ASTM F2260-18
(Test Method)Standard Test Method for Determining Degree of Deacetylation in Chitosan Salts by Proton Nuclear Magnetic Resonance (1H NMR) Spectroscopy
Standard Test Method for Determining Degree of Deacetylation in Chitosan Salts by Proton Nuclear Magnetic Resonance (<sup>1</sup>H NMR) Spectroscopy
SIGNIFICANCE AND USE
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 (DPn) 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,...
SCOPE
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.
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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.
Designation: F2260 − 18
Standard Test Method for
Determining Degree of Deacetylation in Chitosan Salts by
1
Proton Nuclear Magnetic Resonance ( H NMR)
1
Spectroscopy
This standard is issued under the fixed designation F2260; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 2. Referenced Documents
2
2.1 ASTM Standards:
1.1 This test method covers the determination of the degree
F386 Test Method for Thickness of Resilient Flooring Ma-
of deacetylation in chitosan and chitosan salts intended for use
terials Having Flat Surfaces
in biomedical and pharmaceutical applications as well as in
F2103 Guide for Characterization and Testing of Chitosan
Tissue Engineered Medical Products (TEMPs) by high-
1
Salts as Starting Materials Intended for Use in Biomedical
resolution proton NMR ( H NMR). A guide for the character-
and Tissue-Engineered Medical Product Applications
ization of chitosan salts has been published as Guide F2103.
2.2 United States Pharmacopeia Document:
1.2 Thetestmethodisapplicablefordeterminingthedegree
3
USP 35-NF30 <761> Nuclear Magnetic Resonance
of deacetylation (% DDA) of chitosan chloride and chitosan
2.3 European Pharmacopoeia Document:
glutamate salts and is valid for % DDA values from 50 up to
European Pharmacopoeia Monograph 2008:1774 Chitosan
and including 99. It is simple, rapid, and suitable for routine
4
Chloride
use. Knowledge of the degree of deacetylation is important for
anunderstandingofthefunctionalityofchitosansaltsinTEMP
3. Terminology
formulations and applications. This test method will assist end
users in choosing the correct chitosan for their particular
3.1 Definitions:
application. Chitosan salts may have utility in drug delivery
3.1.1 chitosan, n—a linear polysaccharide consisting of
applications, as scaffold or matrix material, and in cell and
β(1→4) linked 2-acetamido-2-deoxy-D-glucopyranose (Glc-
tissue encapsulation applications.
NAc) and 2-amino-2-deoxy-D-glucopyranose (GlcN). Chito-
san is a polysaccharide derived by N-deacetylation of chitin.
1.3 The values stated in SI units are to be regarded as
3.1.2 degradation, n—change in the chemical structure,
standard. No other units of measurement are included in this
physical properties, or appearance of a material. Degradation
standard.
ofpolysaccharidesoccursviacleavageoftheglycosidicbonds.
1.4 This standard does not purport to address all of the
It is important to note that degradation is not synonymous with
safety concerns, if any, associated with its use. It is the
decomposition. Degradation is often used as a synonym for
responsibility of the user of this standard to establish appro-
depolymerization when referring to polymers.
priate safety, health, and environmental practices and deter-
3.1.3 degree of deacetylation, n—the fraction or percentage
mine the applicability of regulatory limitations prior to use.
of glucosamine units (GlcN: deacetylated monomers) in a
1.5 This international standard was developed in accor-
chitosan polymer molecule.
dance with internationally recognized principles on standard-
3.1.4 depolymerization, n—reduction in the length of a
ization established in the Decision on Principles for the
polymer chain to form shorter polymeric units.
Development of International Standards, Guides and Recom-
mendations issued by the World Trade Organization Technical
Barriers to Trade (TBT) Committee.
2
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on
1
This test method is under the jurisdiction ofASTM Committee F04 on Medical the ASTM website.
3
and Surgical Materials and Devices and is the direct responsibility of Subcommittee Available from U.S. Pharmacopeia (USP), 12601 Twinbrook Pkwy., Rockville,
F04.42 on Biomaterials and Biomolecules for TEMPs. MD 20852-1790, http://www.usp.org.
4
Current edition approved June 1, 2018. Published August 2018. Originally Available from European Directorate for the Quality of Medicines (EDQM),
ɛ1
approved in 2003. Last previous edition approved in 2012 as F2260 – 03 (2012) . Publications and Services, European Pharmacopoeia, BP 907, F-67029 Strasbourg,
DOI: 10.1520/F2260-18. France.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1
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...
This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
´1
Designation: F2260 − 03 (Reapproved 2012) F2260 − 18
Standard Test Method for
Determining Degree of Deacetylation in Chitosan Salts by
1
Proton Nuclear Magnetic Resonance ( H NMR)
1
Spectroscopy
This standard is issued under the fixed designation F2260; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1
ε NOTE—Editorial changes were made to subsections 2.2, 2.3, and 4.5 in November 2012.
1. Scope
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
1
NMR ( H 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 (% DA)DDA) of chitosan chloride and chitosan
glutamate salts and is valid for % DADDA 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 a 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 safety, health, and healthenvironmental 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.
2. Referenced Documents
2
2.1 ASTM Standards:
F386 Test Method for Thickness of Resilient Flooring Materials Having Flat Surfaces
F2103 Guide for Characterization and Testing of Chitosan Salts as Starting Materials Intended for Use in Biomedical and
Tissue-Engineered Medical Product Applications
2.2 United States Pharmacopeia Document:
3
USP 35-NF30 <761> Nuclear Magnetic Resonance
2.3 European Pharmacopoeia Document:
4
European Pharmacopoeia Monograph 2008:1774 Chitosan Chloride
3. Terminology
3.1 Definitions:
1
This test method is under the jurisdiction of ASTM Committee F04 on Medical and Surgical Materials and Devices and is the direct responsibility of Subcommittee
F04.42 on Biomaterials and Biomolecules for TEMPs.
Current edition approved Oct. 1, 2012June 1, 2018. Published November 2012August 2018. Originally approved in 2003. Last previous edition approved in 20082012
ɛ1
as F2260 – 03 (2008).(2012) . DOI: 10.1520/F2260-03R12E01.10.1520/F2260-18.
2
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
3
Available from U.S. Pharmacopeia (USP), 12601 Twinbrook Pkwy., Rockville, MD 20852-1790, http://www.usp.org.
4
Available from European Directorate for the Quality of Medicines (EDQM), Publications and Services, European Pharmacopoeia, BP 907, F-67029 Strasbourg, France.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1
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F2260 − 18
3.1.1 chitosan, n—a linear polysaccharide consisting of β(1→4) linked 2-acetamido-2-deoxy-D-glucopyranose (GlcNAc) and
2-amino-2-deoxy-D-glucopyranose (GlcN). Chitosan is a polysaccharide derived by N-deacetylation of chitin.
3.1.2 degradation, n—change in the chemical structure, physical properties, or appearance of a material. Degradation of
polysaccharides occurs via cleavage of the glycosidic bonds. It is important to note that degradation
...
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