Spectroscopic Analysis of Salicylate Dianion Generated during Aspirin Titration Based on the Japanese Pharmacopoeia...

The Japanese Pharmacopoeia (JP) method describes the determination of aspirin content by alkaline hydrolysis of the ester bond and neutralization of the carboxyl group, followed by back-titration of the remaining sodium hydroxide (NaOH) with sulfuric acid. Although the monograph does not explicitly address this point, the excess NaOH is expected to deprotonate the phenolic hydroxyl group of salicylic acid, generating the corresponding dianionic species. Despite its presumed formation under the JP assay conditions, this species has not yet been experimentally verified. In this study, we spectroscopically characterized the dianionic form of salicylic acid, the hydrolysis product of aspirin, to determine whether it actually forms during this procedure. Deprotonation of the phenolic hydroxyl group enhances electron delocalization within the aromatic system, inducing marked changes in both absorbance and fluorescence. Under strongly alkaline conditions (pH > 12), the UV-visible spectra (200-400 nm) exhibited predominantly red-shifts. Furthermore, the fluorescence excitation band at 250 nm increased sharply, whereas that at 294 nm remained unchanged. These absorbance and fluorescence spectral changes are consistent with the formation of the salicylate dianion as predicted by the Henderson-Hasselbalch equation. Lastly, to determine whether the dianionic species is present after treatment with NaOH, we measured the absorbance spectra of undiluted reaction mixtures. The observed spectra were well described by combinations of the monoanionic and dianionic reference spectra, supporting the presence of the dianionic form. This work sheds light on salicylate species overlooked in the aspirin titration process and contributes to a mechanistic understanding of the JP titration.