Waters System Solutions for Drugs of Abuse Analysis

Transcription

1 DRUGS OF ABUSE ANALYSIS APPLICATION ATION NOTEBOOK

2 Waters System Solutions for Drugs of Abuse Analysis Waters Corporation has over 4 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.

3 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.

4 Table of Contents Introduction ChromaLynx Application Manager for Systematic Toxicological Analysis TargetLynx Application Manager for Confirmation Analysis General Unknown Screening for Drugs in Biological Samples by LC/MS A rapid and Sensitive Method for the Quantitation of Amphetamines in Human Saliva Opiates: Use or Abuse? Quantification of Opiates in Human Urine Quantification of Morphine, Morphine-3-Glucuronide and Morphine-6-Glucuronide in Biological Samples by LC/MS/MS Development of a Rapid and Sensitive Method for the Quantification of Benzodiazepines in Plasma and Larvae by LC/MS/MS Detection of Nordiazepam and Oxazepam in Calliphora Vicina Larvae using LC/MS/MS Quantitative Analysis of Δ 9 -Tetrahydrocannabinol in Preserved Oral Fluid by LC/MS/MS Simultaneous Analysis of GHB and its Precursors in Urine Using LC/MS/MS Determination of Aconitine in Body Fluids by LC/MS/MS Published References Compound Index 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.

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

6 Systematic Toxicological Analysis or General Unknown Screening (GUS) LC/MS is also a powerful tool for systematic toxicological analysis (STA). Waters has developed the ChromaLynx Application Manager, a unique data processing tool that can search LC/MS libraries based on cone voltage fragmentation. ChromaLynx Application Manager provides automatic deconvolution and exhaustive examination of complex chromatograms to identify individual components including minor and closely-eluting peaks. Individual components are then searched against library spectra and the results are displayed in an easy-to-use browser format. LC/MS is now increasingly used in screening applications. Until recently, the widespread implementation of LC/MS has primarily been limited by the lack of commercially available LC/MS libraries, and the perceived high capital costs of LC/MS systems. In recent years the capital cost of LC/MS has reduced making the technique more widely accessible. A comprehensive LC/MS library is now available for use on Waters LC/MS (single quadrupole) and LC/MS/MS (tandem quadrupole) systems. It is based on a generic chromatographic run using electrospray ionization (ESI) and mass spectra recorded at multiple cone voltages. Controlled, reproducible fragmentation is caused by in-source fragmentation providing spectra of structurally-related fragments ions; the higher the cone voltage, the more fragmentation is observed. Identification of compounds from this type of experiment is based on matching the spectra from multiple cone voltage spectra at a single retention time. The current version of the Waters toxicology library contains spectra from > 5 compounds, and has been tested for use with the Waters ZQ single quadrupole and Quattro micro tandem mass spectrometer systems. GC/MS/MS Waters Corporation also offers GC/MS/MS systems for forensic toxicology. The Quattro micro GC is the most sensitive GC-tandem quadrupole mass spectrometer on the market today. Waters offers GC/MS/MS systems for analytes which have traditionally been submitted for GC/MS analysis. GC/MS/MS offers enhanced sensitivity and specificity over GC/MS and allows for reduced sample clean-up. Waters Quattro micro GC - tandem mass spectrometer system for the most demanding GC applications. 6

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

8 Flexibility and Ease of Use ChromaLynx has been designed to be easy to use while offering flexibility. It consists of a method editor, to set up chromatographic data processing and library search parameters. The method editor can be viewed spreadsheet-style for ease of review and allows editing of parameters for peak location and detection, and subsequent identification using LC/MS and GC/MS libraries. The processed data is displayed in the ChromaLynx browser for ease of review. ChromaLynx can be used with both LC/MS and GC/MS data and can accommodate both exact mass and nominal mass data. Spectral Deconvolution Following acquisition of full scan spectra recorded at multiple cone voltages, ChromaLynx will analyse each chromatogram and extract full scan spectra and extract specific ion chromatograms to detect the presence of components. The key to the exceptional performance of ChromaLynx is a new, proprietary chromatography deconvolution algorithm. This algorithm efficiently locates peaks in a chromatogram and extracts clean mass spectra of eluting components. The extracted mass spectra can then be searched against LC/MS and GC/MS libraries. ChromaLynx has been designed to exploit a unique multi-function electrospray LC/MS library based on in-source collision induced (CID) mass spectra. By recording mass spectra at multiple cone voltages in both positive and negative ion mode extensive information is acquired on samples. To further enhance the library search process, ChromaLynx also uses retention time information as a search parameter. Figure 2: ChromaLynx method editor displaying chromatogram data processing parameter set up. In this example 7 chromatograms will be processed, five recorded in positive Ion mode and two in negative ion mode. Summary ChromaLynx Application Manager sets new standards for the analysis of complex chromatograms resulting from LC/MS or GC/MS analysis of physiological samples such as blood and urine. A unique algorithm enables ChromaLynx to locate peaks in a chromatogram and then automatically compare the mass spectra against library mass spectra. When using LC/MS mass spectra recorded at multiple cone voltages (using in-source CID) combined with retention time information further enhances the component identification processes. 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 Figure 3: Library search method editor enabling automatic library searching of all peaks and filtering of results for retention time and cone voltage used. Visual comparison of component mass spectrum against proposed library match Figure 4: ChromaLynx Browser displaying identified compounds, candidate compounds, results of a library search and total ion chromatograms recorded at different cone voltages. 8

9 TargetLynx Application Manager for Confirmation Analysis Introduction Quantitation using LC/MS/MS is now well established in many forensic, environmental, clinical and veterinary applications. There are often legal, environmental, human health and financial implications arising from the results of quantitative MS analyses. This has led to an increased demand by regulatory and legal authorities for extra confirmatory and quality control checks. Regulatory or statutory methods often require, for example, the monitoring of multiple structurally specific fragment ions, maximum chromatographic peak width and/or retention time. To calculate and check these manually is a labour intensive, timeconsuming and subsequently costly task. TargetLynx automates data acquisition, processing and reporting incorporating a wide range of confirmatory checks allowing samples falling outside user-specified or regulatory thresholds to be easily identified, giving confidence when reporting quantitative results. TargetLynx is able to rapidly identify and flag samples where, for example: Analytes are above a specified concentration Analyte confirmatory ion ratios are outside specified limits One or more analyte signal-to-noise ratios are below a defined value An analyte retention time or relative retention time is outside limits The coefficient of determination (r 2 ) of the calibration curve exceeds a defined value Drugs of Abuse and Forensic Toxicology LC/MS/MS is now increasingly used in forensic toxicology laboratories for drugs of abuse confirmation and quantitation applications. LC/MS/MS is typically used for confirmation analysis, following a positive immunoassay analysis that indicates the presence of a class of drugs or specific drug, or when there is evidence present that a drug is likely to be present in a sample. If the analytical data is required to be used as part of a police investigation or presented in a court of law, it is essential that extensive analytical information is provided to confirm the presence of a suspected drug. TargetLynx is ideal for this application, where the presence of a suspected drug can be confirmed by the presence of a number of different diagnostic ions and MRM transitions. Confirmation analysis using TargetLynx is enhanced as ion ratio measurements and chromatographic retention time information is also incorporated in the analytical procedure Confirmation 1 Confirmation 2 Quantitation m/z 3 Figure 1: Mass Spectrum of the cocaine metabolite Benzoylecgonine.Transition 29/168 is used for quantitation. Two further MRM transitions 29/15 and 29/82 are monitored for additional confirmation by TargetLynx. Flexibility and Ease of Use TargetLynx can be used with LC/MS/MS and GC/MS/MS data. This data can be SIM (Selected Ion Monitoring), MRM (Multiple Reaction Monitoring) or full scan/full spectrum. In the case of full scan/full spectrum data, extracted ion chromatograms are used for quantitation. TargetLynx consists of a method editor, to set up processing parameters, and a browser, to view processed data. The method editor can be viewed spreadsheet-style for ease of review and allows all quantitation parameters (for peak location and detection, calibration curves etc.) and user-defined criteria for confirmatory and QC checks to be set up. 9

10 Processed data is displayed in the TargetLynx browser for ease of review. A variety of sample flags allows easy location and interrogation of samples falling out with the defined confirmatory and QC criteria. Method Setup The Compound Summary Report, Sample Summary Report, Totals Report and Samples Report allow the user to display calibration information per compound, report one compound/sample/totals group per page or split and print summary reports per sample. Data can be exported from the TargetLynx browser as a XML or comma separated text file into a LIMS system. Data Acquisition Data Processing Review Results Reporting Summary TargetLynx Application Manager automates data acquisition, processing and reporting incorporating a wide range of confirmatory checks allowing samples falling outside user-specified or regulatory thresholds to be easily identified, giving confidence when reporting quantitative results. 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 TargetLynx features various reporting options, with reports being printed directly from the Browser file by sample or by compound. Figure 3: TargetLynx browser showing results summary with flags, calibration curve and chromatograms. 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. 1

11 General Unknown Screening for Drugs in Biological Samples by LC/MS Luc Humbert 1, Michel Lhermitte 1, Frederic Grisel 2 1 Laboratoire de Toxicologie & Génopathologie, CHRU Lille, France 2 Waters Corporation, Guyancourt, France Introduction Identification of drugs of abuse and toxicants in biological fluids is currently performed by a variety of analytical techniques including immunoassays and chromatographic techniques such as GC/MS and LC with UV detection. Although these techniques are well established and widely used, they suffer from limitations for many toxicologically important compounds. For example, sensitivity is often a limitation with LC/UV techniques as newer drugs are used at lower therapeutic concentrations. In addition, LC/UV methods can require extensive sample preparation. GC/MS is often referred to as the gold standard in toxicology laboratories, but even GC/MS has significant limitations for toxicology screening applications where rapid sample analysis is a requirement. Many substances encountered in toxicology laboratories are non-volatile, polar or thermally labile and cannot be directly analyzed by GC/MS. These compounds usually require time consuming derivatization prior to analysis. LC/MS, using electrospray ionization (ESI), is ideally suited to polar, non volatile and, thermally unstable compounds and potentially provides a powerful means of identifying many toxicologically relevant compounds rapidly without the need for sample derivatization. Historically, the lack of availability of LC/MS libraries and reliable LC/MS chromatographic deconvolution software has limited the widespread use of this technique for screening applications. However, with the recent development of a unique LC/MS library and ChromaLynx chromatographic deconvolution software, LC/MS can now be considered a powerful and practical alternative to traditional screening methods. Electrospray is a soft ionization technique that mainly leads to protonated molecular ions in positive ion mode and to deprotonated molecular ions in negative ion mode. In order to get more specific structural information, it is possible to induce fragmentation of these molecular ions in the source region of a mass spectrometer. This can be achieved by increasing the voltage applied to the sampling cone area where ions transit from a high pressure region to a low pressure region. Molecular ions then collide with neutral molecules in the source region and fragment into characteristic ions. This is referred to as in source collision induced dissociation (CID). Using this process reproducible LC/MS mass spectra can be used to produce a library of mass spectra. analyte H 3 O + ESI CID H 2 O [analyte+h] + [analyte+h] + ion + + neutral Figure 1. Atmospheric Pressure Ionisation (API) process - This soft ionisation process leads to cations in positive ion mode and anions in negative ion mode which are generally stable. These molecular ions can be fragmented in the source region of LC/MS instruments. Extractor Sample Cone & Cone Gas LC/MS Library Concept The electrospray ionization process, used in LC/MS systems, is very different from the electron impact (EI) Ionization used in GC/MS systems, thereby preventing the use of commercial EI mass spectra libraries such as NIST, Wiley, and Pfleger- Maurer-Weber. Figure 2. InSource-CID An example showing fragmentation of the moleculer ion (m/z 195) of caffeine at 6V in the Quattro micro API ion source. 11

