Correction of the urinary testosterone to epitestosterone ratio measurement in antidoping analyses by chromatographic and mass spectrometric techniques

The ratio of testosterone (T) to epitestosterone (E) determined in urine samples is the main biomarker used to prove T and precursors abused by athletes. Analytically, the correction of the ratio is required by the World Anti-Doping Agency. This work describes a series of experiments aimed to study when it is appropriate to correct the T/E ratio value, using different mass spectrometric techniques since only reports on GC-MS exist. Analyses using external calibrators, controls, and routine samples were performed by three different techniques GC-MS, GC-MSⁿ , and LC-MSⁿ . A statistical comparison of the T/E was performed after the application of two corrections previously published: Isotopic contribution peak area correction (corr_1) and use of a verified internal deuterated internal standard (TD3/ED3) correction (corr_2) and the ratio based on T and E concentrations. The use of external calibration samples introduces biases that influence not only the T and E concentrations but also the T/E ratio, even when both methods of correction are applied. The correction after applying corr_1 method barely contributed to the accuracy of the T/E calculation. Nevertheless, the application of the corr_2 method increased the accuracy between 5% and 6% when comparing theoretical and experimental T/E values. Finally, the best results were obtained by the ratio calculated directly from the estimated concentrations of T and E. Attention must be paid when the T/E is measured by LC-MSⁿ since different acquisition modes produced significantly different results, even in MS/MS when the same transition is used for T and E.