Abstract
Economical, highly robust, selective, precise, and eco-friendly RP-UPLC and spectrophotometric methods were developed and validated for the concurrent estimation of selected pharmaceutical drugs represented in ceftazidime (CFZ) and pyridine (PYD) in their solutions using Agilent Zorbax SB-C18 RRHD (50 × 2.1 mm, 1.8 μm) column at flow rate 0.3 mL/min with wavelength 254 nm. Box-Behnken design (BBD) established Response surface methodology (RSM) to achieve the optimum chromatographic condition with minimal trials conducted. Three independent variables specifically acetonitrile ratio 60-70%, pH 3-7, and temperature 25-35 °C were implemented to evaluate the influences of these variables on the responses as resolution and retention time. Desirability and overlay plots were carried out to adjust the optimal condition that achieved the shortest retention time of less than 2 min and desired resolution of more than 1.5 using a mobile phase consisting of acetonitrile: purified water (70:30, v/v) at pH 5.0 adjusted by 0.1% orthophosphoric acid with the column oven temperature 30 °C and column void volume 0.46 mL. Mean centering of ratio spectra (MCR) and ratio subtraction (RS) methods were effectively applied to resolve drugs' spectral superposition at 220 nm, 255.4 nm, 260.3 nm, and 254.6 nm for CFZ and PYD, respectively. Linearity range was accomplished for UPLC, MCR, and RS methods over the concentration range of 2-100, 1-50,3-30 and 5-30 µg/mL for CFZ and PYD, respectively with correlation coefficient > 0.999 and good recovery results within 98-102%. Six Sigma methodology was achieved using the process capability index (Cpk) to compare the suggested and USP methods showing that both are highly capable with Cpk > 1.33. The proposed method was successfully validated depending on ICH guidelines and ANOVA results and applied for the accelerated stability study.