12 In the current version of the library, this approach has been used for over 5 compounds, which corresponds to approximately 26 mass spectra. These compounds represent 9 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. Figure 3. Extensive structural information is stored for each component in the library as mass spectra can be stored at every significant cone voltage in both positive and negative ion mode. LC Separation Method An identical generic LC method is used both to generate the library mass spectra and for sample analysis. The generic gradient method has been developed based on water and acetonitrile buffered with 5 mm ammonium formate at ph 3. The total run time including system and column re-equilibration is 26 minutes. Positive 9 V Positive 75 V Positive 6 V Positive 45 V Positive 3 V Positive 15 V Figure 4. Loxapine, a tranquilizer agent. Mass spectra recorded at 6 different CV values using in-source CID.The degree of fragmentation increases with the cone voltage. Using in-source CID, it is possible to generate mass spectra exhibiting different fragmentation patterns according to the value of the cone voltage applied in the source. This can be done in both positive and negative ion modes. These spectra can then be used to build a library. ChromaLynx Application Manager Chromatogram examination is at least as important as the content and structure of the library. The chromatogram from a typical toxicological analysis will usually be complex and exhibit dozens of peaks. Compounds of interest can be difficult to identify especially at low concentrations when they can be hidden in the base line or when they closely elute. ChromaLynx application manager includes a unique algorithm to specifically process multifunctional LC/MS data. The process can be ultimately as exhaustive as analyzing each scan for each cone voltage; this enables the detection of the maximum number of components in a chromatogram. Unlike other LC/MS/MS screening techniques, ChromaLynx application manager enables a complete and systematic chromatogram examination. This type of data processing is essential for systematic toxicological screening or general unknown screening. ChromaLynx application manager selects a single mass spectrum at a given scan and extracts up to 8 of the most intense ions and reconstructs corresponding ion chromatograms. These ion chromatograms are then examined and components are detected according to user defined parameters. Detected components are then searched against library spectra. 12

13 1911 7: Scan ES+ TIC 5.39e9 Positive 9 V : Scan ES+ TIC Positive 75 V 5.59e : Scan ES+ TIC Positive 6 V 4.47e : Scan ES+ TIC Positive 45 V 2.72e : Scan ES+ TIC Positive 3 V 3.13e : Scan ES+ TIC Positive 15 V 3.5e : Scan ES- Negative 3 V TIC 4.51e : Scan ES TIC 2.95e9 Area 4 Time Figure 5. Urine extract - one single sample analysis leads to 7 chromatograms. Manual examination of each chromatogram would be time consuming and not feasible for a routine toxicology laboratory. Analysis of the area circled in the chromatogram above by ChromaLynx (figure 6b) illustrates the presence of several peaks that would be missed on examination of the total ion chromatogram. 6a Time Figure 6a. Close-up view of the minutes section of chromatogram area for function 3 acquired in positive 3 V. Here the total ion chromatogram (TIC) indicates that only one component elutes at 13.8 minutes. This area represents only 4 minutes out of the 26 minutes of the whole chromatogram for function 3 recorded in positive ion mode at 3V. ChromaLynx will process all chromatograms to achieve a detailed and efficient screening. ChromaLynx application manager automatically processes data in minutes that would take hours manually. Mass Spectrum Extraction and Library Search Process Once chromatographic components have been detected, ChromaLynx automatically extracts mass spectra of the individual components. This is performed taking into account possible interferences due to closely eluting peaks. It is possible to customize parameters in order to get precise background subtraction depending on the peak width and tolerance on apex determination. Extracted mass spectra of detected components are then compared to library mass spectra. In order to improve the specificity of the screening technique, additional filters have been developed to enhance the quality of the screening and to get more relevant results. Retention time filters as well as cone voltage filters are available and user defined tolerance parameters can be implemented Time b Peak #5 318 RT min Peak # RT 14.1 min Time Figure 6b. Close-up view of the minutes section of chromatogram area for function 3 acquired in positive 3 V. Using extracted ion chromatograms shows that at least three components elute between 13.5 and 13.8 minutes Peak #3 RT min Peak #2 RT min Peak # RT min m/z Figure 7. Chromatogram acquired in positive 3 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

14 Application Example - Polyintoxication A urine sample was taken from a suspected intoxication. It was known that the person was taking a number of prescribed drugs. The urine samples were analyzed by LC/MS to identify the cause of intoxication. Toxicologists were looking for both expected compounds due to the regular treatment and unexpected active substances that may have been taken accidently or deliberately. Method and Instrumentation Analytical Equipment and Instrumentation Waters Toxicology Screening LC/MS System comprising of: ZQ Single Quadrupole Mass Spectrometer Alliance 2695 Separations Module MassLynx 4. Data Station ChromaLynx 4. Application Manager Sample Preparation Liquid/liquid extraction at 2 ph (4.5 & 9.) using dichloromethane/ether/hexane [3:5:2] +.5 isoamylic alcohol. LC Separation Method Waters XTerra MS Column & Precolumn: C 18, 3.5 μm, 2.1 mm id x 15 mm (1 mm for precolumn) Column Oven Temperature: 3 C Mobile Phase based on Water/Acetonitrile with Ammonium Formate 5 ph 3 Gradient: 5 organic to 9 organic from 2 min. to 16 minutes MS Operating Conditions Capillary 3.5 kv in both positive and negative ion modes Source 12 C & Desolvation 25 C Desolvation Gas Flow 35 l/h & Cone Gas Flow 1 l/h Function 1: Full Scan - Negative ESI from 1 to 65 amu in 25 3 Volts Functions 2 to 7: Full Scan - Positive ESI from 1 to 65 amu in 25 from15 Volts to 9 Volts Results From the resultant analysis, 8 out of 9 expected components were successfully identified by the ChromaLynx data processing library search process. Tramadol was the only expected compound that was not detected. In addition, three unexpected compounds were also Detected - Meprobamate, Acepromazine and Bromazepam. It was highly likely that these three compounds were the cause of the intoxication. Candidate Average Fit () # Analyte Name Status Origin 6 Functions 1 Nicotine Unexpected molecule Smoker / Contamination Trimetazidine Expected molecule Medication Acetaminophen Expected molecule Medication Caffeine Expected molecule Medication Quinine Expected molecule Medication Zolpidem Expected molecule Medication Meprobamate Unexpected molecule Unknown Mianserin Expected molecule Medication Acepromazine Unexpected molecule Unknown Bromazepam Unexpected molecule Unknown Hydroxyzine Expected molecule Medication Propoxyphene Expected molecule Medication Tramadol Expected molecule Medication Not found Green Triangles indicate a component Identified with a high degree of confidence Top three candidates are displayed for each component Compounds confidently Identified Comparison with Library spectra Figure 8: ChromaLynx browser showing a list of candidate compounds, chromatogram recorded at different cone voltages and comparison of a unknown spectra against library spectra. Conclusion Using the combination of in-source CID at multiple cone voltages and retention time data results in a library containing detailed information for each compound. With the development of ChromaLynx data chromatographic deconvolution software, LC/MS can now be considered a powerful tool for toxicology screening applications. The unique ChromaLynx deconvolution algorithm ensures that the maximum number of components are detected. The unique algorithm enables low intensity and closely eluting peaks to be detected and identified. The accuracy of the library search process is enhanced by utilizing multiple mass spectra per component and retention time. 14

15 A Rapid and Sensitive Method for the Quantitation of Amphetamines in Human Saliva 1 Michelle Wood*, 2 Gert De Boeck, 2 Nele Samyn, 1 Don Cooper and 1 Michael Morris 1 Waters, Manchester, UK. 2 National Institute of Criminalistics and Criminology (NICC), Belgium. Introduction Ecstasy ( MDMA), EVE ( MDEA) and MDA are amongst the most frequently used recreational drugs. Target analysis of these drugs and other amphetamines in biological samples is of great importance for clinical and forensic toxicologists alike. Plasma and urine are currently the most common matrices investigated. However, due to the invasive nature of such samples (and the associated inconvenience of sample collection) there is an increasing interest in the use of saliva as an alternative marker for drug abuse. Due to the limited volume of sample (usually <1 μl) the traditional methods for amphetamine analysis (i.e. GC/MS) may not be sufficiently sensitive to allow quantitation. In addition, the high viscosity of saliva can lead to problems during solid-phase extraction. Therefore, we have developed an alternative method. Amphetamines were isolated from saliva using a simple methanol clean-up procedure and subsequently analysed using LC/MS/MS. The developed method has a total analysis time (including sample preparation) of less than 15 minutes and allows the simultaneous analysis of several amphetamines in saliva. Limits of detection of 1 ng/ml saliva (or better) were achieved. Methods and Instrumentation LC conditions LC System: Waters Alliance 269 Column: Conventional C 18 (1 x 2.1 mm, 3.5 μm) Mobile phase: (A) =1 mm ammonium acetate (B) = 95 acetonitrile: 5 1 mm ammonium acetate Isocratic elution (85:15) Flow rate:.3 ml/min Injection volume: 1 μl MS conditions Mass spectrometer: Quattro Ultima tandem mass spectrometer. Ionisation mode: ES positive ion Capillary voltage: 1.5 kv MS/MS: Collision gas: Argon at 2.5 x 1-3 mbar Cone Collision Compound Precursor Product Voltage Energy (m/z) (m/z) (V) (ev) MDEA MDEA D Methamphetamine Methamphetamine D Amphetamine Amphetamine D Ephedrine Ephedrine D MDA MDA D MDMA MDMA D Table 1. MRM transitions and conditions for the measurement of several amphetamines and their internal standards. Results and Discussion MRM transitions were determined for six commonly abused amphetamines and their deuterated analogues. The resultant transitions and conditions used are given in Table 1. Examples of MS and product ion spectra are given in Figure 1. Standard curves were prepared by dilution of a mixture of amphetamines in mobile phase followed by LC/MS/MS analysis. Figure 2 shows the MRM chromatograms acquired simultaneously during a single injection (6 out of 12 shown). The typical linearity of response, in the absence of biological matrix, is demonstrated in Figure 3a. 15

16 a b m/z m/z m/z In order to extend the experiment for determination of amphetamines in oral fluid, a series of calibrators (.1-5 ng/ml) were prepared by adding amphetamines to blank saliva. Following isolation from the matrix using a simple methanol extraction procedure (Figure 4), samples were analysed using LC/MS/MS. The amphetamines were quantified by reference to their respective deuterated internal standards. Once again, responses were linear over the range investigated (Figure 3b). In order to assess the feasibility of using oral fluid as an alternative specimen for drug abuse, saliva samples collected from current amphetamine users were analysed using the developed method. Figure 5 shows the resultant MRM chromatograms for one of the oral fluid samples found to be positive for the presence of the amphetamines MDEA, MDMA and MDA. It should be noted that the designer drug MDA is also a metabolite of MDMA and MDEA. The results from 1 individuals are summarised in Figure 6 and demonstrate that, within this particular group, MDMA (Ecstasy) was the most commonly used amphetamine (with concentrations ranging from 3.1 to > 3 ng/ml). The results also demonstrate the trend for multiple, rather than single, drug use MRM of 12 channels ES+ 194>163 c m/z m/z MRM of 12 channels ES+ 18>15 MRM of 12 channels ES+ 166>148 MRM of 12 channels ES+ 136>91 MRM of 12 channels ES+ 15>91 MRM of 12 channels ES+ 28>163 Time Figure 2. MRM chromatograms obtained for a single injection of a mixture of amphetamines(1 ng/ml) in mobile phase (respective internal standards not shown) m/z Compound name: MDEA Coefficient of Determination: Calibration curve: * x Response type: External Std. Area Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None Figure 1. MS (top trace) and product ion spectra (lower trace) for (a) ephedrine, (b) MDEA and (c) MDEA-D5. Response pg/µl Figure 3a. Typical linearity of response for MDEA in the absence of biological matrix. 16

