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.
ZHANG Chun-li
,
ZHOU Xiao-hua
,
SHI Hai-wei
,
YUAN Yao-zuo
,
WANG Ya
,
TANG Hui
,
WANG Bao-cheng
. Structure identification and application of unknown impurities in benzalkonium chloride pharmaceutical excipients*[J]. Chinese Journal of Pharmaceutical Analysis, 2025
, 45(1)
: 164
-174
.
DOI: 10.16155/j.0254-1793.2024-0252
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