Development and validation of an improved Bradford method for determination of insulin from chitosan nanoparticulate systems

Context: Blank chitosan nanoparticles are currently used as reference for the calibration curve, which fails to resolve the supernatant of the nanoparticles in the interference of Coomassie Brilliant Blue G-250 reagent; supernatants are generated at different chitosan nanoparticulate prescriptions, which have different interferences. There are notable errors in the experimental results, and the method is not feasible. Objective: In this study, an improved, rapid, and economic Bradford method was developed and validated. Materials and methods: The pH of the supernatant of blank chitosan nanoparticles was adjusted to 7-9 through adding saturated NaOH. The precipitation (free chitosan) in the solution was separated by centrifuging for about 10 min (4000 r/min). Results: The method eliminated the interference of free chitosan of different prescriptions. The results showed that the method presented a linearity in the range of 50-300 mu g/mL (R-2 = 0.9992), and possessed a good inter-day and intra-day precision based on relative standard deviation values (less than 3.10%). Recovery of the supernatant of blank chitosan nanoparticles was between 98.30 and 99.93%, and the recovery of blank chitosan nanoparticles was between 95.57 and 100.27%. Discussion and conclusion: The method was further tested for determination of the association efficiency of insulin to nanoparticulate carriers composed of chitosan. Encapsulant release under simulated gastrointestinal fluids was evaluated.