/z

m/z0

100

%

0

100

%

105.2 108.1

163.2

124.2 176.1 212.4217.4

214.3

100 110 120 130 140 150 160 170 180 190 200 210 220

100 110 120 130 140 150 160 170 180 190 200 210 220

213.4

135.3

163.2

133.3 213.4

a

b

c

Figure 1. MS (top trace) and product ion spectra (lower trace) for (a) ephedrine, (b) MDEA and (c) MDEA-D5.

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).

Figure 3a. Typical linearity of response for MDEA in the absence of biological matrix.


Figure 5. MRM chromatogram for an oral fluid sample found to be positive for the presence of MDEA, MDMA and MDA.

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.

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.

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.

17

00

50 100 150 200 250 300 350 400 450 500pg/µl

12437615

Compound name: MDEACoefficient of Determination: 0.998354Calibration curve: 23664.8* x + 1238.54Response type: External Std. AreaCurve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

Response

50 μl saliva

200 μL methanol (containing internal standards)

Centrifuge 13,000 rpm(to collect supernatant)

Total analysis time: 15 mins

HPLC/MRM analysis

+

100%0

100%0

100%0

100

0.00 0.50 1.00 1.50

8421488

12912

93918

2.00







2.50 3.00Time

%0

100%0

100%0

1 2 3 4 5 6 7 8 9 10

0

50

100

150

200

250

300

350

Con

cent

ratio

n (n

g/m

L)

Individual #

MethamphetamineAmphetamineEphedrineMDAMDEAMDMA

Figure 3b. Typical linearity of response for saliva containing MDEA.

Figure 4. Schematic overview of the developed LC/MRM technique.


ConclusionsThe 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.

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

020406080

100120140160180200

0 60 180 240 300120

Time after admininistration (minutes)

Mea

nM

DM

Ain

pla

sma

(ng/

mL)

0

200

400

600

800

1000

1200

1400

Mea

nM

DM

Ain

pla

sma

(ng/

mL)

Figure 7. Mean MDMA levels (n=12) in plasma and oral fluid following a single administration of 75 mg MDMA.

Opiates: Use or Abuse? Quantification of Opiates in Human Urine

1Michelle Wood1, Kevin Rush2, Michael Morris1 and Allan Traynor2

Waters Corporation, Manchester, UK 2Medscreen Ltd., London, UK

Introduction 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.

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.

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.

MethodologySample preparationUrine 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 μL urine into 700 μL 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 μL) of the eluant were analysed using LC in conjunction with multiple reaction monitoring (MRM).

LC/MS/MSA 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 LC2790 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.


19

Figure 1: The Asian opium poppy, Papaver somniferum.

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.

Precursor Product Cone Collision Compound ion ion Voltage energy (m/z) (m/z) (V) (eV)

Morphine 286 165 42 38

Morphine - d3 289 165 45 40

Codeine 300 165 45 40

Dihydrocodeine (DHC) 302 199 45 32

6-Monoacetyl morphine ( 6-MAM) 328 165 50 38

6-Monoacetyl morphine ( 6-MAM)-d6 334 165 45 38

6-acetylcodeine 342 165 50 38


ResultsA 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.

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 μL 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).

SummaryWe 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.

ReferencesHayes 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.

Figure 3. MRM chromatograms for: (top to bottom) acetylcodeine, 6-MAM, DHC, codeine and morphine. Responses were obtained with a 10 μL injection of the 5 ng/mL urine calibrator.

Figure 4. Standard curve for 6-MAM. Responses were calculated in reference to the integrated area of the deuterated internal standards.

342>165

328>165

302>199

300>165

286>165

Compound name: 6-MAMCoefficient of Determination: 0.999261Calibration curve: 0.167559* x + 0.0400415Response type: Internal Std. (Ref 4), Area* (IS Conc./IS Area)Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

0 20 80 100 120 140 160 180 200 220 2406040ng/mL0

41.9

Response

CodeineMorphine

6-MAMHeroin Acetylcodeine

CH3

CH3H3C

H3C

H3C

N

H

OO

O

O

CH3CH3

CH3 CH3

CH3CH3

N N

H H

HH

NN

OOHO

OO

OOO OHOHHO

OO

OO

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-6-glucuronide (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.

