反相高效液相色谱法测定血浆游离氨基酸

本文报告了采用高效液相色谱法反相梯度洗脱,邻苯二甲醛和β-巯基乙醇柱前衍生化,荧光检测分血浆游离氨基酸。实验采用线性洗脱,在50分钟内可同时测定18种氨基酸,血浆样品的预处理简单,衍生化反应的时间仅需1分30秒,血浆样品的实际进样量少于1μl。本测定方法的精确度高,各个氨基酸保留时间的变异系数平均为0.89%±0.45%(SD),峰面积的变异系数平均为2.06%±1.76%(SD),各个氨基酸的浓度在15—150μmol/L的范围中,线性关系的相关系数平均为0.985±0.0305(SD)。准确性好,各个氨基酸的回收率平均为97.6%±5.1%(SD)。实验还讨论了氨基酸分离时溶液pH值、柱温、离心速度等因素对分析结果的影响。

Determination of Free Amino Acids in Blood Plasma by Reversed Phase High Performance Liquid Chromatography

Amino acids are usually determined by an amino acid analyser. We tried to analyse free amino acid in plasma with reversed-phase high performance liquid Chromatograhy, using gradient elution, O- phthalaldehyde/2-mercaptoethanol pro column derivatization and flu- orescence detection. The present method is able to determine eighteen plasma amino acids at one run within fifty five minutes. The apparent advantages of this method are simple preparation short period of der- ivatization, small sample size, high precision and accuracy, and high recorvery of each amino acid (97.6%±5.1% SD). We have examined experimentally the factors that could influence chromatographic sepa- ration, such as property and temperature of the column, pH of buffer solution in the mobile phase, and the time and temperature of deriva- tization reaction.