GNPS - A comprehensive lipidomic workflow for multi-cohort population phenotyping using stable isotope dilution targeted liquid chromatography-mass spectrometry

Dysregulated lipid metabolism underpins many chronic diseases including cardiometabolic diseases. Mass spectrometry-based lipidomics is an important tool for understanding mechanisms of lipid dysfunction and is widely applied in epidemiology and clinical studies. With ever-increasing sample numbers, single batch acquisition is often unfeasible, requiring advanced methods that are accurate and robust to batch-to-batch and inter-day analytical variation. Herein, an optimised comprehensive targeted workflow for plasma and serum lipid quantification is presented, combining stable isotope internal standard dilution, automated sample preparation, and ultra-high performance liquid chromatography-tandem mass spectrometry with rapid polarity switching to target 1163 lipid species spanning 20 sub-classes. The resultant method is robust to common sources of analytical variation including blood collection tubes, haemolysis, freeze-thaw cycles, storage stability, analyte extraction technique, inter-instrument variation and batch-to-batch variation with 820 lipids reporting a relative standard deviation of <30% in 1048 replicate quality-control plasma samples acquired across 16 independent batches (total injection count= 6142). However, sample haemolysis of greater than 0.4% impacted lipid concentrations, specifically for phosphatidylethanolamines (PEs). Low inter-instrument variability across two identical LC-MS systems indicated feasibility for intra-/inter-lab parallelisation of the assay. In summary, we have optimised a comprehensive lipidomic protocol to support rigorous analysis for large-scale, multi-batch applications in precision medicine.