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

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 μL, spiked with deuterated internal stan-dards) 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 evapora-tor and then redissolved in 100 μL of mobile phase. Reconstituted samples were briefly vortex mixed before the analysis of 10 μL using LC in conjunction with mul-tiple reaction monitoring (MRM).

LC/MS/MSA 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.

Table 1: MRM transitions and conditions for the measurement of morphine and it’s 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.


21

Precursor Product Cone Collision Compound ion ion Voltage energy (m/z) (m/z) (V) (eV)

Morphine 286 165 45 38 Morphine–d3 289 165 45 40 Morphine–M3G–glucuronide 462 286 45 28 Morphine–M3G–d3-glucuronide 465 289 45 30 Morphine–M6G–glucuronide 462 286 45 28Figure 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.


ResultsA series of calibrators (0.5-500 μg/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.

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 μL injection of the 5 μg/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).

SummaryWe 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.

References1. 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.

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.

Figure 2. MRM chromatogram for morphine (MOR), M3G and M6G. The above responses were obtained with a 10 μL injection of the 5 μg/L plasma calibrator. Due to the isobaric nature of M3G and M6G chromatographic resolution is required to enable identification.

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 μg/L.

22

47.1

0.0100 500

µg/L400

1.06

0.0 10.0 µg/L

8.06.04.02.00.00

3002000

ResponseResponse

Compound name: Morphine-3-glucuronideCorrelation coefficient: r= 0.999883, rˆ 2 = 0.999766Calibration curve: 0.09404 * x + 0.100968Response type: Intermal Std ( Ref 4 ), Area * ( IS Conc. / IS Area )Curve type: Linear, Origin: Exclude, Weighting: 1/x Axis trans: None

M6G

Time

MOR

0

%

100

0

462>286

286>165

%

100

7.004.003.00 5.00 6.001.00 2.00

Gert De Boeck1, Nele Samyn1, Karen Pien2, Patrick Grootaert3 and Michelle Wood4, 1 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.

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 μg/L or better were achieved when just 25 μL plasma was used.

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.

Experimental Conditions

LC/MS/MS conditions

LC System: Waters Alliance® 2690Column: Conventional Phenyl Column (2.1 x 150 mm, 5 μm)Mobile phase : A =10:10:80 acetonitrile: methanol: 20 mM ammonium acetate B = 95:5 acetonitrile: 20 mM ammonium acetate

Flow rate: 0.25 mL/minInjection volume: 10 μL

MS conditions:

Mass spectrometer: Quattro Ultima® Ionisation Mode: ES positive ionCapillary voltage : 3kVMS/MS: MRM analysis (Table 1). Collision gas Argon at 2.5 x 10-3 mbar

Time (min) A (%) B (%) Curve number

0 100 0 1 0.5 75 25 1 8 40 60 7 (concave) 11 40 60 6 (linear) 12 100 0 1 15 100 0 1

Results and DiscussionFigure 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.

A series of calibrators (1, 10, 40, 100, 200, 400 and 800 μg/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 μL injection of the 10 μg/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.


Development of a Rapid and Sensitive Method for the Quantification of Benzodiazepines in Plasma and Larvae by LC/MS/MS

23


Responses were linear, in all cases, over the range investigated (Coefficient of Determination > 0.99).

24

100 120 140 160 180 200 220 240 260 280 300 320m/z0

100

%

280.9

274.0

240.8164.9138.0

205.9226.9

250.9

309.0

100 120 140 160 180 200 220 240 260 280 300 320m/z0

100

%

308.9

180.8

166.9

152.9140.8

280.9

182.8212.9

273.9226.8 254.8

282.9

311.0

312.0

A B

301.1

For all compounds, LOD’s of 0.2 μg/L (or better) and LOQ’s of 1 μg/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 μg/L respectively). Coefficients of variation (%CV’s) were found to be highly satisfactory (<15%).

