Shanghai Center for Systems Biomedicine, College of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, PR China.
J Pharm Biomed Anal. 2010 Aug 1;52(4):589-96. doi: 10.1016/j.jpba.2010.01.046. Epub 2010 Feb 4.
In this paper, we present a tissue metabonomic method with an optimized extraction procedure followed by instrumental analysis with gas chromatography/time-of-flight mass spectrometry (GC/TOFMS) and spectral data analysis with multivariate statistics. Metabolite extractions were carried out using three solvents: chloroform, methanol, and water, with design of experiment (DOE) theory and multivariate statistical analysis. A two-step metabolite extraction procedure was optimized using a mixed solvent of chloroform-methanol-water (1:2:1, v/v/v) and then followed by methanol alone. This approach was subsequently validated using standard compounds and liver tissues. Calibration curves were obtained in the range of 0.50-125.0mug/mL for standards and 0.02-0.25g/mL acceptable for liver tissue samples. For most of the metabolites investigated, relative standard deviations (RSD) were below 10% within a day (reproducibility) and below 15% within a week (stability). Rat liver tissues of carbon tetrachloride-induced acute liver injury models (n=10) and healthy control rats (n=10) were analyzed which demonstrated the applicability of the developed procedure for the tissue metabonomic study.
本文提出了一种组织代谢组学方法，该方法采用了优化的提取程序，随后使用气相色谱/飞行时间质谱（GC/TOFMS）进行仪器分析，并结合多元统计进行光谱数据分析。代谢物提取使用了三种溶剂：氯仿、甲醇和水，并采用实验设计（DOE）理论和多元统计分析。采用氯仿-甲醇-水（1:2:1，v/v/v）混合溶剂进行两步代谢物提取程序优化，然后单独使用甲醇。该方法随后使用标准化合物和肝组织进行了验证。标准品的校准曲线范围为 0.50-125.0μg/mL，肝组织样品的可接受范围为 0.02-0.25g/mL。对于大多数研究的代谢物，日内（重现性）的相对标准偏差（RSD）低于 10%，一周内（稳定性）的 RSD 低于 15%。分析了 10 只四氯化碳诱导的急性肝损伤模型大鼠的肝组织和 10 只健康对照大鼠的肝组织，证明了所开发的方法在组织代谢组学研究中的适用性。
