4.6. Sample Preparation and Instrument Analysis

The method of sample preparation and the conditions of instrument analysis were also based on our previously reported procedures with some modifications [38]. In brief, 1 g of tissue samples (e.g., liver, kidney, gill, and muscle and skin) or 1 mL of plasma was thawed at room temperature and transferred into a 15-mL plastic tube. Then 5 mL McIlvaine buffer (0.04 mol/L sodium dihydrogen phosphate, 0.06 mol/L citric acid monohydrate, and 0.1 mol/L EDTA-Na₂, pH = 4) was added to each tube and vigorously shaken for 30 s. After standing for 10 min, 4.5 mL of acetonitrile containing 3% formic acid was pipetted to each tube, and then 1 g of NaCl was weighted into them following shaking for 30 s. To ensure the maximum amount of the target compound was extracted from samples, the mixture of sample and extractant was sufficiently mixed by ultrasound for 5 min, and then centrifugated at 3500 g for 5 min. The resulting supernatant was decanted into a 10-mL tube. The above procedures were repeated. The obtained upper layer was combined into the same tube added 200 mg of C₁₈ and 1 g of MgSO₄ following shaking 30 s, and centrifuged at 3500 g for 5 min to remove impurities. The cleaned extractant was pipetted into a new 10-mL plastic tube and condensed to dryness by a gentle nitrogen stream at 45 °C. The dry extract was reconstituted by 1 mL 10% acetonitrile-water containing 0.1% formic acid. Finally, the mixture was filtrated by a 0.22 nylon member filter and prepared to analyze by UPLC.

All samples were analyzed by a Waters UPLC (Milford, MA, USA) equipped with a binary solvent manager with a binary solvent pump, a sampler manager with an autosampler, and an ultraviolet detector. The detailed analytical conditions were set in line with our previous studies [15,16].