Optimization of the extraction and analysis of natural androgen steroids and their metabolites in urine by GC/MS and GC/FID

International audienceA multiresidue method was developed for screening, quantification, and confirmation of nine natural androgen steroids and their metabolites in urine. Steroids were first extracted from urine by solid phase extraction, enzymatically deglucuronated, re-extracted using a liquid/liquid extraction for purification, and finally acetylated for GC/MS and GC/FID analysis. Each step of sample preparation, as well as analysis, was optimized: solid phase extraction, liquid/liquid extraction, and derivatization reaction ... Therefore, a rugged sample preparation procedure was developed leading to extracts of sufficient purity (recoveries >66% and few matrix compounds). The whole methodology allowed reliable detection and quantification of the nine steroids at low concentration levels. Linearity and repeatability were established and were found to be satisfactory (R-2 > 0.996, RSD < 11%). Finally, the method was applied to quantify compounds of interest in real samples collected from healthy volunteers and patients treated with 4-androstenedione or dehydroepiandrosterone

L'apport de la spectrométrie de masse haute-résolution à l'obtention d'empreintes moléculaires d'échantillons d'urine. Applications potentielles au dopage.

National audienc

Comparaison des extractions MEPS et SPE pour la quantification par LC-MS/MS d'androgènes et d'oestrogènes sous forme libres et conjugués dans du sérum de rat.

communication par afficheNational audienc

Multi-residue analysis of free and conjugated hormones and endocrine disruptors in rat testis by QuEChERS-based extraction and LC-MS/MS

International audienceEndocrine disrupting compounds (EDCs) are suspected to be responsible for many disorders of the human reproductive system. To establish a causality relationship between exposure to endocrine disruptors and disease, experiments on animals must be performed with improved or new analytical tools. Therefore, a simple, rapid, and effective multi-residue method was developed for the determination of four steroid hormones (i.e., testosterone, androstenedione, estrone, and estradiol), glucuronide and sulfate conjugates of estrone and estradiol and four endocrine disruptors in rat testis (i.e., bisphenol A, atrazine, and active metabolites of methoxychlor and vinclozolin). The sample preparation procedure was based on the Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) approach. An analytical method was then developed to quantify these compounds at ultra-trace levels by liquid chromatography coupled to tandem mass spectrometry. The QuEChERS extraction was optimized with regard to the acetonitrile/water ratio used in the extraction step, the choice of the cleanup method and the acetonitrile/hexane ratio used in the cleanup step. The optimized extraction method exhibited recoveries between 89% and 108% for all tested compounds except the conjugates (31% to 58%). The detection limits of all compounds were below 20 ng g(-1) of wet weight of testis. The method was subsequently applied to determine the levels of hormones and EDCs in seven rat testis samples

Urinary signature of anabolic steroids and glucocorticoids in humans by LC-MS.

International audienceA metabonomic strategy based on LC-MS was employed to investigate the metabolic profile of urine samples from 20 athletes who had been tested positive for corticoids and anabolic steroids and 29 controls. In this aim, different sample preparations and chromatographic conditions were compared. The acquired LC-MS data of doped athletes and controls were subjected to analysis of variance (ANOVA) and principal component analysis (PCA). Using this approach, molecular signature of human urine was obtained showing that metabonomics could be a complementary tool to discriminate different urinary profiles and to track down metabolic changes in humans