Phosphate-specific derivatization for concurrent analysis of phosphate-containing human metabolites in reverse-phase LC-MS

Abstract

Phosphate-containing human metabolites such as nucleotides, acyl-coenzyme A, phospholipids and phosphorylated sugars, etc., play crucial role in life process and are related to the pathogenesis of many diseases. However, the analysis of these phosphate-containing metabolites using reverse-phase (RP) LC-ESI/MS is still challenging because of the highly polar nature of polyanionic phosphate groups and their non-specific binding with column and tubing. Therefore, special chromatographic technologies such as ion-pairing reagents or hydrophilic interaction chromatography (HILIC) are commonly employed to analyze phosphate-containing metabolites. However, these analytical methods still have diverse problems including system contamination, ion suppression and chromatographic resolving power. In this study, we developed a phosphate-specific derivatization method in aqueous phase to analyze phosphate-containing human metabolites in reverse-phase LC-ESI/MS. As results, we optimized the reaction conditions for the phosphate-specific derivatization, and the phosphate specificity was verified via simultaneous derivatization of diverse compounds including aliphatic ？OH, phenolic ？OH, aliphatic ？COOH, phenolic ？COOH, sulfonic ？OH and etc. In addition, an increase in retention time in RPLC and an excellent peak shape of phosphate-containing metabolites were obtained. In conclusion, the developed derivatization method has a potential to be a novel tool for the concurrent analysis of phosphate-containing metabolites in RPLC. It could become a key technology to develop a single analytical LC-MS/MS platform for the concurrent analysis of over 1000 metabolites in targeted metabolomics.