Development and validation of HPLC-DAD method for the simultaneous determination of amoxicillin, metronidazole and...

This work deals with the development, validation and application of an HPLC-DAD method for the determination of a ternary mixture containing amoxicillin (AX), metronidazole (MZ) and the proton pump inhibitor rabeprazole sodium (RB). This triple therapy is used for treatment of Helicobacter pylori infection. Effective chromatographic separation between the three drugs was achieved using Thermo Hypersil BDS-C8 (4.6×250mm, 5μm particle size) column and a mobile phase composed of phosphate buffer pH 7 and acetonitrile (70: 30, by volume). The mobile phase was pumped isocratically at a flow rate of 1 mL/min. Quantification of the analytes was based on measuring their peak areas at 230nm for both AX and RB, and at 319nm for MZ. AX, MZ and RB eluted at retention times 2.36, 3.55 and 8.72min respectively. The reliability and analytical performance of the proposed HPLC procedure were statistically validated with respect to linearity, ranges, precision, accuracy, selectivity, robustness, detection and quantification limits. The linear dynamic ranges were 25-250, 25-250 and 5-50μg/mL for AX, MZ and RB respectively with correlation coefficients>0.9998. The validated method was successfully applied to the analysis of several laboratory-prepared mixtures as well as simulated intestinal fluid samples spiked with the three drugs.