Identification of a Riboflavin–Boric Acid Complex by Electrospray Ionization Mass Spectrometry

Boric acid ingestion in children and adults has been shown to increase urinary riboflavin excretion (1), while high intakes of riboflavin reduce symptoms of boron toxicity in rats, guinea pigs, and chicks (2). Boric acid, the physiological form of boron, exhibits an affinity for molecules containing vicinal diols. Esterification of vicinal diols by boric acid has been reported for NAD+, the active form of vitamin B3, and cADPR, a signaling molecule derived from NAD+ that functions in the Ca2+ signaling pathway and oxidative stress (3, 4, 5). Riboflavin (vitamin B2) contains a ribityl side chain with vicinal diols, and although indirect evidence suggests an interaction with boron, direct evidence has been lacking. In this study, electrospray ionization mass spectrometry (ESI-MS) was used to investigate complex formation between riboflavin and boric acid. In negative-ion mode at pH 10.3, riboflavin produced a dominant ion at m/z 375.3, and the addition of boric acid resulted in a new signal at m/z 401.3, corresponding to a + 26 Da increase relative to riboflavin and assigned to the formation of a 1:1 riboflavin-boric acid complex through boron-diol esterification. This species accounted for approximately 8% of the riboflavin signal, indicating a weak yet measurable binding. These findings provide direct analytical evidence for riboflavin-boric acid complexation. Together with previously reported interactions in humans and animals, these findings highlight the need to define a safe range of boric acid/riboflavin intake, as both are beneficial, but excessive intake of one may impact the status of the other.