Identification of a diastereomer of penicilloic acid during acid-mediated degradation of 6-aminopenicillanic acid

6-Aminopenicillanic acid (6-APA), the core scaffold of penicillin antibiotics, is a key intermediate in the industrial synthesis of β-lactam drugs but it is prone to degradation under acidic conditions due to the strain of the β-lactam ring. Here, the acid-mediated degradation of 6-APA was investigated by real-time ¹H NMR, complemented by HPLC and High-resolution mass spectrometry. An unexpected degradation product was detected that did not fully correspond to the classical β-lactam hydrolysis pathways. Structural analysis revealed that the species corresponds to a diastereomer of penicilloic acid, consistent with epimerization at the C6 position following β-lactam hydrolysis. This integrated analytical approach provides the first direct NMR evidence of a diastereomeric transformation pathway under acidic conditions, offering new insights into the stability of 6-APA that are relevant to pharmaceutical analysis and process control.