Zhang Qi, Wang Guangji, Du Yu, Zhu Lingling, Jiye A
Center of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing, China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2007 Jul 1;854(1-2):20-5. doi: 10.1016/j.jchromb.2007.03.048. Epub 2007 Apr 8.
In this paper, an optimized protocol was established and validated for the metabonomic profiling in rat urine using GC/MS. The urine samples were extracted by methanol after treatment with urease to remove excessive urea, then the resulted supernatant was dried, methoximated, trimethylsilylated, and analyzed by GC/MS. Forty-nine endogenous metabolites were separated and identified in GC/MS chromatogram, of which 26 identified compounds were selected for quantitative analysis to evaluate the linearity, precision, and sensitivity of the method. It showed good linearity between mass spectrometry responses and relative concentrations of the 26 endogenous compounds over the range from 0.063 to 1.000 (v/v, urine/urine+water) and satisfactory reproducibility with intra-day and inter-days precision values all below 15%. The metabonomic profiling method based on GC/MS was successfully applied to urine samples from hyperlipidemia model rats. Obviously, separated clustering of model rats and the control rats were shown by principal components analysis (PCA); time-dependent metabonomic modification was detected as well. It was suggested that metabonomic profiling based on GC/MS be a robust method for urine samples.
本文建立并验证了一种优化的使用气相色谱/质谱（GC/MS）对大鼠尿液进行代谢组学分析的方法。尿液样本先用脲酶处理以去除过量尿素，然后用甲醇提取，所得上清液经干燥、甲氧胺化、三甲基硅烷化后，进行GC/MS分析。在GC/MS色谱图中分离并鉴定出49种内源性代谢物，从中选择26种已鉴定的化合物进行定量分析，以评估该方法的线性、精密度和灵敏度。结果表明，在0.063至1.000（v/v，尿液/尿液+水）范围内，26种内源性化合物的质谱响应与相对浓度之间呈现良好的线性关系，日内和日间精密度值均低于15%，具有令人满意的重现性。基于GC/MS的代谢组学分析方法成功应用于高脂血症模型大鼠的尿液样本。主成分分析（PCA）显示模型大鼠和对照大鼠明显分离聚类，同时检测到随时间变化的代谢组学改变。结果提示基于GC/MS的代谢组学分析是一种用于尿液样本的可靠方法。
