Determination of the chondroitin sulfate disaccharides in dog and horse plasma by HPLC using chondroitinase digestion,precolumn derivatization,and fluorescence detection

A sensitive and selective HPLC method for the determination of the disaccharides of chondroitin sulfate in horse and dog plasma was validated. Chondroitin sulfate is degraded by chondroitinase ABC to three primary unsaturated disaccharides, (1) 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-galactose, (2) 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose, and (3) 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose, when treated with chondroitinase. Plasma samples (0.5 ml) were treated with 50 mU of chondroitinase ABC in 50 microl of 1 mM sodium phosphate buffer (pH 7.0) at 37 degrees C for 6 h. The samples were extracted with 25% trifluoroacetic acid in ethanol. The resultant samples were derivatized with 1% dansylhydrazine in ethanol at 40 degrees C for 3 h. The chromatographic conditions consisted of fluorescence detection (excitation at 350 nm and emission at 530 nm), mu-Bondapack NH(2) (300 x 3.9 mm), and mobile phase of acetonitrile:100 mM acetate buffer, pH 5.6 (76:24), pumped at 1.0 ml/min. The standard curves for each chondroitin disaccharide showed linearity over the selected concentration range (r > or = 0.99). The intraday percentage relative standard deviation was < or =9.5% and the interday precision was < or =6.9% or less. The relative intraday and interday error ranged from -7.3 to 6.6% for each chondroitin disaccharide in the plasma. The extraction recovery was found to be in the range of 90-96%. The validated method accurately quantitated the disaccharides of chondroitin sulfate after administration to dogs and horses.