1、Designation: F2260 03 (Reapproved 2012)1Standard 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 the yea
2、r 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.1NOTEEditorial changes were made to subsections 2.2, 2.3, and 4.5 i
3、n November 2012.1. Scope1.1 This test method covers the determination of the degreeof 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
4、 the character-ization of chitosan salts has been published as Guide F2103.1.2 The 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 suitabl
5、e for routine use.Knowledge of the degree of deacetylation is important for anunderstanding 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 uti
6、lity in drug deliveryapplications, as a scaffold or matrix material, and in cell 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 thesa
7、fety 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 use.2. Referenced Documents2.1 ASTM Standards:2F386 Test Method for Thickness
8、 of Resilient Flooring Ma-terials Having Flat SurfacesF2103 Guide for Characterization and Testing of ChitosanSalts as Starting Materials Intended for Use in Biomedicaland Tissue-Engineered Medical Product Applications2.2 United States Pharmacopeia Document:USP 35-NF30 Nuclear Magnetic Resonance32.3
9、 European Pharmacopoeia Document:European Pharmacopoeia Monograph 2008:1774 ChitosanChloride43. Terminology3.1 Definitions:3.1.1 chitosan, na linear polysaccharide consisting of(14) linked 2-acetamido-2-deoxy-D-glucopyranose (Glc-NAc) and 2-amino-2-deoxy-D-glucopyranose (GlcN). Chito-san is a polysa
10、ccharide derived by N-deacetylation of chitin.3.1.2 degradation, nchange in the chemical 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.
11、 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 monomers) in achitosan polymer molecule.3.1.4 depolymerization, nreduction in the length of apolymer chain to form
12、 shorter polymeric units.4. Significance and Use4.1 The degree of deacetylation of chitosan salts is animportant characterization parameter since the charge density1This test method is under the jurisdiction of ASTM Committee F04 on Medicaland Surgical Materials and Devices and is the direct respons
13、ibility of SubcommitteeF04.42 on Biomaterials and Biomolecules for TEMPs.Current edition approved Oct. 1, 2012. Published November 2012. Originallyapproved in 2003. Last previous edition approved in 2008 as F2260 03 (2008).DOI: 10.1520/F2260-03R12E01.2For referenced ASTM standards, visit the ASTM we
14、bsite, www.astm.org, orcontact ASTM Customer Service at serviceastm.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.4Av
15、ailable from European Directorate for the Quality of Medicines (EDQM),Publications and Services, European Pharmacopoeia, BP 907, F-67029 Strasbourg,France.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1of the chitosan molecule is res
16、ponsible 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 arrangemen
17、t of monomer units in chitin andchitosan. The test method described is primarily based on thework of Vrum et al. (1991),5which represents the firstpublication on routine determination of chemical compositionin chitosans by solution state1H NMR spectroscopy. This testmethod is applicable for determin
18、ing 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 concentration ofchemically distinct protons in the sample, consequently, noassumptions, calibration curves or ca
19、lculations other thandetermination of relative signal intensity ratios are necessary.4.3 In order to obtain well-resolved NMR spectra, depo-lymerization of chitosans to a number average degree ofpolymerization (DPn) of 15 to 30 is required. This reduces theviscosity and increases the mobility of the
20、 molecules.Althoughthere are several options for depolymerization of chitosans, themost convenient procedure is that of nitrous acid degradationin deuterated water. The reaction is selective, stoichiometricwith respect to GlcN, rapid, and easily controlled (Allan 32768 at400 MHz.Typical temperature
21、equilibration time is 15 min and spec-trum acquisition time is approximately 10 min or less.5Vrum, 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 (Chitosans) by High-Fi
22、eld 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, pp.257-272.F2260 03 (2012)126.2.1.2 The use of digital filters and appropriate di
