核磁共振波谱法测定b型流感嗜血杆菌荚膜多糖中的核糖含量
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摘要
目的建立核磁共振波谱(nuclear magnetic resonance,NMR)法测定b型流感嗜血杆菌(Haemophilus influenzae type b,Hib)荚膜多糖中核糖含量的方法。方法利用NMR技术中的氢核磁共振波谱(1H-NMR)的定量功能,对不同浓度的D-核糖进行定量分析,验证该方法的精密度及准确度,并与传统化学方法进行比较。结果 NMR法定量结果相对标准偏差(RSD)均小于1.0%,相对误差除最低浓度20 mmol/L为7.4%外,其余浓度均在1%~3%之间,不同浓度的样品回收率在97.2%~107%之间;化学法定量结果 RSD在0.6%~7.9%之间,相对误差在3%~6.5%之间,不同浓度样品回收率在93.5%~105.3%之间;两种方法检测的3批Hib荚膜多糖中核糖含量无明显差别,均在《中国药典》三部(2010版)规定的320~410 mg/g之间。结论 1H-NMR定量方法取样少,可直接定量,该方法精密度高、准确度高、重现性好,且不破坏多糖结构,可应用于细菌荚膜多糖质量控制的核糖定量。
Objective To develop a method for determination of ribose content in Haemophilus influenzae type b polysaccharide by nuclear magnetic resonance(NMR). Methods D-ribose at various concentrations were analyzed by the quantitative function of1H-NMR. The developed method was verified for precision and accuracy,and compared with the traditional chemical analysis. Results The relative standard deviations(RSD)of results of quantitative NMR were less than 1. 0%,while the relative errors,except that of samples at a concentration of 20 mmol / L(7. 4%),were 1% ~ 3%.The recovery rates of samples at various concentrations were 97. 2% ~ 107%. However,the relative standard deviations(RSDs)of results of chemical analysis were 0. 6% ~ 7. 9%,while the relative errors were 3% ~ 6. 5%,and the recovery rates of samples were 93. 5% ~ 105. 3%. Conclusion The samples for quantitative NMR method are in a small quantity and may be quantified directly. With high precision,reproducibility and accuracy,the method may be used for quantitation of ribose in quality control of bacterial polysaccharide,by which no polysaccharide structure is destroyed.
Objective To develop a method for determination of ribose content in Haemophilus influenzae type b polysaccharide by nuclear magnetic resonance(NMR). Methods D-ribose at various concentrations were analyzed by the quantitative function of1H-NMR. The developed method was verified for precision and accuracy,and compared with the traditional chemical analysis. Results The relative standard deviations(RSD)of results of quantitative NMR were less than 1. 0%,while the relative errors,except that of samples at a concentration of 20 mmol / L(7. 4%),were 1% ~ 3%.The recovery rates of samples at various concentrations were 97. 2% ~ 107%. However,the relative standard deviations(RSDs)of results of chemical analysis were 0. 6% ~ 7. 9%,while the relative errors were 3% ~ 6. 5%,and the recovery rates of samples were 93. 5% ~ 105. 3%. Conclusion The samples for quantitative NMR method are in a small quantity and may be quantified directly. With high precision,reproducibility and accuracy,the method may be used for quantitation of ribose in quality control of bacterial polysaccharide,by which no polysaccharide structure is destroyed.
引文
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[17]Mawas F,Bolgiano B,Rigsby P,et al.Evaluation of the saccharide content and stability of the first WHO International Standard for Haemophilus influenzae b capsular polysaccharide[J].Biologicals,2007,35(4):235-245.
[18]Yan ZL.Quantitative study of main sugars and other main ingredients in honey,sweet wine and raw orange juice by nuclear magnetic resonance[D].Changsha:Hunan Normal University,2009.(in Chinese)阎政礼.蜂蜜、甜型葡萄酒和桔子原汁中主要糖类及其它主成分的NMR定量研究[D].湖南长沙:湖南师范大学,2009.
[19]Chinese Pharmacopoeia Commission(ChPC).Pharmacopoeia of People′s Republic of China(VolⅢ)[S].Beijing:China Med Sci Press,2010.(in Chinese)国家药典委员会.中华人民共和国药典(三部)[S].北京:中国医药科技出版社,2010.
[20]Drew KN,Zajicek J,Bondo G,et al.13C-labeled aldopen-toses:detection and quantitation of cyclic and acyclic forms by heteronuclear 1D and 2D NMR spectroscopy[J].Carbohyd Res,1998,307(3):199-209.
