Immunoextraction–Tandem Mass Spectrometry Method for Measuring Intact Human Chorionic Gonadotropin, Free β-Subunit, and β-Subunit Core Fragment in Urine

Abstract

BACKGROUND Human chorionic gonadotropin (hCG) stimulates testosterone production by the testicles. Because of the potential for abuse, hCG is banned (males only) in most sports and has been placed on the World Anti-Doping Agency list of prohibited substances. Intact hCG, free β-subunit (hCGβ), and β-subunit core fragment (hCGβcf) are the major variants or isoforms in urine. Immunoassays are used by antidoping laboratories to measure urinary hCG. Cross-reactivity with isoforms differs among immunoassays, resulting in widely varying results. We developed a sequential im-munoextraction method with LC-MS/MS detection for quantification of intact hCG, hCGβ, and hCGβcf in urine. METHODS hCG isoforms were immunoextracted with antibody-conjugated magnetic beads and digested with trypsin, and hCGβ and hCGβcf unique peptides were quantified by LC-MS/MS with the corresponding heavy peptides as internal standard. hCG isoform concentrations were determined in urine after administration of hCG, and the intact hCG results were compared to immunoassay results. RESULTS The method was linear to 20 IU/L. Total imprecision was 6.6%-13.7% (CV), recovery ranged from 91% to 109%, and the limit of quantification was 0.2 IU/L. Intact hCG predominated in the urine after administration of 2 hCG formulations. The window of detection ranged from 6 to 9 days. Mean immunoassay results were 12.4-15.5 IU/L higher than LC-MS/MS results. CONCLUSIONS The performance characteristics of the method are acceptable for measuring hCG isoforms, and the method can quantify intact hCG and hCGβ separately. The limit of quantification will allow LC-MS/MS hCG reference intervals to be established in nondoping male athletes for improved doping control.

Urinary human chorionic gonadotropin isoform concentrations in doping control samples

“…As shown in Figure 1, hCG isoforms (hCG, hCGβ, and hCGβcf) were sequentially extracted from the 1 mL of concentrated urine using antibody-conjugated magnetic particles, as previously described. [12] β7 (GeneTex, Irvine, CA, USA) and β1 (Abcam, Cambridge, MA, USA) monoclonal antibodies were conjugated to carboxylate-modified Sera-Mag Magnetic SpeedBeads (GE Healthcare, Piscataway, NJ, USA) according to the manufacturer's instructions. [12] β7 (GeneTex, Irvine, CA, USA) and β1 (Abcam, Cambridge, MA, USA) monoclonal antibodies were conjugated to carboxylate-modified Sera-Mag Magnetic SpeedBeads (GE Healthcare, Piscataway, NJ, USA) according to the manufacturer's instructions.…”

“…[12] Materials and methods The distribution of hCG isoforms was determined in the following urine samples: samples with total hCG below the cut-off of 11.8 pmol/L; samples from an athlete receiving hCG for a medical condition; and samples with total hCG results >11.8 pmol/L and intact hCG result <11.8 pmol/L (atypical results).…”

Comparison of the point of care test (POCT), i-CHROMA™ human chorionic gonadotrophin (HCG), leutinzing hormone (LH) and follicle stimulating hormone (FSH) methods with the other laboratory methods in the Randox International Quality Assessment Scheme (RIQAS)

“…More recently, mass spectrometry-based assays have been developed for the detection and quantification of hCG in serum and urine [4][5][6]. Now qualitative and quantitative serum human chorionic gonadotropin (hCG) tests are used to diagnose pregnancy.…”

Androgens

“…hCG is routinely detected in urine by immunoassays which, unlike LH immunoassay, are validated for human urine having been developed for pregnancy testing and monitoring of trophoblastic tumors [92,93] . Recently, more accurate, sensitive, and specific immunoextraction-MS proteomic methods are becoming available [94] with some proof-of-principle clinical studies [95] . As hCG provides no ergogenic advantage in women, while potentially invading privacy by detecting early pregnancy, hCG testing is confined to men [59] .…”