Compound Precursor ion Product ion Cone Voltage Collision energy (m/z) (m/z) (V) (eV)

Alprazolam 308.8 280.9 70 25 Alprazolam-d5 313.8 285.8 100 25 Clonazepam 315.8 269.8 80 25 Clonazepam-d4 319.9 273.8 100 25 Diazepam 284.9 154.0 60 25 Diazepam-d5 289.8 153.7 80 25Flunitrazepam 313.9 267.9 80 25Flunitrazepam-d7 320.8 274.8 80 25 Lorazepam 320.8 274.7 60 23 Lorazepam-d4† 326.8 280.8 80 23 Nordiazepam 270.9 139.8 80 25 Nordiazepam-d5 275.9 139.8 80 25 Oxazepam 287.0 240.8 60 26 Oxazepam-d5 291.7 245.8 80 26 Prazepam 324.9 270.9 80 25 Prazepam-d5 330.0 276.0 80 25 Temazepam 300.9 255.0 60 25 Temazepam-d5 305.8 259.8 60 25 Triazolam 342.9 307.7 60 25 Triazolam-d4† 349.0 313.9 60 25

Table 1. MRM transitions and conditions for the measurement of 10 benzodiazepines. †Note that due to the isobaric nature between these benzodiazepines and their deuterated analogues alternative precursor ions were utilised. 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 μL injection of the 10 μg/L plasma calibrator.

Figure 2. MS and MS/MS spectra of alprazolam.


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.

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.

Larvae were reared on artificial foodstuff (beefheart) spiked with a range of concentrations of nordiazepam (0, 0.5, 1 and 2 μg/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.

Figure 4. Preparation of larvae for LC/MS/MS analysis.

Figure 5. MRM chromatograms obtained with the analysis of larvae that were reared on artificial foodstuff spiked with Nordiazepam at 0 and 1 μg/g (A and B respectively). Figure C shows the MRM chromatogram for the internal standard i.e. Nordiazepam-d5.

ConclusionWe have developed a simple, rapid method that allows the simultaneous quantification of 10 benzodiazepines in plasma a single chromatographic run. LOD’s were better than 0.2 μg/L when only 25 μL plasma was used. The method involves a simple protein precipitation step with acetonitrile followed by LC/MS/MS analysis.

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.

C

B

A

271>140

276>140

271>140

100

0

%

100

0

%

100

06.00 8.00 10.00

Time

%

Compound name: DiazepamCoefficient of Determination: 0.999378Calibration curve: 1.22307* x + 0.093412Response type: Internal Std (Ref 10), Area* (IS Conc./IS Area)Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

0 100 200 300 400 500 600 700 800µg/L0

979

Response

After 7 days Dry to 100µL

Filter

500µL H2Omix throughly

LC/MS/MSanalysis

(10µL aliquot)

1mL ACN and I.S.(nordiazepam d5 & oxazepam d5)

vortex thoroughty

Detection of Nordiazepam and Oxazepam inCalliphora Vicina Larvae using LC/MS/MS

Karen Pien1; Patrick Grootaert2; Gert De Boeck3; Nele Samyn3; Tom Boonen4; Kathy Vits4; Michelle Wood5; Michael Morris5.1Free 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.

IntroductionIn 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.

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).

In addition, we compared the development of postfeeding larvae and pupae fed on different concentrations of Nordiazepam.

Experimental Conditions

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.

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.

Sample preparation

Individual larvae and pupae samples were prepared for LC/MS/MS as follows; the sample was transferred to a vial containing 500 μL 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 μL and then filtered. A 10 μL aliquot was analysed using LC/MS/MS.

LC/MS/MS

LC Conditions

LC System: Waters Alliance® 2690Column: Conventional Phenyl Column (2.5 x 150 mm, 5 μm)Mobile Phase: A=10:10:80 acetonitrile:methanol: 20 mM ammonium acetate B=95:5 acetonitrile: 20 mM ammonium acetate

Time (min) A (%) B (%) Curve number

0 100 0 1 0.5 75 25 1 8 40 60 7 (concave) 11 40 60 6 (linear) 12 100 0 1 15 100 0 1

Flow Rate: 0.25 mL/minInjection Volume: 10 μL


26

Sample Target Concentration (μg/g)*

Control 0

Nor I (human therapeutic dose) 0.5

Nor II (human lethal dose) 1

Nor III (2x human lethal dose) 2

Table 1: Target concentration of Nordiazepam in larval food. *Concentrations expressed in μg/g food.


