Simple and sensitive cellulose paper-based microextraction of 25 steroid esters from human serum/plasma for doping...

Blood is increasingly used as a biological matrix in the doping control field; however, the limited volume typically available for analysis poses a significant challenge, particularly when multiple tests must be carried out on the same sample. This study proposes a simple and efficient microextraction protocol based on the use of unmodified cellulose for the extraction of 25 steroid esters from just 20 µL of serum or plasma. Sample preparation consisted of spotting 20 µL of serum or plasma on a cellulose card, followed by a double extraction of the target analytes with 500 µL of methanol and subsequent derivatization using Girard's Reagent P. The analysis was carried out using liquid chromatography coupled with tandem mass spectrometry. The analytical workflow was qualitatively validated according to the UNI CEI EN ISO/IEC 17025 standard and the WADA technical rules and notes in terms of selectivity (target analytes were distinguishable from the matrix interferences), sensitivity (limits of detection in the range of 0.05-0.70 ng/mL), carry-over (no signals were detected in the negative sample injected after the positive sample), intra and inter-day stability of the retention times (≤0.5%), matrix effect (13-32%), extract stability (the target analytes were stable for at least 72 h in the autosampler at 10 °C), and extraction yield (43-88%). Plasma samples collected after testosterone undecanoate injection were finally analyzed: the compound was detectable in all three volunteers evaluated for at least 1 month after administration, demonstrating the effectiveness of the newly developed method for doping control purposes.