Microdetermination of chondroitin sulfate in normal human plasma by fluorophore-assisted carbohydrate electrophoresis (FACE).
An inexpensive, simple, sensitive and reproducible analytical method for the quantitative and qualitative evaluation of chondroitin sulfate (CS) from human blood plasma samples by using fluorophore-assisted carbohydrate electrophoresis (FACE) has been developed. After treatment with a nonspecific protease to convert proteins into small peptides, CS from 100 microl of normal human plasma was extracted by using a filter membrane (molecular mass cut-off of 3000 Da) or purification by using an anion-exchange resin. The recovered CS was converted into unsaturated disaccharides through the action of chondroitin ABC lyase, derivatized with 2-aminoacridone by reductive amination in the presence of cyanoborohydride and separated by FACE. The procedure using the purification of plasma CS on the anion-exchange resin produced a cleaner separation and a better resolution of Delta-disaccharides then using microfiltration. The linearity, sensitivity and reproducibility of the method were determined in comparison with HPLC equipped with postcolumn derivatization and fluorescence detection using 2-cyanoacetamide as a fluorogenic reagent. The detection limit was calculated to be 50 ng of CS with a linear response from 50 to 2000 ng. The recovery was found greater than 85% (from 2 to 10 microg CS) with a variation coefficient of approx. 10%. Furthermore, the results obtained from 100 microl plasma were almost identical to those obtained using 20 microl, 50 microl and 200 microl. This method was applied to the characterization of CS in 33 healthy human subjects ageing from 30 to 63 years old.
Volpi
Nicola
N
Department of Biologia Animale, University of Modena and Reggio Emilia, Via Campi 213/D, 41100 Modena, Italy. volpi@unimo.it
Maccari
Francesca
F
D000328:Adult; D002809:Chondroitin Sulfates / Q000097:blood*; D002851:Chromatography, High Pressure Liquid; D004586:Electrophoresis; D005260:Female; D005456:Fluorescent Dyes; D006801:Humans; D008297:Male; D008875:Middle Aged; D012680:Sensitivity and Specificity
D005456:Fluorescent Dyes; D002809:Chondroitin Sulfates