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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 2.A) plot of intensity of reversed phase internal standards Heptsdecanoic 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.g005: Recoveries of HILIC and reversed phase internal standards in experiment 2.A) plot of intensity of reversed phase internal standards Heptsdecanoic 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: As with assessing the performance of the IVDE and instrument methods, the first step in assessing the effect of tissue homogenisation and sample mass is to look at the recovery of the internal standards. As in experiment 1 both HILIC internal standards are seen in positive and negative ionisation modes (Fig 5B and 5C). In the positive mode the CV’s of the internal standard recoveries were 13.5% and 14.7% for serine and valine respectively. In the negative mode CV’s of the internal standard recoveries were 14.9% and 14.4% for serine and valine respectively. In the reversed phase data heptadecanoic acid is measured in the negative mode with a CV of 13.4%, and tripentadecanoin was measured in the positive mode with a CV of 3.8%. The recovery of the HILIC internal standards is more variable in these samples than in experiment 1, suggesting that the tissue homogenisation step is contributing significantly to analytical variability. This is further supported by no increase in the variability of tripentadecanoin which is spiked into the sample after tissue homogenisation. The recovery of the HILIC internal standards in the quality control samples, which are pooled after tissue homogenisation, were more consistent than in the analytical samples, and comparable with experiment 1 with CV’s of 3.8% and 4.8% in positive and 5.3% and 7.1% in negative for serine and valine respectively, further supporting the hypothesis that tissue homogenisation is contributing significantly to the observed variability. With the increased CV’s showing that tissue homogenisation is contributing to an increase in data variability, it is important to assess the effect of the extracted tissue volume on the recovery of the internal standards. Spearman’s correlation was used to assess the relationship between standard recovery and sample mass, this analysis revealed no significant correlations showing that internal standard recovery is independent of the sample mass extracted.


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 2.A) plot of intensity of reversed phase internal standards Heptsdecanoic 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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getmorefigures.php?uid=PMC4390242&req=5

pone.0122883.g005: Recoveries of HILIC and reversed phase internal standards in experiment 2.A) plot of intensity of reversed phase internal standards Heptsdecanoic 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: As with assessing the performance of the IVDE and instrument methods, the first step in assessing the effect of tissue homogenisation and sample mass is to look at the recovery of the internal standards. As in experiment 1 both HILIC internal standards are seen in positive and negative ionisation modes (Fig 5B and 5C). In the positive mode the CV’s of the internal standard recoveries were 13.5% and 14.7% for serine and valine respectively. In the negative mode CV’s of the internal standard recoveries were 14.9% and 14.4% for serine and valine respectively. In the reversed phase data heptadecanoic acid is measured in the negative mode with a CV of 13.4%, and tripentadecanoin was measured in the positive mode with a CV of 3.8%. The recovery of the HILIC internal standards is more variable in these samples than in experiment 1, suggesting that the tissue homogenisation step is contributing significantly to analytical variability. This is further supported by no increase in the variability of tripentadecanoin which is spiked into the sample after tissue homogenisation. The recovery of the HILIC internal standards in the quality control samples, which are pooled after tissue homogenisation, were more consistent than in the analytical samples, and comparable with experiment 1 with CV’s of 3.8% and 4.8% in positive and 5.3% and 7.1% in negative for serine and valine respectively, further supporting the hypothesis that tissue homogenisation is contributing significantly to the observed variability. With the increased CV’s showing that tissue homogenisation is contributing to an increase in data variability, it is important to assess the effect of the extracted tissue volume on the recovery of the internal standards. Spearman’s correlation was used to assess the relationship between standard recovery and sample mass, this analysis revealed no significant correlations showing that internal standard recovery is independent of the sample mass extracted.

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