Determination of diclofenac in rat bile and its interaction with cyclosporin A using on-line microdialysis coupled to liquid chromatography

Diclofenac is a potent inhibitor of prostaglandin synthesis, as well as an established antipyretic and analgesic agent. To determine diclofenac in rat bile and investigate its hepatobiliary excretion, a procedure using rapid and sensitive high-performance liquid chromatography coupled to microdialysis sampling system was developed. A shunt linear microdialysis probe was inserted into the common bile duct between the liver and the duodenum for continuous sampling of the drug from bile fluids following intravenous administration of diclofenac (1 mg/kg). Separation and quantitation of diclofenac in the bile dialysates were achieved using a microbore reversed-phase C18 column (150×1.0 mm I.D.; particle size 5 μm) maintained at ambient temperature. Samples were eluted with a mobile phase containing 100 mM sodium dihydrogenphosphate (pH 3.1)–acetonitrile (30:70, v/v), and the flow-rate of the mobile phase was 0.05 ml/min. The UV detector wavelength was set at 280 nm. The concentration–response relationship from the present method indicated linearity (r2>0.995) over a concentration range of 5–5000 ng/ml for diclofenac. Intra-assay and inter-assay precision and accuracy of diclofenac fell well within the predefined limits of acceptability (≤15%). The diclofenac in rat bile appeared to have a slow elimination phase, with a peak concentration at 20 min following diclofenac administration. The results demonstrated that diclofenac might be secreted into bile in unconjugated form by a canalicular bile acid transporter, and then go through hepatobiliary excretion. These results may provide good clinical evidence showing the value of diclofenac for the treatment of biliary colic. The elimination half-life of diclofenac in the biliary elimination was prolonged by treatment with cyclosporin A, indicating that the drug–drug interaction might affect the hepatobiliary excretion of diclofenac.