A rapid and sensitive modified HPLC method for determination of diclofenac in human plasma and its application in pharmacokinetic studies

Abstract

A sensitive, accurate and rapid reverse phase HPLC method was developed to quantitate plasma levels of diclofenac sodium in human plasma. The drug, internal standard (naproxene) and orthophosphoric acid 1 M, were added to plasma samples and vortexed for 20 sec. A mixture of hexane /isopropyl alcohol ( 90:10 ) was then added and vortexed for 2 min. Samples were centrifuged and the supernatant layer was separated, evaporated to dryness under nitrogen gas stream, reconstituted in mobile phase and an aliquot of 50 μl was analyzed on a μ-bondapack C₁₈ (150 × 4.6mm) column, with 45% acetonitrile in deionised water and 0.5% orthophosphoric acid, (pH = 3.5) at 276 nm.  The standard curve covering 0.005 - 4 μg/ml concentration range, was linear, relative errors were within 0.13 to 16 % and the CV % ranged from 1.24 to 8.75. The limits of quantitation and detection of the method were 0.005 μg/mL and 0.002μg/mL, respectively. The method was suitable for bioavailability and pharmacokinetic studies of diclofenac in humans and applied in a randomized, two-way cross over bioequivalance study of two different diclofenac sodium preparations with twelve subjects and with a one-week washout period