Determination of fluorescent trypanocidal diamidines by quantitative thin-layer chromatography

The fluorescent trypanocidal diamidines 2-(4-amidinophenyl)indole-6-carboxamidine dihydrochloride (I, DAPI), 2-(4-amidinophenyl)benzo[b]thiophene-6-carboxamidine dihydrochloride (II) and 2-(4-amidinophenyl)-1-benzofurane-5-carboxamidine dihydrochloride (III) were determined in plasma, urine, faeces and tissues of experimental animals using quantitative thin-layer chromatography. Samples were extracted with n-octanol after addition of sodium hydroxide and subsequently re-extracted into 0.1 M hydrochloric acid. Chromatography was performed on silica gel plates under nitrogen with n-butanol saturated with 2 M hydrochloric acid. Quantitation was performed by measuring native fluorescence using a fluorodensitometer. The respective diamidines were used as internal standards for each other to ensure precision (coefficient of variation less than 7%) and accuracy of the assay. Calibration curves were linear up to 150 ng/ml of sample solution with detection limits of 10 ng/ml of sample solution for I and III and 50 ng/ml for II. The described method has been successfully used for pharmacokinetic studies in experimental animals.