1、Designation: F 2260 03 (Reapproved 2008)Standard Test Method forDetermining Degree of Deacetylation in Chitosan Salts byProton Nuclear Magnetic Resonance (1H NMR)Spectroscopy1This standard is issued under the fixed designation F 2260; the number immediately following the designation indicates the ye
2、ar 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. Scope1.1 This test method covers the determination of the degre
3、eof deacetylation in chitosan and chitosan salts intended for usein biomedical and pharmaceutical applications as well as inTissue Engineered Medical Products (TEMPs) by high-resolution proton NMR (1H NMR). A guide for the character-ization of chitosan salts has been published as Guide F 2103.1.2 Th
4、e test method is applicable for determining the degreeof deacetylation (% DA) of chitosan chloride and chitosanglutamate salts and is valid for % DA values from 50 up to andincluding 99. It is simple, rapid, and suitable for routine use.Knowledge of the degree of deacetylation is important for anund
5、erstanding of the functionality of chitosan salts in TEMPformulations and applications. This test method will assist endusers in choosing the correct chitosan for their particularapplication. Chitosan salts may have utility in drug deliveryapplications, as a scaffold or matrix material, and in cell
6、andtissue encapsulation applications.1.3 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.4 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the us
7、er of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2F 386 Test Method for Thickness of Resilient FlooringMaterials Having Flat SurfacesF 2103 Guide for Characteriz
8、ation and Testing of ChitosanSalts as Starting Materials Intended for Use in Biomedicaland Tissue-Engineered Medical Product Applications2.2 United States Pharmacopeia Document:USP 24-NF19 Nuclear Magnetic Resonance32.3 European Pharmacopoeia Document:European Pharmacopoeia Monograph 2002:1774 Chito
9、sanChloride43. Terminology3.1 Definitions:3.1.1 chitosan, na linear polysaccharide consisting ofb(14) linked 2-acetamido-2-deoxy-D-glucopyranose(GlcNAc) and 2-amino-2-deoxy-D-glucopyranose (GlcN).Chitosan is a polysaccharide derived by N-deacetylation ofchitin.3.1.2 degradation, nchange in the chemi
10、cal structure,physical properties, or appearance of a material. Degradationof polysaccharides occurs via cleavage of the glycosidic bonds.It is important to note that degradation is not synonymous withdecomposition. Degradation is often used as a synonym fordepolymerization when referring to polymer
11、s.3.1.3 degree of deacetylation, nthe fraction or percentageof glucosamine units (GlcN: deacetylated monomers) in achitosan polymer molecule.3.1.4 depolymerization, nreduction in the length of apolymer chain to form shorter polymeric units.4. Significance and Use4.1 The degree of deacetylation of ch
12、itosan salts is animportant characterization parameter since the charge densityof the chitosan molecule is responsible for potential biologicaland functional effects.4.2 The degree of deacetylation (% DA) of water-solublechitosan salts can be determined by1H nuclear magneticresonance spectroscopy (1
13、H NMR). Several workers havereported on the NMR determination of chemical compositionand sequential arrangement of monomer units in chitin and1This test method is under the jurisdiction of ASTM Committee F04 on Medicaland Surgical Materials and Devices and is the direct responsibility of Subcommitte
14、eF04.42 on Biomaterials and Biomolecules for TEMPs.Current edition approved May 1, 2008. Published June 2008. Originallyapproved in 2003. Last previous edition approved in 2003 as F 2260 03.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at servi
15、ceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3Available from U.S. Pharmacopeia (USP), 12601 Twinbrook Pkwy., Rockville,MD 20852-1790, http:/www.usp.org.4Available from European Directorate for the Quality of Medicine
16、s (EDQM),Publications and Services, European Pharmacopoeia, BP 907, F-67029 Strasbourg,France.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.chitosan. The test method described is primarily based on thework of Vrum et al. (1991),5wh
