Simple, rapid determination of enrofloxacin and ciprofloxacin in bovine milk and plasma by high-performance liquid chromatography with fluorescence detection

Abstract

A rapid and simple procedure for determination of enrofloxacin and ciprofloxacin in bovine milk and plasma is described. Protein precipitation from both milk and plasma samples was achieved by addition of acetonitrile and phosphoric acid. Acetonitrile was removed with methylene chloride, leaving enrofloxacin and ciprofloxacin in the acidic aqueous extract. The aqueous extract was analyzed by high-performance liquid chromatography (HPLC) with fluorescence detection. The limit of quantitation (LOQ) for enrofloxacin and ciprofloxacin in milk was found to be 2 ng/ml. LOQ for enrofloxacin and ciprofloxacin in plasma was found to be 1 ng/ml. Linear calibration curves were obtained with correlation coefficient ( r 2) ≥0.99. Analysis of quality control (QC) samples gave results within ±10% of the nominal values. Inter-assay precision for the analysis of milk QC samples were in the ranges: 4.63–12.49% (for enrofloxacin) and 4.67–9.86% (for ciprofloxacin). Inter-assay precision for the analysis of plasma QC samples were in the ranges: 6.60–17.31% (for enrofloxacin) and 6.14–13.87% (for ciprofloxacin). Intra-assay precision for the analysis of milk QC samples were in the following ranges: 3.65–7.21% (for enrofloxacin) and 1.58–14.28% (for ciprofloxacin). Intra-assay precision for the analysis of plasma QC samples were in the following ranges: 2.17–16.95% (for enrofloxacin) and 3.31–16.31% (for ciprofloxacin). The effectiveness of protein precipitants other than phosphoric acid was investigated. The method described has been applied to a study of the pharmacokinetics of enrofloxacin and ciprofloxacin in lactating dairy cows and beef steers.