Determination of Reductant and Alkylating Agent in the Alkylated Human Immunoglobulin by High Performance Liquid Chromatography

Abstract

High performance liquid chromatography (HPLC) by use of a reverse phase column (TSK LS410K) was applied to determine the residual dithiothreitol, iodoacetamide and oxidized dithiothreitol in the alkylated human immunoglobulin (AHIG). After extraction by ethyl acetate, the above compounds were separated by HPLC with MeOH-H₂O (60 : 40 v/v) as solvent. Determination was completed within 5 min with high reproducibility. Less than 0.5 ppm for each residual substance could be quantitated. The method was quite suitable for the routine analysis of the residual contaminants in AHIG.