Abstract
Introduction: The aim of this study was to analyze whether blood microsampling (µS) can provide equal information on circulating metabolites in a human biological system when compared to a conventional matrix, plasma1. In our study, three blood microsampling (BµS) devices, Whatman dried blood spots (W), Mitra volumetric absorptive sampling device (M), and Capitainer B (C) were compared with plasma using a global untargeted LC-MS/MS assay.
Methods: Venipuncture blood samples were collected from 10 fasting healthy individuals. Twenty μl of the blood from each individual was sampled onto W, C, and V. Liquid whole blood and plasma was also collected. Samples were stored in -80̊C for six months before analysis. The extraction process involved protein precipitation, mixing, sonication, centrifugation, evaporation, and reconstitution with the mobile phase spiked with three internal standards (phenylalanine-c13, butyrylcarnitine-d3, and octanoylcarnitine-d3) at 5 ppm. An aliquot was collected from each individual matrix (devices or plasma) to create a quality control (QC) pool for assessment of within run precision. The samples were analyzed using an untargeted reverse phase LC-time of flight-MS assay in positive ESI mode and data dependent acquisition (DDA). An ACQUITY UPLC HSS T3 1.8 µm, 2.1 x 100 mm column was used to separate the metabolites over a 17 min gradient. Data were preprocessed and analyzed using MSDIAL and XCMS.
Results: There were 3805 common metabolite features (m/z-rt) in all the samples and matrices. Higher numbers of identified features were observed in C, followed by M, W, and plasma. In addition, C had the greatest number of features with higher intensity. Each specimen however showed to have some unique features, 910 were unique to C, 287 to plasma, 94 to M, and 11 to W. Multivariate analysis of the data showed that metabolic fingerprints differ depending on the device and all differ from native plasma. W and M show more similar profiles. Regarding the metabolites detected, 46 were annotated based on an in-house library built from whole blood analysis. Twenty-six were level 1 matched to standards and 22 level 2 identifications. For the annotated metabolites, 33 out of the 46 metabolites had higher intensity in C samples whereas 6 metabolites namely ergothioneine, cysteinylglycine, glutathione (reduced and oxidized), adenosine 5`-monophosphate, and s-4-(2-oxo-butyl)glutathione were not detected in plasma.
Summary: The results suggest that dried blood specimens can provide useful metabolic information similar to plasma. However, differences are observed that need to be further investigated. Based on these results it was shown that the C device had the greatest number of features and the most annotated metabolites with higher intensity. Furthermore, some metabolites that are not detected in the conventional matrix, plasma, can be seen in dried BµS .
Acknowledgement: This research has been funded by the European Union under the HUMAN Marie Curie Doctoral Training Network, Project Number - 101073062.
References
1. de Sá E Silva, D. M., Thaitumu, M., Theodoridis, G., Witting, M., & Gika, H. (2023). Volumetric Absorptive Microsampling in the Analysis of Endogenous Metabolites. Metabolites, 13(10), 1038. https://doi.org/10.3390/metabo13101038
2. Kind, T., Tsugawa, H., Cajka, T., Ma, Y., Lai, Z., Mehta, S. S., Wohlgemuth, G., Barupal, D. K., Showalter, M. R., Arita, M., & Fiehn, O. (2018). Identification of small molecules using accurate mass MS/MS search. Mass spectrometry reviews, 37(4), 513–532. https://doi.org/10.1002/mas.21535