Synthesis, purification and characterization of amoxicillin impurities

Pharmaceutical impurities are chemical molecules that may arise during the manufacturing, storage, or degradation of active pharmaceutical ingredients. Identifying and characterizing these compounds is essential to ensure drug safety, efficacy, and regulatory compliance. This thesis focuses on the isolation and characterization of specific degradation products of amoxicillin, namely closed-ring and open-ring dimers and trimers, which can form at non-extreme conditions. The synthesis of these compounds was performed through a base-catalyzed condensation of amoxicillin in DMF with triethylamine, followed by crystallization, vacuum drying, and purification via preparative HPLC. Open-ring derivatives were obtained by enzymatic hydrolysis using a class A β-lactamase. Analytical characterization was performed through analytical HPLC and mass spectrometry. Lastly, since no toxicity data were available in the literature for these specific degradation products, a predictive toxicity assessment was also performed using two in silico tools: VEGA and Toxtree. The goal of the project was to develop two identification standards: a standard of amoxicillin enriched with closed-ring and open-ring dimers and trimers and a standard containing amoxicillin and all its known impurities. To complete the second mixture, penilloic acids and penicilloic acids of amoxicillin were synthetized. Toxicological data for the latter were already available in the literature.