Received for publication August 9, 2007.
Revised December 17, 2007.
Accepted for publication December 17, 2007.

ARYL-PROPIONAMIDE-DERIVED SELECTIVE ANDROGEN RECEPTOR MODULATORS: LC-MS/MS CHARACTERIZATION OF THE IN VITRO SYNTHESIZED METABOLITES FOR DOPING CONTROL PURPOSES

Abstract

Selective androgen receptor modulators (SARM) is a prominent group of compounds for being misused in sport owing to their advantageous anabolic properties and reduced side-effects. To target the preventive doping control analysis in relevant compounds, the challenge is to predict the metabolic fate of a new compound. For aryl-propionamide-derived SARM an in vitro assay employing microsomal and S9 human liver enzymes was developed to simulate phase-I and phase-II metabolic reactions. In vitro metabolic profiles and structure-metabolic relationship were compared between four structurally modified substrates. Accurate mass measurements were used to characterize the synthesized metabolites, and also collision-induced dissociation was examined to suggest the methodological approach to monitor the prohibited use of aryl-propionamide-derived drug candidates. Subsequent phase-I and phase-II metabolic reactions were successfully combined in one in vitro assay. The main routes of phase-I modifications involved the hydrolysis of ether linkage, mono-hydroxylation and hydrolytic cleavage of the amide bond. Nitro-reduction and deacetylation were reactions observed for substrates possessing the corresponding functionality. SARM metabolites were analyzed in negative ion electrospray ionization (ESI) and detected as deprotonated species [M-H]^-. The main metabolic modifications were observed to occur in the B-ring side, and collision-induced dissociation resulted in the product ions originating from the A-ring side of the compound. These structure-specific ions may be monitored as target ions in the routine doping control.

Key words: analytical chemistry, drug analysis, hepatocytes, mass spectrometry, phase II drug metabolism