Carbon dioxide radical anion (•CO2-) is a powerful reducing agent that can reduce protein disulfide bonds and convert molecular oxygen to superoxide. Therefore, the generation of •CO2- can be detrimental to pharmaceutical formulations. Iron is among the most prevalent impurities in formulations, where Fe(III) chelates of histidine (His) can produce •CO2- upon exposure to near-UV light (Zhang and Schöneich, Eur. J. Pharm. Biopharm. 2023, 190, 231-241). Here, we monitor by spin-trapping in combination with electron paramagnetic resonance spectroscopy and/or high-performance liquid chromatography-mass spectrometry analysis the photochemical formation of •CO2- for a series of common amino acid excipients, including arginine (Arg), methionine (Met), proline (Pro), glutamic acid (Glu), glycine (Gly), aspartic acid (Asp), and lysine (Lys). Our results indicate that in the presence of Fe(III), Asp, and Glu produce significant yields of •CO2- under photoirradiation with near-UV light. Notably, Asp demonstrates the highest efficiency of •CO2- generation compared with that of the other amino acid excipients. Stable isotope labeling indicates that •CO2- exclusively originates from the α-carboxyl group of Asp. Mechanistic studies reveal two possible pathways for •CO2- formation, which involve either a β-carboxyl radical or an amino radical cation intermediate.
Keywords: amino acids; aspartic acid; carbon dioxide radical anion; excipients; photodegradation; radicals.