Fast screening of glycosaminoglycan disaccharides by fluorophore-assisted carbohydrate electrophoresis (FACE): applications to biologic samples and pharmaceutical formulations.

Hyaluronan (HA), chondroitin sulfate (CS), and heparan sulfate (HS) are glycosaminoglycans (GAGs) with a great importance in biological processes as they participate in functional cell properties, such as migration, adhesion, and proliferation. A perturbation of the quantity and/or the sulfation of GAGs is often associated with pathological conditions. In this chapter, we present valuable and validated protocols for the analysis of HA-, CS-, and HS-derived disaccharides after derivatization with 2-aminoacridone and by using the fluorophore-assisted carbohydrate electrophoresis (FACE). FACE is a well-known technique and a reliable tool for a fast screening of GAGs, as it is possible to analyze 16 samples at the same time with one electrophoretic apparatus. The protocols for the gel preparation are based on the variations of the acrylamide/bisacrylamide and buffer concentrations. Different approaches for the extraction and purification of the disaccharides of various biologic samples and pharmaceutical preparations are also stressed.

FACE analysis as a fast and reliable methodology to monitor the sulfation and total amount of chondroitin sulfate in biological samples of clinical importance.

Glycosaminoglycans (GAGs) due to their hydrophilic character and high anionic charge densities play important roles in various (patho)physiological processes. The identification and quantification of GAGs in biological samples and tissues could be useful prognostic and diagnostic tools in pathological conditions. Despite the noteworthy progress in the development of sensitive and accurate methodologies for the determination of GAGs, there is a significant lack in methodologies regarding sample preparation and reliable fast analysis methods enabling the simultaneous analysis of several biological samples. In this report, developed protocols for the isolation of GAGs in biological samples were applied to analyze various sulfated chondroitin sulfate- and hyaluronan-derived disaccharides using fluorophore-assisted carbohydrate electrophoresis (FACE). Applications to biologic samples of clinical importance include blood serum, lens capsule tissue and urine. The sample preparation protocol followed by FACE analysis allows quantification with an optimal linearity over the concentration range 1.0-220.0 µg/mL, affording a limit of quantitation of 50 ng of disaccharides. Validation of FACE results was performed by capillary electrophoresis and high performance liquid chromatography techniques.