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

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(...

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

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 1.418×10⁵~2.281×10⁵respectively.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.