Blood microsampling (BμS) has recently emerged as an interesting approach in the analysis of endogenous metabolites but also in metabolomics applications. Their non-invasive way of use and the simplified logistics that they offer renders these technologies highly attractive in large-scale studies, especially the novel quantitative microsampling approaches such as VAMs or qDBS. Objectives: Herein, we investigate the potential of BµS devices compared to the conventional plasma samples used in global untargeted mass spectrometry-based metabolomics of blood. Methods: Two novel quantitative devices, namely, Mitra, Capitainer, and the widely used Whatman cards, were selected for comparison with plasma. Venous blood was collected from 10 healthy, overnight-fasted individuals and loaded on the devices; plasma was also collected from the same venous blood. An extraction solvent optimization study was first performed on the three devices before the main study, which compared the global metabolic profiles of the four extracts (three BµS devices and plasma). Analysis was conducted using reverse phase LC-TOF MS in positive mode. Results: BµS devices, especially Mitra and Capitainer, provided equal or even superior information on the metabolic profiling of human blood based on the number and intensity of features and the precision and stability of some annotated metabolites compared to plasma. Despite their rich metabolic profiles, BµS did not capture metabolites associated with biological differentiation of sexes. Conclusions: Overall, our results suggest that a more in-depth investigation of the acquired information is needed for each specific application, as a metabolite-dependent trend was obvious.

Introduction: Blood microsampling (BµS) devices collect less than 100 µL of blood, offering a less invasive and more cost-effective alternative to venipuncture. However, its metabolomic comparability to conventional samples remains unclear, and standardized BµS metabolomic workflows are lacking. Objectives: This study evaluated the impact of using three BµS devices (Mitra ^® , Capitainer ^® , and Whatman ^™ 903) on the metabolomic interpretation of human biomonitoring samples. We compared them to conventional samples (plasma and whole blood) and evaluated the interplay of different analytical conditions. Methods: Venous blood from 10 adults (5 males, 5 females) was sampled onto the three devices. First, three agitation conditions (ultrasound, shaker, and homogenizer) were evaluated at three blood concentrations (1.5%, 5.5%, and 11%). The optimized method was then used to compare the metabolite profiles between BµS devices, whole blood, and plasma. Reverse-phase and hydrophilic-interaction chromatography, in positive and negative ionization modes, were combined for liquid chromatography–mass spectrometry (LC-MS) analysis. Results: All agitation conditions and concentrations proved suitable for BµS untargeted metabolomics. Combining different analytical modes and fragmentation ranges proved helpful for maximizing metabolite coverage. BµS-derived metabolite profiles aligned more closely with whole blood than plasma. Some metabolites were more characteristic of a sample type, whereas others were common across sample types. All sample types enabled sex-based differentiation, with metabolites such as amino acids, lipids, and acylcarnitines driving the separation. Conclusions: These findings enhance our understanding of BµS metabolite coverage and highlight its potential in human biomonitoring. The choice of device depends on the application and the metabolites of interest, offering flexibility for clinical use and research.

Introduction: Blood microsampling (BµS) devices collect less than 100 µL of blood, offering a less invasive and more cost-effective alternative to venipuncture. However, its metabolomic comparability to conventional samples remains unclear, and standardized BµS metabolomic workflows are lacking. Objectives: This study compared BµS extraction methods and assessed the metabolite coverage of three BµS devices (Mitra®, Capitainer®, and Whatman™ 903) as an alternative to conventional samples (plasma and whole blood) for human biomonitoring. Methods: Venous blood from 10 adults (5 males, 5 females) was sampled onto the three devices. First, three extraction techniques (ultrasound, shaker, and homogenizer) were evaluated at three blood concentrations (1.5%, 5.5%, and 11%). The optimized method was then used to compare the metabolite profiles between BµS devices, whole blood, and plasma. Reverse-phase and hydrophilic-interaction chromatography, in positive and negative ionization modes, were combined for Liquid Chromatography–Mass Spectrometry (LC-MS) analysis. Results: All extraction techniques and concentrations proved suitable for BµS untargeted metabolomics. Combining different analytical modes and fragmentation ranges proved useful for maximizing metabolite coverage. BµS-derived metabolite profiles aligned more closely with whole blood than plasma. Some metabolites were more characteristic of a sample type, whereas others were common across sample types. All sample types enabled sex-based differentiation, with metabolites such as testosterone sulfate and hydroxyisovaleric acid driving the separation. Conclusions: These findings enhance our understanding of BµS metabolite coverage and highlight its potential in human biomonitoring. The choice of device depends on the application and the metabolites of interest, offering flexibility for clinical use and research.