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.
Department of Pharmacology and Psychiatry, Faculty of Medicine, University of Extremadura, Badajoz, Spain.
Pedro
P
Dorado
Roland
R
Berecz
Macarena C
MC
Cáceres
Adrián
A
LLerena
D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000652:urine*; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D065729:Cytochrome P-450 CYP2C9; D004008:Diclofenac / Q000652:urine*; D005838:Genotype; D006801:Humans; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet
D000894:Anti-Inflammatory Agents, Non-Steroidal; D004008:Diclofenac; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases