咪达唑仑原料药及其注射液中醛基类基因毒性杂质测定与含量比较

Determination and Comparative Analysis of Aldehyde Genotoxic Impurity in Midazolam Active Pharmaceutical Ingredients and Midazolam Injections

目的建立测定咪达唑仑原料药及注射液中醛基类基因毒性杂质M 1-[4-氯-2-(2-氟苯甲酰基)苯基]-2-甲基-1H-咪唑-5-甲醛(简称杂质M)含量的超高效液相色谱串联质谱法,比较我国3家生产企业咪达唑仑原料药及注射液、参比制剂中杂质M的含量。方法色谱柱为Acquity UPLC BEH-C18柱(100 mm×2.1 mm,1.7µm),流动相为含0.1%甲酸的0.01 mol/L甲酸铵水溶液-含0.1%甲酸的乙腈(梯度洗脱),流速为0.4 mL/min,柱温为40℃,进样量为5µL。采用电喷雾电离子源、正离子、多反应监测模式检测,毛细管电压为3.5 kV,脱溶剂温度为350℃,锥孔气流速为60 L/h,脱溶剂气流速为600 L/h。结果杂质M的质量浓度在0.46~9.16 ng/mL(r=0.9992,n=7)和28.62~1144.77 ng/mL(r=0.9953,n=7)范围内与峰面积线性关系良好;检测限为0.03 ng/mL,定量限为0.13 ng/mL;平均加样回收率为98.53%,RSD为4.53%(n=6)。国内3家生产企业的咪达唑仑注射液中杂质M的含量均显著高于原料药(P<0.05);未通过一致性评价的厂家A生产的咪达唑仑注射液中杂质M的含量最高,且显著高于参比制剂(P<0.05)。结论该方法灵敏度高、重复性好,可用于咪达唑仑注射液中杂质M的质量控制。建议未通过一致性评价的生产企业尽快开展一致性评价研究,优化制剂生产工艺,并基于风险评估制订杂质控制策略。

Objective To establish an ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)method for the content determination of 1-[4-chloro-2-(2-fluorobenzoyl)phenyl]-2-methyl-1H-imidazole-5-carbaldehyde(aldehyde-based genotoxic impurity M,referred to as impurity M)in midazolam active pharmaceutical ingredients(API)and midazolam injections,and to compare the content of impurity M in midazolam API and midazolam injections from three Chinese manufacturers,and reference preparations.Methods The chromatographic column was Acquity UPLC BEH-C18 column(100 mm×2.1 mm,1.7µm),the mobile phase was 0.01 mol/L aqueous ammonium formate-acetonitrile containing 0.1%formic acid(gradient elution),the flow rate was 0.4 mL/min,the column temperature was 40℃,and the injection volume was 5µL.The electrospray ion source(ESI)was adopted with positive ion detection and multiple reactionmonitoring(MRM)mode,the capillary voltage was 3.5 kV,the temperature of desolvent was 350℃,the gas flow of the cone hole was 60 L/h,and the flow of the desolvent was 600 L/h.Results The linear ranges of impurity M were 0.46-9.16 ng/mL(r=0.9992,n=7)and 28.62-1144.77 ng/mL(r=0.9953,n=7).The limit of detection was 0.03 ng/mL,and the limit of quantification was 0.13 ng/mL.The average recovery of impurity M was 98.53%with an RSD of 4.53%(n=6).The contents of impurity M in the Midazolam Injection from three domestic manufacturers were significantly higher than those in the midazolam API(P<0.05),and the content of impurity M in the Midazolam Injection produced by manufacturer A,which did not pass the consistency evaluation,was the highest and significantly higher than that in the reference preparation(P<0.05).Conclusion This method has high sensitivity and good repeatability,which can be used for quality control of impurity M in Midazolam Injection.It is recommended that manufacturers that have not passed consistency evaluation conduct consistency evaluation research as soon as possible,optimize preparation production processes,and formulate impurity control strategies based on risk assessment.