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Determination of twelve heparin- and heparan sulfate-derived disaccharides as 2-aminoacridone derivatives by capillary zone electrophoresis using ultraviolet and laser-induced fluorescence detection.
Electrophoresis. 2002 Apr; 23(7-8):1104-9.E

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.

Authors+Show Affiliations

Militsopoulou M
Department of Chemistry, Section of Organic Chemistry, Biochemistry and Natural Products, Laboratory of Biochemistry, University of Patras, Greece.
Lamari FN
No affiliation info available
Hjerpe A
No affiliation info available
Karamanos NK
No affiliation info available

MeSH

AminoacridinesCarbohydrate SequenceDisaccharidesElectrophoresis, CapillaryHeparinHeparitin SulfateLasersMolecular Sequence DataReproducibility of ResultsSensitivity and SpecificitySpectrometry, FluorescenceSpectrophotometry, Ultraviolet

Pub Type(s)

Journal Article

Language

eng

PubMed ID

11981858

Citation

Militsopoulou, Maria, et al. "Determination of Twelve Heparin- and Heparan Sulfate-derived Disaccharides as 2-aminoacridone Derivatives By Capillary Zone Electrophoresis Using Ultraviolet and Laser-induced Fluorescence Detection." Electrophoresis, vol. 23, no. 7-8, 2002, pp. 1104-9.
Militsopoulou M, Lamari FN, Hjerpe A, et al. Determination of twelve heparin- and heparan sulfate-derived disaccharides as 2-aminoacridone derivatives by capillary zone electrophoresis using ultraviolet and laser-induced fluorescence detection. Electrophoresis. 2002;23(7-8):1104-9.
Militsopoulou, M., Lamari, F. N., Hjerpe, A., & Karamanos, N. K. (2002). Determination of twelve heparin- and heparan sulfate-derived disaccharides as 2-aminoacridone derivatives by capillary zone electrophoresis using ultraviolet and laser-induced fluorescence detection. Electrophoresis, 23(7-8), 1104-9.
Militsopoulou M, et al. Determination of Twelve Heparin- and Heparan Sulfate-derived Disaccharides as 2-aminoacridone Derivatives By Capillary Zone Electrophoresis Using Ultraviolet and Laser-induced Fluorescence Detection. Electrophoresis. 2002;23(7-8):1104-9. PubMed PMID: 11981858.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Determination of twelve heparin- and heparan sulfate-derived disaccharides as 2-aminoacridone derivatives by capillary zone electrophoresis using ultraviolet and laser-induced fluorescence detection. AU - Militsopoulou,Maria, AU - Lamari,Fotini N, AU - Hjerpe,Anders, AU - Karamanos,Nikos K, PY - 2002/5/1/pubmed PY - 2002/9/19/medline PY - 2002/5/1/entrez SP - 1104 EP - 9 JF - Electrophoresis JO - Electrophoresis VL - 23 IS - 7-8 N2 - 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. SN - 0173-0835 UR - https://www.unboundmedicine.com/medline/citation/11981858/Determination_of_twelve_heparin__and_heparan_sulfate_derived_disaccharides_as_2_aminoacridone_derivatives_by_capillary_zone_electrophoresis_using_ultraviolet_and_laser_induced_fluorescence_detection_ L2 - https://doi.org/10.1002/1522-2683(200204)23:7/8<1104::AID-ELPS1104>3.0.CO;2-1 DB - PRIME DP - Unbound Medicine ER -