A modified LC-MS/MS method to simultaneously quantify glycerol and mannitol concentrations in human urine for doping control purposes

Glycerol and mannitol have the potential to act as plasma volume expanders and have been prohibited as masking agents by the World Anti-Doping Agency (WADA) accordingly. In this study, an improved strategy was developed and validated for the determination of urinary glycerol and mannitol levels simultaneously using a liquid chromatography/tandem mass spectrometry technique within 7 min in an initial testing procedure. For confirmation, mannitol and all possible hexitols (allitol, altritol, galactitol, iditol and sorbitol) that can occur in human urine were baseline separated. This method made use of the derivatization of glycerol and mannitol by benzoyl chloride followed by analysis via LC-ESI-MS/MS with limited sample preparation. The limit of detection (LOD) for glycerol and mannitol was lower than 50 ng/mL. The limit of quantitation (LOQ) for both substances was below 150 ng/mL. The assay was linear from 0.15 to 1000 μg/mL for glycerol and mannitol in human urine. The coefficients of variation of all inter- and intra-assay determinations at three concentration levels (0.5, 500, 900 μg/mL) were better than 13% for glycerol and under 15% for mannitol. The method also afforded satisfactory results in terms of accuracy, derivatization yield, extraction recovery, matrix effect and specificity for both substances.