The proposed mass fragmentation pathways for bolasterone metabolite identification in rats by high resolution LC-HRMS and GC-MS/MS

Abstract

Bolasterone (7α,17α-dimethyltestosterone) is an anabolic steroid in WADA prohibited list and one of the illegally abused drugs available in a black market. Recently, our group reported the bolasterone metabolites by LC-HRMS in rats and suggested the proposed mass fragmentation pathways. The structure elucidation of metabolites is critical for doping tests, but we often met the difficulty in availability of the authentic standards. Objective of this work is to investigate the mass fragmentation pathways of bolasterone metabolites and compare the spectra in the GC-MS/MS and LC-HRMS. Bolasterone (40 mg/kg) was orally administrated to rats and urine samples were collected by 168 h. The samples were pre-treated for clean-up by liquid-liquid extraction for LC-HRMS analysis in positive mode of ESI, and additionally derivatized with 100 ？L of MSTFA/NH4I/DTE (v/w/w, 500:4:2) for GC-MS/MS. As a result, by LC-HRMS, the parent bolasterone (BOLAL), 16 hydroxylated metabolites (mono-hydroxy form of A1L-A6L; di-hydroxy form of A7L-A13L; tri-hydroxy form of A14L-A16L), 1 glucuronic acid conjugated metabolite (A17L), and 1 metabolite from the reduction of the 4-ene and 3-keto moieties (A18L) were detected. By GC-MS/MS, bolasterone (BOLAG) and its 13 hydroxylated metabolites (mono-hydroxy form of B1G-B6G and di-hydroxy form of B7G-B13G) were observed. The mass fragmentation pathways here were suggested from each MS/MS spectrum of the parent bolastestone (BOLAL, BOLAG), its mono- (A1L, B2G) and di-hydroxylated metabolites (A9L, B8G) as examples. Methyltestosterone (MeTSL, MeTSG) also was included. The pair ions of m/z 173 and 193 indicate that both rings A and B remain intact as in parent bolasterone (BOLAL), while the ions m/z 171 and 189 were common characteristic ions in mono-(A1L) and di-(A9L), indicating the modifications at the B ring. The other characteristic ions such as m/z 121.0648 (C8H9O) generated from ring A of the mono-hydroxylated metabolites and 121.1016 (C9H13) from ring D of the di-hydroxylated metabolites were observed (data not shown here). One diagnostic ion from the GC-MS/MS spectra (in both B2G and B8G) were m/z 281 that indicated one hydroxylation at the B ring. The other ions m/z 403 of B2G (mono-) and m/z 491 of B8G (di-) with m/z 143 indicated an intact (meaning no hydroxylation) D ring that was more vulnerable than the rest of rings to EI ionization. These results may be useful for elucidating bolasterone metabolites or analogs for understanding plausible mass fragmentation pathways from the spectra.