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A novel LIF‐CE method for the separation of hyaluronan‐ and chondroitin sulfate‐derived disaccharides: Application to structural and quantitative analyses of human plasma low‐ and high‐charged chondroitin sulfate isomers
Author(s) -
Zinellu Angelo,
Pisanu Salvatore,
Zinellu Elisabetta,
Lepedda Antonio J.,
Cherchi Gian Mario,
Sotgia Salvatore,
Carru Ciriaco,
Deiana Luca,
Formato Marilena
Publication year - 2007
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200600668
Subject(s) - chondroitin sulfate , chemistry , chondroitin , sulfate , human plasma , chromatography , plasma , glycosaminoglycan , biochemistry , organic chemistry , physics , quantum mechanics
The report describes a rapid and simple CE method using LIF detection for the analysis of unsaturated disaccharides obtained from enzymatic depolymerization of plasma chondroitin sulfate (CS) isomers. The disaccharide reducing groups were labeled with 2‐aminoacridone (AMAC). The fluorotagged products can be separated by reversed‐polarity CE using a sodium acetate buffer, pH 3.8, in the presence of 0.05% methylcellulose. The choice of the appropriate electrophoretic conditions was performed after a deep analysis of the most important parameters affecting analyte separation. In particular, the effect of both run buffer concentration and pH on resolution, efficiency, migration times, and peak area was evaluated. The selected electrophoretic conditions allowed us to separate the CS isomers‐derived Δ‐disaccharides in less than 12 min, also resolving the nonsulfated disaccharides released from CS isomers from those released from hyaluronan (HA). Moreover, these conditions gave a good reproducibility of both the migration times (CV%, 0.25) and the peak areas (CV%, 1.4). Intra‐ and interassay CV were 5.37 and 7.23%, respectively, and analytical recovery was about 86%. The applicability of the above method to the quantitative and structural disaccharide analyses of plasma CS isomers was investigated. Data obtained from 44 healthy human subjects were compared with those obtained by a fluorophore‐assisted carbohydrate electrophoresis (FACE) reference assay, by using the Passing and Bablok regression and Bland–Altman tests. The developed method could represent a good tool for an ultrasensitive analysis of CS isomers in biological samples from different sources, particularly when samples are available in very low amounts.