Optimizing nanoLC-TIMS-TOF and lipidomics sample preparation for low volume of plasma

Lipidomics provides critical insights into disease mechanisms, biomarker discovery, and precision medicine, but conventional LC-MS workflows often require large sample volumes, limiting their application in minimally invasive studies. Here, we establish a nanoflow liquid chromatography (nanoLC) platform coupled with trapped ion mobility spectrometry and time-of-flight mass spectrometry (TIMS-TOF) for comprehensive lipid profiling from as little as 1 μL of plasma. Two commonly employed single-phase extraction protocols (MeOH:MTBE and IPA:H₂O) were scaled to microsample volumes using NIST® SRM® 1950 plasma, and the influence of mobile phase buffer concentration (10, 5, and 2 mM NH₄HCO₂) on lipid coverage and chromatographic performance was systematically evaluated. Multivariate analysis revealed solvent- and buffer-dependent shifts in lipid distributions, while manual annotation and verification based on ion mobility ensured precise lipid identification, even from small volumes. The optimized nanoLC-TIMS-TOF workflow achieved broad lipid class coverage, enhanced detection of low-abundance species, and improved separation of structurally similar lipids using only 1 μL of starting material. Compared to high-flow LC-MS approaches, similar lipid coverage was achieved at lower buffer concentrations, though ammonium adduct formation introduced distinct lipid-specific biases. These results demonstrate the robustness and scalability of nanoLC-TIMS-MS lipidomics for sample-limited applications.