Abstract
A high-performance size-exclusion chromatography (HPSEC), using TSKgel G3000SW column, for the quantitative analysis of FITC-labeled dextran and pullulan in the biological specimen was developed. FITC-labeled dextrans of various molecular weights (Mw=4, 400∼485, 000) were purchased from Sigma Co., whereas pullulans (Mw=5, 300∼611, 000) were labeled with FITC by the method of de Belder and Granath. These derivatives showed a very low degree of substitution at which the labeling of the parent polysaccharides with FITC did not affect their chromatographic behavior. Linear calibration curves were obtained at amounts down to 5 ng when the derivatives were monitored fluorometrically. The FITC-labeled polysaccharides were susceptible to the enzyme catalyzed hydrolysis. A total peak area of the degraded fractions with smaller molecular weights was identical to that obtained with the parent compound. These results indicate that labeling the polysaccharides with FITC and monitoring fluorometrically by HPSEC is a promising method for the pharmacokinetic investigations on the macromolecular conjugates. The applicability of the method was demonstrated by measuring the hepatic level-time profile and biodegradation of a FITC-labeled dextran following injection to mice.
