ASTM F2103-18

Standard Guide for Characterization and Testing of Chitosan Salts as Starting Materials Intended for Use in Biomedical and Tissue-Engineered Medical Product Applications

SIGNIFICANCE AND USE
4.1 This guide contains a listing of those characterization parameters that are directly related to the functionality of chitosan. This guide can be used as an aid in the selection and characterization of the appropriate chitosan or chitosan salt for a particular application. This standard is intended to give guidance in the methods and types of testing necessary to properly characterize, assess, and ensure consistency in the performance of a particular chitosan. It may have use in the regulation of devices containing chitosan by appropriate authorities.  
4.2 The chitosan salts covered by this guide may be gelled, extruded, or otherwise formulated into biomedical devices for use as tissue-engineered medical products or drug delivery devices for implantation as determined to be appropriate, based on supporting biocompatibility and physical test data. Recommendations in this guide should not be interpreted as a guarantee of clinical success in any tissue-engineered medical product or drug delivery application.  
4.3 To ensure that the material supplied satisfies requirements for use in TEMPs, several general areas of characterization should be considered. These include identity of chitosan, physical and chemical characterization and testing, impurities profile, and performance-related tests.
SCOPE
1.1 This guide covers the evaluation of chitosan salts suitable for use in biomedical or pharmaceutical applications, or both, including, but not limited to, tissue-engineered medical products (TEMPS).  
1.2 This guide addresses key parameters relevant for the functionality, characterization, and purity of chitosan salts.  
1.3 As with any material, some characteristics of chitosan may be altered by processing techniques (such as molding, extrusion, machining, assembly, sterilization, and so forth) required for the production of a specific part or device. Therefore, properties of fabricated forms of this polymer should be evaluated using test methods that are appropriate to ensure safety and efficacy.  
1.4 Warning—Mercury has been designated by EPA and many state agencies as a hazardous material that can cause central nervous system, kidney, and liver damage. Mercury, or its vapor, may be hazardous to health and corrosive to materials. Caution should be taken when handling mercury and mercury-containing products. See the applicable product Material Safety Data Sheet (MSDS) for details and EPA’s website (http://www.epa.gov/mercury/faq.htm) for additional information. Users should be aware that selling mercury or mercury-containing products, or both, in your state may be prohibited by state law.  
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.6 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.  
1.7 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.

General Information

• Status: Published
• Publication Date: 31-May-2018
• ICS: 11.100 - Laboratory medicine
• ICS: 11.100.99 - Other standards related to laboratory medicine
• Technical Committee: F04 - Medical and Surgical Materials and Devices
• Drafting Committee: F04.42 - Biomaterials and Biomolecules for TEMPs

Relations

• Replaces: ASTM F2103-11 - Standard Guide for Characterization and Testing of Chitosan Salts as Starting Materials Intended for Use in Biomedical and Tissue-Engineered Medical Product Applications
• Effective Date: 01-Jun-2018
• Refers: ASTM F749-20 - Standard Practice for Evaluating Material Extracts by Intracutaneous Injection in the Rabbit
• Effective Date: 01-Feb-2020
• Refers: ASTM F1903-18 - Standard Practice for Testing for Cellular Responses to Particles <emph type="bdit" >in vitro</emph>
• Effective Date: 01-Oct-2018
• Refers: ASTM D2196-18 - Standard Test Methods for Rheological Properties of Non-Newtonian Materials by Rotational Viscountess
• Effective Date: 01-Jun-2018
• Refers: ASTM F1439-03(2018) - Standard Guide for Performance of Lifetime Bioassay for the Tumorigenic Potential of Implant Materials
• Effective Date: 01-Feb-2018
• Refers: ASTM F756-17 - Standard Practice for Assessment of Hemolytic Properties of Materials
• Effective Date: 01-Mar-2017
• Refers: ASTM F748-16 - Standard Practice for Selecting Generic Biological Test Methods for Materials and Devices
• Effective Date: 01-Apr-2016
• Refers: ASTM F895-11(2016) - Standard Test Method for Agar Diffusion Cell Culture Screening for Cytotoxicity
• Effective Date: 01-Apr-2016
• Refers: ASTM F756-13 - Standard Practice for Assessment of Hemolytic Properties of Materials
• Effective Date: 01-Dec-2013
• Refers: ASTM F1439-03(2013) - Standard Guide for Performance of Lifetime Bioassay for the Tumorigenic Potential of Implant Materials
• Effective Date: 01-Oct-2013
• Refers: ASTM F2602-13 - Standard Test Method for Determining the Molar Mass of Chitosan and Chitosan Salts by Size Exclusion Chromatography with Multi-angle Light Scattering Detection (SEC-MALS)
• Effective Date: 01-Aug-2013
• Refers: ASTM F749-13 - Standard Practice for Evaluating Material Extracts by Intracutaneous Injection in the Rabbit
• Effective Date: 01-Mar-2013
• Refers: ASTM F2260-03(2012)e1 - Standard Test Method for Determining Degree of Deacetylation in Chitosan Salts by Proton Nuclear Magnetic Resonance (¹H NMR) Spectroscopy
• Effective Date: 01-Oct-2012
• Refers: ASTM F813-07(2012) - Standard Practice for Direct Contact Cell Culture Evaluation of Materials for Medical Devices
• Effective Date: 01-Oct-2012
• Refers: ASTM F749-98(2012) - Standard Practice for Evaluating Material Extracts by Intracutaneous Injection in the Rabbit
• Effective Date: 01-Oct-2012

Overview

ASTM F2103-18, Standard Guide for Characterization and Testing of Chitosan Salts as Starting Materials Intended for Use in Biomedical and Tissue-Engineered Medical Product Applications, provides comprehensive guidance for evaluating the properties and suitability of chitosan salts for biomedical and pharmaceutical applications. Chitosan, a linear polysaccharide typically derived from marine sources, exhibits unique properties such as biocompatibility, mucoadhesiveness, and the ability to act as a structural matrix or drug delivery vehicle. This standard is essential for manufacturers, regulators, and researchers developing tissue-engineered medical products (TEMPs) and other implantable biomedical devices utilizing chitosan materials.

Key Topics

• Characterization Parameters: The standard identifies physical and chemical characteristics that impact chitosan’s performance in biomedical contexts, including:

  • Identity confirmation using spectroscopic techniques (FT-IR, NMR)
  • Degree of deacetylation
  • Molecular weight distribution and dispersity
  • Viscosity in aqueous solutions
  • Ash and moisture content
  • Insoluble impurities

• Impurity Profiling: Ensuring high purity is critical for clinical safety. The standard addresses:

  • Endotoxin content, using validated Limulus Amebocyte Lysate (LAL) tests
  • Protein content analysis
  • Heavy metal (elemental impurity) assessment, referencing USP methods
  • Microbiological safety, ensuring low bioburden and sterility

• Processing Effects: The physical and chemical properties of chitosan can be altered by processing techniques such as molding, extrusion, sterilization, and assembly. ASTM F2103-18 recommends re-evaluating properties post-processing to guarantee material safety and efficacy.

