Analysis of diclofenac and its metabolites by high-performance liquid chromatography: relevance of CYP2C9 genotypes in diclofenac urinary metabolic ratios

In humans, diclofenac is metabolised to 4′-hydroxy (OH), 3′-OH and 5-OH metabolites. The polymorphic CYP2C9 is involved in the metabolism of diclofenac to 4′-OH diclofenac and 3′-OH diclofenac. The aim of the present study was to develop a high-performance liquid chromatographic method to simultaneously measure diclofenac and its metabolites in urine, suitable for metabolic studies. After liquid–liquid extraction the compounds were separated in a reversed-phase column and measured by ultraviolet absorption at 282 nm. For all compounds intra-day and inter-day variations were less than 7%, and the limits of quantitation were 0.25 mg/l. No analytical interference with endogenous compounds was found. The relationship between diclofenac metabolic ratios among different CYP2C9 genotypes is reported. The CYP2C9∗3/∗3 subject had the highest diclofenac/4′-OH ratios. However no difference was found between CYP2C9∗2/∗2 and ∗1/∗1 genotypes. The chromatographic method developed was sensitive and reliable for the measurement of diclofenac and its metabolites simultaneously in human urine, and is suitable for use in diclofenac metabolism studies.