Optimization of a vanillin assay for determination of anthocyanins using D-optimal design

Abstract

The vanillin assay is a spectrophotometric method for anthocyanin determination that is simple, quick and inexpensive. The method is preferred because of its high sensitivity, specificity and simplicity; however, the results of this test are influenced by several factors. Hence, a D-optimal experimental design approach was investigated to simultaneously, without loss of information, optimize five factors that influence the vanillin assay: acid normality, vanillin concentration, temperature, time and acid type. Further optimization with a D-optimal design and response surface analysis (RSM) showed that a second-order polynomial model fit the experimental data appropriately. The optimum conditions were determined as follows: the optimal acid type was H₂SO₄ with a normality of 7.00, vanillin concentration of 1.50% w/v, temperature of 22.0 °C, and reaction duration of 21.0 min. Using these conditions, the absorbance maximum predicted using the model was calculated to be 0.71. Good agreement between the observed values and the values predicted by the model confirmed the statistical significance of the model and the model's ability to predict the optimum conditions.