Development of GC–MS/MS method with programmable temperature vaporization large volume injection for monitoring of 17β-estradiol and 2-methoxyestradiol in plasma

Abstract

Monitoring of estradiol and its metabolites in biological samples is essential for the accurate diagnosis of a number of endocrine diseases. In this study, a sensitive, precise and specific GC–MS/MS method for the quantification of 17β-estradiol (17-BE) and its main metabolite, 2-methoxyestradiol (2-MEOE), in plasma was developed and validated. Plasma concentrations of these steroids are currently investigated as diagnostic markers for pre-eclampsia, a systematic disorder of pregnancy and a leading cause of maternal and fetal morbidity and mortality worldwide. The method comprised treatment of the plasma sample by protein precipitation and subsequent isolation of steroids by solid phase extraction, derivatization of steroids by trifluoroacetic anhydride and GC–MS/MS analysis of the derivatized steroids. The large volume (10 μL) injection with the assistance of a Programmed Temperature Vaporization (PTV) injector in solvent split mode allowed a substantial increase in the sensitivity of the method. The ion trap MS was operated in optimized Product Ion Scan. By increasing the damping gas flow in the ion trap from the conventional 0.3 mL min −1 to 2 mL min −1, ion fragmentation was reduced and the instrument response was enhanced substantially. As a result, mass spectra with predominant molecular ions were acquired and molecular ions of the steroids of interest were used as precursor ions thus increasing specificity of the method. Under optimized GC–MS/MS conditions in product ion mode, the Limit of Detection (LOD) of the analyzed steroids ranged from 18.4 pg mL −1 for 17-BE to 5.5 pg mL −1 for 2-MEOE (S/N = 3). The instrument response was linear in the investigated concentration range from 0.1 to 10 ng mL −1 with R 2 > 0.99 for 17-BE and 2-MEOE. The intra-batch accuracy obtained for quality control samples at the concentration levels of 0.1, 1, 3, 7 ng mL −1 ranged from 94.9% to 109.9% for 17-BE and from 99.9% to 104.5% for 2-MEOE.