HPLC法测定发芽糙米中γ-氨基丁酸中不同衍生方式的比较研究

The Comparative Study of Different Derivative Methods in the Determination of γ-Aminobutyric Acid in Germinated Brown Rice Acid by HPLC

针对HPLC法测定发芽糙米中γ-氨基丁酸（GABA）所用衍生剂缺乏系统的比较分析,研究了3种常见衍生剂及其色谱方法比较。以邻苯二甲醛（OPA）、丹磺酰氯（Dansyl-Cl）、6-氨基喹啉基-N-羟基琥珀酰亚氨基氨基甲酸酯（AQC）3种物质作为柱前衍生剂,色谱柱为intertsil ODS-C18柱（前两者使用）、Waters AccQ Tag氨基酸分析柱,均为梯度洗脱,紫外吸收波长分别为332 nm、386 nm、250 nm,线性方程分别为Y=9.0×106X＋2062.5（R2=0.9989）、Y=7.0×106X＋11337（R2=0.9982）、Y=7.1×106X＋10025（R2=0.9999）,检出限分别为0.005 mg/mL、0.01 mg/mL、0.001 mg/mL,同一样品中测得GABA峰面积的RSD分别为1.16%、1.98%、0.94%,加标回收率分别为98.5%～106.9%、95.8%～110.6%、99.4%～103.4%,同一样品中测得GABA含量分别为11.24、10.67、13.47 mg/100 g干重。从上述结果得知,HPLC法测定GABA的3种衍生剂中,测定结果最准确的是AQC,其次是OPA和Dansyl-Cl。AQC作为一种新型衍生剂,在γ-氨基丁酸乃至氨基酸分析中前景广阔。

The derivative agent using in the determination of γ-aminobutyric acid by HPLC lacked systematic comparative analysis. So this paper studied the comparison of three kinds of common derivative agent and chromatographic process. A gradient elution was used for the separation and quantification of GABA precolumn derivatization with OPA, Dansyl-Cl, AQC. The column were intertsil ODS-C18（by the first two）, waters AccQ·Tag for free amino acids. GABA was determined with UV detector at 332 nm, 386 nm, 250 nm, and linear equations were Y=9.0×106X＋2062.5（R2=0.9989）, Y=7.0×106X＋11337（R2=0.9982）, Y=7.1×106X＋10025（R2=0.9999）, and detection limits were 0.005 mg/mL, 0.01 mg/mL, 0.001 mg/mL, and RSD of peak area of GABA measured in the same sample was 1.16%, 1.98%, 0.94%, and the recoveries were 98.5%-106.9%, 95.8%-110.6%, 99.4%-103.4%, GABA content measured in the same sample was 11.24, 10.67, 13.47 mg/ 100 g dry weight, respectively. We learned from the above results, that in three kinds of derivative agentdetermining GABA by HPLC, the most accurate one is AQC, followed by OPA and Dansyl-Cl, AQC, as a new derivative agent, has the broad prospect in γ-aminobutyric acid and even amino acid analysis.