[Determination of 12 halogenated organic pollutants in edible fish by ultra performance liquid chromatography-high resolution mass spectrometry combined with ultrasound-assisted extraction and gel permeation chromatography purification]

Halogenated organic pollutants (HOPs) have attracted considerable attention owing to their persistence, bioaccumulation, and toxicity. The development of methods to detect HOPs in fish is challenging owing to the compositional complexity of fish matrices, which contain high levels of lipids and relatively low concentrations of HOPs. In addition, the lipophilicity of most HOPs renders their extraction difficult. Moreover, the simultaneous determination of multiple HOPs to achieve the high-throughput screening of these analytes is complex. In this study, a reliable and efficient pretreatment method based on ultrasound-assisted extraction, gel permeation chromatography purification, and ultra performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) was developed for the determination of 12 HOPs in edible fish. The procedures of sample extraction and purification and LC-HRMS detection parameters were optimized to improve the performance of the method. Fresh fish samples were thoroughly rinsed with water, and non-edible parts, including the skin, bones, and phosphorus, were removed. The fish were weighed, cut into small pieces, and vacuum freeze-dried for 48 h. Subsequently, a freeze grinder was used to grind the dried fish into a fine powder. Exactly 2 g of the fish powder was weighed, fortified with isotope-labeled internal standards of the HOPs, and allowed to stand for 5 min. Methanol-acetonitrile (1∶1, v/v) was then added, followed by vortex mixing and ultrasonication. After centrifugation, the supernatant was transferred to a fresh tube. The extraction process was repeated twice and all extracts were combined. The extract was evaporated under a gentle nitrogen flow and redissolved in a mixture of ethyl acetate-cyclohexane (1∶1, v/v). The sample mixture was cleaned using gel permeation chromatography, and the eluate was collected and concentrated under a nitrogen flow. Sample residuals were reconstituted with water-methanol (1∶1, v/v) prior to instrumental analysis. Chromatographic separation was performed using an ACQUITY UPLC BEH C18 column (100 mm×2.1 mm, 1.7 μm). Water containing 2 mmol/L NH₄Ac and acetonitrile were used as the mobile phases, and an optimized gradient elution program was applied. Isotope dilution and an internal standard method were used to quantify the HOPs. An electrospray ionization source operated in negative mode was applied to ionize the HOPs, and a full scan together with data-dependent acquisition (DDA) was applied for HRMS. Excellent linearities (R²>0.99) were obtained for all HOPs in the quantification range of 1.0-1000.0 ng/mL. The limits of quantification were 0.5 ng/g. The analytical method was validated using pooled fish samples fortified with HOP standards (4, 40, and 400 ng/g). The recoveries of the HOPs were in the range of 67.6%-133.8%, and the corresponding RSDs were 0.5%-15.6%. A total of 27 commercially available fish samples were analyzed using the developed method, and the results revealed the presence of HOPs in the fish, indicating the practicability of the method for real-world samples. The developed method is rapid, accurate, precise, and suitable for detecting HOPs in fish. This study provides a useful approach for environmental monitoring and food safety assurance by enabling the accurate and efficient analysis of HOPs in commonly consumed fish. Given increasing global concerns over HOPs, the method developed in this study will provide practical technical support for consumers aiming to reduce their exposure to and the adverse impacts of HOPs via fish.