THE OPTIMIZATION OF RP-HPLC CONDITION USING RESPONSE SURFACE METHODOLOGY BOX-BEHNKEN DESIGN FOR SIMULTANEOUS DETERMINATION OF METFORMIN HCL AND GLIMEPIRIDE IN SPIKED PLASMA
Objective: The objective of this study was to develop and validate RP-HPLC method using Box-Behnken Design (BBD) for simultaneous analysis metformin HCl and glimepiride in spiked plasma.
Methods: The chromatographic system used in this research was comprised of acetonitrile-phosphate buffer 0.0125 M + SDS 1 mM as mobile phase and Ascentis® Phenyl C18 (250 x 4.6 mm i.d.; 5 µm) column as stationary phase with UV detector at 210 nm. Three independent variables included phosphate buffer (%), pH and flow rate were optimized using BBD. The observed responses were retention time, peak area and resolution.
Results: The predicted optimum condition of RP-HPLC system consisted of phosphate buffer solution with concentration of 72%, pH at 4.3 and flow rate adjusted at 0.8 ml/min. By using this condition, retention time of glimepiride was more than 18 min, so that it was necessary to modified the flow rate to be 1 mL/min in order to get shorter duration of analysis. This HPLC condition was then applied to analyze of glimepiride and metformin in spiked plasma and validated by assessing parameters of specificity, linearity, accuracy, precision, LLOQ, carryover, dilution integrity and stability. All parameters met the requirements of the EMA, except for stability of glimepiride.
Conclusion: BBD can be used to help optimize the HPLC system and the optimum condition was valid to analyse metformin and glimepiride in spiked plasma by considering the storage time of plasma samples.
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