The central carbon metabolism including glycolysis, the pentose phosphate pathway, and the TCA-cycle is essential for all biological systems. The measurements of metabolites involved in these pathways, known as metabolomics, can provide valuable insights about cell biology for various life science applications including biomedical research. However, the chromatographic separation of these, mostly polar compounds (e.g. amino acids, carboxylic acids, cofactors, nucleotides, sugars, and sugar-phosphates), is still a challenging task. It has been shown that ion pair- reverse phase chromatographic separation is a suitable technique to tackle this challenge. We took advantage of the Agilent metabolomics dynamic MRM database and method to establish a robust workflow for the quantification of almost 200 metabolites. This LC-MS/MS based method employs ion pair- reverse phase chromatographic separation using tributylamine as an ion-pair reagent.  For metabolite detection, a dynamic MRM method is employed. It should be pointed out that methods can be freely modified and all parameters are accessible.Our main aim was to establish a workflow for absolute quantification using an external calibration and internal standardization. First, we comprehensively revised the provided...

Learning Objectives:

1. Usage of the Agilent metabolomics dynamic MRM database and method for analysis of wide range metabolites

2. Practical tips and tricks for the setup of quantitative workflow

For Research Use Only. Not for use in diagnostic procedures.