Development of a gradient high performance liquid chromatography assay for simultaneous analysis of hydrophilic...

A rapid and reliable gradient high performance liquid chromatography (HPLC) method was developed for the simultaneous analysis of a hydrophilic anticancer drug, gemcitabine, and a lipophilic phytochemical, curcumin. A central composite design (CCD) was employed to optimize the gradient elution programme to obtain an acceptable retention time and peak width for curcumin with the aid of the desirability function (DF). Dual wavelengths (gemcitabine, 270nm; curcumin, 420nm) were selected to give maximal specific detection using a diode-array detector. The optimized DF was obtained with a mobile phase (water:acetonitrile) of 95:5 for 4.5min, followed by 30:70 for 5min, which gave retention times within 11min with peak widths <0.10min, respectively. No interference from the formulation ingredients was detected. The method was linear over the range of 0.125-5μM for gemcitabine, and 1.25-50μM for curcumin. The intra-day and inter-day variability were less than 5%, with accuracies between 90 and 110% of the true values. The limit of detection (LOD) for gemcitabine and curcumin was 0.04μM and 0.03μM, respectively. Using this method, release study for a dual-drug loaded formulation revealed a Fickian diffusion process for both. The stability of curcumin was pH dependent in aqueous solutions with a half-life of 3h at 25°C and pH 7.4. This was significantly improved after incorporation into the liposomes with no degradation for at least 3 months. In conclusion, the combination of CCD and DF offers a new approach to optimize a gradient HPLC method with high efficiency. A liposomal carrier system may provide an option for tumour dual-drug therapy with the additional advantage of chemical stabilization of the encapsulated compound.