Detection of new exemestane metabolites by liquid chromatography interfaced to electrospray-tandem mass spectrometry

Exemestane is an irreversible aromatase inhibitor used for anticancer therapy. Unfortunately, this drug is also misused in sports to avoid some adverse effects caused by steroids administration. For this reason exemestane has been included in World Anti-Doping Agency prohibited list. Usually, doping control laboratories monitor prohibited substances through their metabolites, because parent compounds are readily metabolized. Thus metabolism studies of these substances are very important. Metabolism of exemestane in humans is not clearly reported and this drug is detected indirectly through analysis of its only known metabolite: 17β-hydroxyexemestane using liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) and gas chromatography coupled to mass spectrometry (GC–MS). This drug is extensively metabolized to several unknown oxidized metabolites. For this purpose LC–MS/MS has been used to propose new urinary exemestane metabolites, mainly oxidized in C6-exomethylene and simultaneously reduced in 17-keto group. Urine samples from four volunteers obtained after administration of a 25 mg dose of exemestane were analyzed separately by LC–MS/MS. Urine samples of each volunteer were hydrolyzed followed by liquid–liquid extraction and injected into a LC–MS/MS system. Three unreported metabolites were detected in all urine samples by LC–MS/MS. The postulated structures of the detected metabolites were based on molecular formulae composition obtained through high accuracy mass determination by liquid chromatography coupled to hybrid quadrupole-time of flight mass spectrometry (LC–QTOF MS) (all mass errors below 2 ppm), electrospray (ESI) product ion spectra and chromatographic behavior.

► Detection of three new exemestane metabolites by LC–MS/MS.
► Two metabolites with oxidized C6 exomethylene moiety and simultaneously reduced in C17 keto group.
► One 6-hydroxyexemestane metabolite (both isomer forms).
► Identification via exact mass (LC-QTOF), fragment spectra (LC-Qtrap) and retention times.
► Detected metabolites are potential targets to monitor exemestane misuse in doping control by LC–MS/MS.