高效液相色谱法检测乳及乳制品中羟脯氨酸

Determination of hydroxyproline in milk and milk products by high performance liquid chromatography

目的:建立高效液相色谱法检测乳及乳制品中羟脯氨酸的方法。方法:乳制品中的胶原蛋白经过酸水解后生成包括羟脯氨酸在内的氨基酸混合物,用6-氨基喹啉基-N-羟基琥珀酰亚胺基甲酸酯(AQC)试剂衍生后测定羟脯氨酸,以缓冲液-乙腈水溶液为流动相,经过C18柱分离,紫外248 nm检测,以顺式-L-羟脯氨酸为内标定量分析。结果:在2μg/ml～30μg/ml范围内呈良好线性关系,40μg/g、100μg/g和300μg/g三个水平添加平均回收率99.7%～120.5%,精密度RSD<5%,方法定量限为4μg/ml(液态奶),方法检测限为2.0μg/ml(液态奶)和10μg/ml(奶粉,乳酪)。结论:方法灵敏度高,准确度好,实用性强,可用于乳及乳制品中羟脯氨酸的检测。

Objective：To develop a method for determination of Hydroxyproline in milk and milk products by high performance liquid chromatography（HPLC）.Methods： After hydrolysis with hydrochloric acid,the collagen in samples was decomplosed into hydroxyproline,which was then derivatized with AQC（6-aminoquinoly-N-Hydroxysu——ccinimidy carbamate） for the determination.Hydroxyproline was assayed by HPLC with the following coditions：reversed-phase C18 column,UV detective wavelength of 248 nm,flow rate of 1.0 ml/min,and gradient elution of buffer and acetonitrile.Cis-4-Hydroxy-L-proline was used as the internal standard for the quantification.Results： They showed good linearity in the range of 2 μg/ml～30 μg/ml,average recovery of the method was 99.7%～120.5%,RSD5%.The detection limit was 2.0 μg/ml in milk and 10 μg/ml in milk powder and cheese.Conclusion： The HPLC method is high sensitive,accurate and practical for determination of hydroxyproline in milk and milk products.