Simultaneous Identification and Quantification of Dextran 20 and Sucrose in Lyophilized Thrombin Powder by Size Exclusion Chromatography with ELSD

Abstract

A rapid and accurate size exclusion chromatography method for the simultaneous identification and quantification of dextran 20 and sucrose with optical activity in the lyophilized thrombin powder was developed and validated. The assay was conducted on a Hitachi model D-2000 Elite HPLC system with a TOSOH TSKgel G3000 PWxl column (30 cm × 7.8 mm, 7 μm particle size) and an Alltech 3300 evaporative light scattering detector (ELSD). The mobile phase was acetonitrile–water (46:54, v/v) mixture delivered at a flow rate of 0.7 mL min−1 at 25 °C. The ELSD was operated at a nebulizer-gas flow rate of 2.0 L min−1 and drift tube temperature of 90 °C, and the gain was set at one. Afterward, method validation system for the size exclusion chromatography analysis was developed. The linear range was 0.1–1.6 and 0.1–1.0 g L−1 for dextran 20 and sucrose, respectively, and the detection limits were <0.02 g L−1 for dextran 20 and <0.015 g L−1 for sucrose. Inter-day and intra-day variabilities showed that RSD ranged from 0.27 to 4.20%. Recovery validation showed that average recovery was between 96.00 and 103.98%. The developed analytical procedure was successfully applied to determine the contents of dextran 20 and sucrose in the lyophilized thrombin powder.