• Test Methodology: The guide outlines appropriate analytical methods, including:

  • Spectroscopic analysis for identity and structure
  • Chromatographic and viscosity-based methods for molecular weight determination
  • Gravimetric methods for moisture and ash content
  • Microbiological and chemical testing for impurity profiles

• Stability and Sterilization: Evaluating chitosan’s stability under storage and after sterilization (e.g., gamma irradiation, ethylene oxide, filtration) is emphasized for ensuring product performance throughout its lifecycle.

Applications

Chitosan salts characterized per ASTM F2103-18 are commonly used in:

• Tissue-Engineered Medical Products (TEMPs): Chitosan serves as scaffolding for cell growth, supporting tissue regeneration.
• Implantable Drug Delivery Devices: Its ability to enhance drug transport and provide sustained release is valuable in localized therapies.
• Biomedical Implants: Chitosan’s biocompatibility, biodegradability, and physical versatility make it suitable for a range of implantable devices.
• Regulatory Submissions: Data generated using the prescribed characterization and testing methods is valuable for FDA and other regulatory body submissions, supporting product safety and consistency.

Related Standards

For comprehensive evaluation, ASTM F2103-18 references and aligns with a variety of important standards:

• ASTM Standards:

  • D2196 (Rheological Properties)
  • F619 (Extraction of Medical Plastics)
  • F2260 (Degree of Deacetylation by NMR)
  • F2602 (Molar Mass by SEC-MALS)
  • Additional biocompatibility and cytotoxicity practices (e.g., F748, F756, F813, F895, F981)

• ISO Standards:

  • ISO 10993 series (Biological evaluation of medical devices)
  • ISO 13408-1 (Aseptic processing)

• US Pharmacopeia (USP):

  • <85> Bacterial Endotoxins
  • <61>, <71>, <1211> (Microbial Limit, Sterility, Sterility Assurance)
  • <731> (Elemental Impurities)

• AAMI/ANSI Standards:

  • Sterilization and microbiological methods (e.g., ANSI/AAMI/ISO 11737)

Conclusion

ASTM F2103-18 is a critical resource ensuring that chitosan salts used in advanced biomedical and pharmaceutical products are well-characterized, high-purity, and fit for purpose. By standardizing testing and evaluation protocols, it supports product safety, regulatory compliance, and successful clinical outcomes for tissue-engineered and implantable devices.

Standards Content (Sample)

