Analysis of steroids in human urine by on-line coupled liquid chromatography-gas chromatography (LC-GC) with mass spectrometry (MS) and combustion-isotope ratio mass spectrometry (C-IRMS) for anti-doping purpose

International Conference on Sports Medicine and Fitness

March 23-25, 2015 Chicago, USA

Jesús Villén

Posters-Accepted Abstracts: J Sports Med Doping Stud

Abstract :

Anti-doping laboratories accredited by the WADA (World Anti-Doping Agency) must have available methods capable of detecting synthetic steroids at concentrations below 10 ng mLâ??1. Most accredited laboratories use gas chromatographyâ?? combustionâ??isotopic ratio mass spectrometry (GCâ??C-IRMS) to discriminate between natural and synthetic steroids, and gas chromatographyâ??mass spectrometry GC-MS to identify the analyte peak. The method involves laborious sample preparation, including hydrolysis, liquidâ??liquid extraction and acetylization. A further cleaning step of the derivatized steroids is usually carried out by liquid chromatography (LC) to ensure the purity of the steroid. In the analytical methods presented, the Through Oven Transfer Adsorption Desorption (TOTAD) interface was used to couple the last clean up stage of LC with GCâ??IRMS or GC-MS to provide on-line coupled LC-GC-IRMS and LC-GC-MS analytical methods. The methods developed were used to analyse 11-hydroxyandrosterone, 11-ketoetiocholanolone, epitestosterone, testosterone, etiocholanolone, androsterone, 5αAdiol, 5βAdiol and pregnandiol, as well as Boldenone and its principal metabolite. The volumes transferred from the LC to GC range from 700 to 2200 μL, while acetonitrile/water was used as the LC mobile phase. The TOTAD interface eliminates the solvent but retains steroids that are introduced in the GC column. Good sensitivity is achieved, detection limits being below 5 ng mL-1 in all cases, as the whole LC fraction containing the analytes is transferred from LC to GC. The relative standard deviation (RSD) of the absolute peak areas is below 20% for MS detection, and the RSD of the δ13C is about 1.1%, both of which can be considered very good since they represent the variability in the whole process, including sample preparation.