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

Abstract

Lipidomics provides critical insights into disease mechanisms, biomarker discovery, and precision medicine, but conventional LC-MS workflows often require sample volumes that limit their application in minimally invasive studies. Here, we evaluated a nanoflow liquid chromatography workflow coupled with trapped ion mobility spectrometry and time-of-flight mass spectrometry (nanoLC-TIMS-TOF) for positive ion lipid profiling from as little as 1 µL of plasma. Two commonly used single-phase extraction protocols, MeOH: MTBE and IPA: H₂O, were scaled to microsample volumes using NIST® SRM® 1950 plasma. The effects of plasma input volume and mobile-phase ammonium formate concentration (2, 5, and 10 mM) on feature detection and chromatographic performance were evaluated. Descriptive comparisons revealed extraction-, volume-, and buffer-associated differences in the detected feature profiles, while putative lipid annotations were evaluated using MS/MS and ion-mobility information, where available. The optimized workflow enabled broad positive ion lipid class profiling and detection of low intensity features from only 1 µL of starting material. The 1 µL extracts retained the dominant annotated lipid classes observed at higher input volumes, although profiling depth and feature distributions remained dependent on plasma input volume. Among the tested mobile phase conditions, 5 mM ammonium formate produced the highest detected feature count while limiting pressure drift during extended acquisition. These results support the feasibility and practical scalability of positive ion nanoLC-TIMS-MS lipid profiling for sample-limited applications under the evaluated conditions.