Simplified Analysis of Native Steroid Esters in Dried Blood Spots by LC-MS³

Steroidal esters (e.g., testosterone, nandrolone and boldenone esters) belong to the substance class prohibited in professional sport that is particularly frequently abused due to its considerable performance-enhancing effects. Thus, a high number of adverse analytical findings in doping controls is reported every year. Unfortunately, the analysis of steroids that are not exclusively of exogenous nature and, in particular, their differentiation from endogenously produced steroids is very time-consuming and resource-intensive (necessitating isotope-ratio mass spectrometric analysis). The direct detection of the applied (unequivocally exogenous) steroidal esters in blood or dried blood spots (DBS) can offer a much simpler approach. In the present project, the analysis of 17 underivatized steroidal esters (composed of testosterone, nandrolone and boldenone species) is reported, employing liquid chromatography-(low-resolution)-MSⁿ. The results show that all included esters can be selectively and sensitively detected in MS³ mode at sub-ng/mL levels in DBS. The detection limits for most analytes extracted from a single spot were below 0.1 ng/mL, and recovery rates were determined at 40%-80%. The overall procedure was controlled using four different stable isotope-labelled internal standards. Notably, the obtained results are largely independent from the sampling device, and the method works for cellulose-based DMPK cards as well as polymer-based TASSO devices. Proof-of-concept and applicability of the method to authentic samples were demonstrated by analysing post-administration samples collected after an oral administration of 80 mg of testosterone undecanoate.