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Immunoextraction-tandem mass spectrometry method for measuring intact human chorionic gonadotropin, free ?-subunit, and ?-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 ?-subunit (hCG?), and ?-subunit core fragment (hCG?cf) 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 immunoextraction method with LC-MS/MS detection for quantification of intact hCG, hCG?, and hCG?cf in urine. METHODS:hCG isoforms were immunoextracted with antibody-conjugated magnetic beads and digested with trypsin, and hCG? and hCG?cf 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 hCG? separately. The limit of quantification will allow LC-MS/MS hCG reference intervals to be established in nondoping male athletes for improved doping control.

SUBMITTER: Woldemariam GA 

PROVIDER: S-EPMC4334569 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Immunoextraction-tandem mass spectrometry method for measuring intact human chorionic gonadotropin, free β-subunit, and β-subunit core fragment in urine.

Woldemariam Getachew A GA   Butch Anthony W AW  

Clinical chemistry 20140604 8


<h4>Background</h4>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 amo  ...[more]

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