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.
Keywords: alkoxyl radical; carbon dioxide radical anion; citrate; near-UV light; photo-degradation; visible light.