Near-UV and Visible Light Degradation of Iron (III)-Containing Citrate Buffer: Formation of Carbon Dioxide Radical Anion via Fragmentation of a Sterically Hindered Alkoxyl Radical

Citrate is a commonly used buffer in pharmaceutical formulations which forms complexes with adventitious metals such as Fe3+. Fe3+-citrate complexes can act as potent photosensitizers under near-UV and visible light exposure, and recent studies reported evidence for the photo-production of a powerful reductant, carbon dioxide radical anion (•CO2–), from Fe3+-citrate complexes (Subelzu, N.; Schöneich, N., Mol. Pharm.2020, 17, 4163−4179). The mechanisms of •CO2– formation are currently unknown but must be established to devise strategies against •CO2– formation in pharmaceutical formulations which rely on the use of citrate buffer. In this study, we first established complementary evidence for the photolytic generation of •CO2– from Fe3+-citrate through spin trapping and electron paramagnetic resonance (EPR) spectroscopy, and subsequently used spin trapping in conjunction with tandem mass spectrometry (MS/MS) for mechanistic studies on the pathways of •CO2– formation. Experiments with stable isotope-labeled citrate suggest that the central carboxylate group of citrate is the major source of •CO2–. Competition studies with various inhibitors (alcohols and dimethyl sulfoxide) reveal two mechanisms of •CO2– formation, where one pathway involves β-cleavage of a sterically hindered alkoxyl radical generated from the hydroxyl group of citrate