Comprehensive and Reproducible Untargeted Lipidomic Workflow Using LC-QTOF Validated for Human Plasma Analysis

The goal of this work was to develop a label-free, comprehensive, and reproducible high-resolution liquid chromatography–mass spectrometry (LC-MS)-based untargeted lipidomic workflow using a single instrument, which could be applied to biomarker discovery in both basic and clinical studies. For this, we have (i) optimized lipid extraction and elution to enhance coverage of polar and nonpolar lipids as well as resolution of their isomers, (ii) ensured MS signal reproducibility and linearity, and (iii) developed a bioinformatic pipeline to correct remaining biases. Workflow validation is reported for 48 replicates of a single human plasma sample: 1124 reproducible LC-MS signals were extracted (median signal intensity RSD = 10%), 50% of which are redundant due to adducts, dimers, in-source fragmentation, contaminations, or positive and negative ion duplicates. From the resulting 578 unique compounds, 428 lipids were identified by MS/MS, including acyl chain composition, of which 394 had RSD < 30% inside thei...