Drugs of Abuse Analysis Application Notebook

DDRUGSRUGS OOFF AABUSEBUSE AANALYSISNALYSIS AAPPLICATIONPPLICATION NOTEBOOKNOTEBOOK Waters System Solutions for Drugs of Abuse Analysis

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

The MassTrak™ System for routine drugs of abuse confirmation analysis, consisting of an Alliance® HT HPLC system and the Quattro micro™ API incorporating TargetLynx™ Application Manager, sets new standards for sample throughput, sensitivity and ease of use.

The Quattro micro™ API can also be used for toxicology screening applications using the unique ChromaLynx™ chromatographic data processing software and in-source CID libraries.

The MassTrak™ system incorporating the LCT Premier™ XE represents a new powerful solution for forensic toxicology screening applications. The LCT Premier™ XE is based on time of flight (TOF) technology, which combined with the ACQUITY UPLC™ system provides fast, reliable exact mass measurement. The combination of high full scan sensitivity and routine exact mass measurement (< 5 ppm) enables identification of unknown compounds with the highest degree of confidence.

The LCT Premier™ XE, shown here with the ACQUITY UPLC™ System, represents a major advance for screening applications and provides the ultimate sensitivity and speed of analysis.

The MassTrak™ System incorporating the Quattro Premier™ XE raises the performance bar for drugs of abuse analysis. The Quattro Premier™ XE is a high-performance benchtop tandem quadrupole instrument, featuring advanced Travelling Wave (T-Wave™) technology. The ultra fast scanning speed enabled by T-Wave™ technology enables the Quattro Premier™ XE to take full advantage of the exceptionally high chromatographic resolution of the ACQUITY. The combination of the ACQUITY UPLC™ and Quattro Premier™ XE delivers unmatched sensitivity and analysis speed for drugs of abuse confirmation analysis. Chemistries for the Drugs of Abuse Laboratory

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

Introduction...... 5-6

ChromaLynx™ Application Manager for Systematic Toxicological Analysis ...... 7-8

TargetLynx™ Application Manager for Confirmation Analysis ...... 9-10

General Unknown Screening for Drugs in Biological Samples by LC/MS ...... 11-14

A rapid and Sensitive Method for the Quantitation of Amphetamines in Human Saliva ...... 15-18

Opiates: Use or Abuse? Quantification of Opiates in Human Urine ...... 19-20

Quantification of Morphine, Morphine-3-Glucuronide and Morphine-6-Glucuronide in Biological Samples by LC/MS/MS ...... 21-22

Development of a Rapid and Sensitive Method for the Quantification of Benzodiazepines in Plasma and Larvae by LC/MS/MS...... 23-25

Detection of Nordiazepam and Oxazepam in Calliphora Vicina Larvae using LC/MS/MS ...... 26-28

Quantitative Analysis of Δ 9-Tetrahydrocannabinol in Preserved Oral Fluid by LC/MS/MS ...... 29-33

Simultaneous Analysis of GHB and its Precursors in Urine Using LC/MS/MS ...... 34-37

Determination of Aconitine in Body Fluids by LC/MS/MS ...... 38-40

Published References ...... 41-43

Compound Index...... 44

Cautionary Statement: The MassTrak™ systems are CE marked and declared as in vitro diagnostic devices in the European Union under EU directive 98/79/EC, however the application notes described in this document are for Forensic use only, they are not to be used for any medical device diagnostic application.

The utilization of LC/MS (and particularly With LC/MS/MS, amphetamines, opiates, LC/MS/MS) in forensic toxicology laboratories has benzodiazepines, GHB and many other drugs can increased significantly in recent years. The sensitivity, be analyzed without extensive sample pre-treatment rapid analysis, selectivity and simple sample pre- and without derivatization. The sensitivity of treatment requirements have led to LC/MS/MS LC/MS/MS allows the use of small sample volumes. methods being adopted as the first choice for many Thus, volume-limited samples from alternate matrices, important drugs of abuse analysis applications. e.g. hair, sweat and oral fluid can be used in addition to blood, plasma or urine. MassTrak™ In addition to the common illicit drugs such as ™ amphetamines, opiates, cannabis, LSD and cocaine, Systems equipped with the TargetLynx application many prescribed ‘legal’ drugs have a high potential manager enable the quantification and verification for abuse and are knowingly abused or accidentally of drugs of abuse in a single chromatographic run misused e.g. benzodiazepines and the prescribed with a high degree of confidence. opiates, methadone and buprenorphine. The Waters toxicology application group has gained Drugs of abuse analysis is typically a two-part extensive expertise in developing and refining process - an initial screening test is usually followed LC/MS/MS methods for the quantification of a by a confirmatory analysis of putative positive results wide range of drugs of abuse in their application screens. The most widely used screening technique laboratories in Manchester (UK), Paris (France) and is immunoassay while GC/MS is the most utilized Milford (USA) and in collaboration with many forensic technique for confirmation analysis. LC/MS/MS is laboratories in Europe. Some of these methods are now an established technique for confirmation analysis documented in this application notebook. and is increasingly used for screening applications. Areas of focus have been on: Amphetamines from plasma, urine and saliva Basic drugs in saliva Targeted and Confirmatory Analysis THC in saliva Liquid chromatography—tandem mass spectrometry (LC/MS/MS) is now a widely accepted technique Opiates from plasma and urine in forensic toxicology laboratories for quantitative Benzodiazepines from plasma, urine and fly larvae and confirmatory analysis. It is particularly useful for polar, non-volatile and thermally labile compounds GHB from urine that are difficult to analyze by gas chromatography (GC). In addition, the reduced sample pre-treatment requirements and short run times of LC/MS/MS, compared to GC/MS, make this technique attractive for high-throughput laboratories and for laboratories tasked with providing rapid results.

Waters latest mass spectrometer systems, the ACQUITY™ SQD and ACQUITY™ TQD combine ease of use and robustness with the speed and sensitivity of UPLC® technology.

5 ©2007 Waters Corporation. Systematic Toxicological Analysis or General A comprehensive LC/MS library is now available Unknown Screening (GUS) for use on Waters LC/MS (single quadrupole) and LC/MS is also a powerful tool for systematic LC/MS/MS (tandem quadrupole) systems. It is based toxicological analysis (STA). Waters has developed on a generic chromatographic run using electrospray the ChromaLynx™ Application Manager, a unique ionization (ESI) and mass spectra recorded at multiple data processing tool that can search LC/MS libraries cone voltages. Controlled, reproducible fragmentation based on cone voltage fragmentation. ChromaLynx™ is caused by in-source fragmentation providing spectra Application Manager provides automatic deconvolution of structurally-related fragments ions; the higher the and exhaustive examination of complex chromatograms cone voltage, the more fragmentation is observed. to identify individual components including minor and Identification of compounds from this type of experiment closely-eluting peaks. Individual components are then is based on matching the spectra from multiple cone searched against library spectra and the results are voltage spectra at a single retention time. displayed in an easy-to-use browser format. The current version of the Waters toxicology library LC/MS is now increasingly used in screening contains spectra from > 500 compounds, and has been ™ applications. Until recently, the widespread tested for use with the Waters ZQ single quadrupole ™ implementation of LC/MS has primarily been limited and Quattro micro tandem mass spectrometer systems. by the lack of commercially available LC/MS libraries, and the perceived high capital costs of LC/MS systems. In recent years the capital cost of LC/MS has GC/MS/MS reduced making the technique more widely accessible. Waters Corporation also offers GC/MS/MS systems for forensic toxicology. The Quattro micro™ GC is the most sensitive GC-tandem quadrupole mass spectrometer on the market today. Waters offers GC/MS/MS systems for analytes which have traditionally been submitted for GC/MS analysis. GC/MS/MS offers enhanced sensitivity and specificity over GC/MS and allows for reduced sample clean-up.

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

Introduction ChromaLynx™ for the Forensic The need to qualitatively analyze complex mixtures Toxicology Laboratory is frequently encountered in forensic toxicology Chromalynx™ addresses many of the requirements of laboratories. Due to the polar, and non volatile nature the toxicology laboratory for screening applications. of many toxicologically relevant compounds, LC/MS It is designed for automated processing of LC/MS and methods are now increasingly utilised in toxicological GC/MS data and some of the key features are: screening applications. In these applications there is • Detection of all component peaks in a usually a need to detect and identify toxic compounds sample, including peaks not seen in total from complex chromatograms resulting from the ion chromatogram (TIC) traces analysis of biological fluids such as blood and urine. • Spectral deconvolution and peak identification Manually reviewing complex chromatograms to • Automated library searching at multiple detect and identify potential toxic compounds can cone voltages be a laborious, time-consuming and subsequently expensive task. In a manual process, closely eluting • Automatic scoring of the library search or low intensity components can easily be missed. • Combination of retention time and ChromaLynx™ automates this manual task, enabling mass spectra in the library search. rapid detection and identification of compounds • Results displayed in user-friendly browser from complex mixtures. with report generator option When combined with a powerful multi-function LC/MS library, ChromaLynx™ offers the most comprehensive LC/MS solution for screening applications.

Figure 1:

ChromaLynx™ is able to confidently detect and locate closely eluting peaks. Here Cocaine is identified with a high degree of confidence in a very complex area of the Chromatogram.

Total Ion Chromatogram (TIC) traces from 7 functions from a LC/MS analysis of a urine sample. Several components were identified by ChromaLynx™ including, Ecgoninemethyl ester, Morphine, Benzolyecognine, Cocaine and Noscapine

ChromaLynx™ is able to detect and locate low intensity peaks. Peak eluting at 3.14 mins was subsequently identified as Morphine. On visual inspection, there is no conclusive evidence that a significant component elutes at 3.14 minutes. The unique ChromaLynx™ deconvolution algorithm clearly indicates that a component is present and has been confidently identified.

7 ©2007 Waters Corporation. Flexibility and Ease of Use Spectral Deconvolution ChromaLynx™ has been designed to be easy to use Following acquisition of full scan spectra recorded at while offering flexibility. It consists of a method editor, multiple cone voltages, ChromaLynx™ will analyse to set up chromatographic data processing and library each chromatogram and extract full scan spectra search parameters. The method editor can be viewed and extract specific ion chromatograms to detect spreadsheet-style for ease of review and allows editing the presence of components. of parameters for peak location and detection, and The key to the exceptional performance of ChromaLynx™ subsequent identification using LC/MS and GC/MS is a new, proprietary chromatography deconvolution libraries. The processed data is displayed in the algorithm. This algorithm efficiently locates peaks in ChromaLynx™ browser for ease of review. a chromatogram and extracts ‘’clean’’ mass spectra ChromaLynx™ can be used with both LC/MS and of eluting components. The extracted mass spectra GC/MS data and can accommodate both exact mass can then be searched against LC/MS and GC/MS and nominal mass data. libraries. ChromaLynx™ has been designed to exploit a unique multi-function electrospray LC/MS library based on in-source collision induced (CID) mass spectra. By recording mass spectra at multiple cone voltages in both positive and negative ion mode extensive information is acquired on samples. To further enhance the library search process, ChromaLynx™ also uses retention time information as a search parameter.

Summary ChromaLynx™ Application Manager sets new standards Figure 2: ChromaLynx™ method editor displaying chromatogram data processing parameter set up. In this for the analysis of complex chromatograms resulting from example 7 chromatograms will be processed, five recorded LC/MS or GC/MS analysis of physiological samples in positive Ion mode and two in negative ion mode. such as blood and urine. A unique algorithm enables ChromaLynx™ to locate peaks in a chromatogram and then automatically compare the mass spectra against library mass spectra. When using LC/MS mass spectra recorded at multiple cone voltages (using in-source CID) combined with retention time information further enhances the component identification processes.

Green peaks indicate a component identified with a high degree of confidence

List of possible components present. Green indicates confident identification, yellow tentative identifications, red indicates a poor library match

Library search results for component in-source at three different cone voltages.

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

Visual comparison of component mass spectrum against proposed library match Figure 3: Library search method editor enabling automatic Figure 4: ChromaLynx™ Browser – displaying identified compounds, library searching of all peaks and filtering of results for candidate compounds, results of a library search and total ion retention time and cone voltage used. chromatograms recorded at different cone voltages.

