Detection of testosterone misuse: comparison of two chromatographic sample preparation methods for gas chromatographic-combustion/isotope ratio mass spectrometric analysis.

Two chromatographic methods, reversed-phase liquid chromatography (LC) and immunoaffinity chromatography (IAC), were compared in the preparation of purified testosterone extracts suitable for gas chromatography-combustion/isotope ratio mass spectrometry (GC-C-IRMS) analysis. We have shown previously that GC-C-IRMS is a promising means of detection of testosterone misuse in sport. The two clean-up procedures afford sufficient recovery and adequate purity of testosterone. LC presents several advantages over IAC: access to other urinary steroids, longer column life, no need for special equipment and no antibody preparation. For IAC, the antibodies to testosterone must be selected with care for high affinity and low cross-reactivity. Nevertheless, IAC is of some interest in our experiments, the recovery is slightly better for low concentrations of urinary testosterone and IAC does not induce isotopic discrimination even in overloading experiments. This is the first report on sample preparation by IAC prior to GC-C-IRMS and carbon isotope ratio values for urinary epitestosterone. The carbon isotope ratio test can identify users' urines missed by the testosterone to epitestosterone ratio (T/E > 6) test.