Development of intact hCG reference intervals in normal male urine samples for establishing a threshold value for doping control

Code: T14M02AB 

Human chorionic gonadotropin (hCG) stimulates testosterone production by the testicles and is prohibited in males according to the World Anti-Doping Agency list of prohibited substances. Immunoassays are currently used by anti-doping laboratories to measure urinary hCG but results can vary widely among laboratories due to differences in cross-reactivity against different molecular forms of hCG (isoforms). We recently developed a sequential immunoextraction method with liquid chromatography tandem mass spectrometry (LC-MS/MS) detection for quantification of intact hCG, hCG free β-subunit and β-subunit core fragment in urine. Data from hCG excretion studies demonstrated that intact hCG should be monitored for detection of doping with hCG. In order to apply the LC-MS/MS method for confirmation of immunoassay screen positive hCG results a threshold concentration needs to be established. In preliminary studies we found that the current threshold concentration of 5 mIU/mL applied to immunoassays is not appropriate when measured by LC-MS/MS and needs to be significantly lower. 
In the present study we plan to determine the concentration of intact hCG in 600 non-doping male urine samples using the recently developed immunoextraction method with LC-MS/MS detection. Half of the urine samples will be from normal male volunteers and the other half from non-doping male athletes. The data from this study will be immediately used to determine the appropriate threshold concentration for confirming doping with hCG. 

Main Findings:

The concentrations of intact hCG in the male urine samples are provided in the attached file and are graphically displayed in Figure 1. Of the 570 male urine samples, 243 had undetectable intact hCG concentrations (<0.02 IU/L) not displayed on the graph. The remaining 327 urine samples had intact hCG concentrations ranging from 0.02 to 0.50 IU/L.  Of the 590 total samples, 542 (95.1%) had intact hCG concentrations <0.01 IU/L. Of the remaining 28 samples, 27 had intact hCG concentrations >0.01 and <0.26 IU/L. Only sample had an intact hCG concentration of 0.50 IU/L (27 year old, not shown on graph). Given these data, we recommend an extremely conservative intact hCG cutoff of 2 IU/L to be used during confirmation testing for detection of doping with hCG. At a concentration of 2.5 IU/L the imprecision of the method was 11.4%, when 3 separate urine samples were analyzed on 5 different days.