A multi-targeted liquid chromatography–mass spectrometry screening procedure for the detection in human urine of drugs non-prohibited in sport commonly used by the athletes

Abstract

This work presents an analytical method for the simultaneous analysis in human urine of 38 pharmacologically active compounds (19 benzodiazepine-like substances, 7 selective serotonin reuptake inhibitors, 4 azole antifungal drugs, 5 inhibitors of the phosphodiesterases type 4 and 3 inhibitors of the phosphodiesterase type 5) by liquid-chromatography coupled with tandem mass spectrometry. The above substances classes include both the most common “non banned” drugs used by the athletes (based on the information reported on the “doping control form”) and those drugs who are suspected to be performance enhancing and/or act as masking agents in particular conditions. The chromatographic separation was performed by a reverse-phase octadecyl column using as mobile phases acetonitrile and ultra-purified water, both with 0.1% formic acid. The detection was carried out using a triple quadrupole mass spectrometric analyser, positive electro-spray as ionization source and selected reaction monitoring as acquisition mode. Sample pre-treatment consisted in an enzymatic hydrolysis followed by a liquid–liquid extraction in neutral field using tert-butyl methyl-ether. The analytical procedure, once developed, was validated in terms of sensitivity (lower limits of detection in the range of 1–50ngmL−1), specificity (no interferences were detected at the retention time of all the analytes under investigation), recovery (≥60% with a satisfactory repeatability, CV % lower than 10), matrix effect (lower than 30%) and reproducibility of retention times (CV% lower than 0.1) and of relative abundances (CV% lower than 15). The performance and the applicability of the method was evaluated by analyzing real samples containing benzodiazepines (alprazolam, diazepam, zolpidem or zoplicone) or inhibitors of the phosphodiesterases type 5 (sildenafil or vardenafil) and samples obtained incubating two of the phosphodiesterases type 4 studied (cilomilast or roflumilast) with pooled human liver microsomes. All the parent compounds, together with their main phase I metabolites, were clearly detected using the analytical procedures here developed.