The　rare　N-unsubstituted　glucosamine　(GlcNH　3　+　)　residues　in　heparan　sulfate　(HS)　have　important　biological　and　pathophysiological　roles.　Therefore,　the　ability　to　chemically　generate　a　series　of　oligosaccharides,　which　have　a　similar　structure　to　the　naturally-occurring,　GlcNH　3　+　-　containing　oligosaccharides　from　HS,　would　greatly　contribute　to　investigating　their　natural　role　in　HS.　In　this　study,　a　hexasaccharide　library　that　possess　GlcNH　3　+　residues　were　prepared　from　the　chemical　modification　of　the　fully　sulfated　dp6.　Chemical　reaction　conditions　were　optimized　to　generate　different　pattern　of　GlcNH　3　+　-　containing　oligosaccharides,　then　the　structure　of　the　library　was　detected　by　high-performance　liquid　chromatography-ion　trap/time-of-flight　mass　spectrometry　(LC/MS-ITTOF)　analysis.　EIC/MS　and　MS　2　analysis　showed　different　fragmentation　patterns　of　dp6s　with　different　GlcNH　3　+　residues.　This　provides　a　foundation　for　further　identification　and　quantification　of　GlcNH　3　+　-　oligosaccharides　by　mass　spectrum　analysis.　©　2015　Springer　Science+Business　Media　New　York.