基于过甲酸氧化法HPLC-PITC柱前衍生对胱氨酸、蛋氨酸的测定

Determination of Cystine and Methionine by HPLC with PITC Pre-column Derivatization Based on the Oxidation of Performic Acid

通过实验制备了氧化剂过甲酸,胱氨酸、蛋氨酸经过甲酸氧化后分别转化成磺基丙氨酸和蛋氨酸砜,再与PITC进行衍生,对衍生产物采用XB-C18色谱柱(4.6 mm×250 mm,5μm),以乙腈/水(v乙腈/v水=80/20)为流动相A,50 mmol/L乙酸钠(p H=6.5)/乙腈(v乙酸钠/v乙腈=93/7)为流动相B进行梯度洗脱,在波长254 nm、柱温40℃条件下对实际样品的含量测定。结果表明:胱氨酸、蛋氨酸氧化后的衍生产物稳定,连续6次进样峰面积RSD分别为1.81%、1.65%;在187.5~1 500μg/m L范围内呈良好的线性关系,检出限为6.6 mg/L、4.52 mg/L,平均回收率符合实验要求。

Oxidant performic acid was prepared by formic acid and hydrogen peroxide,cystine and methionine were oxidated into sulfo alanine and methionine sulfone by formic acid,respectively,and then derivatived with PITC. The products were separated on a XB-C18（ 4. 6 mm × 250 mm,5 μm） by using gradient elution with acetonitrile-water（ 80/20,v/v） as mobile phase A and 50 m M sodium acetate buffer solution（ p H = 6. 5）-acetonitrile（ 93/7,v/v） as mobile B,and real samples were detected at 254 nm with column temperature of 40 ℃. The results showed that derivation products were stable,whose relative standard deviation（ n = 6） of peak area were 1. 81%,1. 65%,respectively,and had good linear correlation between 187. 5 ~ 1500 μg/m L,whose average recovery met requirement of the experiment.