Introduction If the analytical data is required to be used as part of Quantitation using LC/MS/MS is now well established a police investigation or presented in a court of law, in many forensic, environmental, clinical and veterinary it is essential that extensive analytical information is applications. There are often legal, environmental, provided to confirm the presence of a suspected drug. ™ human health and financial implications arising from TargetLynx is ideal for this application, where the the results of quantitative MS analyses. This has led presence of a suspected drug can be confirmed by to an increased demand by regulatory and legal the presence of a number of different diagnostic ions authorities for extra confirmatory and quality control and MRM transitions. Confirmation analysis using ™ checks. Regulatory or statutory methods often require, TargetLynx is enhanced as ion ratio measurements for example, the monitoring of multiple structurally and chromatographic retention time information is also specific fragment ions, maximum chromatographic incorporated in the analytical procedure. peak width and/or retention time. To calculate and check these manually is a labour intensive, time- 168 consuming and subsequently costly task. 100 Quantitation TargetLynx™ automates data acquisition, processing and reporting incorporating a wide range of confirmatory checks allowing samples falling outside user-specified or regulatory thresholds to be easily identified, giving confidence when reporting 105 Confirmation 1 quantitative results. % Confirmation 2 TargetLynx™ is able to rapidly identify and flag 82 119 samples where, for example: 150 93 122 • Analytes are above a specified concentration 290 0 m/z • Analyte confirmatory ion ratios are outside 50 100 150 200 250 300 specified limits

• One or more analyte signal-to-noise ratios are Figure 1: Mass Spectrum of the cocaine metabolite below a defined value Benzoylecgonine.Transition 290/168 is used for quantitation. • An analyte retention time or relative retention time Two further MRM transitions 290/105 and 290/82 are monitored for additional confirmation by TargetLynx™. is outside limits • The coefficient of determination (r2) of the calibration curve exceeds a defined value Flexibility and Ease of Use TargetLynx™ can be used with LC/MS/MS and Drugs of Abuse and Forensic Toxicology GC/MS/MS data. This data can be SIM (Selected Ion Monitoring), MRM (Multiple Reaction Monitoring) LC/MS/MS is now increasingly used in forensic or full scan/full spectrum. In the case of full scan/full toxicology laboratories for drugs of abuse confirmation spectrum data, extracted ion chromatograms are used and quantitation applications. LC/MS/MS is typically for quantitation. used for confirmation analysis, following a positive immunoassay analysis that indicates the presence of TargetLynx™ consists of a method editor, to set up a class of drugs or specific drug, or when there is processing parameters, and a browser, to view evidence present that a drug is likely to be present in processed data. The method editor can be viewed a sample. spreadsheet-style for ease of review and allows all quantitation parameters (for peak location and detection, calibration curves etc.) and user-defined criteria for confirmatory and QC checks to be set up.

9 ©2007 Waters Corporation. Processed data is displayed in the TargetLynx™ browser The Compound Summary Report, Sample Summary for ease of review. A variety of sample flags allows Report, Totals Report and Samples Report allow the easy location and interrogation of samples falling out user to display calibration information per compound, with the defined confirmatory and QC criteria. report one compound/sample/totals group per page or split and print summary reports per sample. Data can be exported from the TargetLynx™ browser Method Setup as a XML or comma separated text file into a LIMS system. Data Acquisition

Summary Data Processing TargetLynx™ Application Manager automates data acquisition, processing and reporting incorporating a Review Results wide range of confirmatory checks allowing samples falling outside user-specified or regulatory thresholds to be easily identified, giving confidence when reporting Reporting quantitative results. TargetLynx™ provides an easy to use and flexible solution to increase laboratory productivity and improve the quality of quantitative LC/MS/MS or GC/MS/MS data.

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

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

Reporting Customisation and Export of Data Figure 3: TargetLynx™ browser – showing results summary with flags, ™ TargetLynx features various reporting options, with calibration curve and chromatograms. reports being printed directly from the Browser file by sample or by compound. Report formats can be customised and can consist of all or some of the following; the Calibration page, Compound Summary Report, Sample Summary Report, Totals Support, Samples Report and Audit Report. In the Calibration page the user can select how the data is displayed, for example ‘Show Residuals’, ‘Show Response Curve’ and/or ‘Show QC Points’.

Luc Humbert1, Michel Lhermitte1, Frederic Grisel2 1Laboratoire de Toxicologie & Génopathologie, CHRU Lille, France 2Waters Corporation, Guyancourt, France

Introduction Electrospray is a soft ionization technique that mainly Identification of drugs of abuse and toxicants in leads to protonated molecular ions in positive ion biological fluids is currently performed by a variety mode and to deprotonated molecular ions in negative of analytical techniques including immunoassays ion mode. In order to get more specific structural and chromatographic techniques such as GC/MS information, it is possible to induce fragmentation of and LC with UV detection. Although these techniques these molecular ions in the source region of a mass are well established and widely used, they suffer spectrometer. This can be achieved by increasing from limitations for many toxicologically important the voltage applied to the sampling cone area where compounds. For example, sensitivity is often a ions transit from a high pressure region to a low limitation with LC/UV techniques as newer drugs pressure region. Molecular ions then collide with are used at lower therapeutic concentrations. In neutral molecules in the source region and fragment addition, LC/UV methods can require extensive into characteristic ions. This is referred to as in–source sample preparation. GC/MS is often referred to as collision induced dissociation (CID). Using this process the gold standard in toxicology laboratories, but reproducible LC/MS mass spectra can be used to even GC/MS has significant limitations for toxicology produce a library of mass spectra . screening applications where rapid sample analysis is a requirement. Many substances encountered in + toxicology laboratories are non-volatile, polar or H3O H2O thermally labile and cannot be directly analyzed analyte [analyte+H]+ by GC/MS. These compounds usually require time ESI consuming derivatization prior to analysis. [analyte+H]+ CID ion+ + neutral LC/MS, using electrospray ionization (ESI), is ideally suited to polar, non volatile and, thermally unstable Figure 1. Atmospheric Pressure Ionisation (API) process - This soft ionisation process leads to cations in positive ion mode and compounds and potentially provides a powerful means anions in negative ion mode which are generally stable. These of identifying many toxicologically relevant compounds molecular ions can be fragmented in the source region of rapidly without the need for sample derivatization. LC/MS instruments. Historically, the lack of availability of LC/MS libraries and reliable LC/MS chromatographic deconvolution Extractor 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. Sample Cone & Cone Gas

LC/MS Library Concept The electrospray ionization process, used in LC/MS systems, is very different from the electron impact (EI) Ionization used in GC/MS systems, thereby preventing the use of commercial EI mass spectra libraries such as NIST, Wiley, and Figure 2. InSource-CID – An example showing fragmentation Pfleger- Maurer-Weber. of the moleculer ion (m/z 195) of caffeine at 60V in the Quattro micro™ API ion source.

11 ©2007 Waters Corporation. In the current version of the library, this approach has been used for over 500 compounds, which corresponds to approximately 2600 mass spectra. These compounds represent 90% of the intoxication cases encountered in Europe. In addition chromatographic retention times are also stored for each compound in the library. The library is easy to maintain and user appendable.

LC Separation Method An identical generic LC method is used both to generate the library mass spectra and for sample analysis. The generic gradient method has been developed based on water and acetonitrile buffered Figure 3. Extensive structural information is stored for each with 5 mM ammonium formate at pH 3. The total run component in the library as mass spectra can be stored at every time including system and column re-equilibration is significant cone voltage in both positive and negative ion mode. 26 minutes.

Positive ESI @ 90 V ChromaLynx™ Application Manager Chromatogram examination is at least as important Positive ESI @ 75 V as the content and structure of the library. The chromatogram from a typical toxicological analysis Positive ESI @ 60 V 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 Positive ESI @ 45 V hidden in the base line or when they closely elute. ChromaLynx™ application manager includes a unique Positive ESI @ 30 V algorithm to specifically process multifunctional LC/MS data. The process can be ultimately as exhaustive as analyzing each scan for each cone voltage; this Positive ESI @ 15 V enables the detection of the maximum number of components in a chromatogram. Unlike other LC/MS/MS screening techniques, Figure 4. Loxapine, a tranquilizer agent. Mass spectra recorded ChromaLynx™ application manager enables a at 6 different CV values using in-source CID.The degree of complete and systematic chromatogram examination. fragmentation increases with the cone voltage. This type of data processing is essential for systematic toxicological screening or general unknown screening. ChromaLynx™ application manager selects a single Using in-source CID, it is possible to generate mass mass spectrum at a given scan and extracts up to 8 of spectra exhibiting different fragmentation patterns the most intense ions and reconstructs corresponding according to the value of the cone voltage applied ion chromatograms. These ion chromatograms in the source. This can be done in both positive and are then examined and components are detected negative ion modes. These spectra can then be used according to user defined parameters. Detected to build a library. components are then searched against library spectra.

12 ©2007 Waters Corporation. 1911 7: Scan ES+ This area represents only 4 minutes out of the 26 TIC Positive ESI @ 90 V 5.39e9 % 1 minutes of the whole chromatogram for function 3 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 1911 6: Scan ES+ ™ TIC recorded in positive ion mode at 30V. ChromaLynx Positive ESI @ 75 V 5.59e9 % 1 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 will process all chromatograms to achieve a detailed 1911 5: Scan ES+ TIC ™ Positive ESI @ 60 V 4.47e9 and efficient screening. ChromaLynx application % 1 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 1911 4: Scan ES+ manager automatically processes data in minutes that TIC Positive ESI @ 45 V 2.72e9

% would take hours manually. 2 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 1911 3: Scan ES+ TIC Positive ESI @ 30 V 3.13e9 % 2 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 1911 2: Scan ES+ TIC Mass Spectrum Extraction and Library Positive ESI @ 15 V 3.05e9 % 2 Search Process 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 1911 1: Scan ES- TIC Negative ESI @ 30 V 4.51e7

% Once chromatographic components have been detected, 62 Time 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 ChromaLynx™ automatically extracts mass spectra of Figure 5. Urine extract - one single sample analysis leads to 7 the individual components. This is performed taking into chromatograms. Manual examination of each chromatogram account possible interferences due to closely eluting would be time consuming and not feasible for a routine peaks. It is possible to customize parameters in order toxicology laboratory. Analysis of the area circled in the chromatogram above by ChromaLynx™ (figure 6b) illustrates to get precise background subtraction depending on the presence of several peaks that would be missed on the peak width and tolerance on apex determination. examination of the total ion chromatogram. Extracted mass spectra of detected components are then compared to library mass spectra. In order to improve the specificity of the screening 1911 13.08 3: Scan ES+ 706433728 TIC 2.95e9 technique, additional filters have been developed to Area

6a 12.49 enhance the quality of the screening and to get more 501322304 relevant results. Retention time filters as well as cone % voltage filters are available and user defined tolerance 11.67 166387152 14.60 115287184 parameters can be implemented. 13.84 46390212 14.07 20840152 4 Time 11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00 1911 23 14.13 100 1 Figure 6a. Close-up view of the 11-15 minutes section of 4 5 chromatogram area for function 3 acquired in positive ESI @ 30 % V. Here the total ion chromatogram (TIC) indicates that only one 3 Time component elutes at 13.8 minutes. 11.50 12.00 12.50 13.00 13.50 14.00 14.50

318 100 Peak #5 RT 14.13 min 1911 % 12.20 147 279 319 100 130 161 205 233 293 357 381 390 0 6b 11.73 327 100 Peak #4 13.60 11.61 14.77 % RT 14.01 min % 265 328 161 282 383 117 175 205 240 316 367 0

11.15 14.19 11.96 12.73 14.01 14.31 265 13.37 100 Peak #3 3 Time 11.25 11.50 11.75 12.00 12.25 12.50 12.75 13.00 13.25 13.50 13.75 14.00 14.25 14.50 14.75 RT 13.84 min 266 % 101 130 263 383 175 208 224 306 339 367 Figure 6b. Close-up view of the 11 - 15 minutes section of 0 chromatogram area for function 3 acquired in positive ESI @ 30 251 100 Peak #2 V. Using extracted ion chromatograms shows that at least three RT 13.72 min % 252 components elute between 13.5 and 13.8 minutes. 101 227 371 398 145 177 208 301 329 361 0

Peak #1 329 100 RT 13.66 min % 100 190 251 295 361 142 177 242 284 386 393 0 m/z 100 150 200 250 300 350

Figure 7. Chromatogram acquired in positive ESI @ 30 V and corresponding mass spectra of 5 components detected by ChromaLynx™. Automated spectral deconvolution allows extraction of clean mass spectra that can be used for library searching.

