Application of 2-aminopyridine fluorescence labeling in the analysis of invivo and in vitro metabolism of dextran sulfate sodium by size-exclusion high-performance liquid chromatography

The present study describes a size-exclusion high-performance liquid chroma tographic method for the separation and quantification of sulfated polysacc harides, such as dextran sulfate sodium (DSS). Pyridylamination of DSS was achieved without difficulty using 2-aminopyridine as a fluorometric label. In addition, 0.1-0.2 M phosphate buffer (pH 3.0) was found to be the mobile phase which produced the best separation. In vitro enzymatic degradation o f the pyridylamino-DSS (PA-DSS5000, M-r 5000) using alpha -amylase and the in vivo metabolism in the rat feces after oral administration of PA-DSS5000 were then evaluated. Two small peaks of approximately M-r 380 and 600 appe ared after co-incubation with alpha -amylase, indicating PA-DSS5000 may be considerably depolymerized. In vivo, however, PA-DSS5000 excreted in the fe ces was mainly of PA-DSS5000 polymer. No peaks of less than M, 5000 were no t clearly detectable in the feces because of background fluorescence attrib utable to gut lumen contents. This method of fluorometric analysis allows f airly selective detection of sulfated polysaccharides in biological materia ls. (C) 2001 Elsevier Science B.V. All rights reserved.