Single-analytical platform for targeted concurrent analysis of 1,032 metabolites

Abstract

In metabolomics, the detection, structural identification and reliable quantification of as many metabolites as possible are key point. Therefore, comprehensive and sensitive concurrentanalysis of metabolites using a single analytical platform is of great importance. However, it is challenging to develop a sensitive and comprehensive analytical method covering all classes of metabolites in a single-run analysis due to their structural diversity, wide range of polarities, ionization modes and efficiencies, and chromatographic behavior. The aim of this study is to develop a single-analytical LCESI/MS/MS platform for the targeted concurrent analysis of over 1,000 metabolites. To achieve integrated chromatography, concurrent ionization, and reduced non-specific binding, various column and mobile phase compositions were systematically investigated and optimized. Additionally, a phosphate-specific derivatization method was developed to enhance the retention and detection of phosphate-containing metabolites, which typically exhibit poor chromatographic behavior in reversedphase liquid chromatography (RPLC) due to strong non-specific binding with LC-MS system. As results, a high number of metabolites in diverse biological matrices (serum: 634, plasma: 581, urine: 465, whole blood: 641, liver: 701) were identified by the present single-analytical platform using RPLC-ESI-MS/MS coupled with phosphatespecific derivatization. This method was successfully applied to the analysis of liver samples investigating the effects of starvation.