Assessment of human plasma and urine sample preparation for reproducible and high-throughput UHPLC-MS clinical metabolic phenotyping

Southam, Andrew D.; Haglington, Liam D.; Najdekr, Lukáš; Jankevics, Andris; Weber, Ralf; Dunn, Warwick B.

doi:10.1039/d0an01319f

Cited by 40 publications

(45 citation statements)

References 40 publications

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“…MTBE/methanol [3/1] demonstrated high spectral reproducibility, while chloroform/methanol [3/1] extracted high numbers of reproducible features . In a cell line model, isopropanol/water [3/1] and [9/1] had high reproducibility and a comparable yield to biphasic chloroform/methanol/water and MTBE/methanol/water methods with apparent increases in the yield of some lipid classes including phosphatidylinositol (consistent with the findings in plasma); however, this approach has not been tested in tissues.…”

Section: Introductionmentioning

confidence: 96%

“…Monophasic approaches are less common for tissue metabolite and lipid extraction despite their popularity for plasma preparation . For tissue monophasic metabolite extractions, methanol/water [1/1] was shown to be more reproducible than acetonitrile/water [1/1] and methanol/water [1/3] .…”

Section: Introductionmentioning

confidence: 99%

“…Resuspension of dried polar extracts for HILIC UHPLC-MS analysis often matches the initial mobile phase conditions (95/5 acetonitrile/water). , However, HILIC UHPLC-MS performs equally well with other sample solvent compositions (e.g., methanol/water [3/1] and methanol/acetonitrile/water [1.5/1.5/1]) . To maximize the solubility and detection of the extracted compounds, the extraction and resuspension solvents should be the same if they are compatible with the HILIC assay.…”

Section: Introductionmentioning

confidence: 99%

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Characterization of Monophasic Solvent-Based Tissue Extractions for the Detection of Polar Metabolites and Lipids Applying Ultrahigh-Performance Liquid Chromatography–Mass Spectrometry Clinical Metabolic Phenotyping Assays

et al. 2020

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Metabolic phenotyping of tissues uses metabolomics and lipidomics to measure the relative polar and nonpolar (lipid) metabolite levels in biological samples. This approach aims to understand disease biochemistry and identify biochemical markers of disease. Sample preparation methods must be reproducible, sensitive (high metabolite and lipid yield), and ideally rapid. We evaluated three biphasic methods for polar and nonpolar compound extraction (chloroform/methanol/water, dichloromethane/methanol/water, and methyl tert-butyl ether [MTBE]/methanol/water), a monophasic method for polar compound extraction (acetonitrile/methanol/water), and a monophasic method for nonpolar compound extraction (isopropanol/water). All methods were applied to mammalian heart, kidney, and liver tissues. Polar extracts were analyzed by hydrophilic interaction chromatography (HILIC) ultrahigh-performance liquid chromatography−mass spectrometry (UHPLC− MS) and nonpolar extracts by C 18 reversed-phase UHPLC−MS. Method reproducibility and yield were assessed using multiple annotated endogenous compounds (putatively and MS/MS annotated). Monophasic methods had the highest yield and high reproducibility for both polar (positive ion: median relative standard deviation (RSD) < 18%; negative ion: median RSD < 28%) and nonpolar (positive and negative ion: median RSD < 15%) extractions for heart, kidneys, and liver. The polar monophasic method extracted higher levels of lipid than biphasic polar extractions, and these lipids caused minimal detection suppression for other compounds during HILIC UHPLC−MS. The nonpolar monophasic method had similar or greater detection responses of all detected lipid classes compared to biphasic methods (including increased phosphatidylinositol, phosphatidylserine, and cardiolipin responses). Monophasic methods are quicker and simpler than biphasic methods and are therefore most suited for future automation.

