Immunoaffinity trapping of urinary human chorionic gonadotropin and its high-performance liquid chromatographic-mass spectrometric confirmation.

A method has been developed for confirmation of the glycopeptide human chorionic gonadotropin (hCG) in urine. A solid-phase immunoaffinity trapping technique utilizing a monoclonal antibody recognizing both intact hCG and free hCG beta-subunits was developed for the extraction of hCG from urine. Recovery of hCG from a urine matrix was essentially quantitative. The hCG was quantitatively eluted with 6 M guanidine hydrochloride, reductively alkylated with vinylpyridine, and subjected to tryptic digestion. The tryptic digest was analyzed by HPLC-MS. Ions from three tryptic fragments were monitored with selected ion monitoring to provide specific detection of hCG. The signal observed for a concentration of 25 mIU/ml of hCG could be clearly distinguished from background with a signal-to-noise ratio of 12:1.