13 ©2007 Waters Corporation. Application Example - Polyintoxication Tramadol was the only expected compound that was not detected. In addition, three unexpected compounds A urine sample was taken from a suspected were also Detected - Meprobamate, Acepromazine intoxication. It was known that the person was and Bromazepam. It was highly likely that these three taking a number of prescribed drugs. The urine compounds were the cause of the intoxication. samples were analyzed by LC/MS to identify the cause of intoxication. Toxicologists were looking Candidate Average Fit (%) for both expected compounds due to the regular # Analyte Name Status Origin 6 Functions treatment and unexpected active substances that 1 Nicotine Unexpected molecule Smoker / Contamination 56.1 may have been taken accidently or deliberately. 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 Method and Instrumentation 6 Zolpidem Expected molecule Medication 94.7 7 Meprobamate Unexpected molecule Unknown 55.3 Analytical Equipment and Instrumentation 8 Mianserin Expected molecule Medication 67.1 Waters® Toxicology Screening LC/MS System 9 Acepromazine Unexpected molecule Unknown 57.6 comprising of: 10 Bromazepam Unexpected molecule Unknown 53.1 11 Hydroxyzine Expected molecule Medication 88.2 ZQ™ Single Quadrupole Mass Spectrometer 12 Propoxyphene Expected molecule Medication 62.1 Alliance® 2695 Separations Module 13 Tramadol Expected molecule Medication Not found MassLynx™ 4.0 Data Station ChromaLynx™ 4.0 Application Manager

Green Triangles indicate a Sample Preparation component Identified with a high degree of confidence Liquid/liquid extraction at 2 pH (4.5 & 9.0) using dichloromethane/ether/hexane [30:50:20] + 0.5% isoamylic alcohol. LC Separation Method ® Waters XTerra MS Column & Precolumn: C18, Top three candidates are displayed 3.5 μm, 2.1 mm id x 150 mm (10 mm for precolumn) for each component Column Oven Temperature: 30 ˚C Compounds confidently Identified Mobile Phase based on Water/Acetonitrile with Comparison with Library spectra Ammonium Formate 5 mM @ pH 3 Gradient: 5% organic to 90% organic from 2 min. to 16 minutes Figure 8: ChromaLynx™ browser showing a list of candidate MS Operating Conditions compounds, chromatogram recorded at different cone voltages and comparison of a unknown spectra against library spectra. Capillary 3.5 kV in both positive and negative ion modes Source Temperature @ 120 ˚C & Desolvation Conclusion Temperature @ 250 ˚C Using the combination of in-source CID at multiple Desolvation Gas Flow Rate @ 350 l/h & cone voltages and retention time data results in a Cone Gas Flow Rate @ 100 l/h library containing detailed information for each ™ Function 1: Full Scan - Negative ESI from 100 to 650 compound. With the development of ChromaLynx amu in 250 ms @ 30 Volts data chromatographic deconvolution software, LC/MS can now be considered a powerful tool for toxicology Functions 2 to 7: Full Scan - Positive ESI from 100 to screening applications. 650 amu in 250 ms @ from15 Volts to 90 Volts The unique ChromaLynx™ deconvolution algorithm ensures that the maximum number of components are detected. Results The unique algorithm enables low intensity and closely From the resultant analysis, 8 out of 9 expected eluting peaks to be detected and identified. The accuracy components were successfully identified by the of the library search process is enhanced by utilizing ChromaLynx™ data processing library search process. multiple mass spectra per component and retention time.

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

Introduction MS conditions ‘Ecstasy’ (MDMA), ‘EVE’ (MDEA) and MDA are Mass spectrometer: Quattro Ultima® tandem amongst the most frequently used recreational mass spectrometer. drugs. Target analysis of these drugs and other Ionisation mode: ES positive ion amphetamines in biological samples is of great Capillary voltage: 1.5 kV importance for clinical and forensic toxicologists alike. Plasma and urine are currently the most MS/MS: Collision gas: Argon at common matrices investigated. However, due to the 2.5 x 10-3 mbar invasive nature of such samples (and the associated

inconvenience of sample collection) there is an Cone Collision increasing interest in the use of saliva as an alternative Compound Precursor Product Voltage Energy marker for drug abuse. (m/z) (m/z) (V) (eV) Due to the limited volume of sample (usually <100 μL) MDEA 208 163 50 12 the traditional methods for amphetamine analysis MDEA D5 213 163 50 12 (i.e. GC/MS) may not be sufficiently sensitive to Methamphetamine 150 91 50 15 allow quantitation. In addition, the high viscosity of saliva can lead to problems during solid-phase Methamphetamine D14 164 98 50 18 extraction. Therefore, we have developed an Amphetamine 136 91 60 17 alternative method. Amphetamines were isolated from Amphetamine D11 147 98 60 16 saliva using a simple methanol clean-up procedure Ephedrine 166 148 30 12 and subsequently analysed using LC/MS/MS. The developed method has a total analysis time Ephedrine D3 169 151 40 12 (including sample preparation) of less than 15 minutes MDA 180 105 30 22 and allows the simultaneous analysis of several MDA D5 185 168 50 10 amphetamines in saliva. Limits of detection of 1 ng/mL saliva (or better) were achieved. MDMA 194 163 60 12 MDMA D5 199 165 60 13

Table 1. MRM transitions and conditions for the measurement of Methods and Instrumentation several amphetamines and their internal standards. LC conditions LC System: Waters Alliance® 2690 Results and Discussion MRM transitions were determined for six commonly Column: Conventional C18 (100 x 2.1 mm, 3.5 μm) abused amphetamines and their deuterated analogues. The resultant transitions and conditions used are given Mobile phase: (A) =10 mM ammonium acetate in Table 1. Examples of MS and product ion spectra (B) = 95% acetonitrile: 5% 10 mM are given in Figure 1. ammonium acetate Standard curves were prepared by dilution of a Isocratic elution (85:15) mixture of amphetamines in mobile phase followed Flow rate: 0.3 mL/min by LC/MS/MS analysis. Figure 2 shows the MRM Injection volume: 10 μL chromatograms acquired simultaneously during a single injection (6 out of 12 shown). The typical linearity of response, in the absence of biological matrix, is demonstrated in Figure 3a.

15 ©2007 Waters Corporation. In order to extend the experiment for determination 166.2 100 of amphetamines in oral fluid, a series of calibrators (0.1-500 ng/mL) were prepared by adding amphetamines to blank saliva. Following isolation % 148.2 from the matrix using a simple methanol extraction procedure (Figure 4), samples were analysed using 167.2 149.2 108.1 129.1 143.2 176.2 179.2 LC/MS/MS. The amphetamines were quantified 0 m/z a 100 110 120 130 140 150 160 170 180 190 200 210 by reference to their respective deuterated internal 148.4 100 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 166.3 collected from current amphetamine users were analysed

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

100 amphetamines MDEA, MDMA and MDA. It should be 208.4 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 135.1 163.2 209.3 105.1 129.1 176.2 179.2 0 m/z most commonly used amphetamine (with concentrations b 90 100 110 120 130 140 150 160 170 180 190 200 210 ranging from 3.1 to > 3000 ng/mL). The results also 100 163.1 demonstrate the trend for multiple, rather than single, drug use.

% 208.3 100 MRM of 12 channels ES+ % 194>163 0

0 m/z 90 100 110 120 130 140 150 160 170 180 190 200 210 100 MRM of 12 channels ES+ % 180>105 0

100 MRM of 12 channels ES+ % 166>148 0 100 213.4 100 MRM of 12 channels ES+ % 136>91 0

100 MRM of 12 channels ES+ % % 150>91 0

100 MRM of 12 channels ES+ 163.2 214.3 % 208>163 105.2 212.4 Time 108.1 124.2 176.1 217.4 0 0 m/z 0.00 0.50 1.00 1.50 2.00 2.50 3.00 c 100 110 120 130 140 150 160 170 180 190 200 210 220 100 163.2 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). %

Compound name: MDEA 133.3 135.3 213.4 Coefficient of Determination: 0.998354 Calibration curve: 23664.8* x + 1238.54 0 m/z 100 110 120 130 140 150 160 170 180 190 200 210 220 Response type: External Std. Area Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

12437615

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

0 pg/µl 0 50 100 150 200 250 300 350 400 450 500

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

16 ©2007 Waters Corporation. Compound name: MDEA Coefficient of Determination: 0.998354 Calibration curve: 23664.8* x + 1238.54 Response type: External Std. Area Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None 350

12437615 300 250

200

Response 150 100

50 Methamphetamine

Concentration (ng/mL) Amphetamine 0 pg/µl 0 Ephedrine 0 50 100 150 200 250 300 350 400 450 500 1 2 MDA 3 4 MDEA 5 6 7 8 MDMA 9 10 Figure 3b. Typical linearity of response for saliva containing Individual # MDEA. Figure 6. Summary of results obtained from LC/MRM analysis of 10 saliva samples collected from current amphetamine users. N.B. MDMA concentrations ranged from 3 to over 3000 ng/mL.

50 μl saliva

+ 200 μL methanol (containing internal standards) In a separate (controlled) study, blood and saliva were collected from experienced MDMA-users (n=12) at various times following oral administration of Centrifuge 13,000 rpm (to collect supernatant) 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 HPLC/MRM analysis corresponding saliva concentrations were usually higher and ranged from 47 to > 6000 ng/mL. In both matrices peak MDMA levels were generally observed Total analysis time: 15 mins between 2 and 4 hours after administration. Figure 7 Figure 4. Schematic overview of the developed LC/MRM technique. shows the mean MDMA levels in blood and oral fluid and also demonstates the clear relationship between the two matrices.

93918 Saliva spiked with amphetamines were firstly 100 MRM of 12 channels ES+ % 194>163 extracted using methanol. Following centrifugation, 0 12912 supernatants were analysed using LC/MRM analysis. 100 MRM of 12 channels ES+ % 180>105 0 Amphetamines were quantified by reference to their

100 MRM of 12 channels ES+ internal standards. % 166>148 0

100 MRM of 12 channels ES+ % 136>91 0

100 MRM of 12 channels ES+ % 150>91 0

100 8421488 MRM of 12 channels ES+ % 208>163 0 Time 0.00 0.50 1.00 1.50 2.00 2.50 3.00

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

17 ©2007 Waters Corporation. Conclusions 200 1400 The use of oral fluid as a non-invasive alternative 180 1200 160 to blood or urine as a marker for drug use, is an 140 1000 attractive possibility. Collection of this biological 120 800 100 sample requires no special equipment or facilities and 600 (ng/mL)

(ng/mL) 80 can be supervised, thus removing the opportunity for 60 400 40 sample adulteration. 200 Mean MDMA in plasma Mean MDMA in plasma 20 0 0 To this end we have developed a simple, rapid 0 60120 180 240 300 method that allows the simultaneous quantitation Time after admininistration (minutes) of several amphetamines in saliva during a single chromatographic run. The procedure involves the Figure 7. Mean MDMA levels (n=12) in plasma and oral fluid extraction of amphetamines from saliva followed by following a single administration of 75 mg MDMA. 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.

1Michelle Wood 1, Kevin Rush 2, Michael Morris1 and Allan Traynor 2 Waters Corporation, Manchester, UK 2Medscreen Ltd., London, UK

Introduction Methodology Heroin is a highly addictive drug. It is processed Sample preparation from morphine, a naturally occurring substance Urine samples were prepared for LC/MS/MS analysis extracted from the seedpod of the Asian opium by means of a simple, generic solid-phase extraction poppy (Figure 1). Abuse of heroin is associated (SPE) procedure. A Waters Oasis® HLB Extraction with serious health conditions, including fatal Cartridge (1 cc/30 mg) was firstly conditioned with overdose, collapsed veins and an increased risk methanol (1 mL) followed by water (1 mL). Urine of infectious diseases such as hepatitis, HIV/AIDS samples (spiked with deuterated internal standards) for and tuberculosis. Once inside the body it is rapidly SPE were diluted into water (300 μL urine into 700 μL metabolised to morphine (Figure 2), which is then water before applying to the pre-conditioned cartridge). excreted in the urine. The cartridge was washed with 5% methanol before The presence of morphine in urine cannot alone elution of the sample using 1 mL 100% methanol. Ten be used as a marker for illicit heroin abuse since microlitres (10 μL) of the eluant were analysed using LC morphine and codeine (which is also metabolised in conjunction with multiple reaction monitoring (MRM). to morphine) can be found in prescriptive medicines and foods. For example, such medicines are valuable treatments for pain, coughs and diarrhea. Ingestion of LC/MS/MS pastries containing poppy seeds has also been shown A Quattro micro™ triple quadrupole mass spectrometer to lead to the presence of morphine and codeine fitted with ZSpray™ ion interface was used for all in the urine (Hayes et al., 1987). However, the analyses. Ionization was achieved using electrospray intermediate metabolite of heroin, in the positive ionization mode (ES+). Details of the 6-monoacetylmorphine (6-MAM) can be used as a MRM conditions are given in Table 1. specific marker for heroin as it does not result from the metabolism of either morphine or codeine. In addition, LC analyses were performed using a Waters LC acetylcodeine is a known impurity of illicit heroin 2790 separations module. Chromatography was ® synthesis and may be used to distinguish between the achieved using a Waters Nova-Pak CN HP column pharmacologically pure heroin that is used in heroin (3.9 x 75 mm) eluted isocratically with 2 mM maintenance programs and illicit ‘street’ heroin. ammonium acetate:methanol (50:50) containing 0.5% formic acid at a flow rate of 0.3 mL/min. The column We have developed an LC/MS/MS method that temperature was maintained at 30 ˚C. All aspects of allows the simultaneous quantification of several system operation and data acquisition were controlled opiates in urine. The method can also be used to using MassLynx™ software with automated data establish whether morphine present in the urine has processing using the QuanLynx™ program. originated from illicit heroin use.

