Development and Validation of HPLC-DAD Method with Pre-Column PMP Derivatization for Monomeric Profile Analysis of...

The instrumental analysis of complex mixtures of sugars often requires derivatization to enhance the method's selectivity and sensitivity. 1-Phenyl-3-methyl-5-pyrazolone (PMP) is a common sugar derivatization agent used in high-performance liquid chromatography (HPLC). Although many C18 column applications for PMP-sugar derivative analysis have been developed, their transferability is not straightforward due to variations in column chemistry and preparation technology. The aim of this study was to develop and validate an application for Zorbax Extend C18 columns for the analysis of 8 neutral and 2 acidic sugars commonly found in plant polysaccharides. The method was further compared to well-established alditol acetates and m-hydroxydiphenyl methods and employed for sugar profiling of selected agro-industrial wastes. The most influential separation factors were the mobile-phase pH and acetonitrile content, optimized at 8.0 and a 12-17% gradient, respectively. The method showed excellent linearity, repeatability and intermediate precision. High sensitivity was achieved, especially for neutral sugars, with an accuracy error range of 5-10% relative standard deviation. The sugar profiling results were highly correlated to the reference method for neutral sugars. The HPLC method was highly applicable for the evaluation of polysaccharides in selected wastes and showed advantages in terms of simplicity, accuracy in acidic sugar determination and suitability for their simultaneous analysis with neutral sugars.