Urinary human chorionic gonadotropin isoform concentrations in doping control samples

Abstract

Anti-doping laboratories routinely use immunoassays to measure urinary concentrations of human chorionic gonadotropin (hCG). To minimize immunoassay differences and false positive screen results from inactive isoforms (free β-subunit (hCGβ), β-subunit core fragment (hCGβcf)) laboratories now use intact hCG instead of total hCG immunoassays to measure hCG. To determine the distribution of hCG isoforms in urine, we determined the concentrations of intact hCG, hCGβ, and hCGβcf in male urine samples based on immunoassay total hCG concentrations using a sequential immunoextraction and a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. hCG was isolated using antibody-conjugated magnetic beads and unique tryptic peptides were quantified by LC-MS/MS. Negative samples with detectable but low total hCG concentrations (1.2-3.5 pmol/L) had intact and hCGβ concentrations <1.2 pmol/L, and hCGβcf concentrations <2.3 pmol/L by LC-MS/MS. Urine samples from an athlete receiving hCG had intact hCG concentrations ranging from 18.8 to 57.6 pmol/L, hCGβ concentrations <0.7 pmol/L, and hCGβcf concentrations ranging from 94 to 243% of the intact hCG concentration. In 27 atypical samples with total hCG concentrations ranging from 16.7 to 412.7 pmol/L with intact hCG <2.7 pmol/L by immunoassay, all samples had intact hCG concentrations <3.8 pmol/L and hCGβ concentrations <6.2 pmol/L by LC-MS/MS. hCGβcf concentrations by LC-MS/MS varied widely and ranged from 1.03 to 21.9 pmol/L. In summary, total hCG immunoassays significantly overestimate hCG concentrations and can produce false positive results. Although the intact hCG immunoassay slightly overestimates hCG concentrations compared to LC-MS/MS, it can distinguish between cases of hCG use and atypical cases with elevated total hCG concentrations. Copyright © 2016 John Wiley & Sons, Ltd.

Ultrasensitive Detection of Human Chorionic Gonadotropin Using Frequency Locked Microtoroid Optical Resonators

“…The use of performance enhancing drugs during athletic events is prohibited by the World Anti-Doping Association (WADA). To monitor the use of these drugs, antidoping drug tests are routinely performed during athletic events. , These tests can detect minute quantities of chemicals from bodily fluids, such as blood or urine. Mass spectrometry is currently the gold standard , for detecting various doping agents; however, it can have insufficient limits of detection, requires a trained operator, involves specialized enzymes, , and can be expensive, costing in excess of $100 per assay.…”

Urine reference intervals for human chorionic gonadotropin (hCG) isoforms by immunoextraction–tandem mass spectrometry to detect hCG use

“…When compared to hCG immunoassays routinely utilized by WADA‐accredited laboratories to screen for doping with hCG, the LC–MS/MS method produces considerably lower intact hCG concentrations. This has been shown when hCG positive urine samples from excretion studies and atypical urine samples with total hCG concentrations >5 IU/L were analyzed using the Roche intact hCG immunoassay and the LC–MS/MS method . In addition, the Immulite total hCG immunoassay has also been shown to produce hCG concentrations as high as 4.9 IU/L when 1199 urine samples from male athletes were analyzed .…”

“…Since intact hCG is the biologically active form and is detected in the highest concentrations following hCG administration, the upper threshold at 99.9% (1 in 1000) and 99.99% (1 in 10000) for intact hCG would be 0.56 IU/L (1.32 pmol/L) and 0.98 IU/L (2.30 pmol/L), respectively. Based on these data, a threshold of 1.0 IU/L for urinary 9,15 In addition, the Immulite total hCG immunoassay has also been shown to produce hCG concentrations as high as 4.9 IU/L when 1199 urine samples from male athletes were analyzed. 16 These data indicate that separate hCG thresholds are needed depending upon the method used for…”

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

“…For human chorionic gonadotrophin (hCG), threshold levels apply for the intact heterodimeric version of the hormone with 5 IU/L for doping control samples collected from male athletes . Butch and Woldemariam compared the performance of different immunoassays targeting either total hCG (including the intact heterodimer of hCG, hCGβ, and hCGβ core fragment (cf) or intact heterodimeric hCG only with an established LC–MS/MS method . In a total of 27 routine doping control samples that returned total hCG levels between 7.1 and 175.6 IU/L, intact hCG was determined ≤ 1.1 IU/L using immunological test methods and ≤ 1.6 IU/L by means of LC–MS/MS.…”