技术中药注射剂微毒快速测试体系反应条件优化与方法学考察.docVIP

技术中药注射剂微毒快速测试体系反应条件优化与方法学考察 　　[摘要]基于费氏弧菌CS234的Microtox微毒测试体系优化与方法学考察，为该方法更加可靠地应用于中药注射剂综合急性毒性的测试打下基础。采用PlackettBurman法优化设计影响费氏弧菌发光的因素，建立以七水硫酸锌为标准毒物和质量控制样品的反应体系，得到最优发光条件为：费氏弧菌冻干粉平衡15 min后加入复苏液1 mL，混匀10 min，取100 μL菌液测试初始发光度，随后立即加入1 mL复苏液或待测样品，反应10 min后再次测试相应发光强度，上述操作均在（15±1）℃条件下完成；复苏稀释液、渗透压调节液及七水硫酸锌储备液不干扰费氏弧菌发光强度的测定，该方法特异性良好；质量控制样品、定量下限样品批内精密度5%，批间精密度10%，准确度在858%～1032%。七水硫酸锌溶液在386～7727 mg?L-1，标准曲线方程为y=2178lnx-1514，R2=0998。该方法下ZnSO4?7H2O对费氏弧菌的IC50为1990 mg?L-1；对七水硫酸锌储备液及其质量控制样品分别连续考察120，8 h，其RSD2%，提示其在室温及4 ℃保存条件下稳定性良好；pH 45～80时，费氏弧菌发光的波动控制在±10%，该pH可满足绝大多数中药注射剂的测试需要。针对费氏弧菌CS234建立的微毒测试体系进行优化后，具有良好的特异性、稳定性、灵敏度、准确度及适应性，提示可应用于中药注射剂综合急性毒性的测试。 　　[关键词]中药注射剂；微毒测试；急性毒性；费氏弧菌CS234 　　[Abstract]Vibrio fischeri CS234 was used to establish and optimize microtox assay system， laying a foundation for the application of this method in comprehensive acute toxicity test of traditional Chinese medicine （TCM） injections Firstly， the PlackettBurman method was carried out to optimize the factors which would affect Vibrio fischeri CS234 luminescence Secondly， ZnSO4?7H2O was chosen as reference substance to establish its reaction system with quality control samples The optimal luminescence conditions were achieved as follows： ①At a temperature of （15±1） ℃， Vibrio fischeri CS234 lyophilized powders were balanced for 15 min， then， 1 mL resuscitation fluid was added and blended for 10 min 100 μL bacteria suspension was taken to measure the initial luminescence intensity， and then 1 mL resuscitation fluid or test sample was immediately added；after reaction for 10 min， corresponding luminescence intensity was measured again Resuscitation diluent， osmotic pressure regulator and ZnSO4?7H2O stock solution showed no interference to the determination of Vibrio fischeri CS234 luminescence intensity， so this method was of good specificity The withinand betweenbatch precisions of quality controls and the lower limit of quantification （LLOQ） samples were 　　[Key words]traditional Chinese medicine injection