高效液相色谱法测定桂花叶片中游离氨基酸浓度

Determination of free Amino Acids in Fragrans Leaves by Using High Performance Liquid Chromatography

建立了自动在线柱前衍生反相高效液相色谱法同时测定桂花叶片中21种游离氨基酸的方法。样品超纯水超声提取,经阳离子交换树脂柱纯化,有效降低了色素、糖类等杂质对定量的影响,同时减轻了样品对液相色谱柱的损害。以邻苯二甲醛(OPA)与9-芴甲基氯甲酸酯(FMOC)为衍生剂,Agilent Zorbax Eclipse-AAA色谱柱分离,梯度洗脱,荧光检测器检测,在19min内完成桂花叶片中21种氨基酸的分析,双内标法定量。结果 21种氨基酸线性关系、精密度、准确性、重现性良好。标准氨基酸的保留时间和峰面积平均相对标准偏差分别为0.14%和2.99%,加标回收率平均为91.09%。桂花叶片游离氨基酸中,含量最多的是谷氨酸,占总游离氨基酸的24%。本实验方法保证了氨基酸浓度定量的准确可靠性,可用于高等植物叶片中游离氨基酸浓度的测定。

We developed an automated pre-column derivation procedure,to achieve the determination of 21 free amino acids in leaves of fragrans by using reversed-phase high performance liquid chromatography. Ultrapure water was used as extractant; the influence of pigment and carbohydrate on quantification could be reduced effectively with the purification of cation exchange resin,moreover,it could also alleviate the damage of samples to the liquid chromatographic column. The free amino acids in leaves of fragrans were analyzed with OPA and FMOC derivation within 19 min,the amino acids derivatives were separated on an Agilent Zorbax Eclipse-AAA column by a gradient elution method and identified with a fluorescence detector,and determined with the method of double internal standard. We get a good result of linear relationship,relative standard deviation,accuracy and reproducibility for 21 amino acids. The mean relative standard deviation of retention time and area of the chromatogram were 0.14% and 2.99%,respectively. The average recovery of amino acids was 91.09%. The content of aspartic acid amounted to 24% in leaves of fragrans as the most abundant free amino acids. This method ensure the reliability and accuracy of the quantitative analysis of the free amino acids concentrations,it can be used for the determination of the free amino acids concentration in the higher plant.