板蓝根药材中氨基酸含量测定研究

Determination of free amino acids and connected amino acids in Isatidis Radix by HPLC

目的:建立HPLC法测定板蓝根药材中10种游离氨基酸和结合氨基酸含量的方法。方法:采用2,4-二硝基氟苯柱前衍生法及微波水解-柱前衍生法测定板蓝根药材中10种游离氨基酸和水解氨基酸的含量,再通过水解氨基酸与游离氨基酸的差值计算出结合氨基酸的含量。色谱柱为Inertsil ODS-3柱(250 mm×4.6 mm,5μm);流动相A为0.03 mol·L-1乙酸钠缓冲液(pH 6.4,含0.15%三乙胺),流动相B为乙腈,梯度洗脱;检测波长为360 nm;柱温为27℃;流速为0.6 mL·min-1。结果:板蓝根药材中天门冬氨酸等10种游离氨基酸和水解氨基酸在一定范围内,浓度与峰面积呈良好的线性关系,平均回收率在85%～115%范围内,RSD均在3%以内。板蓝根药材中游离氨基酸以精氨酸和脯氨酸含量最高,分别大于20和10mg·g-1;水解氨基酸以精氨酸、脯氨酸、谷氨酸、组氨酸含量为高,分别大于36,11,7和7 mg·g-1;结合氨基酸则以精氨酸、谷氨酸、组氨酸、天冬氨酸含量为高,分别大于17,6,4.5和4.5 mg·g-1。结论:本方法适合用于板蓝根药材中天门冬氨酸等10种氨基酸的游离氨基酸和结合氨基酸的含量测定,方法简便、快速、准确,可用于板蓝根的氨基酸质量控制。

Objective：To develop an HPLC method for the content determination of ten amino acids in Isa- tidis Radix. Methods：The determination was performed with microwave hydrolysis and precolumn derivatization on an Inertsil ODS-3 column（250 mm x 4.6 mm,5 Ixm）, the mobile phase consisted of 0.03 mol-L-1 sodium acetate buffer solution （ pH 6.4） and 0.15 % triethylamine （A） and acetonitrile （B） with linear gradient elution, and the flow rate was O. 6 mL＇min-l. The column temperature was at 27 ~C. The detection wavelength was 360 nm. Results： The linearities of the calibration curves for the ten amino acids in Isatidis Radix were good. And the average recov- eries（ n =6）were 85% ~ 115% ,with RSDs less than 3%. As free amino acids in Isatidis Radix,the contents of ar- ginine and proline were the highest,which were greater than 20 and 10 mg-g-l, respectively; as hydrolyzed amino acids,arginine, proline, glutamic acid, and histidine were the highest, which were greater than 36, I I, 7, and 7 -1 mg. g ,respectively ; as combined amino acids, arginine, glutamic acid, histidine, and aspartic acid were the high- est, which were greater than 17,6,4.5, and 4.5 mg. g- 1, respectively. Conclusion ： The method is simple, rapid, and accurate,and can be used to control the quality of Isatidis Radix.