HPLC–assay of diclofenac in human plasma: application for protein binding study

A rapid and sensitive HPLC-PDA method was developed for the quantification of diclofenac in human plasma using chlorzoxazone as internal standard. The analyte and internal standard were extracted from the plasma sample using simple protein precipitation method. Chromatographic separation was achieved on a Thermo (250 × 4.6 mm, 5 µm) column with a mobile phase consisting 0.1 % formic acid in TDW and acetonitrile (40: 60, v/v with); at a flow rate of 1.0 ml /min. The assay was validated with a linear range of 1.25 – 40 µg /ml for diclofenac using 200 µL of plasma sample. The intra- and inter-day assay precision ranged from 2.03% to 6.69% and 1.63 to 5.24 %, respectively, and intra- and inter- day assay accuracy was between from 1.86 to 6.7% and 2.85 % to 5.18%, respectively. The method was successfully applied to the protein binding studies of diclofenac in human plasma.