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: F2103 − 18
Standard Guide for
Characterization and Testing of Chitosan Salts as Starting
Materials Intended for Use in Biomedical and Tissue-
Engineered Medical Product Applications
This standard is issued under the fixed designation F2103; 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.
INTRODUCTION
Biopolymers from marine sources have been studied and used in commercial applications and
product development for a number of years. Chitosan, a linear polysaccharide consisting of
glucosamine and N-acetyl glucosamine derived mainly from crustacean shells, has been used in many
technical applications such as water purification (as a flocculant), in cosmetics, and recently as a
proposed fat-binding weight control product. In solution, the cationic nature of chitosan gives this
polymer a mucoadhesive property. Chitosan and its salts can be used as a matrix or scaffold material
as well as in non-parenteral delivery systems for challenging drugs. Chitosan salts have been shown
to increase the transport of polar drugs across the nasal epithelial surface. The purpose of this guide
is to identify key parameters relevant for the functionality and characterization of chitosan and
chitosansaltsforthedevelopmentofnewcommercialapplicationsofchitosansaltsforthebiomedical
and pharmaceutical industries.
1. Scope central nervous system, kidney, and liver damage. Mercury, or
its vapor, may be hazardous to health and corrosive to
1.1 This guide covers the evaluation of chitosan salts
materials.Cautionshouldbetakenwhenhandlingmercuryand
suitable for use in biomedical or pharmaceutical applications,
mercury-containing products. See the applicable product Ma-
or both, including, but not limited to, tissue-engineered medi-
terial Safety Data Sheet (MSDS) for details and EPA’s website
cal products (TEMPS).
(http://www.epa.gov/mercury/faq.htm) for additional informa-
1.2 This guide addresses key parameters relevant for the
tion. Users should be aware that selling mercury or mercury-
functionality, characterization, and purity of chitosan salts.
containingproducts,orboth,inyourstatemaybeprohibitedby
1.3 As with any material, some characteristics of chitosan state law.
may be altered by processing techniques (such as molding,
1.5 The values stated in SI units are to be regarded as
extrusion, machining, assembly, sterilization, and so forth)
standard. No other units of measurement are included in this
required for the production of a specific part or device.
standard.
Therefore, properties of fabricated forms of this polymer
1.6 This standard does not purport to address all of the
should be evaluated using test methods that are appropriate to
safety concerns, if any, associated with its use. It is the
ensure safety and efficacy.
responsibility of the user of this standard to establish appro-
1.4 Warning—Mercury has been designated by EPA and
priate safety, health, and environmental practices and deter-
many state agencies as a hazardous material that can cause
mine the applicability of regulatory limitations prior to use.
1.7 This international standard was developed in accor-
dance with internationally recognized principles on standard-
This guide is under the jurisdiction of ASTM Committee F04 on Medical and
Surgical Materials and Devices and is the direct responsibility of Subcommittee
ization established in the Decision on Principles for the
F04.42 on Biomaterials and Biomolecules for TEMPs.
Development of International Standards, Guides and Recom-
Current edition approved June 1, 2018. Published August 2018. Originally
mendations issued by the World Trade Organization Technical
approved in 2001. Last previous edition approved in 2011 as F2103 – 11. DOI:
Barriers to Trade (TBT) Committee.
10.1520/F2103-18.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F2103 − 18
2. Referenced Documents ISO 22442-1 Medical Devices UtilizingAnimal Tissues and
2 Their Derivatives — Part 1:Application of Risk Manage-
2.1 ASTM Standards:
ment
D2196 Test Methods for Rheological Properties of Non-
ISO 22442-2 Medical Devices UtilizingAnimal Tissues and
Newtonian Materials by Rotational Viscometer
Their Derivatives — Part 2: Controls On Sourcing,
F619 Practice for Extraction of Medical Plastics
Collection, and Handling
F748 PracticeforSelectingGenericBiologicalTestMethods
ISO 22442-3 Medical Devices UtilizingAnimal Tissues and
for Materials and Devices
Their Derivatives — Part 3: Validation of the Elimination
F749 Practice for Evaluating Material Extracts by Intracuta-
and/or Inactivation of Viruses and Transmissible Spongi-
neous Injection in the Rabbit
form Encephalopathy (TSE) Agents
F756 Practice for Assessment of Hemolytic Properties of
ISO 80000-9:2009 Quantities and units — Part 9: Physical
Materials
Chemistry and Molecular Physics
F763 Practice for Short-Term Screening of Implant Materi-
2.4 ICH Documents:
als
International Conference on Harmonization (1997) Guid-
F813 Practice for Direct Contact Cell Culture Evaluation of
ance for Industry M3 Nonclinical Safety Studies for the
Materials for Medical Devices
Conduct of Human Clinical Trials for Pharmaceuticals 62
F895 TestMethodforAgarDiffusionCellCultureScreening
FR 62922
for Cytotoxicity
International Conference on Harmonization (1996) Guide-
F981 Practice for Assessment of Compatibility of Biomate-
line for Industry S2A Specific Aspects of Regulatory
rials for Surgical Implants with Respect to Effect of
Genotoxicity Tests for Pharmaceuticals 61 FR 18199
Materials on Muscle and Insertion into Bone
International Conference on Harmonization (1997) Guid-
F1251 Terminology Relating to Polymeric Biomaterials in
ance for Industry S2B Genotoxicity: A Standard Battery
Medical and Surgical Devices (Withdrawn 2012)
for Genotoxicity Testing of Pharmaceuticals 62 FR 62472
F1439 Guide for Performance of Lifetime Bioassay for the
International Conference on Harmonization (1994) Guide-
Tumorigenic Potential of Implant Materials
line for Industry S5A Detection of Toxicity to Reproduc-
F1903 Practice for Testing For Biological Responses to
tion for Medicinal Products 59 FR 48746
Particles In Vitro
International Conference on Harmonization (1996) Guid-
F1904 Practice for Testing the Biological Responses to
ance for Industry S5B Detection of Toxicity to Reproduc-
Particles in vivo
tion for Medicinal Products: Addendum on Toxicity to
F1905 Practice For Selecting Tests for Determining the
Male Fertility 61 FR 15360
Propensity of Materials to Cause Immunotoxicity (With-
International Conference on Harmonization (1996) Guide-
drawn 2011)
line for Industry S1A The Need for Long-term Rodent
F1906 Practice for Evaluation of Immune Responses In
Carcinogenicity Studies of Pharmaceuticals 61 FR 8153
BiocompatibilityTestingUsingELISATests,Lymphocyte
3 International Conference on Harmonization (1998) Guid-
Proliferation, and Cell Migration (Withdrawn 2011)
ance for Industry S1B Testing for Carcinogenicity of
F2260 Test Method for Determining Degree of Deacety-
Pharmaceuticals 63 FR 8983
lation in Chitosan Salts by Proton Nuclear Magnetic
International Conference on Harmonization (1995) Guide-
Resonance ( H NMR) Spectroscopy
line for Industry S1C Dose Selection for Carcinogenicity
F2602 Test Method for Determining the Molar Mass of
Studies of Pharmaceuticals 60 FR 11278
Chitosan and Chitosan Salts by Size Exclusion Chroma-
International Conference on Harmonization (1997) S1C[R]
tography with Multi-angle Light Scattering Detection
Guidance for Industry Addendum to Dose Selection for
(SEC-MALS)
