Abstract
Clonazepam (CLO) plays a significant role in treating seizures, myoclonic seizures, and other clinical infections, while metronidazole (MTZ) is an antiprotozoal, antibacterial drug. The reversed-phase high performance liquid chromatographic (RP-HPLC) technique has provided greater precision and sensitivity over other methods, especially the colorimetric and spectrophotometric. This study aimed to implement a simple method in pure and pharmaceutical tablets for the simultaneous determination of CLO and MTZ. The RPHPLC method's development and optimization for validating a separation method for the simultaneous estimation of both drugs in pharmaceutical formulation involved studying the optimum mobile phase, buffer concentration, and the pH value. Under the chromatographic conditions, RP-HPLC system achieved excellent separation on a Phenomenex HyperClone BDS (250 x 4.60 mm, 130A, and 5μ) at a temperature of 45°C and the following conditions: 65:35% acetonitrile:acetic acid and sodium acetate buffer solution (NaOAc/HAc) as mobile phase (pH 3.5); 20 μL injection volume at a flow rate of 0.75 mL/min and detection wavelength of 310 nm. The proposed RP-HPLC procedure demonstrated high precision (RSD% 1%), as well as the good linear relationship of the calibration graph at concentration ranges of 50-160, 35-100 ppb with a coefficient of determination (r2) of the regression line of 0.9996 and 0.9997 for metronidazole and clonazepam respectively. The proposed method offered excellent validated values for both LOD (4.24 and 3.06 ppb) and LOQ (14.15– 10.21 ppb) for both metronidazole and clonazepam drugs, respectively. The RP-HPLC method has been successfully applied to estimate metronidazole and clonazepam in the well-known commercial pharmaceutical tablets and gave an excellent recovery> 98% for both drugs. The results of the method was compared with the standard pharmacopeial method for both drugs using statistical tests, which indicated no difference in accuracy between the methods
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