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A liquid chromatographic method of tetracycline and its major degradation products on a C8-reversed phase column with acidic mobile phase and fluorescence detection is described. The quantification limit, measured as the amount of sample that gave a signal ten times the peak-to-peak noise of the baseline, was: 0.25 ng for tetracycline (TC) and epitetracycline (ETC), 25 ng for and 4-epianhydrotetracycline (EATC) and 50 ng for anhydrotetracycline (ATC) of injected standard. By means of this liquid chromatography (LC) assay TC, ETC, EATC and ATC as main degradation products of tetracycline, can be separated and determined with good sensitivity and specificity within 15 min.

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The carbazole ring is the basic structure present in the fluorescence derivatization reagents 9-chlorocarbonylcarbazole and 9-carbazolylacetic acid. The fluorescence behaviour of these carbazole derivatives was studied in solvents with different polarities (cyclohexane, ethanol, acetonitrile, water) and at different pH values (4.5 and 8.8). The influence of the low polarity environment afforded by 2-hydroxypropyl-beta-cyclodextrin (HPbeta-CD) is also described. The behaviour of the fluorescent reagents is compared to the model molecules carbazole and 9-methylcarbazole. For all derivatives studied, a bathochromic shift in the fluorescence emission maxima was observed when the solvent polarity was increased. A bathochromic shift was observed in dioxane solutions, which can be ascribed to the peculiar behaviour of this solvent. The changes in the fluorescence intensity in the case of 9-carbazolylacetic acid can be related to the ionization of the carboxylic acid group. Inclusion into the cavity of HPbeta-CD allows emission spectra to be obtained close to those obtained in ethanolic solutions with a remarkable enhancement in the fluorescence intensity, depending on the chemical structure of the carbazole derivative included.

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