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

Table 1: MRM transitions and conditions for the measurement Figure 1: The Asian of several opiates. The conditions for deuterated morphine and opium poppy, 6-MAM (d3 and d6 respectively) were also included for the Papaver somniferum. purpose of internal standardisation.

H H H

H3C CH3 CH 3 CH3 O O O O O O HO O H3C O O O O O CH 3 CH3

N N

H H

O OH O HO H3C O OH

Morphine Codeine

Results A series of calibrators (0.5-250 ng/mL) were prepared 342>165 by adding opiate standards to blank urine. Urine 328>165 samples (either calibrators or unknown samples) were then extracted using the SPE method described above 302>199 prior to LC/MRM analysis. 300>165 Following LC/MRM analysis, the areas under the specific MRM chromatograms were integrated. 286>165 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 Figure 3. MRM chromatograms for: (top to bottom) acetylcodeine, by reference to the integrated area of the deuterated 6-MAM, DHC, codeine and morphine. Responses were obtained with a 10 μL injection of the 5 ng/mL urine calibrator. internal standards. Responses were linear for all compounds (Figure 4 shows a typical standard curve for 6-MAM in urine). Compound name: 6-MAM Coefficient of Determination: 0.999261 Calibration curve: 0.167559* x + 0.0400415 Response type: Internal Std. (Ref 4), Area* (IS Conc./IS Area) Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None 41.9 Summary We describe a sensitive method for the simultaneous analysis of several opiates in urine. The method Response involves a simple SPE purification step prior to analysis using LC/MRM and may be used to identify cases of heroin abuse. 0 ng/mL 0 20 40 60 80 100 120 140 160 180 200 220 240

Figure 4. Standard curve for 6-MAM. Responses were calculated References in reference to the integrated area of the deuterated internal Hayes LW, Krasselt WG and Mueggler PA. Concentrations standards. of morphine and codeine in serum and urine after ingestion of poppy seeds. Clinical. Chemistry. 1987; 33: 806-808.

1Michelle Wood and Michael Morris. Waters Corporation, Manchester, UK.

Introduction Cartridge (1 cc/30 mg) was firstly conditioned with Morphine is a potent analgesic isolated from the 1 mL volumes of each of the following: methanol, opium poppy papaver somniferum and traditionally water and ammonium carbonate (10 mM, pH 8.8). used for the treatment of moderate to severe pain. Samples (100 μL, spiked with deuterated internal stan- Analgesia results from the action of morphine at dards) were made up to a final volume of 1 mL with the opioid receptors of the spinal cord and brain ammonium carbonate before applying to the (Figure 1), where it attenuates both the speed of the pre-conditioned cartridge. The cartridge was then impulse and the perception of pain. washed with 1 mL ammonium carbonate before elution of the sample using 100% methanol (0.5 mL). Eluents In human subjects, morphine is extensively metabolised were dried using a Savant Speedvac Plus evapora- (primarily by conjugation with glucuronic acid) to tor and then redissolved in 100 μL of mobile phase. form morphine-3-glucuronide (M3G) and morphine-6- Reconstituted samples were briefly vortex mixed before glucuronide (M6G). Whilst, the principal metabolite the analysis of 10 μL using LC in conjunction with mul- i.e. M3G, has little or no analgesic effect, M6G has tiple reaction monitoring (MRM). been shown to be highly effective and is believed likely to contribute significantly to the overall effectiveness of morphine1. Hence, quantification of LC/MS/MS both the parent drug and metabolites is desirable for ™ pharmacokinetic studies. A Waters Quattro micro triple quadrupole mass spectrometer fitted with ZSpray™ ion interface was Previously we have described a LC/MS/MS method used for all analyses. Ionisation was achieved using that allows the quantification of morphine and several electrospray in the positive ionisation mode (ES+). 2 other opiates in urine . Here we present a simple Details of the MRM conditions are given in Table 1. method that enables the quantification of morphine

in plasma, whole blood and urine. Furthermore this Precursor Product Cone Collision procedure allows differentiation between two isobaric Compound ion ion Voltage energy (m/z) (m/z) (V) (eV) glucuronide metabolites. 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 Figure 1. Image of Morphine–M6G–glucuronide 462 286 45 28 guinea-pig brain. The red areas Table 1: MRM transitions and conditions for the measurement represent the highest of morphine and it’s metabolites. The deuterated analogues of density of opioid morphine and morphine-3-glucuronide were also included for the receptors; yellow areas purpose of internal standardisation. represent moderate density; whilst blue, LC analyses were performed using a Waters purple and white 2795 separations module. Chromatography was represent low density. achieved using a C18 column (3.9 x 150 mm) eluted isocratically with 0.1% formic acid:acetonitrile (97:3) Methodology at a flow rate of 0.3 mL/min. Column temperature was maintained at 30 ˚C. All aspects of system operation Sample preparation and data acquisition were controlled using MassLynx™ Biological samples were prepared for LC/MS/MS 4.0 software with automated data processing using ™ analysis by means of a simple, solid-phase extraction the QuanLynx program. (SPE) procedure. A Waters Oasis® HLB extraction

100 A series of calibrators (0.5-500 μg/L) were prepared 462>286 in duplicate by adding standards to blank plasma, M6G whole blood or urine. Samples were then extracted % using the SPE method described above prior to 0 LC/MRM analysis. MOR 100 Following LC/MRM analysis, the areas under the 286>165 specific MRM chromatograms were integrated. % Figure 2 shows the extracted MRM chromatogram 0 Time of morphine, M3G and M6G obtained with a 10 μL 1.00 2.00 3.00 4.00 5.00 6.00 7.00 injection of the 5 μg/L plasma calibrator. Opiates were quantified by reference to the integrated area Figure 2. MRM chromatogram for morphine (MOR), M3G and M6G. The above responses were obtained with a 10 μL injection of the deuterated internal standards. Responses were of the 5 μg/L plasma calibrator. Due to the isobaric nature of linear (r = >0.999) over the range investigated for all M3G and M6G chromatographic resolution is required to enable 3 compounds and in each matrix (Figure 3 shows a identification. typical standard curve for M3G in urine).

Compound name: Morphine-3-glucuronide Correlation coefficient: r= 0.999883, rˆ 2 = 0.999766 Calibration curve: 0.09404 * x + 0.100968 Summary Response type: Intermal Std ( Ref 4 ), Area * ( IS Conc. / IS Area ) Curve type: Linear, Origin: Exclude, Weighting: 1/x Axis trans: None We present a sensitive method for the quantification of 47.1 morphine and its glucuronide metabolites. The method involves a simple SPE purification prior to analysis 1.06 using LC/MRM and is suitable for plasma, whole blood or urine samples. Response Response

0.00 µg/L 0.0 2.0 4.0 6.0 8.0 10.0 References 0.0 µg/L 0 100 200 300 400 500 1. The Analgesic Effect of Morphine-6-Glucuronide. R Osborne, P Thomson, S Joel, D Trew, N Patel and M Slevin. Figure 3. Standard curve for M3G in urine. Responses Br J Clin. Pharmacol. 1992. 34 (2) 130-8. (duplicates) were calculated in reference to the integrated area of 2. Opiates: Use or Abuse? Quantification of Opiates in the deuterated internal standards. The inserted figure shows the Human Urine. (Waters Application Brief WAB45). response for the range 0-10 μg/L. 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.

Gert De Boeck1, Nele Samyn1, Karen Pien2, Patrick Grootaert 3 and Michelle Wood 4, 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 Time (min) A (%) B (%) Curve number Benzodiazepines are the most widely prescribed 0 100 0 1 psychoactive drugs in the world for the symptomatic 0.5 75 25 1 treatment of anxiety and sleep disorders. However, 8 40 60 7 (concave) misuse of these compounds has been reported and 11 40 60 6 (linear) they are frequently encountered in postmortem blood 12 100 0 1 analysis (suicide or accidental death). 15 100 0 1 Here we describe the development of a rapid and sensitive LC/MS/MS method for the quantification of Results and Discussion 10 benzodiazepines. Limits of detection of 0.2 μg/L or better were achieved when just 25 μL plasma Figure 1 shows the MS and MS/MS spectra for was used. alprazolam. Table 1 summarizes the MRM transitions and conditions used for this and several other In addition, we present the application of this method benzodiazepines (and their respective deuterated to the analysis of benzodiazepines in Calliphora analogues). The latter were used as internal standards vicina larvae. Insects and their larvae are commonly for quantification purposes. used in the estimation of postmortem interval. Furthermore, they may serve as a reliable alternate A series of calibrators (1, 10, 40, 100, 200, 400 source for toxicological analysis in the absence of and 800 μg/L) were prepared by adding the suitable tissues and fluids that are normally taken for benzodiazepines to drug-free plasma. Plasma this purpose. samples were isolated from the matrix using a simple acetonitrile clean-up procedure (which also incorporates the addition of the internal standards).

Experimental Conditions Figure 2 shows the MRM chromatograms of the benzodiazepines obtained with a 10 μL injection of LC/MS/MS conditions the 10 μg/L plasma calibrator. Quantification was LC System: Waters Alliance® 2690 performed by integration of the area under the specific Column: Conventional Phenyl Column MRM chromatograms. Figure 3 shows a typical (2.1 x 150 mm, 5 μm) standard curve for diazepam in plasma. 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/min Injection volume: 10 μL

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

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 25 Flunitrazepam 313.9 267.9 80 25 Flunitrazepam-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.

308.9 280.9 100 100 AB

311.0

% 180.8 % 309.0

280.9 166.9 274.0

182.8 312.0 282.9 240.8 212.9 164.9 205.9 250.9 140.8 152.9 273.9 226.8 254.8 301.1 138.0 226.9 0 m/z 0 m/z 100 120 140 160 180 200 220 240 260 280 300 320 100 120 140 160 180 200 220 240 260 280 300 320

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

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%).

24 ©2007 Waters Corporation. Compound name: Diazepam Coefficient of Determination: 0.999378 100 Calibration curve: 1.22307* x + 0.093412 Response type: Internal Std (Ref 10), Area* (IS Conc./IS Area) 276>140 Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None C %

979 0

100

B % 271>140 Response

100 0 µg/L 0 100 200 300 400 500 600 700 800 A % 271>140 Figure 3.Typical response for plasma containing diazepam. 0 Time 6.00 8.00 10.00 Diazepam spiked plasma was firstly extracted using acetonitrile prior to analysis using LC/MRM. Benzodiazepines were quantified by reference to their deuterated internal standards. 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 The developed LC/MS/MS was subsequently applied C shows the MRM chromatogram for the internal standard i.e. Nordiazepam-d5. 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. Conclusion Larvae were reared on artificial foodstuff (beefheart) We have developed a simple, rapid method that spiked with a range of concentrations of nordiazepam allows the simultaneous quantification of 10 (0, 0.5, 1 and 2 μg/g). Post-feeding larvae were benzodiazepines in plasma a single chromatographic harvested (after 7 days) for analysis of drug content run. LOD’s were better than 0.2 μg/L when only by LC/MS/MS. Figure 4 outlines the initial sample 25 μL plasma was used. The method involves a simple preparation method used for these specimens. All protein precipitation step with acetonitrile followed by control larvae reared on spiked foodstuff were positive LC/MS/MS analysis. for nordiazepam and the metabolite oxazepam. All The method was subsequently applied to the analysis of control samples were negative. Figure 5 shows the Calliphora vicina larvae in a study designed to assess MRM chromatograms obtained following LC/MS/MS the feasibility of using insects as alternate specimens in analysis of a control larva and a larva positive for the absence of any suitable human tissues. nordiazepam. The method was sufficiently sensitive to The sensitivity was such that it was possible to detect measure benzodiazepines in single larvae whereas benzodiazepines in single larvae whereas previous previous analytical techniques e.g. GC/MS, RIA, TLC methods have required pools. have required pools i.e. typically 20 larvae.

After 7 days 500µL H2O 1mL ACN and I.S. Dry to 100µL mix throughly (nordiazepam d5 & oxazepam d5) vortex thoroughty

LC/MS/MS analysis (10µL aliquot)

Filter

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

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.

