Deep insights into microsampling technologies for global lipidomic profiling of human whole blood using high-throughput LC-MS

• First comprehensive lipidomics study of human whole blood from 3 BµS devices vs plasma. • All-in-one lipid extraction protocol successfully applied to all BµS devices. • Single-step data processing and lipid annotation using targeted peak extraction. • Over 500 lipids detected in the 3 BµS devices and plasma for lipidome comparison. • Extraction recovery, matrix effect, and precision evaluation of BµS devices. In recent years, blood microsampling (BμS) technologies have gained popularity, as they offer simplicity, minimal invasiveness, and suitability for remote sample collection. In this study, the aim was to explore the potential of three BμS devices for reliable untargeted lipidomic profiling using reversed-phase ultra-high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry. Such devices –from the traditional non-volumetric dried blood spot cards (DBS) to the recently developed quantitative DBS (qDBS), and volumetric absorptive microsampling (VAMS)– were compared to conventional plasma samples from ten healthy volunteers. In this novel study, a comprehensive lipidomic workflow was applied, combining an all-in-one extraction protocol and a single-step data processing and lipid annotation using a targeted peak extraction strategy based on a human plasma lipid database. BμS lipidomics analysis was evaluated for recovery, matrix effect and precision. The data acquired for BµS devices yielded consistent profiles, comparable to plasma, observing that the volumetric devices –qDBS and VAMS– were more similar between them compared to DBS. For BμS, more than 500 lipid species from 20 distinct lipid subclasses were annotated with high confidence level. Most deuterium-labeled lipid species studied were successfully recovered in the BµS devices with minimal ion suppression detected. Precision analysis of deuterated lipid species and the 195 endogenous lipids indicated that qDBS and VAMS had higher precision compared to DBS. Differences in lipids between each BµS device and plasma were assessed. Our work presents a robust workflow for lipid profiling using BμS, suitable for taking informed decisions in further experimental designs.