Development and validation of a HPLC method for determination of degree of polymerization of xylo-oligosaccharides

Abstract A reliable reversed-phase HPLC method was developed for high resolution separation and high sensitivity determination of xylo-oligosaccharides (XOS) with degree of polymerization from 2 to 8. The method was carried out on a Kromasil C18 column using pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP) and UV detection at 245 nm. The effects of pH value of mobile phase, volume proportion of acetonitrile, concentration of ammonium acetate buffer and flow rate on the retention time and degree of separation of XOS derivatives were investigated. A satisfactory result was achieved in 25 min with a mobile phase of 10 mmol/L ammonium acetate buffer (pH5.5)-acetonitrile by a gradient elution at 0.8 mL/min. In addition, this method was validated by liquid chromatography–tandem mass spectroscopy (LC–MS) analysis and several uncertain compounds were identified. The proposed HPLC method is suitable for the compositional analysis and quality control of XOS.