离子色谱法测定肝素钠中核苷酸的含量

Determination of Nucleotide in Heparin Sodium by Sax-HPLC

肝素来源于动物的小肠粘膜,在提取过程中杂质核酸会转化为核苷酸,传统的紫外吸光光度法测定核苷酸杂质方法的灵敏度较低,为有效控制肝素钠中的杂质,文章采用C18色谱柱,在室温条件下采用超纯水∶甲醇∶冰醋酸∶正四丁基溴化铵（10%）[457∶40∶2.5∶0.25（体积比）]作为流动相,检测波长260 nm的离子色谱法测定肝素钠中核苷酸（胞苷酸、脱氧胞苷酸、腺苷酸、脱氧腺苷酸、鸟甘酸、脱氧鸟甘酸、尿甘酸、脱氧胸苷酸）的含量,结果测得的8种核苷酸标准曲线的相关系数均大于0.99,回收率为在96.3%~104.8%之间,重复性的RSD均小于2%。因此该方法检测快速,结果准确,重复性好,适用于肝素钠中核苷酸杂质的控制。

Heparin is derived from animal＇s intestinal mucosa,impurity nucleic acid will transfer to nucleotide during the process of purifying.The traditional UV absorbance method has a low sensibility to test nucleotide content.In order to control the impurity（cytidylic acid,deoxycytidylic acid,adenylic acid,deoxyadenylic acid,guanylic acid,deoxyguanylic acid,uridylic acid,deoxythymidylic acid） of heparin sodium effectively,we use SAX-HPLC method,the conditions include C18column,ultra-pure water∶ methanol∶ glacial acetic acid∶ tetrabutylammonium bromide（10%） = 457∶ 40∶ 2.5∶ 0.25（volume） as mobile phase and detection wavelength at 260 nm,room temperature.The eight kind of nucleotides＇ correlation coefficient of calibration curves are all more than 0.99,the recoveries are 96.3% ~ 104.8%,RSDs of reproducibility are all less than 2%.The method is simple and quick and accurate with good reproducibility and can be used for the nucleotide control of heparin sodium.