Do Metabolites Account for Higher Serum Steroid Hormone Levels Measured by RIA Compared to Mass Spectrometry?

Higher circulating estradiol levels are generally obtained using conventional radioimmunoassays (RIA) compared to liquid chromatography/tandem mass spectrometry (LC-MS/MS) assays, and this has been attributed to the presence of estradiol metabolites that cross-react with the antibody used in the RIA. This study aimed to determine which estradiol metabolites may contribute to this effect. LC-MS/MS analysis was performed on 70 serum samples from premenopausal women, after purification by extraction and Celite column partition chromatography as would be used prior to conventional RIA for estradiol. The metabolites estrone and 2-methoxyestradiol accounted for 6.9% and 2.1% of the estradiol fractions in the purified samples overall, but the extent of contamination with these metabolites was greater in the samples containing <50 pg/mL estradiol (14.6% and 3.8% respectively) than in those containing >50 pg/mL estradiol. However, since these metabolites have a < 1% cross-reactivity to the antibody in the estradiol RIA, this level of contamination is too small to account for the differences between RIA and LC-MS/MS measurements of estradiol.