【摘要】 目的 建立伤寒Vi多糖-蛋白结合疫苗原液中己二酰肼（adipic acid dihydrazide,ADH）残留量的反向高效液相色谱（reversed-phase high-performance liquid chromatography,RP-HPLC）检测方法，并进行优化、验证及初步应用，以期用于伤寒Vi多糖-蛋白结合疫苗原液或其他结合疫苗中ADH残留量的检测。方法 采用RP-HPLC法检测伤寒Vi多糖-蛋白结合疫苗原液中ADH残留量，并优化检测波长（全波长）、流动相[10 mmol/L PBS(pH 7.0)+10%乙腈、10 mmol/L PBS(pH 7.0)+0.8%氯化钠溶液+10%甲醇+5 mmol/L乙酸铵溶液]、流速（0.8及1.0 mL/min），验证方法的专属性、线性范围、准确性、精密性，确定检测限及定量限。采用建立的方法检测3批伤寒Vi多糖-蛋白结合疫苗原液中的ADH残留量。结果 确定最佳检测波长为202 nm，流动相为10 mmol/L PBS(pH 7.0)+0.8%氯化钠溶液+10%甲醇+5 mmol/L乙酸铵溶液，流速为1.0 mL/min。该方法能特异性测定结合物原液中的ADH残留，结合疫苗原液中其他成分对ADH峰的检测无干扰；对照品溶液ADH浓度在2～10μg/mL范围内，与峰面积呈良好的线性关系，线性回归方程y=30 617 x+1 296.5，相关系数（R~2）=0.999 7；伤寒Vi多糖-蛋白结合疫苗原液ADH的加标回收率为91.70%～106.00%；精密性验证RSD均<8%；检测限及定量限分别为0.2和0.5μg/mL。3批伤寒Vi多糖-蛋白结合疫苗原液均未检出ADH残留。结论 建立的方法具有良好的专属性、精密性、准确性，且方便快捷，可应用于伤寒Vi多糖-蛋白结合疫苗原液中AOH残留量的检测。更多还原

【Abstract】 Objective To develop a reversed-phase high-performance liquid chromatography(RP-HPLC) method for the determination of adipic acid dihydrazide(ADH) residues in the bulk of typhoid Vi polysaccharide-protein conjugate vaccine,and to optimize,verify and preliminarily apply the method,so as to use it for the determination of ADH residues in typhoid Vi polysaccharide-protein conjugate vaccine bulk or other conjugate vaccines.Methods RP-HPLC was used to detect ADH residues in the bulk of typhoid Vi polysaccharide-protein conjugate vaccine.The detection wavelength(full wavelength),mobile phase(10 mmol/L PBS(pH 7.0)+10% acetonitrile,10 mmol/L PBS(pH 7.0)+0.8% sodium chloride solution+10%methyl alcohol+5 mmol/L ammonium acetate solution) and flow rate(0.8 and 1.0 mL/min) were optimized.The method was then verified for the specificity,linear range,accuracy and precision,and determined for the limit of detection(LOD) and limit of quantitation(LOQ).The ADH residues in three batches of typhus Vi polysaccharide-protein conjugate vaccine bulks were detected by the established method.Results The optimum detection wavelength was determined to be 202 nm,the mobile phase was 10 mmol/L PBS(pH 7.0)+0.8% sodium chloride solution+10% methyl alcohol+5 mmol/L ammonium acetate solution,and the flow rate was 1.0 mL/min.This method could specifically determine ADH residues in conjugate bulk,and other components in conjugate vaccine bulk exhibited no interference to the detection of ADH peak.At the range of 2-10 μg/mL,ADH concentration in the reference solution showed a good linear correlation with the peak area,with the linear regression equation of y=30 617 x+1 296.5,R2=0.999 7.The spiked recovery rates of ADH in the bulks of typhoid Vi polysaccharide-protein conjugate vaccines were 91.70%-106.00%.The RSDs of precision verification were less than 8%.The LOD and LOQ were 0.2 and 0.5 μg/mL,respectively.ADH residues were not detected in the bulks of three batches of typhoid Vi polysaccharide-protein conjugate vaccines.Conclusion The developed method has good specificity,precision and accuracy with convenience and rapidity,which can be used for the determination of ADH residues in the bulk of typhoid Vi polysaccharide-protein conjugate vaccine.更多还原