比浊法检测疫苗中TritonX-100残留量的方法学验证

目的建立检测疫苗中Triton X-100残留量的比浊法,并进行方法学验证。方法将TritonX-100与5%苯酚溶液充分混匀后,室温静置15 min,采用比浊法测定340 nm处吸光度值,与经同样处理的标准品绘制的标准曲线比较,计算样品中残留TritonX-100的浓度。由4名试验人员连续3 d测定12次,评价不同试验人员建立标准曲线的成功率;由同一试验人员在同一试验内和不同时间内及由4名试验人员在不同时间内分别测定低(15μg/ml)、中(25μg/ml)、高(45μg/ml)3个不同浓度的TritonX-100的浓度,验证该方法的精密度和准确度;并检测BSA对试验准确度和精密度的影响。结果 4名试验人员连续3 d的12次检测结果均满足标准曲线的成立条件,成功率为100%;同一试验人员在同一试验内对低、中、高3个不同浓度的TritonX-100溶液重复测定10次,变异系数分别为分别为5.33%、1.19%和1.39%,回收率分别为99.33%、105.60%和110.67%;同一试验人员在不同时间内连续测定3次,变异系数分别为4.94%、7.49%和3.46%,回收率分别为87.75%、93.85%和95.51%,4名试验人员在不同时间内连续测定3次,变异系数在1.73%~12.17%之间,回收率在89.55%~99.26%之间,灵敏度为10μg/ml,具有良好的精密度和准确度;BSA对试验准确度和精密度无显著影响。结论该方法快捷、简便,可灵敏、准确地定量检测TritonX-100的含量,可用于疫苗样品中残留TritonX-100的质量控制。

Verification of turbidimetric method for detection of residual TritonX-100 in vaccines

Objective To develop and validate a turbidimetric method for detection of residual TritonX-100 in vaccines.Methods TritonX-100 was mixed with 5% phenol solution well and incubated at room temperature for 15 min,of which the A340 value was determined by turbidimetric method and compared with that on standard curve plotted using the standard TritonX-100 treated by the same method to calculate the residual TritonX-100 content in samples.Each sample was tested for 12 times in 3 consecutive days by 4 laboratory workers to evaluate the success rate of establishment of standard curve.TritonX-100 samples at low(15 μg / ml),moderate(25 μg / ml)and high(45 μg / ml)concentrations were determined by one laboratory worker in one test and on different working days,as well as by four laboratory workers on different working days,to evaluate the precision and accuracy of the method.The effects of BSA on the precision and accuracy were observed.Results All the results of twelve tests by 4 laboratory workers in 3 working days proved that the standard curve was valid,indicating a success rate of 100%.The coefficients of variation(CVs)of results of TritonX-100 samples at low,moderate and high concentrations determined by one laboratory worker for 10 times in one test were 5.33%,1.19% and 1.39%,while the recovery rates were 99.33%,105.60% and 110.67%,respectively;the CVs of results determined by one laboratory worker for 3 times in different working days were 4.94%,7.49% and 3.46%,while the recovery rates were 87.75%,93.85% and 95.51%,respectively.However,the CVs of results determined by 4 laboratory workers in different working days were 1.73% ~ 12.17%,while the recovery rates were 89.55% ~ 99.26%.The method showed a sensitivity of 10 μg / ml as well as high precision and accuracy.BSA showed no significant effect on the accuracy and precision of the method.Conclusion The developed turbidimetric method was rapid,simple,sensitive and accurate for determination of TritonX-100 content,which may used for quality control vaccine.