Concurrent analysis of Multi-Class 26 prohibited substances in dried blood spot by LC-ESI/MS/MS in doping control

Abstract

Doping testing using dried blood spot (DBS) was approved by the World Anti-Doping Agency (WADA) in 2021. The World Anti-Doping Agency provides a technical document (TDDBS), to standardize the analytical testing and storage of DBS samples. This TDDBS includes the Minimum Harmonized DBS Testing Menu, which classifies prohibited substances into four major categories: Multi-Class Menu, Steroid Esters Menu, Small Peptides Menu, and Erythropoiesis-Stimulating Agents (ERAs). In particular, the Multi-Class Menu includes various classes of substance such as Anabolic Androgenic Steroids (AAS), Hypoxia-inducible factor (HIF) activating agents, Beta-2 Agonists, Aromatase Inhibitors, Anti-Estrogenic Substances, Metabolic Modulators, and Diuretics and Masking Agents. In this study, we developed an LC-ESI/MS/MS based analytical method for concurrent analysis of 26 doping prohibited substances in the Multi-Class Menu. Various extraction solvents and mobile phase compositions were investigated and optimized for simultaneous extraction and highly sensitive analysis of multi-class substances from DBS samples. As a result, the present analytical method satisfied the Minimum Required Performance Levels (MRPL) of 26 prohibited substances in Multi-Class menu required by WADA. Furthermore, the developed method was successfully applied to the analysis of WADA EQAS DBS samples.

This study was supported by an intramural grant from Korea Institute of Science and Technology (No. 2V10481).