Quantitative determination of aceclofenac and its three major metabolites in rat plasma by HPLC ‐ MS / MS

We developed a method for the simultaneous quantification of aceclofenac and its three major metabolites in rat plasma. After protein precipitation with acetonitrile including flufenamic acid as an internal standard ( IS ), aceclofenac, diclofenac, 4′‐hydroxyaceclofenac, 4′‐hydroxydiclofenac, and the IS were chromatographed on a reverse‐phase C 18 analytical column. The isocratic mobile phase of acetonitrile/0.1% formic acid (aq; 9:1 [v/v]) was eluted at 0.3 mL/min. Quantification was performed on a triple‐quadrupole mass spectrometer using electrospray ionization, and the ion transitions were monitored in selective reaction‐monitoring mode. The coefficient of variation in the assay precision was less than 8%, and the accuracy was 92–103%. This method was successfully used to measure the concentrations of aceclofenac and its three major metabolites in rat plasma following the oral administration of a single 20 mg/kg oral dose of aceclofenac.