Intracellular metabolite profiling of platelets: Evaluation of extraction processes and chromatographic strategies

Paglia, Giuseppe; Magnusdottir, Manuela; Thorlacius, Steinunn; Sigurjónsson, Ólafur E.; Guðmundsson, Sveinn; Palsson, Bernhard Ø.; Thiele, Ines

doi:10.1016/j.jchromb.2012.04.026

Cited by 44 publications

(45 citation statements)

References 28 publications

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“…Moreover, extraction protocols are sample dependent [29,30], and specific extraction procedures are recommended for cells [31], serum [18], urine [32], and dried blood spots [15,33], respectively.…”

Section: Europe Pmc Funders Author Manuscriptsmentioning

confidence: 99%

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

et al. 2017

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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.

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Section: Europe Pmc Funders Author Manuscriptsmentioning

confidence: 99%

“…We have previously demonstrated that HILIC-MS methods are suitable for separating polar metabolites [31,19]. Since we are interested in polar metabolites, in this study, we analyzed the metabolomes extracted from VAMS by using a HILIC-MS method designed for the large-scale studies of plasma and serum samples [34].…”

Section: Europe Pmc Funders Author Manuscriptsmentioning

confidence: 99%

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

et al. 2017

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“…So far, the majority of related LC-MS studies use reversed phase [41][42][43][44][45][46][47][48] or hydrophilic interaction stationary phases [12,13,15,16,[49][50][51][52][53][54]. Despite multiple advantages such as robustness and straightforward implementation, reversed phase liquid chromatography (RPLC) typically lacks adequate retention for nonderivatized hydrophilic analytes on silica-based reversed phase stationary phases [33].…”

Section: Discussionmentioning

confidence: 99%

Alkaline conditions in hydrophilic interaction liquid chromatography for intracellular metabolite quantification using tandem mass spectrometry

Teleki

Santiago

Takors

2015

Analytical Biochemistry

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“…The choice of ionization mode is also relevant as particular metabolites only ionize efficiently in a certain mode; the ionization efficiency can also be improved by using either acidic or alkaline mobile phase conditions (Paglia et al 2012). In theory, this means that in order to obtain maximum metabolite coverage, chromatographic and mass spectrometric selectivity needs to be sufficiently high.…”

Section: Lc-ms Analysismentioning

confidence: 99%

LC–MS based global metabolite profiling: the necessity of high data quality

et al. 2016

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LC-MS based global metabolite profiling currently lacks detailed guidelines to demonstrate that the obtained data is of high enough analytical quality. Insufficient data quality may result in the failure to generate a hypothesis, or in the worst case, a false or skewed hypothesis. After assessing the literature, it is apparent that an analytically focused summary and critical discussion related to this subject would be beneficial for both beginners and experts engaged in this field. A particular focus will be placed on data quality, which we here define as the degree to which a set of parameters fulfills predetermined criteria, similar to the established guidelines for targeted analysis. However, several of these parameters are difficult to assess since holistic approaches measure thousands of metabolites in parallel and seldom include predefined knowledge of which metabolites will differ between sample groups. In this review, the following parameters will be discussed in detail: reproducibility, selectivity, certainty of metabolite identification and metabolite coverage. The review systematically describes the generic workflow for LC-MS based global metabolite profiling and highlights how each separate part may affect data quality. The last part of the review describes how data quality can be evaluated as well as identifies areas where additional improvement is needed. In this review, we provide our own analytical opinions in regards to evaluation and, to some extent, improvement of data quality.

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Intracellular metabolite profiling of platelets: Evaluation of extraction processes and chromatographic strategies

Cited by 44 publications

References 28 publications

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

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

Alkaline conditions in hydrophilic interaction liquid chromatography for intracellular metabolite quantification using tandem mass spectrometry

LC–MS based global metabolite profiling: the necessity of high data quality

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