1、Designation: F2260 03 (Reapproved 2012)1F2260 18Standard Test Method forDetermining Degree of Deacetylation in Chitosan Salts byProton Nuclear Magnetic Resonance (1H NMR)Spectroscopy1This standard is issued under the fixed designation F2260; the number immediately following the designation indicates
2、 the year oforiginal adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1 NOTEEditorial changes were made to subsections 2.2, 2.3,
3、and 4.5 in November 2012.1. Scope1.1 This test method covers the determination of the degree of deacetylation in chitosan and chitosan salts intended for use inbiomedical and pharmaceutical applications as well as in Tissue Engineered Medical Products (TEMPs) by high-resolution protonNMR (1H NMR). A
4、 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 chitosanglutamate salts and is valid for % DADDA values from 50 up to and including 99. It is simple,
5、rapid, and suitable for routine use.Knowledge of the degree of deacetylation is important for an understanding of the functionality of chitosan salts in TEMPformulations and applications.This test method will assist end users in choosing the correct chitosan for their particular application.Chitosan
6、 salts may have utility in drug delivery applications, as a scaffold or matrix material, and in cell and tissue encapsulationapplications.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 t
7、o address all of the safety concerns, if any, associated with its use. It is the responsibilityof the user of this standard to establish appropriate safety safety, health, and healthenvironmental practices and determine theapplicability of regulatory limitations prior to use.1.5 This international s
8、tandard was developed in accordance with internationally recognized principles on standardizationestablished in the Decision on Principles for the Development of International Standards, Guides and Recommendations issuedby the World Trade Organization Technical Barriers to Trade (TBT) Committee.2. R
9、eferenced Documents2.1 ASTM Standards:2F386 Test Method for Thickness of Resilient Flooring Materials Having Flat SurfacesF2103 Guide for Characterization and Testing of Chitosan Salts as Starting Materials Intended for Use in Biomedical andTissue-Engineered Medical Product Applications2.2 United St
10、ates Pharmacopeia Document:USP 35-NF30 Nuclear Magnetic Resonance32.3 European Pharmacopoeia Document:European Pharmacopoeia Monograph 2008:1774 Chitosan Chloride43. Terminology3.1 Definitions:1 This test method is under the jurisdiction of ASTM Committee F04 on Medical and Surgical Materials and De
11、vices and is the direct responsibility of SubcommitteeF04.42 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 20082012as F2260 03 (2008).(2012)1. DOI: 10.15
12、20/F2260-03R12E01.10.1520/F2260-18.2 For referencedASTM standards, visit theASTM website, www.astm.org, or contactASTM Customer Service at serviceastm.org. For Annual Book of ASTM Standardsvolume information, refer to the standards Document Summary page on the ASTM website.3 Available from U.S. Phar
13、macopeia (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.This document is not an ASTM standard and is intended on
14、ly to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Becauseit 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 vers
15、ionof the standard as published by ASTM is to be considered the official document.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States13.1.1 chitosan, na linear polysaccharide consisting of (14) linked 2-acetamido-2-deoxy-D-glucopyranose (
16、GlcNAc) and2-amino-2-deoxy-D-glucopyranose (GlcN). Chitosan is a polysaccharide derived by N-deacetylation of chitin.3.1.2 degradation, nchange in the chemical structure, physical properties, or appearance of a material. Degradation ofpolysaccharides occurs via cleavage of the glycosidic bonds. It i
17、s important to note that degradation is not synonymous withdecomposition. Degradation is often used as a synonym for depolymerization when referring to polymers.3.1.3 degree of deacetylation, nthe fraction or percentage of glucosamine units (GlcN: deacetylated monomers) in a chitosanpolymer molecule
18、.3.1.4 depolymerization, nreduction in the length of a polymer chain to form shorter polymeric units.3.1.5 water soluble chitosan salt, nan ionic compound between chitosan molecule (cation) and a negatively charged anion(e.g. glutamate, acetate, lactate, chloride) that is soluble in water.4. Signifi
19、cance and Use4.1 The degree of deacetylation of chitosan salts is an important characterization parameter since the charge density of thechitosan molecule is responsible for potential biological and functional effects.4.2 The degree of deacetylation (% DA)DDA) of water-soluble chitosan salts can be
20、determined by 1H nuclear magneticresonance spectroscopy (1H NMR). Several workers have reported on the NMR determination of chemical composition andsequential arrangement of monomer units in chitin and chitosan. The test method described is primarily based on the work ofVrum et al. (1991),5 which re
21、presents the first publication on routine determination of chemical composition in chitosans bysolution state 1H NMR spectroscopy. This test method is applicable for determining the % DADDA of chitosan chloride andchitosan glutamate salts. It is a simple, rapid, and suitable method for routine use.
