Isolated and Sequential Effects of Sodium Hypochlorite and Hydrogen Peroxide on Dentin Chemical Composition: An In Vitro FTIR and EDX Study

Sodium hypochlorite (NaOCl) remains the gold standard irrigant in endodontics due to its proteolytic and antimicrobial properties, whereas hydrogen peroxide (HP) is widely used for internal bleaching because of its oxidative capacity. Both agents have been associated with chemical and structural alterations in dentin; however, the impact of their sequential application on the organic–mineral balance has not been fully elucidated. Objective: To evaluate whether the isolated and sequential application of 5.25% NaOCl and 37.5% HP induces chemical alterations in dentin by analyzing changes in the organic matrix and mineral phase using Fourier-transform infrared spectroscopy (FTIR) and Energy-dispersive X-ray spectroscopy (EDX). Methods: Twenty-four independent dentin sections (n = 6 per group) from six human third molars were distributed using a tooth-balanced allocation into four groups: Control, NaOCl (5.25%, 15 min), HP (37.5%, 30 min), and sequential NaOCl+HP. FTIR assessed organic (amide I, II, III, CH2) and inorganic (phosphate, carbonate) components through baseline-corrected integrated areas, Full Width at Half Maximum (FWHM), and molecular ratios. Surface elemental composition and the calculated Ca/P atomic ratio were determined by EDX. Multiple sub-measurements per specimen were averaged before statistical analysis. Data were analyzed using Kruskal–Wallis and Mann–Whitney U tests with Bonferroni correction (p < 0.05). Results: FTIR revealed treatment-dependent modifications. NaOCl reduced absorbance in organic-associated bands, indicating collagen degradation, whereas HP altered the mineral phase. The NaOCl+HP group exhibited increased numerical values for integrated band areas, with differences detected in carbonate, phosphate, and amide III bands (p < 0.05), reflecting structural disorganization and modified spectral signal rather than tissue preservation. No differences were detected across the calculated infrared ratios (p > 0.05). EDX showed decreased absolute atomic percentages of Ca, P, and O in the NaOCl+HP group (p < 0.05), indicating structural demineralization, while its stoichiometric Ca/P ratio remained at 1.56. Isolated HP shifted the mineral stoichiometry to the highest numerical Ca/P ratio (1.69; range 1.58–1.80). Fluorine decreased across all treated groups (p < 0.001). Conclusions: Sequential NaOCl and HP application triggers distinct chemical alterations compared to individual treatments, inducing severe structural disorganization of the organic network and absolute mineral depletion of Ca and P. This multi-agent sequence alters dentin stoichiometry, which may compromise the biomechanical integrity of the tissue.