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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 establish a reversed-phase HPLC method for determination of free trace formaldehyde in influenza virus split vaccine. Methods After the derivatization conditions were optimized, the free formaldehyde in the vaccine was lysed by 2,4-dinitrophenylhydrazine (DNPH) -derived influenza virus and the determination of trace formaldehyde was carried out by reverse-phase HPLC. The chromatographic conditions: Column: TC-C18 (250 mm × 4.6 mm, 5μm); mobile phase: acetonitrile-water (45:55); injection volume: 10μl; flow rate: 1.0 ml / min; detection wavelength: 35 ° C. The detection wavelength of DNPH derivative was determined. The established method was validated by linearity, accuracy, precision, stability and reproducibility. The limits of quantitation and detection limit were determined. Take four batches of different manufacturers of influenza virus lysis vaccine, according to the established method, two batches of derivative, two batches of direct formaldehyde detection. The results showed that the derivatization solution was phosphoric acid solution (5 mol / L), the derivatization temperature was 20 ℃, and the final reaction time was 30 min. The detection wavelength of DNPH derivative is 360 nm. The concentration of formaldehyde in the range of 10.02 ~ 250.5 ng / ml, and the peak area showed a good linear relationship, R2 = 0.999 8; 6 formaldehyde formaldehyde DNPH derivative samples of the average recovery was 99.93%, RSD 0.34%; Formaldehyde DNPH derived The average area of the peak area of the sample was 624.15 and the RSD was 0.74%. The average amount of trace formaldehyde detected by the formaldehyde DNPH derivative repeated 6 times was 8.04μg / ml with RSD of 0.98%, stable within 24 h The average free trace formaldehyde in 6 batches of samples was 8.06 μg / ml with a RSD of 0.68%. The minimum detectable limit of trace formaldehyde was 0.01 μg / ml and the limit of quantification was 0.03 μg / ml. Traces of formaldehyde in the four batches of influenza virus split vaccine were lower than 50 μg / ml as prescribed in the third edition of Chinese Pharmacopoeia (2010 edition). Conclusion The reverse-phase HPLC method for the determination of free trace formaldehyde in the influenza virus lytic vaccine established in this paper is simple, efficient, sensitive and traceable and is suitable for the determination of free trace formaldehyde in the vaccine.