Rapid and harmonized analytical workflow for the determination of peptidic and non-peptidic doping agents in dried and liquid blood matrices

Abstract

Recently, methods for detecting small peptides in dried blood spots have been published. These procedures typically involve multiple sample preparation steps, resulting in labor-intensive and costly workflows. In the present study, we report a fast, streamlined, and harmonized analytical workflow to detect 54 prohibited peptidic and non-peptidic compounds in dried blood spots, serum, and plasma. Sample preparation is based on 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. The validation results showed satisfactory performance with respect to selectivity (no interferences were detected at the retention times of the analytes), detection limits (0.05-1.25 ng/mL), carry-over (no signals in the negative sample injected after the positive sample), matrix effect (5-33%), extraction yield (15-80%), and extract stability (the target analytes were stable for at least 72 h in the autosampler at 10 °C). The method was successfully applied to samples containing sub-ng levels of ibutamoren, confirming that the analytical procedure presented in this study is fit for purpose within the doping-control framework. Stability studies showed that all compounds were stable (variation lower than 15%) for at least two months at −20 °C in all the blood matrices considered. At 4 and 22 °C, alexamorelin, AOD9604, buserelin, hGH 176–191, kisspeptin-10 and LHRH were extensively degraded after one week in serum and plasma, whereas BPC-157, TB500, vasopressin, lypressin, and terlipressin showed complete degradation only in serum. In contrast, in dried matrices all compounds remained detectable throughout the entire duration of the study, indicating that samples can be transported and stored under non-refrigerated conditions, thereby reducing costs.