反相高效液相色谱-脉冲安培法测定硫酸奈替米星注射液的有关物质  

Reversed-Phase High Performance Liquid Chromatography Coupled with Pulsed Electrochemical Detection for the Determination of Related Substances in Netilmicin Sulfate Injection

作　　者：赵卫 陈达 赵述强 潘广文 袁耀佐 张玫 胡昌勤[4] 

机构地区：[1]江苏省食品药品监督检验研究院,南京210008 [2]中国药科大学中药学院,南京210009 [3]赛默飞世尔科技(中国)有限公司,上海201203 [4]中国食品药品检定研究院,北京100050

出　　处：《中国药学杂志》2017年第20期1805-1811,共7页Chinese Pharmaceutical Journal

摘　　要：目的建立用于测定硫酸奈替米星注射液有关物质的反相高效液相色谱-脉冲安培法(HPLC-PAD)。方法采用Agilent Proshell 120 SB-C_(18)(4.6 mm×150 mm,2.7μm)色谱柱;梯度洗脱,流动相A为0.2 mol·L^(-1)三氟乙酸溶液(含0.1 mol·L^(-1)氢氧化钠)-乙腈(97∶3),流动相B为0.1%五氟丙酸溶液-乙腈(97∶3),流速为0.8 mL·min^(-1);脉冲安培电化学检测器检测(Ag-Ag Cl参比电极,钛对电极,金工作电极,直径3 mm),四电位检测波形;柱温、检测器温度均为35℃;进样量25μL;柱后加0.8 mol·L^(-1)NaOH溶液,流速0.3 mL·min^(-1)。结果对比2015年版《中国药典》所用方法,本方法检出典型样品中的杂质多达22个,杂质与主峰之间、杂质与杂质之间均可有效分离;奈替米星在0.05~15μg·mL^(-1)内线性关系良好(r=0.999 1);方法的检出限为0.25 ng(相当于0.004%),定量限为1.25 ng(相当于0.02%);最大单个杂质峰面积重复性RSD为0.9%(n=6),总杂质峰面积的重复性RSD为0.8%(n=6);溶液在24 h内稳定。结论新建立的方法分离能力强,专属、灵敏,线性范围宽,重复性准确度好,能有效区分不同原料来源的硫酸奈替米星注射剂,可用于硫酸奈替米星注射液有关物质检查。OBJECTIVE To establish an improved reversed-phase high performance liquid chromatography method coupled with pulsed electrochemical detection for determining the related substances of netilmicin sulfate injection.METHODS Agilent Proshell 120 SB-C_（18） column（4.6 mm×150 mm,2.7 μm） and gradient elution were used.Mobile phase A was 0.2 mol·L^-1 trifluoroacetic acid in 0.1 mol · L^-1 sodium hydroxide solution-acetonitrile（97∶3）,mobile phase B was 0.1% pentafluoropropionic acid-acetonitrile（9∶3）,and the flow rate was 0.8 mL·min^-1.A pulsed electrochemical detector was adopted,and the temperatures of detector and column were kept at 35℃.The working electrode was a gold electrode with diameter of 3 mm and a quadruple-potential waveform（QPW） was selected as detection waveform.The injection volume was 25 μL.NaOH solution of 0.8 mol·L^-1 was added post-column at a flow rate of 0.3 mL·min^-1.RESULTS A total of 28 impurities could be detected and effective separation was achieved in the typical sample and most of which could not be separated in the method of Ch.P2015.The linearity of the calibration curve for netilmicin ranged from 0.25 to 15 μg·mL^-1 with a coefficient of determination equal to 0.999 1.The LOD and LOQ of netilmicin were found to be 0.25 ng and 1.25 ng,respectively.The repeatability RSD（n=6） of the single largest impurity and total impurities were 0.9%and 0.8%,respectively.The sample solution was stable within 24 h.CONCLUSION Compared with previously published investigations,the improved method shows higher sensitivity,better separation ability and good reproducibility,especially for differentiating the origin of bulk drug for netilmicin sulfate injection,thus is more suitable for the determination of related substances of netilmicin sulfate injection.

关 键 词：硫酸奈替米星 注射液 反相高效液相色谱-脉冲安培法 梯度洗脱 有关物质 

分 类 号：R917[医药卫生—药物分析学]