A Sensitive and Specific ESI-Positive LC–MS/MS Method for the Quantitation of Estrogens in Human Serum in Under Three Minutes

Amplifex Diene reagent was employed to derivatize estradiol (E2) to enhance the analyte signal at low picogram concentrations. This derivatization enabled measurement of E2 (and other estrogens) in ESI+ mode, earlier retention times for analytes than other methods, avoidance of MS harmful ammonium fluoride in mobile phases, and an LLOQ below 1 pg/mL. The sample preparation workflow involved liquid–liquid extraction followed by Amplifex Diene derivatization for 10 min at ambient temperature. Samples were chromatographed using a standard C18 column and analyzed using a SCIEX 6500(+) mass spectrometer. The assay calibrators were prepared in-house, traceable to certified reference materials, and ranged from 1.29 to 624 pg/mL. A method comparison to samples from the CDC HoSt program yielded a correlation coefficient of 0.9858 and bias of −1.37%. The LLOQ using certified reference material was 0.66 pg/mL. The intra-run precision was <9.00% for low- and high-level samples, whereas the inter-run precision was 15.2 and 5.43% for low- and high-level samples, respectively. No interference from other clinically relevant steroids was found. Amplifex Diene derivatized E2 and estrone (E1) was found to be stable for over 6 months, both refrigerated and frozen.