Abstract
Microsampling technologies have emerged in bioanalysis and find applications in various fields of biomedical research including metabolomics studies. Although several devices share the same concept for sample collection, variations on their matrices might impact analytical outcomes from biological samples. The aim of the present work is to evaluate the information obtained by the different microsampling devices, namely Dried Blood Spots, Volumetric Absorptive Microsampling and Capitainer cards for the blood global metabolic profile.
To achieve this goal, a whole blood untargeted analysis method was developed, using an UHPLC with a Reverse Phase system, coupled to a TIMS-TOF working in DDA mode. MS conditions were optimized to increase sensitivity and reduce in- source fragmentation. Our aim was to obtain the most detailed chromatographic profile possible, while facilitating trustful annotations. Extraction protocols were optimized based on literature surveys [1][2]. Among the different solvents tested (acetonitrile, methanol and water, both individually and at different proportions), acetonitrile:methanol mixture in a ratio of 70:30 yielded the most favorable outcomes, considering number features, area of annotated peaks and reproducibility. Data processing and analysis was performed by different software (i.e. MSDial, XCMS, Metaboscape).
Each individual device underwent a similar optimization process. Methanol: H2O demonstrated to be the most efficient solvent, when allied to vortex agitation and sonication with beads. To increase sensitivity and improve chromatographic profile, samples underwent evaporation and reconstitution in a reduced volume of water-methanol mixture at a ratio of 95:5.
A similar procedure was done using a GC/MS method. Samples underwent an analogous process for solvent optimization, and derivatization was performed after sample evaporation using Methoxyamine and Trimethylsilane [3]. Finally, we compared the untargeted profiles obtained from ten individuals using the three different microsampling devices and plasma samples, under the optimized analysis conditions at both chromatographic systems.
References
[1] de Sá e Silva, D.M. Thaitumu, M.; Theodoridis, G.; Witting, M.; Gika, H. Volumetric Absorptive Microsampling in the Analysis of Endogenous Metabolites. Metabolites 2023, 13, 1038. https://doi.org/10.3390/metabo13101038
[2] Southam AD , Haglington LD , Najdekr L , Jankevics A , Weber RJM , Dunn WB . Assessment of human plasma and urine sample preparation for reproducible and high-throughput UHPLC-MS clinical metabolic phenotyping. Analyst. 2020145(20):6511-6523. doi:10.1039/d0an01319f
[3] Dunn WB, Broadhurst D, Begley P, et al. Procedures for large-scale metabolic profiling of serum and plasma using gas chromatography and liquid chromatography coupled to mass spectrometry. Nat Protoc. 20116(7):1060-1083. Published 2011 Jun 30. doi:10.1038/nprot.2011.335