Aplicação de FACE (fluorophore assisted carbohydrate electrophoresis) na análise de condroitim sulfato de uso

Abstract

Chondroitin sulfate is a sulfated glycosaminoglycan composed of alternate sequences of D-glucuronic acid and N-acetyl-D-galactosamine. This polysaccharide is widely recommended for treatment of osteoarthritis in Brazil, in association with other drugs. The objective of the present study was to evaluate the quality of chondroitim sulfate raw material used for pharmaceutical recipes. A polyacrylamide gel electrophoresis method for carbohydrate analysis (FACE) was established, so as the production of specific enzymes able to digest chondroitin sulfate. Different unsaturated disaccharides, sulfated monosaccharides and neutral sugars derivatized with 2-aminoacridona were resolved by FACE, using two distinct buffer systems. F. heparinum chondroitinase AC, C and B were fractioned by hydrophobic interaction chromatography, and the activity of these enzymes was characterized by FACE. Chondroitinase AC demonstrated contamination by a sulfatase specific for sulfation at position 4 of hexosamine, which was inhibited by 10 mM sodium fluoride. Glucuronidase contamination with specific activity against non sulfated and 6-sulfated disaccharide was detected in chondroitinase C, and 10 mM D-saccharic acid-1,4-lactone inhibited the degradation of only non sulfated disaccharide. Chondroitin sulfate content in pharmaceutical raw materials was determined by agarose gel electrophoresis and by photometric titration. Only five samples showed content greater than 80%. Chondroitin sulfate average molecular weight in these samples varied from 16 to 26 kDa, and disaccharide analysis by FACE revealed similar proportions between 4-sulfated and 6-sulfated disaccharide. Eleven samples showed less than 20% of chondroitin sulfate, and contaminant analysis by FACE detected the presence of lactose or different polymers of glucose. We concluded that there is a large distortion between the data reported at the certificate of analysis of eleven products and the results obtained in this work. Stricter regulation of this raw material should be enforced.