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 [9010] 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 micro l was analyzed on a micro-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 micro g/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 ?g/mL and 0.002 microg/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