High-performance liquid chromatographic method for the routine determination of diclofenac and its hydroxy and methoxy metabolites from in vitro systems.

A rapid and sensitive high-performance liquid chromatographic method is presented for the determination of diclofenac and its hydroxylated and methoxylated metabolites. The procedure describes extraction of diclofenac and its metabolites from acidified incubation medium into tert.-butylmethyl ether. Separation is achieved with a C18 reversed-phase column and quantification by UV detection at 280 nm. The method employs an internal standard resulting in good accuracy and precision. The limit of detection is 5 ng/ml for diclofenac and 10 ng/ml for its metabolites. One analysis requires no more than twelve minutes so that the assay is very suitable for the determination of a large number of samples.