High sensitivity separation and detection of heparan sulfate disaccharides

J Chromatogr A. 2006 Nov 24;1135(1):52-6. doi: 10.1016/j.chroma.2006.09.064. Epub 2006 Oct 10.

Abstract

Eight Delta-disaccharide standards from heparan sulfate/heparin were derivatized with the fluorophore 4,4-difluoro-5,7- dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, hydrazide (BODIPY) via formation of a Schiff's base and separated using HPAEC on a Propac PA1 column with a linear salt gradient and isocratic 150 mM NaOH. Detection was with an in-line fluorescence detector. The standard deviation (sigma(n-1)) in retention times were 0.7-2% over nine runs. The limit of detection, was 100 fmol (100 x 10(-15)mol) of BODIPY labeled Delta-disaccharides, representing considerably improved detection compared to other fluorophore labeled derivatives and, unlike these, required no further purification steps. Separation and improved detection of BODIPY-Delta-disaccharide conjugates will assist the structural analysis of HS and the development of improved sequencing methodologies.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Boron Compounds / chemistry*
  • Chromatography, High Pressure Liquid
  • Disaccharides / analysis*
  • Disaccharides / chemistry
  • Fluorescent Dyes / chemistry
  • Heparitin Sulfate / analysis*
  • Heparitin Sulfate / chemistry
  • Reproducibility of Results
  • Salts / chemistry
  • Sensitivity and Specificity
  • Sodium Hydroxide / chemistry
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Time Factors

Substances

  • 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene
  • Boron Compounds
  • Disaccharides
  • Fluorescent Dyes
  • Salts
  • Sodium Hydroxide
  • Heparitin Sulfate