Determination of dermatan sulfate and chondroitin sulfate as related substances in heparin by capillary electrophoresis

Abstract

Capillary electrophoresis (CE) was applied to the quantitation of dermatan sulfate (DS) and chondroitin sulfate (CS) as related substances in sodium heparin. The method is based on the selective digestion of either CS and DS contained in the main drug heparin, by using chondroitinase ABC (specific for both DS and CS) and chondroitinase AC (specific for only CS). The unsaturated disaccharides released after exhaustive digestion, can be separated by CE using a 110 mM phosphate buffer, pH 3.5 as the background electrolyte in a fused silica capillary (64.5 cm × 50 μm i.d.) at 40 °C and −30 kV. Since the level of each disaccharide released upon enzymatic digestion corresponds to its content in the native glycosaminoglycan, the amount of CS and DS was determined by proportion with the released disaccharides. In particular, ΔUA → GalNAc-4S Na 2 and ΔUA → GalNAc-6S Na 2 were selected for quantitation of CS and DS because of their significant response and short migration time (less than 7 min).The method was validated for linearity, accuracy, precision and it showed to be able in detecting selectively, DS and CS at impurity level (LOD 0.01%, w/w). The proposed CE approach was finally applied to real samples. The results obtained were found in excellent correlation with those achieved by the analysis of the same samples using the official USP method based on high performance anion exchange chromatography (HPAEC) with pulsed amperometric detector.