衍生毛细管气相色谱法测定流感病毒裂解疫苗中游离甲醛含量

Determination of free formaldehyde content in influenza virus split vaccine by derivatization capillary gas chromatography

目的 采用衍生毛细管气相色谱法测定流感病毒裂解疫苗中游离甲醛含量,并进行验证。方法 应用2,4-二硝基苯肼（2,4-dinitrophenylhydrazine,DNPH）衍生流感病毒裂解疫苗中的游离甲醛,对其衍生化条件进行优化后,采用毛细管气相色谱法直接进样测定。色谱条件：色谱柱为HP-5毛细管色谱柱,初始温度为150℃,保持1 min,以20℃/min的速率升温至250℃,保持10 min;检测器为电子捕获检测器,温度为350℃,尾吹60 ml/min;进样口温度为300℃,隔垫吹扫3 ml/min,分流比50：1;载气为氮气,流量为2.5 ml/min;进样量1μl。确定建立的方法的检测限和定量限,进行线性、专属性、准确度、精密度及稳定性验证,并用建立的方法检测2个企业共11批样品中游离甲醛含量。结果 确定的最佳衍生化条件为：量取对照品溶液和供试品溶液各1 ml,分别置15 ml离心管中,加入DNPH盐酸溶液1 ml,涡旋1 min;超声5 min;60℃衍生45 min;冰浴冷却,加入环己烷2 ml,涡旋萃取1 min;静置分层,收集上层液1 ml,注入气相色谱仪进行检测。该方法的最低检测限为0.1μg/ml,定量限为0.2μg/ml,游离甲醛含量在1～30μg/ml范围内,与峰面积呈良好的线性关系（r2=0.999 8）;该色谱分离条件对甲醛的衍生物甲醛2,4-二硝基苯腙有较好的专属性;3个不同浓度样品平均加样回收率为107%,相对标准偏差（relative standard deviation,RSD）为2.11%;对照品溶液连续进样6次的峰面积平均值为40 652.8,RSD为0.004%;甲醛衍生物溶液冻存7 h内的峰面积平均值为40 257.35,RSD为1.57%。11批流感病毒裂解疫苗的游离甲醛含量均符合《中国药典》三部（2010版）规定,均不高于50μg/ml。结论 建立了衍生毛细管气相色谱法检测流感病毒裂解疫苗中游离甲醛含量,该方法操作简单,精密度、稳定性、准确度好,专属性强,可用于流感病毒裂解疫苗中游离甲醛含量的测定。

Objective To determine the free formaldehyde content in influenza virus split vaccine by derivatization capillary gas chromatography. Methods The free formaldehyde in influenza virus split vaccine was derivatized by 2,4-dinitrophenylhydrazine（DNPH）and,after optimization of condition for derivatizaton,determined by capillary gas chromatography with direct injection. HP-5 capillary chromatographic column was used at an original temperature of 150 ℃which was maintained for 1 min and increased to 250 ℃ at a rate of 20 ℃ / min and further maintained for 10 min.Electron capture detector was used at a temperature of 350 ℃ and a tail blowing of 60 ml / min. The temperature of injection port was 350 ℃,while the rate of septum purge flow was 3 ml / min,and the split flow was 50 ∶ 1. Nitrogen was used as a carrier gas,at a flow rate of 2. 5 ml / min. The sample loading was 1 μl. The developed method was determined for detection and quantitative limits,verified for linearity,specificity,accuracy,precision and stability,and used for determination of the free formaldehyde contents in 11 batches of vaccine manufactured by two manufacturers.Results The condition for derivatization was optimized as follows：1 ml of control and 1 ml of test sample were put into15 ml centrifugal tubes respectively,added with 1 ml of hydrochloric acid solution of DNPH and oscillated in vortex for1 min,then ultrasonicated for 5 min,incubated at 60 ℃ for 45 min for derivatization,cooled down in ice-bath,added with 2 ml of cyclohexane,and extracted in vortex for 1 min. The liquid in container was layered on standing,and 1 ml of supernatant was collected and injected into gas chromatographi c instrument. The minimum detection limit of the method was 0. 1 μg / ml,while the quantitative limit was 0. 2 μg / ml. The free formaldehyde contents in vaccine showed good linear relationship to the peak area within a content range of 1 ~ 30 μg / ml（r2 = 0. 999 8）. The chromatographic condition showed good specificity to DNPH. The mean recovery of samples at three concentrations was 107%,with a relative standard deviation（RSD）of 2. 11%. The mean peak area of control solution injected for 6 times was 40 652. 8,with a RSD of 0. 004%. The mean peak area of derivative of formaldehyde solution within 7 h after storage in frozen was40 257. 35,with a RSD of 1. 57%. All the free formaldehyde contents in 11 batches of influenza virus split vaccine were not more than 50 μg / ml,which met the requirements in Chinese Pharmacopoeia（Volume Ⅲ,2010 edition）. Conclusion The derivatization capillary gas chromatography for determination of free formaldehyde content in influenza virus split vaccine was simple,precise,stable,accurate and specific,which might be used for determination of free formaldehyde content in influenza virus split vaccine.