Detection and characterization of clostebol sulfate metabolites in Caucasian population

Abstract

Anabolic androgenic steroids (AAS) are synthetic testosterone derivatives which undergo extensive metabolism in man. Differences in the excretion of phase II metabolites are strongly associated with inter-individual and inter-ethnic variations. Sulfate metabolites have been described as long-term metabolites for some AAS. Clostebol is the 4-chloro derivative of testosterone and the aim of the present study was the evaluation of clostebol sulfate metabolites in Caucasian population by LC-MS/MS technology. Clostebol was orally administered to four healthy Caucasian male volunteers, and excretion study urines were collected up to 31 days. Several analytical strategies (neutral loss scan, precursor ion scan and selected reaction monitoring acquisitions modes) were applied to detect sulfate metabolites in post-administration samples. Sixteen sulfate metabolites were detected, five of them having detectability times above 10 days (S1a, S2a, S3b, S3g and S4b). Interestingly, metabolite S1a could be detected up to the last collected sample of all excretion studies and it was characterized by LC-MS/MS and GC-MS as 4ξ-chloro-5α-androst-3β-ol-17-one 3β-sulfate. Thus, monitoring of S1a improves the detection time of clostebol misuse with respect to the commonly monitored metabolites, excreted in the glucuronide fraction. Importantly, this new metabolite can be incorporated into recently developed LC-MS/MS screening methods base on the direct detection of phase II metabolites.

Direct quantitation of endogenous steroid sulfates in human urine by liquid chromatography‐electrospray tandem mass spectrometry

“…18,19,[25][26][27] Therefore, unlike glucuronides, there is no universal method for the cleavage of all steroid sulfates and this is an important limitation of the indirect analysis of these conjugates by gas chromatography coupled to mass spectrometry (GC-MS). 13,14,[20][21][22][23][24][28][29][30][31][32][33][34][35] However, the LC-MS reported methods for endogenous sulfates are only focused on some of the possible sulfate metabolites (eg, sulfates of T, E, A, and Etio), and do not cover other metabolites that could be of interest. 27 As a result, in recent years, some methods have been described to directly analyze sulfate metabolites in urine using LC-MS systems.…”

Sulfate metabolites as alternative markers for the detection of 4‐chlorometandienone misuse in doping control

“…A theoretical SRM method was developed in negative mode because of the higher ionization efficiency of these metabolites when working under this mode (Table 1, method 1). [9,19] As precursor ions, the [M-H] À ions of potential sulfate metabolites were calculated considering all phase I metabolites described up to now. boldenone or clostebol).…”

“…First, the characteristic product ion of sulfate metabolites (m/z 97) [30,33] was selected for all metabolites. [19] Finally, the same ion transitions considering 37 Cl isotope were also added into the method ( Table 1, method 1). On the one hand, for metabolites with a 3keto-4-Cl-1,4-diene structure the ion transitions resulting from the NL of a methyl group (15 Da) were incorporated.…”

“…Conventional approaches have focused on the detection of the most abundant metabolites after hydrolysis with β-glucuronidase enzymes to release the phase I metabolite and analysis by gas chromatography-mass spectrometry (GC-MS) or liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). [8][9][10][11][12][13][14][15][16][17][18][19] For some steroids, sulfate metabolites really improved the retrospectivity of the detection. [8][9][10][11][12][13][14][15][16][17][18][19] For some steroids, sulfate metabolites really improved the retrospectivity of the detection.…”

“…Among the 12 potential sulfate metabolites included in the method, peaks at the theoretical ion transitions for metabolites 2, 7, 8, 11, and 12 were detected in postadministration samples together with many other peaks of lower abundance ( Table 2). [9,19] The presence of other sulfates (e.g. No peaks at the same retention time (RT) were observed in pre-administration samples.…”