LIQUID-CHROMATOGRAPHIC ANALYSIS OF 9-AMINOCAMPTOTHECIN IN PLASMA MONITORED BY FLUORESCENCE INDUCED UPON POSTCOLUMN ACIDIFICATION

Through preclinical studies by the Developmental Therapeutics Program of the National Cancer Institute with 9-amino-20(S)-camptothecin (AC), this new investigational anticancer agent will soon enter phase I clinical trials in cancer patients. During initial attempts to monitor the drug in biological fluids, it became evident that the presence of an amino group on the camptothecin A-ring suppressed the intense native fluorescence characteristic of the unsubstituted compound. However, subsequent spectrofluorometric studies revealed that the fluorescence of AC was highly pH-dependent in a manner not typically exhibited by aromatic amines. The uncharged species that exists in neutral and weakly acidic solution is nonfluorescent. Protonation of the C-9 amino group proceeds with the development of fluorescence, the intensity of which is optimum in moderately acidic solution of apparent pH 1.7-2.3. Under more strongly acidic conditions, fluorescent intensity again diminishes due to further protonation at the quinoline nitrogen atom. Therefore, postcolumn acidification prior to fluorescence detection provided a convenient and sensitive method to monitor the elution of AC during liquid chromatography. Following protein precipitation induced by mixing with a solution of the internal standard, camptothecin, in methanol chilled to -70-degrees-C, plasma samples were separated on a 5 mum Ultrasphere ODS column (4.6 mm x 25 cm) using an isocratic mobile phase composed of acetonitrile-methanol-0.1 M ammonium acetate buffer, pH 5.5 (23:10:67, v/v/v) at a flow rate of 1.0 ml/min. Prior to detection, the column effluent was mixed inline with 0.3 M aqueous trifluoroacetic acid at a flow rate of 0.3 ml/min and ambient temperature. Fluorescence was then monitored using an excitation wavelength of 352 nm and a 418 nm emission cutoff filter. Typical retention times of the drug and internal standard were 7 and 12 min, respectively. Employing sample volumes of 50 mul, the lowest plasma concentration of AC included in the standard curve, 13.3 nM (5.0 ng/ml) was quantified with a 7.63% coefficient of variation (n = 10).