Stability of serum metabolites following delayed centrifugation

: Pre-analytical variability represents a critical source of error in laboratory medicine, significantly influencing the accuracy of serum metabolite measurements. In metabolomics, delays in centrifugation can lead to artificial changes in metabolite concentrations due to prolonged interaction between blood cells and serum. Therefore, standardizing the time to centrifugation is essential for ensuring reliable and comparable data. This study investigated the stability of serum metabolites under different centrifugation delays. Venous blood samples were collected from healthy volunteers. For each individual, one sample was processed immediately (0 h), while two additional samples were kept at room temperature and centrifuged after 2 and 6 h. A total of 58 metabolites, including amino acids and acylcarnitines, were quantified using targeted tandem mass spectrometry. Statistical analysis was conducted using repeated-measures ANOVA with Bonferroni correction, and changes were also expressed as percentage differences from baseline. Results showed that a subset of metabolites underwent significant time-dependent alterations with delayed centrifugation. Notably, short- and long-chain acylcarnitines, including acetyl- (C2), 3-hydroxyisovaleryl- (C5-OH), stearoyl- (C18), oleyl- (C18:1), and linoleyl- (C18:2) and some amino acids, including alanine, aspartate, glutamate, glycine, branched-chain, and aromatic amino acids increased progressively at 2 and 6 h. In contrast, most metabolites remained stable, while a few, such as glycyl-proline and carnitine palmitoyltransferase I, showed decreases. Overall, the findings demonstrate that delayed centrifugation affects specific metabolite levels, particularly amino acids and acylcarnitines, while most remain stable. This underscores the importance of prompt and standardized sample processing in metabolomics studies.