Analysis of exemestane and 17β‐hydroxyexemestane in human urine by gas chromatography/mass spectrometry: development and validation of a method using MO‐TMS derivatives

Abstract

Trimethylsilylation of anabolic agents and their metabolites is frequently achieved by using the derivatization mixture N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA)/NH(4)I/2-mercaptoethanol. Nevertheless, artifacts were formed when this mixture was employed in the monitoring of exemestane and its main metabolite 17β-hydroxyexemestane prior to GC-MS analysis. These artifacts were identified as the N-methyltrifluoroacetamide (MTFA) and trimethylsiloxyethylmercapto products of the respective trimethylsilyl (TMS) derivatives. Furthermore, artifact formation was evaluated taking the structure (1,4-diene-3-keto-6-exomethylene) of the compounds into account. Although these artifacts are relevant for investigations regarding the derivatization process and may be of interest in many fields, they are detrimental to cope with the requirements of the World Anti-Doping Agency (WADA) in terms of the limits of detection (LODs) required. To overcome this issue, a method using an alternative derivatization was proposed: formation of methyloxime-TMS derivatives through double derivatization using O-methylhydroxylamine/pyridine and MSTFA/TMS imidazole after enzymatic hydrolysis and liquid-liquid extraction. Samples from an excretion study after administration of exemestane to healthy volunteers were analyzed by the proposed method and detection of both exemestane and its main metabolite was possible. This method showed excellent results for both analytes meeting the LODs required for antiestrogenic agents (50 ng/mL) established by WADA. The method was validated for the main metabolite, it was robust and cost-effective for qualitative and quantitative purposes, with LOD and LOQ of 10 ng/mL and 25 ng/mL, respectively.

Impact of nonsteroidal aromatase inhibitors on steroid profile in a Chinese population

“…Exemestane was previously monitored by its major metabolite for doping control purpose. [24] Using the current method, the drug could not be detected after 25.5 hours (Appendix 6). However, there was overt change in the EWMA of T/estrone, with detection window increasing from 1 to 10 days (Appendix 7).…”

Annual banned‐substance review: analytical approaches in human sports drug testing

“…The representative of aromatase inhibitors referred to as exemestane and particularly its major metabolite 17β‐hydroxyexemestane were implemented in routine doping controls using a modified GC‐MS‐based method . Following a sample preparation procedure employing enzymatic hydrolysis, LLE and double derivatization to obtain the methyloxime‐trimethylsilylated derivative of 17β‐hydroxyexemestane, the target compound was sensitively detected by GC‐MS at a LOD of 10 ng/ml.…”

Quantification of Exemestane Accumulation During Microbial Bioconversion by TLC Image Analysis

“…Number of methods have been employed for quantification of exemestane in samples of human urine by gas chromatography/mass spectrometry [14][15] and liquid chromatography/tandem mass spectrometry [16], in human plasma by LC-MSMS [17], in various pharmaceutical dosage forms and stability testing by HPTLC, HPLC and RP-HPLC [18][19][20][21][22]. All these methods are time-consuming and involve sophisticated instrumentation.…”