Carcinogenicity Studies of Pharmaceuticals:Addition of a
2.2 Ph. Eur. Document:
4 Limit Dose and Related Notes 62 FR 64259
Ph. Eur. Monograph Chitosan Chloride, Nov. 2000
International Conference on Harmonization (ICH) Q1AICH
2.3 ISO Documents:
Harmonized Tripartite Guidance for Stability Testing of
ISO31-8 QuantitiesandUnits—Part8:PhysicalChemistry
New Drug Substances and Products (September 23, 1994)
and Molecular Physics
2.5 FDA Documents:
ISO 13408-1: 1998 Aseptic Processing of Health Care Prod-
FDA Guideline DHHS
ucts — Part 1: General Requirements
FDA Guideline on Validation of the Limulus Amebocyte
Test as an End-Product Endotoxin Test for Human and
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Animal Parenteral Drugs, Biological Products and Health-
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
care Products DHHS, December 1987
Standards volume information, refer to the standard’s Document Summary page on
FDA Interim Guidance for Human and Veterinary Drug
the ASTM website.
Products and Biologicals. Kinetic LAL Techniques
The last approved version of this historical standard is referenced on
www.astm.org.
Available from EDQM, Publications and Services European Pharmacopoeia,
BP 907 226, avenue de Colmar, F-67029 Strasbourg Cedex 1, France. Available from ICH Secretariat, c/o IFPMA, 30 rue de St-Jean, PO Box 758,
Available from International Organization for Standardization (ISO), ISO 1211 Geneva 13, Switzerland.
Central Secretariat, BIBC II, Chemin de Blandonnet 8, CP 401, 1214 Vernier, Available from Food and Drug Administration (FDA), 5600 Fishers Ln.,
Geneva, Switzerland, http://www.iso.org. Rockville, MD 20857, http://www.fda.gov.
F2103 − 18
DHHS, July 15, 1991 3.1.3.1 Discussion—Degradation of polysaccharides occurs
by means of cleavage of the glycosidic bonds, usually by acid
2.6 ANSI Documents:
—catalyzed hydrolysis. Degradation can also occur thermally.
ANSI/AAMI/ISO 11737-1: 1995 Sterilization of Medical
Note that degradation is not synonymous with decomposition.
Devices—Microbiological Methods—Part 1: Estimation
Degradation is often used as a synonym for depolymerization
of Bioburden on Product
when referring to polymers.
ANSI/AAMI/ISO 11737-2: 1998 Sterilization of Medical
Devices—Microbiological Methods—Part 2:Tests of Ste-
3.1.4 degree of deacetylation, n—the fraction or percentage
rilityPerformedintheValidationofaSterilizationProcess
of glucosamine units (deacetylated monomers) in a chitosan
2.7 AAMI Documents:
polymer molecule.
AAMI TIR No. 19—1998 Guidance for ANSI/AAMI/ISO
3.1.5 depolymerization, n—reduction in length of a polymer
10993–7: 1995, Biological Evaluation of Medical
chain to form shorter polymeric units.
Devices—Part 7: Ethylene Oxide Sterilization Residuals
3.1.5.1 Discussion—Depolymerization may reduce the
AAMI/ISO 14160:1998 Sterilization of Single-Use Medical
polymer chain to oligomeric or monomeric units, or both. In
Devices Incorporating Materials of Animal Origin—
chitosan, hydrolysis of the glycosidic bonds is the primary
Validation and Routine Control of Sterilization by Liquid
mechanism.
Chemical Sterilants
AAMI ST67/CDV-2:1999 Sterilization of Medical
3.1.6 dispersity, n—measure of the heterogeneity of sizes of
Devices—Requirements for Products Labeled “Sterile”
molecules or particles in a mixture, calculated by the ratio of
¯ ¯
2.8 United States Pharmacopeia Documents:
M /M .
w n
USP Chapter <85> Biological Tests and Assays: Bacterial
3.1.7 endotoxin,n—pyrogenichighmolarmasslipopolysac-
Endotoxins Tests
charide (LPS) complex associated with the cell wall of
USP Chapter <161> Microbial Limit Tests
gram-negative bacteria.
USP36-NF31 Elemental Impurities
3.1.7.1 Discussion—Though endotoxins are pyrogens, not
USP Chapter <24>
all pyrogens are endotoxins. Endotoxins are specifically de-
USP Chapter <71> Sterility Tests
tected through a Limulus Amebocyte Lysate (LAL) test.
USP Chapter <1211> Sterilization and Sterility Assurance
of Compendial Articles
3.1.8 molecular mass average (molecular weight average),
<731>USP 24/NF19 n—the given molecular weight (M) of a chitosan will always
represent an average of all of the molecules in the population.
2.9 NIST Document:
NIST Special Publication 811 Guide for the Use of the The most common ways to express the M are as the number
¯ ¯
average (M ) and the weight average (M ). The two averages
International System of Units (SI)
n w
are defined by the following equations:
2.10 U.S. Code of Federal Regulations:
21CFR312: Title 21— Code of Federal Regulations, Part
N M
(i i i
H
312 Investigational New Drug Application
M 5
n
N
i
(i
3. Terminology
and
3.1 Definitions:
W M N M
(i i i (i i i
3.1.1 chitosan, n—a linear polysaccharide consisting of
H
M 5 5
w
W N M
β(1→4) linked 2-acetamido-2-deoxy-D-glucopyranose (Glc- (i i (i i i
NAc) and 2-amino-2-deoxy-D-glucopyranose (GlcN).
where:
3.1.1.1 Discussion—Chitosan is a polysaccharide derived
N = number of molecules having a specific molecular
i
by N-deacetylation of chitin.
weight M and
i
3.1.2 decomposition, n—structural changes of chitosans as a
W = weight of molecules having a specific molecular
i
result of exposure to environmental, chemical, or thermal
weight M. In a disperse molecular population the
i
factors, such as temperatures greater than 200°C.
¯ ¯ ¯ ¯
relation M > M is always valid. The ratio M /M is
w n w n
3.1.2.1 Discussion—Decomposition can result in deleteri-
referred to as the dispersity, and will typically be in the
ous changes to the chitosan.
range from 1.5 to 3.0 for commercial chitosans.
3.1.3 degradation, n—change in the chemical structure,
NOTE 1—The term molecular weight (abbreviated MW) is obsolete and
should be replaced by the SI equivalent of either relative molecular mass
physical properties, or appearance of a material.
(M ), which reflects the dimensionless ratio of the mass of a single
r
moleculetoanatomicmassunit(seeISO31-8),ormolarmass(M),which
8 refers to the mass of a mole of a substance and is typically expressed as
Association for the Advancement of Medical Instrumentation, 111 N. Glebe
g/mol. For polymers and other macromolecules, use of the symbols M ,
Rd., Suite 220, Arlington, VA 22201–4795.
w
M , and M continue, referring to mass-average molar mass, number-
Available from U.S. Pharmacopeial Convention (USP), 12601 Twinbrook
n z
Pkwy., Rockville, MD 20852-1790, http://www.usp.org. average molar mass, and z-average molar mass, respectively. For more
Available from National Institute of Standards and Technology (NIST), 100 information regarding proper utilization of SI units, see NIST Special
Bureau Dr., Stop 1070, Gaithersburg, MD 20899-1070, http://www.nist.gov. Publication 811.
Available from U.S. Government Printing Office, Superintendent of
3.1.9 pyrogen, n—any substance that produces fever when
Documents, 732 N. Capitol St., NW, Washington, DC 20401-0001, http://
www.access.gpo.gov. administered parenterally.
F2103 − 18
4. Significance and Use
Chitosan Base Chitosan Chitosan
(as Acetate) Chloride Glutamate
4.1 This guide contains a listing of those characterization
Frequency Frequency Frequency
Bond Bond Bond
-1 -1 -1
parameters that are directly related to the functionality of
(cm ) (cm ) (cm )
O-H and 3362b O-H and 3344b N-H 1555b
chitosan. This guide can be used as an aid in the selection and
N-H N-H
characterization of the appropriate chitosan or chitosan salt for
a particular application. This standard is intended to give Asymmetric 1556 N-H 1605 CH -OH 1396
2 2
carboxylate
guidance in the methods and types of testing necessary to
anion
