Design of experiments and Derringer’s desirability function in optimisation and validation of RP-HPLC method for the analysis of enrofloxacin and its impurities

Using the Design of Experiments methodology (Response-Surface Methodology and Derringer’s Desirability Function), a simple, fast and robust RP-HPLC method was developed for the analysis of enrofloxacin (EFC), its impurity A (fluoroquinolonic acid, FQ) and impurity B (ciprofloxacin, CPX). Gradient elution of samples was performed on a Zorbax Eclipse XDB C18 column (15034.6 mm, 3.5 μm) with a mobile phase consisting of 32mM phosphate buffer pH 3.5 – methanol (0 min-19.6% methanol; 15.5 min-19.6% methanol; 29.5 min-80% methanol; 30 min-19.6% methanol; 35 min-19.6% methanol), delivered at a flow rate of 1.5 mL min1, wavelength of detection 278 nm (for EFX and CFX) and 265 nm for FQ. A good linear response was achieved in the range 15–35 μgmL1 (EFX) and LOQ150% for impurities (CFX and FQ). Other validation parameters were also tested: precision, accuracy, sensitivity and robustness. The developed method was shown to be simple, practical and suitable for the analysis of EFC and its impurities (CPX, FQ) in veterinary drugs.