A population study of urine glycerol concentrations in elite athletes competing in North America

Abstract

Glycerol is an endogenous substance that is on the World Anti-Doping Agency's list of prohibited threshold substances due to its potential use as a plasma volume expansion agent. The WADA has set the threshold for urine glycerol, including measurement uncertainty, at 1.3 mg/mL. Glycerol in circulation largely comes from metabolism of triglycerides in order to meet energy requirements and when the renal threshold is eclipsed, glycerol is excreted into urine. In part due to ethnic differences in postprandial triglyceride concentrations, we investigated urine glycerol concentrations in a population of elite athletes competing in North America and compared the results to those of athletes competing in Europe. 959 urine samples from elite athletes competing in North America collected for anti-doping purposes were analyzed for urine glycerol concentrations by a gas chromatography mass-spectrometry method. Samples were divided into groups according to: Timing (in- or out-of-competition), Class (strength, game, or endurance sports) and Gender. 333 (34.7%) samples had undetectable amounts of glycerol (<1 μg/mL). 861 (89.8%) of the samples had glycerol concentrations ≤20 μg/mL. The highest glycerol concentration observed was 652 μg/mL. Analysis of the data finds the effects of each category to be statistically significant. The largest estimate of the 99.9(th) percentile, from the in-competition, female, strength athlete samples, was 1813 μg/mL with a 95% confidence range from 774 to 4251 μg/mL. This suggests a conservative threshold of 4.3 mg/mL, which would result in a reasonable detection window for urine samples collected in-competition for all genders and sport classes.

Applications and Challenges in Using LC–MS/MS Assays for Quantitative Doping Analysis

“…In September 2013, a glycerol threshold was set at 4.3 mg/ml [2] because the previous threshold (1.3 mg/ml) was more prone to false positives [28]. Glycerol is not expected to have an AAF and an accurate, precise and sensitive method with LC-MS was proposed for measuring it using simple urine sample preparation including a 10-fold urine dilution followed by injection into the LC coupled with high-resolution/high-accuracy MS (LC-HRMS) (dilute-and-shoot).…”

“…THCA and its glucuronide conjugate are major urinary cannabinoid metabolites and the WADA threshold for THCA is 150 ng/ml. Dilute-and-shoot method development and validation for LC-MS/MS to measure urinary THCA has been described in the literature [10][11][12] and this approach permitted more rapid sample preparation with fewer steps prior to LC-MS/ THCA 150 ng/ml 15 ng/ml 10 180 ng/ml [10][11][12][13][14][15][16][17][18][19][20][21] Salbutamol 1.0 μg/ml 0.1 μg/ml 10 1.2 μg/ml [22][23][24] Formoterol 40 ng/ml 6.0 ng/ml 15 50 ng/ml [10,[25][26][27] Glycerol 4.3 mg/ml 0.65 mg/ml 15 5.3 mg/ml [28][29][30] Morphine 1.0 μg/ml 0.15 μg/ml 15 1.3 μg/ml [31][32][33][34][35][36][37][38][39][40][41] Cathine 5.0 μg/ml 0.5 μg/ml 10 6.0 μg/ml [22] Ephedrine 10 μg/ml 0.5 μg/ml 5 11 μg/ml [42][43][44][45][46][47][48][49]…”

Annual banned‐substance review: analytical approaches in human sports drug testing

“…In order to account for potential differences in glycerol elimination originating from endogenous sources, e.g . due to ethnicity, gender, etc., 959 athletes' samples from North America were quantified concerning urinary glycerol concentrations . As a result of the findings presented therein, the above mentioned conservative threshold of 4.3 mg/mL was suggested and adopted since urinary glycerol values observed in post‐administration samples reached up to more than 60 mg/mL.…”

Screening and confirmation of a glycerol‐positive case

“…Nevertheless, glycerol was included in the World Anti‐Doping Agency (WADA) prohibited list from January 2012 onwards, with a threshold of 1.3 mg/mL . In September 2013, the threshold was further increased to 4.3 mg/mL (Decision limit (DL) 5.3 mg/mL) because research has shown that the previous threshold might yield false positive findings . Because of the aforementioned questionable and moderate benefits of glycerol, it was not expected to have an Adverse Analytical Finding (AAF).…”