流感病毒裂解疫苗中游离的痕量甲醛反向HPLC检测方法的建立  

Development of reverse high performance liquid chromatography method for determination of free trace formaldehyde in influenza virus split vaccine

作　　者：王潇 谷丽娟[2] 李井涛 王佳凤 

机构地区：[1]吉林省食品检验所,吉林长春130012 [2]长春生物制品研究所有限责任公司,吉林长春130062

出　　处：《中国生物制品学杂志》2016年第11期1210-1214,共5页Chinese Journal of Biologicals

摘　　要：目的建立测定流感病毒裂解疫苗中游离的痕量甲醛的反向HPLC法。方法优化衍生化条件后,2,4-二硝基苯肼（2,4-dinitrophenylhydrazine,DNPH）衍生流感病毒裂解疫苗中游离的甲醛,利用反向HPLC法进行痕量甲醛的测定,色谱条件：色谱柱：TC-C18（250 mm×4.6 mm,5μm）;流动相：乙腈-水（45∶55）;进样量：10μl;流速：1.0 ml/min;检测波长：360 nm;柱温：35℃。确定DNPH衍生物的检测波长,对建立的方法进行线性、准确度、精密度、稳定性、重现性验证,确定方法的定量限及检出限。取4批不同厂家的流感病毒裂解疫苗,按建立的方法,2批衍生化、2批直接进行痕量甲醛检测。结果确定衍生化溶液为磷酸溶液（5 mol/L）,衍生温度为20℃,30 min为最终体系反应时间。DNPH衍生物的检测波长为360 nm。甲醛浓度在10.02-250.5 ng/ml范围内,与峰面积呈良好的线性关系,R2=0.999 8;6份甲醛DNPH衍生物样品检测的平均回收率为99.93%,RSD为0.34%;甲醛DNPH衍生物样品连续重复进样6次的峰面积均值为724.15,RSD为0.74%;甲醛DNPH衍生物不同时间重复6次检测的痕量甲醛平均为8.04μg/ml,RSD为0.98%,24 h内稳定性好;6份同批样品的游离痕量甲醛平均为8.06μg/ml,RSD为0.68%;痕量甲醛的最低检出限为0.01μg/ml,定量限为0.03μg/ml。4批流感病毒裂解疫苗痕量甲醛均低于《中国药典》三部（2010版）中规定的50μg/ml。结论本文建立的流感病毒裂解疫苗中游离痕量甲醛的反向HPLC检测方法,具有简单、高效、灵敏、痕量等特点,适用于疫苗中游离痕量甲醛的测定。Objective To develop a reverse high performance liquid chromatography（HPLC）method for determination of free trace formaldehyde in influenza virus split vaccine. Methods The condition for derivation was optimized, based on which the free formaldehyde in 2, 4-dinitrophenylhydrazine（DNPH）-derived influenza virus split vaccine was determined by revere HPLC. TC-C18 column（250 mm × 4. 6 mm, 5 μm） was served, using acetonitrile-water（45 ∶ 55） as mobile phase. The sample load, flow rate, detection wavelength and column temperature were 10 μl, 1. 0 ml / min, 360 nm and35 ℃ respectively. The wavelength for detection of DNPH derivative was determined, based on which the developed method was verified for linearity, accuracy, precision, stability and reproducibility, and determined for quantitative and detection limits. Four batches of influenza virus split vaccine from various manufacturers were determined for trace formaldehyde by the method, of which two batches were derived at first, and the other two batches were determined directly. Results The optimal derivation solution, derivation temperature and reaction time were 5 mol / L phosphoric acid, 20 ℃ and 30 min respectively. The detection wavelength for DNPH derivative was 360 nm. The formaldehyde concentration within a range of 10. 02 - 250. 5 ng / ml showed good linear relationship to the peak area（r = 0. 999 8）.The mean recovery rate of six samples of DNPH derivatives was 99. 93%, with a RSD of 0. 34%. The mean peak area of DNPH derivative samples loaded for six times was 724. 15, with a RSD of 0. 74%. The mean content of trace formaldehyde in six tests at various time points was 8. 04 μg / ml, with a RSD of 0. 98%, which was stable within 24 h. However, the mean content of trace formaldehyde in six samples of the same batch was 8. 06 μg / ml, with a RSD of 0. 68%. The minimum detection limit of trace formaldehyde was 0. 01 μg / ml, while the quantitative limit was 0. 03 μg / ml. All the contents of trace formaldehyde in four batches o

关 键 词：流感病毒裂解疫苗 高效液相色谱法 甲醛 痕量分析 

分 类 号：R373.13[医药卫生—病原生物学] O657.72[医药卫生—基础医学]