Dried urine spot strategy coupled with UHPLC-MS/MS for the analysis of troponin activators

A rapid, sensitive, and reliable analytical approach based on dried urine spot (DUS) sampling coupled with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was established and fully validated for the simultaneous quantification of six troponin activators in human urine, following the WADA and FDA analytical guidelines. Key experimental parameters, including filter paper type, punch diameter, extraction solvent, solvent volume, and ultrasonic extraction time, were systematically optimized. Under the optimized conditions, all target analytes exhibited favorable linearity (R² > 0.99) in their respective ranges, and the limits of detection (LODs) were below 1 ng/mL for most compounds. The extraction recoveries of the six troponin activators ranged from 80.16% to 90.34%, and matrix effects were determined to be within the range of 82.76%-111.34%. Both intra-day and inter-day precision values were below 15%. Stability verification revealed that DUS samples possessed distinctly superior long-term stability compared with liquid urine samples under diverse storage conditions. The established DUS-UHPLC-MS/MS method features simple operation, minimal sample consumption, low solvent dosage, and excellent stability, making it well-suited for rapid screening and quantitative confirmation of troponin activator misuse in routine sports anti-doping analysis.