Development and validation of a UHPLC-ESI-MS/MS method for determining estrogenic compounds in surface water at the ultra-trace level

Natural estrogens (estrone: E1, 17β-estradiol: E2, estriol: E3) and synthetic estrogen (17α-ethinylestradiol: EE2) are powerful endocrine disruptors harmful to aquatic wildlife and human at extremely low concentrations. The European Commission included these molecules in the first (2015) and second (2018) surface water Watch List issued under the Water Framework Directive regarding emerging aquatic pollutants, proposing maximum detection limits (LOD) of 0.035 ng/L for EE2 and 0.4 ng/L for E1 and E2. Attaining these limits represents a challenge even with the most up-to-date analytical tools, in particular in surface water. A two-step sample preparation, involving a preliminary extraction of a whole water sample on a solid-phase extraction (SPE) disk and further purification on a Florisil SPE cartridge, was optimized. The cleaned-up extract was then quantified by LC-MS/MS following a derivatization step, which greatly enhanced the response in electrospray ionization positive mode (ESI (+)). This derivatization procedure allowed to obtain at least two transitions for each analyte which were proved to be specific and hence selected for quantification and confirmation. The main goal was to maximize the recoveries in order to achieve the very low LODs required by the European Watch List. The method was fully validated in seven surface water. The LODs calculated for all the seven surface water were below the recommended maximum acceptable levels required by the European Commission.