A merged method for targeted analysis of amino acids and derivatives using parallel reaction monitoring combined with untargeted profiling by HILIC-Q-Orbitrap HRMS.

With continuously increased scan rate and sensitivity, high resolution mass spectrometry (HRMS) allows for both reliable targeted analysis (e.g., parallel reaction monitoring, PRM) and a global overview for discovery-based untargeted profiling (e.g., data dependent acquisition, DDA) to be performed. Based on previous study on PRM for large scale targeted metabolomics quantification, we developed an innovative method merged targeted and untargeted approaches in a single run. In our workflow, the scheduled PRM for targeted analysis of amino acids and derivatives combined with the full scan was acquired in every sample injection by hydrophilic interaction liquid chromatography tandem quadrupole-Orbitrap high resolution mass spectrometry (HILIC-Q-Orbitrap HRMS). The identification of metabolic features from full scan was further performed with DDA methodology on grouped quality control (QC) samples and matched with available database. Specifically, 20 amino acids and 40 derivatives were selected for targeted analysis with optimal chromatographic separation and PRM parameters. All isomers within the selected metabolites were totally separated in the robust HILIC condition. 36 of selected metabolites were well-detected and showed a good linearity and reproducibility in NIST SRM 1950 plasma. Moreover, the absolute quantification performance of targeted PRM method was systematically validated using 10 amino acids with the corresponding stable isotope-labeled internal standards (SIL-IS). Finally, the newly developed method was successfully applied to analysis of the plasma samples from patients of pancreatic benign tumor and pancreatic cancer. The significant reduction of circulating amino acids in patients with pancreatic malignancy was confirmed by targeted PRM method and other amino acids modifications as well as polar metabolites were identified with untargeted profiling. Therefore, we have established a workflow that combines specifically and reliably targeted PRM method as well as broad-coverage untargeted profiling, which provides an innovative strategy for basic and clinical metabolomics study.