Glycerol and mannitol could be abused by athletes for its capability to keep under control their haematocrit or haemoglobin values, following illicit stimulation of erythropoiesis. As a consequence,
glycerol was added to the WADA prohibited list in January 2010 and all forms of glycerol administration are prohibited in sports. In addition, the intravenously administered of mannitol has been
covered in the WADA prohibited list since early 2009. In this study, a novel analytical method is described and validated for the determination of urinary glycerol and mannitol levels using LC-MS/MS
with simple sample preparation. This method also makes the separation of different stereoisomers (allitol, altritol, galactitol, iditol, mannitol, sorbitol) possible and thus enables the
quantification of mannitol accurately.
An Agilent 1290 Series HPLC coupled to an Agilent triple-quadruple 6460C mass spectrometer equipped with an ESI source and multiple reaction monitoring (MRM) were employed. This method made use of
derivatization of glycerol and mannitol by benzoyl chloride in aqueous solution at 50oC for 1 hour followed by analysis via LC-MS/MS within 7 min on an Eclipse XDB-C18 column (2.1 mm × 100 mm, 3.5 μm)
in an initial testing procedure. For confirmation, all possible hexitols that can occur in human urine were baseline separated with mannitol and identified on a Poroshell 120 PFP (2.1 mm × 150 mm, 2.7
μm) column within 20 min.
The method for qualitative and quantitative analysis of glycerol and mannitol in human urine was developed and fully validated in this study. The limits of detection (LODs) for glycerol and mannitol
were 0.3 and 0.1 μg/mL seperately. The limits of quantification (LOQs) were 1.0 and 0.3 μg/mL respectively. The assay was linear from 1.0 to 1000 for glycerol and 0.3 to 1000 μg/mL for mannitol
independently in human urine. The coefficients of variation of all inter- and intra-assay determinations at three concentration levels (3, 500, 900 μg/mL) were less than 13% for glycerol and 15% for
mannitol. The method also afforded satisfactory results in terms of accuracy (82-87, 97-111 and 94-109% for glycerol, 104-118, 96-105 and 95-103% for mannitol at three concentrations), derivatization
yield (92, 96 and 92% for glycerol, 55, 61 and 65% for mannitol at three concentrations), extraction recovery (91, 90 and 94% for glycerol, 66, 75 and 84% for mannitol at three concentrations), matrix
effect (18, 5 and 2% for glycerol, 18, 6 and 3% for mannitol at three concentrations) and specificity for both substances.
The LC-MS/MS assay described is a suitable procedure for separation, detection and quantification of glycerol and mannitol in human urines. It has proven to be selective, linear, accurate and precise
for the two prohibited substances. In comparison to
previously published papers, this approach displayed significant improvements in throughput and ease of use. This method has been found to be applicable for doping-control purpose and applied to our
routine sample analysis.