Laccase-catalyzed reactions of 17β-estradiol in the presence of humic acid: Resolved by high-resolution mass spectrometry in combination with (13)C labeling.

The widespread presence of estrogens in natural waters poses potential threats to the aquatic organisms and human health. It is known that estrogens undergo enzyme-catalyzed oxidative coupling (ECOC) reactions, which may impact their environmental fate and can be used in wastewater treatment to remove estrogens, but little information is available on how natural organic matter (NOM) may influence 17β-estradiol (E2) transformation in ECOC processes. A series of experiments were conducted to examine the transformation of E2 in aqueous solution containing humic acid (HA) as model NOM by laccase-mediated ECOC reactions. The impact of HA on the reaction behaviors and product distribution is systematically characterized. The presence of HA inhibited the extent of E2 self-coupling in laccase-mediated systems, while promoted cross-coupling between E2 and HA. Reconfiguration of humic molecules was also observed and characterized by changes in absorbance at 275 nm and the ratios between A250 nm/A365 nm. In particular, experiments were conducted with un-labeled E2 mixed with (13)C3-labeled E2 at a set ratio, with the products probed using high-resolution mass spectrometry (HRMS). The high m/z accuracy of HRMS enabled the use of isotope ratio as a tracer to identify possible cross-coupling products between E2 and HA. Such a method combining HRMS and isotope labeling provides a novel means for identification of products in a reaction system involving NOM or other complex matrices. These findings provide a basis for optimization of ECOC reactions for estrogen removal, and also help to understand the environmental transformation of estrogens.