Quantitative trace analysis of estriol in human plasma by negative ion chemical ionization gas chromatography–mass spectrometry using a deuterated internal standard

Abstract

A stable isotope dilution gas chromatography–mass spectrometry (GC–MS) assay for the trace level determination of estriol in human plasma is described. Negative ion chemical ionization (NICI) MS is used for highly specific detection. The method involves derivatization of the phenolic hydroxyl to the pentafluorobenzyl ether derivative and subsequent reaction of the remaining hydroxyls with heptafluorobutyric anhydride. This derivative allows detection of the strikingly abundant phenolate ion under NICI conditions. [2,4,17β]- 2H 3-labeled estriol was used as an internal standard. For high-level measurements (>313 ng/l) plasma was directly derivatized by extractive alkylation followed by heptafluorobutylation prior to analysis. A rapid and simple sample work up procedure was elaborated for trace level determinations (>5 ng/l plasma) using solid-phase extraction on C 18 with an absolute recovery of 92.9%. For low-level measurements, the calibration curve was linear in the range of 5 to 625 ng/l ( r=0.99993). Inter-assay analytical precisions (RSDs) were 1.29, 2.30 and 2.89% at 39, 156 and 650 ng/l plasma, respectively. For high-level measurements, calibration curve linearity was observed in the range of 0.313 to 20 μg/l ( r=0.99998). Inter-assay analytical precisions (RSDs) were 5.17, 1.92, 2.57 and 2.74% at 0.313, 0.625, 2.5 and 10 μg/l plasma, respectively. Postmenopausal plasma was used for spiked plasma samples. Sensitivity and specificity of the presented method allows adequate determination of estriol in human plasma samples.