Metabolic study of androsta-1,4,6-triene-3,17-dione in horses using liquid chromatography/high resolution mass spectrometry

A review of designer anabolic steroids in equine sports

“…The identities of the phase I metabolites not matched to standards were tentatively assigned by analysis of the LC‐HRAM product‐ion spectra. The position of hydroxylation in these metabolites was not assigned where standards were not available, although the authors comment on the presence of MS fragments at m/z 149 and 167 being characteristic of D‐ring hydroxylation which suggests C16‐hydroxylation as a major pathway in the metabolism of these compounds . A pair of metabolites resulting from C1‐C2 and C17‐direduction and hydroxylation were observed that had not been previously detected in human studies .…”

“…The metabolism of ATD has also been studied in vivo by a controlled oral administration (800 mg, 2 Thoroughbred geldings) . Following phase I metabolism, fourteen metabolites were identified by LC‐HRAM analysis including: three reduced metabolites (two C17‐reduced and C1‐C2 reduced), three direduced metabolites (C1‐C2 and C17‐direduced, C1‐C2 and C3‐direduced, and boldenone), four reduced and hydroxylated metabolites, and four direduced and hydroxylated metabolites (two C5‐C6 and C17 direduced with C16‐hydroxylation, and two C1‐C2 and C17‐direduced and hydroxylated metabolites).…”

“…Typically, the reaction of glucose‐6‐phosphate with a glucose‐6‐phosphate dehydrogenase (G6PDH) enzyme is used, which regenerates NADH/NADPH as a by‐product of oxidation. This allows for the use of a catalytic amount of these co‐factors in the metabolism reaction . Recent reports have also demonstrated the practicality of using homogenized whole liver tissue to perform in vitro studies, in an effort to closely replicate in vivo metabolism .…”

“…This allows for the use of a catalytic amount of these co‐factors in the metabolism reaction . Recent reports have also demonstrated the practicality of using homogenized whole liver tissue to perform in vitro studies, in an effort to closely replicate in vivo metabolism . These studies have even demonstrated the ability to generate phase II metabolites without the use of expensive phase II co‐factors such as UDP‐glucuronic acid or PAPS .…”

Challenges in detecting substances for equine anti‐doping

“…Both in vitro biotransformation of ATD using homogenized horse liver detected 14 metabolites using LC-HRMS technique, and in vivo ATD administration in horses gave evidence of 12 metabolites with 2 of them detected up to 77 h post-administration in horse urine. [63] However, many of the studies published so far focus on stallions and/or horses to which steroids have been administered intramuscularly or orally, and not on naturally present endogenous AAS (β-boldenone is the most illicit candidate for endogenous presence). A recent study [64] refers to healthy, out-of-competition horses, attempting to improve the knowledge on the natural, endogenous AAS in horse urine in general.…”