柱前衍生HPLC法测定硫酸阿米卡星及其制剂中基因毒性杂质肼的研究

摘要：目的 建立柱前衍生HPLC法测定硫酸阿米卡星及其制剂中基因毒性杂质肼的含量。方法 以5%苯甲醛甲醇溶液为衍生化试剂，采用Agilent Zobax Eclipse XDB-C18色谱柱(2.1 mm×150 mm，5 μm)，流动相A为0.1%甲酸溶液，流动相B为乙腈，进行线性梯度洗脱，流速1.0 mL/min，DAD紫外检测器，检测波长300 nm，柱温25℃，进样量20 μL。结果 肼在0.5022~12.5543 μg/mL浓度范围与峰面积的线性关系良好(r=0.9994)，硫酸阿米卡星、注射用硫酸阿米卡星、硫酸阿米卡星注射液的平均回收率分别为94.8%、94.1%和94.3%。国内企业硫酸阿米卡星、注射用硫酸阿米卡星、硫酸阿米卡星注射液中的肼分别为未检出~1.8 μg/g、未检出~0.2 μg/g、未检出~3.1 μg/g；日本Nichiiko公司的注射用硫酸阿米卡星和硫酸阿米卡星注射液均未检出肼。结论

Determination of genotoxic impurity hydrazine in amikacin sulfate and its preparations by HPLC with precolumn derivatization

Abstract Objective To establish a pre-column derivatization HPLC method for the determination ofhydrazine in amikacin sulfate and its preparations. Methods 5% benzaldehyde methanol solution was used as thederivatization reagent. Agilent Zobax Eclipse XDB-C18 column (2.1 mm×150 mm, 5 μm) was used. The mobilephase A was 0.1% formic acid solution, the mobile phase B was acetonitrile, and linear gradient elution was carriedout according to the attached table. The flow rate was 1.0 mL/min. DAD UV detector was used, and the detectionwavelength was 300 nm. The column temperature was 25℃, and the injection volume was 20 μL. Results Thelinear relationship of hydrazine was found in the concentration range of 0.5022~12.5543 μg/mL(r=0.9994). Therecovery values of amikacin sulfate, amikacin sulfate for injection, and amikacin sulfate injection were 94.8%, 94.1%,and 94.3%, respectively. The content of hydrazine in amikacin sulfate, amikacin sulfate for injection, and amikacinsulfate injection in domestic enterprises were undetected~1.8 μg/g, undetected~0.2 μg/g, and undetected~3.1 μg/g,respectively. No hydrazine was detected in amikacin sulfate for injection and amikacin sulfate injection in nichiikopharmaceutical Co. Ltd. Conclusion The new precolumn derivatization HPLC method was accurate, simple, andspecific. It can be used to control the genotoxic impurity hydrazine in amikacin sulfate and its