Factorial design-assisted reversed phase-high performance liquid chromatography method for simultaneous determination...

A 2³ full factorial design model was used for the development of a new high performance liquid chromatography method with UV detection to estimate three antifungal drugs simultaneously. Fluconazole (FLU), itraconazole (ITR) and terbinafine (TRH) are co-administered for severe fungal infections. They have been determined using MOS-1 Hypersil C₁₈ column and an isocratic eluent; methanol 95% and phosphate buffer 5% with 0.001% triethylamine. The pH was adjusted to 7, and the flow rate was 0.7 ml min^-1. The three drugs were separated within less than 7 min at 210 nm. The developed method gave a linear response over 5-80 µg ml^-1, 5-50 µg ml^-1 and 1-50 µg ml^-1 for FLU, ITR and TRH, respectively. It showed detection limits of 0.88, 0.29 and 0.20 µg ml^-1 and quantification limits of 2.66, 0.88 and 0.60 µg ml^-1 for the three drugs, respectively. The design of the experiment facilitated the optimization of different variables affecting the separation of the three drugs. The sensitivity of the designed method permitted the simultaneous estimation of ITR and TRH in spiked human plasma successfully.