Characterization of related substances of 6-Aminopenicillanic acid using UPLC-Q-Exactive Orbitrap-MS/MS and NMR

6-Aminopenicillanic acid (6-APA), a key intermediate in the synthesis of semi-synthetic penicillin antibiotics, was subjected to forced degradation under thermal, alkaline, acidic, oxidative, and photolytic conditions. The resulting degradation products were identified by ultra high-pressure liquid chromatography-quadrupole-Exactive Orbitrap mass/mass spectrometry (UPLC-Q-Exactive Orbitrap-MS/MS) and nuclear magnetic resonance (NMR) techniques. An oxidative forced degradation of 6-APA, designated RS7, was isolated and purified using semi-preparative liquid chromatography (semi-Prep-LC). Structural characterization confirmed RS7 as penicillamine disulfide. A total of 18 related substances were identified, comprising three process-related substances (including 2 pairs of isomers), and 14 degradation products (including 5 pairs of isomers). The degradation pathways of 6-APA were proposed and summarized. For the first time, the oxidative and reductive degradation products of 6-APA were identified, including the new detection of penicillamine disulfide among its forced degradation products.