Immunoaffinity extraction and tandem mass spectrometric analysis of human chorionic gonadotropin in doping analysis

A confirmatory and quantitative HPLC–tandem mass spectrometry (MS–MS) method for human chorionic gonadotropin hormone (hCG) at concentrations as low as 5 IU/l following immunoaffinity extraction of the glycoprotein from urine was developed. The extraction method involved retention of urinary hCG in the immunoaffinity column via specific antigen–antibody interaction. A variety of eluents were then used to quantitatively elute hCG from the immunoaffinity column. Qualitative and quantitative analysis of hCG were undertaken using MS–MS by identifying the amino acid sequence of the marker peptide βT5 obtained from hCG by tryptic digestion and the peak areas of three product ions b6+, b9+ and y11+, respectively.