17、ich represents the firstpublication on routine determination of chemical compositionin chitosans by solution state1H NMR spectroscopy. This testmethod is applicable for determining the % DA of chitosanchloride and chitosan glutamate salts. It is a simple, rapid, andsuitable method for routine use. Q
18、uantitative1H NMR spec-troscopy reports directly on the relative concentration ofchemically distinct protons in the sample, consequently, noassumptions, calibration curves or calculations other thandetermination of relative signal intensity ratios are necessary.4.3 In order to obtain well-resolved N
19、MR spectra, depoly-merization of chitosans to a number average degree of poly-merization (DPn) of 15 to 30 is required. This reduces theviscosity and increases the mobility of the molecules.Althoughthere are several options for depolymerization of chitosans, themost convenient procedure is that of n
20、itrous acid degradationin deuterated water. The reaction is selective, stoichiometricwith respect to GlcN, rapid, and easily controlled (Allan 32768 at400 MHz.Typical temperature equilibration time is 15 min andspectrum acquisition time is approximately 10 min or less.6.2.1.2 The use of digital filt
21、ers and appropriate digitalsignal processing is recommended for good baseline perfor-mance.6.2.2 Processing:6.2.2.1 Use exponential window with 0.5 Hz line broaden-ing and zero-fill to 64k data points before Fourier transforma-tion.6.2.2.2 Relative areas of proton signals are estimated bynumeric int
22、egration of the relevant1H NMR signals; K1, H1D,H1A, H2D and HAc (for chitosan chloride only) (Figs. 1 and2). Correct phasing and flat baseline is essential for good result.5Vrum, K. M., Anthonsen, M. W., Grasdalen, H., and Smidsrd, O., “Deter-mination of the Degree of N-acetylation and the Distribu
23、tion of N-acetyl Groups inPartially N-deacetylated Chitins (Chitosans) by High-Field N.M.R. Spectroscopy-,”Carbohydr. Res., Vol 211, 1991, pp. 1723.6Allan, G. G. and Peyron, M., “Molecular Weight Manipulation of Chitosan 1:Kinetics of Depolymerization by NitrousAcid,” Carbohydr. Res., Vol 277, 1995,
24、 pp.257-272.F 2260 03 (2008)26.3 CalculationsFor chitosan chloride, signal intensitiesof H1D and H2D may be averaged. Similarly, intensities ofH1A and HAc/3 (3 protons in HAc) may be averaged, to givea better estimate of the relative occurrence of GlcN- andGlcNAc-units. This gives a more precise est
25、imate of % DA.Averaging of the two acetylated signals cannot be performedwith chitosan glutamate, due to severe overlap of HAc withglutamate signals (Figs. 1 and 2).6.3.1 The relative number of GlcN-units in the polymerbefore depolymerization can be expressed as:NOTESignal assignments are indicated
26、in the figure. K1: Proton 1 of chitose. 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 sh
27、ift reference at 0.000 ppm.FIG. 1 Typical1H NMR Spectrum of Chitosan Chloride (% DA = 85)NOTESignal assignments are indicated in the figure (see also Fig. 1). Glutamate contributes with 1H NMR signals at 3.75 ppm, and multiplets centeredat 2.5 and 2.1 ppm, the latter overlapping with HAc.FIG. 2 Typi
28、cal1H NMR Spectrum of Chitosan Glutamate (% DA = 84)F 2260 03 (2008)3D 5 K1 1H1D1H2D!/2 (1)where K1, H1D and H2D are estimates of the correspondingsignal intensities from the1H NMR spectrum (Figs. 1 and 2).6.3.2 The relative number of GlcNAc-units in the polymerbefore depolymerization can be express
29、ed as:A 5H1A 1HAc/3!/2 chitosan chloride! (2)A 5 H1A chitosan glutamate!where H1A and HAc are estimates of the correspondingsignal intensities from the1H NMR spectrum (Figs. 1 and 2).6.3.3 Degree of deacetylation (%) is calculated according tothe following equation:%DA5 Degree of deacetylation %!510
30、0%*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)DPnwill be overestimated by approximately 15 % due topartial saturation of K1 with the experimental parameters givenin this test method. This effect is insignific
31、ant with respect tothe calculated % DA.6.3.5 Chitosans With a Low Degree of Deacetylation (% DA60) Only:6.3.5.1 Chitosans with high content of acetylated groupsmight to some degree be subjected to acid hydrolysis duringdepolymerization with nitrous acid (acid hydrolysis specifi-cally cleaves after a
