邻苯二甲醛柱前衍生化时间对反相高效液相色谱法测定稻米氨基酸含量的影响

Effects of pre-column derivatization time of O-phthalaldehyde on determination of amino acids content in rice by reversed-phase high performance liquid chromatography

中文关键词:  反相高效液相色谱法  稻米  氨基酸  邻苯二甲醛  柱前衍生

英文关键词:reversed-phase high performance liquid chromatography  rice  amino acids  O-phthalaldehyde  pre-column derivatization

中文摘要:

      目的 确定反相高效液相色谱法(reversed-phase high performance liquid chromatography, RP-HPLC)测定稻米氨基酸含量最适柱前衍生化时间。方法 以氨基酸标准品以及稻米氨基酸提取物为对象, 对柱前衍生化时间与氨基酸检测结果之间的关系进行研究。结果 邻苯二甲醛(O-phthalaldehyde, OPA)和3-巯基丙酸与氨基酸的衍生化反应进行到15 min时, 氨基酸的峰面积达到最大值, 不同OPA-氨基酸衍生化产物的稳定性存在很大的差异。在15~300 min的衍生化反应中, 酪氨酸和苯丙氨酸衍生物的峰面积保持在非常稳定的水平上; 而侧链含有羟基的丝氨酸和苏氨酸对衍生化时间特别敏感, 衍生化时间超过15 min后, 其衍生物的峰面积开始快速下降; 当衍生化时间在15~60 min之内时, 15种氨基酸中, 只有酪氨酸、苯丙氨酸、精氨酸、谷氨酸、天冬氨酸、赖氨酸、亮氨酸、异亮氨酸和丙氨酸9种氨基酸的衍生物峰的面积下降幅度小于5%。结论 柱前衍生化时间对稻米氨基酸衍生物峰面积的检测值较为敏感, 衍生化时间控制在15 min之内能有效减小实验误差。

英文摘要:

      Objective To determine the optimum pre-column derivatization time for the determination of amino acids content in rice by reversed-phase high performance liquid chromatography (RP-HPLC). Methods The relationship between pre-column derivatization times and amino acid detection results was studied based on amino acid standard liquid and amino acid extractives from rice. Results When the derivatization reaction was carried out with O-phthalaldehyde (OPA), 3-mercaptopropionic acid and amino acids, the peak area of derivatives reached the maximum when the derivatization time was at 15 min. However, there were great differences in the stability of derivatives from different OPA-amino acids derivatives. When the derivatization reaction was in the range of 15?300 min, the peak areas of derivatives from tyrosine and phenylalanine remained at a very stable level. Nevertheless, the derivatives from serine and threonine with hydroxyl groups in the side chain were particularly sensitive to the derivatization time, after the derivatization time exceeded 15 min, the peak areas of derivatives began to decrease rapidly; when the derivatization time was within 15?60 min, among the 15 amino acids, the peak areas of derivatives from 9 kinds of amino acids including tyrosine, phenylalanine, arginine, glutamate, aspartic acid, lysine, leucine, isoleucine and alanine decreased by less than 5%. Conclusion The detection values of peak areas from amino acid derivatives from rice are sensitive to the pre-column derivatization time, and controlling the derivatization time within 15 min can effectively reduce the experimental errors.