Abstract: Heparan sulfate (HS) and heparin are complex sulfated glycosaminoglycans which have important biological activities in developmental processes, angiogenesis, blood coagulation, cell adhesion, and tumor metastasis. These interactions involve a wide variety of proteins, i.e. enzymes, cytokines, growth factors, extracellular matrix proteins. HS and heparin consist of repeating disaccharide units each composed of an uronic acid (UA) or sulfated at C-2 (UA2S) and a derivative of glucosamine N-acetylglucosamine (GlcNAc), N-sulfated glucosamine (GlcNS), or unsubstituted glucosamine (GlcNH₃⁺) that is variably O sulfated. The characterization of GlcNH₃⁺ containing HS/heparin is fundamental to further elucidating such functional properties. In this study, a method of reversed-phase liquid chromatography-electrospray ionization ion trap-time-of-flight mass spectrometry (RP-LC-ESI-IT-TOF MS) was described for the separation and structural analysis of N-unsubstituted disaccharides from heparin/HS. Firstly, 12 disaccharides of heparin/HS were labeled with 2-aminoacridone (AMAC). Subsequently, the AMAC-labeled disaccharides were separated by RP-LC with varied reversed-phase columns. The results showed that the baseline separation of 12 AMAC-labeled heparin/HS disaccharides could be achieved by the ODS-2 HYPERSIL C18 column with a linear elution comprised by eluent A(40 mmol/L ammonium acetate solution (pH 5.6)) and eluent B (methanol). The mass spectrometer detector voltage was 1.8 kV, and the curved desolvation line and heat block temperatures both were 200 ℃. In mass spectrometry, both the ionization efficiency of each disaccharide, and the sulfate loss in the mass spectrometer ion source were different. In order to correct the quality deviation caused by above situation, the relative quantitative analysis of the 12 AMAC-labeled disaccharides was performed by calibration curves established by analyzing a series of increasing amounts of standard AMAC-labeled disaccharide mixtures(200-400 ng). Linearity was assessed based on the amount of AMAC-labeled disaccharide (x) and the peak areas in extract ion chromatography (y). All calibration curves of AMAC-labeled disaccharide showed a good linear relationship (R²≥0.98). The limits of quantitation were determined by 10 times of the signal-to-noise ratio S/N, which was less than 170 ng for all disaccharides. The N-unsubstituted heparin derivatives (N-sulfated heparin, de-6, N-sulfated heparin and de-2,6,N-sulfated heparin) prepared by our laboratory were analyzed by this method. The results showed that in three N-unsubstituted heparin derivatives, various de-sulfation reactions had successfully converted N-sulfo groups into N-unsubstituted residues. The total N-unsubstituted disaccharides yields were as high as 60.51%, 67.43% and 68.39% for N-sulfated heparin, de-6,N-sulfated heparin and de-2,6,N-sulfated heparin, respectively, and the composition of N-unsubstituted disaccharides in each derivative was significantly different. These results indicated that this method is suitable for analyzing heparin derivatives containing N-unsubstituted disaccharides. At the same time, it is able to be applied to qualitatively and quantitatively analyze the disaccharides containing N-unsubstituted glucosamine in biological samples, and provides an important analysis method for further study on the structure and function of N-unsubstituted glucosamine, which helps to broaden our understanding of the important function of N-unsubstituted glucosamine residues in human health and disease.