Amoxicillin (CAS No. 26787-78-0), also known as hydroxyampicillin, is a widely used broad-spectrum β-lactam penicillin antibiotic. It is unstable under conditions such as high temperature, light exposure, acidic or alkaline environments, and oxidation. Clinical studies have shown that its polymerized impurities may cause allergic reactions. Therefore, related substances generated during production and storage directly affect product quality and clinical safety.
According to the Chinese Pharmacopoeia (2020), related substances of amoxicillin are analyzed using HPLC-UV with a mobile phase consisting of potassium dihydrogen phosphate and acetonitrile. However, the presence of non-volatile phosphate salts makes it incompatible with direct mass spectrometry analysis.
To address this, an online column-switching two-dimensional liquid chromatography (2D-LC) method is applied. Target impurity peaks from the first dimension are transferred via valve switching to a second column, where online desalting is performed. A mass spectrometry-compatible mobile phase is then used for qualitative analysis.
Figure 1 Structural Formula of Amoxicillin (MW 365.4)
Instrument System:
Chromai Two-Dimensional Liquid Chromatography System (dual ternary pump P60, autosampler A10C, column oven C10V2 equipped with two 2-position 6-port valves as standard, UV detector D10)
Chromai Triple Quadrupole Mass Spectrometer (TQ5000)
Conclusion
This approach enables rapid qualitative analysis of six trace impurities in amoxicillin. Structural identification is achieved through MS/MS fragmentation, providing valuable support for quality control of amoxicillin active pharmaceutical ingredients (APIs).
