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

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 (R 2 > 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.