Multiresidue LC-MS screening by using solid-phase extraction and optimized enzymatic hydrolysis in sports doping

Abstract

In this study, we developed a multi-residue tandem analysis method using solid-phase extraction and optimized glucuronidase enzymatic hydrolysis for 351 prohibited substances in sports doping. When athletes take performance enhancing drugs (PEDs), the substances themselves are rarely detected in urine and are mostly excreted in the form of conjugated glucuronic acid. To obtain accurate results for drug analysis, the glucuronide form must be converted back to its parent form using the enzyme glucuronidase. To optimize the extraction efficiency of glucuronides, different reaction conditions consisted of the pH of the extraction solvent, incubation temperature, and hydrolysis time, and solid phase extraction (SPE) was performed under the same conditions. Univariate studies were performed to evaluate the influence of each operational variable as the ratio of morphine area to peak area of morphine-3-β-d after hydrolysis. In addition, the column and mobile phase were optimized to improve the separation efficiency, which significantly enhanced the sensitivity to HIF stabilizers and certain polar substances. The results showed that the sensitivity was improved by at least 2-fold and up to 300-fold over previous assays for several banned drugs, including polar substances such as meldonium and oxilofrine. The developed method was validated for limit of detection (LOD), selectivity, reliability, stability, and matrix effects. The developed method was also used to analyze real doping samples and proved to be a reliable tool as no false negative or positive results were observed.