Development of a gradient high performance liquid chromatography assay for simultaneous analysis of hydrophilic gemcitabine and lipophilic curcumin using a central composite design and its application in liposome development

• First simultaneous analysis HPLC method for gemcitabine and curcumin.
• Application of the central composite design and desirability factor for HPLC.
• Two response factors for optimization were evaluated simultaneously.
• Application of validated HPLC method for liposome development and characterization.

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, 270 nm; curcumin, 420 nm) 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 4.5 min, followed by 30:70 for 5 min, which gave retention times within 11 min with peak widths <0.10 min, respectively. No interference from the formulation ingredients was detected. The method was linear over the range of 0.125–5 μM for gemcitabine, and 1.25–50 μM 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 0.04 μM and 0.03 μM, 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 3 h 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.

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