properly characterize, assess, and ensure consistency in the
performance of a particular chitosan. It may have use in the Symmetric 1406 N-H 1513 C-O-C 1154
carboxylate (glycosidic
regulation of devices containing chitosan by appropriate au-
anion linkage)
thorities.
C-O-C 1153 CH -OH 1379 C-O-C 1085s
4.2 The chitosan salts covered by this guide may be gelled,
(glycosidic (glycosidic
extruded, or otherwise formulated into biomedical devices for
linkage) ring)
use as tissue-engineered medical products or drug delivery
C-O-C 1083s C-O-C 1154 . .
devicesforimplantationasdeterminedtobeappropriate,based
(glycosidic (glycosidic
on supporting biocompatibility and physical test data. Recom-
ring) linkage)
mendations in this guide should not be interpreted as a
... ... C-O-C 1086s . . . . . .
guarantee of clinical success in any tissue-engineered medical
(glycosidic
product or drug delivery application. ring)
The peak designations are: sh: sharp; s: strong; m: medium;
4.3 To ensure that the material supplied satisfies require-
w: weak; and b: broad.
ments for use in TEMPs, several general areas of characteriza-
tion should be considered. These include identity of chitosan,
5.2 Physical and Chemical Characterization of Chitosan:
physical and chemical characterization and testing, impurities
5.2.1 The composition and sequential structure of chitosan
profile, and performance-related tests.
can be a key functional attribute of any chitosan or chitosan
salt. Variations in the composition or the sequential structure,
5. Chemical and Physical Test Methods
or both, may, but not necessarily will, cause differences in
5.1 Identity of Chitosan—The identity of chitosan and
performance of a chitosan in a particular end use. This
chitosan salts can be established by several methods including,
information may be determined by the following method:
1 13
but not limited to the following:
High-resolution H- and C-nuclear magnetic resonance spec-
5.1.1 Chitosan chloride monograph Ph. Eur.
troscopy (NMR).
5.1.2 Fourier Transform Infrared Spectroscopy (FT-IR)—
5.2.2 The degree of deacetylation of chitosan can be estab-
Almost all organic chemical compounds absorb infrared radia-
lished using a number of techniques including, but not limited
tion at frequencies characteristic for the functional groups in
to, the following:
1 13
the compound. A FT-IR spectrum will show absorption bands
5.2.2.1 High-resolution H- and C-Nuclear Magnetic
relating to bond stretching and bending and can therefore serve
Resonance Spectroscopy (NMR)—Chitosan salts should be
as a unique fingerprint of a specific compound. Cast a chitosan
dissolved in D O and partially degraded to a degree of
film from a 0.25 % (weight/volume) solution of chitosan in
depolymerization of 20 to 30 using sodium nitrite before
1 % (volume⁄volume) acetic acid or chitosan salt (dissolved in
recording proton or carbon NMR spectra (see Test Method
water) by drying approximately 500 µL of the sample onto a
F2260).
disposable IR card for3to4hat 60°C. Record a background
5.2.2.2 UV Spectroscopy—This method is based upon that
spectrum between 4000 and 400 cm-1 using 128 scans at a
reported by Muzzarelli et al. The method is actually a
-1
resolution of 4 cm . Record the IR spectrum of a dried blank
quantitative measure of the number of amine functional groups
IR card, then record the IR spectrum of the sample using 128
in the polymer. The method uses a standard curve produced
-1
scans at a resolution of 4 cm , percent transmission mode.
from varying concentrations of N-acetyl glucosamine. The
-1
Label the peaks. Typical representative frequencies (cm ) for
degree of deacetylation is calculated from recordings of the
chitosan are as follows:
first derivative of the UV spectra of N-acetyl glucosamine and
of chitosan samples at 202 nm.
5.2.2.3 Titration—Methods are based on titrating a known
base solution to a known mass of chitosan dissolved in acid.A
graph of pH versus added volume of base added will have two
inflexion points with the first representing the neutralization of
acid and the second the neutralization of the ammonium ion
Vårum, K. M., Anthonsen, M. W., Grasdalen, H., and Smidsrod, O., Carbo-
hydrate Research, Vol 211, 1991, pp. 17–23.
12 14
No suitable commercially available IR cards are available for the IR analysis Muzzarelli, R. A. A., Rochetti, R., Stanic, V., and Weckx, M., Chitin
of chitosan glutamate salt. Alternative methods are under investigation. Handbook, R. A. A. Muzzarelli and M. T. Peters, Ed., Atec Grottammare, 1997.
F2103 − 18
group on the chitosan. The difference in the inflection points is presence of a wide range of chitosan fractions may be an issue.
used to calculate the degree of de-acetylation (1, 2) . In such cases, calculation of the dispersity will be important.
5.2.3 Molecular mass (molecular weight) of a chitosan will
Typically,thisisbetween1.5and3.0forcommercialchitosans.
define certain performance characteristics such as viscosity.As
5.2.4 Depending on the final use and the required perfor-
such and depending on the sensitivity of a particular end use to
mance control, other characterization assays can include, but
these variations, determination of molecular mass directly or
are not limited to the following:
indirectly may be necessary. Commercial chitosans are dis-
5.2.4.1 Viscosity in Aqueous Solution—Viscosity is a liq-
perse with respect to molecular weight (M). Molecular weight
uid’s resistance to flow. The molecular mass of a chitosan will
may be expressed as the number average (M ) or the weight
n
determine the extent to which it will thicken an aqueous
average (M ). Molecular weights may be determined by
w
solution. Therefore, a simple viscosity test may yield informa-
methods such as, but not limited to, the following:
tion on the relative differences in molecular mass among
5.2.3.1 Molecular Weight Determination Based on Intrinsic
chitosan samples. To allow comparison between laboratories,
Viscosity—The intrinsic viscosity describes a polymer’s ability
theviscometerusedmustbecalibratedwithtraceablestandards
to form viscous solutions in water and is directly proportional
(seeTestMethodsD2196).Theviscositymeasuredwilldepend
to the average molecular weight of the polymer. The intrinsic
viscosity is a characteristic of the polymer under specified on several parameters related to how the testing is conducted.
solvent and temperature conditions. It is independent of con- Important parameters to control include, but are not limited to,
centration. The intrinsic viscosity (η) is directly related to the
the following:
molecular weight of a polymer through the Mark-Houwink-
(1) Temperature—The temperature at which the measure-
Sakurada (MHS) equation:
ment is performed is critical. An increase in temperature will,
a
in almost every case, result in a decrease in the viscosity.
@η# 5 KM
Consistent and controlled temperature (that is, with a standard
where:
temperature bath) is critical to achieving reproducible results.
K = a constant,
Typically, the temperature used to measure viscosity can be
M = viscosity derived average molecular weight, and
20°, 25°, or 37°C, or a combination thereof.
a = an empirical constant describing the conformation of
(2) Chitosan Concentration—The moisture content of the
the polymer.
chitosan must be known to prepare correct concentrations of
By measuring the intrinsic viscosity, the viscosity average
chitosan or chitosan salts (see 5.2.4.2). Chitosan can have
molecular weight can be determined if K and a are accurately
about an 8 % moisture by weig
...

