b型流感嗜血杆菌精制多糖分子大小分布及重均分子质量检测方法的改良

Modification of methods for determination of molecular size and molecular weight of Haemophilus influenzae type b polysaccharide

目的改良HPLC(AKTA层析)系统,采用HPLC-MALLS系统检测b型流感嗜血杆菌(Haemophilus influenzae type b,Hib)精制多糖的分子大小分布及重均分子质量(MW),并进行方法的验证。方法采用HPLC(AKTA层析)系统连接示差分析仪检测9批Hib精制多糖供试品的分配系数(KD)及K_(D)值为0.5的回收率(R_(X))。采用HPLC连接18角度静态光散射分析系统MALLS(HPLC-MALLS)检测供试品的M_(W)、K_(D)及R_(X),并对两种系统的测试结果进行相关性分析;验证HPLC-MALLS方法的重复性、准确性及定量限。结果 9批供试品经AKTA层析系统检测,K_(D)为0.29~0.37,RX为78%~90%;经HPLC-MALLS系统检测,KD为0.31~0.38,RX为84%~93%,MW为(1.418~2.281)×10^(5)g/moL,两种系统检测同批供试品的KD值回归方程为Y=0.903 X+0.051,r为0.978,表明检测结果具有显著相关性,两种方法等效。9批供试品经HPLC-MALLS/RI系统重复检测6次,M_(W)、K_(D)及R_(X)的RSD均小于3.8%,多糖回收率及其RSD分别为80%~87%和0.05%~3.46%;定量限为1 mg/mL。结论 HPLC-MALLS系统能与HPLC系统基本对接,在准确检测Hib精制多糖M_(W)的同时还可检测K_(D)及R_(X),且方法的准确性及重复性良好,可作为检测Hib精制多糖分子大小的改良方法。

Objective To modify HPLC (AKTA chromatography) system,determine the molecular size and molecular weight(MW)of Haemophilus influenzae type b(Hib)polysaccharides by HPLC-MALLS system,and verify the methods.Methods Nine batches of Hib polysaccharides were determined for distribution coefficient (K_(D))value and recovery rate(RX)when the K_(D) was 0.5 by HPLC (AKTA chromatography)system linked with differential analyzer.HPLC system was linked with Multi-Angle Laser Scattering(MALLS)analyzer and used for determination of M_(W),K_(D) and R_(X) of test samples.The relationship between determination results by HPLC(AKTA chromatography) and HPLC-MALLS were analyzed.The developed HPLC-MALLS method was verified for reproducibility,accuracy and quantitative limit.Results The determination result of K_(D) values of nine batches of test samples by HPLC (AKTA chromatography)were 0.29~0.37,while the RXvalues were 78%~90%.However,the determination results of KD value,RX value and M_(W) by HPLC-MALLS were 0.31~0.38,84%~93%and M_(W) were 1.418×10^(5)~2.281×10^(5)respectively.The regression equation of K_(D) values of the same batch of samples determined by the two methods was as follows:Y=0.903 X+0.051,with an r value of0.978,indicating significant relationship of determination result and equivalence of the two methods.All the RSDs of determination results of M_(W),K_(D) and R_(X) of nine batches of samples in six repeat tests were less than 3.8%,while t he recovery rates of polysaccharide were 80%~87%with RSDs of 0.05%~3.46%,and the quantitative limit was 1 mg/m L.Conclusion HPLC-MALLS system may be used for the determination of M_(W),K_(D) and R_(X) of purified Hib polysaccharide instead of HPLC system,which is accurate and reproducible.