Development of a rapid method for simultaneous separation of hyaluronic acid, chondroitin sulfate, dermatan sulfate and heparin by capillary electrophoresis

• HA, CS, DS and heparin were separated.
• A new buffer system was employed.
• A fast and steady plasma GAGs extraction method was established.

This study reports the use of diethylenetriamine as background electrolyte for the simultaneous separation of hyaluronan acid, chondroitin sulfate, dermatan sulfate and heparin. The analytes were baseline separated by using an uncoated fused silica capillary at 37 °C with a run time of 23 min. The migration order, with hyaluronan acid at first and heparin at last, was related to the sulfation degree. The effect of salt concentration on resolution and migration order was also investigated. The developed method was applied to the simultaneous determination of hyaluronan acid and chondroitin sulfate in mouse plasma. Interferences in plasma were removed by protein precipitation and glycosaminoglycans were further purified by ethanol precipitation. The method was validated over the concentration range from 50 to 600 μg/mL for hyaluronan acid and 500 to 6000 μg/mL for chondroitin sulfate in mouse plasma. Results from assay validations showed that the method was selective and robust.