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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 a reverse-phase high performance liquid chromatography-pulse amperometry (HPLC-PAD) method for the determination of related substances in netilmicin sulfate injection. Methods The mobile phase A was eluted with 0.2 mol·L -1 trifluoroacetic acid solution (containing 0.1 mol · L -1) of Agilent Proshell 120 SB-C 18 (4.6 mm × 150 mm, 2.7 μm) The mobile phase B was 0.1% pentafluoropropionic acid solution - acetonitrile (97: 3) at a flow rate of 0.8 mL · min -1. The pulse Ampere electrochemical detector (Ag-Ag Cl reference electrode, titanium counter electrode, gold working electrode, diameter 3 mm), four potential detection waveforms; column temperature, detector temperature were 35 ℃; injection volume 25μL; column After adding 0.8 mol·L -1 NaOH solution, the flow rate was 0.3 mL · min -1. Results Compared with the method used in the 2015 edition of “Chinese Pharmacopoeia”, this method detected as many as 22 impurities in typical samples, with effective separation between impurities and main peaks; netilmicin at 0.05-15 μg · The linear range was linear (r = 0.999 1). The limit of detection was 0.25 ng (equivalent to 0.004%) and the limit of quantification was 1.25 ng (equivalent to 0.02%). The peak area of the largest single impurity was repeated The RSD was 0.9% (n = 6) and the RSD of the total impurity peak area was 0.8% (n = 6). The solution was stable within 24 h. Conclusion The newly established method has the advantages of strong separation capability, specificity, sensitivity, wide linear range, good repeatability, and the ability to effectively distinguish netilmicin sulfate injection from different raw materials. It can be used for the related substances examination of netilmicin sulfate injection.