Efficient high performance liquid chromatograph/ultraviolet method for determination of diclofenac and 4′-hydroxydiclofenac in rat serum

A rapid and sensitive high-performance liquid chromatographic method was developed for determination of diclofenac and its major metabolite, 4′-hydroxydiclofenac, in serum from rats treated with diclofenac. The method is simple with a one-step extraction procedure, isocratic HPLC separation, and UV detection at 280 nm. Use of N-phenylanthranilic acid as the internal standard provided good accuracy without interference by endogenous compounds or 5-hydroxydiclofenac, another metabolite of interest. Limits of detection for diclofenac and 4′-hydroxydiclofenac were 0.0225 and 0.0112 μg/ml, respectively. Average extraction efficiencies of diclofenac, 4′-hydroxydiclofenac, and the internal standard were ≥76%. The method was applied to serum collected at 3 h after rats were treated with an experimentally useful dosage range of 3, 10 and 50 mg/kg diclofenac. Recovery (as a percentage of dose) for the 4′-hydroxy metabolite in serum was found to consistently average from 0.10 to 0.12% following each dosage, whereas recovery of diclofenac in serum declined from 0.45 to 0.37%. Thus, the method is suitable for measurement of a major diclofenac metabolite in experimental studies.