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目的采用两步水解及反相高效液相色谱法(reversed phase high performance liquid chromatography,RP-HPLC)分离测定疫苗中聚肌胞(polyinosinic-cytidylic acid,PIC)成分。方法对经蛋白酶K消化、饱和酚抽提、乙醇沉淀回收的含有PIC成分的狂犬病疫苗样品采用KOH碱水解及碱性磷酸酶水解2、6、10 h后,采用RP-HPLC法检测疫苗中的PIC成分,色谱条件:色谱柱Xtimate HPLC Column(C18,4.6 mm×250 mm,5μm);流动相:乙酸铵溶液-甲醇(96∶4),流速:0.8 ml/min;紫外检测波长:254 nm;柱温:25℃;进样量:5μl。结果 PIC经碱性磷酸酶不同时间水解,确定最佳水解时间为6 h;疫苗中的PIC成分被成功分离成肌苷峰和胞苷峰,且分离度良好。结论两步水解及RP-HPLC法可成功分离出疫苗中的PIC成分,为疫苗的质量控制及监管提供参考。
OBJECTIVE To isolate polyinosinic-cytidylic acid (PIC) from vaccine by two-step hydrolysis and reversed phase high performance liquid chromatography (RP-HPLC). Methods The rabies vaccine samples containing PIC component recovered by protease K digestion, saturated phenol extraction and ethanol precipitation were hydrolyzed by KOH alkaline hydrolysis and alkaline phosphatase for 2,6 and 10 h, respectively, and then were detected by RP-HPLC PIC composition, chromatographic conditions: column Xtimate HPLC Column (C18, 4.6 mm × 250 mm, 5 μm); mobile phase: ammonium acetate solution-methanol (96: 4); flow rate: 0.8 ml / min; UV detection wavelength: 254 nm ; Column temperature: 25 ℃; injection volume: 5μl. Results PIC was hydrolyzed by alkaline phosphatase at different times, and the optimal hydrolysis time was 6 h. The PIC components in the vaccine were successfully separated into inosine and cytidine peaks with good resolution. Conclusion Two-step hydrolysis and RP-HPLC method can successfully isolate PIC components in vaccine and provide reference for the quality control and regulation of the vaccine.