多糖蛋白结合疫苗中残留ADH和EDAC同时定量检测的液相色谱串联质谱法的建立

Development of liquid chromatography tandem mass spectrometry for simultaneous determination of residual ADH and EDAC in polysaccharide conjugate vaccines

目的建立同时定量检测己二酰肼(adipic acid dihydrazide,ADH)和N-(3-二甲基氨基丙基)-N′-乙基碳二亚胺[N-(3-dimethylaminopropyl)-N′-ethylcarbodimide,EDAC]的液相色谱串联质谱(liquid chromatography tandem mass spectrometry,LC-MS/MS)法,实现多糖蛋白结合物(肺炎球菌结合物、脑膜炎球菌结合物和b型流感嗜血杆菌结合物)中残留ADH和EDAC的定量研究。方法通过检测EDAC在纯水、中性水溶液[(50 mmol/L磷酸缓冲溶液(pH 6.8)、200 mmol/L氯化钠水溶液和疫苗培养基)]及0.1%甲酸(formic acid,FA)水溶液中的稳定性,确定实现EDAC完全转换为EDU的前处理条件。优化了色谱柱种类对ADH、EDU和EDAC的保留,以及质谱条件如产物离子、碰撞电压、Q1和Q3电压等对ADH和EDU定量灵敏度的影响,建立以C18WCX(2.1 mm×150 mm,5μm)为固定相,pH 2~3的0.1%FA-水和0.1%FA-乙腈为流动相的LC-MS/MS方法,实现ADH和EDAC(实际检测对象为EDAC的转换产物EDU)的高灵敏度定量分析。对建立方法的精密度、准确度、线性范围、检出限(LOD)和定量限(LOQ)进行验证。结果ADH在水溶液中较稳定,EDAC自溶解开始即水解为EDU,且难以完全转换,导致EDAC测定困难;在样品和标准品溶液中加入FA,可实现EDAC完全且快速的转换为EDU。ADH、EDU和EDAC在C18色谱柱上保留弱,在色谱柱的死时间附近出峰,无法实现这些化合物与样品基质的分离,而本文所选C18WCX固定相可实现目标化合物的保留且峰形较好。通过前体离子扫描、产物离子扫描,选择目标化合物的前体离子和响应强度较好的产物离子作为MRM的离子对,并用MRM自动优化程序获得目标化合物的最佳Q1、CE和Q3电压值。建立的方法检测的ADH和EDAC含量与峰面积线性关系良好,R2均大于0.999;ADH的LOD为3.96μg/L、LOQ为15.63μg/L,EDAC的LOD为0.58μg/L、LOQ为1.17μg/L,灵敏度较高;精密度(峰面积RSD<2%)及准确度(回收率为90%~105%)较高。结论建立的LC-MS/MS方法实现了多糖蛋白结合疫苗中残留ADH和EDAC(通过测定EDU定量EDAC)的高灵敏度检测,与《中国药典》三部(2015版)方法相比,该方法预处理更简单,自动化程度更高且灵敏度更好,适合于多种多糖蛋白结合物中ADH和EDAC残留的检测。

Objective To develop a liquid chromatography tandem mass spectrometry(LC-MS/MS)method for simultaneous determination of residual adipic acid dihydrazide(ADH)and N-(3-dimethylaminopropyl)-N′-ethylcarbo-diimide(EDAC)in polysaccharide conjugate vaccines including pneumococcal conjugate vaccine,meningococcal conjugate vaccine and Haemophilus influenzae type b conjugate vaccine.Methods The pretreatment condition for complete change of EDAC to EDU was determined by analysis of stability of EDAC in pure water,neutral aqueous solution(50 mmol/L phosphate buffer,pH 6.8,200 mmol/L sodium chloride aqueous solution and medium)and 0.1%FA aqueous solution.The retention of ADH,EDU and DEAC by various chromatographic columns,as well as influence of chromatographic condition such as product ions,fragmentor voltage,Q1 and Q3 voltages on the quantitative sensitivities of ADH and EDU,were investigated,based on which a LC-MS/MS method was developed by using C18 WCX column(2.1 mm×150 mm,5μm)as stationary phase and the mixture of 0.1%formic acid(FA)-water and 0.1%FA-acetonitrile(pH 2~3)as mobile phase to achieve the highly sensitive quantitative analysis of ADH and EDAC(converted to EDU in practice).The developed method was verified for precision,accuracy,linear range limit of detection(LOD)and limit of quantitation(LOQ).Results ADH was stable in aqueous solution.EDAC was hydrolyzed into EDU starting with dissolution and was difficult to be converted completely,resulting in difficulty in determination,while was converted completely and rapidly after addition with FA into samples and standard.ADH,EDU and EDAC were not separated from vaccine matrix on C18 column due to very weak retention of these compounds on C18 column(eluted nearly dead time).However,A C18 WCX column was used as stationary phase to separate target compounds due to good retentions and peak shape.Precursor ions and product ions of target compounds were obtained by Q1 scan and product scan,and the product ions with high MS responses were used for the optimization of MRM parameters including Q1,CE and Q3 voltage.The ADH and EDAC contents determined by the developed method showed good linear relationship with the peak area,with R2 values of more than 0.999.The LOD and LOQ of ADH were 3.96 and 15.63μg/L,while those of EDAC were 0.58 and 1.17μg/L,respectively,indicating a high sensitivity.The developed method showed a high precision(RSD%of peak area was less than2%)and a high accuracy(the recovery was 90%~105%).Conclusion The LC-MS/MS method for highly sensitive determination of ADH and EDAC(quantified by determination of EDU)in polysaccharide conjugate vaccines was developed,which was more simple in pretreatment,more automatic and more sensitive as compared with the method in Chinese Pharmacopoeia(VolumeⅢ,2015 edition).