In vitro phase I metabolism of selective estrogen receptor modulators in horse using ultra‐high performance liquid chromatography‐high resolution mass spectrometry

Abstract

Selective estrogen receptor modulators (SERMs) are chemicals that possess the anti-oestrogenic activities that are banned 'in' and 'out' of competition by the World Anti-Doping Agency (WADA) in human sports, and by the International Federation of Horseracing Authorities (IFHA) in horseracing. SERMs can be used as performance-enhancing drugs to boost the level of androgens or to compensate for the adverse effects as a result of extensive use of androgenic anabolic steroids (AASs). SERMs have indeed been abused in human sports; hence, a similar threat can be envisaged in horseracing. Numerous analytical findings attributed to the use of SERMs have been reported by WADA-accredited laboratories, including 42 cases of tamoxifen and 2 cases of toremifene in 2014. This paper describes the identification of the in vitro phase I metabolites of tamoxifen and toremifene using ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS), with an aim to identify potential screening targets for doping control in equine sports. A total of 13 and 11 in vitro metabolites have been identified for tamoxifen and toremifene, respectively, after incubation with homogenized horse liver. The more prominent in vitro biotransformation pathways include N-desmethylation, hydroxylation, and carboxylation. In addition, this is the first report of some novel metabolites for both tamoxifen and toremifene with hydroxylation occurring at the N-methyl moiety. To our knowledge, this is the first study of the phase I metabolism of tamoxifen and toremifene in horses using homogenized horse liver.

Process development for the total synthesis of the novel drug metabolite Carboxy toremifene as a standard reference material along with characterization and purity assessment for the Antidoping quality Control Purposes

“…Since 2005, the World Anti‐Doping Agency (WADA) has banned to use SERMs in or out of competition. WADA has listed in section S4 of the WADA list of prohibited substances as “agents with anti‐oestrogenic activity 9,10 . A total of 11 metabolites have been reported in the literature for the toremifene.…”

“…M4d), dihydroxytoremifene (T.O.R. M4a‐c), and carboxy toremifene (TOR M5) have been reported in the literature 9,11 …”

Advances in equine anti‐doping

“…An impressive 14 original research articles are published in this special issue. On the ‘small molecule’ front these cover in vitro phase I metabolic studies of the selective estrogen receptor modulators (SERMs) tamoxifen and toremifene, and population studies of 5‐Aminoimidazole‐4‐carboxamide ribonucleotide (AICAR) in the horse . Equine pharmacokinetic studies are well represented with a focus on methylxanthines, betamethasone, and meldonium …”