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-C18(4.6 mm×150 mm,2.7 μm)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-AgCl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25 μL;柱后加0.8 mol·L-1NaOH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0.05~15 μg·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。
Abstract
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-C18 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 (97:3), 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.P 2015. 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
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Key words
netilmicin sulfate injection /
HPLC-PAD /
quadruple potential-time waveform /
gradient elution /
related substance
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中图分类号：
R917
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参考文献
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