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.
Designation: F2103 − 11 F2103 − 18
Standard Guide for
Characterization and Testing of Chitosan Salts as Starting
Materials Intended for Use in Biomedical and Tissue-
Engineered Medical Product Applications
This standard is issued under the fixed designation F2103; 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.
INTRODUCTION
Biopolymers from marine sources have been studied and used in commercial applications and
product development for a number of years. Chitosan, a linear polysaccharide consisting of
glucosamine and N-acetyl glucosamine derived mainly from crustacean shells, has been used in many
technical applications such as water purification (as a flocculant), in cosmetics, and recently as a
proposed fat-binding weight control product. In solution, the cationic nature of chitosan gives this
polymer a mucoadhesive property. Chitosan and its salts can be used as a matrix or scaffold material
as well as in non-parenteral delivery systems for challenging drugs. Chitosan salts have been shown
to increase the transport of polar drugs across the nasal epithelial surface. The purpose of this guide
is to identify key parameters relevant for the functionality and characterization of chitosan and
chitosan salts for the development of new commercial applications of chitosan salts for the biomedical
and pharmaceutical industries.
1. Scope
1.1 This guide covers the evaluation of chitosan salts suitable for use in biomedical or pharmaceutical applications, or both,
including, but not limited to, tissue-engineered medical products (TEMPS).
1.2 This guide addresses key parameters relevant for the functionality, characterization, and purity of chitosan salts.
1.3 As with any material, some characteristics of chitosan may be altered by processing techniques (such as molding, extrusion,
machining, assembly, sterilization, and so forth) required for the production of a specific part or device. Therefore, properties of
fabricated forms of this polymer should be evaluated using test methods that are appropriate to ensure safety and efficacy.
1.4 Warning—Mercury has been designated by EPA and many state agencies as a hazardous material that can cause central
nervous system, kidney, and liver damage. Mercury, or its vapor, may be hazardous to health and corrosive to materials. Caution
should be taken when handling mercury and mercury-containing products. See the applicable product Material Safety Data Sheet
(MSDS) for details and EPA’s website (http://www.epa.gov/mercury/faq.htm) for additional information. Users should be aware
that selling mercury or mercury-containing products, or both, in your state may be prohibited by state law.
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.6 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.
1.7 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.
This guide 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 March 1, 2011June 1, 2018. Published March 2011August 2018. Originally approved in 2001. Last previous edition approved in 20072011 as
ε2
F2103 – 01F2103 – 11.(2007) . DOI: 10.1520/F2103-11.10.1520/F2103-18.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F2103 − 18
2. Referenced Documents
2.1 ASTM Standards:
D2196 Test Methods for Rheological Properties of Non-Newtonian Materials by Rotational Viscometer
F619 Practice for Extraction of Medical Plastics
F748 Practice for Selecting Generic Biological Test Methods for Materials and Devices
F749 Practice for Evaluating Material Extracts by Intracutaneous Injection in the Rabbit
F756 Practice for Assessment of Hemolytic Properties of Materials
F763 Practice for Short-Term Screening of Implant Materials
F813 Practice for Direct Contact Cell Culture Evaluation of Materials for Medical Devices
F895 Test Method for Agar Diffusion Cell Culture Screening for Cytotoxicity
F981 Practice for Assessment of Compatibility of Biomaterials for Surgical Implants with Respect to Effect of Materials on
Muscle and Insertion into Bone
F1251 Terminology Relating to Polymeric Biomaterials in Medical and Surgical Devices (Withdrawn 2012)
F1439 Guide for Performance of Lifetime Bioassay for the Tumorigenic Potential of Implant Materials
F1903 Practice for Testing For Biological Responses to Particles In Vitro
F1904 Practice for Testing the Biological Responses to Particles in vivo
F1905 Practice For Selecting Tests for Determining the Propensity of Materials to Cause Immunotoxicity (Withdrawn 2011)
F1906 Practice for Evaluation of Immune Responses In Biocompatibility Testing Using ELISA Tests, Lymphocyte Proliferation,
and Cell Migration (Withdrawn 2011)
F2260 Test Method for Determining Degree of Deacetylation in Chitosan Salts by Proton Nuclear Magnetic Resonance ( H
NMR) Spectroscopy
F2602 Test Method for Determining the Molar Mass of Chitosan and Chitosan Salts by Size Exclusion Chromatography with
Multi-angle Light Scattering Detection (SEC-MALS)
2.2 Ph. Eur. Document:
Ph. Eur. Monograph Chitosan Chloride, Nov. 2000
2.3 ISO Documents:
ISO 1099331-8 Biological Evaluation of Medical DevicesQuantities and Units — Part 8: Physical Chemistry and Molecular
Physics
ISO 13408-1: 1998 Aseptic Processing of Health Care Products — Part 1: General Requirements
ISO 10993-122442-1 Biological Evaluation of Medical Devices—Part 1: Evaluation and TestingMedical Devices Utilizing
Animal Tissues and Their Derivatives — Part 1: Application of Risk Management
ISO 10993-3—Part 3:22442-2 Tests for Genotoxicity, Carcinogenicity and Reproductive ToxicityMedical Devices Utilizing
Animal Tissues and Their Derivatives — Part 2: Controls On Sourcing, Collection, and Handling
ISO 10993-9—Part 9:22442-3 Framework for Identification and Quantification of Potential Degradation ProductsMedical
Devices Utilizing Animal Tissues and Their Derivatives — Part 3: Validation of the Elimination and/or Inactivation of Viruses
and Transmissible Spongiform Encephalopathy (TSE) Agents
ISO 10993-17—Part 17: Methods for Establishment of Allowable Limits for Leachable Substances Using Health-Based Risk
Assessment
ISO 13408-1: 1998:80000-9:2009 Aseptic Processing of Health Care Products—Part 1: General Requirements Quantities and
units — Part 9: Physical Chemistry and Molecular Physics
2.4 ICH Documents:
International Conference on Harmonization (1997) Guidance for Industry M3 Nonclinical Safety Studies for the Conduct of
Human Clinical Trials for Pharmaceuticals 62 FR 62922
International Conference on Harmonization (1996) Guideline for Industry S2A Specific Aspects of Regulatory Genotoxicity
Tests for Pharmaceuticals 61 FR 18199
International Conference on Harmonization (1997) Guidance for Industry S2B Genotoxicity: A Standard Battery for
Genotoxicity Testing of Pharmaceuticals 62 FR 62472
International Conference on Harmonization (1994) Guideline for Industry S5A Detection of Toxicity to Reproduction for
Medicinal Products 59 FR 48746
International Conference on Harmonization (1996) Guidance for Industry S5B Detection of Toxicity to Reproduction for
Medicinal Products: Addendum on Toxicity to Male Fertility 61 FR 15360
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
The last approved version of this historical standard is referenced on www.astm.org.
Available from EDQM, Publications and Services European Pharmacopoeia, BP 907 226, avenue de Colmar, F-67029 Strasbourg Cedex 1, France.
Available from American National Standards Institute (ANSI), 25 W. 43rd St., 4th Floor, New York, NY 10036, http://www.ansi.org.International Organization for
Standardization (ISO), ISO Central Secretariat, BIBC II, Chemin de Blandonnet 8, CP 401, 1214 Vernier, Geneva, Switzerland, http://www.iso.org.
Available from ICH Secretariat, c/o IFPMA, 30 rue de St-Jean, PO Box 758, 1211 Geneva 13, Switzerland.
F2103 − 18
