OPA柱前衍生反相高效液相色谱法测定氨基酸含量

Determination of Amino Acids by Precolumn Derivatization with o Phthaldialdehyde(OPA) and Reversed Phase High Performance Liquid Chromatography

建立了邻苯二甲醛（ＯＰＡ）手动柱前衍生反相高效液相色谱法测定样品中氨基酸含量的方法。以邻苯二甲醛（ＯＰＡ）／３－巯基丙酸（３－ＭＰＡ）为衍生试剂进行衍生，ＯＤＳ柱分离，３４０ｎｍ检测，在４０ｍｉｎ内１８种氨基酸全部得到基线分离。测定牛血清白蛋白（ＢＳＡ）的氨基酸组成和小鼠血清中的游离氨基酸，取得满意的结果。

A manual pre column derivatization procedure has been developed for the determination of amino acids in sample by reversed phase high performance liquid chromatography. OPA/3 mercaptopropionic acid (3 MPA) were used as derivatization reagents and separation of OPA amino acids derivatives was performed on a 5μm partical size ODS column. A phosphate buffer of 10mmol/L pH7.2(PB) containing 0.3% tetrahydrofuran(THF) was used as mobile phase (A) and a mixture of PB methanol acetonitrile(50∶35∶15) as mobile phase (B). A single linear gradient program was employed and the signals were detected at 340nm with UV detector. Under the above conditions all 18 amino acids in standard solution were seperated to baseline in 40 minutes. The precision of analysis was observed from the reproducibility of the retention times and peak areas of eight consecutive injections of 18 amino acids standard solution and the results showed that both retention times and peak areas were highly reproduceable with the RSD 0.09% 0.48% for retention times and 1.2% 4.9% for peak areas. The linearity of UV response was tested by injecting of derivatized amino acid with different concentrations from 25 to 250 μmol/L for each of 18 amino acids. The r values were 0.996 0.999. External standardization method was used for the quantitation. The method was applied to analyze amino acid composition of bovine serum albumin (BSA) and the results were in consistence with literature values. Free amino acids in rat plasma were also determined and good results were obtained.