We developed an LC–MS/MS method to simultaneously detect methyltestosterone (MeT) and testosterone propionate (TP) in dried blood spots and a confirmation analysis method for MeT. Both substances were simultaneously detected after transdermal application of over-the-counter drug. Fingertip puncture sampling could lead to contamination from MeT and TP remaining on the fingertip.

ABSTRACT

In the past few years, doping tests have been performed with dried blood spots (DBS) as test samples; this sampling method is minimally invasive and requires minimal storage space. Steroid esters are stable in dried blood, allowing the direct analysis of testosterone esters. In Japan, the cream-based formulation containing 17α-methyltestosterone (MeT) and testosterone propionate (TP) is available as an over-the-counter drug for hair growth. Here, we investigated whether the transdermal uptake of this formulation could be detected using the existing method for steroid ester analysis in DBS. A method that was previously validated to detect and identify TP could also detect MeT. We developed a high throughput liquid chromatography–tandem mass spectrometry method to identify MeT in DBS. The limits of detection and identification of MeT were 0.1 ng/mL and 0.6 ng/mL, respectively. Five male human subjects received the cream-based formulation of MeT and TP transdermally. We then collected blood samples by finger pricking and made DBS on cellulose paper. Using this method, MeT was detected and identified up to 97 h after the final application. TP was also detected up to 97 h and identified up to 49 h after the final application. The developed method provides a direct confirmation of the presence of the drug. Moreover, using cream preparations could lead to contamination from MeT and TP remaining on the fingertip skin during fingertip puncture sampling.