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Metabolomic method: UPLC-q-ToF polar and non-polar metabolites in the healthy rat cerebellum using an in-vial dual extraction.

Ebshiana AA, Snowden SG, Thambisetty M, Parsons R, Hye A, Legido-Quigley C - PLoS ONE (2015)

Bottom Line: To date however only a small number of metabolomic studies have been applied to studying the metabolite composition of tissue samples, this is due, in part to a number of technical challenges including scarcity of material and difficulty in extracting metabolites.The aim of this study was to develop a method for maximising the biological information obtained from small tissue samples by optimising sample preparation, LC-MS analysis and metabolite identification.The described metabolomics method includes a database for 200 metabolites, retention time, mass and relative intensity, and presents the basal metabolite composition for brain tissue in the healthy rat cerebellum.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmaceutical Sciences, King's College London, Franklin-Wilkins Building, 150 Stamford Street, London, SE1 9NH, United Kingdom.

ABSTRACT
Unbiased metabolomic analysis of biological samples is a powerful and increasingly commonly utilised tool, especially for the analysis of bio-fluids to identify candidate biomarkers. To date however only a small number of metabolomic studies have been applied to studying the metabolite composition of tissue samples, this is due, in part to a number of technical challenges including scarcity of material and difficulty in extracting metabolites. The aim of this study was to develop a method for maximising the biological information obtained from small tissue samples by optimising sample preparation, LC-MS analysis and metabolite identification. Here we describe an in-vial dual extraction (IVDE) method, with reversed phase and hydrophilic liquid interaction chromatography (HILIC) which reproducibly measured over 4,000 metabolite features from as little as 3mg of brain tissue. The aqueous phase was analysed in positive and negative modes following HILIC separation in which 2,838 metabolite features were consistently measured including amino acids, sugars and purine bases. The non-aqueous phase was also analysed in positive and negative modes following reversed phase separation gradients respectively from which 1,183 metabolite features were consistently measured representing metabolites such as phosphatidylcholines, sphingolipids and triacylglycerides. The described metabolomics method includes a database for 200 metabolites, retention time, mass and relative intensity, and presents the basal metabolite composition for brain tissue in the healthy rat cerebellum.

No MeSH data available.


Related in: MedlinePlus

Recoveries of HILIC and reversed phase internal standards in experiment 1.A) plot of intensity of reversed phase internal standards Heptadecanoic acid (negative) and Tripentadecanoin (positive), B) plot of intensity of HILIC internal standards in positive ionisation mode, C) plot of intensity of HILIC internal standards in negative ionisation mode, D) average intensity and coefficient of variance of all internal standards.
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pone.0122883.g003: Recoveries of HILIC and reversed phase internal standards in experiment 1.A) plot of intensity of reversed phase internal standards Heptadecanoic acid (negative) and Tripentadecanoin (positive), B) plot of intensity of HILIC internal standards in positive ionisation mode, C) plot of intensity of HILIC internal standards in negative ionisation mode, D) average intensity and coefficient of variance of all internal standards.

Mentions: The first step in assessing the precision of the in-vial dual extraction (IVDE) and both the reversed phase and HILIC methods was to determine the recovery for four internal standards (Fig 3). In the HILIC method both internal standards were measured in both the positive and negative ionisation modes. In the positive data the recovery of internal standards are highly consistent with coefficient of variation (CV) of 2.4% and 3.7% (Fig 3B) for the serine and valine standards respectively. In the negative mode, recovery is more variable than the positive mode with CV’s of 9.1% and 5.7% (Fig 3C) for serine and valine respectively. In the reversed phase method heptadecanoic acid was measured in the negative mode and tripentsdecanoin was measured in the positive. The recovery of both standards was consistent with CV’s of 2.5% and 4.4% for heptadecanoic acid and tripentadenanoin respectively. The standard recoveries suggests that the IVDE and both HILIC and reversed phase methods have good precision with all internal standard measurements having CV’s less than 15% [33], with mass spectrometry in the negative mode adding more variability than the positive mode.