17 Response Compound name: MDEA Coefficient of Determination: Calibration curve: * x Response type: External Std. Area Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None pg/µl Figure 3b. Typical linearity of response for saliva containing MDEA μl saliva + 2 μl methanol (containing internal standards) Centrifuge 13, rpm (to collect supernatant) HPLC/MRM analysis Total analysis time: 15 mins Figure 4. Schematic overview of the developed LC/MRM technique MRM of 12 channels ES+ 194>163 MRM of 12 channels ES+ 18>15 MRM of 12 channels ES+ 166>148 Concentration (ng/ml) Individual # 1 Methamphetamine Amphetamine Ephedrine MDA MDEA MDMA Figure 6. Summary of results obtained from LC/MRM analysis of 1 saliva samples collected from current amphetamine users. N.B. MDMA concentrations ranged from 3 to over 3 ng/ml. In a separate (controlled) study, blood and saliva were collected from experienced MDMA-users (n=12) at various times following oral administration of 75 mg MDMA. Samples were analysed using GC/MS (blood) and LC/MRM (oral fluid). Blood concentrations of MDMA ranged from 21 to 295 ng/ml. The corresponding saliva concentrations were usually higher and ranged from 47 to > 6 ng/ml. In both matrices peak MDMA levels were generally observed between 2 and 4 hours after administration. Figure 7 shows the mean MDMA levels in blood and oral fluid and also demonstates the clear relationship between the two matrices. Saliva spiked with amphetamines were firstly extracted using methanol. Following centrifugation, supernatants were analysed using LC/MRM analysis. Amphetamines were quantified by reference to their internal standards. 1 MRM of 12 channels ES+ 136> MRM of 12 channels ES+ 15>91 MRM of 12 channels ES+ 28> Time Figure 5. MRM chromatogram for an oral fluid sample found to be positive for the presence of MDEA, MDMA and MDA. 17

18 Conclusions The use of oral fluid as a non-invasive alternative to blood or urine as a marker for drug use, is an attractive possibility. Collection of this biological sample requires no special equipment or facilities and can be supervised, thus removing the opportunity for sample adulteration. To this end we have developed a simple, rapid method that allows the simultaneous quantitation of several amphetamines in saliva during a single chromatographic run. The procedure involves the extraction of amphetamines from saliva followed by LC/MRM analysis and is less time-consuming and labour-intensive than the existing GC/MS method. 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. Mean MDMA in plasma (ng/ml) Time after admininistration (minutes) Mean MDMA in plasma (ng/ml) Figure 7. Mean MDMA levels (n=12) in plasma and oral fluid following a single administration of 75 mg MDMA. 18

19 Opiates: Use or Abuse? Quantification of Opiates in Human Urine 1 Michelle Wood 1, Kevin Rush 2, Michael Morris 1 and Allan Traynor 2 Waters Corporation, Manchester, UK 2 Medscreen Ltd., London, UK Introduction Heroin is a highly addictive drug. It is processed from morphine, a naturally occurring substance extracted from the seedpod of the Asian opium poppy (Figure 1). Abuse of heroin is associated with serious health conditions, including fatal overdose, collapsed veins and an increased risk of infectious diseases such as hepatitis, HIV/AIDS and tuberculosis. Once inside the body it is rapidly metabolised to morphine (Figure 2), which is then excreted in the urine. The presence of morphine in urine cannot alone be used as a marker for illicit heroin abuse since morphine and codeine (which is also metabolised to morphine) can be found in prescriptive medicines and foods. For example, such medicines are valuable treatments for pain, coughs and diarrhea. Ingestion of pastries containing poppy seeds has also been shown to lead to the presence of morphine and codeine in the urine (Hayes et al., 1987). However, the intermediate metabolite of heroin, 6-monoacetylmorphine ( 6-MAM) can be used as a specific marker for heroin as it does not result from the metabolism of either morphine or codeine. In addition, acetylcodeine is a known impurity of illicit heroin synthesis and may be used to distinguish between the pharmacologically pure heroin that is used in heroin maintenance programs and illicit street heroin. We have developed an LC/MS/MS method that allows the simultaneous quantification of several opiates in urine. The method can also be used to establish whether morphine present in the urine has originated from illicit heroin use. Methodology Sample preparation Urine samples were prepared for LC/MS/MS analysis by means of a simple, generic solid-phase extraction (SPE) procedure. A Waters Oasis HLB Extraction Cartridge (1 cc/3 mg) was firstly conditioned with methanol (1 ml) followed by water (1 ml). Urine samples (spiked with deuterated internal standards) for SPE were diluted into water (3 μl urine into 7 μl water before applying to the pre-conditioned cartridge). The cartridge was washed with 5 methanol before elution of the sample using 1 ml 1 methanol. Ten microlitres (1 μl) of the eluant were analysed using LC in conjunction with multiple reaction monitoring (MRM). LC/MS/MS A Quattro micro triple quadrupole mass spectrometer fitted with ZSpray ion interface was used for all analyses. Ionization was achieved using electrospray in the positive ionization mode (ES + ). Details of the MRM conditions are given in Table 1. LC analyses were performed using a Waters LC 279 separations module. Chromatography was achieved using a Waters Nova-Pak CN HP column (3.9 x 75 mm) eluted isocratically with 2 mm ammonium acetate:methanol (5:5) containing.5 formic acid at a flow rate of.3 ml/min. The column temperature was maintained at 3 C. All aspects of system operation and data acquisition were controlled using MassLynx software with automated data processing using the QuanLynx program. Precursor Product Cone Collision Compound ion ion Voltage energy (m/z) (m/z) (V) (ev) Morphine Morphine - d Codeine Dihydrocodeine (DHC) Monoacetyl morphine ( 6-MAM) Monoacetyl morphine ( 6-MAM)-d acetylcodeine Figure 1: The Asian opium poppy, Papaver somniferum. Table 1: MRM transitions and conditions for the measurement of several opiates. The conditions for deuterated morphine and 6-MAM (d3 and d6 respectively) were also included for the purpose of internal standardisation. 19

20 CH 3 CH 3 CH 3 Heroin N 6-MAM N Acetylcodeine N H H H H 3 C O O O O CH 3 O HO O O CH 3 O H 3 C O O O CH3 O CH 3 N CH 3 N H H HO O OH H 3 C O O OH Morphine Codeine Results A series of calibrators (.5-25 ng/ml) were prepared by adding opiate standards to blank urine. Urine samples (either calibrators or unknown samples) were then extracted using the SPE method described above prior to LC/MRM analysis. Following LC/MRM analysis, the areas under the specific MRM chromatograms were integrated. Figure 3 shows the MRM chromatograms of various opiates obtained with a 1 μl injection of the 5 ng/ml urine calibrator. The opiates were quantified by reference to the integrated area of the deuterated internal standards. Responses were linear for all compounds (Figure 4 shows a typical standard curve for 6-MAM in urine). Summary We describe a sensitive method for the simultaneous analysis of several opiates in urine. The method involves a simple SPE purification step prior to analysis using LC/MRM and may be used to identify cases of heroin abuse. 342> >165 32>199 3> >165 Figure 3. MRM chromatograms for: (top to bottom) acetylcodeine, 6-MAM, DHC, codeine and morphine. Responses were obtained with a 1 μl injection of the 5 ng/ml urine calibrator. Compound name: 6-MAM Coefficient of Determination: Calibration curve: * x Response type: Internal Std. (Ref 4), Area* (IS Conc./IS Area) Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None 41.9 Response ng/ml References Hayes LW, Krasselt WG and Mueggler PA. Concentrations of morphine and codeine in serum and urine after ingestion of poppy seeds. Clinical. Chemistry. 1987; 33: Figure 4. Standard curve for 6-MAM. Responses were calculated in reference to the integrated area of the deuterated internal standards. 2

21 Quantification of Morphine, Morphine-3-Glucuronide and Morphine-6-Glucuronide in Biological Samples by LC/MS/MS 1 Michelle Wood and Michael Morris. Waters Corporation, Manchester, UK. Introduction Morphine is a potent analgesic isolated from the opium poppy papaver somniferum and traditionally used for the treatment of moderate to severe pain. Analgesia results from the action of morphine at the opioid receptors of the spinal cord and brain (Figure 1), where it attenuates both the speed of the impulse and the perception of pain. In human subjects, morphine is extensively metabolised (primarily by conjugation with glucuronic acid) to form morphine-3-glucuronide (M3G) and morphine-6- glucuronide (M6G). Whilst, the principal metabolite i.e. M3G, has little or no analgesic effect, M6G has been shown to be highly effective and is believed likely to contribute significantly to the overall effectiveness of morphine 1. Hence, quantification of both the parent drug and metabolites is desirable for pharmacokinetic studies. Previously we have described a LC/MS/MS method that allows the quantification of morphine and several other opiates in urine 2. Here we present a simple method that enables the quantification of morphine in plasma, whole blood and urine. Furthermore this procedure allows differentiation between two isobaric glucuronide metabolites. Methodology Sample preparation Figure 1. Image of guinea-pig brain. The red areas represent the highest density of opioid receptors; yellow areas represent moderate density; whilst blue, purple and white represent low density. Biological samples were prepared for LC/MS/MS analysis by means of a simple, solid-phase extraction (SPE) procedure. A Waters Oasis HLB extraction Cartridge (1 cc/3 mg) was firstly conditioned with 1 ml volumes of each of the following: methanol, water and ammonium carbonate (1 mm, ph 8.8). Samples (1 μl, spiked with deuterated internal standards) were made up to a final volume of 1 ml with ammonium carbonate before applying to the pre-conditioned cartridge. The cartridge was then washed with 1 ml ammonium carbonate before elution of the sample using 1 methanol (.5 ml). Eluents were dried using a Savant Speedvac Plus evaporator and then redissolved in 1 μl of mobile phase. Reconstituted samples were briefly vortex mixed before the analysis of 1 μl using LC in conjunction with multiple reaction monitoring (MRM). LC/MS/MS A Waters Quattro micro triple quadrupole mass spectrometer fitted with ZSpray ion interface was used for all analyses. Ionisation was achieved using electrospray in the positive ionisation mode (ES + ). Details of the MRM conditions are given in Table 1. Precursor Product Cone Collision Compound ion ion Voltage energy (m/z) (m/z) (V) (ev) Morphine Morphine d Morphine M3G glucuronide Morphine M3G d3-glucuronide Morphine M6G glucuronide Table 1: MRM transitions and conditions for the measurement of morphine and it s metabolites. The deuterated analogues of morphine and morphine-3-glucuronide were also included for the purpose of internal standardisation. LC analyses were performed using a Waters 2795 separations module. Chromatography was achieved using a C 18 column (3.9 x 15 mm) eluted isocratically with.1 formic acid:acetonitrile (97:3) at a flow rate of.3 ml/min. Column temperature was maintained at 3 C. All aspects of system operation and data acquisition were controlled using MassLynx 4. software with automated data processing using the QuanLynx program. 21

22 Results A series of calibrators (.5-5 μg/l) were prepared in duplicate by adding standards to blank plasma, whole blood or urine. Samples were then extracted using the SPE method described above prior to LC/MRM analysis. Following LC/MRM analysis, the areas under the specific MRM chromatograms were integrated. Figure 2 shows the extracted MRM chromatogram of morphine, M3G and M6G obtained with a 1 μl injection of the 5 μg/l plasma calibrator. Opiates were quantified by reference to the integrated area of the deuterated internal standards. Responses were linear (r = >.999) over the range investigated for all 3 compounds and in each matrix (Figure 3 shows a typical standard curve for M3G in urine) MOR Figure 2. MRM chromatogram for morphine (MOR), M3G and M6G. The above responses were obtained with a 1 μl injection of the 5 μg/l plasma calibrator. Due to the isobaric nature of M3G and M6G chromatographic resolution is required to enable identification. M6G 462> > Time Summary We present a sensitive method for the quantification of morphine and its glucuronide metabolites. The method involves a simple SPE purification prior to analysis using LC/MRM and is suitable for plasma, whole blood or urine samples. Compound name: Morphine-3-glucuronide Correlation coefficient: r= , rˆ 2 = Calibration curve:.944 * x Response type: Intermal Std ( Ref 4 ), Area * ( IS Conc. / IS Area ) Curve type: Linear, Origin: Exclude, Weighting: 1/x Axis trans: None 47.1 Response 1.6 Response References 1. The Analgesic Effect of Morphine-6-Glucuronide. R Osborne, P Thomson, S Joel, D Trew, N Patel and M Slevin. Br J Clin. Pharmacol (2) Opiates: Use or Abuse? Quantification of Opiates in Human Urine. (Waters Application Brief WAB45). M Wood, K Rush*, M Morris and A Traynor*. Clinical Applications Development Group, UK Limited, Manchester, UK. *Medscreen Ltd., 1A Harbour Quay, 1 Prestons Rd, London... µg/l µg/l Figure 3. Standard curve for M3G in urine. Responses (duplicates) were calculated in reference to the integrated area of the deuterated internal standards. The inserted figure shows the response for the range -1 μg/l. 22

