目的 篩選最佳的體外原代心肌（PC）細(xì)胞代謝組學(xué)樣品預(yù)處理方法，對超高效液相色譜-飛行時(shí)間質(zhì)譜聯(lián)用技術(shù)（UPLC/Q-TOF-MS）的色譜-質(zhì)譜條件優(yōu)化，為建立體外心肌毒性代謝組學(xué)研究方法奠定基礎(chǔ)。方法 利用UPLC/Q-TOF-MS技術(shù)，分別考察0.05%胰酶消化后冰甲醇提取和刮刀刮取后6種不同溶劑提取的心肌細(xì)胞樣品預(yù)處理方法，考察5種流動(dòng)相梯度洗脫方案和正、負(fù)離子檢測模式下的基峰離子流圖，利用質(zhì)量控制樣本（QC）進(jìn)行儀器精密度、方法精密度和樣本穩(wěn)定性試驗(yàn)等方法學(xué)考察。結(jié)果 最佳的心肌細(xì)胞樣品預(yù)處理方法為：0.05%胰蛋白酶-0.02%EDTA消化后-80℃甲醇直接提取。最佳的流動(dòng)相洗脫程序?yàn)椋? 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。最佳的質(zhì)譜檢測模式為：正離子模式。本方法的儀器精密度、方法精密度和樣本穩(wěn)定性試驗(yàn)以相對峰面積比值的RSD計(jì)分別為4.9%～14.9%、8.6%～17.8%和5.2%～16.3%，以保留時(shí)間的RSD計(jì)均<1.0%，符合檢測要求。結(jié)論 確定了最佳的心肌細(xì)胞樣品預(yù)處理方法及UPLC/Q-TOF-MS分析條件，為建立和完善體外心肌毒性代謝組學(xué)研究提供參考依據(jù)。

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.

重大新藥創(chuàng)制科技重大專項(xiàng)（2012ZX09505001-001）