Determination of polyanionic macromolecules by size-exclusion chromatography

Area of Science:

• Biochemistry
• Analytical Chemistry
• Pharmacology

Background:

• Dextran sulfate is a negatively charged polysaccharide crucial in biological and pharmaceutical research.
• Accurate quantification of dextran sulfate and similar polyanions in biological matrices is challenging.
• Existing methods often require extensive sample preparation and lack specificity.

Purpose of the Study:

• To extend a previously developed method for dextran sulfate determination in rat serum.
• To adapt the method for the sensitive quantitation of various glycosaminoglycans and macromolecular polyanions.
• To offer a more efficient and selective analytical approach compared to traditional spectrophotometric assays.

Main Methods:

• High-performance size-exclusion chromatography (HPSEC) for molecular size fractionation.
• Post-column complexation with 1,9-dimethylmethylene blue (DMMB) for sensitive detection.
• Metachromatic shift analysis of the dye-analyte complex to determine absorbance maxima.

Main Results:

• The method successfully quantifies dextran sulfate in rat serum.
• Various glycosaminoglycans and macromolecular polyanions were determined using the adapted chromatographic conditions.
• The technique demonstrated enhanced selectivity and reduced sample preparation requirements compared to spectrophotometric methods.
• Molecular mass information was provided as part of the analytical output.

Conclusions:

• The extended method offers a simple, sensitive, and selective procedure for quantitating dextran sulfate and related polyanions.
• HPSEC coupled with DMMB detection provides significant advantages over conventional assays.
• The method is adaptable for the determination of additional compounds through modifications in eluent composition, dye concentration, and detection wavelength.