Development and Validation of a Reversed-Phase Liquid Chromatographic Method for Analysis of Rofecoxib in Human Plasma

Abstract

High performance liquid chromatographic method was developed
for the estimation of rofecoxib in human plasma. Varying
amounts of rofecoxib (5 to 400 ng) and fixed quantity of nimesulide
(500 ng) were added to blank plasma (0.5 mL) and extracted with
dichloromethane. The mixture was centrifuged, organic layer was
separated, evaporated to dryness under vacuum and reconstituted
with 0.1 mL of methanol. Twenty microlitres of this solution was
injected into reverse phase C-18 column using a mobile phase
consisting of methanol and water (consisting of 0.04% triethylamine
and 0.15% glacial acetic acid) in the ratio of 50: 50 v/v and the
eluents were monitored at 272 nm. The method was validated for
its linearity, precision and accuracy. The calibration curve was linear
in the range of 5 to 400 ng/0.5 mL of plasma. The intra- and
inter-day variation was found to be less than 2.3% indicating that
the method is highly precise. The mean recovery of rofecoxib from
plasma samples containing 50 or 100 ng/mL of the drug was
95.66%. There was no interference of either the drug metabolites
or plasma constituents with the proposed HPLC method for the
estimation of rofecoxib in human plasma. Due to its simplicity,
sensitivity, high precision and accuracy, the proposed HPLC method
may be used for biopharmaceutical and pharmacokinetic evaluation
of rofecoxib formulations.