Crocheted ETFE-reactor for on-line post-column photoderivatization of diclofenac in high-performance liquid chromatography.
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.
Kuhlmann
O
O
Martin-Luther-University Halle-Wittenberg, Department of Biochemistry/Biotechnology, Halle, Germany.
Krauss
G J
GJ
D001082:Aqueous Humor / Q000737:chemistry; D001331:Automation / Q000379:methods; D002227:Carbazoles / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004008:Diclofenac / Q000032:analysis* / Q000097:blood / Q000528:radiation effects; D005456:Fluorescent Dyes / Q000032:analysis*; D005466:Fluorocarbons; D006801:Humans; D011138:Polytetrafluoroethylene / Q000031:analogs & derivatives; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence; D014466:Ultraviolet Rays
D002227:Carbazoles; D005456:Fluorescent Dyes; D005466:Fluorocarbons; C071095:carbazole-1-acetic acid; D004008:Diclofenac; C040378:poly(ethylene-tetrafluoroethylene); D011138:Polytetrafluoroethylene; C015531:tetrafluoroethylene