目的: 建立阿莫西林胶囊中热降解聚合物杂质的分析方法。方法: 采用高效凝胶排阻色谱法（HPSEC 法）,使用Shimpack Bio Diol-60（4.6 mm×300 mm,3 μm）二醇基高纯硅胶色谱柱,进样量为20 μL 并以254 nm 为检测波长,进行阿莫西林和聚合物杂质的分离。通过优化HPSEC 中流动相pH、缓冲盐浓度、有机相比例等色谱条件,结合阿莫西林13 个杂质对照品,考察不同杂质的出峰规律。采用高效二维液相色谱法（2D-HPLC 法）,使用500 μL 样品环将HPSEC 分离所得聚合物杂质通过中心切割转移到二维反相色谱中脱盐,二维采用质谱兼容流动相,高分辨质谱分析确认结构。结果: HPSEC 法中流动相pH 是影响阿莫西林与聚合物峰分离的关键因素。杂质对照品在HPSEC 的保留时间研究表明色谱分离未严格按照相对分子质量大小的顺序进行。通过对聚合物杂质K 和J 的质谱裂解行为研究揭示了β- 内酰胺类抗生素的一般裂解规律。2D-HPLC 法对阿莫西林热降解溶液深入分析,主峰前聚合物峰在二维进一步分离出14 个化合物,为阿莫西林二聚体、三聚体、四聚体及其异构体。结论: HPSEC 与RP-HPLC 结合组成的二维色谱高分辨质谱系统可作为更精准的聚合物发现的手段。
Objective： To establish a method for determining the polymer impurities in amoxicillin heat degradation. Methods： The analysis was carried out by high performance size exclusion chromatography（ HPSEC） using a Shimpack Diol-60 column（ 4.6 mm×300 mm,3 μm）. The detection wavelength was 254 nm. The injection volume was 20 μL. An amoxicillin heat degradation solution was used to optimize the HPSEC conditions,including mobile phase pH,mobile phase type and organic concentration. In the optimized condition,13 impurities standards were injected to assess the specificity of HPSEC. Using high performance two-dimensional liquid chromatography （2D-HPLC）,the polymer impurities separated by HPSEC were transferred to second dimensional reversed-phase chromatography for desalting by 500 μL sample loop. Mass spectrometry compatible mobile phase was used in the second dimensional HPLC,and the structure was identified by high resolution mass spectrometry. Results： pH of mobile phase was the key factor affecting the separation of amoxicillin and polymer impurity. The retention time of impurity standards in HPSEC showed that the chromatographic separation was not strictly in accordance with the order of molecular weight. Fragmentation characters of polymer impurity J and K were studied and the general fragmentation pathways of β-lactam antibiotics were induced. 2D-HPLC coupled with QTOF was used to identify 14 polymer impurities,and their possible structures were deduced,including amoxicillin dimers,trimers and tetramers and their isomers. Conclusion： HPSEC method combined with 2D-HPLC high resolution mass spectrometry can provide a useful tool for characterization of polymer impurities in β-lactam antibiotics.
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