Detection of Lower Molecular Mass Peptides, Steroid Esters, and Multi‐Class Substances in a Single Dried Blood Spot for Comprehensive Initial Testing in Doping Control

Dried blood spots (DBS) have become an increasingly important matrix in anti‐doping testing, providing, among other advantages, direct access to circulating parent compounds. However, the limited amount of blood available from a single DBS represents a considerable analytical challenge, particularly following the inclusion of lower molecular mass peptides into the current World Anti‐Doping Agency (WADA) guidelines for DBS testing. Existing analytical methods are primarily dedicated to peptide 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 workflow for the simultaneous detection of lower molecular mass peptides, anabolic steroid esters, and prohibited non‐threshold substances without minimum reporting level (MRL) from a single DBS, collected on both Tasso‐M20 devices and cellulose‐based DMPK‐C cards. Method validation was performed according to current WADA requirements for initial testing procedures and included the evaluation of selectivity, reliability, limits of detection, extract stability, carryover, and robustness. The developed method fulfilled all validation criteria for both DBS collection devices and enabled the sensitive detection of all investigated intact peptides together with steroid esters and multi‐class prohibited substances. The presented workflow enables, for the first time, the simultaneous detection of lower molecular mass peptides, steroid esters, and multi‐class prohibited substances from a single DBS spot. This integrated approach reduces sample consumption, simplifies laboratory workflows, and preserves additional DBS material for confirmatory or complementary analyses, providing a practical and flexible strategy for routine DBS anti‐doping testing.