在10.0 g猪肉样品中加入含3%(体积分数)氨水的乙酸乙酯溶液25 mL,涡旋1 min混匀,离心5 min,所得沉淀用上述方法再重复提取一次。合并两次提取液,于40℃减压浓缩至近干,用10 mL磷酸盐缓冲溶液(pH 6.0)溶解残渣。所得溶液过活化好的HLB固相萃取柱,用10 mL水淋洗,真空抽柱1 min,用6 mL甲醇洗脱。收集洗脱液,于40℃氮吹至近干,加入体积比8∶2的正庚烷-异丙醇混合溶液1 mL,涡旋3 min溶解残渣,过0.22 μm有机滤膜,滤液供超高效合相色谱仪分析。采用CHIRALPAK AD-3手性色谱柱作固定相,设置柱温40℃,系统背压13.8 MPa,以不同体积比的超临界二氧化碳和含0.5%(体积分数)氨水的甲醇溶液的混合溶液为流动相进行梯度洗脱,在检测波长224 nm处用外标法测定(-)-氟苯尼考、(+)-氟苯尼考及它们的代谢物氟苯尼考胺的含量。结果显示:3种目标化合物的质量浓度在0.50~20.00 mg·L^-1内与其对应的峰面积呈线性关系,测定下限为0.05 mg·kg^-1[(-)-氟苯尼考和(+)-氟苯尼考]和0.1 mg·kg^-1(氟苯尼考胺)。以阴性猪肉样品为基质进行3个浓度水平的加标回收试验,3种目标化合物的回收率为81.2%~107%,测定值的相对标准偏差(n=6)为5.0%~9.0%。方法用于20份猪肉样品的分析,仅在1份样品中检出(-)-氟苯尼考(248 μg·kg^-1)。

Ethyl acetate solution (25 mL) containing 3% (φ) aqueous ammonia was added into 10.0 g of pork sample, the mixture was vortexed for 1 min, centrifuged for 5 min, and the precipitation was extracted once again as the method described above. The two extracts were combined and evaporated to nearly dry under vacuum at 40℃, and the residue was redissolved with 10 mL of phosphate buffer solution (pH 6.0). The HLB solid phase column activated beforehand was used for loading the solution, washed with 10 mL of water, drained by vacuum for 1 min, and eluted with 6 mL of methanol. The eluate was collected and dried by nitrogen at 40℃ to nearly dry. The residue was dissolved by vortex for 3 min with 1 mL of mixed solution of n-heptane and isopropanol at volume ratio of 8:2, filtered through a 0.22 μm filter membrane and the filtrate was analyzed by ultra-performance convergence chromatography. CHIRALPAK AD-3 chiral column was used as the stationary phase with column temperature of 40℃ and the system back pressure of 13.8 MPa. The mixture of supercritical carbon dioxide and methanol solution containing 0.5% (φ) aqueous ammonia at different volume ratios was used as the mobile phase for gradient elution. (-)-Florfenicol, (+)-florfenicol and their metabolite florfenicol amine were determined by external standard method at wavelength of 224 nm. It was shown that the mass concentrations of the 3 targets were linearly related with their corresponding peak areas in the range of 0.50-20.00 mg·L^-1, and the lower limits of determination were 0.05 mg·kg^-1[for (-)-florfenicol and (+)-florfenicol] and 0.1 mg·kg^-1 (for florfenicol amine). Test for the spiked recovery was made on the negative pork samples at the 3 concentration levels, giving recoveries of the 3 targets in the range of 81.2%-107%, and RSDs (n=6) of the determined values ranged from 5.0% to 9.0%. The proposed method was used for the analysis of 20 pork samples, and (-)-florfenicol (248 μg·kg^-1) was detected in one sample.