Production and certification of a freeze-dried reference material for human urinary steroids to ensure quality of longitudinal profiling data

Records of elite athletes’ steroid profiles are maintained by the World Anti-Doping Agency (WADA) to facilitate detection of doping with endogenous steroids. As the steroid measurements comprising these profiles are obtained in a variety of laboratories throughout an athlete’s career. It is critical to ensure their metrological traceability to a fixed reference, which permits meaningful identification of any changes. Certified reference materials (CRMs) are an important tool to ensure the comparability of measurement results. The National Measurement Institute, Australia, has prepared a freeze-dried urine CRM, NMIA MX017, with property values for the mass fractions and mass concentrations of the six steroids specified by WADA as markers for the urinary steroid profile. Its reference values are traceable to the units (kg and m) of the international system of units (SI), and it is now available to be used by WADA-accredited laboratories as a replacement for a previous, exhausted CRM. The certification of NMIA MX017 was performed using a high accuracy reference method developed specifically for this material. A two-dimensional clean-up of the urine matrix by high-performance liquid chromatography provided interference-free quantification of the analytes and their deuterium-labelled analogues by gas chromatography with tandem mass spectrometry. Confirmatory analysis by gas chromatography with high-resolution mass spectrometry was used to verify the absence of bias due to potential influences by matrix coextractives or contaminants. The characterisation of NMIA MX017 and the approach used to obtain a rigorous estimate of the measurement uncertainty of the property values of the CRM are described.