23 Development of a Rapid and Sensitive Method for the Quantification of Benzodiazepines in Plasma and Larvae by LC/MS/MS Gert De Boeck 1, Nele Samyn 1, Karen Pien 2, 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 Benzodiazepines are the most widely prescribed psychoactive drugs in the world for the symptomatic treatment of anxiety and sleep disorders. However, misuse of these compounds has been reported and they are frequently encountered in postmortem blood analysis (suicide or accidental death). Here we describe the development of a rapid and sensitive LC/MS/MS method for the quantification of 1 benzodiazepines. Limits of detection of.2 μg/l or better were achieved when just 25 μl plasma was used. In addition, we present the application of this method to the analysis of benzodiazepines in Calliphora vicina larvae. Insects and their larvae are commonly used in the estimation of postmortem interval. Furthermore, they may serve as a reliable alternate source for toxicological analysis in the absence of suitable tissues and fluids that are normally taken for this purpose. Experimental Conditions LC/MS/MS conditions LC System: Waters Alliance 269 Column: Conventional Phenyl Column (2.1 x 15 mm, 5 μm) Mobile phase : A =1:1:8 acetonitrile: methanol: 2 mm ammonium acetate B = 95:5 acetonitrile: 2 mm ammonium acetate Time (min) A () B () Curve number (concave) (linear) Results and Discussion Figure 1 shows the MS and MS/MS spectra for alprazolam. Table 1 summarizes the MRM transitions and conditions used for this and several other benzodiazepines (and their respective deuterated analogues). The latter were used as internal standards for quantification purposes. A series of calibrators (1, 1, 4, 1, 2, 4 and 8 μg/l) were prepared by adding the benzodiazepines to drug-free plasma. Plasma samples were isolated from the matrix using a simple acetonitrile clean-up procedure (which also incorporates the addition of the internal standards). Figure 2 shows the MRM chromatograms of the benzodiazepines obtained with a 1 μl injection of the 1 μg/l plasma calibrator. Quantification was performed by integration of the area under the specific MRM chromatograms. Figure 3 shows a typical standard curve for diazepam in plasma. Flow rate: Injection volume:.25 ml/min 1 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 1-3 mbar 23

24 Responses were linear, in all cases, over the range investigated (Coefficient of Determination >.99). Compound Precursor ion Product ion Cone Voltage Collision energy (m/z) (m/z) (V) (ev) Alprazolam Alprazolam-d Clonazepam Clonazepam-d Diazepam Diazepam-d Flunitrazepam Flunitrazepam-d Lorazepam Lorazepam-d Nordiazepam Nordiazepam-d Oxazepam Oxazepam-d Prazepam Prazepam-d Temazepam Temazepam-d Triazolam Triazolam-d Table 1. MRM transitions and conditions for the measurement of 1 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 1 μl injection of the 1 μg/l plasma calibrator. 1 A B m/z m/z Figure 2. MS and MS/MS spectra of alprazolam. For all compounds, LOD s of.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, 4 and 2 μg/l respectively). Coefficients of variation (CV s) were found to be highly satisfactory (<15). 24

25 Compound name: Diazepam Coefficient of Determination: Calibration curve: * x Response type: Internal Std (Ref 1), Area* (IS Conc./IS Area) Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None C 1 276> Response B 271>14 µg/l Figure 3.Typical response for plasma containing diazepam. Diazepam spiked plasma was firstly extracted using acetonitrile prior to analysis using LC/MRM. Benzodiazepines were quantified by reference to their deuterated internal standards. The developed LC/MS/MS was subsequently applied to the analysis of Calliphora vicina larvae in a study to assess the feasibility of using insects and their larvae as alternate specimens in the absence of any suitable human specimens for toxicological analysis. Larvae were reared on artificial foodstuff (beefheart) spiked with a range of concentrations of nordiazepam (,.5, 1 and 2 μg/g). Post-feeding larvae were harvested (after 7 days) for analysis of drug content by LC/MS/MS. Figure 4 outlines the initial sample preparation method used for these specimens. All control larvae reared on spiked foodstuff were positive for nordiazepam and the metabolite oxazepam. All control samples were negative. Figure 5 shows the MRM chromatograms obtained following LC/MS/MS analysis of a control larva and a larva positive for nordiazepam. The method was sufficiently sensitive to measure benzodiazepines in single larvae whereas previous analytical techniques e.g. GC/MS, RIA, TLC have required pools i.e. typically 2 larvae. A 1 Figure 5. MRM chromatograms obtained with the analysis of larvae that were reared on artificial foodstuff spiked with Nordiazepam at and 1 μg/g (A and B respectively). Figure C shows the MRM chromatogram for the internal standard i.e. Nordiazepam-d5. Conclusion Time >14 We have developed a simple, rapid method that allows the simultaneous quantification of 1 benzodiazepines in plasma a single chromatographic run. LOD s were better than.2 μg/l when only 25 μl plasma was used. The method involves a simple protein precipitation step with acetonitrile followed by LC/MS/MS analysis. The method was subsequently applied to the analysis of Calliphora vicina larvae in a study designed to assess the feasibility of using insects as alternate specimens in the absence of any suitable human tissues. The sensitivity was such that it was possible to detect benzodiazepines in single larvae whereas previous methods have required pools. After 7 days 5µL H 2O 1mL ACN and I.S. Dry to 1µL mix throughly (nordiazepam d5 & oxazepam d5) vortex thoroughty LC/MS/MS analysis (1µL aliquot) Filter Figure 4. Preparation of larvae for LC/MS/MS analysis. 25

26 Detection of Nordiazepam and Oxazepam in Calliphora Vicina Larvae using LC/MS/MS Karen Pien 1 ; Patrick Grootaert 2 ; Gert De Boeck 3 ; Nele Samyn 3 ; Tom Boonen 4 ; Kathy Vits 4 ; Michelle Wood 5 ; Michael Morris 5. 1 Free University of Brussels, Belgium; 2 Royal Institute of Natural Sciences, Brussels, Belgium; 3 National Institute of Criminalistics and Criminology (NICC), Section Toxicology, Brussels, Belgium; 4 National Institute of Criminalistics and Criminology, Brussels, Belgium; 5 Waters, Manchester, UK. Introduction In addition to their use in the estimation of postmortem interval, insects may serve as reliable alternate source for toxicological analyses in the absence of tissues and fluids normally taken for such purpose. To date, a variety of compounds have been measured in fly larvae and pupae using different analytical procedures i.e. (Radio-Immunoassay (RIA), Gas Chromatography (GC) and Thin-Layer Chromatography (TLC)). In these studies a minimum of 1g (approximately 2 larvae) was needed to detect the toxic compound. In this study we used LC/MS/MS (Liquid Chromatography-Tandem Mass Spectrometry) to detect the benzodiazepine Nordiazepam and its metabolite Oxazepam, in single larvae of the Calliphora vicina. Benzodiazepines are prescribed for the symptomatic treatment of anxiety and sleep disorders. They are frequently encountered in postmortem blood analysis (suicide or accidental deaths). In addition, we compared the development of postfeeding larvae and pupae fed on different concentrations of Nordiazepam. Experimental Conditions Study design Flies and larvae were from a stock colony of Calliphora vicina maintained in an environmental chamber at C and 6-7 humidity with cyclical artificial lighting simulating 16 h daylight and 8 h darkness. Larvae were reared on artificial food (beef heart) spiked with a range of concentrations of Nordazepam (Table 1). Post-feeding larvae were harvested from day 4 till day 8. Thirty larvae were boiled and conserved (in a mixture of ethanol and acetic acid) prior to measurement of length. Another 3 were used for toxicological analysis. These were weighed and then killed, by freezing to -2 C. The larvae were stored at -2 C until analysis. Sample preparation Individual larvae and pupae samples were prepared for LC/MS/MS as follows; the sample was transferred to a vial containing 5 μl water and vortex-mixed thoroughly. One millilitre of acetonitrile (containing deuterated internal standards) was then added and the samples mixed for a further minute. The mixture was evaporated to ~1 μl and then filtered. A 1 μl aliquot was analysed using LC/MS/MS. Sample LC/MS/MS LC Conditions LC System: Waters Alliance 269 Column: Conventional Phenyl Column (2.5 x 15 mm, 5 μm) Mobile Phase: A=1:1:8 acetonitrile:methanol: 2 mm ammonium acetate B=95:5 acetonitrile: 2 mm ammonium acetate Time (min) A () B () Curve number (concave) (linear) Flow Rate:.25 ml/min Injection Volume: 1 μl Target Concentration (μg/g)* Control Nor I (human therapeutic dose).5 Nor II (human lethal dose) 1 Nor III (2x human lethal dose) 2 Table 1: Target concentration of Nordiazepam in larval food. *Concentrations expressed in μg/g food. 26

27 MS Conditions Mass Spectrometer: Quattro Ultima triple quadrupole Ionisation Mode: ES + Capillary Voltage: 3kV Results All larvae, pupae and food spiked with Nordiazepam were positive for the drug, whereas all control samples were negative. Figures 1 and 2 show the larvae Nordiazepam and Oxazepam concentrations from days 4-8. Figure 3 shows the MRM chromatograms obtained following the LC/MS/MS analysis of a control larva and a Nordiazepam positive larva. Compound Precursor Ion Product Ion Cone Voltage Collision Energy (m/z) (m/z) (V) (ev) Nordiazepam Nordiazepam-d Oxazepam Oxazepam-d Table 2: MRM transitions and conditions for them LC/MS/MS analysis of Nordiazepam and Oxazepam. Deuterated analogues were also included as internal standards. Peak concentrations of Nordiazepam were measured on day 4 for NOR I, II and III, followed by a precipitous fall of larval Nordiazepam concentrations. From day 7, Nordiazepam was not detectable in a single larva. Peak concentrations of Oxazepam were measured on day 5 for NOR II and III and at day 6 for NOR I. Low concentrations of Oxazepam were still measured at day 8. In this study, two patterns of development were observed; the post-feeding larvae fed on Control, NOR I and NOR III food regime developed at approximately the same rate and each demonstrated wandering-phase behaviour at day 6, pupation at day 8 and emerging of adult flies at day 18. Figure 1: Concentration of Nordiazepam in larva reared (for 4-8 days) on foodstuff spiked with Nordiazepam. Mean concentrations are plotted (± 1SD). C 276>14 B 271>14 271>14 Figure 2: Concentration of Oxazepam in larva reared (for 4-8 days) on foodstuff spiked with Nordiazepam. Mean concentrations are plotted (± 1SD). A Figure 3: MRM chromatograms obtained with the analysis of larvae that were reared on artificial foodstuff spiked with Nordiazepam at and 1 μg/g (A and B respectively). Figure C shows the MRM chromatogram for the internal standard i.e. Nordiazepam d5. 27

