Determination of diclofenac in micro-whole blood samples by high-performance liquid chromatography with electrochemical detection. Application in a pharmacokinetic study.

A rapid and sensitive method for the determination of diclofenac (CAS 15307-86-5) in whole blood samples by high-performance liquid chromatography with amperometric detection has been developed. This method was then used to study the pharmacokinetics of oral diclofenac sodium in the rat. The method includes a single extraction of acidified whole blood with ethyl acetate. Extracts were analyzed on a reversed-phase column eluted with a mixture of acetonitrile and 0.075 mol/l sodium acetate solution (pH 3.3) and detected amperometrically at + 1.1 V against Ag/AgCl. Retention times for diclofenac and the internal standard (naproxen) were 3.5 and 6 min, respectively. The method was linear in the range of 25 to 2000 ng/ml and the detection limit of the method was 10 ng/ml, using 100 microliters of whole blood sample. Employing this method, the oral pharmacokinetics of diclofenac in the rat was studied. Wistar male rats received an oral dose of 1, 3.2 or 10 mg/kg of diclofenac and blood samples were drawn at selected times during 12 h. After administration of diclofenac, a rapid increase of circulating concentrations was observed reaching a maximum in about 10 min. Then concentration decayed with a half-life of about 15 h. It is concluded that the method here reported is adequate for realization of pharmacokinetic studies of diclofenac in small species.