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.
JIANG Jun, JI Feng, CHEN Zhen-he, DONG Jing, YANG Xiao-bo, LI Xiao-dong, CAO Lei
. Analysis of the polymer impurities in amoxicillin capsules heat degradation by 2D-HPLC and the exploration of mass spectrometry fragmentation mechanism[J]. Chinese Journal of Pharmaceutical Analysis, 2021
, 41(7)
: 1244
-1258
.
DOI: 10.16155/j.0254-1793.2021.07.16
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