1、Designation: F 2259 03 (Reapproved 2008)Standard 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 indicate
2、s 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. Scope1.1 This test method covers the determination of t
3、he com-position and 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
4、, a linear polymer 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 (t
5、hat is, NG1). Knowledgeof 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 matr
6、ix material for TEMPs, in celland tissue encapsulation applications, and in drug deliveryformulations.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, i
7、f 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:2E 386 Practice for Data Presentation Relating
8、to High-Resolution Nuclear Magnetic Resonance (NMR) Spectros-copyF 2064 Guide for Characterization and Testing of Alginatesas Starting Materials Intended for Use in Biomedical andTissue-Engineered Medical Products Application2.2 United States Pharmacopeia Document:USP 24-NF19 Nuclear Magnetic Resona
9、nce33. Terminology3.1 Definitions:3.1.1 alginate, na polysaccharide substance extractedfrom brown algae, mainly occurring in the cell walls andintercellular spaces of brown seaweed and kelp. Its mainfunction is to contribute to the strength and flexibility of theseaweed plant. Sodium alginate, and i
10、n particular calciumcross-linked alginate gels are used in Tissue EngineeredMedical Products (TEMPs) as biomedical matrices, controlleddrug delivery systems, and for immobilizing living cells.3.1.2 degradation, nchange in the chemical structure,physical properties, or appearance of a material. Degra
11、dationof 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 polymers.3.1.3 depolymerization, nreduction in the length of apolymer chain
12、to form shorter polymeric units.4. Significance and Use4.1 The composition and sequential structure of alginatedetermines the functionality of alginate in an application. Forinstance, the gelling properties of an alginate are highlydependent upon the monomer composition and sequentialstructure of th
13、e polymer. Gel strength will depend upon theguluronic acid content (FG) and also the average number ofconsecutive guluronate moieties in G-block structures (NG1).4.2 Chemical composition and sequential structure of algi-nate can be determined by1H- and13C-nuclear magnetic reso-nance spectroscopy (NM
14、R).Ageneral description of NMR can1This 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 May 1, 2008. Published June 2008
15、. Originallyapproved in 2003. Last previous edition approved in 2003 as F 2259 03.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary pag
16、e onthe ASTM website.3Available from U.S. Pharmacopeia (USP), 12601 Twinbrook Pkwy., Rockville,MD 20852-1790, http:/www.usp.org.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.be found in of the USP 24-NF19. The NMR method-ology and
17、assignments are based on data published by Gras-dalen et al. (1979, 1981, 1983).4,5,6The NMR technique hasmade it possible to determine the monad frequencies FM(frac-tion of mannuronate units) and FG(fraction of guluronateunits), the four nearest neighboring (diad) frequencies FGG,FMG,FGM,FMM, and t
18、he eight next nearest neighboring (triad)frequencies FGGG,FGGM,FMGG,FMGM,FMMM,FMMG,FGMM,FGMG. Knowledge of these frequencies enables number aver-ages of block lengths to be calculated. NGis the numberaverage length of G-blocks, and NG1is the number averagelength of G-blocks from which singlets (-MGM
19、-) have beenexcluded. Similarly, NMis the number average length ofM-blocks, and NM1is the number average length of M-blocksfrom which singlets (-GMG-) have been excluded.13C NMRmust be used to determine the M-centered triads and NM1.This test method describes only the1H NMR analysis ofalginate. Algi
20、nate can be well characterized by determining FGand NG1.4.3 In order to obtain well-resolved 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 todepolym
21、erize the alginate samples. Freeze-drying, followed bydissolution in 99 % D2O, and another 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 chelat
22、or, other agents such as EDTA and citratemay be used. Such interactions may lead to line 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 resi
23、dual water toan area outside that of interest.5. Materials5.1 Chemicals:5.1.1 Alginate sample.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 o
24、fpolymerization (DPn) is:DPn=(M+G+red-a+red-b)/(red-a + red-b)7. Range, Standard Deviation, 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 sequentialpar
25、ameters (monads, diads, and triads) as well. Consequently,sequential parameters should 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
26、 content:68 %” (standard deviation 61 %). M-rich alginates should bereported with mannuronic 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 approximatel
27、y 10 %.Consequently, NG1should be reported with 1 decimal place,and the standard deviation 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,
28、the range of the method is considered tospan the interval of FGvalues from 0.30 to 0.75. 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:FIG. 1 Th
29、e Region of the1H NMR Spectrum of Alginate Used for Quantitative AnalysisF 2259 03 (2008)37.6.1 AccuracyThis parameter is limited by how well theNMR instrument is regularly maintained and controlled. Thereare no reference samples for a true value of the fraction ofguluronate in alginate.7.6.2 Specif
30、icityIf 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 NMR peak area is proportional to thenumber of protons represented by that peak.7.7 Further recommendations for NM
31、R 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 guidanc
32、e in the characterization ofsodium alginate used in such applications.X2. BACKGROUNDX2.1 Alginate 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 seque
33、ntial arrange-ment along the polymer chain vary widely, depending on theorigin of the alginate. 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 a
34、lternatingsequence of M and G units (MG-blocks) co-exist. Thus, thealginate molecule cannot be described 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 NM
35、Rspectroscopy that alginate has no regular repeating unit.FIG. X2.1 Alginate StructureF 2259 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 d
36、etermination 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 any time by the responsible technical committee and must be reviewed every five years andif not revised, either reapproved
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38、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 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).F 2259 03 (2008)5
