Simultaneous Detection of Steroid Esters and Multi‐Class Substances in Dried Blood Spots by LC‐HRMS/MS From a Single Spot for Human Doping Control

ABSTRACT The direct detection of intact anabolic steroid esters in blood constitutes unequivocal evidence of exogenous administration and therefore represents a valuable target for anti‐doping testing. Dried blood spots (DBSs) offer several practical advantages over conventional blood sampling and may improve the stability of steroid esters during sample transport and storage. Existing analytical methods are primarily dedicated to targeted steroid ester analysis and do not address the simultaneous detection of other prohibited substances within routine anti‐doping workflows. The aim of the present study was to develop and validate a comprehensive LC‐HRMS/MS method for the simultaneous detection of 15 testosterone, nandrolone, and boldenone esters along with approximately 85 non‐threshold substances without minimum reporting level (MRL) in DBS samples, collected using Tasso‐M20 devices and cellulose‐based DMPK cards. Method validation was performed according to current World Anti‐Doping Agency (WADA) requirements and included selectivity, reliability, limit of detection, autosampler stability, carryover, and robustness. As proof of concept, testosterone undecanoate was successfully detected in authentic DBS samples following oral administration. The presented method fulfills the current WADA requirements and enables, for the first time, the combined detection of steroid esters and multi‐class prohibited substances from a single spot. This approach reduces sample consumption, simplifies laboratory workflows, and represents a practical strategy for routine DBS‐based anti‐doping testing.