Citrate is a commonly used buffer in pharmaceutical formulations which forms complexes with adventitious metals such as Fe 3+ . Fe 3+ -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 ( • CO 2 − ), from Fe 3+ -citrate complexes (Subelzu, N.; Schöneich, N., Mol. Pharm. 2020, 17, 4163−4179). The mechanisms of • CO 2 − formation are currently unknown but must be established to devise strategies against • CO 2 − 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 • CO 2 − from Fe 3+ -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 • CO 2 − formation. Experiments with stable isotope-labeled citrate suggest that the central carboxylate group of citrate is the major source of • CO 2 − . Competition studies with various inhibitors (alcohols and dimethyl sulfoxide) reveal two mechanisms of • CO 2 − formation, where one pathway involves β-cleavage of a sterically hindered alkoxyl radical generated from the hydroxyl group of citrate.