22、Quantitative 1H NMR spectroscopy reportsdirectly on the relative concentration of chemically distinct protons in the sample, consequently, no assumptions, calibration curvesor calculations other than determination of relative signal intensity ratios are necessary.4.3 In order to obtain well-resolved
23、 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 severaloptions for depolymerization of chitosans, the most convenient procedure is that of
24、 nitrous acid degradation in deuterated water.The reaction is selective, stoichiometric with respect to GlcN, rapid, and easily controlled (Allan 32768 at400 MHz.Typical temperature equilibration time is 15 min and spectrum acquisition time is approximately 10 min or less.6.2.1.2 The use of digital
25、filters and appropriate digital signal processing is recommended for good baseline performance.6.2.2 Processing:6.2.2.1 Use exponential window with 0.5 Hz line broadening and zero-fill to 64k data points before Fourier transformation.6.2.2.2 Relative areas of proton signals are estimated by numeric
26、integration of the relevant 1H NMR signals; K1, H1D, H1A,H2D and HAc (for chitosan chloride only) (Figs. 1 and 2). Correct phasing and flat baseline isare essential for good results.6.3 CalculationsFor chitosan chloride, signal intensities of H1D and H2D may be averaged. Similarly, intensities of H1
27、AandHAc/3 (3 protons in HAc) may be averaged, to give a better estimate of the relative occurrence of GlcN- and GlcNAc-units. Thisgives a more precise estimate of % DA.DDA. Averaging of the two acetylated signals cannot be performed with chitosanglutamate, due to severe overlap of HAc with glutamate
28、 signals (Figs. 1 and 2).6.3.1 The relative number of GlcN-units in the polymer before depolymerization can be expressed as:D5K11H1D1H2D!/2 (1)where K1, H1D and H2D are estimates of the corresponding signal intensities from the 1H NMR spectrum (Figs. 1 and 2).6.3.2 The relative number of GlcNAc-unit
29、s in the polymer before depolymerization can be expressed as:A5H1A1HAc/3!/2 chitosan chloride! (2)A5H1A chitosan glutamate!where H1A and HAc are estimates of the corresponding signal intensities from the 1H NMR spectrum (Figs. 1 and 2).6.3.3 Degree of deacetylation (%) is calculated according to the
30、 following equation:%DA5Degree of deacetylation %!5100%*D/D1A! (3)%DDA5Degree of deacetylation %!5100%*D/D1A! (3)6.3.4 The number average degree of polymerization (DPn) may be estimated as a control of the degradation as:DPn5K11A1D!/K1 (4)F2260 183DPn will be overestimated by approximately 15 % due
31、to partial saturation of K1 with the experimental parameters given in thistest method. This effect is insignificant with respect to the calculated % DA.DDA.6.3.5 Chitosans With a Low Degree of Deacetylation (% DADDA 60) Only:NOTE 1Signal assignments are indicated in the figure. K1: Proton 1 of chito
32、se. H1D: Proton 1 of GlcN-units. H1A: Proton 1 of GlcNAc-units. K3:Proton 3 of chitose (not used for calculations). HDO: Solvent signal (residual protons from deuterated water). H2D: Proton 2 of GlcN-units. HAc:Acetylprotons (3) of GlcNAc-units. TMSP: Chemical shift reference at 0.000 ppm.FIG. 1 Typ
33、ical 1H NMR Spectrum of Chitosan Chloride (% DADDA = 85)NOTE 1Signal assignments are indicated in the figure (see also Fig. 1). Glutamate contributes with 1H NMR signals at 3.75 ppm, and multipletscentered at 2.5 and 2.1 ppm, the latter overlapping with HAc.FIG. 2 Typical 1H NMR Spectrum of Chitosan