32、cetylated units). Such depolymerizationcan be identified by the presence of H1a reducing-end signals(termed “red-a”) from GlcNAc-units at 5.2 ppm (doublet) inthe1H NMR spectrum. For maximum accuracy, one shouldinclude this signal in the expression for the relative number ofGlcNAc-units given above,
33、noting that the a-anomer accountsfor roughly23 of the anomer population. Consequently, forthese chitosans, the relative number of GlcNAc-units is:A 51.5 red2a 1 H1A 1HAc/3!/2 chitosan chloride! (5)A 5 H1A 11.5 red2a chitosan glutamate!6.3.5.2 For chitosans with low degree of deacetylation (%DA 60),
34、ignoring this note will typically introduce an error of1 to 2 units in the calculated % DA (for example, % DA isassigned a value too high by 1 to 2 percentage units).7. Range, Standard Deviation, and Reporting Results7.1 Standard deviations for repeatability and intermediateprecision have been found
35、 to be similar. The standard deviationof the method has been determined after validation to be lessthan 61 percentage unit.7.2 The determination of low degrees of deacetylation byNMR is limited by the solubility of the sample. Experimentalresults indicate that the method is valid for % DAvalues high
36、erthan 50. The method may be used to measure high degrees ofdeacetylation. Consequently, the range of the method is con-sidered to be valid for % DA values from 50 up to andincluding 99.7.3 Non-Applicable Method Parameters:7.3.1 AccuracyThis parameter is limited by how well theNMR instrument is regu
37、larly maintained and controlled. % DAis obtained by comparing the signal intensities from the twocomponents, acetylated and deacetylated units. No standard isrequired and recovery is not relevant. There are no referencesamples for a true value of the degree of deacetylation inchitosan.7.3.2 Specific
38、ityIf there should be any impurities in thesample, unexpected proton signals will be shown in the spectra.7.3.3 LinearityNot relevant since NMR spectroscopy isquantitative. Each proton NMR peak area is proportional to thenumber of protons represented by that peak.7.4 Further recommendations for NMR
39、data presentationcan be found in Practice E 386.APPENDIXES(Nonmandatory Information)X1. RATIONALEX1.1 The use of naturally occurring biopolymers forbiomedical and pharmaceutical applications and in TissueEngineered Medical Products (TEMPs) is increasing. This testmethod is designed to give guidance
40、in characterizing thedegree of deacetylation of chitosan salts used in suchapplications.X2. BACKGROUNDX2.1 Chitosan is a linear, binary polysaccharide consistingof b(14) linked 2-acetamido-2-deoxy-D-glucopyranose(GlcNAc; acetylated unit) and 2-amino-2-deoxy-D-glucopyranose (GlcN; deacetylated unit).
41、 The two differentmonosaccharides differ only by the substitution at carbon 2;GlcNAc contains an N-acetylated amino group, whereas GlcNcontains only the amino-group (it is said to be deacetylated).Thus, the degree of deacetylation (in %) is a measure of thefraction of GlcN-units in the chitosan chai
42、n.F 2260 03 (2008)4ASTM International takes no position respecting the validity of any patent rights asserted in connection with any item mentionedin this standard. Users of this standard are expressly advised that determination of the validity of any such patent rights, and the riskof infringement
43、of such rights, are entirely their own responsibility.This standard is subject to revision at any time by the responsible technical committee and must be reviewed every five years andif not revised, either reapproved or withdrawn. Your comments are invited either for revision of this standard or for
44、 additional standardsand should be addressed to ASTM International Headquarters. Your comments will receive careful consideration at a meeting of theresponsible technical committee, which you may attend. If you feel that your comments have not received a fair hearing you shouldmake your views known
45、to the ASTM Committee on Standards, at the address shown below.This standard is copyrighted by ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959,United States. Individual reprints (single or multiple copies) of this standard may be obtained by contacting ASTM at the aboveaddress or at 610-832-9585 (phone), 610-832-9555 (fax), or serviceastm.org (e-mail); or through the ASTM website(www.astm.org).FIG. X2.1 Chitosan StructureF 2260 03 (2008)5
