柱后衍生化-HPLC法检测硫酸新霉素的有关物质

目的建立硫酸新霉素有关物质检查的柱后衍生化-HPLC-荧光检测法,并与新建的HPLC-电化学检测法进行比较。方法色谱柱为COSMOSIL 5C18-PAQ(4.6mm×250mm,3.5um);以pH3.4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)]-乙脂(95.5)为流动相A,pH3.4缓冲液-乙腈(60:40)为流动相B;梯度洗脱;柱温为30℃;流速为1.0mL/min。柱后衍生化试液为邻苯二甲醛溶液[pH值为(10.4±0.1)1:流速为0.3mL/min。电化学检测法采用脉冲安培检测器,金电极为工作电极,四电位波形工作模式。结果柱后衍生化法与电化学检测法均检出24个杂质。柱后衍生化法的检测限和定量限分别为1.1和3.5ng。结论柱后衍生化-HPLC法操作简便,灵敏度高,重复性好,可作为硫酸新霉素有关物质检查的常规方法。

Detection of the related substances in neomycin sulphate by post-column derivatization HPLC

Objective To establish an HPLC method with post-column derivatization for detection of the related substances in neomycin sulfate. To compare the analytical performance with a new HPLC method with pulsed amperometric detector (PAD) established for the analysis of neomycin sulfate. Methods A COSMOSIL 5C18-PAQ column (4.6mm×250mm, 3.5μm) was used, the mobile phase A was buffer (containing 20mmol/L 1-heptanesulfonate sodium salt and 110mmol/L sodium sulfate, pH3.4±0.1)-acetonitrile (95:5) and the mobile phase B was buffer- acetonitrile (60:40); the elution was in the gradient form; the column temperature was 30℃ and the flow rate was 1.0mL/min. The o-phthalaldehyde (OPA) solution pH(10.4±0.1) was used for the post-column derivatization at a flow rate of 0.3mL/min. A pulsed amperometric detector (PAD) was applied in the electrochemical method with golden working electrode in a four-potential working mode. Results According to the results of the two detection methods, 24 impurities could be separated. The limit of detection and the limit of quantitation for the post column derivatization method were 1.1 and 3.5ng, respectively. Conclusion Post column derivatization method was proved to be user-friendly, sensitive, and repeatable, and can be applied to the detection of related substances in neomycin