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Novel reverse-phase ion pair-high performance liquid chromatography separation of heparin, heparan sulfate and low molecular weight-heparins disaccharides and oligosaccharides.

Journal of chromatography. A (2013-03-05)
Fabio Galeotti, Nicola Volpi
ABSTRACT

In this study, by using tetrabutylammonium bisulfate as ion-pairing reagent, we were able to separate all the main heparin/heparan sulfate disaccharides generated by the action of heparinases along with the main Hep tetrasaccharide possessing a 3-O-sulfate group on the sulfoglucosamine unit and resistant to enzymatic action. Moreover, this novel HPLC method was able to separate and quantify uncommon disaccharides/oligosaccharides present in low molecular weight-heparins produced by chemical treatment with nitrous acid, dalteparin, or benzylation followed by alkaline hydrolysis, enoxaparin. Additionally, this procedure yields a sensitivity ∼4-times higher compared to conventional strong-anion exchange-HPLC separation. This was obtained by a common UV detector at 232 nm avoiding the use of complex procedures capable of increasing sensitivity by post-column derivatization. Finally, it is worth mentioning that disaccharide/oligosaccharide composition by HPLC and UV detection is a common analytical approach in quality control laboratories to evaluate heparins and low molecular weight-heparins structure and quality during their extraction and production. This simple HPLC approach offers high resolution and sensitivity for the rapid differentiation of pharmaceutical native heparins and derivatives and for the compositional analysis of small amounts of samples derived from biological sources at a glycosaminoglycans level of a few hundred nanogram.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Tetrabutylammonium hydroxide solution, 1.0 M in methanol
Sigma-Aldrich
Tetrabutylammonium hydroxide solution, 40 wt. % in H2O
Sigma-Aldrich
Tetrabutylammonium hydrogensulfate, 97%
Sigma-Aldrich
Tetrabutylammonium phosphate monobasic solution, 1.0 M in H2O
Sigma-Aldrich
Tetrabutylammonium cyanide, 95%
Sigma-Aldrich
Tetrabutylammonium hydroxide solution, 54.0-56.0% in H2O
Sigma-Aldrich
Tetrabutylammonium hydroxide solution, technical, ~40% in H2O (~1.5 M)
Sigma-Aldrich
Tetrabutylammonium perchlorate, ≥95.0% (T)
Supelco
Tetrabutylammonium perchlorate, for electrochemical analysis, ≥99.0%
Supelco
Tetrabutylammonium hydroxide solution, ~40% in water, suitable for ion chromatography
Supelco
Tetrabutylammonium phosphate monobasic solution, suitable for ion pair chromatography, LiChropur, concentrate, ampule
Sigma-Aldrich
Tetrabutylammonium cyanide, technical, ≥80%
Sigma-Aldrich
Tetrabutylammonium bisulfate solution, ~55% in H2O
Sigma-Aldrich
Tetrabutylammonium fluoride solution, 75 wt. % in H2O
Sigma-Aldrich
Tetrabutylammonium nitrate, 97%
Sigma-Aldrich
Tetrabutylammonium fluoride solution, 1.0 M in THF
Sigma-Aldrich
Tetrabutylammonium bromide solution, 50 wt. % in H2O
Sigma-Aldrich
Tetrabutylammonium iodide, reagent grade, 98%
Sigma-Aldrich
Tetrabutylammonium bromide, ACS reagent, ≥98.0%
Supelco
Tetrabutylammonium bromide, suitable for ion pair chromatography, LiChropur, ≥99.0%
Sigma-Aldrich
Tetrabutylammonium chloride, ≥97.0% (NT)
Sigma-Aldrich
Tetrabutylammonium bisulfate, puriss., ≥99.0% (T)
Supelco
Tetrabutylammonium iodide, suitable for ion pair chromatography, LiChropur, ≥99.0%
Supelco
Tetrabutylammonium bisulfate, suitable for ion pair chromatography, LiChropur, ≥99.0%
Supelco
Tetrabutylammonium chloride, suitable for ion pair chromatography, LiChropur, ≥99.0%
Sigma-Aldrich
Tetrabutylammonium iodide, ≥99.0% (AT)
Supelco
Tetrabutylammonium bisulfate solution, suitable for ion pair chromatography, LiChropur, concentrate, ampule
Sigma-Aldrich
Tetrabutylammonium phosphate monobasic, puriss., ≥99.0% (T)
Sigma-Aldrich
Tetrabutylammonium azide