Quantitative analysis of hyaluronan in vitreous humor using capillary electrophoresis

Capillary electrophoresis was used for the separation and quantitative analysis of the glycosaminoglycan hyaluronan in human and bovine vitreous with detection by UV absorbance at 200 nm. Calibration was carried out using standards made up from known concentrations of hyaluronan of umbilical cord origin. The purity of the standard was examined by 1H NMR (nuclear magnetic resonance). Concentrations as low as 25 micrograms/mL could be detected by capillary electrophoresis. Confirmation that the signal was due to hyaluronan was obtained by depolymerization of the native mucopolysaccharide by hyaluronidase. This resulted in loss of the hyaluronan peak and appearance of several new peaks corresponding to the oligomeric fragments which had shorter migration times. Capillary electrophoresis is a reproducible and sensitive technique for the quantification and characterisation of hyaluronan in vitreous samples.