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目的建立衍生化毛细管气相色谱法测定重组乙型肝炎疫苗[酿酒酵母或中国仓鼠卵巢细胞(Chinese hamster ovary,CHO)]中游离甲醛含量。方法应用2,4-二硝基苯肼(2,4-dinitrophenylhydrazine,2,4-DNPH)衍生重组乙型肝炎疫苗中的游离甲醛后,环己烷萃取衍生物,HP-5毛细管色谱柱分离,电子捕获检测器测定,外标法定量。色谱条件:初始温度为150℃,保持1 min,再以20℃·min-1的速率升温至250℃,保持10 min;检测器温度为350℃,尾吹60 m L·min-1;进样口温度为300℃,分流比为50∶1;载气为N2;进样量为1μL。结果游离甲醛质量浓度在1.01~30.3 mg·L-1内,与峰面积呈良好的线性关系,回归方程为A=3 968.5ρ-284.1(r=0.999 6)。低、中、高3个浓度的平均加样回收率为99.1%,RSD值为2.87%。结论衍生化毛细管气相色谱法可用于重组乙型肝炎疫苗中游离甲醛含量的质量控制。
Objective To establish a derivatized capillary gas chromatographic method for the determination of free formaldehyde in recombinant hepatitis B vaccine [Saccharomyces cerevisiae or Chinese hamster ovary (CHO)]. Methods The free formaldehyde in recombinant hepatitis B vaccine was induced by 2,4-dinitrophenylhydrazine (2,4-DNPH). After cyclohexane extraction and HP-5 capillary column separation, , Electron capture detector determination, external standard method. Chromatographic conditions: the initial temperature of 150 ℃, holding 1 min, and then at a rate of 20 ℃ · min-1 to 250 ℃, for 10 min; detector temperature of 350 ℃, tail blowing 60 m L · min-1; Sample temperature is 300 ℃, split ratio of 50: 1; carrier gas is N2; injection volume of 1μL. Results The free formaldehyde concentration ranged from 1.01 to 30.3 mg · L-1, with a good linear relationship with the peak area. The regression equation was A = 3968.5ρ-284.1 (r = 0.999 6). The average recoveries of low, medium and high concentrations were 99.1% and the RSD was 2.87%. Conclusion Derivatization capillary gas chromatography can be used for the quality control of free formaldehyde in recombinant hepatitis B vaccine.