1、Designation: F 2260 03Standard 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 year oforiginal adop
2、tion or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method covers the determination of the degreeof deacetylation
3、 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 The test method is
4、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 anunderstanding of the
5、 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 andtissue encapsu
6、lation applications.1.3 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to
7、 use.2. Referenced Documents2.1 ASTM Standards:E 386 Practice for Data Presentation Relating to High-Resolution Nuclear Magnetic Resonance (NMR) Spectros-copy2F 2103 Guide for the Characterization and Testing of Chi-tosan Salts as Starting Material Intended for Use inBiomedical and Tissue Engineered
8、 Medical Product Appli-cations32.2 United States Pharmacopeia Document:USP 24-NF19 Nuclear Magnetic Resonance42.3 European Pharmacopoeia Document:European Pharmacopoeia Monograph 2002:1774 ChitosanChloride53. Terminology3.1 Definitions:3.1.1 chitosan, na linear polysaccharide consisting ofb(14) link
9、ed 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 chemical structure,physical properties, or appearance of a material. Degradationof polysaccharides occurs via c
10、leavage 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 polymers.3.1.3 degree of deacetylation, nthe fraction or percentageof glucosamine units (GlcN: deacetylated monom
11、ers) 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 chitosan salts is animportant characterization parameter since the charge densityof the chitosan molecule is
12、 responsible for potential biologicaland functional effects.4.2 The degree of deacetylation (% DA) of water-solublechitosan salts can be determined by1H nuclear magneticresonance spectroscopy (1H NMR). Several workers havereported on the NMR determination of chemical compositionand sequential arrang
13、ement 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 SubcommitteeF04.42 on Biomaterials and Biomolecules for TEMPs.Current edition approved Apr. 10, 2003. Published May 2
14、003.2Annual Book of ASTM Standards, Vol 03.06.3Annual Book of ASTM Standards, Vol 13.01.4Available from U.S. Pharmacopeia (USP), 12601 Twinbrook Pkwy., Rockville,MD 20852.5Available from European Directorate for the Quality of Medicines (EDQM),Publications and Services, European Pharmacopoeia, BP 90
15、7, 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),6which represents the firstpublication on routine determination of c
16、hemical 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. Quantitative1H NMR spec-troscopy reports directly on the relative
17、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 NMR spectra, depoly-merization of chitosans to a number average de
18、gree 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 nitrous acid degradationin deuterated water. The reaction is sele
19、ctive, 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 filters and appropriate digitalsignal processing is recommended for
20、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 integration of the relevant1H NMR signals; K1, H1D,H1A, H2D and HAc
21、 (for chitosan chloride only) (Figs. 1 and2). Correct phasing and flat baseline is essential for good result.6Vrum, K. M., Anthonsen, M. W., Grasdalen, H., and Smidsrd, O., “Deter-mination of the Degree of N-acetylation and the Distribution of N-acetyl Groups inPartially N-deacetylated Chitins (Chit
22、osans) by High-Field N.M.R. Spectroscopy-,”Carbohydr. Res., 211, 1991, pp. 1723.7Allan, G. G. and Peyron, M., “Molecular Weight Manipulation of Chitosan 1:Kinetics of Depolymerization by Nitrous Acid,” Carbohydr. Res., 277, 1995, pp.257-272.F22600326.3 CalculationsFor chitosan chloride, signal inten
23、sitiesof 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 estimate of % DA.Averaging of the two acetylated signals cannot be performedwith ch
24、itosan 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 in the figure. K1: Proton 1 of chitose. H1D: Proton 1 of GlcN-units. H1A: Proton
25、 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 Typical1H NMR Spectrum of Chitosan Chloride (%
26、 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 Typical1H NMR Spectrum of Chitosan Glutamate (% DA = 84)F2260033D 5 K1 1H1D1H2D!/2 (
27、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 expressed as:A 5H1A 1HAc/3!/2 chitosan chloride! (2)A 5 H1A chitosan glutamate!where H1A and HAc
28、 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 %!5100%*D/D1A! (3)6.3.4 The number average degree of polymerization (DPn)may be estimated as a
29、 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 insignificant with respect tothe calculated % DA.6.3.5 Chitosans With a Low Degree of Deacetylation
30、 (% 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 acetylated units). Such depolymerizationcan be identified by the presence of H1a reducing-
31、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, noting that the a-anomer accountsfor roughly23 of the anomer population. Consequently, fo
32、rthese 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), ignoring this note will typically introduce an error of1 to 2 units in the calculated % D
33、A (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 to be similar. The standard deviationof the method has been determined after validation
34、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 % DA values higherthan 50. The method may be used to measure high degrees ofdeacetylation. Consequently,
35、 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 regularly maintained and controlled. % DAis obtained by comparing the signal intensities fro
36、m 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 SpecificityIf there should be any impurities in thesample, unexpected proton signals will be sho
37、wn 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 data presentationcan be found in Practice E 386.APPENDIXES(Nonmandatory Information)X1.
38、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 in characterizing thedegree of deacetylation of chitosan salts used in such applica-tion
39、s.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). The two differentmonosaccharides differ only by the substitution at carbon 2;GlcNAc c
40、ontains 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 chain.F2260034ASTM International takes no position respecting the validity of any patent r
41、ights 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 of such rights, are entirely their own responsibility.This standard is subject to revision at a
42、ny 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 additional standardsand should be addressed to ASTM International Headquarters. Your comments
43、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 to the ASTM Committee on Standards, at the address shown below.This standard is copyrighted by
44、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 StructureF2260035
