Quantification of Five Compounds with Heterogeneous Physicochemical Properties (Morphine, 6-Monoacetylmorphine, Cyamemazine, Meprobamate and Caffeine) in 11 Fluids and Tissues, Using Automated Solid-Phase Extraction and Gas Chromatography–Tandem Mass Spectrometry

Journal of Analytical Toxicology 2014;38:256 –264 doi:10.1093/jat/bku029 Article Quantification of Five Compounds with Heterogeneous Physicochemical Properties (Morphine, 6-Monoacetylmorphine, Cyamemazine, Meprobamate and Caffeine) in 11 Fluids and Tissues, Using Automated Solid-Phase Extraction and Gas Chromatography –Tandem Mass Spectrometry Fabien Bévalot1,2*, Charline Bottinelli1, Nathalie Cartiser3, Laurent Fanton2 and Jérôme Guitton3,4 1 Laboratoire LAT LUMTOX, 71 Avenue Rockefeller, Lyon 69003, France, 2Institut de Médecine Légale, Université de Lyon, Université Claude Bernard Lyon 1, 8 Avenue Rockefeller, 69373 Lyon cedex 08, France, 3Laboratoire de Toxicologie, ISPB-Faculté de Pharmacie, Université de Lyon, Université Claude Bernard Lyon 1, 8 Avenue Rockefeller, 69373 Lyon cedex 08, France and 4Laboratoire de Pharmacologie-Toxicologie, Centre Hospitalier Lyon-Sud, Hospices Civils de Lyon, 165 Chemin Grand Revoyet, 69495 Pierre Bénite cedex, France *Author to whom correspondence should be addressed. Email: An automated solid-phase extraction (SPE) protocol followed by gas chromatography coupled with tandem mass spectrometry was developed for quantification of caffeine, cyamemazine, meprobamate, morphine and 6-monoacetylmorphine (6-MAM) in 11 biological matrices [blood, urine, bile, vitreous humor, liver, kidney, lung and skeletal muscle, brain, adipose tissue and bone marrow (BM)]. The assay was validated for linearity, within- and between-day precision and accuracy, limits of quantification, selectivity, extraction recovery (ER), sample dilution and autosampler stability on BM. For the other matrices, partial validation was performed (limits of quantification, linearity, within-day precision, accuracy, selectivity and ER). The lower limits of quantification were 12.5 ng/mL(ng/g) for 6-MAM, morphine and cyamemazine, 100 ng/mL(ng/g) for meprobamate and 50 ng/ mL(ng/g) for caffeine. Analysis of real-case samples demonstrated the performance of the assay in forensic toxicology to investigate challenging cases in which, for example, blood is not available or in which analysis in alternative matrices could be relevant. The SPE protocol was also assessed as an extraction procedure that could target other relevant analytes of interest. The extraction procedure was applied to 12 molecules of forensic interest with various physicochemical properties (alimemazine, alprazolam, amitriptyline, citalopram, cocaine, diazepam, levomepromazine, nordazepam, tramadol, venlafaxine, pentobarbital and phenobarbital). All drugs were able to be detected at therapeutic concentrations in blood and in the alternate matrices. Introduction In forensic toxicology, analyzing matrices (fluids and tissues) other than blood are widely agreed to be an important issue in various situations such as exsanguinations, putrefaction, postmortem redistribution, etc. (1). Pharmacokinetic and toxicological data (distribution, accumulation, metabolism, toxic concentration, etc.) are necessary to improve the interpretation of analyte concentrations found. The reliability of data from experimental studies and routine analysis of real postmortem cases depends on assay performance, and more specifically on the limits of quantification, accuracy and precision. Advances in analytical technology, especially in the field of mass spectrometry, have significantly improved the specificity and sensitivity of drug detection in complex samples, matrix effects; however, they remain an acknowledged pitfall. Ideally, assays need to be adapted to and validated for each matrix. In routine laboratory practice, however, processing different matrices are not always practical, as testing would involve analysis of multiple standard curves and controls depending on the matrix under investigation. A better approach is to organize testing as to limit the number of different assays required. This approach reduces both the time needed for staff training and the costs associated with supplies, standards and instrument maintenance. In this context, an assay was developed based on automated solid-phase extraction (SPE) followed by gas chromatography coupled with tandem mass spectrometry (GC – MS-MS) for the quantification of five different compounds [caffeine, cyamemazine, meprobamate, morphine and 6-monoacetylmorphine (6-MAM)] with heterogeneous physicochemical properties representative of the kinds of drugs encountered in real-life forensic toxicology (Table I). The SPE protocol proved effective in removing interference and was more reproducible than multistep manual liquid – liquid extraction. Moreover, SPE can be automated, which is important in view of the large number of samples to be processed in validation protocols and in routine practice. Liquid chromatography coupled with tandem mass spectrometry is prone to matrix effects that are difficult to control in postmortem toxicology, especially with complex samples (6); GC – MS-MS using labeled isotopes as internal standards (ISs) and matrix-matched calibration, in contrast, showed well-adapted performance. The assay was validated in 11 biological matrices [blood, urine, bile, vitreous humor (VH), liver, kidney, lung, skeletal muscle, brain, adipose tissue (AT) and bone marrow (BM)] following a cross-validation plan in compliance with FDA guidelines (7). The assay was first applied to real samples obtained from a human autopsy. Secondly, it was assessed as a standard process for further development of targeted analysis and screening. For this purpose, an extended application test evaluated the performance of the automated SPE protocol, without modification, in 12 drugs frequently encountered in forensic toxicology (alimemazine, alprazolam, amitriptyline, citalopram, cocaine, diazepam, levomepromazine, nordazepam, tramadol, venlafaxine, pentobarbital and phenobarbital) in blood, BM and lung samples. The present study reports a validated GC – MS-MS procedure with an automated SPE protocol to quantify five drugs of forensic interest in 11 biological matrices. The interest of the finding is illustrated by two applications: analysis of human samples from a real autopsy case and assessment of the protocol as a starter set for the development of further analytical tools. # The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: Table I Physicochemical Properties and Classification of the Molecules Studied Molecules logP pKa Vd (L/kg) ATC Classification 6-MAM Caffeine – 0a – 0.8b (amine) – 0.4 – 0.6b Cyamemazine (data from levomepromazine) 4.7a 9.2c 23 –42b Meprobamate 0.7a 0.7b Morphine 0.89d 4.2b (carisoprodol) 8.0c – Psychostimulant xanthine derivative Antipsychotic, phenothiazine with aliphatic side chain Anxiolytic, carbamate 2.0 – 5.0b Analgesic, opioid a Ref. (2). Ref. (3). Ref. (4). d Ref. (5). ATC, World Health Organization Anatomical Therapeutic Chemical classification system; Vd, Volume of distri (...truncated)