title: High performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) for the sensitive determination of hyaluronan oligosaccharides.
abstract: High performance anion exchange chromatography (HPAEC) with pulsed amperometric detection (PAD) was optimized for the analysis of oligosaccharides derived from the extracellular matrix component hyaluronan. Using this sensitive approach, the separation of oligosaccharides consisting of two (molecular weight ca. 0.8 kDa) up to 25-30 (molecular weight: ca. 9.5-11.4 kDa) disaccharide moieties was possible. Standard oligosaccharides (comprising 2-4 repetitive disaccharides) were detectable at very low amounts of 0.2-0.3 pmol (20-30 nM). Including 10 min of column equilibration, a complex mixture of low molecular weight hyaluronan can be analyzed within 40 min. The HPAEC method was successfully applied to the study of the size-dependency of both the action of bovine testicular hyaluronidase (BTH) and the precipitation of hyaluronan by cetyltrimethylammonium bromide (CTAB), a physicochemical reaction often used for the determination of hyaluronan and hyaluronidase activity.
authors.affiliation: University of Regensburg, Institute of Pharmacy, 93040 Regensburg, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Rothenhöfer
authors.affiliation: University of Regensburg, Institute of Analytical Chemistry, Chemo- and Biosensors, 93040 Regensburg, Germany.
authors.forename: Marco
authors.initials: M
authors.lastname: Grundmann
authors.affiliation: University of Regensburg, Institute of Pharmacy, 93040 Regensburg, Germany.
authors.forename: Günther
authors.initials: G
authors.lastname: Bernhardt
authors.affiliation: University of Regensburg, Institute of Analytical Chemistry, Chemo- and Biosensors, 93040 Regensburg, Germany.
authors.forename: Frank-Michael
authors.initials: FM
authors.lastname: Matysik
authors.affiliation: University of Regensburg, Institute of Pharmacy, 93040 Regensburg, Germany. Electronic address: armin.buschauer@ur.de.
authors.forename: Armin
authors.initials: A
authors.lastname: Buschauer
mesh_terms: D000818:Animals; D002417:Cattle; D000077286:Cetrimonium; D002593:Cetrimonium Compounds; D002852:Chromatography, Ion Exchange / Q000379:methods*; D019075:Electrophoresis, Capillary; D006820:Hyaluronic Acid / Q000032:analysis* / Q000378:metabolism; D006821:Hyaluronoglucosaminidase / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D057230:Limit of Detection; D016014:Linear Models; D008297:Male; D009844:Oligosaccharides / Q000032:analysis* / Q000378:metabolism; D015203:Reproducibility of Results; D019346:Sodium Acetate; D012972:Sodium Hydroxide; D013696:Temperature
chemical_list: D002593:Cetrimonium Compounds; D009844:Oligosaccharides; D019346:Sodium Acetate; D012972:Sodium Hydroxide; D006820:Hyaluronic Acid; D006821:Hyaluronoglucosaminidase; D000077286:Cetrimonium
keywords: Bovine testicular hyaluronidase; Capillary electrophoresis; High performance anion exchange chromatography; Hyaluronan oligosaccharides; Pulsed amperometric detection; Turbidimetric hyaluronidase assay