硫酸卡那霉素及注射液的有关物质测定方法研究

Research of Analytical Method of Related Substances in Kanamycin Sulfate and its Injection

目的新建硫酸卡那霉素及注射液有关物质的高效液相色谱-柱后衍生化-荧光检测方法。方法色谱柱为Agilent ZORBAX SB-C_(18)(4.6mm×250mm,5μm);pH3.4的缓冲液(取庚烷磺酸钠一水合物4.35g和无水硫酸钠16g,加水溶解并稀释至1000mL,用冰醋酸调节pH值至3.4±0.1)-甲醇(95∶5)为流动相A,甲醇为流动相B,梯度洗脱条件:0-10min,A为90%→80%;10-20min,A为80%;20-30min,A为80%→60%;30-40min,A为60%;40-41min,A为60%→90%;41-51min,A为90%;柱温为35℃;流速为1.0mL/min。配有柱后衍生化装置,衍生化试液为邻苯二甲醛溶液;流速为0.5mL/min;衍生化反应温度为35℃;荧光激发波长为340nm,发射波长为455nm。结果在选定条件下,有关物质分离良好,精密度、重复性、回收率均满足分析要求。卡那霉素浓度与峰面积线性关系良好(R2>0.99),检出限可达到1.5ng。采用该方法对单硫酸卡那霉素、硫酸卡那霉素以及硫酸卡那霉素注射液进行了测定,检出的有关物质个数及含量均高于现行标准的结果。结论该法较以往测定硫酸卡那霉素有关物质的方法专属性更强,能更有效控制药品的质量。

Objective To establish a method of HPLC with post-column derivatization for the determination of the related substances in kanamycin sulfate raw materials and its injection.Methods Gradient elution was used for the analysis.C_(18)(Agilent ZORBAX SB-C_(18),4.6 mm×250 mm,5 Pm)column wasused.The mobile phase A was buffer(containing 1-heptanesulfonate sodium salt and sodium sulfate)-methanol(95:5),and the mobile phase B was methanol.The gradient elution program was as follows:0-10min,A:90%→80%;10-20min,A:80%;20-30min,A:80%→60%;30-40min,A:60%;40-41min,A:60%→90%;41-51min,A:90%.The column temperature was maintained at 35℃,the flow rate was 1.0 mL/min,the OPA solution was used for the post-colum derivatization at a flow rate of 0.5nL/min The temperature of the postcolumn reaction was 35℃.The fluorescence was detected by an excitation and emission wavelength of 340 and 455nm respectively.Results By using this novel method,the related substances were well separated The precision,the repeatability and the recoveries of this method were all acceptable.There was a good linear relationship between the kanamycin concentration and the peak area(P>0.99).The detection limit could reach 1.5ng.This method was applied to determine kanamycin monosulfate,kanamycin sulfate and kanamycin sulfate injection The detected number and content of related substances were higher than the current standard results.Conclusion This method is more specific than the exising method for determination of related substances of kanamycin,which can more effectively control the quality of the drug.