Journal of Chromatography A, 1304 (2013) 52–60 Contents lists available at SciVerse ScienceDirect Journal of Chromatography A j our nal homep age: www.elsevier.com/locate/chroma In-vial dual extraction liquid chromatography coupled to mass spectrometry applied to streptozotocin-treated diabetic rats. Tips and pitfalls of the method  Joanna Godzien a,b , Michal Ciborowski a,c , Luke Whiley d,e , Cristina Legido-Quigley d , Francisco J. Ruperez a , Coral Barbas a,∗ a Center for Metabolomics and Bioanalysis (CEMBIO), Facultad de Farmacia, Campus Monteprincipe, Universidad CEU San Pablo, 28668 Boadilla del Monte, Madrid, Spain b Department of Molecular Biology, Faculty of Mathematics and Natural Sciences, The John Paul II Catholic University of Lublin, Krasnicka 102, 20-718 Lublin, Poland c Department of Physical Chemistry, Medical University of Bialystok, Kilinskiego 1, 15-089 Bialystok, Poland d Institute of Pharmaceutical Sciences, King’s College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, United Kingdom e Institute of Psychiatry, King’s College London, De Crespigny Park, London SE5 8AF, United Kingdom a r t i c l e i n f o Article history: Received 12 November 2012 Received in revised form 9 May 2013 Accepted 6 July 2013 Available online 11 July 2013 Keywords: In-vial dual extraction (IVDE) Metabolomics Lipidomics LC–MS Plasma Diabetes a b s t r a c t 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 L 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. © 2013 Elsevier B.V. All rights reserved. 1. 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 com- prehensive and quantitative analysis of all metabolites in a cell, tissue or organism [1]. However, in practice, the primary focus is on  Presented at the 29th International Symposium on Chromatography, Torun, Poland, 9–12 September 2012. ∗ Corresponding author at: Pharmacy Faculty, Campus Monteprincipe, Universi- dad CEU San Pablo, 28668 Boadilla del Monte, Madrid, Spain. Tel.: +34 913724711; fax: +34 913724712. E-mail address: cbarbas@ceu.es (C. Barbas). metabolic fingerprinting, a technique that analyzes all detectable analytes in a given sample with subsequent classification and iden- tification of differentially expressed metabolites, which define the sample class. Comprehensive metabolomics investigations are pri- marily a challenge for analytical chemistry because of numerous analytes with very diverse physico-chemical properties and differ- ent 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 spectrome- try (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 con- centration of metabolites that are eventually analyzed by mass spectrometry. It is this range of molecular variation that currently 0021-9673/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2013.07.029