title: Metabolomic method: UPLC-q-ToF polar and non-polar metabolites in the healthy rat cerebellum using an in-vial dual extraction.
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.
authors: Institute of Pharmaceutical Sciences, King's College London, Franklin-Wilkins Building, 150 Stamford Street, London, SE1 9NH, United Kingdom.
authors: Amera A
authors: AA
authors: Ebshiana
authors: Institute of Psychiatry, Department of Old Age Psychiatry, King's College London, De Crespigny Park, London, SE5 8AF, United Kingdom.
authors: Stuart G
authors: SG
authors: Snowden
authors: Clinical and Translational Neuroscience Unit, Laboratory of Behavioural Neuroscience, National Institute on Aging, Baltimore, Maryland, United States of America.
authors: Madhav
authors: M
authors: Thambisetty
authors: Institute of Pharmaceutical Sciences, King's College London, Franklin-Wilkins Building, 150 Stamford Street, London, SE1 9NH, United Kingdom.
authors: Richard
authors: R
authors: Parsons
authors: Institute of Psychiatry, Department of Old Age Psychiatry, King's College London, De Crespigny Park, London, SE5 8AF, United Kingdom.
authors: Abdul
authors: A
authors: Hye
authors: Institute of Pharmaceutical Sciences, King's College London, Franklin-Wilkins Building, 150 Stamford Street, London, SE1 9NH, United Kingdom.
authors: Cristina
authors: C
authors: Legido-Quigley
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism*; D002531:Cerebellum / Q000737:chemistry / Q000378:metabolism*; D002853:Chromatography, Liquid; D055442:Metabolome; D055432:Metabolomics; D051381:Rats; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D015415:Biomarkers