International Conference on Harmonization (1996) Guideline for Industry S1A The Need for Long-term Rodent Carcinogenicity
Studies of Pharmaceuticals 61 FR 8153
International Conference on Harmonization (1998) Guidance for Industry S1B Testing for Carcinogenicity of Pharmaceuti-
cals 63 FR 8983
International Conference on Harmonization (1995) Guideline for Industry S1C Dose Selection for Carcinogenicity Studies of
Pharmaceuticals 60 FR 11278
International Conference on Harmonization (1997) S1C[R] Guidance for Industry Addendum to Dose Selection for
Carcinogenicity Studies of Pharmaceuticals: Addition of a Limit Dose and Related Notes 62 FR 64259
International Conference on Harmonization (ICH) Q1A ICH Harmonized Tripartite Guidance for Stability Testing of New Drug
Substances and Products (September 23, 1994)
2.5 FDA Documents:
FDA Guideline DHHS
FDA Guideline on Validation of the Limulus Amebocyte Test as an End-Product Endotoxin Test for Human and Animal
Parenteral Drugs, Biological Products and Healthcare Products DHHS, December 1987
FDA Interim Guidance for Human and Veterinary Drug Products and Biologicals. Kinetic LAL Techniques DHHS, July 15,
2.6 ANSI Documents:
ANSI/AAMI/ISO 11737-1: 1995 Sterilization of Medical Devices—Microbiological Methods—Part 1: Estimation of Bioburden
on Product
ANSI/AAMI/ISO 11737-2: 1998 Sterilization of Medical Devices—Microbiological Methods—Part 2: Tests of Sterility
Performed in the Validation of a Sterilization Process
2.7 AAMI Documents:
AAMI TIR No. 19—1998:19—1998 Guidance for ANSI/AAMI/ISO 10993–7: 1995, Biological Evaluation of Medical
Devices—Part 7: Ethylene Oxide Sterilization Residuals
AAMI/ISO 14160—1998:14160:1998 Sterilization of Single-Use Medical Devices Incorporating Materials of Animal Origin—
Validation and Routine Control of Sterilization by Liquid Chemical Sterilants
AAMI ST67/CDV-2: 1999:ST67/CDV-2:1999 Sterilization of Medical Devices—Requirements for Products Labeled “Sterile”
2.8 EN United States Pharmacopeia Documents:
EN 12442-1USP Chapter <85> Animal Tissues and Their Derivative Utilized in the Manufacture of Medical Devices—Part 1:
Analysis and Management of RiskBiological Tests and Assays: Bacterial Endotoxins Tests
EN 12442-Part 3:USP Chapter <161> Validation of the Elimination and/or Inactivation of Virus and Transmissible
AgentsMicrobial Limit Tests
USP36-NF31 Elemental Impurities
USP Chapter <24>
USP Chapter <71> Sterility Tests
USP Chapter <1211> Sterilization and Sterility Assurance of Compendial Articles
<731>USP 24/NF19
2.9 NIST Document:
NIST Special Publication 811 Guide for the Use of the International System of Units (SI)
2.10 U.S. Code of Federal Regulations:
21CFR312: Title 21— Code of Federal Regulations, Part 312 Investigational New Drug Application
3. Terminology
3.1 Definitions:
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).
3.1.1.1 Discussion—
Chitosan is a polysaccharide derived by N-deacetylation of chitin.
3.1.2 decomposition, n—structural changes of chitosans as a result of exposure to environmental, chemical, or thermal factors,
such as temperatures greater than 200°C.
Available from Food and Drug Administration (FDA), 5600 Fishers Ln., Rockville, MD 20857, http://www.fda.gov.
Association for the Advancement of Medical Instrumentation, 111 N. Glebe Rd., Suite 220, Arlington, VA 22201–4795.
Available from European Committee for Standardization, CEN Management Centre, 36 rue de Stassart, B-1050 Brussels, Belgium.U.S. Pharmacopeial Convention
(USP), 12601 Twinbrook Pkwy., Rockville, MD 20852-1790, http://www.usp.org.
Available from National Institute of Standards and Technology (NIST), 100 Bureau Dr., Stop 1070, Gaithersburg, MD 20899-1070, http://www.nist.gov.
Available from U.S. Government Printing Office, Superintendent of Documents, 732 N. Capitol St., NW, Washington, DC 20401-0001, http://www.access.gpo.gov.
F2103 − 18
3.1.2.1 Discussion—
Decomposition can result in deleterious changes to the chitosan.
3.1.3 degradation, n—change in the chemical structure, physical properties, or appearance of a material.
3.1.3.1 Discussion—
Degradation of polysaccharides occurs by means of cleavage of the glycosidic bonds, usually by acid —catalyzed hydrolysis.
Degradation can also occur thermally. Note that degradation is not synonymous with decomposition. Degradation is often used as
a synonym for depolymerization when referring to polymers.
3.1.4 degree of deacetylation, n—the fraction or percentage of glucosamine units (deacetylated monomers) in a chitosan
polymer molecule.
3.1.5 depolymerization, n—reduction in length of a polymer chain to form shorter polymeric units.
3.1.5.1 Discussion—
Depolymerization may reduce the polymer chain to oligomeric or monomeric units, or both. In chitosan, hydrolysis of the
glycosidic bonds is the primary mechanism.
3.1.6 dispersity, n—measure of the heterogeneity of sizes of molecules or particles in a mixture, calculated by the ratio of
M¯ /M¯ .
w n
3.1.7 endotoxin, n—pyrogenic high molar mass lipopolysaccharide (LPS) complex associated with the cell wall of
gram-negative bacteria.
3.1.7.1 Discussion—
Though endotoxins are pyrogens, not all pyrogens are endotoxins. Endotoxins are specifically detected through a Limulus
Amebocyte Lysate (LAL) test.
3.1.8 molecular mass average (molecular weight average), n—the given molecular weight (Mw)(M) of a chitosan will always
represent an average of all of the molecules in the population. The most common ways to express the MwM are as the number
average (M¯ ) and the weight average (M¯ ). The two averages are defined by the following equations:
n w
F2103 − 18
N M
(i i i
H
M 5
n
N
(i i
and
W M N M
(i i i (i i i
H
M 5 5
w
W N M
i i i
(i (i
where:
N = number of molecules having a specific molecular weight M and
i i
w = weight of molecules having a specific molecular weight M . In a polydisperse molecular population the relation M¯ > M¯
i i w n
is always valid. The coefficient M¯ /M¯ is referred to as the polydispersity index, and will typically be in the range 1.5
w n
to 3.0 for commercial chitosans.
W = weight of molecules having a specific molecular weight M . In a disperse molecular population the relation M¯ > M¯ is
i i w n
always valid. The ratio M¯ /M¯ is referred to as the dispersity, and will typically be in the range from 1.5 to 3.0 for
w n
commercial chitosans.
NOTE 1—The term molecular weight (abbreviated MW) is obsolete and should be replaced by the SI equivalent of either relative molecular mass (M ),
r
which reflects the dimensionless ratio of the mass of a single molecule to an atomic mass unit (see ISO 31-8), or molar mass (M), which refers to the
mass of a mole of a substance and is typically expressed as g/mol. For polymers and other macromolecules, use of the symbols M ,M , and M continue,
w n z
referring to mass-average molar mass, number-average molar mass, and z-average molar mass, respectively. For more information regarding proper
utilization of SI units, see NIST Special Publication 811.
3.1.9 pyrogen, n—any substance that produces fever when administered parenterally.
4. Significance and Use
4.1 This guide contains a listing of those characterization parameters that are directly related to the functionality of chitosan.
This guide can be used as an aid in the selection and characterization of the appropriate chitosan or chitosan salt for a particular
application. This standard is intended to give guidance in the methods and types of testing necessary to properly characterize,
assess, and ensure consistency in the performance of a particular chitosan. It may have use in the regulation of devices containing
chitosan by appropriate authorities.
4.2 The chitosan salts covered by this guide may be gelled, extruded, or otherwise formulated into biomedical devices for use
as tissue-engineered medical products or drug delivery devices for implantation as determined to be appropriate, based on
