Systematic analysis of glycerol: colourimetric screening and gas chromatography–mass spectrometric confirmation

Abstract

Glycerol is a naturally occurring polyol in the human body, essential for several metabolic processes. It is widely used in the food, pharmaceutical, and medical industries and in clinical practice as a plasma volume expander (PVE). Athletes, however, may use glycerol to mask the presence of forbidden substances or to enhance performance, inclusively through hyperhydration achieved by glycerol ingestion with added fluid. These practices are considered doping, and are prohibited by the World Anti-Doping Agency (WADA). Therefore, glycerol was introduced in the prohibited list. Doping through glycerol ingestion can readily be identified by detection of elevated glycerol concentrations in urine. In this paper, a protocol for the fast detection of glycerol in urine is proposed. It consists of a previous visual colourimetric screening, followed by a quantitative/qualitative confirmation analysis by mass spectrometry. The screening procedure involves a reaction in which polyhydric alcohols are oxidized by periodate to formic acid and formaldehyde, which is detected by the addition of a fuchsin solution. For the subsequent qualitative/quantitative confirmation analysis, a gas chromatography-mass spectrometry based approach with a non-deuterated internal standard and a drying step of only 10 min is proposed. The linear correlation was demonstrated within WADA´s threshold range. The calculated RSD were 2.1% for within-day precision and 2.8% for between-day precision. The uncertainty estimation was calculated, and a value of 2.7% was obtained. The procedure may also be used for the analysis of other polyols in urine, as for example the PVE mannitol.

Doping control analysis at the Rio 2016 Olympic and Paralympic Games

“…[19] Approximately 1.2% of the samples were submitted to dedicated LC-MS methods developed for the confirmation of these analytes. All confirmations were negative.…”

“…The XXXI Summer Olympic Games (August [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21]2016) and the XV Paralympic Games (September 7-18, 2016) were held in Rio de Janeiro, marking the first time that these events were organised in South America. Doping control analyses have been conducted in Brazil since 1989, were incorporated into the International Olympic Committee (IOC) accreditation in 2002, and were absorbed by the World Anti-Doping Agency (WADA) accreditation system in 2005.…”

Screening and confirmation of a glycerol‐positive case

“…Papers describing glycerol detection by GC‐MS are ubiquitous . Hence the first idea was to screen for glycerol by this latter technique by incorporating glycerol into a GC‐MS method used for the detection of anabolic steroids .…”

“…Extracted ion chromatograms and corresponding full scan mass spectra for glycerol after confirmatory GC‐MS analysis: (a) blank sample; (b) QC‐sample spiked at 4.3 mg/mL; (c) AAF‐sample. All chromatograms were scaled to the peak found in the AAF (c) to illustrate the low background originating from the endogenous occurrence of glycerol in blank urine…”

Fast Ephedrine Quantification by Gas Chromatography Mass Spectrometry

“…The samples were analyzed by the method described by Sardela et al 21 for quantification of glycerol. Briefly, ten microliters of urine sample were mixed with 190 µL of a freshly prepared internal standard (I.S.)…”