Bias estimation in the certification of steroid reference materials for carbon isotope delta measurements via elemental analyser and gas chromatography-combustion-isotope ratio mass spectrometry.

RATIONALE: Two new certified reference materials (CRMs) have been prepared providing three steroids certified for stable carbon isotope delta values, δ(13 C) ‰. These materials have been designed to assist anti-doping laboratories in validating their calibration method or to be employed as calibrant for stable carbon isotope measurements of Boldenone, Boldenone Metabolite 1 and Formestane. These CRMs will allow for accurate and traceable analysis in compliance with World Anti-Doping Agency (WADA) Technical Document TD2021IRMS. METHODS: Certification was performed using an elemental analyser-isotope ratio mass spectrometry (EA-IRMS) primary reference method on the bulk carbon isotope ratios of nominally pure steroid starting materials. EA-IRMS analyses were carried out on a Flash EA Isolink CN coupled via a Conflo IV to a Delta V plus mass spectrometer. Confirmation analysis was performed by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) using a Trace 1310 GC coupled to a Delta V plus mass spectrometer via GC Isolink II. RESULTS: Based on the EA-IRMS analysis, the materials were certified with δ(13 C) values of -30.38‰ (Boldenone), -29.71‰ (Boldenone Metabolite 1) and 30.71‰ (Formestane). Noting that the assumption of 100% purity in the starting materials has the potential to introduce bias, this was investigated using GC-C-IRMS analysis and theoretical modelling based on purity assessment data. CONCLUSIONS: Careful application of this theoretical model was shown to provide reasonable estimates of uncertainty while avoiding the introduction of errors associated with analyte-specific fractionation during GC-C-IRMS analysis.

Certification of steroid carbon isotope ratios in a freeze-dried human urine reference material.

An accurate method for the measurement of carbon isotope ratios of steroids in human urine has been developed at the National Measurement Institute, Australia (NMIA) for the certification of a freeze-dried human urine reference material (CRM NMIA MX005). The method measures δ(13)C values by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) analysis following hydrolysis, solvent extraction and high performance liquid chromatography (HPLC) purification. Reference δ(13)C values for testosterone metabolites etiocholanolone, androsterone, and endogenous reference compounds (ERCs) 11ß-hydroxyandrosterone and pregnanediol were determined, as well as information δ(13)C values for testosterone, epitestosterone, 11-oxoetiocholanolone, and a range of differences (Δ(13)C) between testosterone metabolites and ERCs. The measurement uncertainty was rigorously evaluated with expanded uncertainties for the reference δ(13)C values between 1.1 and 1.6 ‰ at the 95% coverage level.