MS Conditions

Mass Spectrometer: Quattro Ultima™ triple quadrupoleIonisation Mode: ES+ Capillary Voltage: 3kV

Results

All larvae, pupae and food spiked with Nordiazepam were positive for the drug, whereas all control samples were negative.

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 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.

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.

27

Compound Precursor Ion Product Ion Cone Voltage Collision Energy (m/z) (m/z) (V) (eV)

Nordiazepam 278 91 30 30

Nordiazepam-d5 325 109 38 25

Oxazepam 315 86 28 18

Oxazepam-d5 327 270 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.

Figure 1: Concentration of Nordiazepam in larva reared (for 4-8 days) on foodstuff spiked with Nordiazepam. Mean concentrations are plotted (± 1SD).

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

276>140

271>140

271>140

C

B

A

Figure 3: MRM chromatograms obtained with the analysis of larvae that were reared on artificial foodstuff spiked with Nordiazepam at 0 and 1 μg/g (A and B respectively). Figure C shows the MRM chromatogram for the internal standard i.e. Nordiazepam d5.


In contrast, the development of larvae fed with the NOR II regime was 1 day later in all stages.

Post-feeding larval length is shown in Table 3; no significant differences were observed.

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.

Discussion and Conclusions

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.

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.

28

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

Day 4 Day 5 Day 6 Day 7 Day 8

Control 69.5 84.5 78.2 86.2 71.4

Nor I 73.4 89 78.5 87.5 80.5

Nor II 110.8 105.8 101.7 96 92.9

Nor III 82.5 83.5 83.5 84 83.1

Table 3: Mean post-feeding larval length (mm).

Table 4: Mean post-feeding larval weight (mg).

1Marleen Laloup1, Maria del Mar Ramirez Fernandez1, Michelle Wood2, Gert De Boeck1, Cecile Henquet3, Viviane Maes4, Nele Samyn1

1National Institute of Criminalistics and Criminology (N.I.C.C), Brussels, Belgium; 2Waters Corp., Manchester, UK;.3Maastricht University, The Netherlands4, Free University of Brussels, Brussels, Belgium


29

IntroductionCannabis 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. Δ9-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.

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 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.

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®.

Methods and InstrumentationCalibrators 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 manufacturer’s 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.

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 μg 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).

The study protocol was approved by the ethics committee of the University Hospital of Maastricht in the Netherlands.

Internal standard solution

An internal standard (IS) working solution of THC-d3 at a concentration of 10 ng/mL was prepared in methanol.

Quantitative Analysis of Δ9- Tetrahydrocannabinolin Preserved Oral Fluid


Sample preparationExtraction was performed using either 100 or 500 μL of the collected specimen. When using 500 μL, 50 μL of the IS working solution and 4 mL of hexane were added; when only 100 μL of oral fluid was used, an additional 400 μL 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 μL of mobile phase.

LC conditions

LC system: Waters® Alliance® SystemColumn: Waters XTerra® MS C18 column (2.1 x 150 mm, 3.5 μm) at 40 ˚CMobile phases: (A): 1 mM ammonium formate (B): methanol Isocratic elution 10:90 (A:B)Flow rate: 0.2 mL/minInjection volume: 20 μL

Mass spectrometry conditions

Mass spectrometer: Waters Quattro Premier™ tandem mass spectrometerIonisation mode: ES+

Capillary voltage: 2 kVSource temperature: 120 ˚CDesolvation gas: Nitrogen at 700 L/Hr, 280 ˚CMS/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>193.1Collision gas: Argon at 3.5 x 10-3 mbar

Results and DiscussionFigure 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 μL and 500 μL 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.

