冻干b型流感嗜血杆菌结合疫苗中蔗糖含量检测方法的建立及应用  

Development and application of a method for determination of sucrose content in freeze-dried Haemophilus influenzae type b conjugate vaccine

作　　者：强鹏侠 姜珊 卓晓琴 蔡文博 雒丽红 冯潇 傅元欣 冯德杰 QIANG Peng-xia;JIANG Shan;ZHUO Xiao-qin;CAI Wen-bo;LUO Li-hong;FENG Xiao;FU Yuan-xin;FENG De-jie(The Quality Control Department in Lanzhou Institute of Biological Products Co.,Ltd.,Center for Gansu Provincial Vaccine Engineering Research,Lartzhou 730046,Gansu Province,China)

机构地区：[1]兰州生物制品研究所有限责任公司质量检定室,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》2021年第4期38-43,共6页Progress In Microbiology and Immunology

摘　　要：目的建立用高效液相色谱法(high performance liquid chromatography, HPLC)检测冻干b型流感嗜血杆菌结合疫苗中蔗糖含量的方法,并对方法进行初步应用。方法样品经离心除去蛋白质,上清液采用氨基柱分离,示差折光检测器进行检测,依外标法建立校正曲线,并对建立的方法进行验证及初步应用。结果蔗糖质量浓度在2.5~40.0 mg/mL范围内,r2均>0.999;5.0、10.0、20.0 mg/mL不同质量浓度的3个蔗糖对照品溶液平均回收率分别为99.9%、100.2%、100.7%;最小检出限和定量限分别为0.005、0.050 mg/mL;分别改变柱温及流速,即柱温从30℃改变为32℃,流速从1.5 mL/min改变为1.3 mL/min时,对蔗糖含量检测无明显影响,表明该方法具有良好的耐用性。初步应用结果显示,建立的方法可准确检测疫苗的蔗糖含量,RSD最大仅为0.6%,精密度良好,具有很好的重复性。结论建立了用HPLC检测冻干b型流感嗜血杆菌结合疫苗中蔗糖含量的方法,该方法线性范围良好、重复性好、定量准确,适用于冻干b型流感嗜血杆菌结合疫苗中蔗糖含量的检测。Objective To develop and preliminary application a HPLC method for determination of sucrose content in lyophilized Haemophilus influenzae type b conjugate vaccine. Methods Test samples were centrifuged to remove the protein, and the supernatant was separated by aminocolumn chromatography and monitored by refractive index detector(RID), based on which a correction curve was plotted by external standard method, and the developed method was verified. Results The sucrose concentration at a range of 2.5-40.0 mg/mL showed good linear relationship, the correlation coefficients r2 was more than 0.999;The mean recovery rates of three sucrose reference solutions at low, moderate and high concentrations were 99.9%, 100.2% and 100.7%, respectively. The minimum and quantitative detection limits of the developed method were 0.005 mg/mL and 0.050 mg/mL, respectively. When the column temperature was changed from 30 ℃ to 32 ℃ and the flow rate was changed from 1.5 mL/min to 1.3 mL/min, there was no significant effect on the determination of sucrose content, indicating that the method had good durability. The preliminary application results showed that the established method can accurately detect the sucrose content of the vaccine, with the maximum RSD of only 0.6% and good precision and repeatability. Conclusion A HPLC method for determination of sucrose contents in lyophilized Haemophilus influenzae type b conjugate vaccine was developed. The method has good linear range, good repeatability and accurate quantification, which is suitable for the determination of sucrose content in lyophilized Haemophilus influenzae type b conjugate vaccine.

关 键 词：冻干b型流感嗜血杆菌结合疫苗 蔗糖 高效液相色谱法 

分 类 号：K378.41[历史地理—历史学]