Determination of twelve heparin- and heparan sulfate-derived disaccharides as 2-aminoacridone derivatives by capillary zone electrophoresis using ultraviolet and laser-induced fluorescence detection

Abstract

In quest for high sensitivities, we developed an ultrahigh capillary electrophoresis (CE) method for the structural analysis of heparin and heparan sulfate (HS) in biologic samples. Heparin and HS were digested with an equi-unit mixture of heparin lyases I, II and III and the obtained Delta-disaccharides were derivatized with the fluorophore 2-aminoacridone. All known twelve non-, mono-, di- and trisulfated Delta-disaccharides were completely resolved in a single run, using 50 mM phosphate buffer, pH 3.5, and reversed polarity at 30 kV. Relative standard deviation in migration times and peak areas as well as day-to-day variance ranged from 0.9 to 2.4%, suggesting a reproducible and precise method. Detection of 2-aminoacridone (AMAC)-derivatives of Delta-disaccharides by UV at 255 nm showed 2.8 and 10 times higher sensitivity than that of derivatized and non-derivatized ones at 232 nm. Laser-induced fluorescence detection with an Ar-ion laser source showed an approximately 100 times higher sensitivity than that obtained at 232 nm of the non-derivatized species. Application of this method to quantitative analysis of Delta-disaccharides derived from porcine intestinal mucosa heparin and bovine kidney HS showed excellent agreement with previously published methods, suggesting an accurate method. The developed method can be easily applied for the disaccharide analysis of heparin/HS at the attomole level with high accuracy, for distinguishing between heparin and HS and may be of value for studying their interactions with matrix effective molecules.

Wild blueberry (Vaccinium angustifolium) consumption affects the composition and structure of glycosaminoglycans in Sprague-Dawley rat aorta☆

“…Resolution and analysis were carried out on an uncoated fused-silica capillary tube (50 Am i.d., 64.5 cm total length, 56 cm effective length) at 258C, using 50 mM phosphate buffer, pH 3.5 for determination of HSderived D-disaccharides [24,25] and 50 mM phosphate buffer, pH 3.0, for the determination of HA/GalAG-derived D-disaccharides [26,27], at 30 kV, as it has been described. The operating buffer was filtrated through a 0.2 Am membrane filter.…”

Quantitative determination of disaccharide content in digested unfragmented heparin and low molecular weight heparin by direct-infusion electrospray mass spectrometry

“…Several of the methods compared here employ UV absorbance detection of disaccharides at 232 nm [12,16]. To improve the detection sensitivity, other groups employed a fluorophore-derivatization step [13,14]. However, such a step adds time and variability to sample preparation.…”

“…When digested with heparinases I, II, and III, the same preparation of heparin was determined to contain IS (83-85%), IIS/IIIS (11-12%), and IA (4-5%). While previous works [12][13][14][15][16][17] utilized a mix of all three heparinases in their digests prior to analysis, the work presented here compared the use of heparinases I and II versus I, II, and III for the analysis of heparin and LMWH from porcine intestinal mucosa to determine if the addition of heparinase III had an observable effect on the results. As stated above, digestion of native porcine heparin with heparinases I, II, and III revealed peaks representative of disaccharide IA, which were not observed with heparinases I and II alone.…”

“…This type of data provides information on levels of overall disaccharide sulfation, as well as possible contamination of pharmaceutical native heparin and LMWH preparations with other, less sulfated GAGs. Much of the work previously performed to determine relative heparin disaccharide content has utilized various forms of separation, including capillary electrophoresis (CE) and highperformance liquid chromatography (HPLC), often coupled to UV absorbance detection [12][13][14][15][16]. More recently, several groups have utilized tandem mass spectrometry (MS n ) without any prior sample separation to analyze and quantitate relative proportions of various disaccharides in heparin disaccharide standard mock mixes or heparin digests [17,18].…”

Dietary manganese affects the concentration, composition and sulfation pattern of heparan sulfate glycosaminoglycans in Sprague-Dawley rat aorta

“…Resolution and analysis were carried out on an uncoated fused-silica capillary tube (50 lm i.d., 64.5 cm total length, 56 cm effective length) at 25°C, using 50 mmol/l phosphate buffer, pH 3.5 for determination of HS-derived D-disaccharides (Karamanos et al 1996;Militsopoulou et al 2002) and 50 mmol/l phosphate buffer, pH 3.0, for the determination of HA/GalAG-derived D-disaccharides (Karamanos et al 1994;Lamari et al 1999), at 30 kV, as it has been described. The operating buffer was filtered through a 0:2 lm membrane filter.…”