supporting biocompatibility and physical test data. Recommendations in this guide should not be interpreted as a guarantee of
clinical success in any tissue-engineered medical product or drug delivery application.
4.3 To ensure that the material supplied satisfies requirements for use in TEMPs, several general areas of characterization should
be considered. These include identity of chitosan, physical and chemical characterization and testing, impurities profile, and
performance-related tests.
5. Chemical and Physical Test Methods
5.1 Identity of Chitosan—The identity of chitosan and chitosan salts can be established by several methods including, but not
limited to the following:
5.1.1 Chitosan chloride monograph Ph. Eur.
5.1.2 Fourier Transform Infrared Spectroscopy (FT-IR)—Almost all organic chemical compounds absorb infrared radiation at
frequencies characteristic for the functional groups in the compound. A FT-IR spectrum will show absorption bands relating to
bond stretching and bending and can therefore serve as a unique fingerprint of a specific compound. Cast a chitosan film from a
0.25 % (w/v)(weight/volume) solution of chitosan (inin 1 % (volume ⁄volume) acetic acid)acid or chitosan salt (dissolved in water)
by drying approximately 500 μL of the sample onto a disposable IR card for 3 to 4 h at 60°C. Record a background spectrum
-1
between 4000 and 400 cm-1 using 128 scans at a resolution of 4 cm . Record the IR spectrum of a dried blank IR card, then record
-1
the IR spectrum of the sample using 128 scans at a resolution of 4 cm , percent transmission mode. Label the peaks. Typical
-1
representative frequencies (cm ) for chitosan are as follows:
No suitable commercially available IR cards are available for the IR analysis of chitosan glutamate salt. Alternative methods are under investigation.
F2103 − 18
Chitosan Base Chitosan Chitosan
(as Acetate) Chloride Glutamate
Frequency Frequency Frequency
Bond Bond Bond
-1 -1 -1
(cm ) (cm ) (cm )
O-H and 3362b O-H and 3344b N-H 1555b
N-H N-H
Asymmetric 1556 N-H 1605 CH -OH 1396
2 2
carboxylate
anion
Symmetric 1406 N-H 1513 C-O-C 1154
carboxylate (glycosidic
anion linkage)
C-O-C 1153 CH -OH 1379 C-O-C 1085s
(glycosidic (glycosidic
linkage) ring)
C-O-C 1083s C-O-C 1154 . . . . . .
(glycosidic (glycosidic
ring) linkage)
. . . . . . C-O-C 1086s . . . . . .
(glycosidic
ring)
The peak designatorsdesignations are: sh: sharp; s: strong; m: medium; w: weak; and b: broad.
5.2 Physical and Chemical Characterization of Chitosan:
5.2.1 The composition and sequential structure of chitosan can be a key functional attribute of any chitosan or chitosan salt.
Variations in the composition or the sequential structure, or both, may, but not necessarily will, cause differences in performance
of a chitosan in a particular end use. This information may be determined by the following method: High-resolution H-
and C-nuclear magnetic resonance spectroscopy (NMR).
5.2.2 The degree of deacetylation of chitosan can be established using a number of techniques including, but not limited to, the
following:
1 13
5.2.2.1 High-resolution H- and C-Nuclear Magnetic Resonance Spectroscopy (NMR)—Chitosan salts should be dissolved in
D O and partially degraded to a degree of depolymerization of 20 to 30 using sodium nitrite before recording proton or carbon
NMR spectra.spectra (see Test Method F2260).
5.2.2.2 Determination of the Degree of Deacetylation by UV Spectroscopy—This method is based upon that reported by
Muzzarelli et al. The method is actually a quantitative measure of the number of amine functional groups in the polymer. The
method uses a standard curve produced from varying concentrations of N-acetyl glucosamine. The degree of deacetylation is
calculated from recordings of the first derivative of the UV spectra of N-acetyl glucosamine and of chitosan samples at 202 nm.
5.2.2.3 Titration—Methods are based on titrating a known base solution to a known mass of chitosan dissolved in acid. A graph
of pH versus added volume of base added will have two inflexion points with the first representing the neutralization of acid and
the second the neutralization of the ammonium ion group on the chitosan. The difference in the inflection points is used to calculate
the degree of de-acetylation (1, 2) .
5.2.3 Molecular mass (molecular weight) of a chitosan will define certain performance characteristics such as viscosity. As such
and depending on the sensitivity of a particular end use to these variations, determination of molecular mass directly or indirectly
may be necessary. Commercial chitosans are polydispersedisperse with respect to molecular weight ((M).M ). Molecular weight
W
may be expressed as the number average (M ) or the weight average (M ). Molecular weights may be determined by methods
Nn Ww
such as, but not limited to, the following:
5.2.3.1 Molecular Weight Determination Based on Intrinsic Viscosity—The intrinsic viscosity describes a polymer’s ability to
form viscous solutions in water and is directly proportional to the average molecular weight of the polymer. The intrinsic viscosity
is a characteristic of the polymer under specified solvent and temperature conditions. It is independent of concentration. The
intrinsic viscosity (η) is directly related to the molecular weight of a polymer through the Mark-Houwink-Sakurada (MHS)
equation:
a
@η# 5 KM
where:
K = a constant,
M = viscosity derived average molecular weight, and
Vårum, K. M., Anthonsen, M. W., Grasdalen, H., and Smidsrod, O., Carbohydrate Research, Vol 211, 1991, pp. 17–23.
Muzzarelli, R. A. A., Rochetti, R., Stanic, V., and Weckx, M., Chitin Handbook, R. A. A. Muzzarelli and M. T. Peters, Ed., Atec Grottammare, 1997.
The boldface numbers in parentheses refer to a list of references at the end of this guide.
F2103 − 18
a = an empirical constant describing the conformation of the polymer.
By measuring the intrinsic viscosity, the viscosity average molecular weight can be determined if K and a are accurately known
for the sample: log [η] = log K + a(log M), where M is the molecular weight. The intrinsic viscosity is experimentally determined
by measuring the relative viscosity in a Ubbelohde capillary viscometer. The measurements should be performed in a solvent
containing 0.1M NaCl (a non-gelling, monovalent salt) at a constant temperature of 20°C, and at a sufficiently low chitosan
concentration. Automatic operation and data acquisition are preferred.
5.2.3.2 Molecular Weight and PolydispersityDispersity Determination by Size Exclusion Chromatography with Multiple Angle
Laser Light Scattering Detection (SEC-MALLS)—As there are no chitosan standards currently available, refractive index detectors
cannot be adequately calibrated. It is not sufficient to only use pullulan standards as a calibration material. Therefore, the method
of choice is to use refractive index coupled to MALLS. For separation of the chitosan into different molecular weight fractions,
a hydrophilic column with the appropriate pore size is required. Such columns include, but are not limited to, those mentioned in
the following techniques. The precision of these techniques must be determined as results can vary by 10 to 20 %. Typical methods
using these techniques include, but are not limited to: using 0.01M sodium acetate/acetic acid buffer, pH 5.5 as the mobile phase
with separation using TSK 3000, TSK 4000, and TSK 5000 columns.columns (see Test Method F2602) .
5.2.3.3 Polydispersity—Dispersity—Depending on the end use and the sensitivity of the application to the molecular mass, the
presence of a wide range of chitosan fractions may be an issue. In such cases, calculation of the polydispersitydispersity will be
important. Typically, this is between 1.5 and 3.0 for commercial chitosans.
5.2.4 Depending on the final use and the required performance control, other characterization assays can incl
...