The role of the acquisition methods in the analysis of natural and synthetic steroids and cholic acids by gas chromatography-mass spectrometry

Abstract

An exhaustive GC-MS acquisition study was performed, for the simultaneous analysis of natural and synthetic steroids and cholic acids (in order to insert them into the last tierce of our multiresidue analysis system), such as androsterone, β-estradiol, transdehydroandro-sterone, transdehyroandrosterone, mestranol, dihydrotestosterone, ethinylestradiol, testosterone, norethisterone, estriol, 4-androstene-3,17-dione, gestodene, levonorgestrel, etonogestrel, coprostanol, progesterone, cholesterol, medroxyprogesterone-acetate, lithocholic acid, stigmasterol, cholic acid, chenodeoxycholic acid, β-sitosterol, ursodeoxycholic acid, 3-hydroxy-7-ketocholic acid and dehydrocholic acid, in total 26 compounds. As novelties to the field, for the trimethylsilyl (TMS) oxime ether/ester derivatives of steroids and cholic acids, at first, a tandem mass spectrometric (MS/MS), multiple reaction monitoring (MRM) type acquisition method has been developed in a single run; also for the first time, the three acquisition techniques, the full scan (FS), the selective ion monitoring (SIM), in our case the multiple ion monitoring (MIM) and the currently optimized MRM methods, have been compared; all three, in parallel, under strictly the same derivatization/instrumental conditions, both in matrix free solutions and municipal wastewater from two Hungarian wastewater treatment plants (WWTPs). Critical evaluation of the three acquisition protocols was collated on their analytical performances and validated under the same conditions. The data of six point calibration curves for FS, MIM and MRM methods, showed that both R² (0.9995, 0.9858, 0.9975) and RSD (5.3, 5.8, 5.0), for two parallel derivatizations, each injected three times, proved to be independent of the acquisition processes. Whereas, for the method limit of quantification (LOQ) and the instrument limit of quantification (ILQ) values showed considerable differences. LOQ data, were decreasing in the FS, MIM, MRM line (expressed in ng/L), for all steroids and cholic acids. The same trend was determined in terms of the ILQ values. The practical utility of the optimized acquisition techniques was confirmed by the quantitation of the steroids and cholic acids contents of wastewater samples. Results confirmed the importance of the MRM acquisition method, even in comparison to the MIM one: with particular interest in selected cases: avoiding the extreme overestimation of the β-estradiol (156-1325%) and that of the ethinylestradiol (582-831%) concentrations in the wastewater samples.