Dried Blood Spots for Doping Purpose—Two‐Step Protocol for Analysis of Non‐Threshold Substances and Anabolic Steroid Esters From One Spot/Pebble

Analytical performance in anti-doping science continues to evolve through the identification of novel metabolites, improved detection sensitivity, and the use of alternative biological matrices. In recent years, dried blood spots (DBS) have gained increasing attention in doping control due to advantages such as improved analyte stability and simplified, cost-effective sample collection, shipment, and storage. However, DBS sampling is inherently limited by the small sample volume and the restricted number of spots available for analysis. Therefore, sensitive and comprehensive analytical strategies are required to maximize the information obtained from a single DBS or Tasso M20 devices. This study aimed to develop and validate a two-step analytical workflow enabling the detection of 91 target compounds, including selected non-threshold substances (NTS) and anabolic steroid esters (ASE), in two dried blood matrices: cellulose-based DBS cards and volumetric absorptive microsampling devices (Tasso M20 pebbles). The protocol comprises extraction with a methanol-acetonitrile mixture (4:1, v/v), followed by LC-MS/MS analysis for NTS. Subsequently, the extract is evaporated, derivatized with Girard P reagent, and analyzed by LC-MS/MS for ASE detection. The validated method proved suitable for qualitative detection of the selected prohibited substances.