柱前衍生-反相高效液相色谱法测定氨基酸

RP-HPLC Determination of Amino Acids with Precolumn Derivatization

向氨基酸溶液中加入对甲氧基苯磺酰氯(MOBS-Cl)试剂,在55℃水浴中加热35min使氨基酸衍生化,所得含有氨基酸衍生物的溶液用于反相高效液相色谱分析。选用ODS C_(18)柱(4.6mm×150mm,5.0μm)为固定相,以不同体积比的15mmol·L^(-1)磷酸二氢钠溶液和乙腈为流动相进行梯度淋洗,以235nm作为检测波长,提出了测定氨基酸的柱前衍生-反相高效液相色谱法。17种氨基酸衍生物的峰面积与其浓度均在0.01～5.0mmol·L^(-1)范围内呈线性关系,检出限(3S/N)在0.0010～0.0050mmol·L^(-1)之间。取5.0,25.0,50.0g·L^(-1)氨基酸标准溶液从衍生化操作开始分别重复测定5次,根据所测得峰面积值计算其相对标准偏差均小于5.0%。

Solution of amino acid was mixed with the derivatizing reagent, 4-methoxybenzenesulfonyl chloride （MOBS-Cl）, and derivatized for 35 min at 55 ℃ in a water bath. The solution containing the derivative of amino acid was used for RP-HPLC analysis, using ODS C18 column （4.6 mm× 150 mm, 5.0 μm） as stationary phase and mixtures of 15 mmol · L^-1 NaH2PO4 solution and acetonitrile in different ratios as mobile phase in the gradient elution. UV detection at the wavelength of 235 mn was adopted in the determination. Linear relationships between values of peak area and concentration of the 17 amino acids were obtained in the same range from 0. 01 to 5.0 mmol · L^-1. Detection limits （3S/N） found for this method were in the range of 0. 001 0-0. 005 0 nmol · L^-1. Precision of the method was tested with 5.0, 25.0, 50.0 g · L^-1 of the standard amino acid solutions for 5 determinations including the precolumn derivatization process, values of RSDrs calculated from data of peak area were less than 5.0%.