An Analytical Approach by Tri-Combination of Gradient UFLC, Response Surface Methodology and Green Chemistry Principle for Simultaneous Quantification of Azelnidipine and Chlorthalidone in Rabbit Plasma

In this study, a sustainable and eco-friendly method is developed to quantify azelnidipine and chlorthalidone in rabbit plasma by gradient liquid chromatography based on green chemistry principle and analytical quality by design. The separation was achieved on a Shim pack C18 (25 cm x 5 cm x 4.6 mu m) column with L1 packing. The mobile phase compromised of ethanol and 50-Mm ammonium acetate buffer (pH.6) at flow rate of 0.6 mL/min with 25-min runtime. The resolution and asymmetric factor were identified as critical analytical attributes (CAAs). The screening studies employing Control Noise Experimentation revealed that mobile phase pH, flow rate and ethanol concentration at 6 and 15 min significantly affected the CAAs method. The critical method parameters were optimized using Central Composition design. Chromatogram showed peak of the drugs at retention time of 9.03 min for chlorthalidone and 16.83 min for azelnidipine. The greenness score of the analytical method was found to be 1876.43 using analytical method greenness score calculator. The validation of the developed method was done which showed linearity at the range of 16-520 ng/mL, with R2 of 0.9992 and 0.9996 for azelnidipine and chlorthalidone, respectively, furthermore accuracy, precision, recovery and stability studies are carried out.