RESPONSE SURFACE METHODOLOGY ASSISTED ULTRAPERFORMANCE LIQUID CHROMATOGRAPHIC METHOD OPTIMIZATION FOR THE SIMULTANEOUS ESTIMATION OF SIX FAT-SOLUBLE VITAMINS IN TABLET DOSAGE FORM USING A DEVELOPED AND VALIDATED UPLC-Q-TOF/MS METHOD

In this study, the liquid chromatographic (LC) parameters were optimized using response surface methodology (RSM) as a novel approach to achieve optimal separation of six vitamers of vitamin D and K during simultaneous estimation. Analytes were separated using an Accucore C18 column (50 x 4.6 mm, 2.6 µm), 0.1% aqueous formic acid (pH = 3.5), and methanol as mobile phase components. The Box-Behnken design (BBD) predicted the best combination of the selected critical quality attributes such as organic solvent composition in the mobile phase (90%), mobile phase flow rate (0.42 mL/min), and column oven temperature (40oC). Multiple regression analysis was used to fit the experimental data from 17 sample runs to a second-order polynomial equation. The adjusted coefficient of determination (R2) for three desired responses were 0.983 (retention time of K3 = R1), 0.988 (resolution between D2 and D3 = R2), and 0.992 (retention time of K2-7 = R3), all with significant probability values (p