高效液相色谱测定小米中叶酸含量方法优化

Optimization of determination of folic acid in foxtail millet by HPLC

关键词:  叶酸  小米  三酶法  高效液相色谱法  响应面优化

Key words:  folic acid  foxtail millet  trienzyme  HPLC  response surface tests

摘要:

针对目前小米中叶酸提取及测定方法研究较少，尤其是对适用于规模化提取、测定叶酸含量的研究不够深入的问题，采用高效液相色谱法对小米中叶酸含量进行测定。对影响叶酸提取及测定效果的三酶法和高效液相色谱法进行单因素试验和响应面优化。结果表明:小米中叶酸提取的最佳条件为，提取液添加量1 mL，淀粉酶20 μL，蛋白酶150 μL，大鼠血清25 μL；最佳测定条件为，流速1 mL/min，流动相pH=7，检测波长280 nm、色谱柱柱温30 ℃。使用优化后的三酶法和高效液相色谱法对小米中叶酸进行提取并测定，测定过程中重复性的相对标准偏差(RSD)为0.50%。综上，三酶法和高效液相色谱法是一种可用于大规模高效提取并精确定量小米中叶酸含量的方法。

Abstract:

Currently, there were only a few studies on the extraction and determination of folic acid in foxtail millet, especially the there was lack of in-depth research suitable for large-scale extraction and determination. The single factor test and response surface optimization were carried out for tri-enzymatic extraction method. High performance liquid chromatography(HPLC)analysis was performed to determine the folic acid content. The results showed that the optimum conditions for folic acid extraction were as follows: For 1 mL of extracting solution, 20 μL of α-amylase, 150 μL of protease, 25 μL of rat serum; For the determination of folic acid, The mobile phase of HPLC was at flow rate 1 mL/min, pH=7, and the detection wavelength was 280 nm, the column maintained at 30 ℃. The relative standard deviation(RSD)of repeatability was 0. 5%. In summary, compared with HPLC, the tri-enzymatic extraction method can be used for large-scaleand high-efficient extraction and for accurate quantification of folic acid in foxtail millet.