Introduction Sample preparation In addition to their use in the estimation of postmortem Individual larvae and pupae samples were prepared interval, insects may serve as reliable alternate source for LC/MS/MS as follows; the sample was transferred for toxicological analyses in the absence of tissues to a vial containing 500 μL water and vortex-mixed and fluids normally taken for such purpose. To date, thoroughly. One millilitre of acetonitrile (containing a variety of compounds have been measured in fly deuterated internal standards) was then added and larvae and pupae using different analytical procedures the samples mixed for a further minute. The mixture i.e. (Radio-Immunoassay (RIA), Gas Chromatography was evaporated to ~100 μL and then filtered. A 10 μL (GC) and Thin-Layer Chromatography (TLC)). In these aliquot was analysed using LC/MS/MS. studies a minimum of 1g (approximately 20 larvae) was needed to detect the toxic compound. Sample Target Concentration In this study we used LC/MS/MS (Liquid (μg/g)* Chromatography-Tandem Mass Spectrometry) Control 0 to detect the benzodiazepine Nordiazepam and its metabolite Oxazepam, in single larvae Nor I (human therapeutic dose) 0.5 of the Calliphora vicina. Benzodiazepines are Nor II (human lethal dose) 1 prescribed for the symptomatic treatment of Nor III (2x human lethal dose) 2 anxiety and sleep disorders. They are frequently Table 1: Target concentration of Nordiazepam in larval food. encountered in postmortem blood analysis *Concentrations expressed in μg/g food. (suicide or accidental deaths). In addition, we compared the development of postfeeding larvae and pupae fed on different concentrations of Nordiazepam. LC/MS/MS

LC Conditions ® Experimental Conditions LC System: Waters Alliance 2690 Column: Conventional Phenyl Column Study design (2.5 x 150 mm, 5 μm) Flies and larvae were from a stock colony of Mobile Phase: A=10:10:80 Calliphora vicina maintained in an environmental acetonitrile:methanol: chamber at 18-24 ˚C and 60-70 % humidity with 20 mM ammonium acetate cyclical artificial lighting simulating 16 h daylight B=95:5 acetonitrile: 20 mM and 8 h darkness. ammonium acetate Larvae were reared on artificial food (beef heart) Time (min) A (%) B (%) Curve number spiked with a range of concentrations of Nordazepam 0 100 0 1 (Table 1). Post-feeding larvae were harvested from day 0.5 75 25 1 4 till day 8. Thirty larvae were boiled and conserved 8 40 60 7 (concave) (in a mixture of ethanol and acetic acid) prior to 11 40 60 6 (linear) measurement of length. Another 30 were used for 12 100 0 1 toxicological analysis. These were weighed and then 15 100 0 1 killed, by freezing to -20 ˚C. The larvae were stored at -20 ˚C until analysis. Flow Rate: 0.25 mL/min Injection Volume: 10 μL

26 ©2007 Waters Corporation. MS Conditions Compound Precursor Ion Product Ion Cone Voltage Collision Energy (m/z) (m/z) (V) (eV) Mass Spectrometer: Quattro Ultima™ Nordiazepam 278 91 30 30 triple quadrupole Nordiazepam-d5 325 109 38 25 Ionisation Mode: ES+ Oxazepam 315 86 28 18 Capillary Voltage: 3kV Oxazepam-d5 327 270 35 25

Table 2: MRM transitions and conditions for them LC/MS/MS analysis Results of Nordiazepam and Oxazepam. Deuterated analogues were also included as internal standards. All larvae, pupae and food spiked with Nordiazepam were positive for the drug, whereas all control Peak concentrations of Nordiazepam were measured samples were negative. on day 4 for NOR I, II and III, followed by a Figures 1 and 2 show the larvae Nordiazepam and precipitous fall of larval Nordiazepam concentrations. Oxazepam concentrations from days 4 - 8. Figure 3 From day 7, Nordiazepam was not detectable in a shows the MRM chromatograms obtained following single larva. the LC/MS/MS analysis of a control larva and a Peak concentrations of Oxazepam were measured Nordiazepam positive larva. on day 5 for NOR II and III and at day 6 for NOR I. Low concentrations of Oxazepam were still measured at day 8. In this study, two patterns of development were observed; the post-feeding larvae fed on Control, NOR I and NOR III food regime developed at approximately the same rate and each demonstrated wandering-phase behaviour at day 6, pupation at day 8 and emerging of adult flies at day 18.

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

271>140 B

271>140

A Figure 2: Concentration of Oxazepam in larva reared (for 4-8 days) on foodstuff spiked with Nordiazepam. Mean concentrations are plotted (± 1SD). Figure 3: MRM chromatograms obtained with the analysis of larvae that were reared on artificial foodstuff spiked with Nordiazepam at 0 and 1 μg/g (A and B respectively). Figure C shows the MRM chromatogram for the internal standard i.e. Nordiazepam d5.

27 ©2007 Waters Corporation. In contrast, the development of larvae fed with the Discussion and Conclusions NOR II regime was 1 day later in all stages. We have developed a method that allows the Post-feeding larval length is shown in Table 3; detection of Nordiazepam and its metabolite no significant differences were observed. Oxazepam in single larvae. Larval drug concentrations showed a stepwise increase with increasing drug concentrations in the foodstuff. Day 4 Day 5 Day 6 Day 7 Day 8 It was clear that Nordiazepam was metabolized Control 16.7 17.8 15.2 15.8 15.9 to Oxazepam, which was still detectable at day 8. Nor I 16.9 17.7 16.3 15.6 15.2 Nordiazepam was detectable until day 6. Nor II 17.2 17.3 16.7 15.5 15.8 Control maggots were negative. Nor III 16.9 17.1 16.9 15.8 15.1 No differences were seen on the post-feeding larval

Table 3: Mean post-feeding larval length (mm). length, but differences in post- feeding larval weight and development were seen in the NOR II larvae.

Day 4 Day 5 Day 6 Day 7 Day 8 The reason of this disturbance is not yet understood, but is presumably because larval physiology is Control 69.5 84.5 78.2 86.2 71.4 disturbed to a greater extent by this drug level. This Nor I 73.4 89 78.5 87.5 80.5 study indicates that an estimation of the postmortem Nor II 110.8 105.8 101.7 96 92.9 interval based on the length of the post-feeding Nor III 82.5 83.5 83.5 84 83.1 larvae of Calliphora vicina, which have fed on

Table 4: Mean post-feeding larval weight (mg). 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 Post-feeding larval weight is shown in Table 4: puparial stages. 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.

1Marleen Laloup1, Maria del Mar Ramirez Fernandez1, Michelle Wood 2, Gert De Boeck1, Cecile Henquet 3, 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

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 Instrumentation Calibrators and quality control (QC) samples Oral fluid samples used for the preparation of blanks, calibrators and QC samples were obtained from healthy volunteers and collected with the Intercept® collection device (OraSure Technologies, Bethlehem, Introduction PA) according to the manufacturer’s instructions. Cannabis is the collective term for the psychoactive Briefly, after gently wiping the collector pad between substances of the Cannabis sativa plant (Figure 1) gum and cheek for approximately 2 minutes the device and one of the most frequently used illicit drugs in is placed in the supplied vial and sealed. Following the western world. Δ9-Tetrahydrocannabinol (THC), centrifugation, the recovered fluid was spiked with the main psychoactive constituent of cannabis, is THC to yield a series of calibrators ranging from deposited in the oral cavity during cannabis smoking. 0.1 to 100 ng/mL. QC samples were also prepared Over the last few years there has been an increasing by spiking control oral fluid with THC. interest in the use of oral fluid to document drug Authentic samples use. The advantage of this specimen over the more traditional matrices e.g. urine and blood, is that Oral fluid samples were collected by the police at collection is almost non-invasive, relatively easy roadblocks, the purpose of which, was to intercept to perform, and may be achieved under close drivers who were driving under the influence of drugs. supervision to prevent adulteration or substitution of The samples were collected at the roadside using the the sample. same procedure as described for the blank samples. LC/MS/MS is a technique that lends itself well to the An additional series of authentic samples were high-throughput determination of multiple analytes in obtained from volunteers with a history of cannabis oral fluid samples due to its high specificity, sensitivity use. Once a week, and over 2 consecutive weeks, and short analysis times1,2. subjects received either a placebo cigarette (where the THC had been extracted) or a marijuana cigarette The Intercept® is a FDA cleared oral fluid collection which contained 300 μg cannabis per kg). Samples device that is used on a large scale in the U.S. for were collected 0.5 hour prior to drug administration workplace testing3. It is also the device of choice to and at various times following drug administration collect the samples in a current joint roadside study (0.25, 0.5, 1, 1.25, 1.5 hour). between the European Union and the U.S. to detect driving under the influence of drugs4. The study protocol was approved by the ethics committee of the University Hospital of Maastricht in The Intercept® collection system utilises a variety of the Netherlands. ingredients to ensure stability and to maintain the integrity of the sample. However, these ingredients Internal standard solution can also cause interferences e.g. ion suppression An internal standard (IS) working solution of THC-d3 during LC/MS/MS analysis in the absence of a at a concentration of 10 ng/mL was prepared in 5 suitable clean-up method . methanol.

29 ©2007 Waters Corporation. Sample preparation Results and Discussion Extraction was performed using either 100 or 500 μL Figure 2 shows the MRM chromatograms obtained of the collected specimen. When using 500 μL, 50 μL following the analysis of a sample enriched with THC of the IS working solution and 4 mL of hexane were and the internal standard i.e. THC-d3. added; when only 100 μL of oral fluid was used, an The usefulness of the liquid/liquid extraction step additional 400 μL of deionised water was added. was assessed by a comparison of the effect of the After mechanical shaking (30 min) and centrifugation matrix both before and after sample clean-up. Matrix (10 min at 3000 g), the organic phase was collected effects were monitored throughout the whole of the and then evaporated to dryness at 40 ˚C under chromatographic run by performing post-column infusion nitrogen. The extract was reconstituted in 100 μL of experiments6. The effect on THC response obtained mobile phase. following the injection of a sample prior to extraction LC conditions and the same sample after extraction of 100 μL and 500 μL of oral fluid are given in Figure 3. The results LC system: Waters® Alliance® System clearly demonstrate the usefulness of the liquid-liquid Column: Waters XTerra® MS C column 18 extraction step prior to LC/MS/MS analysis. (2.1 x 150 mm, 3.5 μm) at 40 ˚C Mobile phases: (A): 1 mM ammonium formate (B): methanol Isocratic elution 10:90 (A:B) Flow rate: 0.2 mL/min Injection volume: 20 μL

Mass spectrometry conditions Mass spectrometer: Waters Quattro Premier™ tandem mass spectrometer Ionisation mode: ES+ Capillary voltage: 2 kV Source temperature: 120 ˚C Figure 2. MRM chromatograms obtained with a single injection of a 100 μL extracted oral fluid sample enriched with 5 ng/mL THC Desolvation gas: Nitrogen at 700 L/Hr, 280 ˚C and 5 ng/mL THC-d3. The figure shows the response for THC- MS/MS: THC m/z 315.2>193.1 d3 (top trace) and for the two transitions of THC (quantifier and qualifier middle and bottom trace respectively). Peak intensity is (quantification ion) m/z shown in the top right-hand corner of each chromatogram. 315.2>259.3 (qualifier ion) THC-d3 m/z 318.2>196.1 Cannabinol m/z 311.2>223.1 Cannabidiol m/z 315.2>193.1 Collision gas: Argon at 3.5 x 10-3 mbar

Figure 3. Evaluation of the matrix effect on THC response of an injection of a mobile phase control (A), a blank sample prior extraction (B), the reconstituted extract after extraction of 100 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.