30

Figure 2. MRM chromatograms obtained with a single injection of a 100 μL extracted oral fluid sample enriched with 5 ng/mL THC and 5 ng/mL THC-d3. 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 chromatogram.

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 μL (C) and the reconstituted extract after 500 μL 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.


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 μL 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.

Linear responses (r = >0.999, 1/x weighting) were obtained up to 100 ng/mL when 100 μL of sample was extracted and up to 10 ng/mL when 500 μL 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 μL respectively of the collected sample was extracted.

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 μL 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.

The stability of the samples post extraction was assessed by repeated injections of extracted samples

over a period of 15 hours. No instability was noted over the course of this experiment.

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).

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.

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.

31

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.

Linearity Data slope* intercept* CV of slope (% over 5 r2 (range of 5 Sensitivity Data volume oral fluid consecutive days) consecutive days) LOQ (ng/mL)

100μL 1.0635 0.0209 2.9 0.9993-0.9999 0.5 500μL 5.3976 -0.0009 4.1 0.9992-0.9999 0.1


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.

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.

32

Table 2. Precision and accuracy data for THC for the extraction of 100 μL and 500 μL 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

Intra-assay Precision Interassay Precision Concentration Mean Concentration Mean Concentration Volume Oral of QC Found %CV Accuracy (%) Found %CV Accuracy (%) Fluid (ng/mL) (ng/mL) (ng/mL)

100 μL 2.5 2.5 3.6 -1.0 2.4 2.9 -2.5

25.0 24.8 5.4 -0.7 24.0 5.4 -4.1

500 μL 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

Sample THC (ng/mL) Sample THC (ng/mL)

1 5.7 25 60.2 2 7.0 26 3.9 3 4.6 27 52.2 4 18.5 28 25.4 5 2.5 29 193.5 6 95.8 30 111.2 7 0.3 31 7.3 8 84.7 32 14.6 9 0.3 33 1.9 10 0.5 34 4.7 11 4.5 35 100.0 12 3.9 36 23.0 13 31.9 37 57.1 14 50.8 38 88.6 15 34.6 39 3.9 16 56.0 40 375.8 17 81.1 41 3.7 18 11.9 42 4.4 19 107.4 43 4.2 20 92.1 44 4.2 21 10.0 45 4.2 22 17.6 46 4.1 23 94.8 47 4.0 24 37.2 48 4.4

Table 3. Results obtained applying the method to 48 oral fluid samples collected by the police at the roadside.

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.

THC(ng/mL)


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 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 μL sample.

The LOQ is sufficiently low to meet the requirements of SAMHSA (2 ng/mL) for oral fluid testing.Pharmacokinetic studies may require lower LOQ’s; these requirements can be met by using larger volumes of oral fluid.

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.

References1. 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) 321–330.

2. R. Dams, C.M. Murphy, R.E. Choo, W.E. Lambert, A.P. De Leenheer and M.A. Huestis, Anal. Chem. 75 (2003) 798–804.

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.

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.

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 Wood1, Marleen Laloup2, Nele Samyn2, Michael Morris1, Peter Batjoens3 and Gert De Boeck2

1 Waters Corp., Manchester, UK; 2National Institute of Criminalistics and Criminology (N.I.C.C), Brussels, Belgium; 3Waters Corp., Brussels, Belgium.

IntroductionGamma-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 1,4-BD in urine.

Methods and Instrumentation

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

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.

Authentic Samples

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: Waters Alliance® 2795Column: Waters Atlantis® dC18 column (3 x 100 mm, 5 μm) at 35 ˚CMobile phases: (A): 0.1% aqueous formic acid (B): methanolIsocratic elution: 90:10 (A:B)Flow rate: 200 μL/min.Inj. volume: 20 μL

Mass Spectrometry ConditionsMass spectrometer: Quattro micro® mass spectrometerIonisation mode: ES+

Capillary voltage: 3.5 kVSource Temperature: 120 ˚CDesolvation gas: Nitrogen at 700 L/Hr, 350 ˚CMS/MS: Collision gas (argon) at 5 x 10-3 mbar


34

Liquid Ecstasy

Fantasy

Easy Lay

Blue nitro


Results and Discussion

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.

