Abstract
Two chemometric assisted UV spectrophotometric and one RPLC-PDA (Reversed Phase Liquid Chromatography with Photo Diode Array Detector) methods were developed and validated for the simultaneous in vitro analysis of Isoniazid (INH), Rifampicin (RIFA) and Piperine (PIP) in their ternary mixture and pharmaceutical formulation. The two chemometric methods applied were inverse least square (ILS) and classical least square (CLS). These approaches were successfully applied to quantify each drug in their mixture using the information included in the UV absorption spectra of appropriate solutions in the wavelength range 220-360nm with the intervals of 10nm (Δλ=10nm) at 15 wavelength points. For the chemometric calibration, 20 ternary solutions were prepared as training set and 10 ternary solutions were prepared as validation set. The chemometric methods do not require any separation step. The chromatographic separation was achieved on a reversed-phase, Phenomenex Luna C18 column (250X4.6 mm, 5 m particle size). Gradient elution was carried out with a mobile phase of 0.05M Disodium hydrogen phosphate buffer pH -7.0 (solution-A) and Acetonitrile (solution-B). The total run time was 12 min. Quantitation was achieved with PDA detection at 290nm for INH, RIF and PIP based on peak area. Linearity was observed over concentration range of 30-270 µg mL-1 for INH, 20-180 µg mL-1 for RIFA and 1-9 µg mL-1 for PIP. The developed methods were successfully applied for laboratory prepared mixtures as well as commercial capsule formulation for INH, RIFA and PIP concentration. The results obtained for pharmaceutical formulation by ILS and CLS methods were compared with gradient RPLC-PDA method and a good agreement was found.
