Abstrakt

Fully validated diclofenac HPLC assay

Rajaa F.Hussein, Muhammad M.Hammami

Asimple, sensitive high-performance liquid chromatography (HPLC) assay for diclofenac measurement in human plasma was fully validated and diclofenac stability was studied. After one-step extraction of 1 ml plasma with 5.0 ml of tert. butyl methyl ether and reconstitution in mobile phase, naproxen (internal standard, IS) and diclofenac eluted at 3.9 and 8.3 minutes, respectively, on a Nova-Pak C18 4-m cartridge at room temperature (RT), and were detected using a 996 photodiode array detector set at 276 nm. The mobile phase, 0.2% glacial acetic acid (pH = 3.0) and acetonitrile (51:49, v/v),was delivered at 2.0ml/min. Calibration curveswere linear in the range 0.02-1.92 g/ml, and intra- and inter- run coefficients of variationwere  5.3%and  10.1%, respectively. Extraction recovery and intra- and interrun biaswere = 86%,  12%, and 10%, respectively.Diclofenacwas stable in plasma for 5 hours at RT ( 97%), 7 weeks at -200C (= 93%), and after 3 cycles of freeze at -200C and thaw at RT ( 91 %). In extracted samples, diclofenac was stable for 16 hours at RT (97%) and 48 hours at -200C ( 99 %). Stock solution of diclofenac (1 mg inmethanol) was stable for 24 hours at RT (104%) and 7 weeks at -200C (91%). The assaywas successfully used to determine plasma diclofenac level after the ingestion of a therapeutic dose. The data indicate that the described assay is suitable for therapeutic drug monitoring and bioequivalence studies in humans.