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).

35

A

B

C

Compound Precursor Product Cone Collision ion ion Voltage energy (m/z) (m/z) (V) (eV)

GHB 105 87 10 7

GHB-d6 111 93 10 7

GBL 87 45 25 15

GBL-d6 93 49 25 15

1,4-BD 91 73 12 5

Figure 2: Product ion spectra for GHB (A), GBL (B) and 1,4-BD (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.

1.8 x 105

4.2 x 105

4.3 x 104

1.8 x 105

4.2 x 105

4.3 x 104

GHB

1,4,-BD

GBL

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.

Compound Mean Recovery %CV Mean Recovery %CV (mg/L) (%) (mg/L) (%)

Precision (n=5)

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

Intra-assay (n=5)

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

Intrerassay (n=5)

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

Low Control High Control (4 mg/L) (40 mg/L

Table 2: Precision and analytical recovery data for GHB and its precursors in urine.


Fig. 4: LC/MS analysis of the hydroxybutyric acid isomers. Ion chromatograms obtained following the analysis of gamma-hydroxybutyric 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 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.

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).

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 time-consuming, labour-intensive GC/MS method.

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.

ConclusionsTo 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.

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;

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.

2. It involves fewer manipulations and is less time-consuming.

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

A

B

C


References1. 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 90’s. 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. Gamma- hydroxybutyrate ( 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 Áhydroxybutyric 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 Köhler11 Institute of Legal Medicine, University Hospital Münster, Röntgenstrasse, Münster, Germany2 Clinical Applications Group, Waters Corporation, Simonsway, Manchester M22 5PP, UK.

IntroductionPlants 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.

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.

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.

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

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.

Methods and InstrumentationSample 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 pre-conditioned 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.

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 μL LC mobile phase.


38

Figure 1: Aconitum napellus (monkshood) (A) and Aconitum vulparia (wolfsbane) (B).


LC/MS/MSA Quattro micro™ 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.

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 μm) and a XTerra® RP8 analytical column (2.1 x 150 mm, 3.5 μm). 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 μL/min. The injection volume was 10 μL 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).

ResultsA 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.

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.

Figure 2: MRM chromatograms for a blood calibrator spiked at 0.1 ng aconitine/g blood. Peak intensity is given in the top right-hand corner of the trace.

SummaryWe 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.

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.

39

4.00

4.80

2.00 6.00 8.00 10.00

100

%

Time

MRM of 3 Channels ES+646.4>586.5646.4>526.4646.4>368.4

2.37e3

Aconitine concentration Case no. Blood [ng/g] Stomach content [ng/g] Urine [ng/mL]

1 10.0 3.0 Not available

2 12.1 Not available 180.0

Table 1: Concentrations of aconitine in autopsy samples from two cases of fatal aconite intoxication.


References1. List PH, Hörhammer 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, Köhler H. Determination of aconitine in body fluids by LC-MS-MS. Int. J. Legal Med. 118: 289-293 (2004).

40

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.

2.00 4.00 6.00 8.00 10.00Time

100

%

4.80 MRM of 3 Channels ES+

646.4>586.5646.4>526.4646.4>368.4

1.83e5


Quantitative Analysis of Δ9-Tetrahydrocannabinol in Preserved Oral Fluid by Liquid Chromatography–Tandem Mass SpectrometryMarleen Laloupa, Maria del Mar Ramirez Fernandeza, Michelle Wood b,Gert De Boecka, Cécile Henquet c, Viviane Maesd, Nele Samynaa 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

AbstractA rapid and sensitive method for the analysis of Δ9-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 work-place drug testing. The method comprised a simple liquid–liquid extraction with hexane, followed by liquid chromatography–tandem 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 combi-nation of retention time, and two precursor-product ion transitions. The use of the liquid–liquid 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 μL of oral fluid. The method was linear over the range investigated (0.5–100 ng/mL and 0.1-10 ng/mL when 100 and 500 μL, 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 μL, 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.

