| Glycosaminoglycan is a large class of glycosamine-containing linear polysaccharides including heparin/heparan sulfate, chondroitin/dermatan sulfate, keratan sulfate, hyaluronic acid, chitin, and chitosan. Heparin/heparan sulfate consists of repeating uronic acid and glucosamine disaccharides on which varying O-sulfation and N-acetylation/N-sulfation are common. Chondroitin/dermatan sulfate contains sulfated repeating disacharide composed of N-acetylgalactosamine and uronic acid. Hyaluronic acid is a non-sulfated glycosaminoglycan consisting of repeating N-acetylglucosamine and glucuronic acid disaccharides. Keratan sulfate is special in that its repeating disaccharide is N-acetylglucosamine and galactose instead of uronic acid. Moreover, keratan sulfate is modified by sulfates at both N-acetylglucosamine and galactose residues. Chitin is composed of N-acetylglucosamine as the second most abundant biopolymer on Earth. Chitin is synthesized and degraded by insects, fungi, bacteria, and marine invertebrate, which is amounted to billions of tons annually. Chitosan, a chemically deacetylated chitin, has attracted great attention for its better physical and chemical properties that have been wildly used in agriculture, industry, and medical field.Heparin has been used as a major anticoagulant for 80 years in nearly every major surgery, in life-threatening thrombosis event, and in hemodialysis patients, which makes heparin a time-honored and essential drug in modern medicine. Over 600 million pigs are used to produce more than 200 metric tons of heparin and other glycosaminoglycans every year to meet the anticoagulation needs of the world. Other glycosaminoglycans are also clinically approved drugs such as Danaparoid, Sulodexide, chondroitin sulfate in Europe for treating arthritis. Moreover, chondroitin sulfate is the second bestselling nutraceuticals in the US. Hyaluronic acid has anti-adhesion and other properties, which has been used in ophthalmic microsurgery, drug release, tissue engineering, and cosmetic industries. Extensive studies in the past 20 years have shown that chitosan and chitosan derivatives have anti-microbial, anti-fungal, pro-coagulant, immune stimulating, hypo-lipidemia, and anti-tumor activities. As a result, over 37 clinical trials are ongoing world widely for chitosan-based drugs.Glycosaminoglycan-based drugs are different from other drugs in that its molecular targets are usually outside the cells. Glycosaminoglycans not only regulate the circulatory system, the nerve system, and the immune system but also involve in cell adhesion, recognition and signal transduction. Good safety profile and low toxicity make them great drug candidates. However, as biological drugs, the quality control of glycosaminoglycan-based drugs remains to be a big challenge because such drugs are always a mixture of polysaccharides with different molecular weight, different degree of sulfation, and chemical structures.Structural analysis of glycosaminoglycan remains to be the best way to characterize glycosaminoglycan-based drugs. Analyzing disaccharide compositions of glycaosaminoglycan after enzymatic drgradation followed by HPLC analysis have been established for all kinds of glycaosaminoglycans. However, such methods assume a complete digestion of all glycosaminoglycans into disaccharides by glycosaminoglycan degradation enzymes. However, enzyme activities are variable depending on its purity, specific activity, and storage conditions. More importantly, none of the enzymes could degrade any glycosaminoglycan completely into disaccharides based on published reports. Therefore, it is highly desirable to develop a better glycosaminoglycan degradation method.It has been known that either N-sulfated or free amine-containing glycosamine can be quantitatively cleaved by low pH or high pH nitrous acid, respectively. However, such method has never developed for quality control of glycosaminoglycan-based drugs. Therefore, we established nitrous acid (HONO) degradation protocol for both animal glycosaminoglycans and chitosan. According to the reaction mechanism, only anion sugar can be degraded by nitrous acid. The glucosidic bonds via a reaction sequence initiated by nitrosation of the amino group of the sugar followed by loss of N2 with a ring contraction of the D-glucosamine via deamination reaction will turn into 2,5-anhydro-D-mannose coupled to elimination of the aglycone, resulting in glycosidic bond cleavage and the formation of new reducing