28 In contrast, the development of larvae fed with the NOR II regime was 1 day later in all stages. Post-feeding larval length is shown in Table 3; no significant differences were observed. Day 4 Day 5 Day 6 Day 7 Day 8 Control Nor I Nor II Nor III Table 3: Mean post-feeding larval length (mm). Day 4 Day 5 Day 6 Day 7 Day 8 Control Nor I Nor II Nor III Table 4: Mean post-feeding larval weight (mg). Post-feeding larval weight is shown in Table 4: although no significant differences were seen in larvae reared on Control, NOR I and NOR III food regimes, the mean weight of larvae fed on NOR II was significantly higher. This observation was also confirmed in a second rearing experiment. Discussion and Conclusions We have developed a method that allows the detection of Nordiazepam and its metabolite Oxazepam in single larvae. Larval drug concentrations showed a stepwise increase with increasing drug concentrations in the foodstuff. It was clear that Nordiazepam was metabolized to Oxazepam, which was still detectable at day 8. Nordiazepam was detectable until day 6. Control maggots were negative. No differences were seen on the post-feeding larval length, but differences in post- feeding larval weight and development were seen in the NOR II larvae. The reason of this disturbance is not yet understood, but is presumably because larval physiology is disturbed to a greater extent by this drug level. This study indicates that an estimation of the postmortem interval based on the length of the post-feeding larvae of Calliphora vicina, which have fed on tissues containing Nordiazepam, will have no error. However an error, of up to 24 hours, can be made if the estimation is based on duration of larvaland puparial stages. 28

29 Quantitative Analysis of Δ 9 - Tetrahydrocannabinol in Preserved Oral Fluid 1 Marleen Laloup 1, Maria del Mar Ramirez Fernandez 1, Michelle Wood 2, Gert De Boeck 1, Cecile Henquet 3, Viviane Maes 4, Nele Samyn 1 1 National Institute of Criminalistics and Criminology (N.I.C.C), Brussels, Belgium; 2 Waters Corp., Manchester, UK;. 3 Maastricht University, The Netherlands 4, 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. Introduction Cannabis is the collective term for the psychoactive substances of the Cannabis sativa plant (Figure 1) and one of the most frequently used illicit drugs in the western world. Δ 9 -Tetrahydrocannabinol (THC), the main psychoactive constituent of cannabis, is deposited in the oral cavity during cannabis smoking. Over the last few years there has been an increasing interest in the use of oral fluid to document drug use. The advantage of this specimen over the more traditional matrices e.g. urine and blood, is that collection is almost non-invasive, relatively easy to perform, and may be achieved under close supervision to prevent adulteration or substitution of the sample. LC/MS/MS is a technique that lends itself well to the high-throughput determination of multiple analytes in oral fluid samples due to its high specificity, sensitivity and short analysis times 1,2. The Intercept is a FDA cleared oral fluid collection device that is used on a large scale in the U.S. for workplace testing 3. It is also the device of choice to collect the samples in a current joint roadside study between the European Union and the U.S. to detect driving under the influence of drugs 4. The Intercept collection system utilises a variety of ingredients to ensure stability and to maintain the integrity of the sample. However, these ingredients can also cause interferences e.g. ion suppression during LC/MS/MS analysis in the absence of a suitable clean-up method 5. 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, PA) according to the manufacturer s instructions. Briefly, after gently wiping the collector pad between gum and cheek for approximately 2 minutes the device is placed in the supplied vial and sealed. Following centrifugation, the recovered fluid was spiked with THC to yield a series of calibrators ranging from.1 to 1 ng/ml. QC samples were also prepared by spiking control oral fluid with THC. Authentic samples Oral fluid samples were collected by the police at roadblocks, the purpose of which, was to intercept drivers who were driving under the influence of drugs. The samples were collected at the roadside using the same procedure as described for the blank samples. An additional series of authentic samples were obtained from volunteers with a history of cannabis use. Once a week, and over 2 consecutive weeks, subjects received either a placebo cigarette (where the THC had been extracted) or a marijuana cigarette which contained 3 μg cannabis per kg). Samples were collected.5 hour prior to drug administration and at various times following drug administration (.25,.5, 1, 1.25, 1.5 hour). The study protocol was approved by the ethics committee of the University Hospital of Maastricht in the Netherlands. Internal standard solution An internal standard (IS) working solution of THC-d3 at a concentration of 1 ng/ml was prepared in methanol. 29

30 Sample preparation Extraction was performed using either 1 or 5 μl of the collected specimen. When using 5 μl, 5 μl of the IS working solution and 4 ml of hexane were added; when only 1 μl of oral fluid was used, an additional 4 μl of deionised water was added. After mechanical shaking (3 min) and centrifugation (1 min at 3 g), the organic phase was collected and then evaporated to dryness at 4 C under nitrogen. The extract was reconstituted in 1 μl of mobile phase. LC conditions LC system: Column: Mobile phases: Flow rate: Injection volume: Waters Alliance System Waters XTerra MS C 18 column (2.1 x 15 mm, 3.5 μm) at 4 C (A): 1 mm ammonium formate (B): methanol Isocratic elution 1:9 (A:B).2 ml/min 2 μl Mass spectrometry conditions Mass spectrometer: Waters Quattro Premier tandem mass spectrometer Ionisation mode: ES + Capillary voltage: 2 kv Source temperature: 12 C Desolvation gas: Nitrogen at 7 L/Hr, 28 C MS/MS: THC m/z 315.2>193.1 (quantification ion) m/z 315.2>259.3 (qualifier ion) THC-d3 m/z 318.2>196.1 Collision gas: Cannabinol m/z 311.2>223.1 Cannabidiol m/z 315.2>193.1 Argon at 3.5 x 1-3 mbar Results and Discussion Figure 2 shows the MRM chromatograms obtained following the analysis of a sample enriched with THC and the internal standard i.e. THC-d3. The usefulness of the liquid/liquid extraction step was assessed by a comparison of the effect of the matrix both before and after sample clean-up. Matrix effects were monitored throughout the whole of the chromatographic run by performing post-column infusion experiments 6. The effect on THC response obtained following the injection of a sample prior to extraction and the same sample after extraction of 1 μl and 5 μl of oral fluid are given in Figure 3. The results clearly demonstrate the usefulness of the liquid-liquid extraction step prior to LC/MS/MS analysis. Figure 2. MRM chromatograms obtained with a single injection of a 1 μl extracted oral fluid sample enriched with 5 ng/ml THC and 5 ng/ml THC-d3. The figure shows the response for THCd3 (top trace) and for the two transitions of THC (quantifier and qualifier middle and bottom trace respectively). Peak intensity is shown in the top right-hand corner of each chromatogram. 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 1 μl (C) and the reconstituted extract after 5 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. 3

31 To assess method linearity, limit of quantitation (LOQ), precision, accuracy and analytical recovery a series of oral fluid calibrators were prepared and a 1 or 5 μl aliquot extracted with hexane prior to analysis using LC/MS/MS. Quantification was achieved by integration of the area under the specific MRM chromatogram. For THC, the response was calculated in reference to the integrated area of THC-d3. Linear responses (r = >.999, 1/x weighting) were obtained up to 1 ng/ml when 1 μl of sample was extracted and up to 1 ng/ml when 5 μl sample was extracted. Linearity and sensitivity data are summarised in Table 1. The limit of quantification was defined as the concentration of the lowest calibrator which was calculated to be within ± 2 of the nominal value and with a CV less than 2. This criteria was met by the lowest calibrator i.e..5 and.1 ng/ml when either 1 or 5 μl respectively of the collected sample was extracted. Intra-assay and interassay variation (as CV) were all found to be highly satisfactory at <6 (Table 2). Analytical recovery was estimated by comparing the responses of a 5 ng/ml calibrator (using 1 μl of oral fluid) when the non-deuterated compounds were added before the extraction step (n= 3) with those obtained when the non-deuterated analytes were added after sample preparation (n= 3). The recovery was found to be satisfactory at 85.6 ±.5 The stability of THC in oral fluid collected by the Intercept device was assessed by spiking oral fluid with THC at 3 different concentrations (1, 1 and 1 ng/ml) and then monitoring the stability at 4 C and at room temperature over a period of 48 hours. No statistical significant differences could be observed for the three different concentrations in both conditions. The stability of the samples post extraction was assessed by repeated injections of extracted samples over a period of 15 hours. No instability was noted over the course of this experiment. Cannabidiol and cannabinol are two components that are also naturally-occuring in the Cannabis sativa plant. Since the m/z for the precursor mass of cannabinol is different to that of THC, it does not interfere in its quantitation. On the other hand, the protonated molecular species of cannabidiol i.e. m/z is the same as that of THC. Furthermore it shows the same product ions after collision induced dissociation. Thus chromatographic separation is essential to distiguish between these 2 isobaric compounds. Analysis of standards showed cannabidiol to be chromatographically resolved from THC (Figure 4). The utility of the LC/MS/MS method was demonstrated by the analysis of 12 authentic samples collected from volunteers who smoked a placebo or marijuana cigarette. Figure 5 shows the values for THC in oral fluid collected after smoking the marijuana cigarette; mean values are plotted as a function of time. All specimens collected prior to smoking were negative, with the exception of 3 samples where concentrations were very low (maximum 2.2 ng/ml). Peak concentrations occurred.5 hour after smoking. Thereafter concentrations decreased steadily. There was considerable inter-individual variation in the observed concentrations; this has also been reported by other authors 7 and may also be as a result of the lack of exact volume measurement in the device. Forty eight samples were also collected from drivers intercepted at Belgian roadblocks. Table 3 summarises the quantitative results for all positive samples and Figure 6 shows a MRM chromatogram for one such marijuana user; the presence of cannabidiol was also noted (at 3.28 min) in this specimen. Linearity Data slope* intercept* CV of slope ( over 5 r 2 (range of 5 Sensitivity Data volume oral fluid consecutive days) consecutive days) LOQ (ng/ml) 1μL μL 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

32 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) 1 μl μl Table 2. Precision and accuracy data for THC for the extraction of 1 μl and 5 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) Figure 4. LC/MS/MS analysis of THC-d3 (top trace), THC, cannabidiol (middle trace) and cannabinol (bottom trace). Peak intensity is shown in the top right-hand corner of each trace 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.5 h,.25 h,.5 h, 1 h, 1.25 h and 1.5 h after smoking. Concentrations plotted on the Y-axis are expressed as ng/ml. The central box represents the values from the lower to upper quartile (25 to 75 percentile). The middle line represents the median. The horizontal line extends from the minimum to the maximum value, excluding outside (not present) and far out values (cross marker) which are displayed as separate points. Figure 6. Typical MRM chromatograms obtained following the analysis of an authentic oral fluid specimen obtained from a driver in a roadside setting. The calculated concentrations was 5.7 ng/ml. The figure shows the response for THC-d3 (top trace) and for the two transitions of THC (quantifier and qualifier middle and bottom trace respectively). Peak intensity is shown in the top right-hand corner of each trace. 32

33 Conclusions To the very best of our knowledge, the method presented here is the first demonstration of the use of LC/MS/MS for the analysis of THC in oral fluid samples collected with the Intercept device. The method is simple and comprises simple liquid/liquid extraction followed by LC/MS/MS. The method demonstrates high recovery, excellent precision and accuracy when using either 1 or 5 μl sample. The LOQ is sufficiently low to meet the requirements of SAMHSA (2 ng/ml) for oral fluid testing. Pharmacokinetic studies may require lower LOQ s; these requirements can be met by using larger volumes of oral fluid. The method was successfully applied to the analysis of samples collected in a controlled cannabis smoking study and to samples collected at the roadside by Belgian police. References 1. K.A. Mortier, K.E. Maudens, W.E. Lambert, K.M. Clauwaert, J.F. Van Boxlaer, D.L. Deforce, C.H. Van Peteghem and A.P. De Leenheer, J. Chromatogr. B 779 (22) R. Dams, C.M. Murphy, R.E. Choo, W.E. Lambert, A.P. De Leenheer and M.A. Huestis, Anal. Chem. 75 (23) E.J. Cone, L. Presley, M. Lehrer, W. Seiter, M. Smith, K.W. Kardos, D. Fritch, S. Salamone, S. Niedbala, J. Anal. Toxicol. 26 (22) EU Project ROSITA Roadside Testing Assessment M. Wood, M. Laloup, M. Ramirez Fernandez, K.M. Jenkins, M.S. Young, J.G. Ramaekers, G. De Boeck, N. Samyn, Forensic Sci. Int, in press. 6. R. Bonfiglio, R.C. King, T.V. Olah, K. Merkle, Rapid Commun. Mass Spectrom. 13 (1999) R.S Niedbala, K.W. Kardos, D.F. Fritch, S. Kardos, T. Fries, J. Waga, J. Robb, E.J. Cone, J. Anal. Toxicol. 25 (21)

