Metabolic and lipidomic stability in EDTA plasma from hospitalized patients: impact of refrigerated storage time and additional freeze—thaw cycle

BACKGROUND: Global metabolomics and lipidomics are increasingly applied in clinical research. Storage variability can compromise analyte stability and omics data quality, especially in clinical settings. While previous studies have focused on healthy individuals, the stability of the metabolome and lipidome in patient samples remains underexplored. OBJECTIVE: To identify metabolites and lipids most susceptible to be affected by post-centrifugation storage time and a single additional freeze-thaw cycle in EDTA plasma from hospitalized patients. METHODS: EDTA plasma samples from 20 patients acutely hospitalized in a medical ward were collected (K2EDTA, 5 mL, with gel) and stored at 4 °C for 0, 24, and 72 h post-centrifugation. All samples underwent one additional freeze-thaw cycle and were analyzed using global liquid chromatography-mass spectrometry (LC-MS) metabolomics and lipidomics. RESULTS: Approximately 90% of the global metabolome and lipidome remained stable and robust to the pre-analytical factors induced. Metabolomic profiles showed a storage time-dependent increase in differentially abundant features, while most lipidomic alterations occurred within the first 24 h. A total of 116 annotated compounds exceeded a Cohen's d effect size threshold of ± 0.25, including lactate, hypoxanthine, oxoproline, fatty acid(20:4), and lysophosphatidylcholine(16:0). CONCLUSION: The plasma metabolome and lipidome are largely robust to common storage conditions in patient samples. However, refrigerated plasma storage time post-centrifugation and an additional freeze-thaw cycle can induce biologically significant changes in specific metabolites and lipids. This study is among the first to evaluate metabolomic and lipidomic stability using clinical samples and our data supports a sample collection that can be easily implemented in clinical laboratory workflows.