34、 Glutamate (% DADDA = 84)F2260 1846.3.5.1 Chitosans with high content of acetylated groups (i.e., low DDA) might to some degree be subjected to acid hydrolysisduring depolymerization with nitrous acid (acid hydrolysis specifically cleaves after acetylated units). Such depolymerization canbe identifi
35、ed by the presence of H1 reducing-end signals (termed “red-a”) from GlcNAc-units at 5.2 ppm (doublet) in the 1H NMRspectrum. For maximum accuracy, one should include this signal in the expression for the relative number of GlcNAc-units givenabove, noting that the -anomer accounts for roughly 23 of t
36、he anomer population. Consequently, for these chitosans, the relativenumber of GlcNAc-units is:A51.5red2a1H1A1HAc/3!/2 chitosan chloride! (5)A5H1A11.5red2a chitosan glutamate!6.3.5.2 For chitosans with low degreedegrees of deacetylation (% DADDA 60), ignoring this note will typically introduce anerr
37、or of 1 to 2 units in the calculated % DADDA(for example, % DADDAis assigned a value too high by 1 to 2 percentage units).7. Range, Standard Deviation, and Reporting Results7.1 Standard deviations for repeatability and intermediate precision have been found to be similar. The standard deviation ofth
38、e method has been determined after validation to be less than 61 percentage unit.7.2 The determination of low degrees of deacetylation by NMR is limited by the solubility of the sample. Experimental resultsindicate that the method is valid for % DADDA values higher than 50. The method may be used to
39、 measure high degrees ofdeacetylation. Consequently, the range of the method is considered to be valid for % DADDA values from 50 up to and including99.7.3 Non-Applicable Method Parameters:7.3.1 AccuracyThis parameter is limited by how well the NMR instrument is regularly maintained and controlled.
40、% DADDAis obtained by comparing the signal intensities from the two components, acetylated and deacetylated units. No standard is requiredand recovery is not relevant. There are no reference samples for a true value of the degree of deacetylation in chitosan.7.3.2 SpecificityIf there should be any i
41、mpurities in the sample, unexpected proton signals will be shown in the spectra.7.3.3 LinearityNot relevant since NMR spectroscopy is quantitative. Each proton NMR peak area is proportional to thenumber of protons represented by that peak.7.4 Further recommendations for NMR data presentation can be
42、found in Practice F386.APPENDIXES(Nonmandatory Information)X1. RATIONALEX1.1 The use of naturally occurring biopolymers for biomedical and pharmaceutical applications and in Tissue EngineeredMedical Products (TEMPs) is increasing. This test method is designed to give guidance in characterizing the d
43、egree ofdeacetylation of chitosan salts used in such applications.X2. BACKGROUNDX2.1 Chitosan is a linear, binary polysaccharide consisting of (14) linked 2-acetamido-2-deoxy-D-glucopyranose (GlcNAc;acetylated unit) and 2-amino-2-deoxy-D-glucopyranose (GlcN; deacetylated unit). The two different mon
44、osaccharides differ onlyby the substitution at carbon 2; GlcNAc contains an N-acetylated amino group, whereas GlcN contains only the amino-group (itis said to be deacetylated). Thus, the degree of deacetylation (in %) is a measure of the fraction of GlcN-units in the chitosan chain.F2260 185REFERENC
45、ES(1) Sarmento, Bruno, and Jos das Neves, eds., “Chitosan-based systems for biopharmaceuticals: delivery, targeting and polymer therapeutics,” JohnWiley or through the ASTM website(www.astm.org). Permission rights to photocopy the standard may also be secured from the Copyright Clearance Center, 222Rosewood Drive, Danvers, MA 01923, Tel: (978) 646-2600; http:/ X2.1 Chitosan StructureF2260 186
