Experimental design and optimization

Method-1: Box-Behnken design (BBD) with response surface methodology (RSM) was employed to optimize reaction (saxagliptin-MBTH) conditions for colorimetric method development. The effect of three method variables (at high, low levels), such as the concentration of MBTH (A1): 0.8, 1.2% w/v; concentration of ferric chloride (B1): 1, 5% w/v; reaction time (C1): 10-30 min; on the response (Y1) i.e., absorbance of bluish-green coloured complex was studied at 600 nm in spectrophotometer. Randomized order in experimentation was followed to abate the bias effects of uncontrolled variables for 17 experimental runs under BBD using working standard solution-2 (0.1 µg/mL).

Method-2: Central composite design (CCD) with RSM was exploited to optimize experimental conditions for colorimetric estimation of saxagliptin, upon derivatization with ninhydrin reagent. Working standard solution-1 (10 µg/mL) was used for experimentation. This method optimization was premeditated with 15 experimental runs under CCD (4 factorial points, 5 center points and 6 axial points) to study the influence and interaction of three method parameters (at high, low levels) namely, concentration of ninhydrin reagent (A2): 0.8, 1.2% w/v; concentration of sodium hydroxide (B2): 0.2, 0.6 M; heating time (C2): 5, 15 min; on the response (Y2) i.e. absorbance of Ruhemann’s purple, measured at 585 nm in a spectrophotometer.