HPLC method for Residual 2-Bromoethylamine Hydrobromide in Hemoglobin-based Oxygen Carriers Derived with 4-Methoxybenzenesulfonyl Chloride

Abstract

A stable high-performance liquid chromatography-UV (HPLC) method was developed for the separation and quantification of residual 2-bromoethylamine hydrobromide (BEA) in hemoglobin-based oxygen carriers (HBOCs). Residual BEA was derived by 4-Methoxybenzenesulfonyl chloride (MOBS-Cl) at 20 °C for 5 min in a pyridine solution. With this organic reaction system, MOBS-Cl hydrolysis in aqueous phase can be avoided in the process of derivatization, and glycine (Gly), a protein stabilizer, does not dissolve and extract from the organic reaction system, thus avoiding the serious interference of Gly on BEA determination. After derivatization, the derivative product was analyzed by HPLC with a mobile phase of 35% acetonitrile and 65% 0.01 mol/L ammonium acetate solution (v:v) and detected at 240 nm. The linear range was 0.41–40.98 μg/mL, with a correlation coefficient R2 of >0.9976. Excellent method reproducibility was observed by intra- and inter-day precision, with a relative standard deviation of <3%. The detection limit (S/N = 3) was found to be <0.02 μg/mL. This method can be used for routine determination of residual 2-bromoethylamine hydrobromide in hemoglobin-based oxygen carriers and other biological medicines. The proposed method also provides a new strategy for the separation and determination of other amino compounds which can dissolve in organic phase.