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

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.

In this study, an improved, rapid, and economic Bradford method was developed and validated.

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).

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 microg/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%.

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.