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目的建立检测水痘减毒活疫苗中乙二胺四乙酸二钠(EDTA-2Na)残留量的高效液相色谱法(High Performance Liquid Chromatography,HPLC),并进行验证。方法采用TC-C18色谱柱(250 mm×4.6 mm);流动相:磷酸二氢铵,四丁基氢氧化铵,乙腈溶液;流速:0.8 ml/min;检测波长:257 nm;进样量:20μl。对该方法的系统适用性、专属性、准确性、线性、检测限及定量限及耐用性进行验证。应用建立的方法检测4批水痘减毒活疫苗原液中EDTA-2Na的含量。结果试验的目标峰理论塔板数均在21 000以上,分离度在4.9以上,拖尾因子约1.0;含EDTA-2Na的原液与FeCl_3溶液反应后的色谱图保留时间在11.002 min处出现吸收峰,不含EDTA-2Na的原液未见该峰;EDTA-2Na标准液的浓度在0~40μg/ml范围内,与峰面积呈良好线性关系,回归方程为:y=20.268 11 x+1.276 36,r=0.990 99;加EDTA-2Na的4种浓度的疫苗原液与Fe Cl3溶液反应后的回收率为98.0%~102.0%,RSD为1.55%;最低检测限为0.063μg/ml,最小定量限为0.125μg/ml;流动相pH为2.30、2.36、2.50及7.0时的保留时间分别为11.130、11.303、11.482和12.775。流动相保存1、3、5、7 d的保留时间分别为11.130、11.179、11.259和11.313。4批水痘减毒活疫苗原液中EDTA-2Na的含量分别为0.318 634、0.347 011、0.158 735和0.106 435 ng/μl。结论该方法具有良好的线性、专属性、准确性及耐用性,可用于水痘减毒活疫苗中EDTA-2Na残留量的检测。
Objective To establish and validate the high performance liquid chromatography (HPLC) for the determination of EDTA-2Na residues in live attenuated varicella vaccine. Methods The mobile phase was ammonium dihydrogen phosphate, tetrabutylammonium hydroxide and acetonitrile. The flow rate was 0.8 ml / min. The detection wavelength was 257 nm. The injection volume was 20μl. The system applicability, specificity, accuracy, linearity, limit of detection, limit of quantification and durability of this method were verified. The established method was used to detect the content of EDTA-2Na in four batches of live attenuated vaccine of varicella. The results of the experimental target peak theoretical plate number of more than 21 000, the resolution of more than 4.9, tailing factor of about 1.0; containing EDTA-2Na FeCl_3 solution and the reaction solution after the chromatographic retention time at 11.002 min at the absorption peak , EDTA-2Na did not show this peak in the stock solution without EDTA-2Na. The concentration of EDTA-2Na in the range of 0-40 μg / ml showed a good linear relationship with the peak area. The regression equation was: y = 20.268 11 x + 1.276 36, r = 0.99099. The recoveries of four concentrations of EDTA-2Na vaccine solution and FeCl3 solution were 98.0% ~ 102.0%, RSD was 1.55%, the minimum detection limit was 0.063μg / ml, the minimum limit of quantification was 0.125μg / ml; the retention times of mobile phase at pH 2.30, 2.36, 2.50 and 7.0 were 11.130, 11.303, 11.282 and 12.775, respectively. The retention times of mobile phase at 1, 3, 5 and 7 d were 11.130, 11.179, 11.259 and 11.313.4 respectively. The content of EDTA-2Na in the live attenuated vaccine was 0.318 634, 0.347 011, 0.158 735 and 0.106 435 ng / μl. Conclusion The method has good linearity, specificity, accuracy and durability and can be used for the detection of EDTA-2Na residues in live attenuated varicella vaccine.