34 Simultaneous Analysis of GHB and its Precursors in Urine Using LC/MS/MS Michelle Wood 1, Marleen Laloup 2, Nele Samyn 2, Michael Morris 1, Peter Batjoens 3 and Gert De Boeck 2 1 Waters Corp., Manchester, UK; 2 National Institute of Criminalistics and Criminology (N.I.C.C), Brussels, Belgium; 3 Waters Corp., Brussels, Belgium. Liquid Ecstasy Fantasy Introduction Gamma-hydroxybutyrate ( GHB) is a metabolite of gamma-aminobutyric acid (GABA) and plays the role of a central neurotransmitter and neuromodulator. Since GHB is a normal component of mammalian metabolism, it is present in all tissues of the body. Typical urinary GHB concentrations are < 1 mg/l 1,2. In some countries GHB is used clinically as an intravenous anaesthetic and as a treatment for narcolepsy, alcoholism and opiate withdrawal. Over the last few years, GHB has been gaining popularity amongst club-goers as the recreational drug (Figure 1) where it is taken for its ability to produce feelings of euphoria and to enhance sexuality 3-5. As a result of its potent prosexual effects, GHB has also been increasingly implicated in drug-facilitated sexual assault 6,7. Ingestion of the chemical precursors of GHB i.e. gamma-butyrolactone (GBL) and 1,4-butanediol (1,4-BD) also results in similar physiological effects since they are rapidly converted to GHB in the body 8. Raised awareness of the effects of these drugs and their potential for misuse, in addition to their ease of availability, has resulted in a dramatic increase in the demand for their analytical determination in both biological specimens and putative drug preparations. The purpose of this study was to develop and validate a sensitive LC/MS/MS procedure that would enable the simultaneous quantification of GHB, GBL and 1,4- BD in urine. Methods and Instrumentation Easy Lay Blue nitro concentrations;, 1, 2, 5, 1, 2, 5 and 8 mg/l. Low and high QC samples were prepared by spiking control urine with the drugs to yield concentrations of 4 and 4 mg/l, respectively. Authentic Samples One hundred and eighty two authentic human urine samples were collected from club-goers attending a post dance-club chill-out venue and were the result of 2 separate raids by the Belgian Police Department. The samples were analysed for GHB and the precursors using the newly-developed LC/MS/MS procedure. For comparative purposes, the samples were also analysed for GHB using a routinely used GC/MS procedure. Sample Preparation Urine samples were diluted (1:2) with an internal standard solution ( GHB-d6 and GBL-d6, at a concentration of 2 mg/l in deionised water). LC Conditions LC system: Waters Alliance 2795 Column: Waters Atlantis dc 18 column (3 x 1 mm, 5 μm) at 35 C Mobile phases: (A):.1 aqueous formic acid (B): methanol Isocratic elution: 9:1 (A:B) Flow rate: 2 μl/min. Inj. volume: 2 μl Mass Spectrometry Conditions Mass spectrometer: Quattro micro mass spectrometer Ionisation mode: ES + Capillary voltage: 3.5 kv Source Temperature: 12 C Desolvation gas: Nitrogen at 7 L/Hr, 35 C MS/MS: Collision gas (argon) at 5 x 1-3 mbar Samples Calibrators and quality control (QC) samples Control urine was spiked with GHB, GBL and 1,4-BD to yield a series of calibrators at the following 34

35 Results and Discussion Multiple reaction monitoring (MRM) transitions were determined for GHB, the precursors and the internal standards i.e. GHB-d6 and GBL-d6 (Table 1). Figure 2 shows some examples of product ion spectra. 1.8 x x 15 GHB 1,4,-BD 1.8 x x 15 Compound Precursor Product Cone Collision ion ion Voltage energy (m/z) (m/z) (V) (ev) GHB GHB-d GBL GBL-d ,4-BD Table 1: MRM transitions and conditions for the measurement of GHB, GBL, 1,4-BD and their deuterated internal standards. 4.3 x 14 GBL 4.3 x 14 Figure 3: MRM chromatograms obtained with a single injection of a control urine sample (left-hand column) prepared by the dilution method and the same sample enriched with 1 mg/l of GHB, GBL and 1,4-BD (right-hand column). Peak intensity is shown in the top right-hand corner of each trace. B A C A series of urine calibrators was prepared. Following preparation i.e. simple dilution, the samples were analysed using LC/MS/MS. Figure 3 shows the MRM chromatograms obtained following the analysis of a control urine sample and the same sample enriched with GHB, GBL and 1,4-BD. Quantification was achieved by integration of the area under the specific MRM chromatogram. For GHB and GBL, responses were calculated in reference to the integrated area of their respective deuterated internal standards. For 1,4-BD the response was calculated by reference to that of GHB-d6. Linear responses were obtained for GHB and 1,4-BD over the range investigated (1-8 mg/l). GBL produced a linear response over the range 1-5 mg/l. Precision, intra-assay and interassay variation (as CV) were all found to be highly satisfactory at < 7. Analytical recoveries ranged from 9-17 (Table 2). Figure 2: Product ion spectra for GHB (A), GBL (B) and 1,4- BD (C). Pure standards (5 mg/l) were infused into the mass spectrometer and the cone voltage (CV) optimised for the precursor ion*. CID was then performed and product ion spectra acquired under optimum conditions for the most abundant product ion. Compound Precision (n=5) Low Control High Control (4 mg/l) (4 mg/l Mean Recovery CV Mean Recovery CV (mg/l) () (mg/l) () GHB GBL ,4-BD Intra-assay (n=5) GHB GBL ,4-BD Intrerassay (n=5) GHB GBL ,4-BD Table 2: Precision and analytical recovery data for GHB and its precursors in urine. 35

36 A Only two, of these seven samples, were above the recommended interpretive cut-off concentration of 1 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 C Fig. 4: LC/MS analysis of the hydroxybutyric acid isomers. Ion chromatograms obtained following the analysis of gammahydroxybutyric acid ( GHB) only (A) and GHB in the presence of alpha and beta-hydroxybutyric acid (traces B and C respectively). Peak intensity is shown in the top right-hand corner of each trace and is the sum of the responses obtained for both the protonated and the sodiated species species i.e. m/z The limit of quantification was defined as the concentration of the lowest calibrator which was calculated to be with in ± 2 of the nominal value and with a CV less than 2. For all of the analytes of interest, this criteria was met by the 1 mg/l calibrator and was sufficient to determine endogenous levels of GHB in the urine. To investigate any potential interference in GHB quantification by its naturally occuring isomers i.e. alpha and beta-hydroxybutyric acid, standards were analysed using the developed LC/MS/MS method. Both compounds were shown to be chromatographically resolved from GHB and thus would not interfere in the quantification of the latter (Figure 4). The utility of the LC/MS/MS method was demonstrated by the analysis of one hundred and eighty-two authentic urine samples. Seven samples contained GHB at concentrations > 2 mg/l. The same seven samples were independently identified by the more timeconsuming, labour-intensive GC/MS method. Conclusions To the very best of our knowledge, the method presented here is the first demonstration of the use of LC/MS/MS for the simultaneous analysis of GHB and its precursors in urine samples. The method is simple and rapid (total analysis time of <12 mins). The method offers sufficient sensitivity to enable the measurement of endogenous levels of GHB and to identify exogenous ingestion. The LC/MS/MS results obtained following the analysis of authentic samples, correlated with the more labour-intensive, time-consuming (~1 hour) GC/MS method. The procedure offers several advantages over other published techniques; 1. It enables the simultaneous quantification of the GHB and the precursors in a single analysis; this can facilitate the identification of the chemical basis of any seized putative drug preparations or if present in the biological specimen, can provide information of the chemical nature of the ingested drug. 2. It involves fewer manipulations and is less timeconsuming. Although the data presented here indicate that the actual prevalence of GHB-positives might be quite low, the hype and publicity surrounding these drugs has led to a dramatic increase in the number of requests for their analysis in biological samples (and particularly in urine). The simplicity and speed of the described LC/MS/MS technique, should prove a useful means to meet this current increased demand on laboratories. 36

37 References 1. Elliott SP. Gamma hydroxybutyric acid ( GHB) concentrations in humans and factors affecting endogenous production. Forensic Sci. Int 23;133: 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 22;26: 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: Ropero-Miller JD and Goldberger BA. Recreational drug current trends in the 9 s. Clin. Lab Med 1998;18: Bellis MA, Hughes K, Bennett A and Thomson R. The role of an international nightlife resort in the proliferation of recreational drugs. Addiction 23;98: ElSohly MA and Salamone SJ. Prevalence of drugs used in cases of alleged sexual assault. J. Anal. Toxicol 1999;23: Ferrara SD, Frison G, Tedeschi L and LeBeau MA. Gammahydroxybutyrate ( GHB) and related products. In: LeBeau MA and Mozayani A, eds. Drug-Facilitated Sexual Assault (DFSA): A Forensic Handbook. London: Academic Press, 21: 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: Fieler EL, Coleman DE and Baselt RC. GHB concentrations in pre and post-mortem blood and urine [Letter]. Clin. Chem 1998;44:

38 Determination of Aconitine in Body Fluids by LC/MS/MS Justus Beike 1, Lara Frommherz 1, Michelle Wood 2, Bernd Brinkmann 1 and Helga Köhler 1 1 Institute of Legal Medicine, University Hospital Münster, Röntgenstrasse, Münster, Germany 2 Clinical Applications Group, Waters Corporation, Simonsway, Manchester M22 5PP, UK. Introduction Plants of the genus Aconitum L (family of Ranunculaceae) are known to be among the most toxic plants of the Northern Hemisphere and are widespread across Europe, Northern Asia and North America. Two plants from this genus are of particular importance: the blue-blooded Aconitum napellus L. (monkshood) which is cultivated as an ornamental plant in Europe and the yellow-blooded Aconitum vulparia Reich. (wolfsbane) which is commonly used in Asian herbal medicine 1 (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 effects 2-4. These effects can be attributed to the presence of the alkaloids; the principal alkaloids are aconitine, mesaconitine, hypaconitine and jesaconitine. The use of the alkaloids as a homicidal agent has been known for more than 2 years. Although intoxications by aconitine are rare in the Western Hemisphere, in traditional Chinese medicine, the use of aconite-based preparations is common and poisoning has been frequently reported. Poisoning has occurred both during clinical use and also as consequence of accidental ingestion e.g. by eating plant material or Aconitum preparations 5, 6. The use of aconite tubers for suicide and homicide purposes has also been reported 7. The first symptoms of aconitine poisoning appear ~2 min to 2 hours after oral uptake and include paraesthesia, sweating and nausea. This leads to severe vomiting, colicky diarrhea, intense pain and then paralysis of the skeletal muscles. Following the onset of life-threatening arrhythmia, including ventricular tachycardia and ventricular fibrillation, death finally occurs as a result of respiratory paralysis or cardiac arrest 5-7. Clearly in the case of suspected aconitine intoxication there is a need for rapid analytical techniques to enable prompt diagnosis and treatment. To this end we have developed a simple LC/MS/MS method for the determination of aconitine in various body fluids 8. The method was fully validated for the determination of aconitine from whole blood samples and applied in two cases of fatal poisoning. Figure 1: Aconitum napellus (monkshood) (A) and Aconitum vulparia (wolfsbane) (B). Methods and Instrumentation Sample preparation Biological samples were prepared for LC/MS/MS by means of a solid-phase extraction (SPE) procedure. Blood and tissue samples (.5 g each) were mixed with 3 ml of.15 M phosphate buffer ph 6., homogenised and centrifuged at 5 g for 1 min. The supernatants were decanted and loaded on a prepared SPE cartridge. Cartridges were preconditioned with 3 ml methanol, 3 ml water and 1 ml of.15 M phosphate buffer ph 6.. Samples were allowed to pass through the cartridge under gravity, before an initial wash step (3 ml water followed by 1 ml.1 M HCl) was performed. Two further washing steps i.e. 2 ml dichloromethane, followed by 2 ml methanol, were performed before elution of the aconitine. Cartridges were dried under vacuum between each of the 3 wash steps. Aconitine was eluted (2 x 1.5 ml) with a mixture of dichloromethane:2-propanol:25 aqueous ammonia (8:2:2). Eluents were pooled and evaporated to dryness under a stream of nitrogen at 4 C before reconstitution with 1 μl LC mobile phase. 38

