Validation of an HPLC-MS method for simultaneous determination of seven troponin activators in human urine and serum...

Troponin activators have been classified as S0 (non-approved substances) by the World Anti-Doping Agency (WADA) since 2024. A rapid, sensitive, and reliable high-performance liquid chromatography-mass spectrometry (HPLC-MS) method was developed for the determination of seven troponin activators (GSK854, Nelutroctiv, Tirasemtiv, RPI-194, EMD57033, 3-chlorodiphenylamine, Reldesemtiv) in urine and serum. For urine samples, liquid-liquid extraction (LLE) was used for pretreatment. The method demonstrated limits of detection (LODs) of 0.04-0.36 ng/mL, with accuracy ranging from 80.7 to 114.5% and precision with RSDs below 14.6% for all analytes. The concentrations of the analytes remained within approximately 3.0-4.5 ng/mL over 7 days under various storage conditions, indicating generally acceptable stability, although compound-dependent degradation was observed, particularly for 3-chlorodiphenylamine. For serum samples, pretreatment combined acetonitrile precipitation and QuEChERS. The LOD was 0.007-0.52 ng/mL, with accuracy ranging from 74.0 to 118.5% and precision (RSD) below 12.1%, and good linearity in the calibration range. This method is expected to contribute significantly to anti-doping control.