Enzymatic methods have been developed for the determination of small quantities of isomeric chondroitin sulfates (ChS-A, ChS-B, and ChS-C) in mixtures with other mucopolysaccharides. These methods utilize chondroitinase-ABC, chondroitinase-AC, chondro-4-sulfatase, and chondro-6-sulfatase to catalyze reactions that produce specific disaccharide fragments. Radioactivity measurements allow precise and rapid determination of these sulfates, even in as little as 4 ml of normal urine. The methods are also applicable to the analysis of chondroitin sulfates in the presence of hyaluronic acid, chondroitin, heparin sulfate, keratosulfate, and heparin. The three isomeric chondroitin sulfates differ in the position of sulfate ester groups in the N-acetylgalactosamine residue. ChS-A and ChS-C both contain D-glucuronic acid, N-acetylgalactosamine, and sulfate residues in equimolar quantities, but differ in sulfate position (position 4 in ChS-A and position 6 in ChS-C). ChS-B contains an N-acetylgalactosamine 4-sulfate residue but is distinguished by its hexuronic acid component (1-iduronic acid). Due to their structural similarity, few reliable methods exist for determining individual isomers in mixtures. The methods described here use enzymatic reactions to separate and quantify the isomers. For example, chondroitinase-ABC and chondroitinase-AC produce specific disaccharide fragments, which are then analyzed using chromatography or colorimetric methods. The disaccharides are separated by paper chromatography, and their absorbance is measured to determine their concentrations. The methods also involve the use of sulfatases to produce inorganic sulfate or ΔDi-OS, which can be quantified using the Morgan-Elson reaction. The methods allow for the determination of ChS-A, ChS-B, and ChS-C in as little as 3 μg in mixtures and in urine samples. The procedures are sensitive, specific, and can be applied to labeled materials, enabling the determination of small quantities of chondroitin sulfates by measuring radioactivity alone. The methods have been validated using synthetic mixtures and have shown good linearity and recovery rates. They are also applicable to the analysis of chondroitin sulfates in tissues and urine, with the ability to detect as little as 0.005 μmol of uronic acid in urine. The methods are efficient, require minimal manipulation, and provide accurate results for the quantification of isomeric chondroitin sulfates.Enzymatic methods have been developed for the determination of small quantities of isomeric chondroitin sulfates (ChS-A, ChS-B, and ChS-C) in mixtures with other mucopolysaccharides. These methods utilize chondroitinase-ABC, chondroitinase-AC, chondro-4-sulfatase, and chondro-6-sulfatase to catalyze reactions that produce specific disaccharide fragments. Radioactivity measurements allow precise and rapid determination of these sulfates, even in as little as 4 ml of normal urine. The methods are also applicable to the analysis of chondroitin sulfates in the presence of hyaluronic acid, chondroitin, heparin sulfate, keratosulfate, and heparin. The three isomeric chondroitin sulfates differ in the position of sulfate ester groups in the N-acetylgalactosamine residue. ChS-A and ChS-C both contain D-glucuronic acid, N-acetylgalactosamine, and sulfate residues in equimolar quantities, but differ in sulfate position (position 4 in ChS-A and position 6 in ChS-C). ChS-B contains an N-acetylgalactosamine 4-sulfate residue but is distinguished by its hexuronic acid component (1-iduronic acid). Due to their structural similarity, few reliable methods exist for determining individual isomers in mixtures. The methods described here use enzymatic reactions to separate and quantify the isomers. For example, chondroitinase-ABC and chondroitinase-AC produce specific disaccharide fragments, which are then analyzed using chromatography or colorimetric methods. The disaccharides are separated by paper chromatography, and their absorbance is measured to determine their concentrations. The methods also involve the use of sulfatases to produce inorganic sulfate or ΔDi-OS, which can be quantified using the Morgan-Elson reaction. The methods allow for the determination of ChS-A, ChS-B, and ChS-C in as little as 3 μg in mixtures and in urine samples. The procedures are sensitive, specific, and can be applied to labeled materials, enabling the determination of small quantities of chondroitin sulfates by measuring radioactivity alone. The methods have been validated using synthetic mixtures and have shown good linearity and recovery rates. They are also applicable to the analysis of chondroitin sulfates in tissues and urine, with the ability to detect as little as 0.005 μmol of uronic acid in urine. The methods are efficient, require minimal manipulation, and provide accurate results for the quantification of isomeric chondroitin sulfates.