title: Determination of the degree of acetylation and the distribution of acetyl groups in chitosan by HPLC analysis of nitrous acid degraded and PMP labeled products.
abstract: Chitin is one of the most abundant polysaccharides on earth. It consists of repeating β-1,4 linked N-acetylated glucosamine (A) units. Chitosan is an N-deacetylated product of chitin. Chitosan and its derivatives have broad medical applications as drugs, nutraceuticals, or drug delivery agents. However, a reliable analytical method for quality control of medically used chitosans is still lacking. 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 Ms. Online UV detection allowed quantification of all M-containing UV peaks whereas online MS analysis directly identified 11 different kinds of mono-, di-, tri-, and oligosaccharides that correlated each oligosaccharide with specific UV peak after HPLC separation. The DA (degree of acetylation) for chitosans was calculated based on the A/(A+M) value derived from the UV data. This newly developed method had several advantages for quality control of chitosan: 1. the experimental procedures were extensively optimized; 2. the reliability of the method was confirmed by online LC-MS analysis; 3. the DA value was obtainable based on the UV data after HPLC analysis, which was comparableto that of (1)H NMR and conductometric titration analyses; 4. finally and most importantly, this method could be used to obtain the DA as well as chemical acetylation/deacetylation mechanisms for chitosan by any laboratory equipped with a HPLC and an online UV detector.
authors.affiliation: School of Medicine and Pharmacy, Ocean University of China, Qingdao, China.
authors.forename: Zhangrun
authors.initials: Z
authors.lastname: Han
authors.affiliation: School of Medicine and Pharmacy, Ocean University of China, Qingdao, China.
authors.forename: Yangyang
authors.initials: Y
authors.lastname: Zeng
authors.affiliation: School of Medicine and Pharmacy, Ocean University of China, Qingdao, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Lu
authors.affiliation: School of Medicine and Pharmacy, Ocean University of China, Qingdao, China. Electronic address: lijuanzhang@ouc.edu.cn.
authors.forename: Lijuan
authors.initials: L
authors.lastname: Zhang
mesh_terms: D000107:Acetylation; D000983:Antipyrine / Q000031:analogs & derivatives* / Q000737:chemistry; D048271:Chitosan / Q000032:analysis* / Q000737:chemistry* / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid; D000077553:Edaravone; D005944:Glucosamine / Q000737:chemistry; D008358:Mannose / Q000031:analogs & derivatives / Q000737:chemistry; D009608:Nitrous Acid / Q000737:chemistry*; D009844:Oligosaccharides / Q000737:chemistry; D013194:Staining and Labeling
chemical_list: D009844:Oligosaccharides; C035767:2,5-anhydromannose; D048271:Chitosan; D005944:Glucosamine; D008358:Mannose; D000077553:Edaravone; D009608:Nitrous Acid; D000983:Antipyrine
keywords: 1-Phenyl-3-methyl-5-pyrazolone (PMP); 2,5-Anhydromannose (M); Chitosan; Degree of acetylation; Distribution of acetyl groups in chitosan; Nitrous acid (HONO)