30 ©2007 Waters Corporation. To assess method linearity, limit of quantitation (LOQ), over a period of 15 hours. No instability was noted precision, accuracy and analytical recovery a series over the course of this experiment. of oral fluid calibrators were prepared and a 100 or Cannabidiol and cannabinol are two components that 500 μL aliquot extracted with hexane prior to analysis are also naturally-occuring in the Cannabis sativa plant. using LC/MS/MS. Quantification was achieved Since the m/z for the precursor mass of cannabinol by integration of the area under the specific MRM is different to that of THC, it does not interfere in chromatogram. For THC, the response was calculated its quantitation. On the other hand, the protonated in reference to the integrated area of THC-d3. molecular species of cannabidiol i.e. m/z 315.2 is Linear responses (r = >0.999, 1/x weighting) were the same as that of THC. Furthermore it shows the obtained up to 100 ng/mL when 100 μL of sample same product ions after collision induced dissociation. was extracted and up to 10 ng/mL when 500 μL Thus chromatographic separation is essential to sample was extracted. Linearity and sensitivity data are distiguish between these 2 isobaric compounds. summarised in Table 1. The limit of quantification was Analysis of standards showed cannabidiol to be defined as the concentration of the lowest calibrator chromatographically resolved from THC (Figure 4). which was calculated to be within ± 20% of the The utility of the LC/MS/MS method was demonstrated nominal value and with a % CV less than 20%. This by the analysis of 102 authentic samples collected criteria was met by the lowest calibrator i.e. 0.5 and from volunteers who smoked a placebo or marijuana 0.1 ng/mL when either 100 or 500 μL respectively of cigarette. Figure 5 shows the values for THC in the collected sample was extracted. oral fluid collected after smoking the marijuana Intra-assay and interassay variation (as % CV) were cigarette; mean values are plotted as a function of all found to be highly satisfactory at <6% (Table 2). time. All specimens collected prior to smoking were Analytical recovery was estimated by comparing the negative, with the exception of 3 samples where responses of a 5 ng/mL calibrator (using 100 μL of concentrations were very low (maximum 2.2 ng/mL). oral fluid) when the non-deuterated compounds were Peak concentrations occurred 0.5 hour after smoking. added before the extraction step (n= 3) with those Thereafter concentrations decreased steadily. There obtained when the non-deuterated analytes were was considerable inter-individual variation in the added after sample preparation (n= 3). The recovery observed concentrations; this has also been reported was found to be satisfactory at 85.6 ±0.5% by other authors7 and may also be as a result of the The stability of THC in oral fluid collected by the lack of exact volume measurement in the device. Intercept® device was assessed by spiking oral fluid Forty eight samples were also collected from drivers with THC at 3 different concentrations (1, 10 and intercepted at Belgian roadblocks. Table 3 summarises 100 ng/mL) and then monitoring the stability at 4 ˚C the quantitative results for all positive samples and and at room temperature over a period of 48 hours. Figure 6 shows a MRM chromatogram for one such No statistical significant differences could be observed marijuana user; the presence of cannabidiol was also for the three different concentrations in both conditions. noted (at 3.28 min) in this specimen. The stability of the samples post extraction was assessed by repeated injections of extracted samples

Linearity Data 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

Table 1. Linearity and sensitivity data for THC in oral fluid.Samples were prepared by the liquid-liquid extraction method as described in the text. *Reported values are the mean of five determinations over 5 consecutive days.

31 ©2007 Waters Corporation. 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

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

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 Figure 4. LC/MS/MS analysis of THC-d3 (top trace), THC, 19 107.4 43 4.2 cannabidiol (middle trace) and cannabinol (bottom trace). Peak 20 92.1 44 4.2 intensity is shown in the top right-hand corner of each trace. 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.

THC (ng/mL)

Figure 5. Box- and whisker plots of THC levels in oral fluid samples following smoking of a single marijuana cigarette. Oral fluid samples were taken prior to administration i.e. at –0.5 h, 0.25 h, 0.5 h, 1 h, 1.25 h and 1.5 h after smoking. Concentrations plotted on the Y-axis are expressed as ng/mL. Figure 6. Typical MRM chromatograms obtained following the The central box represents the values from the lower to upper analysis of an authentic oral fluid specimen obtained from a quartile (25 to 75 percentile). The middle line represents the driver in a roadside setting. The calculated concentrations was median. The horizontal line extends from the minimum to the 5.7 ng/mL. The figure shows the response for THC-d3 (top trace) maximum value, excluding “outside” (not present) and “far out” and for the two transitions of THC (quantifier and qualifier middle values (cross marker) which are displayed as separate points. and bottom trace respectively). Peak intensity is shown in the top right-hand corner of each trace.

32 ©2007 Waters Corporation. Conclusions References To the very best of our knowledge, the method 1. K.A. Mortier, K.E. Maudens, W.E. Lambert, K.M. Clauwaert, J.F. Van Boxlaer, D.L. Deforce, C.H. Van Peteghem and presented here is the first demonstration of the use A.P. De Leenheer, J. Chromatogr. B 779 (2002) 321–330. of LC/MS/MS for the analysis of THC in oral fluid 2. R. Dams, C.M. Murphy, R.E. Choo, W.E. Lambert, ® samples collected with the Intercept device. The A.P. De Leenheer and M.A. Huestis, Anal. Chem. 75 method is simple and comprises simple liquid/liquid (2003) 798–804. extraction followed by LC/MS/MS. The method 3. E.J. Cone, L. Presley, M. Lehrer, W. Seiter, M. Smith, demonstrates high recovery, excellent precision and K.W. Kardos, D. Fritch, S. Salamone, S. Niedbala, accuracy when using either 100 or 500 μL sample. J. Anal. Toxicol. 26 (2002) 541. 4. EU Project ROSITA Roadside Testing Assessment. The LOQ is sufficiently low to meet the requirements http://www.rosita.org. of SAMHSA (2 ng/mL) for oral fluid testing. 5. M. Wood, M. Laloup, M. Ramirez Fernandez, K.M. Jenkins, Pharmacokinetic studies may require lower LOQ’s; M.S. Young, J.G. Ramaekers, G. De Boeck, N. Samyn, these requirements can be met by using larger Forensic Sci. Int, in press. volumes of oral fluid. 6. R. Bonfiglio, R.C. King, T.V. Olah, K. Merkle, Rapid Commun. The method was successfully applied to the analysis Mass Spectrom. 13 (1999) 1175. of samples collected in a controlled cannabis smoking 7. R.S Niedbala, K.W. Kardos, D.F. Fritch, S. Kardos, study and to samples collected at the roadside by T. Fries, J. Waga, J. Robb, E.J. Cone, J. Anal. Toxicol. 25 (2001) 289. Belgian police.

Michelle Wood 1, 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.

concentrations; 0, 1, 2, 5, 10, 20, 50 and 80 mg/L. Easy Lay Low and high QC samples were prepared by spiking Liquid Ecstasy control urine with the drugs to yield concentrations of Fantasy 4 and 40 mg/L, respectively. Blue nitro Authentic Samples One hundred and eighty two authentic human urine samples were collected from club-goers attending a Introduction post dance-club ‘chill-out’ venue and were the result Gamma-hydroxybutyrate (GHB) is a metabolite of 2 separate raids by the Belgian Police Department. of gamma-aminobutyric acid (GABA) and plays The samples were analysed for GHB and the the role of a central neurotransmitter and precursors using the newly-developed LC/MS/MS neuromodulator. Since GHB is a normal component of procedure. For comparative purposes, the samples mammalian metabolism, it is present in all tissues of the were also analysed for GHB using a routinely used body. Typical urinary GHB concentrations are GC/MS procedure. < 10 mg/L1,2. In some countries GHB is used clinically as an intravenous anaesthetic and as a treatment for Sample Preparation narcolepsy, alcoholism and opiate withdrawal. Over Urine samples were diluted (1:20) with an internal the last few years, GHB has been gaining popularity standard solution (GHB-d6 and GBL-d6, at a amongst club-goers as the recreational drug (Figure 1) concentration of 2 mg/L in deionised water). where it is taken for its ability to produce feelings of euphoria and to enhance sexuality3-5. As a result LC Conditions of its potent prosexual effects, GHB has also been LC system: Waters Alliance® 2795 ® increasingly implicated in drug-facilitated sexual Column: Waters Atlantis dC18 column assault 6,7. Ingestion of the chemical precursors of GHB (3 x 100 mm, 5 μm) at 35 ˚C i.e. gamma-butyrolactone (GBL) and 1,4-butanediol Mobile phases: (A): 0.1% aqueous formic acid (1,4-BD) also results in similar physiological effects (B): methanol since they are rapidly converted to GHB in the body8. Isocratic elution: 90:10 (A:B) Raised awareness of the effects of these drugs and Flow rate: 200 μL/min. their potential for misuse, in addition to their ease of availability, has resulted in a dramatic increase in Inj. volume: 20 μL the demand for their analytical determination in both Mass Spectrometry Conditions biological specimens and putative drug preparations. Mass spectrometer: Quattro micro® mass spectrometer The purpose of this study was to develop and validate a sensitive LC/MS/MS procedure that would enable Ionisation mode: ES+ the simultaneous quantification of GHB, GBL and 1,4- Capillary voltage: 3.5 kV BD in urine. Source Temperature: 120 ˚C Desolvation gas: Nitrogen at 700 L/Hr, 350 ˚C MS/MS: Collision gas (argon) at Methods and Instrumentation 5 x 10-3 mbar Samples Calibrators and quality control (QC) samples Control urine was spiked with GHB, GBL and 1,4-BD to yield a series of calibrators at the following

standards i.e. GHB-d6 and GBL-d6 (Table 1). Figure 2 4.2 x 105 4.2 x 105 shows some examples of product ion spectra. 1,4,-BD

Compound Precursor Product Cone Collision 4.3 x 104 GBL 4.3 x 104 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 Figure 3: MRM chromatograms obtained with a single injection GBL-d6 93 49 25 15 of a control urine sample (left-hand column) prepared by the 1,4-BD 91 73 12 5 dilution method and the same sample enriched with 10 mg/L Table 1: MRM transitions and conditions for the measurement of of GHB, GBL and 1,4-BD (right-hand column). Peak intensity is GHB, GBL, 1,4-BD and their deuterated internal standards. shown in the top right-hand corner of each trace.

A series of urine calibrators was prepared. Following A 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 B 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%

C (Table 2).

Low Control High Control Compound (4 mg/L) (40 mg/L 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) Figure 2: Product ion spectra for GHB (A), GBL (B) and 1,4- GHB 3.9 98 3.2 42.7 107 3.5 GBL 3.7 93 3.2 36.1 90 2.9 BD (C). Pure standards (5 mg/L) were infused into the mass 1,4-BD 4.0 100 2.2 40.0 100 3.1 spectrometer and the cone voltage (CV) optimised for the Intrerassay (n=5) precursor ion*. CID was then performed and product ion GHB 4.1 103 5.3 40.0 100 3.4 spectra acquired under optimum conditions for the most GBL 4.0 100 6.6 39.8 100 6.3 abundant product ion. 1,4-BD 3.9 98 3.8 40.5 101 4.7

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

35 ©2007 Waters Corporation. Only two, of these seven samples, were above the recommended interpretive cut-off concentration of A 10 mg/L and were 956 mg/L and 1411 mg/L, respectively. These two samples were also positive for GBL. None of the authentic urine samples contained 1,4-BD.

B Conclusions To the very best of our knowledge, the method presented here is the first demonstration of the use of LC/MS/MS for the simultaneous analysis of GHB C and its precursors in urine samples. The method is simple and rapid (total analysis time of <12 mins). The method offers sufficient sensitivity to enable the measurement of endogenous levels of GHB and to identify exogenous ingestion. Fig. 4: LC/MS analysis of the hydroxybutyric acid isomers. Ion chromatograms obtained following the analysis of gamma- The LC/MS/MS results obtained following the hydroxybutyric acid (GHB) only (A) and GHB in the presence of analysis of authentic samples, correlated with the alpha and beta-hydroxybutyric acid (traces B and C respectively). more labour-intensive, time-consuming (~1 hour) 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 GC/MS method. and the sodiated species species i.e. m/z 105 + 127. The procedure offers several advantages over other published techniques; The limit of quantification was defined as the 1. It enables the simultaneous quantification of the concentration of the lowest calibrator which was GHB and the precursors in a single analysis; calculated to be with in ± 20% of the nominal value this can facilitate the identification of the chemical and with a % CV less than 20%. For all of the basis of any seized putative drug preparations or analytes of interest, this criteria was met by the 1 mg/L if present in the biological specimen, can provide calibrator and was sufficient to determine endogenous information of the chemical nature of the ingested levels of GHB in the urine. drug. To investigate any potential interference in GHB 2. It involves fewer manipulations and is less time- quantification by its naturally occuring isomers i.e. consuming. alpha and beta-hydroxybutyric acid, standards Although the data presented here indicate that the were analysed using the developed LC/MS/MS actual prevalence of GHB-positives might be quite low, method. Both compounds were shown to be the hype and publicity surrounding these drugs has led chromatographically resolved from GHB and thus to a dramatic increase in the number of requests for would not interfere in the quantification of the latter their analysis in biological samples (and particularly (Figure 4). in urine). The simplicity and speed of the described The utility of the LC/MS/MS method was demonstrated LC/MS/MS technique, should prove a useful means to by the analysis of one hundred and eighty-two meet this current increased demand on laboratories. 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.

36 ©2007 Waters Corporation. References 1. Elliott SP. Gamma hydroxybutyric acid ( GHB) concentrations in humans and factors affecting endogenous production. Forensic Sci. Int 2003;133:9-16. 2. LeBeau MA, Christenson RH, Levine B, Darwin WD and Huestis MA. Intra- and inter individual variations in urinary concentrations of endogenous gamma-hydroxybutyrate. J. Anal. Toxicol 2002;26:340-346. 3. Laborit H. Correlations between protein and serotonin synthesis during various activities of the central nervous system (slow and desynchronized sleep, learning and memory, sexual activity, morphine tolerance, aggressiveness and pharmacological action of sodium gamma-hydroxybutyrate). Research Communications in Chemical Pathology and Pharmacology 1972;3:51-81. 4. Ropero-Miller JD and Goldberger BA. Recreational drug current trends in the 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.

