AN EXPERIMENTAL DESIGN APPROACH FOR OPTIMIZATION OF MODIFIED COLORIMETRIC FIRST-ORDER DERIVATIVE METHOD FOR ESTIMATION OF SERRALYSIN IN BULK AND PHARMACEUTICAL FORMULATION

Abstract

Objective: The aim of the present work is to use experimental design to screen and optimize experimental variables for developing a colorimetric first-order derivative method for determining content of serralysin (SER) using biuret and Folin–Ciocalteu phenol reagent for stable color development. The method is based on the reaction of peptide bond in the protein with Biuret reagent in alkaline medium and further reaction of remaining tryptophan and tyrosine residues with Folin–Ciocalteu Phenol reagent to form a stable blue-colored complex (first-order derivative λmax 620 nm).Materials and Methods: A two-level full factorial design was utilized to screen the effect of Volume of NaOH (A), volume of biuret reagent (B), volume of Folin–Ciocalteu phenol reagent (C), and concentration of NaOH (D) on the formation of blue-colored SER - reagent complex (response - absorbance). A box Behnken experimental design with response surface methodology was then utilized to evaluate the main interaction and quadratic effects of these factors on the selected response.Results: With the help of a response surface plot and contour plot, the optimum values of the selected factors were determined and used for further experiments. These values were volume of NaOH (A) of 1.0 mL, volume of biuret reagent (B) of 0.25 mL, and volume of Folin–Ciocalteu phenol reagent (C) of 10 μL. The proposed method was validated according to the ICH Q2(R1) method validation guidelines. The developed colorimetric first-order derivative method was found to be simple, accurate, rapid, sensitive, precise, and economic. Further optimization of the method with experimental design approach makes it convenient for use in laboratory.Conclusion: The results of present study have clearly shown that an experimental design approach may be effectively applied to the optimization of a modified visible spectrophotometric method for estimation of SER in bulk and in pharmaceutical formulation with the least number of experimental runs possible. The method can be employed successfully for the estimation of SER in both bulk and tablet dosage form.