Harmonized LC-HRMS workflow detects 54 doping agents, including BPC-157 and TB500, in dried blood spots with enhanced stability.

Detecting prohibited peptidic and non-peptidic doping agents in sports is crucial, but current analytical methods often involve complex, labor-intensive, and costly sample preparation, particularly for small peptides. Traditional liquid blood matrices also pose challenges for sample transport and long-term storage due to degradation and refrigeration requirements. There's a significant need for streamlined, harmonized workflows that can efficiently analyze a broad spectrum of compounds across various blood matrices, especially those that allow for simpler logistics.

Researchers developed a fast, harmonized analytical workflow to detect 54 prohibited peptidic and non-peptidic compounds in dried blood spots (DBS), serum, and plasma. Sample preparation involved a single microextraction step using 500 µL of a methanol/water (8:2, v/v) mixture. Detection was performed using liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS). The method's performance was validated for selectivity, detection limits, carry-over, matrix effect, extraction yield, and extract stability.

The validated LC-HRMS method demonstrated satisfactory performance across key parameters. Selectivity showed no interferences, and detection limits ranged from 0.05-1.25 ng mL-1. Matrix effects were 5-33%, and extraction yields were 15-80%. Extract stability confirmed target analytes were stable for at least 72 h in the autosampler at 10 °C. All compounds were stable (variation lower than 15%) for at least two months at -20 °C in all blood matrices. However, at 4 °C and 22 °C, alexamorelin, AOD9604, buserelin, hGH 176-191, kisspeptin-10, and LHRH extensively degraded after one week in serum and plasma. In contrast: > BPC-157, TB500, vasopressin, lypressin, and terlipressin showed complete degradation only in serum but remained detectable in dried blood spot matrices throughout the entire study duration.