1、Designation: F2259 10 (Reapproved 2012)1Standard Test Method forDetermining the Chemical Composition and Sequence inAlginate by Proton Nuclear Magnetic Resonance (1H NMR)Spectroscopy1This standard is issued under the fixed designation F2259; the number immediately following the designation indicates
2、 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.1NOTEEditorial changes were made to subsections 2.2 and 4.2
3、 in November 2012.1. Scope1.1 This test method covers the determination of the 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 c
4、haracter-ization of alginate has been published as Guide F2064.1.2 Alginate, a linear polymer composed of -D-mannuronate (M) and its C-5 epimer -L-guluronate (G) linkedby -(14) glycosidic bonds, is characterized by calculatingparameters such as mannuronate/guluronate (M/G) ratio, gu-luronic acid con
5、tent (G-content), and average length of blocksof consecutive G monomers (that is, NG1). Knowledge ofthese 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 th
6、eir particular application. Alginate mayhave utility as a scaffold or matrix 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.
7、1.4 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 use.2. Referenced D
8、ocuments2.1 ASTM Standards:2E386 Practice for Data Presentation Relating to High-Resolution Nuclear Magnetic Resonance (NMR) Spec-troscopyF2064 Guide for Characterization and Testing of Alginatesas Starting Materials Intended for Use in Biomedical andTissue-Engineered Medical Products Application2.2
9、 United States Pharmacopeia Document:USP 35-NF30 Nuclear Magnetic Resonance33. Terminology3.1 Definitions:3.1.1 alginate, npolysaccharide obtained from some ofthe more common species of marine algae, consisting of aninsoluble mix of calcium, magnesium, sodium, and potassiumsalts.3.1.1.1 DiscussionAl
10、ginate exists in brown algae as itsmost abundant polysaccharide, mainly occurring in the cellwalls and intercellular spaces of brown seaweed and kelp.Alginates main function is to contribute to the strength andflexibility of the seaweed plant. Alginate is classified as ahydrocolloid. The most common
11、ly used alginate is sodiumalginate. Sodium alginate and, in particular, calcium cross-linked alginate gels are used in Tissue Engineered MedicalProducts (TEMPs) as biomedical matrices, controlled drugdelivery systems, and for immobilizing living cells.3.1.2 degradation, nchange in the chemical struc
12、ture,physical properties, or appearance of a material. Degradation1This 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
13、Oct. 1, 2012. Published November 2012. Originallyapproved in 2003. Last previous edition approved in 2010 as F2259 10. DOI:10.1520/F2259-10R12E01.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards
14、 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.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United S
15、tates1of 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
16、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
17、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 alg-inate can be determined by1H- and13C-nuclear magneticresonance spectroscopy (NMR)
18、.Ageneral description of NMRcan be found in of the USP 35-NF30. The NMRmethodology and assignments are based on data published byGrasdalen et al. (1979, 1981, 1983).4,5,6The NMR techniquehas made it possible to determine the monad frequencies FM(fraction of mannuronate units) and FG(fraction of gulu
19、ronateunits), the four nearest neighboring (diad) frequencies FGG,FMG,FGM,FMM, and the 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 o
20、f G-blocks, and NG1is the number averagelength of G-blocks from which singlets (-MGM-) 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-centere
21、d triads and NM1.This test method describes only the1H NMR analysis ofalginate. Alginate 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 algina
22、te 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 another freeze-drying beforedissolution in 99.9 % D2O yields samples with low1H2Ocontent. TTHA is used as a chelator to prevent trace
23、s 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 broadeningand selective loss of signal intensity.4.4 Samples are analyzed at a temperature of 80 6 1C.Elevated sample temperatu
24、re 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 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 a
25、re 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 Deviation, and Reporting Results7.1 Data suggest that a suitable value for repeatability andintermediate precision (as measured by the
26、standard deviation,FIG. 1 The Region of the1H NMR Spectrum of Alginate Used for Quantitative AnalysisF2259 10 (2012)13SD) for FGis 0.01. This value applies for all other sequentialparameters (monads, diads, and triads) as well. Consequently,sequential parameters should be reported with 2 significant
27、decimals 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 mannuronic acid content as a percen
28、tage, 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 deviation for NG1should be calculate
29、d 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. If this testmethod is to be
30、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 and controlled. Thereare no refe
31、rence 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 NMR peak area is proportional
32、 to thenumber of protons represented by that peak.7.7 Further recommendations for NMR data presentationcan be found in Practice E386.APPENDIXES(Nonmandatory Information)X1. RATIONALEX1.1 The use of naturally occurring biopolymers for bio-medical and pharmaceutical applications and in Tissue Engi-nee
33、red Medical Products (TEMPs) is increasing. This testmethod is designed to give guidance 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 -D-mannuronic acid (M)and -L-guluronic
34、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. The uronic acid residues are distributedalong the polymer chain in a pattern of blocks, where ho-mopolymeric blocks of
35、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 described by the monomer com-position alone. NMR characterization of the sequence of M andG residues in the alginate cha
36、in 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 StructureF2259 10 (2012)14ASTM International takes no position respecting the validity of any patent rights asserted in connection with an
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