反相离子对色谱法测定注射用核糖核酸酶解产物中的5′-核苷酸

Quantitation of 5′-nucleotides in enzymatic hydrolysis products of ribonucleic acid for injection by reversed phase ion-pair high performance liquid chromatography

目的建立反相离子对色谱法同时测定注射用核糖核酸酶解产物中4种5'-核苷酸的方法,根据核苷酸的含量计算样品中核糖核酸的含量。方法用蛇毒磷酸二酯酶将样品中的核糖核酸水解为5'-核苷酸,采用岛津VP-ODS(250 mm×4.6 mm,5μm)色谱柱,流动相A为甲醇-水(5:95)[含20 mmol L-1KH2PO4和1%(v/v)四丁基氢氧化铵(TBAOH)溶液,pH=6.5],流动相B为甲醇-水(80:20)[含1%(v/v)TBAOH溶液,pH=6.5],梯度洗脱,检测波长为254 nm。结果建立的色谱法能将4种核苷酸良好分离,4种核苷酸在0.50～15μg mL-1线性关系良好,相关系数分别为0.999 8、0.999 9、0.999 9、0.999 9;平均回收率(n=6)均在95.1%～106.3%,RSD均<3.5%。结论该方法专属性好,具有一定的准确性,可用于检测注射用核糖核酸的含量,测定结果可为注射用核糖核酸的质量研究和评价提供依据。

Objective To establish a quantitative method by reversed phase ion-pair HPLC to simultaneously determine 4 types of 5′-nucleotides in enzymatic hydrolysis products in ribonucleic acid for injection.The concentrations of the 5′-nucleotides were summed to obtain the original concentration of ribonucleic acid in the injections.Methods The sample was hydrolyzed by snake venom phosphodiesterase（SVP） to generate 5′-nucleotides.The separation was achieved on a SHIMADZU VP-ODS column（250 mm×4.6 mm,5 μm） proceded with a C18reversed phase cartridge guard column with the mobile phase consisting of buffer A [20 mmol L-1 KH2PO4 buffer,1% tetrabutylammonium hydroxide（TBAOH）（10% aqueous solution）,adjusted to pH = 6.5 and 5% methanol] and buffer B [1% TBAOH（10% aqueous solution）,adjusted to pH = 6.5 and 80% methanol] with a gradient elution.Chromatogram was obtained at 254 nm.Results All the 5′-nucleotides were well separated when an optimized elution condition was applied.The calibration curves of 5′-nucleotides showed good linearity at 0.50-15 μg mL-1,and the correlation coefficient was 0.999 8,0.999 9,0.999 9,and 0.999 9,respectively.The average recovery was 95.1%-106.3% with RSD 3.5%（n = 6）.Conclusion With good reproducibility,specificity and precision,this method can be used for the determination of nucleotides hydrolyzed in ribonucleic acid for injection.