鉴于韩国已对鱼肉中胆汁酸的残留量进行了相关规定,提出了题示方法。取鱼肉样品5.0 g,加入100 μg·L^-1胆酸-d₄(胆酸内标物)、脱氧胆酸-d₄(脱氧胆酸及脱氢胆酸的内标物)混合内标溶液0.1 mL,乙腈20 mL,混匀后超声提取10 min,离心5 min,重复提取一次。合并上清液,旋干,残渣用50%(体积分数,下同)甲醇溶液1 mL超声溶解5 min,加入正己烷5 mL,离心5 min,弃掉正己烷层。保留相过活化好的HLB固相萃取柱,用3 mL水淋洗,抽干柱子,用3 mL甲醇洗脱。收集前5 mL洗脱液,于40℃氮吹至干,残渣用50%甲醇溶液1 mL复溶,过0.45 μm滤膜,滤液按照优化的仪器工作条件测定。以Waters ACQUITY UPLC BEH C₁₈色谱柱为固定相,以不同体积比的水和甲醇的混合液为流动相进行梯度洗脱,分离后的目标物用串联质谱仪在电喷雾离子源负离子(ESI^-)模式下电离,多反应监测(MRM)模式下检测,内标法定量。结果显示:胆酸、脱氧胆酸和脱氢胆酸标准曲线的线性范围均为10~500 μg·L^-1,检出限为0.003 mg·kg^-1。对阴性鱼肉样品进行3个浓度水平的加标回收试验,回收率为82.2%~115%,测定值的相对标准偏差(n=6)为2.6%~14%。

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%.