title: A multi-analytical approach to better assess the keratan sulfate contamination in animal origin chondroitin sulfate.
abstract: Chondroitin sulfate is a glycosaminoglycan widely used as active principle of anti-osteoarthritis drugs and nutraceuticals, manufactured by extraction from animal cartilaginous tissues. During the manufacturing procedures, another glycosaminoglycan, the keratan sulfate, might be contemporarily withdrawn, thus eventually constituting a contaminant difficult to be determined because of its structural similarity. Considering the strict regulatory rules on the pureness of pharmaceutical grade chondrotin sulfate there is an urgent need and interest to determine the residual keratan sulfate with specific, sensitive and reliable methods. To pursue this aim, in this paper, for the first time, we set up a multi-analytical and preparative approach based on: i) a newly developed method by high performance anion-exchange chromatography with pulsed amperometric detection, ii) gas chromatography-mass spectrometry analyses, iii) size exclusion chromatography analyses coupled with triple detector array module and on iv) strong anion exchange chromatography separation. Varied KS percentages, in the range from 0.1 to 19.0% (w/w), were determined in seven pharmacopeia and commercial standards and nine commercial samples of different animal origin and manufacturers. Strong anion exchange chromatography profiles of the samples showed three or four different peaks. These peaks analyzed by high performance anion-exchange with pulsed amperometric detection and size exclusion chromatography with triple detector array, ion chromatography and by mono- or two-dimensional nuclear magnetic resonance revealed a heterogeneous composition of both glycosaminoglycans in terms of sulfation grade and molecular weight. High molecular weight species (>100 KDa) were also present in the samples that counted for chains still partially linked to a proteoglycan core.
authors.affiliation: Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, University of Campania-L.Vanvitelli, ex Second University of Naples, Via De Crecchio 7, 80138, Naples, Italy. Electronic address: odilefrancesca.restaino@unina2.it.
authors.forename: Odile Francesca
authors.initials: OF
authors.lastname: Restaino
authors.affiliation: Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, University of Campania-L.Vanvitelli, ex Second University of Naples, Via De Crecchio 7, 80138, Naples, Italy. Electronic address: rosario.finamore@unina2.it.
authors.forename: Rosario
authors.initials: R
authors.lastname: Finamore
authors.affiliation: Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, University of Campania-L.Vanvitelli, ex Second University of Naples, Via De Crecchio 7, 80138, Naples, Italy. Electronic address: paola.diana@unina2.it.
authors.forename: Paola
authors.initials: P
authors.lastname: Diana
authors.affiliation: Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, University of Campania-L.Vanvitelli, ex Second University of Naples, Via De Crecchio 7, 80138, Naples, Italy. Electronic address: marimars84@hotmail.it.
authors.forename: Mariacarmela
authors.initials: M
authors.lastname: Marseglia
authors.affiliation: Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, University of Campania-L.Vanvitelli, ex Second University of Naples, Via De Crecchio 7, 80138, Naples, Italy. Electronic address: mariovitiello.ita@gmail.com.
authors.forename: Mario
authors.initials: M
authors.lastname: Vitiello
authors.affiliation: Department of Chemical Sciences, University of Naples Federico II, Complesso Universitario Monte S. Angelo, Via Cintia 4, 80126, Naples, Italy. Electronic address: angela.casillo@unina.it.
authors.forename: Angela
authors.initials: A
authors.lastname: Casillo
authors.affiliation: Department of Chemical Sciences, University of Naples Federico II, Complesso Universitario Monte S. Angelo, Via Cintia 4, 80126, Naples, Italy. Electronic address: emiliano.bedini@unina.it.
authors.forename: Emiliano
authors.initials: E
authors.lastname: Bedini
authors.affiliation: Department of Biology, University of Naples Federico II, Complesso Universitario Monte S. Angelo, Via Cintia 4, 80126, Naples, Italy. Electronic address: michelangelo.parrilli@unina.it.
authors.forename: Michelangelo
authors.initials: M
authors.lastname: Parrilli
authors.affiliation: Department of Chemical Sciences, University of Naples Federico II, Complesso Universitario Monte S. Angelo, Via Cintia 4, 80126, Naples, Italy. Electronic address: mariamichela.corsaro@unina.it.
authors.forename: Maria Michela
authors.initials: MM
authors.lastname: Corsaro
authors.affiliation: Department of Chemical Sciences, University of Naples Federico II, Complesso Universitario Monte S. Angelo, Via Cintia 4, 80126, Naples, Italy. Electronic address: marco.trifuoggi@unina.it.
authors.forename: Marco
authors.initials: M
authors.lastname: Trifuoggi
authors.affiliation: Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, University of Campania-L.Vanvitelli, ex Second University of Naples, Via De Crecchio 7, 80138, Naples, Italy. Electronic address: mario.derosa@unina2.it.
authors.forename: Mario
authors.initials: M
authors.lastname: De Rosa
authors.affiliation: Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, University of Campania-L.Vanvitelli, ex Second University of Naples, Via De Crecchio 7, 80138, Naples, Italy. Electronic address: chiara.schiraldi@unina2.it.
authors.forename: Chiara
authors.initials: C
authors.lastname: Schiraldi
mesh_terms: D000818:Animals; D002809:Chondroitin Sulfates / Q000032:analysis*; D004340:Drug Contamination; D007632:Keratan Sulfate / Q000032:analysis*; D011509:Proteoglycans
chemical_list: D011509:Proteoglycans; D002809:Chondroitin Sulfates; D007632:Keratan Sulfate
keywords: Chondroitin sulfate; GC-MS; HPAE-PAD; Keratan sulfate; Pharmacopeia methods; SEC-TDA