ABSTRACT
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<0.0001), indicating a high significance for the regression model. Q-ToF/MS detection was interfaced with an electrospray combined ionization source. The optimized detection parameters delivered specific, sensitive, linear, accurate, precise, and robust quantification of all six analytes in the tablet dosage form.
Subject(s)
Vitamin D , Vitamins , Tablets , Chromatography, Liquid , MethanolABSTRACT
By considering the current regulatory requirement for an analytical method development, a reversed phase high performance liquid chromatographic method for routine analysis of etofenamate in dosage form has been optimized using analytical quality by design approach. Unlike routine approach, the present study was initiated with understanding of quality target product profile, analytical target profile and risk assessment for method variables that affect the method response. A liquid chromatography system equipped with a C18 column (250×4.6 mm, 5 µ), a binary pump and photodiode array detector were used in this work. The experiments were conducted based on plan by central composite design, which could save time, reagents and other resources. Sigma Tech software was used to plan and analyses the experimental observations and obtain quadratic process model. The process model was used for predictive solution for retention time. The predicted data from contour diagram for retention time were verified actually and it satisfied with actual experimental data. The optimized method was achieved at 1.2 ml/min flow rate of using mobile phase composition of methanol and 0.2% triethylamine in water at 85:15, % v/v, pH adjusted to 6.5. The method was validated and verified for targeted method performances, robustness and system suitability during method transfer.
