1、Designation: F 2259 03Standard Test Method forDetermining the Chemical Composition and Sequence inAlginate by Proton Nuclear Magnetic Resonance (1H NMR)Spectroscopy1This standard is issued under the fixed designation F 2259; the number immediately following the designation indicates the year oforigi
2、nal adoption 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 com-position a
3、nd monomer sequence of alginate intended for use inbiomedical 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 alginate has been published as Guide F 2064.1.2 Alginate, a linear polyme
4、r composed of b-D-mannuronate (M) and its C-5 epimer a-L-guluronate (G)linked by b-(14) glycosidic bonds, is characterized bycalculating parameters such as mannuronate/guluronate (M/G)ratio, guluronic acid content (G-content), and average length ofblocks of consecutive G monomers (that is, NG1). Kno
5、wledgeof these parameters is important for an understanding of thefunctionality of alginate in TEMP formulations and applica-tions. This test method will assist end users in choosing thecorrect alginate for their particular application. Alginate mayhave utility as a scaffold or matrix material for T
6、EMPs, in celland tissue encapsulation applications, and in drug deliveryformulations.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 an
7、d determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:E 386 Practice for Data Presentation Relating to High-Resolution Nuclear Magnetic Resonance (NMR) Spectros-copy2F 2064 Guide for Characterization and Testing of Alginatesas Starting Materi
8、als Intended for Use in Biomedical andTissue-Engineered Medical Products Application32.2 United States Pharmacopeia Document:USP 24-NF19 Nuclear Magnetic Resonance43. Terminology3.1 Definitions:3.1.1 alginate, na polysaccharide substance extractedfrom brown algae, mainly occurring in the cell walls
9、andintercellular spaces of brown seaweed and kelp. Its mainfunction is to contribute to the strength and flexibility of theseaweed plant. Sodium alginate, and in particular calciumcross-linked alginate gels are used in Tissue EngineeredMedical Products (TEMPs) as biomedical matrices, controlleddrug
10、delivery systems, and for immobilizing living cells.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 withdecompos
11、ition. Degradation is often used as a synonym fordepolymerization when referring to polymers.3.1.3 depolymerization, nreduction in the length of apolymer chain to form shorter polymeric units.4. Significance and Use4.1 The composition and sequential structure of alginatedetermines the functionality
12、of alginate in an application. Forinstance, the gelling properties of an alginate are highlydependent upon the monomer composition and sequentialstructure of the polymer. Gel strength will depend upon theguluronic acid content (FG) and also the average number ofconsecutive guluronate moieties in G-b
13、lock structures (NG1).4.2 Chemical composition and sequential structure of algi-nate can be determined by1H- and13C-nuclear magnetic reso-nance spectroscopy (NMR). A general description of NMR canbe found in of the USP24-NF19. The NMR methodol-ogy and assignments are based on data published by Grasd
14、alenet al. (1979, 1981, 1983).5,6,7The NMR technique has made it1This 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 Ap
15、r. 10, 2003. Published May 2003.2Annual 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 20852.5Grasdalen, H., Larsen, B., and Smidsrd, O., “A P.M.R. Study of theComposition and Sequence of Uronat
16、e Residues in Alginates.,” Carbohydr. Res., 68,1979, pp. 23-31.6Grasdalen, H., Larsen, B., and Smidsrd, O., “13C-NMR Studies of MonomericComposition and Sequence in Alginate,” Carbohydr. Res., 89, 1981, pp. 179-191.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken,
17、 PA 19428-2959, United States.possible to determine the monad frequencies FM(fraction ofmannuronate units) and FG(fraction of guluronate units), thefour nearest neighboring (diad) frequencies FGG,FMG,FGM,FMM, and the eight next nearest neighboring (triad) frequenciesFGGG,FGGM,FMGG,FMGM,FMMM,FMMG,FGM
18、M,FGMG.Knowledge of these frequencies enables number averages ofblock lengths to be calculated. NGis the number average lengthof G-blocks, and NG1is the number average length ofG-blocks from which singlets (-MGM-) have been excluded.Similarly, NMis the number average length of M-blocks, andNM1is the
19、 number average length of M-blocks from whichsinglets (-GMG-) have been excluded.13C NMR must be usedto determine the M-centered triads and NM1. This test methoddescribes only the1H NMR analysis of alginate. Alginate canbe well characterized by determining FGand NG1.4.3 In order to obtain well-resol
