新型衍生试剂柱前衍生氨基酸的高效液相色谱分析

Determination of Amino Acids by High Performance Liquid Chromatography with o-Nitrobenzenesulfonyl Chloride as Derivatization Reagent

以邻硝基苯磺酰氯为标记试剂,RP-HPLC为分析模式,建立了一种新的氨基酸衍生化方法。通过液质联用对产物进行定性,研究并确定了最佳衍生化条件：衍生温度25℃,缓冲液pH 9.0,衍生时间10 min。实验建立了20种氨基酸的HPLC分离方法：选用Kromasil C18柱,流动相A为30 mmol/L的NH4Ac溶液（pH7.5）,B相为乙腈;采用梯度洗脱,检测波长275 nm,室温。20种氨基酸在0.025-6.4 mmol/L范围内线性关系良好,相关系数在0.998 0-0.999 8之间,检出限为0.01-0.113 nmol。

A novel derivatization method for the determination of amino acids has been developed by use of o-nitrobenzenesulfonyl chloride.The optimized conditions of derivatization were as following：the reaction temperature 25 ℃;the pH value 9 and the reaction time 10 min.The chromatographic conditions were as following：C18 column（5 μm,4.6 mm×250 mm）,the detection wavelength 275 nm,flow rate 1.0 mL·min^-1,mobile phase A：30 mmol/L NH4Ac,mobile phase B：ACN,gradient elution.The calibration curves for 20 amino acids were linear ranging from 0.025 to 6.4 mmol/L.The linear correlation coefficients were between 0.998 0 and 0.999 8.The limits of detection were 0.01-0.113 nmol.The method was simple,rapid and reliable,and suitable for the derivatization of amino acids and other substances which have amino group.