Indirect Detection of Glycosaminoglycans by Capillary Electrophoresis using Anionic Dyes

Glycosaminoglycans (GAGs), a class of polysaccharides with an average molar mass around 15,000 g/mole, are composed of long linear chains of sulfonated disaccharides, making them highly negatively charged. Many have medical uses such as heparin which is widely used as a clinical anticoagulant. Other GAGs include chondroitin sulfate and dermatan sulfate which are commonly used as commercial supplements. In 2007, a potentially fatal contaminant known as oversulfated chondroitin sulfate (OSCS) was identified in heparin samples and as a result, NMR as well as capillary electrophoresis (CE) approaches were developed for quality control. After CE separation, direct UV detection at 205 nm is straightforward for GAGs however use of a dye could provide more selectivity.  Cationic dyes that ion-pair with heparin were found to be problematic, causing capillary plugging.

            Although CE of low molecular weight surfactants with indirect detection is well established, such application has apparently not been applied to high molar mass anionic biopolymers. The anionic dye Fast Green FCF has been evaluated as a CE indirect detection reagent for GAGs.  The CE separation of heparin and OSCS can be separated on a 50 micron x 72 cm capillary (effective length 47 cm) coated with 0.1% Carbowax 20 M (to reduce baseline noise and to improve sensitivity) in about 15 min with detection at 625 nm using a 20 mM glutamic acid buffer with 1.75 mM Fast Green FCF. Linearity of response between 1 and 3 mg/mL heparin has been achieved.  The indirect displacement method showed a limit of detection for heparin of less than 1 mg/ml using potassium bromide as an internal standard.

            Other anionic dyes are under consideration that might offer a closer match in mobility with heparin, improving detectability. The transfer ratio of heparin will be calculated when using the anionic dyes indigo trisulfate (ITS) and bright red 80 (BR) buffered in 20 mM glutamic acid and compared to that of the fast green FCF. CE of various mixtures of OSCS, heparin, and other GAGs will be performed with indirect detection using each dye.