以组分百白破疫苗为基础的联合疫苗中气管细胞毒素定量分析液相色谱串联质谱联用方法的建立及验证  

Development and validation of liquid chromatography tandem mass spectrometry for quantitative analysis of tracheal cytotoxin in combined vaccines based on DTacP

作　　者：卫辰[1] 吴燕[1] 龙珍 黄元礼[1] 王丽婵 马霄[1] WEI Chen;WU Yan;LONG Zhen;HUANG Yuan-li;WANG Li-chan;MA Xiao(National Institutes for Food and Drug Control,Key Laboratory of the Ministry of Health far Research on Quality and Standardization of Biotech Products,Beijing 102629,China;不详)

机构地区：[1]中国食品药品检定研究院卫生部生物技术产品检定方法及其标准化重点实验室,北京102629 [2]岛津企业管理(中国)有限公司,北京100020

出　　处：《中国生物制品学杂志》2020年第12期1421-1425,1430,共6页Chinese Journal of Biologicals

基　　金：国家科技重大专项(2018ZX097380052017);国家自然科学基金青年科学基金(81701978)。

摘　　要：目的建立定量分析以组分百白破疫苗为基础的联合疫苗中百日咳鲍特氏菌气管细胞毒素(tracheal cytotoxin,TCT)的液相色谱串联质谱(liquid chromatography tandem mass spectrometry,LC-MS/MS)方法,并进行验证及初步应用。方法HPLC条件:色谱柱BioC18,流动相A为0.1%甲酸水溶液(V/V),流动相B为0.1%甲酸乙腈溶液(V/V),进样量为5μL,柱温为30℃,流速为0.3 mL/min;MS条件:电喷雾离子源正电子模式,电压为3.0 KV,离子源温度为300℃,脱溶剂管温度为250℃,雾化器流速为3 L/min,干燥气流速为10 L/min,采用多反应监测模式(multi reaction monitoring mode,MRM)检测TCT。验证方法的线性范围、灵敏度、精密度及准确度。采用该方法对国内外公司生产的组分百白破联合疫苗33批成品、63批原液、80批纯化蛋白进行TCT筛查及定量分析。结果TCT标准溶液在20.66~661 ng/L范围内,与TCT的定量高子通道922.3>719.3峰面积线性良好,线性方程为Y=3555.9 X-3368,R2=0.996;检出限为2.58 ng/L,定量限为10.33 ng/L;20.65、82.625及330.5 ng/L TCT标准溶液重复检测5次TCT峰面积的RSD分别为2.23%、4.61%和4.40%;66.1及33.05 ng/L的加标样品回收率分别为104.1%和94.4%。所有组分百日咳联合疫苗成品及原液样品均无TCT检出,纯化蛋白样品仅有一家公司的1批黏附素(pertactin,PRN)蛋白的裂解菌体浓缩液检出65.5 ng/L TCT,其后序工艺样品均未检出。结论成功建立了定量分析组分百白破联合疫苗中TCT的LC-MS/MS方法,该方法灵敏度高,准确度、精密度及线性良好,可用于我国组分百白破联合疫苗的TCT定量定性检测。Objective To develop,validate and preliminarily apply a liquid chromatography tandem mass spectrometry(LC-MS/MS)method for quantitative analysis of tracheal cytotoxin(TCT)of B.pertussis in combined vaccine based on DTacP.Methods The condition for HPLC was as follows:BioC18 chromatographic column was adopted,serving 0.1%(V/V)formic acid-aqueous solution as mobile phase A and 0.1%(V/V)formic acid-acetonitrile solution as mobile phase B.The sample load was 5μL,while the column temperature was 30℃,and the flow rate was 0.3 mL/min.However,the condition for MS was as follows:positron mode with electrospray ion source was adopted.The voltage was3.0 kV,while the temperatures of ion source and desolvent tube were 300 and 250℃,and the flow rates of atomizer and dry airflow were 3 and 10 L/min,respectively.The TCT was determined by multi reaction monitoring mode(MRM).The developed method was validated for linear range,sensitivity,precision and accuracy,and used for screening and quantitative analysis of TCT in 33 batches of final product,63 batches of bulk and 80 batches of purified protein of domestic and imported DTacP.Results The concentration of TCT standard at a range of 20.66~661 ng/L showed good linear relationship to the peak area of quantitativeion channel 922.3>719.3,with a linear equation of Y=3555.9 X-3368 and a R2 value of 0.996.The detection limit and quantitative limit of the developed method were 2.58 and 10.33 ng/L respectively.The RSDs of determination results of peak areas of TCT standard solutions at concentrations of 20.65,82.625 and 330.5 ng/L in five tests were 2.23%,4.61%and 4.40%,while the recovery rates of spike samples at concentrations of 66.1 and 33.05 ng/L were 104.1%and 94.4%,respectively.No TCT was detected in all the final products and bulks of DTacP,while only 65.5 ng/L TCT was detected in the lysed bacterial concentrate during the purification of one batch of PRN protein from one manufacturer.However,no TCT was detected in the subsequent process of the protein.Conclusion The LC-MS/MS

关 键 词：组分百白破疫苗 气管细胞毒素 液相色谱串联质谱 定量分析 

分 类 号：R917[医药卫生—药物分析学]