Bioanalysis of diclofenac as its fluorescent carbazole acetic acid derivative by a post-column photoderivatization high-performance liquid chromatographic method

A sensitive and selective bioanalytical liquid chromatographic method for diclofenac is described. The drug was detected as a flourescent derivative, which was demonstrated by 1H NMR and mass spectrometric studies to be carbazole acetic acid. Diclofenac was derivatized by UV irradiation of the substance performed as a post-column photoreaction. The reactor was a PTFE capillary wound around a 254-nm UV lamp. Diclofenac was isolated from the plasma samples by precipitation of the proteins with acetonitrile. A 50-microliters volume of the supernatant was injected onto a Nucleosil C18 column. The mobile phase was 32% acetonitrile in pH 6.6 buffer. Carbazole acetic acid was detected by a fluorescence detector using an excitation wavelength of 288 nm and an emission wavelength of 360 nm. The recovery was 92%, the standard curve was linear in the range 10-5500 ng diclofenac per ml plasma, and the relative standard deviation at 10 and 5000 ng of diclofenac per ml plasma was 9.0% and 3.3%, respectively. The limit of detection was 6 ng/ml at an injection volume of 50 microliters. Chromatograms of human and rat plasma containing diclofenac are shown.