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1.1 This test method covers the determination of the degree of deacetylation in chitosan and chitosan salts intended for use in biomedical and pharmaceutical applications as well as in Tissue Engineered Medical Products (TEMPs) by high-resolution proton NMR (¹H NMR). A guide for the characterization of chitosan salts has been published as Guide F2103.

1.2 The test method is applicable for determining the degree of deacetylation (% DDA) of chitosan chloride and chitosan glutamate salts and is valid for % DDA values from 50 up to and including 99. It is simple, rapid, and suitable for routine use. Knowledge of the degree of deacetylation is important for an understanding of the functionality of chitosan salts in TEMP formulations and applications. This test method will assist end users in choosing the correct chitosan for their particular application. Chitosan salts may have utility in drug delivery applications, as scaffold or matrix material, and in cell and tissue encapsulation applications.

1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.

1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.

1.5 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

4.1 The degree of deacetylation of chitosan salts is an important characterization parameter since the charge density of the chitosan molecule is responsible for potential biological and functional effects.

4.2 The degree of deacetylation (% DDA) of water-soluble chitosan salts can be determined by ¹H nuclear magnetic resonance spectroscopy (¹H NMR). Several workers have reported on the NMR determination of chemical composition and sequential arrangement of monomer units in chitin and chitosan. The test method described is primarily based on the work of Vårum et al. (1991),5 which represents the first publication on routine determination of chemical composition in chitosans by solution state ¹H NMR spectroscopy. This test method is applicable for determining the % DDA of chitosan chloride and chitosan glutamate salts. It is a simple, rapid, and suitable method for routine use. Quantitative ¹H NMR spectroscopy reports directly on the relative concentration of chemically distinct protons in the sample, consequently, no assumptions, calibration curves or calculations other than determination of relative signal intensity ratios are necessary.

4.3 In order to obtain well-resolved NMR spectra, depolymerization of chitosans to a number average degree of polymerization (DP_n) of ~15 to 30 is required. This reduces the viscosity and increases the mobility of the molecules. Although there are several options for depolymerization of chitosans, the most convenient procedure is that of nitrous acid degradation in deuterated water. The reaction is selective, stoichiometric with respect to GlcN, rapid, and easily controlled (Allan & Peyron, 1995).6 The reaction selectively cleaves after a GlcN-residue, transforming it into 2,5-anhydro-D-mannose (chitose), consequently, depletion of GlcN after depolymerization is expected. On the other hand, the chitose unit displays characteristic ¹H NMR signals the intensity of which may be estimated and utilized in the calculation of % DDA, eliminating the need for correction factors. Using the intensity of the chitose signals, the number average degree of polymerization can easily be calculated as a control of the depolymerization.

4.4 Samples are equilibrated and analyzed at a temperature of 90 ± 1°C. Elevated sample temperature contributes to reducing sample viscosity and repositions the proton signal of residual water to an area outside that of interest. While samples are not stored at 90°C but only analyzed at this elevated temperature, the NMR tubes should be sealed with a stopper to avoid any evaporation. At a sample pH* of 3.8-4.3 (see 6.1.5 below), artifactual deacetylation of the sample does not occur during the short equilibration and analysis time.

4.5 A general description of NMR can be found in <761> of the USP 35-NF30.