Metabolomic method: UPLC-q-ToF polar and non-polar metabolites in the healthy rat cerebellum using an in-vial dual extraction.

Ebshiana AA, Snowden SG, Thambisetty M, Parsons R, Hye A, Legido-Quigley C - PLoS ONE (2015)

Recoveries of HILIC and reversed phase internal standards in experiment 1.A) plot of intensity of reversed phase internal standards Heptadecanoic acid (negative) and Tripentadecanoin (positive), B) plot of intensity of HILIC internal standards in positive ionisation mode, C) plot of intensity of HILIC internal standards in negative ionisation mode, D) average intensity and coefficient of variance of all internal standards.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4390242&req=5

pone.0122883.g003: Recoveries of HILIC and reversed phase internal standards in experiment 1.A) plot of intensity of reversed phase internal standards Heptadecanoic acid (negative) and Tripentadecanoin (positive), B) plot of intensity of HILIC internal standards in positive ionisation mode, C) plot of intensity of HILIC internal standards in negative ionisation mode, D) average intensity and coefficient of variance of all internal standards.
Mentions: The first step in assessing the precision of the in-vial dual extraction (IVDE) and both the reversed phase and HILIC methods was to determine the recovery for four internal standards (Fig 3). In the HILIC method both internal standards were measured in both the positive and negative ionisation modes. In the positive data the recovery of internal standards are highly consistent with coefficient of variation (CV) of 2.4% and 3.7% (Fig 3B) for the serine and valine standards respectively. In the negative mode, recovery is more variable than the positive mode with CV’s of 9.1% and 5.7% (Fig 3C) for serine and valine respectively. In the reversed phase method heptadecanoic acid was measured in the negative mode and tripentsdecanoin was measured in the positive. The recovery of both standards was consistent with CV’s of 2.5% and 4.4% for heptadecanoic acid and tripentadenanoin respectively. The standard recoveries suggests that the IVDE and both HILIC and reversed phase methods have good precision with all internal standard measurements having CV’s less than 15% [33], with mass spectrometry in the negative mode adding more variability than the positive mode.

Bottom Line: To date however only a small number of metabolomic studies have been applied to studying the metabolite composition of tissue samples, this is due, in part to a number of technical challenges including scarcity of material and difficulty in extracting metabolites.The aim of this study was to develop a method for maximising the biological information obtained from small tissue samples by optimising sample preparation, LC-MS analysis and metabolite identification.The described metabolomics method includes a database for 200 metabolites, retention time, mass and relative intensity, and presents the basal metabolite composition for brain tissue in the healthy rat cerebellum.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmaceutical Sciences, King's College London, Franklin-Wilkins Building, 150 Stamford Street, London, SE1 9NH, United Kingdom.

ABSTRACT
Unbiased metabolomic analysis of biological samples is a powerful and increasingly commonly utilised tool, especially for the analysis of bio-fluids to identify candidate biomarkers. To date however only a small number of metabolomic studies have been applied to studying the metabolite composition of tissue samples, this is due, in part to a number of technical challenges including scarcity of material and difficulty in extracting metabolites. The aim of this study was to develop a method for maximising the biological information obtained from small tissue samples by optimising sample preparation, LC-MS analysis and metabolite identification. Here we describe an in-vial dual extraction (IVDE) method, with reversed phase and hydrophilic liquid interaction chromatography (HILIC) which reproducibly measured over 4,000 metabolite features from as little as 3mg of brain tissue. The aqueous phase was analysed in positive and negative modes following HILIC separation in which 2,838 metabolite features were consistently measured including amino acids, sugars and purine bases. The non-aqueous phase was also analysed in positive and negative modes following reversed phase separation gradients respectively from which 1,183 metabolite features were consistently measured representing metabolites such as phosphatidylcholines, sphingolipids and triacylglycerides. The described metabolomics method includes a database for 200 metabolites, retention time, mass and relative intensity, and presents the basal metabolite composition for brain tissue in the healthy rat cerebellum.

No MeSH data available.


Related in: MedlinePlus