Dried Blood Spots for Doping Controls - Development of a comprehensive Initial Testing Procedure with fully automated sample preparation.

Currently, primarily urine, whole blood, and serum samples are analyzed for doping-relevant substances in professional sports, but recently dried blood spots (DBS) have been introduced as complementary matrix, offering advantageous features e.g. the minimally invasive sampling procedure. In order to cope with the increased application of DBS, a comprehensive Initial Testing Procedure (ITP) was developed, optimized and validated, comprising a total of 233 substances representing all groups of the World Anti-Doping Agency's (WADA's) Prohibited List. The sample preparation is conducted by employing a fully automated system using an efficient flow-through extraction of a 4 mm diameter spot followed by LC-HRMS/MS analysis. The procedure was successfully validated in terms of selectivity, limit of detection, reproducibility, carryover, robustness with respect to an alternative manual sample preparation, an alternative dried blood collection device and the sample extract stability, and thus found to meet the required criteria of relevant guidelines published by WADA for routine application. As proof-of-concept, DBS samples were analyzed after the administration of the glucocorticoids prednisone and dexamethasone, as well as the stimulant pseudoephedrine and the beta-blocker propranolol. All substances were detected in post-administration samples for at least 4 hours and up to 24 hours after intake, depending on the collection time period, using the developed testing procedure. In particular, for substances that are only banned in-competition, data obtained from DBS samples can be useful for the interpretation of adverse analytical findings. In conclusion, the developed ITP accounts for the anticipated increasing relevance of DBS in anti-doping analysis in the future and provides a foundation for optimized approaches for specific substance classes.