Determination of process-related impurities in N-acetylglucosamine prepared by chemical and enzymatic methods: structural elucidation and quantification

Abstract

beta-N-acetylglucosamine (beta-AG) is a monosaccharide distributed widely in living organisms with various pivotal roles. The presence of particulates and impurities can affect the safety and efficacy of a product for its intended duration of use. Thus, the current study was carried out to identify and quantify the potentially-harmful process related impurities; namely alpha-N,6-diacetylglucosamine (alpha-DAG) and alpha-N-acetylglucosamine (alpha-AG), derived from the chemical and enzymatic synthesis of beta-AG. The impurities were characterized using a high resolution mass spectrometry, a nuclear magnetic resonance spectroscopy, and liquid chromatography-tandem mass spectrometry (LC/MS/MS). The developed method showed a good linearity (R (2) a parts per thousand yen 0.998), satisfactory precision (a parts per thousand currency sign6.1 % relative standard deviation), intra- and inter-day accuracy (88.20-97.50 %), extraction recovery (89.30-110.50 %), matrix effect (89.70-105.20 %), and stability (92.70-101.60 %). The method was successfully applied to determine the level of alpha-DAG that was 3.04 and 0.07 % of the total beta-AG, following chemical and enzymatic methods, respectively. It can be concluded that the enzymatic rather than the chemical method is more efficient for the synthesis of beta-AG. Characterization of impurities heeds the signal for acquiring and evaluating data that establishes biological safety.