Pre-analytic evaluation of volumetric absorptive microsampling and integration in a mass spectrometry-based metabolomics workflow

Abstract

Volumetric absorptive microsampling (VAMS) is a novel approach that allows single-drop (10 μL) blood collection. Integration of VAMS with mass spectrometry (MS)-based untargeted metabolomics is an attractive solution for both human and animal studies. However, to boost the use of VAMS in metabolomics, key pre-analytical questions need to be addressed. Therefore, in this work, we integrated VAMS in a MS-based untargeted metabolomics workflow and investigated pre-analytical strategies such as sample extraction procedures and metabolome stability at different storage conditions. We first evaluated the best extraction procedure for the polar metabolome and found that the highest number and amount of metabolites were recovered upon extraction with acetonitrile/water (70:30). In contrast, basic conditions (pH 9) resulted in divergent metabolite profiles mainly resulting from the extraction of intracellular metabolites originating from red blood cells. In addition, the prolonged storage of blood samples at room temperature caused significant changes in metabolome composition, but once the VAMS devices were stored at - 80 °C, the metabolome remained stable for up to 6 months. The time used for drying the sample did also affect the metabolome. In fact, some metabolites were rapidly degraded or accumulated in the sample during the first 48 h at room temperature, indicating that a longer drying step will significantly change the concentration in the sample. Graphical abstract Volumetric absorptive microsampling (VAMS) is a novel technology that allows single-drop blood collection and, in combination with mass spectrometry (MS)-based untargeted metabolomics, represents an attractive solution for both human and animal studies. In this work, we integrated VAMS in a MS-based untargeted metabolomics workflow and investigated pre-analytical strategies such as sample extraction procedures and metabolome stability at different storage conditions. The latter revealed that prolonged storage of blood samples at room temperature caused significant changes in metabolome composition, but if VAMS devices were stored at - 80 °C, the metabolome remained stable for up to 6 months.

Keywords: Mass spectrometry; Metabolomics; Volumetric absorptive microsampling.

Fig. 1

Evaluation of extraction procedures for the integration of VAMS with untargeted metabolomics. a Total count features in positive mode obtained for each specific group after processing the data by using our XCMS. b Total count features in negative mode obtained for each specific group after processing the data by using our XCMS. c Overlaid base peak chromatograms and feature count along the chromatogram obtained using different extraction procedures in positive mode. d Overlaid base peak chromatograms and feature count along the chromatogram obtained using different extraction procedures in negative mode. e Principal component analysis performed on features obtained in positive mode. f Principal component analysis performed on features obtained in negative mode. Color code: yellow represents samples extracted using the ACN solution; orange represents samples extracted using the ACN-H₂O solution; violet represents samples extracted using the MeOH solution; red represents samples extracted using the pH 2 solution; green represents samples extracted using the pH 7 solution; blue represents samples extracted using the pH 9 solution

Fig. 2

Evaluation of the extraction procedures for the recovery of different classes of metabolites. a Comparison of extraction procedure for the analysis of carboxylic acid. b Comparison of extraction procedure for the analysis of RBC intracellular metabolites. c Comparison of extraction procedure for the analysis of other metabolites. d Comparison of extraction procedure for the analysis of amino acids. e Comparison of extraction procedure for the analysis of carnitines. f Comparison of extraction procedure for the analysis of phospholipids

Fig. 3

Evaluation of the number of consecutive steps necessary for extracting the whole metabolome. a Percent of extracted metabolites in each extraction step for selected classes of metabolites. Amino acids, n = 37; phosphorylated compounds, n = 24; carboxylic acids, n = 20; carnitines, n = 10; lipids n = 21; other compounds, n = 21. The percent of extracted metabolites was calculated based on the sum of the signals obtained in each extraction step. b Overlaid percent of extracted metabolites in each extraction step for selected classes of metabolites

Fig. 4

Evaluation of sample stability at different storage conditions. a Experimental design. b Principal component analysis performed on tentatively identified metabolites (n = 103)

Fig. 5

Heatmap performed on annotated metabolites extracted from VAMS sample stored at different conditions

Fig. 6

Time profiles of selected metabolites stored at different storage conditions. The average of three technical replicates is plotted in each chart