Measurement of estradiol, estrone, and testosterone in postmenopausal human serum by isotope dilution liquid chromatography tandem mass spectrometry without derivatization

• We present a high-throughput sample preparation method for serum steroids.
• We present a sensitive mass spectrometric method for quantitating steroids.
• LOD, LOQ, sLOQ: E1 (0.1 pg, 0.3 pg, 1 pg/ml), E2 (0.3 pg, 1 pg, 3 pg/ml), T (0.3 pg, 1 pg, 30 pg/ml).
• Comparison between ELISA and LC–MS/MS with postmenopausal samples.
• LC–MS/MS able to accurately and reproducibly quantitate samples ELISA cannot.

BackgroundA high-throughput, sensitive, specific, mass spectrometry-based method for quantitating estrone (E1), estradiol (E2), and testosterone (T) in postmenopausal human serum has been developed for clinical research. The method consumes 100 μl human serum for each measurement (triplicates consume 300 μl) and does not require derivatization. We adapted a commercially available 96-well plate for sample preparation, extraction, and introduction into the mass spectrometer on a single platform.MethodsSteroid extraction from serum samples and mass spectrometer operational parameters were optimized for analysis of estradiol and subsequently applied to other analytes. In addition to determining the limit of detection (LOD) and limit of quantitation (LOQ) from standard curves, a serum LOQ (sLOQ) was determined by addition of known steroid quantities to serum samples. Mass spectrometric method quantitative data were compared to results using a state-of-the-art ELISA (enzyme-linked immunosorbent assay) using stored serum samples from menopausal women.ResultsThe LOD, LOQ, sLOQ was (0.1 pg, 0.3 pg, 1 pg/ml) for estrone, (0.3 pg, 1 pg, 3 pg/ml) for estradiol, and (0.3 pg, 1 pg, 30 pg/ml) for testosterone, respectively. Mass spectrometry accurately determined concentrations of E2 that could not be quantified by immunochemical methods. E1 concentrations measured by mass spectrometry were in all cases significantly lower than the ELISA measurements, suggesting immunoreactive contaminants in serum may interfere with ELISA. The testosterone measurements broadly agreed with each other in that both techniques could differentiate between low, medium and high serum levels.ConclusionsWe have developed and validated a scalable, sensitive assay for trace quantitation of E1, E2 and T in human serum samples in a single assay using sample preparation method and stable isotope dilution mass spectrometry.