高效液相色谱法测定卤肉制品中酸性橙Ⅱ染料

Determination of acid orange Ⅱ in cooked meat by HPLC

目的建立操作简便、灵敏度高的高效液相色谱测定卤肉制品中酸性橙Ⅱ的方法。方法样品经均质,取2 g粉碎样品,乙醇震荡提取5 min,提取液经聚酰胺固相萃取柱分离净化。采用C18色谱柱(250 mm×4.6 mm×5μm)分离,以甲醇-乙酸铵溶液梯度洗脱,流速为1 ml/min,设定柱温30℃,进样体积为10μl,二极管阵列检测器检测酸性橙Ⅱ(λ=484 nm)并测定卤肉中酸性橙Ⅱ的含量及加标回收率,并作方法学论证。结果酸性橙Ⅱ在0.1μg/ml^20μg/ml的范围内,线性关系良好,线性方程为y=34.94x-2.697,r=0.9998。酸性橙Ⅱ最低检出浓度为0.08μg/ml。样品添加浓度为0.5 mg/kg^15 mg/kg时,方法回收率为96%~99%,相对标准偏差为3.39%~7.31%。结论用HPLC法检测非法添加于卤肉中的酸性橙Ⅱ的含量,该操作简便,定量准确可靠。

Objective To establish an easy-handling and high sensitive method of HPLC for quantitation of acid orangeⅡ in cooked meat.Methods Samples were processed by homogenized,2 grams of sample was extracted with ethanol for 5 minutes with vibration.The extract was separated and purified by polyamide solid phase extraction column.HPLC separation was achieved by using C18（250 mm × 4.6 mm × 5 μm） with an mobile phase consisting of methanol and acetate buffer,and gradient program was used at a flow rate of 1 ml/min.The column temperature was set at 30 ℃ and the injection volume was 10 μl.The detection wavelength was at 484 nm for acid orangeⅡ,and then selecting the best chromatograph conditions to determine the concentration of acid orangeⅡ in cooked meat and the added standard recoveries of it.Then do the HPLC methodology demonstration using the selected chromatograph conditions.Results This HPLC method exhibited a linear relation within 0.1 μg/ml- 20 μg/ml acid orangeⅡ content.The regression equation was y = 34.94x-2.697（r = 0.9998） and the minimum detectable limit of this method was 0.1 μg/ml.Furthermore,the recoveries of basic orange were 96% - 99%,the relative standard deviations（RSDs） were 3.39% - 7.31% while the contents of added standard were 0.5 mg/kg - 10 mg/kg.Conclusion This is a simple,accurate and reliable HPLC method for determination of acid orangeⅡ in cooked meat.