Determination of Residues of Cholic Acid, Deoxycholic Acid and Dehydrochloric Acid in Fish by Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry after Purification with Liquid-Liquid Extraction Combined with Solid Phase Extraction

In view of the fact that South Korea has made relevant regulations on the residual amount of bile acids in fish, the title method was proposed. Fish sample (5.0 g) was mixed with 0.1 mL of 100 μg·L^-1 mixed internal standard solution of cholic acid-d₄ (internal standard of cholic acid) and deoxycholic acid-d₄ (internal standard of deoxycholic acid and dehydrocholic acid), and 20 mL of acetonitrile was added. The mixture was extracted by ultrasound for 10 min, centrifuged for 5 min, and the extraction was repeated once again. After the supernatants were combined, spin-dried, the residue was dissolved in 1 mL of 50% (volume fraction, the same balow) methanol solution by ultrasound for 5 min. n-Hexane (5 mL) was added, the mixed solution was centrifuged for 5 min, n-hexane layer was discarded, and the solution retained was passed through the activated HLB solid phase extraction column. After the column was rinsed with 3 mL of water and drained, 3 mL of methanol was added for elution. The first 5 mL of the eluate was collected and blown to dry with nitrogen at 40℃. The residue was redissolved with 1 mL of 50% methanol solution, passed through a 0.45 μm filter membrane, and the filtrate was analyzed according to the optimized working conditions of the instrument. The Waters ACQUITY UPLC BEH C₁₈ chromatographic column was used as the stationary phase, and the mixture of water and methanol at different volume ratios was used as the mobile phase for gradient elution. Targets obtained was ionized by tandem mass spectrometer in ESI^- mode, detected in MRM mode and quantified by internal standard method. It was shown that the linear ranges of the standard curves of cholic acid, deoxycholic acid and dehydrocholic acid were 10-500 μg·L^-1, and the detection limits were 0.003 mg·kg^-1. Test for the spiked recovery was made on the negative fish samples at the 3 concentration levels, giving values of recovery in the range of 82.2%-115%, and RSDs (n=6) of the determined values ranged from 2.6% to 14%.