An Improved Metabolomics Workflow Enables Untargeted Data Acquisition and Targeted Data Analysis Using Liquid Chromatography–Mass Spectrometry

In this study, we present an improved metabolomics methodological framework that synergistically integrates untargeted data acquisition with targeted data analysis using chemical derivatization combined with ultrahigh performance liquid chromatography–quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF MS) analysis. Data-independent acquisition (DIA)-based mass spectrometry (MS1) data were used to conduct conventional untargeted analysis for biomarker discovery. The data-dependent acquisition (DDA) method was applied to obtain high-quality tandem mass spectrometry (MS2) information for quantitative data analysis. 1-Aminopiperidine (1AP) served as the derivatization reagent for sample preparation, which selectively reacts with carboxyl-containing compounds. Fatty acid (FA) standards were used to examine the derivatization reaction, and the results of LC-MS analysis showed that protonated FA + 1AP-H₂O was the precursor. The ion at m/z 84.08, along with the product ion from a neutral loss of 45.02 Da, emerged as characteristic fragments, facilitating compound annotation and quantitative analysis. Method validation results demonstrated the proposed method with excellent repeatability, stability, and linearity. The lung tissue samples were successfully analyzed using this method, which was further employed to evaluate the therapeutic efficacy of Zhuye Shigao Decoction (ZSD) against lipopolysaccharide (LPS)-induced acute pneumonia in mice.