Journal of Chromatography A, 1082 (2005) 15–24

Simultaneous Analysis of Gamma-Hydroxybutyric Acid and its Precursors in Urine using Liquid Chromatography–Tandem Mass SpectrometryMichelle Wooda,?, Marleen Laloupb, Nele Samyn b, Michael R. Morris a, Ernst A. de Bruijnc, Robert A. Maesc, Michael S. Youngd, Viviane Maese, 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

AbstractWe 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 chromatography–tandem mass spectrometry (LC/MS/MS) analysis. Chromatographic separation was achieved using an Atlantis® dC18 column, eluted with a mixture of formic acid and metha-nol. The method was linear from 1–80 mg/L for GHB and 1,4-BD and from 1–50 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) 83–90

Development of a Rapid and Sensitive Method for the Quantitation of Amphetamines in Human Plasma and Oral Fluid by LC/MS/MSM. 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

AbstractTarget analysis of amphetamines in biological samples is of great impor-tance for clinical and forensic toxicologists alike. At present, most labora-tories analyze such samples by gas chromatography–mass 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 μL of biological sample, has a total analysis time of less than 20 min (including sample preparation) and enables the simultane-ous quantitation of 3,4-methylenedioxymethamphetamine, 3,4-methylene-dioxyamphetamine, 3,4-methylenedioxyethylamphetamine, amphetamine, methamphetamine, and ephedrine in a single chromatographic run. Limits of detection of 2 μg/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/MSM. 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

AbstractLiquid chromatography–tandem mass spectrometry (LC/MS/MS) is emerg-ing 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 putre-fied tissue. Four sample pretreatment methods for isolating the benzodiaz-epines 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 μg/g nordiazepam. The larvae were harvested at day 5 for analysis of drug content. The method was sufficiently

Published References


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/MSJ. Beike1, L. Frommherz1, M. Wood2, B. Brinkmann1 and H. Köhler1(1) Institute of Legal Medicine, University Hospital Münster, Röntgenstrasse 23, 48149 Münster, Germany (2) Waters Corporation, MS Technologies Centre, Atlas Park, Manchester, United Kingdom

Abstract 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 C8 cation exchange columns followed by liquid chromatogra-phy-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 per-formed 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 0.5–25 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).

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 Wooda, Marleen Laloupb, Maria del Mar Ramirez Fernandezb, Kevin M. Jenkinsc, Michael S. Youngc, Jan G. Ramaekersd, Gert De Boeckb and Nele Samynb,aWaters Corporation, MS Technologies Centre, Manchester, UK bFederal Public Service Justice, National Institute of Criminalistics and Criminology (NICC), Vilvoordsesteenweg 100, 1120 Brussels, Belgium cWaters Corporation, Milford, MA, USA dExperimental Psychopharmacology Unit, Brain and Behaviour Institute, Maastricht University, Maastricht, The Netherlands

AbstractWe 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-treat-ment. 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.

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 (2–200 μg/L) with an excel-lent 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 μg/L. Limits of quantitation were estimated to be at 2 μg/L with limits of detection ranging from 0.2 to 0.5 μg/L, which meets the requirements of SAMHSA for oral fluid testing in the workplace. The method was subsequent-ly 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.

Forensic Science International, Volume 150, Issues 2-3 , 10 June 2005, Pages 227-238

Recent Applications of LC/MS in Forensic ScienceG. De Boeck1, M. Wood2 and N. Samyn1

1National Institute of Criminalistics and Criminology, Brussels, Belgium,2Micromass UK Limited, Wythenshawe, UK.

