Enrofloxacin assay validation and pharmacokinetics following a single oral dose in chickens R. G. DA SILVA* F. G. R. REYES   J. R. SARTORI à & S. RATH* *Department of Analytical Chemistry, Institute of Chemistry and   Department of Food Science, Faculty of Food Engineering, State University of Campinas, Campinas, SP; à Department of Animal Breeding and Nutrition, College of Veterinary Medicine and Animal Science, UNESP – Sa ˜o Paulo State University, Botucatu, SP, Brazil da Silva, R. G., Reyes, F. G. R., Sartori, J. R., Rath, S. Enrofloxacin assay validation and pharmacokinetics following a single oral dose in chickens. J. vet. Pharmacol. Therap. 29, 365–372. The pharmacokinetics of enrofloxacin (ENRO), a fluoroquinolone antimicrobial agent, was studied in male broiler chickens (Cobb) after single oral adminis- tration of 10 mg of ENRO/kg b.w. A high-performance liquid chromatography- photodiode array detector (DAD) (HPLC-DAD) method was developed and validated and used for quantitation of ENRO and its major metabolite ciprofloxacin in plasma. The HPLC analyses were carried out using a cationic-octadecyl mixed column and 0.05 mol/L phosphate buffer (pH 2.5)/ acetonitrile as mobile phase. The sample preparation of plasma consisted of the precipitation of proteins followed by solid phase extraction on cationic- octadecyl mixed cartridges. The method was validated considering linear range, linearity, selectivity, sensitivity, limit of detection (LOD), limit of quantitation (LOQ), intra- and inter-day precisions and accuracy. The LOD and LOQ for both fluoroquinolones were 60 and 200 ng/mL for plasma. The plasma concentration vs. time graph was characteristic of a two-compartment open model. The maximal plasma concentration of 1.5 ± 0.2 mg/mL was achieved at 9 ± 2 h. The elimination half-life and the mean residence time of ENRO were 1.5 ± 0.2 and 15.64 h, respectively. The area under the concentration–time curve was calculated as 35 ± 4 mgÆh/mL. (Paper received 14 March 2006; accepted for publication 19 May 2006) Susanne Rath, Department of Analytical Chemistry, Institute of Chemistry, State University of Campinas, PO Box 6154, 13084-971 Campinas, SP, Brazil. E-mail: raths@iqm.unicamp.br INTRODUCTION Fluoroquinolones (FQ) are considered the most important group of synthetic antibacterial agents since the discovery of sulfona- mides (Posyniak et al., 1999), with a broad spectrum of activity against gram-positive and gram-negative bacteria. These com- pounds are used in human medicine and in preventive and therapeutic treatments in farm animals, especially pigs and chickens (Ovando et al., 1999). The first FQ approved for use in food animals in the US were sarafloxacin in 1995 and enrofloxacin (ENRO) in 1996 (Anderson et al., 2003). Enrofloxacin (Fig. 1a) is a FQ that was developed exclusively for veterinary use in cattle, pigs, dogs, cats, etc. (Altreuther, 1987) and for the treatment of respiratory disease in chickens and turkeys (Anderson et al., 2003). After administration, ENRO is partly de-ethylated to ciprofloxacin (CIPRO) (Fig. 1b) in vivo, which is also pharmacologically active and is employed in human medicine (Rao et al., 2002). US Food and Drug Administration (FDA) withdrew approval for ENRO for the purpose of treating bacterial infections in poultry because of scientific data that showed that the use of ENRO in poultry caused resistance to Campylobacter, a bacterium that causes food- borne illness (US FDA, 2005). Similar to other FQ, the bactericidal activity of ENRO is mediated by affecting bacterial DNA-gyrase. Its principal appli- cations have been in the treatment of gastrointestinal and respiratory infections caused by gram-negative bacteria (Knoll et al., 1999). The pharmacokinetic behavior of ENRO have been determined in goats (Rao et al., 2002), pigs and rabbits (Manceau et al., 1999), dogs (Kung et al., 1993), fish (Lewbart et al., 1997; Intorre et al., 2000) and chickens (Anado ´n et al., 1995; Knoll et al., 1999; Ovando et al., 1999; Schneider & Donoghue, 2004), and variations among animal species have been observed. The Joint Expert Committee of Food Additives and Contami- nants, at its 48th Meeting in 1997, established an acceptable J. vet. Pharmacol. Therap. 29, 365–372, 2006. Ó 2006 The Authors. Journal compilation Ó 2006 Blackwell Publishing Ltd 365