Characterization of equine liver microsome‐generated metabolites of growth hormone secretagogue small molecule ibutamoren

Abstract

Interest in growth hormone secretagogues has intensified during the past several years based on capable, ever-widening investigational applications of recombinant growth hormone in animals and humans. Ibutamoren is a potent, long-acting, selective and orally active non-peptide growth hormone secretagogue, which has a great potential for abuse as a performance-enhancing agent in sports.Methods: To support drug metabolism and pharmacokinetic studies of chiral pharmaceuticals, it is necessary to combine the resolving power of high-performance liquid chromatography with the sensitivity of mass spectrometric techniques. This paper describes the metabolic conversion of ibutamoren using equine liver microsomes and metabolite characterization using a QExactive high-resolution mass spectrometer.Results: A total of 32 metabolites for ibutamoren (20 phase I and 12 phase II) were detected. The important findings of the current research are as follows: (1) the growth hormone secretagogue ibutamoren was prone to oxidation, resulting in corresponding hydroxylated metabolites; (2) in ibutamoren, the dissociation of the phenyl ring and 2-amino-2-methylpropanamide side chain was also observed; (3) the glucuronic acid conjugates of mono-, di-and trihydroxylated analogues were detected; and (4) no sulfonic acid conjugated metabolites were observed in this study of ibutamoren. Conclusions:The reported data help in the speedy detection of the growth hormone secretagogue ibutamoren and reveal its illegal use in competitive sports.

Equine metabolism of the growth hormone secretagogue MK‐0677 in vitro and in urine and plasma following oral administration

“…All the metabolites detected in this study were reported in the previously published in vitro work 21 ; structural interpretations of these analytes were consistent with what is reported in the current study. However, some additional oxygenated metabolites were also detected in that study, 21 including an additional metabolite mono‐oxygenated at the piperidine ring, a metabolite di‐oxygenated at the piperidine ring and a metabolite di‐oxygenated at the benzyl ring. Furthermore, five tri‐oxygenated metabolites were detected that were not observed in this study.…”

“…Figure 1 shows the acquired MS/MS spectrum and the proposed identities of the observed fragment ions. The fragmentation of MK‐0677 using positive ESI‐MS/MS has previously been reported, 17,21 and the major ions are consistent with what was observed in this study. The base peak ion was observed at theoretical m/z 91.0542, representing the benzyl moiety.…”

“…The fragment at theoretical m/z 188.1308 was suggested to represent a radical cation consisting of the spiro[indole‐3,4′‐piperidine], potentially formed by the loss of the methylsulfonyl group from the m/z 267.1162 ion. All these ions were observed in the previous work, 17,21 but structures were not postulated for the ions at m/z 91.0542 or 188.1308. An additional ion observed in this study at theoretical m/z 131.0730 was postulated to represent further fragmentation of the spiro[indole‐3,4′‐piperidine] structure, to give a radical cation consisting of the indole ring system with an additional double bond.…”

“…These metabolites had been produced in vitro but at very low levels that were not detectable by LC‐HRMS analysis. In a previous in vitro study, only a single isomer of M6 was reported and no equivalent M7 metabolites were detected 21 …”

“…Analytical methods have also been developed for the detection of MK‐0677 in human doping samples 18–20 . More recently, the equine metabolism has been investigated in vitro using equine liver microsomes, with a total of 20 phase I and 12 phase II metabolites that were detected 21 . Knowledge of species‐specific metabolism profiles allows the refinement of screening targets in doping control.…”