IntroductionThe 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


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

aDrugs and Toxicology, Section Toxicology, National Institute of Criminalistics and Criminology, Vilvoordsesteenweg 100, 1120, Brussels, Belgiumb Micromass Ltd., Manchester, UK cExperimental Psychopharmacology Unit, Brain and Behaviour Institute, Maastricht University, Maastricht, The Netherlands dDepartment of Psychology, University of Groningen, Groningen, The Netherlands eLaboratory of Clinical Biology–Toxicology, Ghent University Hospital, Ghent, Belgium

AbstractIn a double-blind placebo controlled study on psychomotor skills important for car driving (Study 1), a 75 mg dose of ±3,4-methylenedioxymethamphet-amine ( 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 μL 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 4–5 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 inter-subject 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. Forensic Science International, Volume 128, Issues 1-2, 14 August 2002, Pages 90-97

Toxicological data and growth characteristics of single post-feeding larvae and puparia of Calliphora vicina (Diptera: Calliphoridae) obtained from a controlled nordiazepam studyKaren 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 Institute of Natural Sciences, Brussels, Belgium (4)Section Micro-traces, National Institute of Criminalistics and Criminology (NICC), Brussels, Belgium (5)Micromass UK Limited, Wythenshawe Manchester, UK

Abstract Larvae of the Calliphora vicina (Diptera: Calliphoridae) were reared on arti-ficial food spiked with different concentrations of nordiazepam. The dynam-ics 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 pupar-ia. 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. 190-193To order any of these reprints contact your local Waters office, or go to www.waters.com/clinical

43


6-MonoacetylMorphine ( 6-MAM) . . . . . . . . . . . . . 19, 20

Alprazolam . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Amphetamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Amphetamines . . . . . . . . . . . . . . . . . . . 4, 5, 15, 17, 41

Benzodiazepines . . . . . . . . . . . . . . 4, 5, 23, 25, 26, 41

Cannabidiol . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30, 31

Cannabinol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

Clonazepam . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Codeine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Diazepam . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24, 25

Dihydrocodeine (DHC) . . . . . . . . . . . . . . . . . . . . . . . 19

Ephedrine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

GHB . . . . . . . . . . . . . . . . . . . 4, 5, 34, 35, 36, 37, 41

Heroin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Lorazepam . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

MDA . . . . . . . . . . . . . . . . . . . . . . . . 15, 16, 17, 42, 43

MDEA . . . . . . . . . . . . . . . . . . . . . . . . . . 15, 16, 17, 43

MDMA . . . . . . . . . . . . . . . . . . . 15, 16, 17, 18, 42, 43

Methamphetamine . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Morphine . . . . . . . . . . . . . . . . . . . . . . . 4, 7, 19, 21, 22

Morphine-3-Glucuronide . . . . . . . . . . . . . . . . . . . . .4, 21

Morphine-6-Glucuronide . . . . . . . . . . . . . . . . . .4, 21, 22

Nordiazepam . . . . . . . . . . . . . . . . . . . . . 4, 24, 25, 26

Oxazepam . . . . . . . . . . . . . . . . . . . . 4, 24, 26, 27, 28

Prazepam . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Temazepam . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Triazolam . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Δ9-Tetrahydrocannabinol . . . . . . . . . . . . . . . . . .4, 29, 41

Compound Index


Notes

45


Notes

46


Notes

47

Austria and European Export

(Central South Eastern Europe, CIS

and Middle East) 43 1 877 18 07

Australia 61 2 9933 1777

Belgium 32 2 726 1000

Brazil 55 11 5094 3788

Canada 1 800 252 4752 x2205

China 86 10 8586 8899

CIS/Russia +7 495 3367000

Czech Republic 420 2 617 1 1384

Denmark 45 46 59 8080

Finland 358 9 5659 6288

France 33 1 30 48 72 00

Germany 49 6196 400600

Hong Kong 852 29 64 1800

Hungary 36 1 350 5086

India and India Subcontinent91 80 2837 1900

Ireland 353 1 448 1500

Italy 39 02 27 421 1

Japan 81 3 3471 7191

Korea 82 2 820 2700

Mexico 52 55 5524 7636

The Netherlands 31 76 508 7200

Norway 47 6 384 60 50

Poland 48 22 833 4400

Puerto Rico 1 787 747 8445

Singapore 65 6273 1221

Spain 34 93 600 9300

Sweden 46 8 555 11 500

Switzerland 41 62 889 2030

Taiwan 886 2 2543 1898

United Kingdom44 208 238 6100

All other countries:Waters Corporation U.S.A.1 508 478 20001 800 252 4752

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