毛细管电泳电化学发光法检测鱼露中的L-羟脯氨酸含量

Determination of L-Hydroxyproline content in Fish Sauce by Capillary Electrophoresis coupled with Electrochemiluminescence

基于毛细管电泳分离-三联吡啶钌(Ru(bpy)_3^(2+))电化学发光检测联用技术,建立了鱼露中L-羟脯氨酸检测的一种新方法。重点考查检测池中环境条件(检测电位,吡啶钌溶液浓度和p H)、进样条件(进样电压和进样时间)及运行条件(运行电压,运行缓冲溶液浓度和pH)等因素对L-羟脯氨酸分离及发光强度的影响。结果表明,最佳测定条件为:光电倍增管电压800 V;检测电位1.20 V;Ru(bpy)_3^(2+)浓度6 mmol/L(溶解于50 mmol/L p H 8.00的PBS);进样参数10 k V×12 s;运行电压14 k V;运行缓冲溶液50 mmol/L PBS(p H8.50)。本体系中电化学发光强度与L-羟脯氨酸浓度呈现良好的线性关系,线性方程为I=3.496 9c–11.988 0,线性范围10~500 mg/L,相关系数r=0.999 8(n=6),检出限为2.26 mg/L(S/N=3)。用该方法测定鱼露中的L-羟脯氨酸,其日内迁移时间和峰高的相对标准偏差(RSD)分别为0.17%和0.26%,其日间迁移时间和峰高的RSD分别为0.43%和0.57%,加标回收率96.67%~99.65%。该方法具有分析速度快,分离效果好,灵敏度高,重现性好等特点,适用于水产品及其制品中L-羟脯氨酸含量的快速检测。

A novel method was developed for determination and separation of L - hydroxyproline in fish sauce by capil- lary eleetrophoresis coupled with electrochemiluminescence （CE - ECL ） based on trisbipyridineruthenium [ Ru （bpy） 3 ^2＋. Several experiment parameters of L - hydroxyproline separation and ECL intensity were optimized, inclu- ding detection cell conditions （detection potential, concentration of Ru（bpy）3^2＋ and pH of detection cell）, injection parameters （injection voltage and injection time） and running parameters （running voltage, concentration and pH of running buffer）. The optimal conditions were as follows ： 1.20 V detection potential, 6 mmol/L Ru （ bpy ） 3^2 ＋ （ 50 mmol/L PBS, pH 8.00） , 10 kV × 12 s injection parameters , 14 kV running voltage , and 50 mmol/L of PBS （ pH 8.50）. The ECL intensity has a linear relation with the concentration of L - hydroxyproline. The linear equation was ： I = 3.496 9c - 11.988 0. The correlation coefficient （r） was 0.999 8 （n = 6） and detection limit was 2.26 mg/L （S/N = 3 ）. The intra- day relative standard deviation （RSD） of migration time and peak height were 0.17% and 0.26% and the inter- day relative standard deviation （RSD） of migration time and peak height were 0.43% and 0.57% respectively with this method to detect L - hydroxyproline in fish sauce. The recovery was between 96.67% 99.65%. The method is suitable for rapid detection of L- hydroxyproline content in fish sauce, based on its rapid a- nalysis, good separation, high sensitivity, and good reproducibility.