sugars. Nitrous acid degradation products can be labeled by 1-phenyl-3-methyl-5-pyrazolone (PMP) in a 1:2 ratio. PMP has a strong UV absorbance at 245 nm that can be used for the detection of PMP-labled compounds by high performance liquid Chromatography (HPLC). Both glucosamine (GlcN) and galactosamine (GalN) in glycosaminoglycans can be cleaved by nitrous acid to generate 2,5 man nose (M) from glucosamine(GlcN) and 2,5 dehydration talose (T) from galactosamine (GalN). We have optimized both nitrous acid degradation and PMP labeling conditions by using GlcN as a model compound initially and further optimized reaction conditions extensively by using different GAGs.The nitrous acid degraded and PMP labeled products of glycosaminoglycans were separated HPLC and detected by ESI-MS. By employing three different desulfation strategies on heparin and the heparin pentasaccharide Fondaparinux coupled with tandem mass spectrometry, we successful assigned each eluted peak detected by MS analysis with specific mono-, di-, and tetrasaccharide structures. We used nitrous acid degradation and PMP labeling method to analyze low molecular weight heparin from three different drug manufacturers. We also compared the traditional enzymatic degradation-based disaccharide analysis results of chondroitin/dermatan sulfate, heparin/heparin sulfate with our newly established method.One way to control quality of chitosan is measuring the degree of acetylation (DA). In current study, nitrous acid was used to cleave all glucosamine residues in chitosan into 2,5-anhydromannose (M) or M at the reducing end of di-, tri-, and oligosaccharides. PMP, i.e. 1-phenyl-3-methyl-5-pyrazolone, was used to label all the M. Online UV detection allowed quantification of all M-containing UV peak whereas online MS analysis directly identified 11 different kinds of mono-, di-, tri-, and oligosaccharides that linked each oligosaccharide with specific UV peak after HPLC separation. The DA for chitosans was then calculated based on the A/(A+M) value derived from the UV data. The accuracy of the DA measurement was improved compared to that of direct 1H-NMR and conductometric titration analyses. The sulfated chitosans with different substitution site and degree of sulfations could be identified by our method. Our data showed that unlike acid hydrolysis, the nitrous acid deamination cleavage 1-4 glycosidic bond did not cause loss of sulfate and sulfated chitosan was cleaved into anhydromannose with either one or two covalently linked sulfate groups. The HPLC system separated the non-sulfated, mono-sulfated, and disulfated M residues with base line resolution. The sulfation positions of the chitosan was also determined by 13C-NMR that was consistent with the results obtained by the newly developed method.To take advantage of the quantitative PMP labeling of degraded glycosaminoglycan products, we decided to synthesize different stable isotope tagged PMP. One was 1-phenyl-3-methyl (D3)-5-pyrazolone D3PMP synthesized from deuterated acetic acid. The other one was 1-phenyl-(D5)-3-methyl-5-pyrazolone D5PMP synthesized from D5-aniline. Three PMP with different molecular weight were used to qualitative and quantitative analysis of low molecular weight heparins from different manufacturers for the quality control purposes. The same strategy was also used to simultaneously compare glycosaminoglycan structures purified from different pig tissues and organs.In summary, we developed a LC-MS method that could be used for analysis of chemical degraded, PMP labeled glycosaminoglycan structures for quality control of such compounds. The convenient and low cost of the newly developed chemical degradation method was a good replacement of the high cost method by employing glycosaminoglycan degradation enzymes. This method was not only applicable for structural analyses of all kinds of glycosaminoglycans but also could be used for analyzing a mixture of glycosaminoglycans. For chitosan, a HPLC with an online UV detector was sufficient to measure the degree of acetylation and to provide useful chitosan structural information. The newly developed method also allowed to label 3 kinds of chemical degradation of glycosaminoglycan samples with 3 different PMPs. The qualitative and quantitative comparison of 3 distinct PMP tagged glycosaminoglycan samples simultaneously by LC-MS analysis provided a novel way to control the quality of glycosaminoglycan-based drugs. |
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