母乳中多不饱和脂肪酸的RP-HPLC测定方法研究

Determination of polyunsaturated fatty acid in human breast milk by reverse phase high performance liquid chromatography

目的:建立测定母乳中亚油酸(Linoleic acid,LA)、α-亚麻酸(α-Linolenic acid,ALA)、花生四烯酸(Arachidonicacid,AA)、二十二碳六烯酸(Docosahexaenoic acid,DHA)的高效液相色谱法。方法:经液-液萃取并衍生提取母乳中的不饱和脂肪酸后,采用Agilent SB-C18(4.6mm×150mm,5μm),流动相为乙腈:水=83:17,流速1.0ml/min,柱温30℃,紫外检测波长254nm进行。结果:ALA在50～350μg/ml、LA在200～1500μg/ml、DHA在10～80μg/ml和AA在20～160μg/ml范围内浓度与峰面积呈良好的线性关系,r均大于0.999;前三者的最低检测限均为0.2μg/ml,后者为0.4μg/ml;高、中、低浓度的平均回收率在92.65%～100.70%以内,精密度RSD≤5.67%。结论:本方法快速、准确且灵敏,满足临床和不同生物样品中多不饱和脂肪酸的含量测定。

Objective：To establish a simultaneous determination of linoleic acid（LA）,α-linolenic acid（ALA）,arachidonic acid（AA）and docosahexaenoic acid（DHA）in human breast milk by reverse phase high performance liquid chromatography（RP-HPLC）.Methods：The method of liquid-liquid extraction and derivation from human breast milk was used in a HPLC analysis.The column was Agilent SB-C18（4.6 mm×150 mm,5 μm）.The mobile phase was acetonitrile：water（83：17,v/v）at 1.0 ml/min with UV detection at 254 nm and the column temperature was 30℃.Results：The standard curves were linear over the ranges of 50 ～350 μg/ml for α-linolenicacid,200～1500μg/mlforlinoleicacid,10～80 μg/ml for docosahexaenoic acid and 20 ～160 μg/ml for arachidonic acid,respectively.The good linearity can be obtained over their concentration range（r〉0.999）.The minimumdetectable limits of ALA,LA and DHA were all 0.2 μg/ml,and AA was 0.4 μg/ml.The average recovery of the method was 92.65%～100.70% and the RSD was lower than 5.67%.Conclusion：The HPLC method in this assay is rapid,direct and sensitive,which can be successfully applied to quantity the detection of polyunsaturated fatty acid of different biological samples as well as in clinic.