Targeted Metabolomics: The LC-MS/MS Based Quantification of the Metabolites Involved in the Methylation Biochemical Pathways

Abstract

Biochemical methylation reactions mediate the transfer of the methyl group regulating vital biochemical reactions implicated in various diseases as well as the methylation of DNA regulating the replication processes occurring in living organisms. As a finite number of methyl carriers are involved in the methyl transfer, their quantification could aid towards the assessment of an organism’s methylation potential. An Hydrophilic Interaction Chromatography-Liquid Chromatography Multiple Reaction Monitoring (HILIC-LC-MRM) mass spectrometry (MS) methodology was developed and validated according to Food & Drug Administration (FDA), European Medicines Agency (EMA), and International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) for the simultaneous determination of nine metabolites i.e., B12, folic acid, 5-methyltetrahydrofolate, S-adenosylmethionine, S-adenosylhomocysteine, betaine, phosphocholine, N,N-dimethylglycine, and deoxythymidine monophosphate in human blood plasma. The sample pretreatment was based on a single step Solid-phase extraction (SPE) methodology using C18 cartridges. The methodology was found to accurately quantitate the analytes under investigation according to the corresponding dynamic range proposed in the literature for each analyte. The applicability of the method was assessed using blood donor samples and its applicability demonstrated by the assessment of their basal levels, which were shown to agree with the established basal levels. The methodology can be used for diagnostic purposes as well as for epigenetic screening.

Keywords

• methylation
• Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)
• solid phase extraction
• validation