HPLC with charged aerosol detector (CAD) as a quality control platform for analysis of carbohydrate polymers

Abstract

Abstract Objective QC analysis of carbohydrates has been historically cumbersome due to lengthy and laborious derivatization techniques and the requirement of complimentary instrumentation. HILIC-CAD has emerged as an effective platform for direct monosaccharide composition analysis of complex carbohydrates without derivatization. Although, several neutral sugars have been separated and detected using HILIC-CAD, there has not been any report on acidic and amino sugar analysis using this method. In this study, we developed a gradient method for simultaneous analysis of acidic, amino and select neutral monosaccharides. As an application of the HILIC-CAD method, we performed composition analysis of commercially purchased hyaluronic acid products. Additionally, since CAD is suitable for SEC experiments, we tested the homogeneity of hyaluronic acids using a SEC-CAD method. Results We separated common uronic acids (GlcA, GalA, LIdoA and Neu5Ac), amino sugars (GlcN, GalN and GlcNAc) and select neutral sugars (LRha, LFuc, Man and Gal) using a gradient HILIC-CAD method. The optimized gradient method demonstrated good linearity (R2 > 0.99), precision (RSD < 8%), LOD (< 85 ng/mL) and LOQ (< 280 ng/mL). HILIC-CAD analysis of commercially purchased hyaluronic acid products indicated that samples were composed of GlcNAc and GlcA. Additionally, SEC-CAD chromatograms indicated the heterogeneous nature of the samples.

Keywords

• HILIC
• HPSEC
• QC of carbohydrates
• Hyaluronic acid
• Uronic acids
• Amino sugars