20、ved NMR spectra, it isnecessary to reduce the viscosity and increase the mobility ofthe molecules by depolymerization of alginate to a degree ofpolymerization of about 20 to 50. Acid hydrolysis is used todepolymerize the alginate samples. Freeze-drying, followed bydissolution in 99 % D2O, and anothe
21、r freeze-drying beforedissolution in 99.9 % D2O yields samples with low1H2Ocontent. TTHA is used as a chelator to prevent traces ofdivalent cations to interact with alginate. While TTHA is amore effective chelator, other agents such as EDTA and citratemay be used. Such interactions may lead to line
22、broadeningand selective loss of signal intensity.4.4 Samples are analyzed at a temperature of 80 6 1C.Elevated sample temperature contributes to reducing sampleviscosity and repositions the proton signal of residual water toan area outside that of interest.5. Materials5.1 Chemicals:5.1.1 Alginate sa
23、mple.5.1.2 Deionized water (Milli-Q Plus or equivalent; conduc-tivity 1=(FGFMGM)/FGGMNM=FM/FMG6.3.2.2 If reducing end signals are integrated (“red-a” and“red-b”), then the estimate of the number average degree ofpolymerization (DPn) is:DPn=(M+G+red-a + red-b)/(red-a + red-b)7. Range, Standard Deviat
24、ion, and Reporting Results7.1 Data suggest that a suitable value for repeatability andintermediate precision (as measured by the standard deviation,SD) for FGis 0.01. This value applies for all other sequentialparameters (monads, diads, and triads) as well. Consequently,sequential parameters should
25、be reported with 2 significantdecimals and a standard deviation of 0.01, for example, FG=0.68 6 0.01.7.2 G-rich alginates should be reported with guluronic acidcontent as a percentage, for example, “guluronic acid content:68 %” (standard deviation 61 %). M-rich alginates should bereported with mannu
26、ronic acid content as a percentage, forexample, “mannuronic acid content: 66 %” (standard deviation61 %).7.3 For NG1, the overall quality of the data suggests toreport a relative standard deviation of approximately 10 %.Consequently, NG1should be reported with 1 decimal place,and the standard deviat
27、ion for NG1should be calculated as10 % of the measured value, reported with 1 decimal place, forexample, NG1= 13.9 6 1.4.7.4 Block lengths NGand NMhave a relative standarddeviation of 0.1 or FG0.9). Consequently, the range of the method is considered tospan the interval of FGvalues from 0.30 to 0.75
28、. If this testmethod is to be used to characterize alginate anticipated tohave an FGbelow or above the stipulated interval, thenadditional validation may be necessary.7.6 Non-Applicable Method Parameters:7.6.1 AccuracyThis parameter is limited by how well theNMR instrument is regularly maintained an
29、d controlled. Thereare no reference samples for a true value of the fraction ofguluronate in alginate.7.6.2 SpecificityIf there should be any impurities in thesample, unexpected proton signals will be shown in the spectra.7.6.3 LinearityNot relevant since NMR spectroscopy isquantitative. Each proton
30、 NMR peak area is proportional to thenumber of protons represented by that peak.7.7 Further recommendations for NMR data presentationcan be found in Practice E 386.FIG. 1 The Region of the1H NMR Spectrum of Alginate Used for Quantitative AnalysisF2259033APPENDIXES(Nonmandatory Information)X1. RATION
31、ALEX1.1 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 the characterization ofsodium alginate used in such applications.X2. BACKGROUNDX2.1 Alginat
32、e is a family of non-branched binary copoly-mers of 1-4 glycosidically linked b-D-mannuronic acid (M)and a-L-guluronic acid (G) residues. The relative amount ofthe two uronic acid monomers and their sequential arrange-ment along the polymer chain vary widely, depending on theorigin of the alginate.
33、The uronic acid residues are distributedalong the polymer chain in a pattern of blocks, where ho-mopolymeric blocks of G residues (G-blocks), homopolymericblocks of M residues (M-blocks) and blocks with alternatingsequence of M and G units (MG-blocks) co-exist. Thus, thealginate molecule cannot be d
34、escribed by the monomer com-position alone. NMR characterization of the sequence of M andG residues in the alginate chain is needed in order to calculateaverage block lengths. It has also been shown by NMRspectroscopy that alginate has no regular repeating unit.FIG. X2.1 Alginate StructureF2259034AS
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