目的: 参照ICH Q3A的要求,对代表性药用辅料苯扎氯铵样品中含量>0.1%的未知杂质进行结构鉴定及来源归属。方法: 运用新建立的适合于苯扎氯铵有关物质结构推定的LC-MS方法,采用Acclaim 120 C18（250 mm×4.6 mm,5 μm）色谱柱,以甲醇（A）-20 mmol · L-1的甲酸铵水溶液（pH 3.5） （B）为流动相,梯度洗脱,流速1 mL · min-1,柱后分流（1 ∶ 3）;采用电喷雾离子源,选择正离子模式进行扫描,采集代表性样品中主成分和未知杂质的质谱信息,借助“诊断碎片离子延伸策略”对未知杂质结构进行推定,通过比较未知杂质与对照品的色谱质谱行为,对未知杂质进行结构确证,并对色谱系统中检出杂质进行来源归属和毒性预测。结果: 采用新建立的LC-MS方法,在苯扎氯铵代表性样品中共检出5个未知杂质,并推定了结构,利用商业途径获得的2个杂质对照品及定向合成的另外2个杂质对照品对其中4个杂质结构进行了确证,杂质D为二苄基二甲基氯化铵,杂质E为甲基二苄胺,杂质G为N-甲基-N-十二烷基苄胺,杂质H为N,N-二苄基-N-甲基十二烷-1-氯化铵,杂质F可能是分子式为C15H17N的同分异构体。Nexus 2.6.0软件预测结果显示,上述杂质均为5类杂质,无基因毒性;此外,还利用杂质对照品,对USP苯扎氯铵有关物质测定方法中检测的4个未知杂质进行了定位。结论: 本文对苯扎氯铵潜在杂质的结构及安全风险进行了系统研究,对完善国内外药典标准及提高苯扎氯铵的质量控制水平具有指导意义。
Objective: To identify the structure and determine the source of unknown impurities present at levels greater than 0.1% in representative pharmaceutical excipient benzalkonium chloride samples, in accordance with ICH Q3A guidelines. Methods: A new LC-MS method suitable for the structure prediction of related substances of benzalkonium chloride was established. The separation was performed on an Acclaim 120 C18 column (250 mm×4.6 mm, 5 μm) using a mobile phase consisting of methanol (mobile phase A) and 20 mmol · L-1 ammonium formate aqueous solution (pH 3.5, mobile phase B) with gradient elution at a flow rate was 1 mL · min-1. The post-column split was set to 1 ∶ 3. MS data were collected in positive ion mode using an electrospray ionization (ESI) source. The structures of unknown impurities were inferred using a “Diagnostic fragment ion extension strategy” and confirmed by comparing the chromatographic and mass spectrometric behaviors of the impurities with those of reference substances. Additionally, the source attribution and genotoxicity prediction of the detected impurities were performed. Results: A total of five unknown impurities were detected in a representative sample of benzalkonium chloride by the newly established LC-MS method, and their structures were inferred. The structures of four impurities were confirmed using two commercially available reference substances and two reference substances synthesized in a directionally oriented manner. Impurity D was identified as N-benzyl-N,N-dimethyl-1-phenylmethanaminium chloride. Impurity E as N-benzyl-N-methyl-1-phenylmethanamine. The impurity G as N-benzyl-N-methyldodecan-1-amine, and impurity H as N,N-dibenzyl-N-methyldodecane-1-ammonium chloride. Impurity F was hypothesized to be an isomer of the formula C15H17N. Predictions using Nexus 2.6.0 software indicated that all the above impurities fell into category 5 and had no genotoxic potential. Furthermore, the method successfully located four unknown impurities detected by the USP method in benzalkonium chloride related substances using impurity reference standards. Conclusion: This study systematically examines the structure and safety risks of potential impurities in benzalkonium chloride, providing valuable insights for enhancing quality control standards and pharmacopoeia criteria both domestically and internationally.
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