Development of a sensitive and robust online dual column liquid chromatography-tandem mass spectrometry method for the analysis of natural and synthetic estrogens and their conjugates in river water and wastewater

An online ultra-high-performance-liquid chromatography-triple quadrupole tandem mass spectrometry (UHPLC-MS/MS) method for detection and quantification of natural and synthetic estrogens and their conjugates in aqueous matrices was developed. Target compounds include the natural estrogen estradiol (E2) and its main metabolites estrone (E1) and estriol (E3), the synthetic estrogens ethinylestradiol (EE2) and diethylstilbestrol (DES) and their conjugates estrone 3-sulfate (E1-3S), estriol 3-sulfate (E3-3S), estradiol 17-glucuronide (E2-17G), estrone 3-glucuronide (E1-3G), and estriol 16-glucuronide (E3-16G). After pH adjustment, sample filtration and addition of internal standards (IS), water samples (5 mL) were preconcentrated on a Hypersil GOLD aQ column after which chromatographic separation was achieved on a Kinetex C18 column using methanol and water as a mobile phase. The experimental parameters, such as sample loading flow rate, elution time, the percentage of organic solvent in the aqueous-organic eluent mixture, pH, and volume of analyzed samples, were optimized in detail. The benefits of the method compared to previously published methods include minimum sample manipulation, lower detection limits, reduced total analysis time, and overall increased method accuracy and precision. Method detection limits (MDLs) are in subnanogram per liter, complying with the requirements of the EC Decision 2015/495 (Watch list) for hormones listed therein. Applicability of the developed method was confirmed by analysis of river and raw wastewater samples taken directly from urban sewerage systems before being discharged into the river.