Sensitive quantitative analysis of phosphorylated primary metabolites using selective metal oxide enrichment and GC- and IC- MS/MS

In this study, we present a novel selective cleanup/enrichment method based on metal oxide solid phase extraction combined with quantitative gas chromatography-tandem mass spectrometry and ion exchange chromatography-tandem mass spectrometry for the analysis of phosphorylated metabolites in yeast cell extracts relevant to biotechnological processes. Following screening of several commercially available metal oxide-based enrichment materials, all steps of the enrichment process (loading, washing and elution) were optimized for both the selective enrichment of 12 phosphorylated compounds from the glycolysis and pentose phosphate pathways, and the simultaneous removal of highly abundant matrix components such as organic acids and sugars. The full analytical workflow was then validated to meet the demands of accurate quantification of phosphorylated metabolites in yeast (Pichia pastoris) cell extracts using the best performing material and cleanup/enrichment method combined with quantification strategies based on internal standardization with isotopically labeled internal standards and external calibration. A good recovery (>70%) for 5 of the 12 targeted phosphorylated compounds with RSDs of less than 6.0% was obtained while many sugars, organic acids and amino acids were removed (>99% of glucose, and >95% of aspartate, succinate, glutamate, alanine, glycine, serine, threonine, proline, and valine). The use of isotopically labeled internal standards added to the samples prior to SPE, enables accurate quantification of the metabolites as it compensates for errors introduced during sample pretreatment and GC-MS or LC-MS analysis. To the best of our knowledge, this is the first time an effective and selective metal oxide-based affinity chromatography cleanup/enrichment method was designed and applied successfully for intracellular phosphorylated metabolites.