Effect of microbial degradation on steroid profile and IRMS analysis : A case study

Effect of microbial degradation on the steroid profile in urine samples has been well investigated but not much is known about its effects on carbon isotope ratios (CIR)[1-3]. Transportation of urine samples, collected for doping control analysis does not always meet ideal conditions of storage which may stimulate bacterial contamination affecting urinary steroids concentration. According to WADA technical document TD2004 EAAS (valid till Dec. 2013), the presence of bacterial degradation and free steroids invalidate the urine sample for reporting Adverse Analytical Findings (AAF)[4]. This paper presents result of one sample received from an international client in 2013, which showed signs of bacterial degradation (presence of high concentration of 4-androstendione, 5αand 5β-androstanedione and a T/E ratio > 10). Further investigation showed that most of the endogenous steroids excreted were in free form (more than 50%) implying a strong indication of degradation. The influence of bacterial degradation on CIR of different urinary steroids was investigated in the combined, unconjugated and conjugated fractions. The peak heights and δ13C values of urinary steroids in the combined fraction were comparable with those observed in the free fraction. The GC/C/IRMS analysis was consistent with exogenous origin of endogenous steroids, meeting the criteria to report an AAF. This sample could not be reported as AAF as all the target compounds were present in un-conjugated form, thereby not meeting WADA criteria of reporting AAF[4]. Further work is in progress with more number of samples to ascertain that CIR is independent of influence of bacterial growth affecting steroid profile. Introduction Urine samples exposed to high temperatures and/or improper storage conditions in hot climate countries may be subjected to bacterial degradation [1]. The effect of degradation processes on steroid concentrations and diagnostic ratios are well investigated but limited work is carried out to know its effect on CIR[1-3]. WADA technical document TD2004 EAAS for reporting and evaluation of T/E-ratios invalidate specimens containing more than 5% of free testosterone (T) and/or epitestosterone (E) for reporting Adverse Analytical Findings (AAF)[4]. A doping control urine sample showing T/E ratio greater than 4 (12.2) in the initial testing procedure showed strong bacterial degradation. Inspite of steroid profile being invalid, the present study was carried out to understand the impact of strong bacterial degradation on GC/C/IRMS analysis. Experimental Chemicals and Reference Standards Reference standards of endogenous steroids were procured from Sigma-Aldrich, USA and National Measurement Institute (NMI), Australia. C-18 sample preparation cartridges were procured from Ranbaxy Fine Chemicals Ltd, India, βglucuronidase enzyme (E. Coli) was from Roche Diagnostics, USA. All other reagents/solvents were of High Performance Liquid Chromatography Grade (HPLC-grade) or analytical grade. Urine Samples A batch of 26 samples, received from an international client in 2013, was processed according to routine screening procedure for anabolic steroids. Out of 26 samples, one sample showed strong signs of bacterial degradation and a T/E ratio