Automated solid-phase extraction for concentration and clean-up of female steroid hormones prior to liquid chromatography–electrospray ionization–tandem mass spectrometry: An approach to lipidomics

A method for determination of free and glucuronide-conjugated female steroid hormones in urine at the pg mL−1 level is here presented. For this purpose, a dual approach with or without β-glucuronidase hydrolysis has been developed to succeed in this analysis. The target analytes were two progestogens – progesterone and pregnenolone – and three endogenous estrogens – estradiol, estriol and estrone. Separation and detection were carried out by liquid chromatography electrospray ionization and tandem mass spectrometry (LC–ESI–MS–MS) with a triple quadrupole (qQq) mass detector. The determination step was optimized by multiple reaction monitoring for highly selective identification and sensitive quantification of female hormones in a complex sample such as human urine. As these compounds are present in urine at very low concentration (ng mL−1 level), a preconcentration and clean-up step by solid-phase extraction was automatically carried out prior to the chromatographic step in order to improve the sensitivity of the method. This sample pretreatment was performed using a lab-on-valve (LOV) manifold which provided preconcentration factors ranging from 59.1 to 72.3 for 10 mL urine. The detection and quantification limits were in the ranges 1.8–18 pg and 6–61 pg on-column, respectively, with precision values from 1.93 to 10.99%, expressed as relative standard deviation. These results enable to conclude the suitability of the LOV–LC–qQq approach for determination of the lipidomic profiling of the main female steroid hormones in a difficult matrix as human urine. The method can be potentially applied to the clinical and other metabolomic areas.