甲型肝炎灭活疫苗(MRC-5细胞)中乙二胺四乙酸二钠残留量HPLC检测方法的建立及验证

Development and validation of HPLC for determination of residual EDTA disodium salt in inactivated hepatitis A vaccine(MRC-5 cells)

目的建立检测甲型肝炎(hepatitis A,HA)灭活疫苗(MRC-5细胞)中乙二胺四乙酸二钠(EDTA-2Na)残留量的HPLC法,并进行验证。方法色谱条件:选用反相色谱柱;流动相为水相-有机相,水相为四丁基氢氧化铵的磷酸盐溶液,有机相为乙腈;流速为1.0 mL/min;检测波长为257 nm;进样量为20μL;柱温为30℃;保留时间为10 min。筛选色谱柱并优化流动相条件(水相与有机相的比例、pH及磷酸盐)。验证方法的适用性、专属性、线性范围、准确度、精密度、耐用性、定量限和检测限。采用该方法检测3批HA灭活疫苗原液供试品中EDTA-2Na残留量。结果最适色谱条件:采用美国Thermo公司的C18色谱柱(250 mm×4.6 mm,5μm),水相与有机相比例为9∶1,pH为2.4,磷酸盐为0.01 mol/L磷酸二氢铵。EDTA-2Na对照品色谱峰的理论塔板数大于10 000,分离度大于1.5;仅对照品有色谱峰,且无其他色谱峰干扰;对照品在0.2~20μg/mL范围内线性关系良好,回归方程为Y=20 482.18 X+0.512 6,R^(2)=0.999 9;准确性验证中,浓度为2、5、10μg/mL的加标样品平均回收率分别为97.44%、100.6%及100.4%,RSD为1.6%;5μg/mL的加标样品重复性及中间精密度的RSD分别为1.4%和1.6%;定量限及检测限分别为0.2和0.05μg/mL;不同pH(2.0、2.4、3.0)流动相的色谱峰面积的RSD分别为0.24%、0.31%及0.18%,相对误差(RE)分别为99.25%、99.12%及99.33%。3批供试品均未检出EDTA-2Na。结论成功建立了检测HA灭活疫苗中EDTA-2Na残留量的HPLC法,该方法具有良好的专属性、线性范围、准确度、精密度及耐用性,为HA灭活疫苗的质量控制研究奠定了基础。

Objective To develop,optimize and validate a HPLC method for determination of residual EDTA disodium salt(EDTA-2Na) content in inactivated hepatitis A(HA) vaccine(MRC-5 cells).Methods The condition for HPLC was as follows:reverse chromatographic column was adopted,using the mobile phase consisting of phosphate solution containing tetrabutylammonium hydroxide as aqueous phase and acetonitrile as the organic phase at a flow rate of 1.0 mL/min and a detection wavelength of 257 nm.The injection volume was 20 μL,while the column temperature was 30 ℃ and the retention time was 10 min.The chromatographic column was screened,and the ratio of aqueous phase to organic phase,p H value as well as phosphate in the mobile phase were optimized.The developed method was validated for suitability,specificity,linear range,accuracy,precision,durability,limit of quantitation(LOQ)and limit of detection(LOD),by which the residual EDTA-2Na contents in three batches of bulk of hepatitis A vaccine were determined.Results Th e optimal column for HPLC was C18 chromatographic column(Thermo,250 mm × 4.6 mm,5 μm),while the optimal ratio of aqueous phase to organic phase was 9 ∶ 1,the optimal pH value was 2.4,and the optimal phosphate was 0.01 mol/L ammonium dihydrogen phosphate.The theoretical plate number of chromatographic peak of reference for EDTA-2Na was more than 10 000,while the separation degree was more than 1.5.Only the chromatographic peak of reference was observed,without interference from other chromatographic peaks.The linear range of the developed method for reference was 0.2~20 μg/mL,with an equation of Y = 20 482.18X + 0.512 6(R^(2) = 0.999 9).The mean recovery rates of spike samples at concentrations of 2,5 and 10 μg/mL in validation for accuracy were 97.44%,100.6% and 100.4%respectively,with a RSD of 1.6%.The RSDs of test results for reproducibility and intermediate precision of spike samples were 1.4% and 1.6%,while the LOQ and LOD were 0.2 and 0.05 μg/mL,respectively.The RSDs of chromatographic peak areas of mobile phases at pH values of 2.0,2.4 and 3.0 were 0.24%,0.31% and 0.18%,with relative errors(REs)of 99.25%,99.12% and 99.33%,respectively.However,no EDTA-2Na was detected in three batches of test samples.Conclusion The HPLC method for determination of residual EDTA-2Na content in inactivated hepatitis A vaccine(MRC-5 cells)was successfully developed,which showed good specificity,linear range,accuracy,precision and durability.It laid a foundation of study on quality control of inactivated hepatitis A vaccine.