Dataset Information

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

ABSTRACT: A 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?l human serum for each measurement (triplicates consume 300?l) 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.Steroid 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.The LOD, LOQ, sLOQ was (0.1pg, 0.3pg, 1pg/ml) for estrone, (0.3pg, 1pg, 3pg/ml) for estradiol, and (0.3pg, 1pg, 30pg/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.We 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.

SUBMITTER: Wooding KM

PROVIDER: S-EPMC4366052 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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

Wooding Kerry M KM Hankin Joseph A JA Johnson Chris A CA Chosich Justin D JD Baek Sung W SW Bradford Andrew P AP Murphy Robert C RC Santoro Nanette N

Steroids 20150121

<h4>Background</h4>A 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 100μl human serum for each measurement (triplicates consume 300μl) 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 plat ...[more]

PMID: 25617740

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Project description:The inaccuracy of routine serum 25-hydroxyvitamin D measurements hampers the interpretation of data in patient care and public health research. We developed and validated a candidate reference measurement procedure (RMP) for highly accurate quantitation of two clinically important 25-hydroxyvitamin D metabolites in serum, 25-hydroxyvitamin D2 [25(OH)D2] and 25-hydroxyvitamin D3 [25(OH)D3]. The two compounds of interest together with spiked deuterium-labeled internal standards [d 3-25(OH)D2 and d 6-25(OH)D3] were extracted from serum via liquid-liquid extraction. The featured isotope-dilution LC-MS/MS method used reversed-phase chromatography and atmospheric pressure chemical ionization in positive ion mode. A pentafluorophenylpropyl-packed UHPLC column together with isocratic elution allowed for complete baseline resolution of 25(OH)D2 and 25(OH)D3 from their structural C-3 isomers within 12 min. We evaluated method trueness, precision, potential interferences, matrix effects, limits of quantitation, and measurement uncertainty. Calibration materials were, or were traceable to, NIST Standard Reference Materials 2972. Within-day and total imprecision (CV) averaged 1.9 and 2.0% for 25(OH)D3, respectively, and 2.4 and 3.5% for 25(OH)D2, respectively. Mean trueness was 100.3% for 25(OH)D3 and 25(OH)D2. The limits of quantitation/limits of detection were 4.61/1.38 nmol/L for 25(OH)D3 and 1.46/0.13 nmol/L for 25(OH)D2. When we compared our RMP results to an established RMP using 40 serum samples, we found a nonsignificant mean bias of 0.2% for total 25(OH)D. This candidate RMP for 25(OH)D metabolites meets predefined method performance specifications (?5% total CV and ?1.7% bias) and provides sufficient sample throughput to meet the needs of the Centers for Disease Control and Prevention Vitamin D Standardization Certification Program. Graphical abstract Bias assessment using NIST standard reference materials. Legend CDC mean mass fractions (ng/g) ± U 95 (6 replicates per mean). NIST-certified mass fractions (ng/g) ± U 95 from the Certificates of Analysis.

Dataset Information

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

Publications

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

Similar Datasets

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