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Use of Liver Homogenates for Rapid Generation of Phase I Metabolites to Facilitate Characterization of Emerging Drugs of Abuse by High Resolution Liquid Chromatography-Mass Spectrometry
Use of Liver Homogenates for Rapid Generation of Phase I Metabolites to Facilitate Characterization of Emerging Drugs of Abuse by High Resolution Liquid Chromatography-Mass Spectrometry
Submitted on 03 Apr 2017

Anna Holderbaum, Chris T. Elliott, Tom Buckley, Mark H. Mooney
Institute for Global Food Security, School of Biological Sciences, Queen's University Belfast
This poster was presented at Pittcon 2017
Poster Views: 781
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Poster Abstract
Microsomal or S9 fractions are typically used to produce drug metabolites for preliminary in vitro metabolism investigations and the development of targeted mass spectrometric methods. However, liver microsomes or S9 fractions are not readily available for many species and the production thereof is tedious, time-consuming and specialized equipment is needed. Simple, rapid and cost-effective in vitro strategies that can promptly respond to analytical challenges in the control and monitoring of emerging illegal drug use in the livestock sector are therefore required. The aim of this work was to investigate the potential of bovine liver homogenates to rapidly generate and characterise phase I metabolites of selective androgen receptor modulator (SARM) compounds. Bovine liver homogenate was generated by homogenization of fresh pooled liver tissue followed by low-speed centrifugation. In contrast, bovine liver microsomes were isolated from the same tissue samples through homogenization, followed by medium and high-speed centrifugation. After incubation of homogenate or microsomes (1 mg/mL) with a representative SARM compound (ostarine) and NADPH, metabolite compounds were concentrated by protein precipitation and liquid-liquid extraction. Analytes were then separated and identified by ultra high performance liquid chromatography (UHPLC) coupled to quadrupole time-of-flight mass spectrometry (QToF-MS). Parent compound (ostarine) and associated metabolites, including hydroxy-, bishydroxy-, O-dephenyl, O-dephenyl-demethyl ostarine, 4-hydroxybenzonitrile and bishydroxybenzonitrile, were detected following incubations with either homogenate or microsomal preparations. The use of liver homogenate was demonstrated to be an effective alternative approach for use within in vitro drug metabolism based strategies facilitating the development of food safety focused targeted mass spectrometry detection methods.

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