In Vitro Metabolism Studies on Methylenedioxy-Substituted Amphetamines Using Human Liver Microsomes and LC/MS/MS with Chemical Derivatization

Yokchue, T. and Anderson, R. (2015) In Vitro Metabolism Studies on Methylenedioxy-Substituted Amphetamines Using Human Liver Microsomes and LC/MS/MS with Chemical Derivatization. In: International Association of Forensic Toxicologist Meeting, Atlanta, GA, USA, 18-23 Oct 2015,

Abstract

The abuse of β-keto derivatives of MDMA such as butylone and methylone has been reported since the mid-2000s. The forensic toxicologist faces problems with these drugs, because of lack of information on their metabolism and unavailability of reference standards of the metabolites and the parent drugs themselves. Ethical considerations usually preclude human metabolism studies under controlled conditions and in vitro methods provide potential alternatives. A few previous studies showed that β-keto derivatives of MDMA have similar metabolic pathways to MDMA, resulting in metabolites formed by demethylenation, demethylenation plus O-methylation (4-OH-3-MeO metabolites) and N-demethylation. Another challenge is the analysis of these polar metabolites using conventional C18 HPLC columns, resulting in low retention volumes. One rapid and simple solution to this is chemical derivatization. Conversion of metabolites to more hydrophobic compounds by derivatization can improve separations by reversed-phase HPLC and increase sensitivity in mass spectrometry. Aims: To identify metabolites of butylone and methylone in humans using human liver microsomes (HLM) in vitro and reversed-phase LC/MS/MS with derivatization. MDMA was used in this study as a model compound as its metabolites are commercially available. Method: Pooled human liver microsomes were incubated with NADPH regenerating system and MDMA, butylone or methylone in 0.1 M phosphate buffer pH 7.4 in a shaking incubator at 37oC for 90 min. Each reaction was stopped by the addition of ice-cold acetonitrile and extracts were derivatised with acetic anhydride/pyridine (3:2 v:v) for 30 min at 60oC. Derivatised metabolites were identified by LC/MS/MS using multiple reaction monitoring (MRM). Ten MDMA positive urines were also analysed to compare the metabolite patterns with those obtained in vitro with HLM. Results: Three phase-I metabolites (both major and minor metabolites) of MDMA, butylone and methylone were detected. For MDMA, 3,4-dihydroxymethamphetamine derivative (HHMA-3Ac) was identified by MRM transitions at m/z 308>266, 308>224 and 308>151, 4-hydroxy-3-methoxymethamphetamine derivative (HMMA-2Ac) at m/z 280>238, 280>165 and 280>137 and MDA-Ac at m/z 222>163, 222>135 and 222>105. For methylone, dihydroxymethcathinone (DHMC), a major metabolite, was identified by MRM transitions at m/z 322>280 and 322>178 (DHMC-3Ac). Nor-methylone (a minor metabolite) was identified by MRM transitions at m/z 236>146 and 236>118 (bk-MDC-Ac). Another metabolite, 4-hydroxy-3-methoxymethcathinone (4-HMMC) was identified by MRM transitions at m/z 294>234 and 294>160 (4-HMMC-2Ac). Furthermore, a major metabolite of butylone (demethylenation metabolite) was identified by MRM transitions at m/z 336>294 and 336>174 (butylone-M dihydroxy-3Ac). Nor-butylone was identified by MRM transitions at m/z 250>160 and 250>132 (bk-BDB-Ac). Finally, the 4-OH-3-MeO metabolite was identified by MRM transitions at m/z 308>248 and 308>174 (butylone-M (demethylenyl-methyl-) 2Ac). Derivatives of MDMA and its metabolites were stable in the HPLC mobile phase for 30 h at room temperature and were quantified in all 10 positive human urine samples. HMMA was the major metabolite in human urine samples whereas HHMA was the major metabolite by HLM. However, HHMA is an intermediate metabolite leading to HMMA. This result confirms that demethylenation of the methylenedioxy ring, followed by catechol-O-methyltransferase (COMT)- catalysed methylation is a major metabolic pathway of MDMA in humans. Conclusions: Human liver microsomes can be used to simulate drug metabolism in humans and to provide chromatographic and mass spectrometric data on metabolites. Acetate derivatives result in higher molecular weights, providing more specific ions for identification, and in decreased polarities of metabolites, improving their analysis on reversed phase C18 columns. This method would be suitable for routine analysis of urine to detect and confirm abuse of methylenedioxy-substituted amphetamines.

• Item Type: Conference Proceedings
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Anderson, Dr Robert
• Authors: Yokchue, T., and Anderson, R.
• College/School: College of Medical Veterinary and Life Sciences > School of Medicine, Dentistry & Nursing

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