Workflow optimization for integrated mass spectrometry-based metabolomics and lipidomics from dried blood microsamples

Blood microsampling has attracted interest as a viable alternative to conventional venipuncture for a wide range of applications including metabolomics. It offers several advantages such as minimal invasiveness, ease of collection, suitability for multiple sampling and longitudinal designs1. This study aims to optimize a liquid chromatography-mass spectrometry-based workflow enabling both untargeted metabolomics and lipidomics analyses from a single dried blood spot (DBS). Workflow optimization was also performed for targeted metabolomics on DBS using the TMIC MEGA assay. For the untargeted workflow the optimization was performed on three commercially available microsampling devices: Capitainer, Whatman (whole blood) and Telimmune (plasma). Among the five extraction solutions tested, pure methanol provided the best compromise for simultaneous extraction of polar metabolites and lipids from the same spot. A two-step consecutive extraction protocol was developed: methanol followed by water improved the recovery of more polar metabolite classes and was successfully applied in a clinical study on cardiac rehabilitation2. Short-term stability of polar metabolites and lipids was assessed at room temperature (RT) for up to five days. Stability of all evaluated compound classes was preserved for up to five days at RT in Capitainer, delivering the best performance. Regarding targeted metabolomics workflow optimization, the manufacturer’s protocol was modified for both Panel A and Panel B. Compared to the initial tests, an increased metabolite coverage was observed in DBS with 323 detected metabolites. Longitudinal comparison with paired serum samples showed that relative temporal changes were preserved in DBS. Overall, this study suggests that methanol extraction enables integrated metabolomics and lipidomics analysis from a single spot and a two-step approach can further enhance polar metabolite coverage. Targeted protocol optimization on the TMIC MEGA assay also improved metabolome coverage in DBS, highlighting the importance of tailored device selection and extraction protocol optimization based on study aims, matrices, and analytical scope.