[关键词]
[摘要]
目的 筛选最佳的体外原代心肌(PC)细胞代谢组学样品预处理方法,对超高效液相色谱-飞行时间质谱联用技术(UPLC/Q-TOF-MS)的色谱-质谱条件优化,为建立体外心肌毒性代谢组学研究方法奠定基础。方法 利用UPLC/Q-TOF-MS技术,分别考察0.05%胰酶消化后冰甲醇提取和刮刀刮取后6种不同溶剂提取的心肌细胞样品预处理方法,考察5种流动相梯度洗脱方案和正、负离子检测模式下的基峰离子流图,利用质量控制样本(QC)进行仪器精密度、方法精密度和样本稳定性试验等方法学考察。结果 最佳的心肌细胞样品预处理方法为:0.05%胰蛋白酶-0.02%EDTA消化后-80℃甲醇直接提取。最佳的流动相洗脱程序为:0 min,2.0% A;2 min,25.0% A;6 min,40.0% A;12 min,90.0% A;保持2 min;16 min,2.0% A;保持2 min。最佳的质谱检测模式为:正离子模式。本方法的仪器精密度、方法精密度和样本稳定性试验以相对峰面积比值的RSD计分别为4.9%~14.9%、8.6%~17.8%和5.2%~16.3%,以保留时间的RSD计均<1.0%,符合检测要求。结论 确定了最佳的心肌细胞样品预处理方法及UPLC/Q-TOF-MS分析条件,为建立和完善体外心肌毒性代谢组学研究提供参考依据。
[Key word]
[Abstract]
Objective To choose the optimum sample pretreatment method for primary cardiomoycytes and chromatography-mass spectrometry conditions, lay the foundation for establishing the metabonomics research method for in vitro cardiotoxicity. Methods By UPLC/Q-TOF-MS technique, the sample pretreatment methods that trypsinization by 0.05% trypsase first then extraction with cold methanol or scraping with scraper first then extraction with different organic solvents were investigated. The base peak ion chromatograms were detected under five different gradient mobile phase elution solutions and positive and negative ion detection modes. Methodology examinations of instrument precision, method precision and sample stability test were performed by quality control samples. Results Trypsinization by 0.05% trypsase-0.02% EDTA first then extraction directly with -80℃ methanol was the optimum sample pretreatment method of cardiomyocytes for metabonomic study. The optimum mobile phase elution program was 0 min-2.0% A,2 min-25.0%A,6 min-40.0% A,12 min-90.0% A,maintain 2 min,16 min-2.0% A,maintain 2 min. The optimum detection mode for mass spectrometry was positive ion detection mode. The instrument precision, method precision, and sample stability1.0% by RSD of retention time, which conforms to the requirement. Conclusion In this study, the optimum sample pretreatment method and UPLC/Q-TOF-MS analytical conditions were determined, which provides references for establishing and improving the metabonomics research method for in vitro cardiotoxicity.
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[基金项目]
重大新药创制科技重大专项(2012ZX09505001-001)