Development and validation of an improved 3-methyl-2-benzothiazolinone hydrazone method for quantitative determination of reducing sugar ends in chitooligosaccharides.

ABSTRACT

An accurate and sensitive analytical method for detecting and quantifying reducing sugar ends (RSE) in chitooligosaccharides (COSs) is the key quality parameter for evaluating their structure-function relationship and potential applications. In this work, we develop and validate a novel colorimetric assay with high accuracy and precision for determining RSE content using 3-methyl-2-benzothiazolinone hydrazone (MBTH). Under optimal conditions, the stoichiometry is verified using mono-, di-, and tri- glucosamine hydrochlorides, and the dilution ratio does not interfere with the RSE content measured at 590 nm. The regression equation of glucosamine reveal a good linear relationship (R2 = 0.9999). The detection limit, quantification limit, mean relative standard deviation (RSD), and recovery are 2.28 µM, 9.11 µM, 1.90%, and 98.0%, respectively. The newly developed method is potentially useful for monitoring COS hydrolysis, number average molecular weight, and chitosanase activity.