Two‐dimensional high performance liquid chromatography purification of underivatized urinary testosterone and metabolites for compound‐specific stable carbon isotope analysis

Testosterone doping in sports is detected through the measurement of the carbon isotopic signature (δ13C) of testosterone and its metabolites in urine. A critical step in achieving accurate and precise δ13C values during compound‐specific stable carbon isotope analysis (CSIA) is the removal of interfering matrix components. To this end, the World Anti‐Doping Agency (WADA) recommends the use of high‐performance liquid chromatography (HPLC) as a method of sample pretreatment. We provide a description of an automated two‐dimensional HPLC (2D‐HPLC) purification method for urine extracts that has made possible the CSIA of underivatized steroids, requiring only 36 min per sample. Eight urinary steroids including testosterone (T) and dehydroepiandrosterone (DHEA) and four of their metabolites as well as two endogenous reference compounds were collected during HPLC purification. Comparative GC chromatograms are used to contrast the efficiency of two‐dimensional (2D) purification to a previously established 1D‐HPLC method. The 2D purification leads to improved sample purity while simultaneously decreasing the analysis time, allowing for unprecedented sample throughput. Precision of δ13C for all analyzed compounds in negative and positive controls was 0.5‰ or better, which is comparable with the precision of pure reference materials at similar intensities. A novel two‐dimensional HPLC method for the purification of urine extracts prior to IRMS analysis leads to improved sample purity while drastically improving sample throughput over traditional one‐dimensional approaches.