Evaluation of metabolite stability in dried blood spot stored at different temperatures and times

Dried blood spot (DBS) sampling offers significant advantages over conventional blood collection methods, such as reduced sample volume, minimal invasiveness, suitability for home-based sampling, and ease of transport. However, understanding the effects of variable storage temperatures and times on metabolite stability is crucial due to varying intervals and delivery conditions between sample collection and metabolomics analysis. To minimize biological variances, all samples were collected from the same individual simultaneously and stored at three different temperatures (4 °C, 25 °C, and 40 °C) for diverse time points (3, 7, 14, and 21 days). Metabolic profiling was conducted an untargeted gas chromatography-mass spectrometry (GC-MS) and ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS)-based multi-platform metabolomics. Principal component analysis (PCA) showed alterations in metabolite stability at different temperatures, with phosphatidylcholines (PCs) and triglycerides (TAGs) as the first principal component (PC1). Specifically, we identified 69 metabolites that remained stable across all three temperatures over the 21-day period, while 78 metabolites exhibited instability. Furthermore, linear correlations between metabolite intensity and storage time were observed. Overall, our study elucidated the influence of storage temperature and time on specific metabolite stability in DBS samples, providing valuable insights for study design, biomarker selection, and data improvement.