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Analytica chimica acta

Bulk derivatization and cation exchange restricted access media-based trap-and-elute liquid chromatography-mass spectrometry method for determination of trace estrogens in serum.


PMID 25597805

Abstract

Estrone (E1), estradiols (α/β-E2), and estriol (E3) are four major metabolically active estrogens exerting strong biological activities at very low circulating concentrations. This paper reports a sensitive and efficient method with automated, on-line clean-up and detection to determine trace estrogens in a small volume of serum samples using liquid chromatography-electrospray ionization-tandem mass spectrometry directly, without off-line liquid-liquid or solid-phase extraction pretreatments. Serum aliquots (charcoal stripped fetal bovine serum, 100 μL) were spiked with four estrogen standards and their corresponding isotope-labeled internal standards, then bulk derivatized with 2-fluoro-1-methyl-pyridium p-toluenesulfonate (2-FMP) to establish the calibration curves and perform method validation. Calibration was established in the concentration ranges of 5-1000 pg mL(-1), and demonstrated good linearity of R(2) from 0.9944 to 0.9997 for the four derivatized estrogens. The lower detection limits obtained were 3-7 pg mL(-1). Good accuracy and precision in the range of 86-112% and 2.3-11.9%, respectively, were observed for the quality control (QC) samples at low, medium, and high concentration levels. The stability tests showed that the derivatized serum samples were stable 8h after derivatization at room temperature and at least to 48 h if stored at -20 °C. The method was applied to measure trace estrogens in real human and bovine serum samples, and three of four estrogen compounds studied were observed and quantified.