目的:基于二维高效液相色谱-四极杆-飞行时间串联质谱技术(2D-LC-Q TOF/MS),鉴定喷昔洛韦乳膏有关物质的结构。方法:采用ODS(250 mm×4.6 mm, 5 μm)色谱柱,以0.15%甲酸10 mmol·L-1甲酸铵缓冲溶液-乙腈为流动相,进行一维梯度洗脱,对喷昔洛韦乳膏及其依照ICH原则强制降解样品的有关物质进行分离,并分别富集各有关物质;再采用Phenomenex Luna SCX(250 mm×4.6 mm, 5 μm)色谱柱,以0.1%甲酸20 mmol·L-1甲酸铵缓冲溶液-乙腈为流动相,进行二维梯度洗脱,实现各有关物质与喷昔洛韦乳膏基质的良好分离。利用电喷雾正离子化-四极杆-飞行时间串联质谱高分辨测定各有关物质母离子及其子离子的准确质量和元素组成,并解析鉴定其结构。结果:在所建立的2D-LC-Q TOF/MS分析条件下,喷昔洛韦与其有关物质分离良好,检测出喷昔洛韦乳膏及其强制降解样品中21个主要有关物质。根据其色谱保留行为、光谱特征、质谱特征及与其他核苷类药物已知有关物质的差异,结合合成和制剂工艺路线分析,可鉴定出它们的结构,它们均为首次被鉴定出的有关物质。结论:研究结果为喷昔洛韦乳膏的质量控制提供了参考依据。
Objective: To identify the structures of the related substances in penciclovir cream by two-dimensional liquid chromatography quadrupole time-of-flight tandem mass spectrometry (2D-LC-Q TOF/MS) technique. Methods: The first-dimension separation of the related substances in penciclovir cream and its stressed samples according to ICH were carried out on an ODS (250 mm×4.6 mm, 5 μm) column with gradient elution by 0.15% formic acid 10 mmol·L-1 ammonium formate buffer solution and acetonitrile as mobile phases, and each related substance was enriched separately. The second-dimension gradient elution was performed on a Phenomenex Luna SCX (250 mm×4.6 mm, 5 μm) column with 0.1% formic acid 20 mmol·L-1 ammonium formate buffer solution and acetonitrile as mobile phases for each of the related substances to achieve good separation with the matrix of penciclovir cream. The accurate mass and elemental composition of the parent ions and their product ions of the related substances were determined by positive electrospray-ionization quadrupole time-of-flight high resolution mass spectrometry, and the structures of all the related substances were elucidated. Results: Under the established 2D-LC-Q TOF/MS analytical conditions, penciclovir and its related substances were adequately separated, and 21 major related substances were detected and identified in the penciclovir cream and its stressed samples. According to their chromatographic retention behavior, spectral characteristics, mass spectrometry characteristics and their differences from other known related substances of nucleoside drugs and combing with synthesis and formulation process route, their structures can be identified all of which were identified for the first time. Conclusion: The results can provide reference for the quality control of penciclovir cream.
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