目的: 对采用现有各国药典中甲硝唑有关物质方法考察甲硝唑强光照射影响因素试验出现的物料不平衡(甲硝唑降解程度明显高于有关物质增长幅度)的原因进行调查。方法: 用液相色谱-二极管阵列检测-串联质谱法(LC PDA-MS)对强光破坏的甲硝唑水溶液及其复方阴道洗剂的光降解产物进行定性鉴别和紫外光谱研究;用推导结构(N-(2-羟乙基)-5-甲基-l,2,4-二唑-3-甲酰胺)的杂质对照品,通过与甲硝唑光降解杂质保留时间和提取PDA光谱的一致性对比,及与甲硝唑氢谱的平行比较,对推导的甲硝唑光降解杂质结构进行确证;根据定性和光谱研究结果,依据现有药典方法优化了能同时检测该光降解杂质的方法并进行全面方法学验证。结果: 甲硝唑在溶液、紫外光照条件下容易发生光降解,该杂质最大吸收约在230 nm,而在各论项下有关物质方法中设置的315/319 nm处吸收强度很弱。已上市甲硝唑注射液(需要缓慢输液)在室温光照条件下放置48 h或在5 000 lx条件下处理2 d和5 d后,光降解产物的量从“未检出”快速增加至0.15%、0.36%和0.88%,均超过本品的鉴定限(0.10%)和界定限(0.15%)。用230 nm检测并引入梯度洗脱、在各论项方法基础上优化建立的方法,能同时检测该光降解杂质和甲硝唑特定杂质2-甲基-5-硝基咪唑,方法的专属性、灵敏度、精密度、溶液的稳定性等指标均满足要求。结论: 通过对甲硝唑光降解杂质的结构鉴定和光谱特征研究,揭示了采用现有药典检查方法考察甲硝唑光破坏样品出现的物料不平衡原因;建立了能同时控制该光降解杂质和甲硝唑现有特定杂质的优化方法;甲硝唑注射液慢速输液过程存在较大的光降解风险,需引起关注。

Objective: To investigate causes of mass imbalance in metronidazole light stressed irradiation test, whose degradation percentages of metronidazole were much higher than the growth rate of related substances by using metronidazole related substances method in pharmacopoeia of various countries. Methods: The photolytic degradant of metronizazole aqueous solution and its compound vaginal lotion destroyed by strong light were qualitatively identified and studied by liquid chromatography with tandem photo diode array detection and mass spectrometry (LC PDA-MS) method. Using the impurity reference substance with the deduced structure (N-(2-hydroxyethyl) -5-methyl-l, 2,4-oxadiazole-3-formamide), the deduced structure of metronidazole photo degradation impurity was confirmed by comparing the retention time of metronidazole photodegradation impurity and the consistency of extracted PDA spectrum with metronidazole hydrogen spectrum. According to the qualitative and spectral research results, the method that could simultaneously detect the photodegradation impurity was optimized according to the existing pharmacopoeia methods, and a comprehensive methodological validation was carried out. Results: Metronidazole in aqueous solution was prone to photo degradation under the conditions of UV irradiation, and the maximum absorption of this impurity was about 230 nm, while the absorption intensity at 315/319 nm set in the method of related substances under various topics was very weak. After the marketed metronidazole injection (requiring slow infusion) was placed for 48 h under room temperature and light conditions or treated for 2 and 5 days under 5 000 lx conditions,the contents of the photodegradant were rapidlyincreased from not observed (<0.01%) to 0.15%, 0.36% and 0.88%, respectively, all higher than the identification threshold (0.10%) and the qualification threshold (0.15%) of metronidazole injection. The proposed method optimized and established on the basis of various methods could simultaneously detect the photodegradation impurity and metronidazole specific impurity 2-methyl-5-nitroimidazole at 230 nm and introduced gradient elution. The specificity, sensitivity, precision, stability of solutions and other validation characteristic(ICH Q2) of the method met the requirements. Conclusion: Through the structural identification and spectral characteristics of metronidazole photo degradation impurities, the reason caused the mass imbalance of light stressed metronidazole detected by current compendial methods is revealed. An optimized method that could simultaneously control the photodegradation impurity and the existing specific impurity of metronidazole is established; There is a great risk of photodegradation in the slow infusion process of metronidazole injection, which needs attention.