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. KeywordsMetabolomics; Volumetric absorptive microsampling; Mass spectrometry ✉ Giuseppe Paglia beppepaglia@gmail.com. Compliance with ethical standardsThis study was performed in accordance with the ethical standards. The local ethics committee (Comitato etico del comprensorio sanitario di Bolzano) approved the study, and all participants provided written informed consent.

Metabolic Signature of Dietary Iron Overload in a Mouse Model

“…Whole blood was collected by means of VAMS and metabolites were extracted as previously described [17]. Briefly, VAMS tips were dried for 2 h and stored at −80 °C until metabolomics analysis.…”

“…As such, metabolomics is a dynamic approach of multiple biochemical pathway analysis, that also takes into account genetics, aging, environmental factors and lifestyle, thus allowing to identify specific metabolic signatures typical of a phenotype [13,14,15]. Metabolomic analysis can be performed in different tissues [16] and biofluids [17,18], and in combination with the information coming from the periphery tissue metabolites, it offers the possibility to better integrate and characterize systemic and tissue specific regulation of enzymatic pathways.…”

“…Back in the 1960s Guthrie and Susi developed a system for the screening of phenylketonuria in newborn infants where only a few drops of whole blood, collected on filter paper, called dried blood spots (DBS), were sufficient for the diagnosis [19,20]. Nowadays, the use of DBS is very common in clinics [21,22,23] nevertheless its application is limited by several factors, including the influence of hematocrit (HCT), the volume of the blood spotted onto the filter paper, and spot homogeneity [17].…”

“…We have recently validated the use of a commercially available tool, allowing for volumetric absorptive microsampling (VAMS), in combination with a mass spectrometry (MS)-based untargeted metabolomics workflow [17]. This technology, similar to DBS sampling, is a novel system that allows accurate and representative single-drop blood (10 µL) collection [24,25] and overcomes the issues with hematocrit.…”

Tranexamic Acid Quantification in Human Whole Blood Using Liquid Samples or Volumetric Absorptive Microsampling Devices

“…When compared with liquid whole blood samples prepared with the same method, the average ratio of the peak area between VAMS devices and liquids samples was 0.6, suggesting that the extraction recovery was not optimal. The sample preparation method was therefore modified according to previous reports [27,28]: samples were vortexed for 30 s, then put in ultrasonic bath for 30 min, shaken for 20 min and centrifuged at 17,000 × g for 10 min. With adding this step of ultrasonic bath, the peak area ratio between VAMS and liquid samples was improved to about 0.8.…”