The aim of this paper is to review chromatographic and mass-spectrometric methods and underline the best analytical approaches for successful analysis of various hyaluronic acid species in different types of samples. Hyaluronan-degrading enzymes and chemical depolymerization produce di- or oligosaccharides suitable for hyaluronan quantification or structural characterization of hyaluronan derivatives. Efficient purification and pre-column derivatization of hyaluronan disaccharides by reductive amination allow subnanogram quantification in biological samples. The chromatographic separation is capable to distinguish all glycosaminoglycans disaccharides and to resolve hyaluronan fragments with 2-40 monomers. Using electrospray ionization or matrix assisted laser desorption ionization, hyaluronan fragments up to 8 kDa or 41 kDa, respectively, can be observed. One- or two-dimensional chromatographic separation with higly sensitive mass-spectrometric detection is an indispensable tool for revealing substituent position, extent of modification and substitution patterns of chemically modified hyaluronan derivatives. It is essential for studying structure-biological function relationships of hyaluronan and its derivatives.

Contipro a s Dolní Dobrouč 401 56102 Dolní Dobrouč Czech Republic

Contipro a s Dolní Dobrouč 401 56102 Dolní Dobrouč Czech Republic; Department of Analytical Chemistry Faculty of Science Palacký University 17 listopadu 12 77146 Olomouc Czech Republic

Department of Analytical Chemistry Faculty of Science Palacký University 17 listopadu 12 77146 Olomouc Czech Republic; Institute of Microbiology v v i The Czech Academy of Sciences Videnska 1083 Prague Czech Republic

Department of Analytical Chemistry Regional Centre of Advanced Technologies and Materials Faculty of Science Palacký University 17 listopadu 12 Olomouc 77147 Czech Republic; Institute of Microbiology v v i The Czech Academy of Sciences Videnska 1083 Prague Czech Republic

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