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Section: Introductionmentioning

confidence: 96%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Characterization of Monophasic Solvent-Based Tissue Extractions for the Detection of Polar Metabolites and Lipids Applying Ultrahigh-Performance Liquid Chromatography–Mass Spectrometry Clinical Metabolic Phenotyping Assays

et al. 2020

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“…Metabolites were extracted from the pig saliva samples using an extraction procedure targeting primarily polar metabolites, similarly to recently published methods (Palmer, Cooper and Dunn, 2019; Southam et al ., 2020). Saliva samples were thawed on ice and briefly vortexed (10 seconds).…”

Section: Methodsmentioning

confidence: 99%

Saliva metabolome alterations after acute stress

Morgan

Birkler

Shaham-Niv

et al. 2022

Preprint

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Major stress has systemic effects on the body that can have adverse consequences for physical and mental health. However, the molecular basis of these damaging effects remains incompletely understood. Here we use a longitudinal approach to characterise the acute systemic impact of major psychological stress in a pig model. We perform untargeted metabolomics on non-invasively-obtained saliva samples from pigs before and 24-hours after transfer to the novel physical and social environment of a slaughterhouse. The main molecular changes occurring include decreases in amino acids, B-vitamins, and amino acid-derived metabolites synthesized in B-vitamin-dependent reactions, as well as yet-unidentified metabolite features. Decreased levels of several of the identified metabolites are implicated in the pathology of human psychological disorders and neurodegenerative disease, suggesting a possible neuroprotective function. Our results provide a fingerprint of the acute effect of psychological stress on the metabolome and suggest candidate biomarkers with potential roles in stress-related disorders.

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“…In the absence of a universally accepted procedure for biomarker discovery by mass spectrometry-based metabolomics, each data production facility uses its own optimized procedure [18]. To achieve broader metabolite coverage, samples are often analyzed several times by complementary LC-HRMS(/MS) methods [63,[75][76][77]. A large portion of the detected signals remains, however, structurally uncharacterized, and thus, metabolite identification still represents a major bottleneck in metabolomics [4,78].…”

Section: The Issue Of Metabolome Coverage and Accurate Measurementmentioning

confidence: 99%

Metabolomics for personalized medicine: the input of analytical chemistry from biomarker discovery to point-of-care tests

et al. 2021

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Metabolomics refers to the large-scale detection, quantification, and analysis of small molecules (metabolites) in biological media. Although metabolomics, alone or combined with other omics data, has already demonstrated its relevance for patient stratification in the frame of research projects and clinical studies, much remains to be done to move this approach to the clinical practice. This is especially true in the perspective of being applied to personalized/precision medicine, which aims at stratifying patients according to their risk of developing diseases, and tailoring medical treatments of patients according to individual characteristics in order to improve their efficacy and limit their toxicity. In this review article, we discuss the main challenges linked to analytical chemistry that need to be addressed to foster the implementation of metabolomics in the clinics and the use of the data produced by this approach in personalized medicine. First of all, there are already well-known issues related to untargeted metabolomics workflows at the levels of data production (lack of standardization), metabolite identification (small proportion of annotated features and identified metabolites), and data processing (from automatic detection of features to multi-omic data integration) that hamper the inter-operability and reusability of metabolomics data. Furthermore, the outputs of metabolomics workflows are complex molecular signatures of few tens of metabolites, often with small abundance variations, and obtained with expensive laboratory equipment. It is thus necessary to simplify these molecular signatures so that they can be produced and used in the field. This last point, which is still poorly addressed by the metabolomics community, may be crucial in a near future with the increased availability of molecular signatures of medical relevance and the increased societal demand for participatory medicine. Graphical abstract

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Assessment of human plasma and urine sample preparation for reproducible and high-throughput UHPLC-MS clinical metabolic phenotyping

Abstract: Clinical metabolic phenotyping employs metabolomics and lipidomics to detect and measure hundreds to thousands of metabolites and lipids within human samples. This approach aims to identify metabolite and lipid changes...

Cited by 40 publications

References 40 publications

Characterization of Monophasic Solvent-Based Tissue Extractions for the Detection of Polar Metabolites and Lipids Applying Ultrahigh-Performance Liquid Chromatography–Mass Spectrometry Clinical Metabolic Phenotyping Assays

Characterization of Monophasic Solvent-Based Tissue Extractions for the Detection of Polar Metabolites and Lipids Applying Ultrahigh-Performance Liquid Chromatography–Mass Spectrometry Clinical Metabolic Phenotyping Assays

Saliva metabolome alterations after acute stress

Metabolomics for personalized medicine: the input of analytical chemistry from biomarker discovery to point-of-care tests

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