Identification of in-vitro and in-vivo metabolites of bolasterone (7α,17α-dimethyltestosterone) by ultra high-performance liquid chromatography/orbitrap mass spectrometer

Abstract

Identification of in-vitro and in-vivo metabolites of bolasterone (7α,17α-dimethyltestosterone) by ultra high-performance liquid chromatography/orbitrap mass spectrometer Anca Raluca Muresan1,2, Khandoker Asiqur Rahaman1,2, Farzana Binte Rafique1,2, and Oh-Seung Kwon1,2,* 1Doping Control Center, Korea Institute of Science and Technology, Seoul, 02792, Korea, 2Division of Bio-Medical Science & Technology, KIST School, Korea University of Science and Technology, Seoul 02792, Korea *, Corresponding author: oskwon@kist.re.kr Bolasterone (7α,17α-dimethyltestosterone) is a typical steroid drug that is often illegally used in different kinds of sports disciplines. Identification of phase I and II metabolites is a useful tool for the confirmation of various metabolic pathways in order to develop long-term biomarkers for anti-doping analysis. This study aimed to investigate phase I and phase II metabolites of designer steroid bolasterone (MW 316) through in-vitro (rat liver microsome) and in-vivo conditions (urine after oral administration 40 mg/kg to Sprague Dawley rats), and to identify metabolites by an ultra high-performance liquid chromatography/Orbitrap mass spectrometer (Q-Exactive). Full scan and further various dd-MS/MS modes were used to elucidate more detailed structural information for the metabolites. We have characterized several metabolites based on their retention times in chromatograms, and characteristic ionization from full MS and MS/MS spectra. A total of 16 metabolites (15 phase I, and 1 phase II) were identified. In-vitro were found 6 mono-hydroxylated (M8-M13 as m/z 333 of [M+H]+), 5 di-hydroxylated (M1, M3-M7, M14 as m/z 493 of [M+H]+), along with 1 resulting from reduction (M15, as m/z 285 of [M+H-2H2O]+), and 1 glucuronide-conjugated metabolite (M16, as m/z 493 of [M+H]+). In-vivo, a total of 8 di-hydroxylated metabolites (M1-M7, M14 as m/z 349 of [M+H]+) were found. Based on the charac