An End-to-End Targeted Metabolomics Workflow

Abstract
 

The metabolome refers to all the small molecules produced by cells or an organism during metabolism. As such, metabolomics data can be used as a direct functional readout of metabolic activity and physiological status. Targeted metabolomics aims to routinely detect and quantify a predefined group of metabolites likely to be involved in biological processes of interest. This application note presents a robust workflow that combines sample preparation and instrumental analysis solutions for targeted metabolomics, and can be applied to plasma and mammalian cell sample types. Automated sample preparation was achieved by combining the Agilent Bravo Metabolomics Sample Prep platform and Agilent Captiva EMR–Lipid plates. Metabolite separation was performed using the Agilent 1290 Infinity II Bio LC ultra-high performance liquid chromatography system coupled with an Agilent InfinityLab Poroshell 120 HILIC-Z column. Metabolite detection were performed using the Agilent 6495 Triple Quadrupole LC/MS system coupled with a custom database of 500 metabolites made using the Agilent MassHunter Optimizer software. The dynamic multiple reaction monitoring (dMRM) method was used with different numbers of ion transitions but can hold one transition for each of the 500 metabolites and can operate reproducibly at a 1 ms dwell time. Additionally, data analysis was performed using the Agilent MassHunter Quantitative Analysis 10 and Mass Profiler Professional (MPP) software. The results presented here demonstrate that this method can be used to efficiently separate metabolites from multiple compound classes in a reproducible way. Moreover, the 6495 Triple Quadrupole LC/MS allows for the detection of metabolites with great sensitivity, even at low dwell times. In summary, this is a reproducible and easy-to-use method that can be customized to fit specific needs and is suited to researchers with varying metabolomics expertise.