A sensitive and selective bioanalytical method for
diclofenac using reversed-phase HPLC and fluorescence detection is described.
Diclofenac was detected as its fluorescent derivative after on-line post-column photoderivatization. Irradiation with UV light of
diclofenac in aqueous solutions leads to the sequential loss of both
chlorine substituents and ring closure. The major product,
carbazole-1-acetic acid, was detected by a fluorescence detector using an excitation wavelength of 286 nm and an emission wavelength of 360 nm. The self-made reactor was a crocheted
ethylene and
tetrafluoroethylene (ETFE, named
TEFZEL) capillary, 20 m in length,
wound directly around a 253.7 nm UV lamp. The capillary was crocheted in order to overcome peak widening. Chromatographic separation was achieved by using a Regis SPS 100 RP-8 column (5 microm; 150 mm x 4.6 mm i.d.) and a LiChrospher 100 RP-18 (5 microm) guard column from E. Merck. The detection limit was 1 ng ml(-1) at an injection volume of 20 microl. Daily relative standard deviations (RSD) were 5.5%, (73 ng
diclofenac/ml, n = 9), and 5.1% (405 ng
diclofenac/ml, n = 6), respectively. Chromatograms of human aqueous humor and human serum containing
diclofenac, and figures showing the time dependent increase/decrease of the photoderivatization product, are shown.