多剂量装双价HPV疫苗中防腐剂含量反相-高效液相色谱法的建立及验证

Development and verification of RP⁃HPLC for determination of preservative content in multi⁃dose bivalent HPV vaccine

目的建立反相-高效液相色谱(reverse phase-high-performanceliquidchromatography,RP-HPLC)法检测双价人乳头状瘤病毒(humanpapillomavirus,HPV)疫苗中3种候选防腐剂(尼泊金甲酯、苯甲醇和间甲酚)的含量,并对方法进行验证及初步应用。方法采用WatersAcquityPeptideBEHC18300A(150mm×2.1mm,1.7μm)色谱柱;流动相A(0.1%三氟乙酸水溶液),流动相B(0.1%三氟乙酸乙腈溶液)进行梯度洗脱;流速为0.2mL/min;柱温为40℃,检测波长为254nm。对建立的方法进行专属性、线性、准确性、精密性、稳定性验证,并采用该方法检测含3种防腐剂的双价HPV疫苗中各防腐剂的浓度。结果在选定的条件下,尼泊金甲酯、苯甲醇和间甲酚有不同的出峰时间(分别为14.6、7.2和15.3min),不受样品中其余组分和检测试剂的影响。尼泊金甲酯在0.005%~0.020%范围内线性关系良好(R²=0.9973),平均回收率为99.60%,RSD为2.31%(n=5);苯甲醇在0.1%~0.5%范围内线性关系良好(R²=0.9999),平均回收率为100.46%,RSD为2.70%(n=5);间甲酚在0.4%~2.0%范围内线性关系良好(R^(2)=0.9994),平均回收率为100.12%,RSD为0.74%(n=5)。尼泊金甲酯和间甲酚于2~8℃放置3d,苯甲醇放置2d,检测结果的RSD均<5%,稳定性良好。使用建立的方法检测HPV双价疫苗样品中的3种防腐剂浓度,均满足±20%的误差范围内。结论建立的RP-HPLC法操作简便,专属性强,准确性、精密性、稳定性良好,能有效对双价HPV疫苗中尼泊金甲酯、苯甲醇和间甲酚3种防腐剂进行检测和质控。

Objective To develop a reverse phase⁃high⁃performance liquid chromatography(RP⁃HPLC)method for the determination of three candidate preservatives(methyl paraben,benzyl alcohol and m⁃cresol)in bivalent human papilloma⁃virus(HPV)vaccine,and to verify and preliminarily apply the method.Methods Waters Acquity Peptide BEH C18300Å(150 mm×2.1 mm,1.7µm)chromatographic column was used;mobile phase A(0.1%trifluoroacetic acid aqueous solution)and mobile phase B(0.1%trifluoroacetic acid acetonitrile solution)were used for gradient elution;the flow rate was 0.2 mL/min,column temperature was 40℃,and the detection wavelength was 254 nm.The specificity,linearity,accuracy,precision and stability of the developed method were verified,and the concentration of each preservative in bivalent HPV vaccine containing three preservatives was detected using this method.Results Under the selected chroma⁃tographic conditions,the peak times of methyl paraben,benzyl alcohol and m⁃cresol were different(14.6 min,7.2 min and 15.3 min respectively),which was not affected by other components in the sample and detection reagents.The linearity of methyl paraben was good in the range of 0.005%⁃0.020%(R^(2)=0.9973),the average recovery was 99.60%,and the RSD was 2.31%(n=5);the linear relationship of benzyl alcohol was good in the range of 0.1%⁃0.5%(R^(2)=0.9999),the average recovery was 100.46%,and the RSD was 2.70%(n=5);the linear relationship of m⁃cresol was good in the range of 0.4%⁃2.0%(R^(2)=0.9994),the average recovery was 100.12%,and the RSD was 0.74%(n=5).When methyl paraben and m⁃cresol were placed at 2⁃8℃for 3 d and benzyl alcohol for 2 d,the RSDs of the detection results were all less than 5%,and the stability was good.The developed method was used to detect the concentration of three preservatives in bivalent HPV vaccine samples,all the results of which met the error range of±20%.Conclusion The developed RP⁃HPLC method is simple for operation with good specificity,accuracy,precision and stability,which can effectively detect and control the quality of three preservatives,methyl paraben,benzyl alcohol and m⁃cresol,in bivalent HPV vaccines.