Detection of stanozolol O‐ and N‐sulfate metabolites and their evaluation as additional markers in doping control

Abstract

Stanozolol (STAN) is one of the most frequently detected anabolic androgenic steroids in sports drug testing. STAN misuse is commonly detected by monitoring metabolites excreted conjugated with glucuronic acid after enzymatic hydrolysis or using direct detection by liquid chromatography-tandem mass spectrometry (LC-MS/MS). It is well known that some of the previously described metabolites are the result of the formation of sulfate conjugates in C17, which are converted to their 17-epimers in urine. Therefore, sulfation is an important phase II metabolic pathway of STAN that has not been comprehensively studied. The aim of this work was to evaluate the sulfate fraction of STAN metabolism by LC-MS/MS to establish potential long-term metabolites valuable for doping control purposes. STAN was administered to six healthy male volunteers involving oral or intramuscular administration and urine samples were collected up to 31 days after administration. Sulfation of the phase I metabolites commercially available as standards was performed in order to obtain MS data useful to develop analytical strategies (neutral loss scan, precursor ion scan and selected reaction monitoring acquisitions modes) to detect potential sulfate metabolites. Eleven sulfate metabolites (M-I to M-XI) were detected and characterized by LC-MS/MS. This paper provides valuable data on the ionization and fragmentation of O-sulfates and N-sulfates. For STAN, results showed that sulfates do not improve the retrospectivity of the detection compared to the previously described long-term metabolite (epistanozolol-N-glucuronide). However, sulfate metabolites could be additional markers for the detection of STAN misuse. Copyright © 2016 John Wiley & Sons, Ltd.

Identification and characterization of novel long‐term metabolites of oxymesterone and mesterolone in human urine by application of selected reaction monitoring GC‐CI‐MS/MS

“…The ‘steroid profile’, a set of endogenous steroids used as markers for testosterone abuse, is analysed concurrently and the use of E. coli is a regulatory requirement for these steroids. Recent publications however have illustrated the potential advantage of detecting sulfates for long‐term detection of steroid abuse, which resulted in a further increased research focus . In this light, the excretion form of the two new long‐term metabolites, oxyM9 and mestM22, was investigated.…”

“…Recent publications however have illustrated the potential advantage of detecting sulfates for long-term detection of steroid abuse, which resulted in a further increased research focus. [38][39][40][41][42][43][44][45] In this light, the excretion form of the two new long-term metabolites, oxyM9 and mestM22, was investigated. Excretion urine samples were prepared with β-glucuronidase (from E. coli), β-glucuronidase/aryl sulfatase (from H. pomatia) and without enzyme, resulting in the free + glucuronide fraction, free + glucuronide + sulfate fraction and free steroid fraction, respectively.…”

Annual banned‐substance review: Analytical approaches in human sports drug testing

“…Focusing exclusively on sulfoconjugation reactions of stanozolol and its phase I metabolites, Balcells et al identified 11 compounds in post‐administration stanozolol samples . Deduced from ESI–MS/MS data of synthesized O ‐conjugated sulfates of stanozolol and 3 hydroxylated phase I metabolites, the in vivo formation of different isomers of sulfoconjugated 16‐hydroxystanozolol, 16‐oxostanozolol, 4‐hydroxy‐16‐oxostanozolol, and stanozolol as N ‐sulfates were postulated.…”

“…Glucuronoconjugated metabolites of metandienone were the subject of a study by Esquivel et al.., who investigated the presence of glucuronides in excretion study urine samples and the potential resistance of these conjugates against commonly employed enzymatic hydrolysis conditions . Utilizing the same LC–ESI–MS/MS conditions reported by Balcells et al, with modified SRM transitions adapted to predicted metandienone‐derived phase II metabolites, a total of 13 conjugates was determined. One of these metabolites (of yet unknown structure) was not deconjugated by β‐glucuronidase, but its relevance for extended retrospectivity in doping controls was found limited due to a 4‐day post‐administration detection window.…”

Ionization and collision induced dissociation of steroid bisglucuronides

“…Study of phase II metabolism of anabolic androgenic steroids (AASs) in sports drug testing by LC‐MS/MS is one of the fields with great research activity in the last years . Antidoping laboratories have to improve the detection capabilities of the forbidden AAS, and studies on steroid metabolism have to be performed to identify new metabolites that could expand the detection window after their administration.…”