Serum Estradiol Quantified by Isotope Dilution–Gas Chromatography/Mass Spectrometry

Estradiol measurements are important in evaluations of ovarian function, infertility, and menopause (1). The assays are challenging because physiologic concentrations of estradiol are typically <100 ng/L in plasma of adult men and postmenopausal women and in both sexes during infancy and childhood (1). Immunoassays are widely used, and they provide high sensitivity and short analysis times (2). Other methods are needed to determine the accuracy of immunoassays. Our aim was to develop a practical gas chromatography–mass spectrometry (GC/MS) method to quantify serum estradiol and use it to evaluate immunoassay accuracy.Isotope dilution–GC/MS is widely regarded as the most accurate technique for organic analytes of interest in clinical chemistry (3). Several GC/MS methods have been reported for the quantification of estradiol in biological sources (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). The complexity of samples requires isolation and purification of estradiol before derivatization for GC/MS analysis.The most common separation methods for estradiol involve solvent extraction followed by further purification with Sephadex LH-20 (4)(5), strong ionic exchangers (6)(7)(8)(9), weak ionic exchanger (9)(10)(11), HPLC (12)(13), affinity chromatography (14), or their combinations. These methods, however, have limitations. The HPLC method for estradiol isolation cannot handle multiple samples simultaneously. The affinity column (14) is not commercially available and is very expensive to make. Other methods generally cannot sufficiently remove serum matrix materials to ensure rugged performance.Here we report a GC/MS method for the quantification of serum estradiol in which estradiol is separated from the ether extract of serum samples by fractionation with a polystyrene divinylbenzene resin that has strong anion- exchange and adsorption properties. The method eliminates the use of HPLC in sample preparation and …