Sabin株脊髓灰质炎灭活疫苗(Vero细胞)中游离甲醛含量柱前衍生-高效液相色谱检测方法的建立及验证  

Establishment and validation of a pre-column derivatization-high performance liquid chromatography method for determination of free formaldehyde content in Sabin strain inactivated poliovirus vaccine(Vero cells)

作　　者：杨欢 非成瑞 王思捷 申雪 杨力 吴凡 YANG Huan;FEI Chengrui;WANG Sijie;SHEN Xue;YANG Li;WU Fan(Yunnan Institute of Supervision and Inspection for Food and Drug,Public Service Platform for Industrial Technology Foundation of the Ministry of Industry and Information Technology,Kunming 650106,Yunnan Province,China)

机构地区：[1]云南省食品药品监督检验研究院工业和信息化部产业技术基础公共服务平台,云南昆明650106

出　　处：《中国生物制品学杂志》2025年第1期53-60,共8页Chinese Journal of Biologicals

基　　金：云南省科技厅重大科技专项计划(202002AA100008)。

摘　　要：目的建立Sabin株脊髓灰质炎灭活疫苗(Vero细胞)中游离甲醛含量柱前衍生-高效液相色谱检测方法,并进行验证及应用,以期为测定疫苗中游离甲醛含量提供新的方法。方法样品用2,4-二硝基苯肼衍生后,经C18色谱柱(5μm,120?,4.6 mm×250 mm)分离,分别对检测波长、流动相比例、流速、衍生时间、温度、缓冲液和衍生容器等检测条件进行优化,并对方法进行专属性、线性、重复性、准确性、检测限和定量限、耐用性验证。采用优化的方法检测20批脊髓灰质炎灭活疫苗(Vero细胞)中游离甲醛含量。结果色谱条件:以乙腈∶水体积分数70∶30为流动相,流速0.6 mL/min,波长352 nm进行测定;衍生条件:加入2,4-二硝基苯肼乙腈溶液0.5 mL和pH 5.0的缓冲液0.25 mL,60℃水浴20 min。该色谱分离条件能够有效分离2,4-二硝基苯肼及甲醛衍生物,且乙醛对测定结果无影响;甲醛标准品浓度在0.05~100μg/mL范围内与峰面积线性关系良好,r为0.9999;重复性试验中6次检测结果的相对标准偏差(relative standard deviation,RSD)为0.36%;9份加标供试品溶液甲醛含量回收率在102.0%~107.0%之间;检出限和定量限分别为0.025和0.05μg/mL;样品衍生后48 h能够稳定存在,耐用性较好。同一批次样品结果重复性较好,甲醛含量在4.5~9.9μg/剂之间。结论建立的方法具有测定范围宽、线性好、准确性高等优点,能够对Sabin株脊髓灰质炎灭活疫苗(Vero细胞)中游离甲醛进行准确定量,可作为疫苗产品游离甲醛含量的辅助检测方法,对疫苗批签发和质量监管具有重要意义。Objective To establish a pre-column derivatization-high performance liquid chromatography(HPLC)method for the determination of free formaldehyde in Sabin strain inactivated poliovirus vaccine(IPV)(Vero cells),validate and apply the method,so as to provide a new method for the determination of free formaldehyde in vaccines.Methods The sample was derivatized with 2,4-dinitrophenylhydrazine and loaded onto a C18 chromatographic column(5μm,120?,4.6 mm×250 mm).The detection wavelength,mobile phase ratio,flow rate,derivatization time,temperature,buffer solution,and derivatization container were optimized for the separation conditions.The specificity,linearity,repeatability,accuracy,limit of detection(LOD),limit of quantitation(LOQ)and robustness of the method were verified.The content of free formaldehyde in 20 batches of IPV(Vero cells)was detected by using the optimized method.Results Chromatographic conditions:acetonitrile and water in a 70∶30 volume ratio as mobile phase,flow rate 0.6 mL/min,determination at a wavelength of 352 nm.Derivatization conditions:0.5 mL of acetonitrile solution containing 2,4-dinitrophenylhydrazine and 0.25 mL of pH 5.0buffer were added,followed by a 20 min incubation in 60℃water bath.This chromatographic separation conditions effectively separated 2,4-dinitrophenylhydrazine and formaldehyde derivatives,and the acetaldehyde had no effect on the determination results.In the range of 0.05-100μg/mL,formaldehyde standard concentration exhibited a good linear relationship with the peak area,with the r value of 0.9999.The relative standard deviations(RSDs)of six test results in the repeatability test was 0.36%.The recovery rates of formaldehyde content in nine samples were between 102.0%and 107.0%.The LOD and LOQ were 0.025 and 0.05μg/mL,respectively.The sample remained stable for 48 h after derivation,showing good robustness.The results of the same batch of samples had good repeatability,and the formaldehyde content was between 4.5-9.9μg/dose.Conclusion The established method has the

关 键 词：高效液相色谱法 脊髓灰质炎灭活疫苗 甲醛 含量测定 

分 类 号：R373-22[医药卫生—病原生物学] O657-72[医药卫生—基础医学]