[2]Hutter J,Pasetti MF,Sanogo D,et al.Naturally acquired and conjugate vaccine-induced antibody to Haemophilus influenzae type b(Hib)polysaccharide in Malian children:serological assessment of the Hib immunization program in Mali[J].Am J Trop Med Hyg,2012,86(6):1026-1031.
[3]Weber M,Hellriegel C,Rueck A,et al.Useing high-performance quantitative NMR(HP-qNMR誖)for certifying traceable and highly accurate purity values of organic reference materials with uncertainties<0.1%[J].Accredit Qual Assur,2013,18(2):91-98.
[4]Krüger MB,Selle C,Heller D,et al.Determination of gas concentrations in liquids by nuclear magnetic resonance:hydrogen in organic solvents[J].J Chem Engi Data,2012,57(6):1737-1744.
[5]Walker GS,Ryder TF,Sharma R,et al.Validation of isolated metabolites from drug metabolism studies as analytical standards by quantitative NMR[J].Drug Metab Dispos,2011,39(3):433-440.
[6]Holzgrabe U,Malet-Martino M.Analytical challenges in drug counterfeiting and falsification-the NMR approach[J].J Pharm Biomed Anal,2011,55(4):679-687.
[7]Schicho R,Shaykhutdinov R,Ngo J,et al.Quantitative metabolomic profiling of serum,plasma,and urine by1H NMR spectroscopy discriminates between patients with inflammatory bowel disease and healthy individuals[J].Prot Res,2012,11(6):3344-3357.
[8]Carrola J,Rocha CM,Barros AS,et al.Metabolic signatures of lung cancer in biofluids:NMR-based metabonomics of urine[J].Prot Res,2011,10(1):221-230.
[9]Barding GA Jr,Salditos R,Larive CK.Quantitative NMR for bioanalysis and metabolomics[J].Anal Bioanalyt Chem,2012,404(4):1165-1179.
[10]Garrido R,Puyada A,Fernández A,et al.Quantitative proton nuclear magnetic resonance evaluation and total assignment of the capsular polysaccharide Neisseria meningitidis serogroup X[J].JPharmBiomedAnal,2012,70(1):295-300.
[11]Wang X,Ren KM,Chen XH,et al.Application of NMR spectrum in identification of pneumococcal capsular polysaccharides[J].Prog Microbiol Immunol,2013,41(3):18-23.(in Chinese)王玺,任克明,陈晓航,等.核磁共振波谱法在肺炎球菌荚膜多糖检定中的应用[J].微生物学免疫学进展,2013,41(3):18-23.
[12]Jones C,Lemercinier X.Use and validation of NMR assays for the identity and O-acetyl content of capsular polysaccharides from Neisseria meningitidis used in vaccine manufacture[J].J Pharm Biomed Anal,2002,30(4):1233-1247.
[13]Lemercinier X,Jones C.Full 1H NMR assignment and detailed O-acetylation patterns of capsular polysaccharides from Neisseria meningitidis used in vaccine production[J].Carbohyd Res,1996,296(1):83-96.
[14]Zhang Q,Li XD,Yang HX.Application of nuclear magnetic resonance in pharmaceutical standards[J].Chin J Pharm Anal,2012,32(3):545-549.(in Chinese)张琪,李晓东,杨化新.核磁共振技术在药品标准领域中的应用进展[J].药物分析杂志,2012,32(3):545-549.
[15]Europeanpharmacopoeia[S].7.0thed.812.
[16]World Health Organization.Recommendations to assure the quality,safety and efficacy of pneumococcal conjugate vaccine[R].ReplacementofTRS,2010,927:Annex2.
[17]Mawas F,Bolgiano B,Rigsby P,et al.Evaluation of the saccharide content and stability of the first WHO International Standard for Haemophilus influenzae b capsular polysaccharide[J].Biologicals,2007,35(4):235-245.
[18]Yan ZL.Quantitative study of main sugars and other main ingredients in honey,sweet wine and raw orange juice by nuclear magnetic resonance[D].Changsha:Hunan Normal University,2009.(in Chinese)阎政礼.蜂蜜、甜型葡萄酒和桔子原汁中主要糖类及其它主成分的NMR定量研究[D].湖南长沙:湖南师范大学,2009.
[19]Chinese Pharmacopoeia Commission(ChPC).Pharmacopoeia of People′s Republic of China(VolⅢ)[S].Beijing:China Med Sci Press,2010.(in Chinese)国家药典委员会.中华人民共和国药典(三部)[S].北京:中国医药科技出版社,2010.
[20]Drew KN,Zajicek J,Bondo G,et al.13C-labeled aldopen-toses:detection and quantitation of cyclic and acyclic forms by heteronuclear 1D and 2D NMR spectroscopy[J].Carbohyd Res,1998,307(3):199-209.