Evaluation of Three Sample Preparation Methods for LC-HRMS Suspect Screening of Contaminants of Emerging Concern in Effluent Wastewater

Suspect screening is a valuable tool for studying the pollution footprint of environmental samples, but the sample preparation (SP) method significantly affects the results. In this study, three SP methods─lyophilization, direct injection, and online solid-phase extraction (SPE)─were evaluated for suspect screening of contaminants of emerging concern (CECs) in effluent wastewater using liquid chromatography-high resolution mass spectrometry (LC-HRMS). LC-HRMS was performed with a Q-ToF mass analyzer in both positive and negative electrospray ionization modes, and pollutants were identified using the NORMAN SusDat database. A total of 119 CECs were identified with high confidence (all above level 2a). Of these, 115 were detected using lyophilization, 37 with direct injection, and 49 with online SPE. Principal component analysis revealed distinct patterns for each SP method. In all cases, the pollution profile was dominated by pharmaceuticals, followed by industrial chemicals. Articaine, a local anesthetic previously unreported in the aquatic environment, was the most abundant compound found. Industrial chemicals included phthalates, flame retardants, benzotriazoles, and perfluoroalkyl substances, among others. The remaining identified CECs were personal care products, pesticides, and drugs of abuse. Compound prioritization, based on abundance, persistence, mobility, bioaccumulation potential, and toxicity, identified the antibiotics clindamycin and tiamulin, the analgesic flufenamic acid, the musk fragrance metabolite galaxolidone, and the antidepressant venlafaxine metabolite, O-desmethyl venlafaxine, as top priority. These results highlight the importance of applying an appropriate SP method to obtain a comprehensive CEC footprint in suspect screening analysis and demonstrate the suitability of lyophilization for wastewater characterization.