柱前衍生液相色谱法与柱后衍生液相色谱法测定疫苗中游离甲醛残留量的对比研究  

Comparative study of pre-column derivatization liquid chromatography and post-column derivatization liquid chromatography for the determination of free formaldehyde residues in vaccines

作　　者：陈宇堃[1] 刘丛丛[1] 梁蔚阳[1] CHEN Yukun;LIU Congcong;LIANG Weiyang(Guangdong Institute for Drug Control,NMPA Key Laboratory for Quality Control of Blood Products,Guangdong Drug Administration Key Laboratory of Quality Control and Research of Blood Products,Guangzhou 510663,China)

机构地区：[1]广东省药品检验所,国家药品监督管理局血液制品质量控制重点实验室,广东省药品监督管理局血液制品质量控制研究重点实验室,广州510663

出　　处：《中国药品标准》2022年第2期169-180,共12页Drug Standards of China

基　　金：国家药典委员会药品标准制修订研究课题(2019S13);广东省医学科学技术研究基金项目(B2021371);广东省药品监督管理局科技创新项目(2021ZDZ02)。

摘　　要：目的:建立柱前衍生液相色谱法与柱后衍生液相色谱法测定疫苗中游离甲醛残留量,并考察2种方法测定结果的一致性程度。方法:柱前衍生液相色谱法色谱条件:岛津LC-20AT液相色谱仪(SPD-20A紫外检测器),采用Kromasil 100-5-C_(18)(250 mm×4.6 mm)色谱柱,流动相为60%乙腈溶液,流速0.8mL·min^(-1),柱温40℃,测定波长360 nm。柱后衍生液相色谱法色谱条件:岛津LC-20AT液相色谱仪(SPD-M20A二极管阵列检测器和vector衍生装置),采用纳谱AQ-C_(18)(250 mm×4.6 mm)色谱柱,流动相为0.2%(V/V)磷酸溶液,流速1.0 mL·min^(-1),柱温25℃,检测波长412 nm;衍生溶液为醋酸缓冲液,流速0.5 mL·min^(-1),温度100℃。分别对2种方法的精密度、重复性、加样回收率等项目进行考察,并对测定结果进行显著性检验(F检验和t检验)。结果:柱前衍生液相色谱法线性范围为0.025~100μg·mL^(-1)(R=0.9999,n=12),精密度RSD为0.06%,重复性RSD为0.3%~1.4%,平均加样回收率为97.3%~104.8%(RSD为0.7~2.9%),定量限为0.02μg·mL^(-1),检出限为0.01μg·mL^(-1)。柱后衍生液相色谱法线性范围为0.025~100μg·mL^(-1)(R=0.9999,n=12),精密度RSD为0.02%,重复性RSD为0.07%~3.5%,平均加样回收率为105.6%~114.6%(RSD为0.3%~1.9%),定量限为0.02μg·mL^(-1),检出限为0.006μg·mL^(-1)。对21批样品测定结果的F检验和t检验结果表明两者无显著性差异。结论:2种方法操作简便,专属性强,灵敏度高,结果准确,可用于疫苗中游离甲醛残留量测定。Objective:To establish a pre-column derivatization liquid chromatography method and a post-column derivatization liquid chromatography method for the determination of residual amount of free formaldehyde in vaccines.Consistency of the results of two methods was investigated.Methods:The pre-column derivatization liquid chromatography was performed on a Shimadzu LC-20AT liquid chromatograph(SPD-20A UV detector).Separation was accomplished on a Kromasil 100-5-C_(18)(250 mm×4.6 mm)column with a mobile phase of 60%acetonitrile solution at a flow rate of 0.8 mL·min^(-1) at 40℃and the detection wavelength was 360 nm.The post-column derivatization liquid chromatography was performed on a Shimadzu LC-20AT liquid chromatograph(SPD-M20A diode array detector and vector derivative device).Separation was accomplished on a Chrom Core AQ-C_(18)(250 mm×4.6 mm)column with a mobile phase of 0.2%(V/V)phosphoric acid solution at a flow rate of 1.0 mL·min^(-1) at 25℃and the detection wavelength was 412 nm.The derivatization solution was acetate buffer,the flow rate was 0.5 mL·min^(-1),and the temperature was 100℃.The precision,repeatability and sample recovery of the two methods were investigated,and the experiment results were tested for significance by F-test and t-test.Results:The pre-column derivatization liquid chromatography had good linearity in the range of 0.025-100μg·mL^(-1)(R=0.9999,n=12).RSD values of precision and repeatability were 0.06%and 0.3%-1.4%,respectively.The average recoveries were 97.3%-104.8%with RSD of 0.7%-2.9%.The limit of quantitation was 0.02μg·mL^(-1),and the limit of detection was 0.01μg·mL^(-1).The post-column derivatization liquid chromatography had good linearity in the range of 0.025-100μg·mL^(-1)(R=0.9999,n=12).RSD values of precision and repeatability were 0.02%and 0.07%-3.5%,respectively.The average recoveries were 105.6%-114.6%with RSD of 0.3%-1.9%.The limit of quantitation was 0.02μg·mL^(-1),and the limit of detection was 0.006μg·mL^(-1).The F-test and the t-test results

关 键 词：疫苗 游离甲醛 柱前衍生 柱后衍生 

分 类 号：R921.2[医药卫生—药学]