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Solid phase extraction and nanoflow liquid chromatography-nanoelectrospray ionisation mass spectrometry for improved global urine metabolomics Andrew J Chetwynd, Alaa Abdul-Sada, and Elizabeth M Hill Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac503769q • Publication Date (Web): 18 Dec 2014 Downloaded from http://pubs.acs.org on December 20, 2014
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Analytical Chemistry
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Solid phase extraction and nanoflow liquid chromatography-
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nanoelectrospray ionisation mass spectrometry for improved global urine
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metabolomics
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Andrew J. Chetwynd, Alaa Abdul-Sada, Elizabeth M. Hill*
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School of Life Sciences, University of Sussex, Falmer, Brighton UK BN1 9QG
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*To whom correspondence should be addressed:
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Tel: +44 1273678382
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Fax: Email:
[email protected] 11
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Keywords: urine, metabolomics, SPE, nanoESI, nanoflow, nanospray, UHPLC-MS
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Word count 7978 including figures.
1 ACS Paragon Plus Environment
Analytical Chemistry
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Abstract
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Global urine metabolomics is a rapidly expanding field with the potential to discover
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biomarkers of disease and exposure. To date most methods focus on rapid sample
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preparation, using neat or diluted urine, together with high throughput analyses, and are
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poorly suited for detection of low abundance metabolites present in urine samples. In this
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study, novel methods have been developed to analyse urine by splitless nanoflowUHPLC-
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nanoESI-TOFMS (nUHPLC-nESI-TOFMS) after pre-concentration by solid phase extraction
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(SPE), thus enabling significant improvements in analytical sensitivity and coverage of the
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urinary metabolome. In initial work, urine samples were extracted by both anion and cation
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exchange mixed mode polymeric SPE cartridges and qualitatively compared with those using
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conventional sample preparations using UHPLC-ESI-TOFMS analyses. Compared with neat
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or diluted urine samples, SPE concentration of urine resulted in detection of additional
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metabolites including bile acids, lipids, pharmaceuticals and markers of lifestyle, with little
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loss of other components of the metabolome. Analyses of SPE preparations by nUHPLC-
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nESI-TOFMS revealed excellent retention time repeatability with 3000 urinary metabolites have been characterised, a number that is expected to grow as
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more sensitive analytical techniques are developed.9 These metabolites are typically 10 fold pre-concentration of sample extracts
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which may lead to improved detection of low abundance metabolites using non-targeted LC-
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MS techniques.19
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Ultra-high performance LC-electrospray- ionisation-time-of-flight MS (UHPLC-ESI-
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TOFMS) using reverse phase chromatography is a commonly used high resolution method
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allowing separation of moderately polar and apolar metabolite classes. UHPLC technology
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uses columns containing 1.7 µm sized particles resulting in better resolution of compounds
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compared with traditional high-performance LC. TOFMS enables high resolution (