Justus Beike1, Lara Frommherz1, Michelle Wood2, Bernd Brinkmann1 and Helga Köhler1 1 Institute of Legal Medicine, University Hospital Münster, Röntgenstrasse, Münster, Germany 2 Clinical Applications Group, Waters Corporation, Simonsway, Manchester M22 5PP, UK.

Introduction the determination of aconitine in various body fluids8. Plants of the genus Aconitum L (family of The method was fully validated for the determination Ranunculaceae) are known to be among the most of aconitine from whole blood samples and applied toxic plants of the Northern Hemisphere and are in two cases of fatal poisoning. 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 Figure 1: Aconitum napellus (monkshood) (A) are aconitine, mesaconitine, hypaconitine and and Aconitum vulparia (wolfsbane) (B). jesaconitine. The use of the alkaloids as a homicidal agent has Methods and Instrumentation been known for more than 2000 years. Although Sample preparation intoxications by aconitine are rare in the Western Biological samples were prepared for LC/MS/MS Hemisphere, in traditional Chinese medicine, the by means of a solid-phase extraction (SPE) procedure. use of aconite-based preparations is common and Blood and tissue samples (0.5 g each) were mixed poisoning has been frequently reported. Poisoning with 3 mL of 0.15 M phosphate buffer pH 6.0, has occurred both during clinical use and also as homogenised and centrifuged at 5000 g for 10 consequence of accidental ingestion e.g. by eating min. The supernatants were decanted and loaded plant material or Aconitum preparations5, 6. The use on a prepared SPE cartridge. Cartridges were pre- of aconite tubers for suicide and homicide purposes conditioned with 3 mL methanol, 3 mL water and has also been reported 7. 1 mL of 0.15 M phosphate buffer pH 6.0. Samples The first symptoms of aconitine poisoning appear were allowed to pass through the cartridge under ~20 min to 2 hours after oral uptake and include gravity, before an initial wash step (3 mL water paraesthesia, sweating and nausea. This leads to followed by 1 mL 0.01 M HCl) was performed. severe vomiting, colicky diarrhea, intense pain Two further washing steps i.e. 2 mL dichloromethane, and then paralysis of the skeletal muscles. Following followed by 2 mL methanol, were performed before the onset of life-threatening arrhythmia, including elution of the aconitine. Cartridges were dried ventricular tachycardia and ventricular fibrillation, under vacuum between each of the 3 wash steps. death finally occurs as a result of respiratory Aconitine was eluted (2 x 1.5 mL) with a mixture of paralysis or cardiac arrest5-7. dichloromethane:2-propanol:25% aqueous ammonia Clearly in the case of suspected aconitine intoxication (80:20:2). Eluents were pooled and evaporated to there is a need for rapid analytical techniques to dryness under a stream of nitrogen at 40 ˚C before enable prompt diagnosis and treatment. To this end reconstitution with 100 μL LC mobile phase. we have developed a simple LC/MS/MS method for

38 ©2007 Waters Corporation. LC/MS/MS In two forensic cases of suspected aconitine A Quattro micro™ tandem mass spectrometer fitted intoxication, aconitine was detected in the blood with Z-Spray™ ion interface was used for all analyses. samples and also in the stomach content and urine Ionisation was achieved using electrospray in the of the deceased (Table 1). Figure 3 shows the positive ionisation mode (ES+). Detection of aconitine chromatogram of the blood sample of aconite victim was performed using multiple reaction monitoring no 2. At the time of autopsy the body was already (MRM). The transition MRM transition m/z 646.4 in an advanced state of putrefaction. Despite these > m/z 586.5 was used for quantification purposes difficult circumstances, the chromatogram shows a and a further two transitions i.e. m/z 646.4 > m/z strong signal for aconitine. 526.4 and m/z 646.4 > m/z 368.4 were monitored for confirmatory purposes. 4.80 MRM of 3 Channels ES+ ® LC analyses were performed using an Alliance 2695 100 646.4>586.5 separations module (Waters). Chromatography was 646.4>526.4 ® achieved using a Waters XTerra RP8 pre-column 646.4>368.4 ® (2.1 x 10 mm, 3.5 μm) and a XTerra RP8 analytical % 2.37e3 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 Time with 1 M acetic acid) and methanol (50:50) at 2.00 4.00 6.00 8.00 10.00 200 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 Figure 2: MRM chromatograms for a blood calibrator spiked at using MassLynx™ NT 4.0 software with automated 0.1 ng aconitine/g blood. Peak intensity is given in the top right- data processing using the QuanLynx™ program hand corner of the trace. (Waters).

Aconitine concentration Summary Case no. Blood [ng/g] Stomach content [ng/g] Urine [ng/mL] 1 10.0 3.0 Not available We have developed a rapid and sensitive method for 2 12.1 Not available 180.0 the quantification of aconitine in biological specimens. The method involves a simple SPE purification prior to Table 1: Concentrations of aconitine in autopsy samples from two analysis using LC/MRM. cases of fatal aconite intoxication. The utility of the method was demonstrated by its application to authentic samples in 2 fatal cases of Results suspected aconitine poisoning. Blood, urine and stomach contents were collected during autopsy and A series of calibrators (0.1 – 25 ng/g) were prepared analysed using the developed LC/MS/MS method. in duplicate by adding aconitine standards to control Aconitine could be detected in the blood of both blood. Samples were then extracted, using the victims, in the stomach content of one individual and SPE method described above, prior to LC/MS/MS in the urine of the other. 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.

Time

2.00 4.00 6.00 8.00 10.00

Figure 3: MRM chromatograms of the blood sample from the victim in case 2, with 12.1 ng aconitine/g. The chromatograms show no interferences although the body was in an advanced state of putrefaction at the time of the autopsy.

References 1. List PH, 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).

Quantitative Analysis of Δ9-Tetrahydrocannabinol in Development of a Rapid and Sensitive Method for the Preserved Oral Fluid by Liquid Chromatography–Tandem Quantitation of Amphetamines in Human Plasma and Oral Mass Spectrometry Fluid by LC/MS/MS Marleen Laloup a, Maria del Mar Ramirez Fernandez a, Michelle Wood b, M. Wood[1], G. De Boeck[2], N. Samyn[2], M. Morris[1], D.P. Cooper[1], R.A.A. Gert De Boeck a, Cécile Henquet c, Viviane Maesd, Nele Samyna Maes[3], and E.A. De Bruijn[3] a National Institute of Criminalistics and Criminology (NICC), Section Toxicology, [1]Micromass U.K. Limited, Atlas Park, Simonsway, Wythenshawe, Manchester Vilvoordsesteenweg 98, 1120 Brussels, Belgium b Waters Corporation, M22 5PP, United Kingdom; MS Technologies Centre, Manchester, UK c Department of Psychiatry and [2]National Institute of Criminalistics and Criminology (NICC), Section Toxicology, Neuropsychology, South Limburg Mental Health Research and Teaching Network, Vilvoordsesteenweg 98, 1120 Brussels, Belgium; and EURON, Maastricht University, Maastricht, The Netherlands dDepartment of [3]Utrecht Institute of Pharmaceutical Sciences (UIPS), Department of Human Clinical Chemistry-Toxicology, Academic Hospital, Free University of Brussels, Toxicology, University of Utrecht, Sorbonnelaan 16, 3584 CA Utrecht, Brussels, Belgium The Netherlands Abstract Abstract A rapid and sensitive method for the analysis of Δ9-Tetrahydrocannabinol Target analysis of amphetamines in biological samples is of great impor- (THC) in preserved oral fluid was developed and fully validated. Oral fluid tance for clinical and forensic toxicologists alike. At present, most labora- was collected with the Intercept, a Food and Drug Administration (FDA) tories analyze such samples by gas chromatography–mass spectrometry. approved sampling device that is used on a large scale in the U.S. for work- However, this procedure is labor-intensive and time-consuming, particularly place drug testing. The method comprised a simple liquid–liquid extraction as a preliminary extraction and derivatization are usually unavoidable. with hexane, followed by liquid chromatography–tandem mass spectrometry Here we describe the development of an alternative method. Amphetamines (LC/MS/MS) analysis. Chromatographic separation was achieved using a were isolated from human plasma and oral fluid using a simple methanol XTerra MS C18 column, eluted isocratically with 1mM ammonium formate– precipitation step and subsequently analyzed using reversed-phase liquid methanol (10:90, v/v). Selectivity of the method was achieved by a combi- chromatography– tandem mass spectrometry. Quantitation of the drugs nation of retention time, and two precursor-product ion transitions. The use of was performed using multiple reaction monitoring. The developed method, the liquid–liquid extraction was demonstrated to be highly effective and led which requires only 50 μL of biological sample, has a total analysis time of to significant decreases in the interferences present in the matrix. Validation less than 20 min (including sample preparation) and enables the simultane- of the method was performed using both 100 and 500 μL of oral fluid. The ous quantitation of 3,4-methylenedioxymethamphetamine, 3,4-methylene- method was linear over the range investigated (0.5–100 ng/mL and dioxyamphetamine, 3,4-methylenedioxyethylamphetamine, amphetamine, 0.1-10 ng/mL when 100 and 500 μL, respectively, of oral fluid were used) methamphetamine, and ephedrine in a single chromatographic run. Limits of with an excellent intra-assay and inter-assay precision (relative standard detection of 2 μg/L or better were obtained. The method has been validated deviations, RSD <6%) for quality control samples spiked at a concentration and subsequently applied to the analysis of plasma and oral fluid samples of 2.5 and 25 ng/mL and 0.5 and 2.5 ng/mL, respectively. Limits of collected from current drug users. quantification were 0.5 and 0.1 ng/mL when using 100 and 500 μL, Journal of Analytical Toxicology, Volume 27, Number 2, March 2003, pp. 78-87 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. Development of a Rapid and Sensitive Method for the Journal of Chromatography A, 1082 (2005) 15–24 Quantitation of Benzodiazepines in Calliphora vicina Larvae and Puparia by LC/MS/MS M. Wood[1], M. Laloup[2], K. Pien[3], N. Samyn[2], M. Morris[1], R.A.A. Maes[4], E.A. de Bruijn[4], V. Maes[5], and G. De Boeck[2] Simultaneous Analysis of Gamma-Hydroxybutyric Acid and [1]Waters Corporation, MS Technologies Centre, Atlas Park, Manchester, United its Precursors in Urine using Liquid Chromatography–Tandem Kingdom; [2]National Institute of Criminalistics and Criminology (NICC), Section Mass Spectrometry Toxicology, Brussels, Belgium; [3]Department of Anatomo-Pathology, Academic Hospital, Free University of Brussels, Belgium; [4]Utrecht Institute of Pharmaceutical Michelle Wood a,?, Marleen Laloup b, Nele Samyn b, Michael R. Morris a, Sciences (UIPS), Department of Human Toxicology, University of Utrecht, The Ernst A. de Bruijn c, Robert A. Maes c, Michael S. Young d, Viviane Maes e, Netherlands; and [5]Department of Clinical Chemistry-Toxicology, Academic Gert De Boeck b aWaters Corporation, MS Technologies Centre, Micromass UK Hospital, Free University of Brussels, Belgium Ltd., Atlas Park, Simonsway, Wythenshawe, Manchester M22 5PP, UK b National Institute of Criminalistics and Criminology (N.I.C.C.), Section Abstract Toxicology, Vilvoordsesteenweg 98, 1120 Brussels, Belgium c Department of Liquid chromatography–tandem mass spectrometry (LC/MS/MS) is emerg- Human Toxicology, Utrecht Institute of Pharmaceutical Sciences (UIPS), University of Utrecht, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands 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 Abstract sensitive method for the simultaneous quantitation of 10 benzodiazepines in We have developed a rapid method that enables the simultaneous analysis of Calliphora vicina (Diptera: Calliphoridae) larvae and puparia. The use of gamma-hydroxybutyrate ( GHB) and its precursors, i.e. gamma-butyrolactone larvae for toxicological analyses offers some technical advantages over putre- (GBL) and 1,4-butanediol (1,4-BD) in urine. The method comprised a simple fied tissue. Four sample pretreatment methods for isolating the benzodiaz- dilution of the urine sample, followed by liquid chromatography–tandem mass epines out of larvae were evaluated. A simple homogenization, followed by spectrometry (LC/MS/MS) analysis. Chromatographic separation was achieved acetonitrile precipitation yielded the highest recoveries. Puparia were ® using an Atlantis dC18 column, eluted with a mixture of formic acid and metha- pulverized and extracted by ultrasonification in methanol. All extracts were nol. The method was linear from 1–80 mg/L for GHB and 1,4-BD and from subsequently analyzed using reversed-phase LC/MS/MS. Larvae and 1–50 mg/L for GBL. The limit of quantification was 1 mg/L for all analytes. puparia calibrators containing benzodiazepines at concentrations ranging The procedure, which has a total analysis time (including sample preparation) from 25 to 750 pg/mg and 50 to 500 pg/mg, respectively, were prepared of less than 12 min, was fully validated and applied to the analysis of 182 and analyzed. The method was demonstrated to be linear over the ranges authentic urine samples; the results were correlated with a previously published investigated. Limits of detection were from 1.88 to 5.13 pg/mg larva and GC/MS procedure and revealed a low prevalence of GHB-positive samples. from 6.28 to 19.03 pg/mg puparium. The developed method was applied Since no commercial immunoassay is available for the routine screening of to the determination of nordiazepam and its metabolite oxazepam in larvae GHB, this simple and rapid method should prove useful to meet the current and puparia of the Calliphora vicina fly that had been reared on artificial increased demand for the measurement of GHB and its precursors. foodstuff (beef heart) spiked with 1 μg/g nordiazepam. The larvae were Journal of Chromatography A, 1056 (2004) 83–90 harvested at day 5 for analysis of drug content. The method was sufficiently

