An improved HPAEC-PAD method for the determination of D-glucuronic acid and 4-O-methyl-D-glucuronic acid from polymeric and oligomeric xylan

Glucuronic acid (GlcA) is an abundant substituent in hardwood xylan, and it is often found in its methylated form as methyl glucuronic acid (MeGlcA). GlcA and MeGlcA are sugar acids, bound to the xylose backbone at position O-2, and their presence can affect the digestibility of the polymer. Currently, detection of released GlcA or MeGlcA from synthetic substrates such as pNP-glucuronic acid can be achieved with colorimetric assays, whereas analysis from natural substrates such as xylan is more complicated. High performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) with an isocratic elution profile has been proposed for quantification of uronic acids in acid-hydrolysed wood samples. However, achieving sufficient separation for comprehensive analysis of hardwood-related xylan components, particularly MeGlcA remains challenging with this methodology. This study offers modified protocols for improved separation by introducing gradient elution profiles to effectively separate hydrolysed hardwood-related compounds, including MeGlcA, and GlcA within a single analytical run. The method showed excellent reproducibility and a standard curve of MeGlcA assured first order linearity in a wide range of concentrations, making the method excellent for quantification.