23、gitalsignal 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 integration of the releva
24、nt1H NMR signals; K1, H1D,H1A, H2D and HAc (for chitosan chloride only) (Figs. 1 and2). Correct phasing and flat baseline is essential for goodresults.6.3 CalculationsFor chitosan chloride, signal intensitiesof H1D and H2D may be averaged. Similarly, intensities ofH1A and HAc/3 (3 protons in HAc) ma
25、y 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 chitosan glutamate, due to severe overlap of HAc withglutamate signals (Figs. 1 and 2).6.3.1 The rel
26、ative number of GlcN-units in the polymerbefore depolymerization can be expressed as:D 5 K11H1D1H2D!/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
27、 can be expressed as:A 5 H1A1HAc/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 deac
28、etylation %! 5 100%*D/D1A! (3)6.3.4 The number average degree of polymerization (DPn)may be estimated as a control of the degradation as:DPn5 K11A1D!/K1 (4)DPnwill be overestimated by approximately 15 % due topartial saturation of K1 with the experimental parameters givenin this test method. This ef
29、fect is insignificant 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-cal
30、ly cleaves after acetylated units). Such depolymerizationcan be identified by the presence of H1 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-un
31、its given above, noting that the -anomer accountsfor roughly23 of the anomer population. Consequently, forthese chitosans, the relative number of GlcNAc-units is:A 5 1.5red 2 a1H1A1HAc/3!/2 chitosan chloride! (5)A 5 H1A11.5red 2 a chitosan glutamate!NOTE 1Signal assignments are indicated in the figu
32、re. 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 shift referen
33、ce at 0.000 ppm.FIG. 1 Typical1H NMR Spectrum of Chitosan Chloride (% DA = 85)F2260 03 (2012)136.3.5.2 For chitosans with low degree of deacetylation (%DA 60), ignoring this note will typically introduce an error of1 to 2 units in the calculated % DA (for example, % DA isassigned a value too high by
34、 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 to be lessthan 61 percentage unit.7.2 The determina
35、tion of low degrees of deacetylation byNMR is limited by the solubility of the sample. Experimentalresults indicate that the method is valid for % DAvalues higherthan 50. The method may be used to measure high degrees ofdeacetylation. Consequently, the range of the method is con-sidered to be valid
36、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 from the twocomponents, acetylated and deacetylated uni
37、ts. 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 shown in the spectra.7.3.3 LinearityNot relevant since
38、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 F386.NOTE 1Signal assignments are indicated in the figure (see also Fig. 1). Glutamate contributes
39、 with 1H NMR signals at 3.75 ppm, and multipletscentered at 2.5 and 2.1 ppm, the latter overlapping with HAc.FIG. 2 Typical1H NMR Spectrum of Chitosan Glutamate (% DA = 84)F2260 03 (2012)14APPENDIXES(Nonmandatory Information)X1. RATIONALEX1.1 The use of naturally occurring biopolymers for bio-medica
40、l and pharmaceutical applications and in Tissue Engi-neered Medical Products (TEMPs) is increasing. This testmethod is designed to give guidance in characterizing thedegree of deacetylation of chitosan salts used in such applica-tions.X2. BACKGROUNDX2.1 Chitosan is a linear, binary polysaccharide co
41、nsistingof (14) linked 2-acetamido-2-deoxy-D-glucopyranose (Gl-cNAc; acetylated unit) and 2-amino-2-deoxy-D-glucopyranose(GlcN; deacetylated unit). The two different monosaccharidesdiffer only by the substitution at carbon 2; GlcNAc contains anN-acetylated amino group, whereas GlcN contains only the
42、amino-group (it is said to be deacetylated). Thus, the degree ofdeacetylation (in %) is a measure of the fraction of GlcN-unitsin the chitosan chain.FIG. X2.1 Chitosan StructureF2260 03 (2012)15ASTM International takes no position respecting the validity of any patent rights asserted in connection w
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45、tion 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 ASTM International, 100 Barr H
46、arbor 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). Permission rights to photocopy the standard may also be secured from the ASTM website (www.astm.org/COPYRIGHT/).F2260 03 (2012)16