39 LC/MS/MS A Quattro micro tandem mass spectrometer fitted with Z-Spray ion interface was used for all analyses. Ionisation was achieved using electrospray in the positive ionisation mode (ES+). Detection of aconitine was performed using multiple reaction monitoring (MRM). The transition MRM transition m/z > m/z was used for quantification purposes and a further two transitions i.e. m/z > m/z and m/z > m/z were monitored for confirmatory purposes. LC analyses were performed using an Alliance 2695 separations module (Waters). Chromatography was achieved using a Waters XTerra RP 8 pre-column (2.1 x 1 mm, 3.5 μm) and a XTerra RP 8 analytical column (2.1 x 15 mm, 3.5 μm). The column was maintained at 4 C and eluted isocratically with.1 ammonium acetate (adjusted to ph 6. with 1 M acetic acid) and methanol (5:5) at 2 μl/min. The injection volume was 1 μl and a total run time of 1 min was used. All aspects of system operation and data acquisition were controlled using MassLynx NT 4. software with automated data processing using the QuanLynx program (Waters). Aconitine concentration Case no. Blood [ng/g] Stomach content [ng/g] Urine [ng/ml] Not available Not available 18. Table 1: Concentrations of aconitine in autopsy samples from two cases of fatal aconite intoxication. Results A series of calibrators (.1 25 ng/g) were prepared in duplicate by adding aconitine standards to control blood. Samples were then extracted, using the SPE method described above, prior to LC/MS/MS analysis. In two forensic cases of suspected aconitine intoxication, aconitine was detected in the blood samples and also in the stomach content and urine of the deceased (Table 1). Figure 3 shows the chromatogram of the blood sample of aconite victim no 2. At the time of autopsy the body was already in an advanced state of putrefaction. Despite these difficult circumstances, the chromatogram shows a strong signal for aconitine MRM of 3 Channels ES > > > e3 Time Figure 2: MRM chromatograms for a blood calibrator spiked at.1 ng aconitine/g blood. Peak intensity is given in the top righthand corner of the trace. Summary We have developed a rapid and sensitive method for the quantification of aconitine in biological specimens. The method involves a simple SPE purification prior to analysis using LC/MRM. The utility of the method was demonstrated by its application to authentic samples in 2 fatal cases of suspected aconitine poisoning. Blood, urine and stomach contents were collected during autopsy and analysed using the developed LC/MS/MS method. Aconitine could be detected in the blood of both victims, in the stomach content of one individual and in the urine of the other. Following analysis, the areas under the specific MRM chromatograms were integrated. The response was linear (r 2 =.999) over the range investigated. The limit of detection (LOD) of the assay was estimated at.1 ng/g blood. Figure 2 shows the responses for the quantifier and qualifier ions of aconitine obtained with a calibrator spiked at the LOD. 39

40 1 4.8 MRM of 3 Channels ES > > > e5 Time 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, , Springer Berlin, Heidelberg. 2. Hikino H, Konno C, Takata H, Yamada Y, Yamada C, Ohizumi Y, Sugio K, Fujimura H (198). Antinflammatory principles of Aconitum roots. J Pharmacobiodyn 3: Desai HK, Hart BP, Caldwell RW, Jianzhong-Huang JH, Pelletier SW (1998). Certain norditerpenoid alkaloids and their cardiovascular action. J Nat Prod 61: Ameri A (1998). The effects of Aconitum alkaloids on the central nervous system. Prog Neurobiol 56: 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: 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: Ito K, Tanaka S, Funayama M, Mizugaki M (2). Distribution of Aconitum Alkaloids in body fluids and tissues in a suicidal case of aconite ingestion. J Analytical Toxicol 24: 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: (24). 4

41 Published References Quantitative Analysis of Δ 9 -Tetrahydrocannabinol in Preserved Oral Fluid by Liquid Chromatography Tandem Mass Spectrometry Marleen Laloup a, Maria del Mar Ramirez Fernandez a, Michelle Wood b, Gert De Boeck a, Cécile Henquet c, Viviane Maesd, Nele Samyna a National Institute of Criminalistics and Criminology (NICC), Section Toxicology, Vilvoordsesteenweg 98, 112 Brussels, Belgium b Waters Corporation, MS Technologies Centre, Manchester, UK c Department of Psychiatry and Neuropsychology, South Limburg Mental Health Research and Teaching Network, EURON, Maastricht University, Maastricht, The Netherlands d Department of Clinical Chemistry-Toxicology, Academic Hospital, Free University of Brussels, Brussels, Belgium Abstract A rapid and sensitive method for the analysis of Δ 9 -Tetrahydrocannabinol (THC) in preserved oral fluid was developed and fully validated. Oral fluid was collected with the Intercept, a Food and Drug Administration (FDA) approved sampling device that is used on a large scale in the U.S. for workplace drug testing. The method comprised a simple liquid liquid extraction with hexane, followed by liquid chromatography tandem mass spectrometry (LC/MS/MS) analysis. Chromatographic separation was achieved using a XTerra MS C 18 column, eluted isocratically with 1mM ammonium formate methanol (1:9, v/v). Selectivity of the method was achieved by a combination of retention time, and two precursor-product ion transitions. The use of the liquid liquid extraction was demonstrated to be highly effective and led to significant decreases in the interferences present in the matrix. Validation of the method was performed using both 1 and 5 μl of oral fluid. The method was linear over the range investigated (.5 1 ng/ml and.1-1 ng/ml when 1 and 5 μl, respectively, of oral fluid were used) with an excellent intra-assay and inter-assay precision (relative standard deviations, RSD <6) for quality control samples spiked at a concentration of 2.5 and 25 ng/ml and.5 and 2.5 ng/ml, respectively. Limits of quantification were.5 and.1 ng/ml when using 1 and 5 μl, respectively. In contrast to existing GC/MS methods, no extensive sample clean-up and time-consuming derivatization steps were needed. The method was subsequently applied to Intercept samples collected at the roadside and collected during a controlled study with cannabis. Journal of Chromatography A, 182 (25) Simultaneous Analysis of Gamma-Hydroxybutyric Acid and its Precursors in Urine using Liquid Chromatography Tandem Mass Spectrometry Michelle Wood a,?, Marleen Laloup b, Nele Samyn b, Michael R. Morris a, Ernst A. de Bruijn c, Robert A. Maes c, Michael S. Young d, Viviane Maes e, Gert De Boeck b a Waters Corporation, MS Technologies Centre, Micromass UK Ltd., Atlas Park, Simonsway, Wythenshawe, Manchester M22 5PP, UK b National Institute of Criminalistics and Criminology (N.I.C.C.), Section Toxicology, Vilvoordsesteenweg 98, 112 Brussels, Belgium c Department of Human Toxicology, Utrecht Institute of Pharmaceutical Sciences (UIPS), University of Utrecht, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands Abstract We have developed a rapid method that enables the simultaneous analysis of gamma-hydroxybutyrate ( GHB) and its precursors, i.e. gamma-butyrolactone (GBL) and 1,4-butanediol (1,4-BD) in urine. The method comprised a simple dilution of the urine sample, followed by liquid chromatography tandem mass spectrometry (LC/MS/MS) analysis. Chromatographic separation was achieved using an Atlantis dc 18 column, eluted with a mixture of formic acid and methanol. The method was linear from 1 8 mg/l for GHB and 1,4-BD and from 1 5 mg/l for GBL. The limit of quantification was 1 mg/l for all analytes. The procedure, which has a total analysis time (including sample preparation) of less than 12 min, was fully validated and applied to the analysis of 182 authentic urine samples; the results were correlated with a previously published GC/MS procedure and revealed a low prevalence of GHB-positive samples. Since no commercial immunoassay is available for the routine screening of GHB, this simple and rapid method should prove useful to meet the current increased demand for the measurement of GHB and its precursors. Journal of Chromatography A, 156 (24) 83 9 Development of a Rapid and Sensitive Method for the Quantitation of Amphetamines in Human Plasma and Oral Fluid by LC/MS/MS M. Wood [1], G. De Boeck [2 ], N. Samyn [2], M. Morris [1], D.P. Cooper [1], R.A.A. Maes [3], and E.A. De Bruijn [3] [1] Micromass U.K. Limited, Atlas Park, Simonsway, Wythenshawe, Manchester M22 5PP, United Kingdom; [2] National Institute of Criminalistics and Criminology (NICC), Section Toxicology, Vilvoordsesteenweg 98, 112 Brussels, Belgium; and [3] Utrecht Institute of Pharmaceutical Sciences (UIPS), Department of Human Toxicology, University of Utrecht, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands Abstract Target analysis of amphetamines in biological samples is of great importance for clinical and forensic toxicologists alike. At present, most laboratories analyze such samples by gas chromatography mass spectrometry. However, this procedure is labor-intensive and time-consuming, particularly as a preliminary extraction and derivatization are usually unavoidable. Here we describe the development of an alternative method. Amphetamines were isolated from human plasma and oral fluid using a simple methanol precipitation step and subsequently analyzed using reversed-phase liquid chromatography tandem mass spectrometry. Quantitation of the drugs was performed using multiple reaction monitoring. The developed method, which requires only 5 μl of biological sample, has a total analysis time of less than 2 min (including sample preparation) and enables the simultaneous quantitation of 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxyethylamphetamine, amphetamine, methamphetamine, and ephedrine in a single chromatographic run. Limits of detection of 2 μg/l or better were obtained. The method has been validated and subsequently applied to the analysis of plasma and oral fluid samples collected from current drug users. Journal of Analytical Toxicology, Volume 27, Number 2, March 23, pp Development of a Rapid and Sensitive Method for the 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] [1] Waters Corporation, MS Technologies Centre, Atlas Park, Manchester, United Kingdom; [2] National Institute of Criminalistics and Criminology (NICC), Section Toxicology, Brussels, Belgium; [3] Department of Anatomo-Pathology, Academic Hospital, Free University of Brussels, Belgium; [4] Utrecht Institute of Pharmaceutical Sciences (UIPS), Department of Human Toxicology, University of Utrecht, The Netherlands; and [5] Department of Clinical Chemistry-Toxicology, Academic Hospital, Free University of Brussels, Belgium Abstract Liquid chromatography tandem mass spectrometry (LC/MS/MS) is emerging as the tool of choice for rapid analysis and the detection of biologically active compounds in complex mixtures. We describe the development of a sensitive method for the simultaneous quantitation of 1 benzodiazepines in Calliphora vicina (Diptera: Calliphoridae) larvae and puparia. The use of larvae for toxicological analyses offers some technical advantages over putrefied tissue. Four sample pretreatment methods for isolating the benzodiazepines out of larvae were evaluated. A simple homogenization, followed by acetonitrile precipitation yielded the highest recoveries. Puparia were pulverized and extracted by ultrasonification in methanol. All extracts were subsequently analyzed using reversed-phase LC/MS/MS. Larvae and puparia calibrators containing benzodiazepines at concentrations ranging from 25 to 75 pg/mg and 5 to 5 pg/mg, respectively, were prepared and analyzed. The method was demonstrated to be linear over the ranges investigated. Limits of detection were from 1.88 to 5.13 pg/mg larva and from 6.28 to 19.3 pg/mg puparium. The developed method was applied to the determination of nordiazepam and its metabolite oxazepam in larvae and puparia of the Calliphora vicina fly that had been reared on artificial foodstuff (beef heart) spiked with 1 μg/g nordiazepam. The larvae were harvested at day 5 for analysis of drug content. The method was sufficiently 41

