柱前衍生RP-HPLC法同时测定天花粉中天门冬氨酸与谷氨酸的含量

Content Determination of Aspartic Acid and Glutamic Acid in Trichosanthis Radix by Pre-column Derivatization RP-HPLC

目的:建立同时测定天花粉中天门冬氨酸与谷氨酸含量的方法。方法:采用柱前衍生反相高效液相色谱法。在碱性条件下,氨基酸与异硫氰酸苯酯反应,生成异硫氰酸苯-氨基酸的衍生物。色谱柱为Venusil-AA氨基酸分析柱(250mm×4.6mm,5μm),流动相A为0.05mol·L-1醋酸钠溶液(pH6.5)、B为乙腈-水(80:20,V/V),梯度洗脱,检测波长为248nm。结果:天门冬氨酸与谷氨酸的进样量分别在12.16～486.40ng(r=0.9996)与21.08～843.20ng(r=0.9997)范围内与各自峰面积积分值呈良好的线性关系;平均加样回收率分别为98.5%(RSD=1.43%,n=9)与98.8%(RSD=1.19%,n=9)。结论:本方法简单、准确、、重复性好,可用于天花粉中天门冬氨酸与谷氨酸的含量测定。

OBJECTIVE： To establish the method for the content determination of aspartic acid and glutamic acid in Trichosan- this Radix. METHODS： Pre-column derivatization RP-HPLC method was adopted. In alkaline conditions, amino acids reacted with phenyl isothiocyanate to obtain the derivatives of benzene isothiocyanate-amino acid complex. The Venusil-AA amino acid analysis （250mm×4.6mm,5μm） column was used and the detection wavelength was set at 248 nm. The mobile phase A was 0.05 mol.L^-1 sodium acetate solution （pH6.5） and the mobile phase B consisted of acetonitrile-water （80 ： 20, V/V） （gradient elution ）. RESULTS： The linear range of aspartic acid was 12.16-486.40 ng（r=0.999 6） and that of glutamic acid was 21.08-843.20 ng（r=0.999 7） ; average recoveries were 98.5%（RSD=1.43%,n=9）and 98.8%（RSD=1.19%,n=9）. CONCLUSION： This method is simple, ac- curate and reproducible for the content determination of aspartic acid and glutamic acid in Trichosanthis Radix.