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.

Gas chromatography/mass spectrometry characterization of urinary metabolites of danazol after oral administration in human.

Danazol (17alpha-pregna-2,4-dien-20-yno [2,3-d]-isoxazol-17beta-ol), is a synthetic derivative of ethisterone, structurally related to stanozolol. For this reason its use as doping agent has been investigated. Danazol (Runch) (200 mg) were orally administered to two healthy male volunteers. Urine samples were collected up to 1-week post-dose. Four new metabolites have been identified in addition to the five previously reported. We propose the monitorization of 6beta-hydroxy-2-hydroxymethyl-1,2-dehydroethisterone and 6beta,16epsilon-dihydroxy-2epsilon-hydroxymethyl-ethisterone by free fraction analysis. In a same way, we proposed to detect the principal isomer of a mono-hydroxylated metabolite of 6beta-hydroxy-2epsilon-hydroxymethylethisterone in the conjugated fraction. We conclude that new metabolites can be included for the detection of danazol abuse since the main metabolite ethisterone is excreted relatively fast in urine.