Physicochemical stability and bactericidal efficacy of a novel premixed triple antibiotic paste during storage: an in vitro study

Background: Triple antibiotic paste (TAP) is a potent intracanal medicament, but its clinical use is often hindered by preparation variability. A premixed, injectable TAP using a propylene glycol (PG) and carboxymethylcellulose (CMC) vehicle was developed to improve preparation accuracy, handling convenience, and storage stability. This study evaluated the physicochemical stability and bactericidal efficacy of this novel TAP formulation compared to the conventional PG and macrogol (MG) formulation over a 24-week storage period. Methods: TAP (10 mg/mL) was prepared using ciprofloxacin, metronidazole, and minocycline in either PG + MG or PG + CMC vehicles. Formulations were evaluated immediately and after 6, 12, and 24 weeks of storage at 4 °C. Physical stability was assessed by visual inspection of appearance and consistency, while chemical stability was analyzed using UV–visible spectrophotometry across wavelengths of 200–500 nm. Bactericidal activity against Enterococcus faecalis was evaluated using an infected root canal model through bacterial culture and LIVE/DEAD confocal laser scanning microscopy (CLSM). Results: Although both formulations exhibited progressive darkening and increased viscosity during storage, UV spectrophotometry confirmed that antibiotic concentrations remained stable throughout the 24-week period. No culturable bacteria were detected in any TAP-treated groups at any time point. While CLSM analysis showed a significant reduction in bacterial viability compared to controls (p < 0.05), a modest decrease in bactericidal efficacy within dentinal tubules was observed after storage. No significant differences in antimicrobial performance were found between the two formulations (p > 0.05). Conclusion: Both TAP/PG + MG and TAP/PG + CMC maintained chemical stability and effective bactericidal activity for up to 24 weeks. The premixed TAP/PG + CMC formulation provides a clinically practical and standardized alternative to conventional formulations without compromising long-term therapeutic efficacy.