Molar substitution of clinically used acetylated starch by size exclusion chromatography using multiple detection

An aqueous size exclusion chromatography - high performance chromatography (SEC-HPLC) system for the analysis of acetyl starch (ACS) is outlined, using a combined dual detection by an UV-VIS-detector and a refractive index detector (RI). This HPLC system enables the simultaneous determination of the molar substitution (MS) and of the concentration of acetyl starch - a new starch ester suited for plasma volume replacement - in unknown biological samples. An adequate sample preparation prevents interferences of proteins and of other substances containing ester groups. The calibration of the system was performed with six well defined ACS-standards ((Mw = 200,000 Da, MS 0.35-0.83), i. e. abbreviated 200/0.35) and with a sample of partially degraded non substituted amylopectin (Mw = 300 000 Da, MS = 0). The method was validated for a concentration range of 0.5 to 5.0 mg/mL ACS. The recovery of MS of ACS from blood plasma was 99%. The investigations with different fractions of a-amylase degraded ACS 200/0.5 demonstrated that the determination of MS is independent of the molar mass distribution of the ACS preparation used. The evaluated method was applied to blood plasma samples of male Volunteers which received intravenous infusion of solutions containing ACS 200/0.5 or ACS 200/0.7. In contrast to the situation following hydroxyethyl starch (HES) no rise of MS has been observed after the infusion of ACS 200/0.5 and ACS 200/0.7. During the degradation of ACS 200/0.5 molecules in the body no alteration of the molar substitution occurs. A slight decrease in the molar substitution was detected after infusion of high-substituted ACS 200/0.7. These results indicates a different metabolic pathway of ACS in contrast to HES. This might cause a more rapid complete degradation of ACS in the body.