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目的:建立核磁共振(nuclear magnetic resonance,NMR)方法检测肺炎球菌多糖衍生物中吡啶硼烷残留量的方法。方法:建立1H-NMR检测方法:脉冲程序为水峰压制,温度为298 K(25℃)。具体参数设置如下:中心频率约为4.7 ppm左右;图谱宽度为7 211 Hz;采样时间为2.272 s;弛豫延迟时间为2 s;扫描次数为512次;信号经傅里叶转换后加入1.0 Hz的窗口函数,检测结果使用Topspin 3.0软件进行处理。并验证该方法的专属性、检测限及耐用性。结果:该方法检测结果显示溶剂对所测组分无干扰;吡啶硼烷与肺炎球菌多糖衍生物有各自明显的特征峰,两者混合之后信号无交叉重叠,并都可以明显检测到各自的信号,说明方法专属性良好。吡啶硼烷浓度在1%~0.01%范围内均可以明显检测到Py-BH3信号,且S/N良好。最终最低检测限定为0.02%。改变试验温度化学位移及信噪比未发生明显变化,方法显示耐用性良好。结论:建立的1H-NMR检测方法检测肺炎球菌多糖衍生物中吡啶硼烷残留具有极高的专属性和耐用性,可用于肺炎球菌结合疫苗中吡啶硼烷残留量的测定。
Objective: To establish a method for the determination of pyridine borane in pneumococcal polysaccharide derivatives by nuclear magnetic resonance (NMR) method. Methods: To establish 1H-NMR detection method: the pulse program is water peak suppression, the temperature is 298 K (25 ℃). The specific parameters are set as follows: the center frequency is about 4.7 ppm, the width of the spectrum is 7 211 Hz, the sampling time is 2.272 s, the relaxation delay time is 2 s, the number of scanning times is 512 times, the signal is Fourier transformed and then added to 1.0 Hz The window function, the test results using Topspin 3.0 software for processing. And verify the method’s specificity, limit of detection and durability. Results: The results of this method showed that the solvent had no interference with the measured components. Pyridine borane and pneumococcal polysaccharide derivatives had distinct characteristic peaks. After mixed, the signals did not overlap each other and both of them could obviously detect their own signals , Indicating that the method is good. The Py-BH3 signal could be detected obviously with the concentration of pyridine borane in the range of 1% ~ 0.01%, and the S / N was good. The final minimum detection limit is 0.02%. Changes in test temperature chemical shift and signal to noise ratio did not change significantly, the method showed good durability. CONCLUSION: The established 1H-NMR detection method for detecting pyridine borane residues in pneumococcal polysaccharide derivatives has high specificity and durability and can be used for the determination of pyridine borane residues in pneumococcal conjugate vaccine.