肺炎球菌多糖衍生物中1,4-丁二胺残留量核磁共振检测方法的建立及验证  

Development and verification of a nuclear magnetic resonance method for determination of residual 1,4-butanediamine content in pneumococcal polysaccharide derivatives

作　　者：杜娇 周海飞 李献林 马凯凯 张钰奇 马钰 杨英英 杨田瑶 李阿妮 罗树权 DU Jiao;ZHOU Hai-fei;LI Xian-lin;MAKai-kai;ZHANG Yu-qi;MA Yu;YANG Ying-ying;YANG Tian-yao;LI A-ni;LUO Shu-quan(The First ResearchDepartment,Lanzhou Institute of Biological Products Co.,Lid.,Center for Gansu Provincial Vaccine Engineering Research,Lanzhou 730046,Gansu Province,China)

机构地区：[1]兰州生物制品研究所有限责任公司第一研究室,甘肃省疫苗工程技术研究中心,甘肃兰州730046

出　　处：《微生物学免疫学进展》2022年第5期21-27,共7页Progress In Microbiology and Immunology

基　　金：国家科技重大专项重大新药创制(2018ZX09738-006)。

摘　　要：目的建立核磁共振(nuclear magnetic resonance,NMR)法检测肺炎球菌多糖衍生物中1,4-丁二胺残留量的方法,并对方法进行验证。方法建立氢谱(^(1)H-NMR)检测方法:脉冲程序为zg30,温度为298 K(24.85℃),图谱宽度为7211 Hz,扫描次数为512次,中心频率为4.70 ppm,弛豫延迟时间为9 s;对该方法进行线性、专属性、准确度、精密度、耐用性验证,并确定该方法的定量限。结果以1,4-丁二胺中H-1和H-4位质子峰为特征峰,该方法检测1,4-丁二胺专属性较好。在0.05~5.00 mg/mL范围内,1,4-丁二胺质量浓度与其对应的峰面积呈良好的线性关系,相关系数R^(2)=0.999。1,4-丁二胺添加实验回收率在92.73%~99.51%之间;建立的方法重复性及中间精密度均良好,不同质量浓度1,4-丁二胺测定值RSD均小于5%;该方法的定量限为0.01 mg/mL;改变温度和扫描次数后1,4-丁二胺的检测结果差异均无统计学意义(P>0.05)。结论建立的~1H-NMR检测方法简便、快速、准确,经验证各项指标均符合质控要求,可用于肺炎球菌多糖衍生物中1,4-丁二胺残留量的测定。Objective To develop and verify a nuclear magnetic resonance(NMR)method for determination of residual 1,4-butanediamine content in pneumococcal polysaccharide derivatives.Methods The pulse program was zg30.The ^(1)H-NMR spectra were collected at 298 K with a spectral width of 7211 Hz,accumulating 512 scans,center frequency 4.70 ppm,and the relaxation delay time was 9 s.The developed method was verified for its linearity,specificity,accuracy,precision and ruggedness,and determined its quantitative detection limit.Results The integrated areas of H-1 and H-4 proton signals in 1,4-butanediamine was took as the characteristic peak.The developed method showed high specificity for determination of 1,4-butanediamine content and showed good linear relationship with its corresponding peak area in the range of 0.05~5.00 mg/mL,and the R^(2)=0.999.The recovery rates of 1,4-butanediamine in spike test were between 92.73%~99.51%;The method has good repeatability and intermediate precision,the RSDs of different concentrations of 1,4-butanediamine determination results were less than 5%.The minimum quantification limit of this method was 0.01 mg/mL,The determination results showed no significant difference(P>0.05)after adjusting the temperature and the number of scans.Conclusion The developed~1H-NMR method was simple,rapid,accurate,and all the verification results met the requirements of quality control,which is suitable for the determination of the residual 1,4-butanediamine in pneumococcal polysaccharide derivatives.

关 键 词：肺炎球菌 多糖衍生物 1 4-丁二胺 核磁共振 残留量 验证 

分 类 号：R392[医药卫生—免疫学]