An experimental design approach for optimization of spectrophotometric method for estimation of cefixime trihydrate using ninhydrin as derivatizing reagent in bulk and pharmaceutical formulation

The aim of the present work is to use experimental design to screen and optimize experimental variables for developing a spectrophotometric method for determining cefixime trihydrate content using ninhydrin as a derivatizing reagent. The method is based on the reaction of the amino group of cefixime with ninhydrin in an alkaline medium to form a yellow-colored derivative (λmax 436 nm). A two-level full factorial design was utilized to screen the effect of ninhydrin reagent concentration (X1), volume of ninhydrin reagent (X2), heating temperature (X3) and heating time (X4) on the formation of the cefixime-ninhydrin complex Y (absorbance). One way ANOVA and Pareto ranking analyses have shown that the ninhydrin reagent concentration (X1), volume of ninhydrin reagent (X2) and heating temperature (X3) were statistically significant factors (P < 0.05) affecting the formation of the cefixime-ninhydrin complex Y (absorbance). A Box-Behnken experimental design with response surface methodology was then utilized to evaluate the main, interaction and quadratic effects of these three factors on the selected response. 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 a ninhydrin reagent concentration (X1) of 0.2% w/v, volume of ninhydrin reagent (X2) of 1 mL and heating temperature (X3) of 80 °C. The proposed method was validated according to the ICH Q2 (R1) method validation guidelines. The results of the present study have clearly shown that an experimental design concept may be effectively applied to the optimization of a spectrophotometric method for estimating the cefixime trihydrate content in bulk and pharmaceutical formulation with the least number of experimental runs possible.