Potential detection of low-dose transdermal testosterone administration in blood, urine, and saliva.

Administration of low amounts of endogenous hormones - so-called micro-dosages - are supposed to represent a major challenge in doping analysis. To model such a situation, we have studied transdermal administrations of 2.4 mg/24 h testosterone patches and examined various steroid concentrations in blood, urine, and saliva of 11 volunteers. Multiple samples were collected at t = 0, 3, 6, 9, 24, 48, and 72 h in four different phases, i.e., all combinations with/without physical exercise and with/without testosterone. Testosterone was analyzed by enzyme-linked-immuno-assay as well as by mass spectrometry and validated in an accredited anti-doping laboratory. Circadian controls with and without exercise did not provoke prominent alterations of whole, free, and salivary testosterone. Testosterone application for 24 h led to a significant (all p < 0.001) mean increase above controls: total testosterone (median: 5.2 vs. 8.0 ng/mL), free testosterone (median: 11.3 vs. 15.6 pg/mL), and salivary testosterone (median: 62.4 vs. 99.9 pg/mL). Additionally, all three testosterone measurements indicated significant correlations to each other (all r > 0.538, all p < .001). Circadian-matching showed peaking testosterone values after 6 h and 9 h, reaching highest augmentation up to 252.6 ± 123.5% in saliva after 9 h. After removal of the testosterone patch, all testosterone levels in blood, saliva, and urine returned to baseline within 24 h. Different techniques of hormone detection (enzyme-linked immunosorbent assay (ELISA), gas chromatography-tandem mass spectrometry (GC-MS/MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS)) indicated significant correlations. Results indicate that saliva, blood, and urine exhibit comparable hormone augmentation during micro-dose testosterone application, indicating a possible consideration in future doping analysis. The inter-individual variability was high in all biofluids, requiring the use of an individual biological passport rather than statistical values.