42 sensitive to allow the detection of nordiazepam and oxazepam in a single larvae. Journal of Analytical Toxicology, Volume 27, Number 7, October 23, pp Determination of Aconitine in Body Fluids by LC/MS/MS J. Beike 1, L. Frommherz 1, M. Wood 2, B. Brinkmann 1 and H. Köhler 1 (1) Institute of Legal Medicine, University Hospital Münster, Röntgenstrasse 23, Münster, Germany (2) Waters Corporation, MS Technologies Centre, Atlas Park, Manchester, United Kingdom Abstract A very sensitive and specific method was developed for the determination of aconitine, the main toxic alkaloid from plants of the genus Aconitum L., in biological samples. The method comprised solid-phase extraction using mixed-mode C 8 cation exchange columns followed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). Chromatographic separation was achieved with a RP 8 column. Detection of aconitine was achieved using electrospray in the positive ionisation mode and quantification was performed using multiple reaction monitoring with m/z as precursor ion, i.e. [M+H]+ of aconitine and m/z 586.5, m/z and m/z as product ions after collision-induced dissociation. The method was fully validated for the analysis of blood samples: the limit of detection and the limit of quantitation were.1 ng/g and.5 ng/g, respectively. Within the linear calibration range of.5 25 ng/g, analytical recovery was In two fatal cases with suspected aconite intoxication, aconitine could be detected in blood samples at concentrations of 1. and 12.1 ng/g. In one case, aconitine could also be detected in the stomach content (3 ng/g) and in the other in the urine (18 ng/ml). International Journal of Legal Medicine, Volume 118, Number 5, October 24, pp Quantitative Analysis of Multiple Illicit Drugs in Preserved Oral Fluid by Solid-Phase Extraction and Liquid Chromatography Tandem Mass Spectrometry Michelle Wood a, Marleen Laloup b, Maria del Mar Ramirez Fernandez b, Kevin M. Jenkins c, Michael S. Young c, Jan G. Ramaekers d, Gert De Boeck b and Nele Samyn b, a Waters Corporation, MS Technologies Centre, Manchester, UK b Federal Public Service Justice, National Institute of Criminalistics and Criminology (NICC), Vilvoordsesteenweg 1, 112 Brussels, Belgium c Waters Corporation, Milford, MA, USA d Experimental Psychopharmacology Unit, Brain and Behaviour Institute, Maastricht University, Maastricht, The Netherlands Abstract We present a validated method for the simultaneous analysis of basic drugs which comprises a sample clean-up step, using mixed-mode solid-phase extraction (SPE), followed by LC/MS/MS analysis. Deuterated analogues for all of the analytes of interest were used for quantitation. The applied LC gradient ensured the elution of all the drugs examined within 14 min and produced chromatographic peaks of acceptable symmetry. Selectivity of the method was achieved by a combination of retention time, and two precursor-product ion transitions for the non-deuterated analogues. Oral fluid was collected with the Intercept, a FDA approved sampling device that is used on a large scale in the US for workplace drug testing. However, this collection system contains some ingredients (stabilizers and preservatives) that can cause substantial interferences, e.g. ion suppression or enhancement during LC/MS/MS analysis, in the absence of suitable sample pre-treatment. The use of the SPE was demonstrated to be highly effective and led to significant decreases in the interferences. Extraction was found to be both reproducible and efficient with recoveries >76 for all of the analytes. Furthermore, the processed samples were demonstrated to be stable for 48 h, except for cocaine and benzoylecgonine, where a slight negative trend was observed, but did not compromise the quantitation. In all cases the method was linear over the range investigated (2 2 μg/l) with an excellent intra-assay and inter-assay precision (coefficients of variation <1 in most cases) for QC samples spiked at a concentration of 4, 12 and 1 μg/l. Limits of quantitation were estimated to be at 2 μg/l with limits of detection ranging from.2 to.5 μg/l, which meets the requirements of SAMHSA for oral fluid testing in the workplace. The method was subsequently applied to the analysis of Intercept samples collected at the roadside by the police, and to determine MDMA and MDA levels in oral fluid samples from a controlled study. Forensic Science International, Volume 15, Issues 2-3, 1 June 25, Pages Recent Applications of LC/MS in Forensic Science G. De Boeck 1, M. Wood 2 and N. Samyn 1 1 National Institute of Criminalistics and Criminology, Brussels, Belgium, 2 Micromass UK Limited, Wythenshawe, UK. Introduction The term forensic science covers those professions that are involved in the application of the social and physical sciences to the criminal justice system. Forensic experts are obliged to explain the smallest details of the methods used, to substantiate the choice of the applied technique and to give their unbiased conclusions. The final result of the work of the forensic scientist, the expert evidence, exerts a direct influence on the fate of a given individual. This burden is a most important stimulus and one that determines the way of thinking and acting in forensic sciences. Consequently, the methods applied in forensic laboratories should assure a very high level of reliability and must be subjected to extensive quality assurance and rigid quality control programmes. 1 Legal systems are based on the belief that the legal process results in justice a belief that has come under some question in recent years. Of course, the forensic scientist cannot change scepticism and mistrust single-handedly. He or she can, however, contribute to restoring faith in the judicial processes by using science and technology in the search for facts in civil, criminal and regulatory matters. The ability of mass spectrometry (MS) to extract chemical fingerprints from microscopic levels of analyte is invaluable in this quest, enabling the legally defensible identification and quantification of a wide range of compounds. Recent years have seen the development of powerful technologies that have provided forensic scientists with new analytical capabilities, which were unimaginable only a few years ago. Gas chromatography GC/MS, liquid chromatography LC/MS, isotope ratio MS and inductively coupled plasma-ms have become routine tools to enable detection and characterization of minute quantities in what can often be very complex matrices. In LC/MS, there has been an explosion in the range of new products available for solving many analytical problems, particularly those applications in which non-volatile, labile and/or high molecular weight compounds are being analysed. Many analysts and laboratories have reached the point at which they are considering the acquisition of LC/MS instrumentation. According to Willoughby et al. LC/MS has progressed from the innovators stage through the early adaptors and on to the early majority stage, and is now open to specialists from a variety of disciplines. This has been as a direct result of the introduction of robust, user-friendly atmospheric pressure ionization (API)-MS instruments at an affordable price. LCGC Europe, Nov 2, 22 42

43 Plasma, oral fluid and sweat wipe ecstasy concentrations in controlled and real life conditions Nele Samyn a, Gert De Boecka, Michelle Wood b, Caroline T. J. Lamersc, Dick De Waardd, Karel A. Brookhuisd, Alain G. Verstraetee and Wim J. Riedelc a Drugs and Toxicology, Section Toxicology, National Institute of Criminalistics and Criminology, Vilvoordsesteenweg 1, 112, Brussels, Belgium b Micromass Ltd., Manchester, UK c Experimental Psychopharmacology Unit, Brain and Behaviour Institute, Maastricht University, Maastricht, The Netherlands d Department of Psychology, University of Groningen, Groningen, The Netherlands e Laboratory of Clinical Biology Toxicology, Ghent University Hospital, Ghent, Belgium Abstract In a double-blind placebo controlled study on psychomotor skills important for car driving (Study 1), a 75 mg dose of ±3,4-methylenedioxymethamphetamine ( MDMA) was administered orally to 12 healthy volunteers who were known to be recreational MDMA-users. Toxicokinetic data were gathered by analysis of blood, urine, oral fluid and sweat wipes collected during the first 5 hours after administration. Resultant plasma concentrations varied from 21 to 295 ng/ml, with an average peak concentration of 178 ng/ml observed between 2 and 4 hours after administration. MDA concentrations never exceeded 2 ng/ml. Corresponding MDMA concentrations in oral fluid, as measured with a specific LC/MS/MS method (which required only 5 μl of oral fluid), generally exceeded those in plasma and peaked at an average concentration of 1215 ng/ml. A substantial intra- and inter-subject variability was observed with this matrix, and values ranged from 5 to 6982 ng/ml MDMA. Somewhat surprisingly, even 4 5 hours after ingestion, the MDMA levels in sweat only averaged 25 ng/wipe. In addition to this controlled study, data were collected from 19 MDMA-users who participated in a driving simulator study (Study 2), comparing sober non-drug conditions with MDMA-only and multiple drug use conditions. In this particular study, urine samples were used for general drug screening and oral fluid was collected as an alternative to blood sampling. Analysis of oral fluid samples by LC/MS/MS revealed an average MDMA/ MDEA concentration of 1121 ng/ml in the MDMA-only condition, with large intersubject variability. This was also the case in the multiple drug condition, where generally, significantly higher concentrations of MDMA, MDEA and/ or amphetamine were detected in the oral fluid samples. Urine screening revealed the presence of combinations such as MDMA, MDEA, amph, cannabis, cocaine, LSD and psilocine in the multiple-drug condition. Forensic Science International, Volume 128, Issues 1-2, 14 August 22, Pages 9-97 Toxicological data and growth characteristics of single postfeeding larvae and puparia of Calliphora vicina (Diptera: Calliphoridae) obtained from a controlled nordiazepam study Karen Pien 1, Marleen Laloup 2, Miriam Pipeleers-Marichal 1, Patrick Grootaert 3, Gert De Boeck 2, Nele Samyn 2, Tom Boonen 4, Kathy Vits 4 and Michelle Wood 5 (1) Department of Pathology Academic Hospital, Free University of Brussels, Brussels, Belgium (2) Section Toxicology, National Institute of Criminalistics and Criminology (NICC), Brussels, Belgium (3) Department Entomology, Royal Belgian Institute of Natural Sciences, Brussels, Belgium (4) Section Micro-traces, National Institute of Criminalistics and Criminology (NICC), Brussels, Belgium (5) Micromass UK Limited, Wythenshawe Manchester, UK Abstract Larvae of the Calliphora vicina (Diptera: Calliphoridae) were reared on artificial food spiked with different concentrations of nordiazepam. The dynamics of the accumulation and conversion of nordiazepam to its metabolite oxazepam in post-feeding larvae and empty puparia were studied. Analysis was performed using a previously developed liquid chromatography-tandem mass spectrometry (LC/MS/MS) method. This method enabled the detection and quantitation of nordiazepam and oxazepam in single larvae and puparia. Both drugs could be detected in post-feeding larvae and empty puparia. In addition, the influence of nordiazepam on the development and growth of post-feeding larvae was studied. However, no major differences were observed for these parameters between the larvae fed on food containing nordiazepam and the control group. To our knowledge, this is the first report describing the presence of nordiazepam and its metabolite, oxazepam, in single Calliphora vicina larvae and puparia. International Journal of Legal Medicine, Volume 118, Number 4, August 24, pp To order any of these reprints contact your local Waters office, or go to 43

44 Compound Index 6-MonoacetylMorphine ( 6-MAM) , 2 Alprazolam Amphetamine Amphetamines , 5, 15, 17, 41 Benzodiazepines , 5, 23, 25, 26, 41 Cannabidiol , 31 Cannabinol Clonazepam Codeine Diazepam , 25 Dihydrocodeine (DHC) Ephedrine GHB , 5, 34, 35, 36, 37, 41 Heroin Lorazepam MDA , 16, 17, 42, 43 MDEA , 16, 17, 43 MDMA , 16, 17, 18, 42, 43 Methamphetamine Morphine , 7, 19, 21, 22 Morphine-3-Glucuronide , 21 Morphine-6-Glucuronide , 21, 22 Nordiazepam , 24, 25, 26 Oxazepam , 24, 26, 27, 28 Prazepam Temazepam Triazolam Δ 9 -Tetrahydrocannabinol , 29, 41 44

45 Notes 45

46 Notes 46

47 Notes 47

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