Analysis of Diclofenac and Four of Its Metabolites in Human Urine by HPLC

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Abstract
An HPLC method for the determination of diclofenac (DCF) and four of its metabolites (3′-hydroxydiclofenac, 4′-hydroxydiclofenac, 5-hydroxydiclofenac, and 3′-hydroxy-4′-methoxydiclofenac) in human urine is described. Following base hydrolysis, the samples were neutralized and extracted. Evaporated extracts were reconstituted in mobile phase containing ascorbic acid, and chromatographed, using flow-rate programming, on a reversed-phase column. Absolute recovery (average), was at least 78% for diclofenac and ranged from 75 to 85% for the four metabolites. Standard curves showed linearity over the range of concentrations of 0.2 to 40 ug/mL, using 0.25 mL of urine. Specificity was demonstrated by examining chromatograms of extracts of blank urine from 8 volunteers and 24 study subjects. Good accuracy was observed for all compounds over the concentration range of 0.2 to 40 ug/mL using 0.25 mL of urine. Based on accuracy and precision criteria, the limit of quantitation for all 5 analytes was 0.4 ug/mL, using 0.25 mL of urine. Analysis of urine from subjects with normal and reduced renal function who received diclofenac orally demonstrated that total diclofenac and metabolites excreted in the urine represented approximately 31% and 4% of an oral dose of diclofenac, respectively.