Development of gradient HPLC-DAD method for assay of ternary mixture containing amebicide and analgesic drugs

Validated gradient high-performance liquid chromatography-diode array detection (HPLC-DAD) method has been developed for simultaneous determination of diloxanide furoate (DIL), mebeverine hydrochloride (MBV), and metronidazole (MET) in their pharmaceutical preparation. This method was based on a reversed-phase HPLC separation of the cited drugs on a C-18 column (150 mm × 4.6 mm, 5 μm) with a gradient elution system of 0.05 M phosphate buffer with 1% (v/v) of triethylamine adjusted to pH 3.0-methanol as the mobile phase at the flow rate of 1.0 mL min -1. The separation was carried out at ambient temperature. Quantitation was achieved with DAD detection at 260 nm for DIL and MEB, and at 320 for MET based on peak area with linear calibration curves at concentration ranges 4–30, 1–20, and 1–30 μg mL -1 for DIL, MEB, and MET, respectively. The proposed chromatographic method was successfully applied to the determination of the investigated drugs in pharmaceutical preparation. It was validated in compliance with International Conference on Harmonization (ICH) guidelines, in terms of linearity, accuracy, precision, robustness, limits of detection, and quantitation and other aspects of analytical validation.