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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 content of free formaldehyde in influenza virus lysed vaccine by capillary gas chromatography and to verify the results. Methods Free formaldehyde was isolated from 2,4-dinitrophenylhydrazine (DNPH) -derived influenza virus vaccine and its derivatisation conditions were optimized. The sample was determined by capillary gas chromatography. Chromatographic conditions: The column was HP-5 capillary column, the initial temperature was 150 ℃, hold 1 min, the rate of 20 ℃ / min to 250 ℃, for 10 min; detector for electronic capture detector temperature of 350 ℃, tail gas 60 ml / min; inlet temperature 300 ℃, septum purge 3 ml / min, split ratio 50: 1; carrier gas is nitrogen, flow rate 2.5 ml / min; The limits of detection and the limits of quantification of the established method were determined. The linearity, specificity, accuracy, precision and stability of the method were validated. The free formaldehyde content of 11 batches of samples from 2 enterprises was tested by the established method. The best derivatization conditions for the determination of the results were as follows: 1 ml of the reference solution and the test solution were respectively set in a 15 ml centrifuge tube, 1 ml of DNPH hydrochloric acid solution was added and vortexed for 1 min; sonicated for 5 min; Derived 45 min; cooled in an ice bath, cyclohexane was added 2 ml, vortex extraction 1 min; static stratification, the supernatant was collected 1 ml, injected into the gas chromatograph for detection. The method has the lowest detection limit of 0.1μg / ml, the limit of quantification of 0.2μg / ml, the free formaldehyde content of 1 ~ 30μg / ml range, and the peak area showed a good linear relationship (r2 = 0.999 8) 2,4-dinitrophenylhydrazone, a formaldehyde derivative, showed good specificity. The average recoveries of three different concentrations of samples were 107% and the relative standard deviation (RSD) was 2.11% ; The average peak area of reference solution 6 times of continuous injection was 40 652.8, the RSD was 0.004%; the average peak area of formaldehyde derivative solution within 7 h was 40 257.35, RSD was 1.57%. 11 batches of influenza virus split vaccine free formaldehyde content are in line with “Chinese Pharmacopoeia” three (2010 edition) requirements, are not higher than 50μg / ml. Conclusions Derived capillary gas chromatography was developed for the determination of free formaldehyde in influenza virus split vaccine. The method is simple in operation, good in precision, stability, accuracy and specificity and can be used for the determination of free formaldehyde in influenza virus split vaccine.