Hušek Petr, Švagera Zdeněk, Hanzlíková Dagmar, Karlínová Iva, Řimnáčová Lucie, Zahradníčková Helena, Šimek Petr
University Hospital Ostrava, Institute of Laboratory Diagnostics, Department of Biochemistry, Ostrava, Czech Republic.
Czech Academy of Sciences, Biology Centre, Institute of Entomology, Analytical Biochemistry & Metabolomics, České Budějovice, Czech Republic.
Methods Mol Biol. 2018;1738:159-181. doi: 10.1007/978-1-4939-7643-0_11.
A simple analytical workflow is described for gas chromatographic-mass spectrometry (GC-MS)-based metabolomic profiling of protic metabolites, particularly amino-carboxylic species in biological matrices. The sample preparation is carried out directly in aqueous samples and uses simultaneous in situ heptafluorobutyl chloroformate (HFBCF) derivatization and dispersive liquid-liquid microextraction (DLLME), followed by GC-MS analysis in single-ion monitoring (SIM) mode. The protocol involves ten simple pipetting steps and provides quantitative analysis of 132 metabolites by using two internal standards. A comment on each analytical step and explaining notes are provided with particular attention to the GC-MS analysis of 112 physiological metabolites in human urine.
描述了一种简单的分析工作流程，用于基于气相色谱-质谱联用（GC-MS）的质子代谢物代谢组学分析，特别是生物基质中的氨基羧酸类物质。样品制备直接在水性样品中进行，采用七氟丁基氯甲酸酯（HFBCF）原位衍生化和分散液液微萃取（DLLME）同步进行，随后在单离子监测（SIM）模式下进行GC-MS分析。该方案包括十个简单的移液步骤，并使用两种内标对132种代谢物进行定量分析。针对每个分析步骤提供了注释和解释说明，特别关注了人尿液中112种生理代谢物的GC-MS分析。
