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 W 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 mu g CS) with a variation coefficient of approx. 10%. Furthermore, the results obtained from 100 mu 1 plasma were almost identical to those obtained using 20 mu l, 50 mu l and 200 mu l. This method was applied to the characterization of CS in 33 healthy human subjects ageing from 30 to 63 years old.
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|Anno di pubblicazione:||2005|
|Titolo:||Microdetermination of chondroitin sulfate in normal human plasma by fluorophore-assisted carbohydrate electrophoresis (FACE)|
|Autori:||N. Volpi; F. Maccari|
|Appare nelle tipologie:||Articolo su rivista|
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