Quantitative analysis of urinary glycerol levels for doping control purposes using gas chromatography-mass spectrometry

The administration of glycerol to endurance athletes results in an increased fluid retention and improved performance, particularly under hot and humid conditions. Consequently, glycerol is considered relevant for sports drug testing and methods for its detection in urine specimens are required. A major issue in this regard is the natural occurrence of trace amounts of glycerol in human urine, which necessitates a quantitative analysis and the determination of normal urinary glycerol levels under various sporting conditions. A quantitative method was established using a gas chromatography/isotope-dilution mass spectrometry-based approach that was validated with regard to tower limit of detection (0.3 mu g mL(-1)), Lower limit of quantification (0.9 mu g mL(-1)), specificity, linearity (1.0-98.0 mu g mL(-1)), intraday and interday precision (<20% at 2.4, 24.1 and 48.2 mu g mL(-1)) as well as accuracy (92-110%). Sample aliquots of 20 mu L were enriched with five-fold deuterated glycerol, dried and derivatised using N-methyt-trimethylsilyltrifluoroacetamide (MSTFA) before analysis. The established method was applied to a total of 1039 doping control samples covering various sport disciplines 1349 endurance samples, 286 strength sport samples, 325 game sport samples and 79 other samples) in- and out-of-competition, which provided quantitative information about the glycerol content commonly observed in elite athletes' urine samples. About 85% of all specimens yielded glycerol concentrations <20.0 mu g mL(-1) and few reached values up to 132.6 mu g mL(-1). One further sample, however, was found to contain 2690 mu g mL(-1), which might indicate the misuse of glycerol, but no threshold for urinary glycerol concentrations has been established yet due to the Lack of substantial data. Based on the results obtained from the studied reference population, a threshold for glycerol levels in urine set at 200 mu q mL(-1) is suggested, which provides a tool. to doping control laboratories to test for the misuse of this agent in elite and amateur sport.