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 (150 3 4.6 mm,
3.5 μm) with a mobile phase consisting of 32 mM 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 μg mL1 (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.
