Development and verification of a reversed-phase high-performance liquid chromatography for determination of adipic acid dihydrazide residues in typhoid Vi polysaccharide-protein conjugate vaccine bulk
10.13200/j.cnki.cjb.004511
- VernacularTitle:伤寒Vi多糖-蛋白结合疫苗原液中己二酰肼残留量反向高效液相色谱检测方法的建立及验证
- Author:
REN Zhenyun
- Publication Type:Journal Article
- Keywords:
Typhoid Vi polysaccharide-protein conjugate vaccine;
Adipic acid dihydrazide(ADH);
Reversed-phase high-performance liquid chromatography(RP-HPLC)
- From:
Chinese Journal of Biologicals
2025;38(06):699-704
- CountryChina
- Language:Chinese
-
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.
