In-vial dual extraction liquid chromatography coupled to mass spectrometry applied to streptozotocin-treated diabetic rats. Tips and pitfalls of the method

doi:10.1016/j.chroma.2013.07.029

Journal of Chromatography A

Volume 1304, 23 August 2013, Pages 52-60

https://doi.org/10.1016/j.chroma.2013.07.029 Get rights and content

Highlights

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For the first time “in vial dual extraction” is applied in a metabolomic study.
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The sample, plasma from control and diabetic rats, is splitted into lipophilic and hydrophilic phases.
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Advisory steps necessary for the proper implementation are included.
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Glycerolipids and glycerophospholipids are among the most important discriminant metabolites.

Abstract

The aim of metabolomics studies is the comprehensive and quantitative analysis of all metabolites in a cell, tissue or organism. This approach requires sample preparation methods to be fast, reproducible and able to extract a wide range of analytes with different polarities, as well as analytical platforms able to detect the extracted metabolites. Recently, we have developed a one-step extraction method consisting of a lipophilic and hydrophilic layer within a single vial insert, in-vial dual extraction (IVDE). In order to check possible application of this method to real biological case, analysis of plasma samples obtained from three streptozotocin-induced diabetic and three control rats was performed. Analytical validity of the method was proved by the calculation (in quality control samples) of relative standard deviation (RSD) for detected metabolites. The percentage of metabolites with RSD < 30% was 93% for Fatty acyls, 80% for Glycerolipids, 93% for Glycerophospholipids, 68% for Sterol lipids, and 91% for Sphingolipids. IVDE allowed for selection of more than 600 different features discriminating two studied groups. For around 40% of these masses putative identification was possible. Adequate, with several considerations described within this paper, application of IVDE method enables wide metabolite coverage from a single 20 μL plasma aliquot. Within the features putatively identified, glycerolipids and glycerophospholipids arose as the most important groups of compounds discriminating diabetic rats from controls. All discriminating metabolites give an idea of the large metabolic differences that can be present in non-controlled type 1 diabetes.

Introduction

Metabolomics is currently extensively employed to improve the understanding of biological changes induced by endogenous or exogenous factors. The aim of the methodology is the comprehensive and quantitative analysis of all metabolites in a cell, tissue or organism [1]. However, in practice, the primary focus is on metabolic fingerprinting, a technique that analyzes all detectable analytes in a given sample with subsequent classification and identification of differentially expressed metabolites, which define the sample class. Comprehensive metabolomics investigations are primarily a challenge for analytical chemistry because of numerous analytes with very diverse physico-chemical properties and different abundance levels, present in individual samples. Traditionally mass spectrometry has provided the most encouraging potential as an analytical tool for this type of investigation[2].

Currently, liquid chromatography coupled to mass spectrometry (LC–MS) is becoming a popular reference tool for metabolomics analysis, with biological extraction and separation methods employed to bias the number, physiochemical properties, and concentration of metabolites that are eventually analyzed by mass spectrometry. It is this range of molecular variation that currently creates a large challenge for global untargeted studies [3]. Consequently, metabolomics applications require sample preparation methods that are fast, reproducible and able to extract a wide range of analytes with different polarities [4], whilst being simultaneously compatible with the instrumental technique. Therefore, analytical strategies, which allow for increased coverage of metabolites determined in one sample, are currently desirable. Recently, we have developed a one-step extraction method consisting of a lipophilic and hydrophilic layer within a single vial insert, termed as in-vial dual extraction (IVDE) [5]. For the lipophilic phase, a 60 min lipid profiling HPLC-QTOF method was developed, enabling the separation and identification of fatty acids, glycerolipids, glycerophospholipids, sphingolipids, and sterols. The aqueous phase of the extract was analyzed with a standard method for metabolic fingerprinting. The method allows the total number of features recovered to be over 4500 from a single 20 μL plasma aliquot. However, to avoid analytical oversights and achieve high reproducibility of results, we have identified several considerations that are recommended to be taken into account. Here we discuss these suggestions necessary for improved implementation of IVDE, as well as detailed treatment procedures for both sample extraction and data analysis. Additionally, a practical application of this novel method to study metabolic changes in rat plasma evoked by streptozotocin-induced diabetes is shown. Results presented within this paper have a character of a preliminary study. The aim of conducted analysis was to validate previously developed IVDE method in terms of the robustness, reliability and utility. Before analysis of large set of samples authors wanted to find out pitfalls and drawbacks of the method; and find out if based on detected metabolites any differences between compared groups (that make sense from biological point of view) can be found.

Section snippets

Reagents and materials

Ultrapure water, used to prepare all the aqueous solutions was obtained “in-house” from a Milli-Qplus185 system (Millipore, Billerica, MA, USA). LC–MS grade methanol, acetonitrile and analytical grade formic acid were purchased from Fluka Analytical (Sigma–Aldrich Chemie GmbH, Steinheim, Germany). Analytical grade ammonia hydroxide (30% ammonium in high purity water) was acquired from Panreac Quimica SA (Barcelona, Spain) and analytical grade methyl-tert-butyl-ether (MTBE) was from

Results and discussion

The essence of IVDE method is preparing the sample, extracting it within the vial producing the partition between two non-miscible solvents, and injecting each layer independently in ad hoc chromatographic methods. A 20 μl aliquot of plasma was extracted and dispersed between ether and aqueous phase and analyzed employing two different chromatographic methods optimized for each type of extracted metabolites. Such strategy allows decreasing matrix effects, increase resolution and compound

Conclusion

Application of IVDE for analysis of plasma samples obtained from streptozotocin-induced diabetic and control rats allowed for selection of more than 600 different features discriminating two studied groups. Moreover for around 40% of these masses putative identification was possible, based on public-access databases. Adequate, with several considerations described within this paper, application of IVDE method enables for wide metabolite coverage from a single 20 μL plasma aliquot. Metabolites

Acknowledgements

Authors acknowledge EADS-CASA and Spanish Ministry of Economy and Competitiveness (MINECO) CTQ2011-23562, and the NHS National Institute of Health Research (NIHR) Biomedical Research Centre (BRC) for mental health at the South London and Maudsley (SLaM).

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^☆: Presented at the 29th International Symposium on Chromatography, Torun, Poland, 9–12 September 2012.

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In-vial dual extraction liquid chromatography coupled to mass spectrometry applied to streptozotocin-treated diabetic rats. Tips and pitfalls of the method☆

Highlights

Abstract

Introduction

Section snippets

Reagents and materials

Results and discussion

Conclusion

Acknowledgements

Trac

J. Chromatogr. A

Anal. Biochem.

J. Lipid Res.

Food Chem. Toxicol.

Clin. Biochem.

Trends Microbiol.

J. Lipid Res.

J. Chromatogr. B Anal. Technol. Biomed. Life Sci.

Proteomics

Mass Spectrom. Rev.

Anal. Chem.

Anal. Chem.

Metabolomics