柱前衍生化反相高效液相色谱法测定板蓝根中的氨基酸

Determination of Amino Acids in Isatis indigotica Fort by Reversed-Phase High Performance Liquid Chromatography with Pre-column Derivatization

建立了板蓝根中指标性成分精氨酸、脯氨酸和谷氨酸的柱前衍生化反相高效液相色谱分析方法.以2,4-二硝基氟苯为衍生化试剂,将氨基酸在碱性条件下衍生化,然后直接进样进行高效液相色谱测定.采用的色谱条件为:色谱柱Sinochrom ODS-BP柱(250 mm×4.6 mm i.d.,5 μm),流动相醋酸钠缓冲溶液(pH 6.4)-乙腈(体积比为850∶150),检测波长360 nm,采用外标法定量.该文同时对谷氨酸、精氨酸和脯氨酸进行定性,并对精氨酸和脯氨酸进行定量.精氨酸和脯氨酸的进样量分别为0.627～5.016 μg和0.874～7.000 μg时,其峰面积与进样量呈良好的线性关系(r分别为0.999 6和0.999 5).精氨酸和脯氨酸的平均回收率分别为98.5% (相对标准偏差(RSD)为2.5% (n=8))和98.4% (RSD为2.3% (n=8)).对板蓝根样品进行了测定,结果表明,板蓝根中精氨酸的含量最高,脯氨酸次之,谷氨酸含量很少.该法简便、准确、可靠,适用于板蓝根药材的质量控制.

A reversed-phase high performance liquid chromatographic method with pre-column derivatization for the determination of amino acids, which were known as the characteristic constituents of Isatis indigotica Fort was established. The amino acids in alkalescence were derivatized with 2,4-dinitro-fluorobenzene （DNFB）. A reproducible method for simultaneous qualitative analysis of glutamic acid （ Glu）, argirine （Arg） and proline （Pro） and quantitative analysis of Arg and Pro in Isatis indigotica Fort has been established. NaAc buffer （ pH 6.4 ） -acetonitrile （85： 15, v/v） as mobile phase and a Sinochrom ODS-BP column were used. The detector was operated at 350 nm. The linear regressions of the standard curves were determined for Arg and for Pro. The method was carried out over the range of 0. 527 -5. 016 μg for Arg and 0. 874 - 7. 000 μg for Pro. The recoveries were 98.5% and 98. 4% with the relative standard deviations of 2.5% and 2.3% for Arg and Pro respectively. The results indicate that among the three amino acids in Isatis indigotica Fort, Arg content was the highest, Pro the second and Glu the lowest. The method has good accuracy and repeatability and it can be used for the quality control of Isatis indigotica Fort.