Certification of steroid carbon isotope ratios in a freeze dried urine

Code: 07C24LM

A key component in identifying the illegal use of the natural hormone testosterone for performance enhancement is the ability of WADA-accredited laboratories to accurately determine the very small changes that occur in carbon isotope ratios after doping. This project aims to enhance a previously prepared Certified Reference Material (CRM) of testosterone in urine by providing a very well characterised reference value for the carbon isotope ratios of steroids related to testosterone abuse in the CRM. The availability of a CRM with well-defined carbon isotope ratios traceable to international standards with low uncertainty will permit laboratories to unequivocally demonstrate the reliability of their methods and ensure comparability of results from different laboratories. This material will be the first certified reference material of this type available in a urine matrix.

Main Findings

The δ 13C reference values assigned in this project to key urinary steroids in the urine matrix CRM, NMIA, MX005 will assure accredited laboratories meet the requirements of WADA Technical Document TD2004EAAS in determining whether results of isotope ratio measurements are consistent with administration of a steroid. The CRM can be used as a quality control and calibration material for isotope ratio measurements at key delta values of interest. The carbon isotope ratios of androsterone and etiocholanolone in the CRM are excellent positive and negative controls, respectively, for the prescribed threshold of 3% difference with respect to endogenous reference steroids. Despite the expanded uncertainty being relatively large, these two steroids are above and below the threshold at the 95% level of confidence. In addition, the certified delta value for androsterone of -27.9% is very close to that at which the results of an analysis must be reported as "inconclusive" when the low concentration of ERCs prevents determination of a delta difference. The use of the CRM in anti-doping programs will lead to improved comparability of results between laboratories for longitudinal studies.