Initial testing procedure based on microextraction followed by LC-HRMS analysis to determine 107 xenobiotics and their metabolites in serum/plasma

Abstract

A simple and sensitive microextraction protocol based on the use of unmodified cellulose was developed for the simultaneous extraction of 107 prohibited compounds and their metabolites from 20 µL of serum and plasma. Sample preparation consisted of spotting 20 µL of serum/plasma onto a cellulose card, followed by extraction of the analytes with 500 µL of a methanol/acetonitrile (1 : 1, v/v) mixture for 20 min. The extracts were analyzed by liquid chromatography coupled to high-resolution mass spectrometry. The entire workflow was validated in terms of selectivity (no interferences were detected at the retention times of the target analytes), sensitivity (limits of detection in the range of 0.08–7.50 ng mL⁻¹) carry-over (no signals in the negative sample injected after the positive sample at high concentration), matrix effect (10–28%), extraction yield (42–89%), and extract stability (the analytes were stable for at least 72 h in the autosampler at 10 °C). The method was successfully applied to the analysis of samples containing the compounds at low nanogram per milliliter range, demonstrating its effectiveness for doping control purposes. Stability studies showed that the compounds were stable for at least 3 months at −20 and 4 °C in serum and plasma samples. In contrast, at 22 °C several thiazide-based compounds were completely degraded after 4 weeks; FG2216 was no longer detectable after 7 weeks; S6 and RAD140 were no longer detectable after 9 weeks, whereas trenbolone was completely degraded after 14 weeks. The other compounds were still visible for the entire study period, with variations in the range of 37–56%.