41 ©2007 Waters Corporation. sensitive to allow the detection of nordiazepam and oxazepam in a single Furthermore, the processed samples were demonstrated to be stable for 48 larvae. h, except for cocaine and benzoylecgonine, where a slight negative trend Journal of Analytical Toxicology, Volume 27, Number 7, October 2003, was observed, but did not compromise the quantitation. In all cases the pp. 505-512 method was linear over the range investigated (2–200 μg/L) with an excellent intra-assay and inter-assay precision (coefficients of variation <10% in most cases) for QC samples spiked at a concentration of 4, 12 and 100 Determination of Aconitine in Body Fluids by LC/MS/MS 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 J. Beike1, L. Frommherz1, M. Wood2, B. Brinkmann1 and H. Köhler1 (1) Institute of Legal Medicine, University Hospital Münster, Röntgenstrasse 23, SAMHSA for oral fluid testing in the workplace. The method was subsequent- 48149 Münster, Germany (2) Waters Corporation, MS Technologies Centre, ly applied to the analysis of Intercept® samples collected at the roadside by Atlas Park, Manchester, United Kingdom the police, and to determine MDMA and MDA levels in oral fluid samples Abstract from a controlled study. A very sensitive and specific method was developed for the determination Forensic Science International, Volume 150, Issues 2-3 , 10 June 2005, of aconitine, the main toxic alkaloid from plants of the genus Aconitum L., Pages 227-238 in biological samples. The method comprised solid-phase extraction using mixed-mode C8 cation exchange columns followed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). Chromatographic separation Recent Applications of LC/MS in Forensic Science 1 2 1 was achieved with a RP8 column. Detection of aconitine was achieved using G. De Boeck , M. Wood and N. Samyn electrospray in the positive ionisation mode and quantification was per- 1National Institute of Criminalistics and Criminology, Brussels, Belgium, formed using multiple reaction monitoring with m/z 646.4 as precursor ion, 2Micromass UK Limited, Wythenshawe, UK. i.e. [M+H]+ of aconitine and m/z 586.5, m/z 526.4 and m/z 368.4 Introduction as product ions after collision-induced dissociation. The method was fully The term “forensic science” covers those professions that are involved in the validated for the analysis of blood samples: the limit of detection and the application of the social and physical sciences to the criminal justice system. limit of quantitation were 0.1 ng/g and 0.5 ng/g, respectively. Within the Forensic experts are obliged to explain the smallest details of the methods linear calibration range of 0.5–25 ng/g, analytical recovery was 79.9%. used, to substantiate the choice of the applied technique and to give their In two fatal cases with suspected aconite intoxication, aconitine could be unbiased conclusions. The final result of the work of the forensic scientist, the detected in blood samples at concentrations of 10.0 and 12.1 ng/g. In one expert evidence, exerts a direct influence on the fate of a given individual. case, aconitine could also be detected in the stomach content (3 ng/g) and This burden is a most important stimulus and one that determines the way in the other in the urine (180 ng/mL). of thinking and acting in forensic sciences. Consequently, the methods International Journal of Legal Medicine, Volume 118, Number 5, October applied in forensic laboratories should assure a very high level of reliability 2004, pp. 289-293 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 Quantitative Analysis of Multiple Illicit Drugs in Preserved Oral — a belief that has come under some question in recent years. Of course, Fluid by Solid-Phase Extraction and Liquid Chromatography– the forensic scientist cannot change scepticism and mistrust single-handedly. Tandem Mass Spectrometry He or she can, however, contribute to restoring faith in the judicial processes Michelle Wooda, Marleen Laloupb, Maria del Mar Ramirez Fernandez b, by using science and technology in the search for facts in civil, criminal and Kevin M. Jenkinsc, Michael S. Young c, Jan G. Ramaekersd, Gert De Boeck b regulatory matters. The ability of mass spectrometry (MS) to extract chemical b and Nele Samyn , fingerprints from microscopic levels of analyte is invaluable in this quest, aWaters Corporation, MS Technologies Centre, Manchester, UK enabling the legally defensible identification and quantification of a wide bFederal Public Service Justice, National Institute of Criminalistics and Criminology (NICC), Vilvoordsesteenweg 100, 1120 Brussels, Belgium range of compounds. Recent years have seen the development of powerful cWaters Corporation, Milford, MA, USA dExperimental Psychopharmacology technologies that have provided forensic scientists with new analytical Unit, Brain and Behaviour Institute, Maastricht University, Maastricht, The capabilities, which were unimaginable only a few years ago. Gas Netherlands chromatography GC/MS, liquid chromatography LC/MS, isotope ratio MS Abstract and inductively coupled plasma-MS have become routine tools to enable We present a validated method for the simultaneous analysis of basic drugs detection and characterization of minute quantities in what can often be very which comprises a sample clean-up step, using mixed-mode solid-phase complex matrices. In LC/MS, there has been an explosion in the range of extraction (SPE), followed by LC/MS/MS analysis. Deuterated analogues new products available for solving many analytical problems, particularly for all of the analytes of interest were used for quantitation. The applied those applications in which non-volatile, labile and/or high molecular weight LC gradient ensured the elution of all the drugs examined within 14 min compounds are being analysed. Many analysts and laboratories have and produced chromatographic peaks of acceptable symmetry. Selectivity reached the point at which they are considering the acquisition of LC/MS of the method was achieved by a combination of retention time, and two instrumentation. According to Willoughby et al. LC/MS has progressed from precursor-product ion transitions for the non-deuterated analogues. Oral fluid the “innovators” stage through the “early adaptors” and on to the “early was collected with the Intercept®, a FDA approved sampling device that is majority” stage, and is now open to specialists from a variety of disciplines. used on a large scale in the US for workplace drug testing. However, this This has been as a direct result of the introduction of robust, user-friendly collection system contains some ingredients (stabilizers and preservatives) atmospheric pressure ionization (API)-MS instruments at an affordable price. that can cause substantial interferences, e.g. ion suppression or enhancement LCGC Europe, Nov 2, 2002 during LC/MS/MS analysis, in the absence of suitable sample pre-treatment. The use of the SPE was demonstrated to be highly effective and led to significant decreases in the interferences. Extraction was found to be both reproducible and efficient with recoveries >76% for all of the analytes.

42 ©2007 Waters Corporation. Plasma, oral fluid and sweat wipe ecstasy concentrations in Toxicological data and growth characteristics of single post- controlled and real life conditions feeding larvae and puparia of Calliphora vicina (Diptera: Nele Samyna, Gert De Boecka, Michelle Woodb, Caroline T. J. Lamersc, Dick De Calliphoridae) obtained from a controlled nordiazepam study Waardd, Karel A. Brookhuisd, Alain G. Verstraetee and Wim J. Riedelc Karen Pien1 , Marleen Laloup2, Miriam Pipeleers-Marichal1, Patrick Grootaert3, Gert De Boeck2, Nele Samyn2, Tom Boonen4, Kathy Vits4 and Michelle Wood5 a Drugs and Toxicology, Section Toxicology, National Institute of Criminalistics (1)Department of Pathology Academic Hospital, Free University of Brussels, b and Criminology, Vilvoordsesteenweg 100, 1120, Brussels, Belgium Brussels, Belgium (2)Section Toxicology, National Institute of Criminalistics and c Micromass Ltd., Manchester, UK Experimental Psychopharmacology Criminology (NICC), Brussels, Belgium (3)Department Entomology, Royal Belgian Unit, Brain and Behaviour Institute, Maastricht University, Maastricht, The Institute of Natural Sciences, Brussels, Belgium (4)Section Micro-traces, National d Netherlands Department of Psychology, University of Groningen, Groningen, Institute of Criminalistics and Criminology (NICC), Brussels, Belgium (5)Micromass e The Netherlands Laboratory of Clinical Biology–Toxicology, Ghent University UK Limited, Wythenshawe Manchester, UK Hospital, Ghent, Belgium

Abstract Abstract Larvae of the Calliphora vicina (Diptera: Calliphoridae) were reared on arti- In a double-blind placebo controlled study on psychomotor skills important ficial food spiked with different concentrations of nordiazepam. The dynam- for car driving (Study 1), a 75 mg dose of ±3,4-methylenedioxymethamphet- ics of the accumulation and conversion of nordiazepam to its metabolite amine ( MDMA) was administered orally to 12 healthy volunteers who were oxazepam in post-feeding larvae and empty puparia were studied. Analysis known to be recreational MDMA-users. Toxicokinetic data were gathered by was performed using a previously developed liquid chromatography-tandem analysis of blood, urine, oral fluid and sweat wipes collected during the first mass spectrometry (LC/MS/MS) method. This method enabled the detection 5 hours after administration. Resultant plasma concentrations varied from 21 and quantitation of nordiazepam and oxazepam in single larvae and pupar- to 295 ng/mL, with an average peak concentration of 178 ng/mL observed ia. Both drugs could be detected in post-feeding larvae and empty puparia. between 2 and 4 hours after administration. MDA concentrations never In addition, the influence of nordiazepam on the development and growth exceeded 20 ng/mL. Corresponding MDMA concentrations in oral fluid, as of post-feeding larvae was studied. However, no major differences were measured with a specific LC/MS/MS method (which required only 50 μL of observed for these parameters between the larvae fed on food containing oral fluid), generally exceeded those in plasma and peaked at an average nordiazepam and the control group. To our knowledge, this is the first report concentration of 1215 ng/mL. A substantial intra- and inter-subject variability describing the presence of nordiazepam and its metabolite, oxazepam, in was observed with this matrix, and values ranged from 50 to 6982 ng/mL single Calliphora vicina larvae and puparia. MDMA. Somewhat surprisingly, even 4–5 hours after ingestion, the MDMA International Journal of Legal Medicine, Volume 118, Number 4, August levels in sweat only averaged 25 ng/wipe. 2004, pp. 190-193 In addition to this controlled study, data were collected from 19 MDMA-users To order any of these reprints contact your local Waters office, or go to who participated in a driving simulator study (Study 2), comparing sober www.waters.com/clinical 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

6-MonoacetylMorphine (6-MAM) ...... 19, 20 Lorazepam ...... 24 Alprazolam ...... 24 MDA ...... 15, 16, 17, 42, 43 Amphetamine ...... 15 MDEA ...... 15, 16, 17, 43 Amphetamines ...... 4, 5, 15, 17, 41 MDMA ...... 15, 16, 17, 18, 42, 43 Benzodiazepines ...... 4, 5, 23, 25, 26, 41 Methamphetamine ...... 15 Cannabidiol ...... 30, 31 Morphine ...... 4, 7, 19, 21, 22 Cannabinol ...... 30 Morphine-3-Glucuronide ...... 4, 21 Clonazepam ...... 24 Morphine-6-Glucuronide ...... 4, 21, 22 Codeine ...... 19 Nordiazepam ...... 4, 24, 25, 26 Diazepam ...... 24, 25 Oxazepam ...... 4, 24, 26, 27, 28 Dihydrocodeine (DHC) ...... 19 Prazepam ...... 24 Ephedrine ...... 15 Temazepam ...... 24 GHB ...... 4, 5, 34, 35, 36, 37, 41 Triazolam ...... 24 Heroin ...... 19 Δ9-Tetrahydrocannabinol ...... 4, 29, 41

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ChromaLynx, TargetLynx, MassTrak, ZQ, Quattro micro, Alliance, MassLynx, XTerra, Quattro Ultima, ZSpray, Nova-Pak, Oasis and QuanLynx are trademarks of Waters Corporation. Intercept™ is a trademark of OraSure Technologies. All other trademarks are the property of their respective owners. ©2007 Waters Corporation Printed in the U.S.A. January 2007 720001808EN MC-UG