Quantitative analysis of glycerol levels in human urine by liquid chromatography–tandem mass spectrometry

• Developed a novel LC–MS/MS method for the quantitation of glycerol in human urine.
• High specificity, sensitivity, throughput and wide linearity range (1–1000 μg/mL).
• Modifications in sample pretreatment make the quantitation more precise and accurate.
• Shorter run time and easy workup.

Glycerol has the latent capacity to act as a plasma volume expander and disguise blood doping practices. Therefore, it has been prohibited in sports as a masking agent by the World Anti-Doping Agency (WADA) since January 2010 and a urinary threshold (1 mg/mL) was recommended recently [1]. The purpose of this study was to establish and validate a novel quantitative method for the determination of urinary glycerol concentrations using a liquid chromatography–tandem mass spectrometry approach. This simple yet highly specific method made use of the derivatization of glycerol by benzoyl chloride in aqueous solution at 40 °C followed by analysis via LC–ESI-MS/MS without sample pre-concentration or cleanup. The assay was linear over the concentration range of 1.0–1000 μg/mL for glycerol in human urine. The lower limit of detection (LLOD) and lower limit of quantitation (LLOQ) were 0.3 μg/mL and 1.0 μg/mL, respectively. The intra- and inter-day precision of the method at three concentration levels (3, 500 and 900 μg/mL) was less than 12.2%. The method also afforded satisfactory results in terms of accuracy, derivatization yield, extraction recovery, matrix effect and specificity. The method has been successfully applied to the detection of glycerol in “Quality Assurance Program” samples provided by the World Association of Anti-Doping Scientists (WAADS) and routine doping-control samples in our laboratory.