Analysis of the disaccharides derived from hyaluronic acid and chondroitin sulfate by capillary electrophoresis with sample stacking

Abstract

Analysis of the disaccharides derived from hyaluronic acid and chondroitin sulfate by capillary electrophoresis with sample stacking CE conditions for monitoring the unsaturated disaccharides of hyaluronic acid (di-HA) and chondroitin sulfate (di-CS) using an alkaline tetraborate buffer, electrokinetic sample injection, and UV absorption detection at 232 nm are reported. Separations were performed in an uncoated fused-silica capillary having reversed polarity and reversed electroosmosis generated with the addition of CTAB to the buffer. The influence of various separation parameters, including the concentration of CTAB, buffer pH, concentration of tetraborate, and applied voltage, on the resolution of the two disaccharides was investigated. Baseline separation was obtained with 25 mM tetraborate at pH 10.0 and having 0.05 mM CTAB. Chloride and phosphate in the sample are beneficial for the stacking of the disaccharides, with di-HA forming a much sharper peak than di-CS. Using samples prepared in 25 mM Tris-HCl (pH 7.5) and electrokinetic injection at the cathode at -10 kV for 40 s, linear relationships between the corrected peak area and the concentration of the disaccharides have been found in the ranges of 1.0-400.0 and 0.1-1.0 lg/mL (0.2-1.0 lg/mL for di-CS), with correlation coefficients being A0.9933 in all cases. The RSDs of detection times and corrected peak areas were between 1.13-1.24 and 1.57-2.13%, respectively. Applied to human serum samples that were prepared by ethanol precipitation and depolymerization of the two polysaccharides with chondroitinase ABC reveals comigration of endogenous compounds with di-HA and a sample-dependent detection time. The di-HA content in the serum sample can be estimated via subtraction of the blank peak that is obtained without enzymatic hydrolysis.

Metabolism and biochemical/physiological roles of chondroitin sulfates: analysis of endogenous and supplemental chondroitin sulfates in blood circulation

“…However, capillary electrophoresis has not been applied to the determination of blood CS disaccharides so far. The only study in the literature that reports the separation of ∆di-nonS HA and ∆di-nonS CS after treatment of blood GAGs with chondroitinase ABC does not provide information on the concentration of CS, probably because of the inability to separate and identify the other CS-derived disaccharides (Yang et al, 2005). CE conditions for monitoring ∆di-nonS HA and ∆di-nonS CS were an alkaline tetraborate buffer, reversed polarity and reversed electroosmosis, generated with the addition of CTAB to the buffer, electrokinetic sample injection and UV detection at 232 nm.…”

“…CE conditions for monitoring ∆di-nonS HA and ∆di-nonS CS were an alkaline tetraborate buffer, reversed polarity and reversed electroosmosis, generated with the addition of CTAB to the buffer, electrokinetic sample injection and UV detection at 232 nm. Analysis of enzymically depolymerized HA in human serum revealed a sharp peak containing di-HA and endogenous interferences, such that the di-HA content could only be estimated via subtraction of the blank peak obtained with the undepolymerized serum (Yang et al, 2005).…”

Capillary electrophoresis for total glycosaminoglycan analysis

“…Following GAG depolymerization, the separation of disaccharide product often relies on strong anion exchange, reversed-phase, reversed-phase ion-pair, normal phase, high-performance liquid chromatography (HPLC) and hydrophilic interaction chromatography, capillary electrophoresis (CE) and fluorophore assisted carbohydrate electrophoresis [12–42]. Mass spectrometry, ultraviolet and fluorescence detection methods are then used for analyte detection and quantification.…”

“…LIF detection of AMAC-derivatized GAG disaccharides is up to 100-fold more sensitive than UV detection of the non-derivatized GAG disaccharides at 232 nm [32]. Disaccharides derived from CS/DS, HS/HP and HA are typically analyzed by CE-UV or CE-LIF in a separate CE experiments [30–42]. This requires multiple, repetitive and tedious sample preparation steps, including GAG extraction from biological sample, GAG-depolymerization, disaccharide labeling, and disaccharide analysis.…”

Capillary electrophoresis of complex natural polysaccharides

“…The unsaturated disaccharide produced by chondroitin ABC lyase has been separated by CE and quantified by UV without any derivatization [64] and HA plasma has been analyzed, following derivatization with AMAC, using LIF detection [65]. In addition to the above-mentioned applications of HA analysis, CE has been used for quantitative HA analysis in vitreous humor [66], in pharmaceutical formulations [67, 68], and to determine hyaluronate lyase activity in bee and snake venom [69].…”