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

Abstract

A sensitive and selective bioanalytical liquid chromatographic method for diclofenac is described. The drug was detected as a fluorescent 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-μl volume of the supernatant was injected onto a Nucleosil C 18 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 μl. Chromatograms of human and rat plasma containing diclofenac are shown.