Injectable drugs manufactured in E. coli must be tested for host residual DNA (hr DNA) impurity in ensuring drug purity and safety. Because of low allowable hr DNA as impurity, highly sensitive methods are needed. Dro...Injectable drugs manufactured in E. coli must be tested for host residual DNA (hr DNA) impurity in ensuring drug purity and safety. Because of low allowable hr DNA as impurity, highly sensitive methods are needed. Droplet digital PCR (ddPCR) is a new method where the reaction is partitioned into about 20,000 nanoliter-sized droplets and each droplet acts as individual PCR reaction. After completion of end-point PCR, droplets are analyzed for fluorescence and categorized as positive or negative and DNA quantified using Poisson statistics. Here we describe development of a direct E. coli hr DNA dd PCR method where the drug is directly added to the ddPCR reaction. We show that the ddPCR method has acceptable precision and high accuracy, works with different biologic drugs, and compared to qPCR shows higher tolerance of drug matrices. The method does not require DNA extraction or standard curves for quantification of hr DNA in unknown samples.展开更多
目的:建立直接进样气相色谱法测定微量乙酸乙酯含量的分析方法。方法:通过对仪器参数进行优化,包括进样口温度、初始柱温、柱流速、分流比,确定最佳分析条件:色谱柱SH-PolarWax(30 m×0.32 mm 0.25μm),程序升温:初始温度为45℃,保...目的:建立直接进样气相色谱法测定微量乙酸乙酯含量的分析方法。方法:通过对仪器参数进行优化,包括进样口温度、初始柱温、柱流速、分流比,确定最佳分析条件:色谱柱SH-PolarWax(30 m×0.32 mm 0.25μm),程序升温:初始温度为45℃,保持4min,以10℃/min升温至90℃,再以20℃/min升温至200℃;进样口温度220℃,分流比10∶1,流速1.00 m L/min。结果:在优化的色谱条件下,微量乙酸乙酯测试方法在0.01~1.00μg/mL范围内线性良好,相关系数(r)大于0.999,回收率在90%~110%范围内,RSD(n=3)均小于5%,检出限为0.004μg/mL,定量限为0.012μg/mL。结论:该方法具有检测时间短、灵敏度高的特点,研究结果可为乙酸乙酯含量检测方法研究提供数据支撑和参考。展开更多
文摘Injectable drugs manufactured in E. coli must be tested for host residual DNA (hr DNA) impurity in ensuring drug purity and safety. Because of low allowable hr DNA as impurity, highly sensitive methods are needed. Droplet digital PCR (ddPCR) is a new method where the reaction is partitioned into about 20,000 nanoliter-sized droplets and each droplet acts as individual PCR reaction. After completion of end-point PCR, droplets are analyzed for fluorescence and categorized as positive or negative and DNA quantified using Poisson statistics. Here we describe development of a direct E. coli hr DNA dd PCR method where the drug is directly added to the ddPCR reaction. We show that the ddPCR method has acceptable precision and high accuracy, works with different biologic drugs, and compared to qPCR shows higher tolerance of drug matrices. The method does not require DNA extraction or standard curves for quantification of hr DNA in unknown samples.
文摘目的:建立直接进样气相色谱法测定微量乙酸乙酯含量的分析方法。方法:通过对仪器参数进行优化,包括进样口温度、初始柱温、柱流速、分流比,确定最佳分析条件:色谱柱SH-PolarWax(30 m×0.32 mm 0.25μm),程序升温:初始温度为45℃,保持4min,以10℃/min升温至90℃,再以20℃/min升温至200℃;进样口温度220℃,分流比10∶1,流速1.00 m L/min。结果:在优化的色谱条件下,微量乙酸乙酯测试方法在0.01~1.00μg/mL范围内线性良好,相关系数(r)大于0.999,回收率在90%~110%范围内,RSD(n=3)均小于5%,检出限为0.004μg/mL,定量限为0.012μg/mL。结论:该方法具有检测时间短、灵敏度高的特点,研究结果可为乙酸乙酯含量检测方法研究提供数据支撑和参考。
