PHOSPHORIMETRIC DETECTION IN HPLC VIA TRIVALENT LANTHANIDES - HIGH-SENSITIVITY TIME-RESOLVED LUMINESCENCE DETECTION OF TETRACYCLINES USING EU3+ IN A MICELLAR POST COLUMN REAGENT

The detection of tetracyclines in HPLC by sensitized Europium phosphorescence has been reinvestigated using a dedicated LC detector with time-resolved luminescence capability. A micellar Eu3+ post column reagent was developed which contained 1% Triton X-100 and 200uM tri-n-octyl phosphine oxide buffered at pH 10.5. This reagent provided optimized time-resolved detection and was found to be compatible with a several mobile phases used in reversed phase chromatography of tetracyclines. The sensitivity and quantitative linearity of the technique was determined using standard fluorometric and time-resolved modes of detection with both grating and filter emission monochromation. Peak areas were linear with concentration from 2 ug on column to the limit of detection. Subnanogram limits of detection were obtained for both tetracycline and oxytetracycline. The relative sensitivity of the technique for compounds tested was oxytetracycline = tetracycline > doxycycline > chlortetracycline > > minocycline. The high selectivity of this method promises to be useful in simplifying chromatograms from complex matrices such as physiological fluids and may also provide positive evidence for the presence of tetracyclines or structural analogs in a number of sample types.