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Article
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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Analytical Chemistry
1
Solid phase extraction and nanoflow liquid chromatography-
2
nanoelectrospray ionisation mass spectrometry for improved global urine
3
metabolomics
4
5
6
Andrew J. Chetwynd, Alaa Abdul-Sada, Elizabeth M. Hill*
7
School of Life Sciences, University of Sussex, Falmer, Brighton UK BN1 9QG
8
*To whom correspondence should be addressed:
9
Tel: +44 1273678382
10
Fax: Email: [email protected]
11
12
Keywords: urine, metabolomics, SPE, nanoESI, nanoflow, nanospray, UHPLC-MS
13
Word count 7978 including figures.
1 ACS Paragon Plus Environment
Analytical Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
14
Abstract
15
Global urine metabolomics is a rapidly expanding field with the potential to discover
16
biomarkers of disease and exposure. To date most methods focus on rapid sample
17
preparation, using neat or diluted urine, together with high throughput analyses, and are
18
poorly suited for detection of low abundance metabolites present in urine samples. In this
19
study, novel methods have been developed to analyse urine by splitless nanoflowUHPLC-
20
nanoESI-TOFMS (nUHPLC-nESI-TOFMS) after pre-concentration by solid phase extraction
21
(SPE), thus enabling significant improvements in analytical sensitivity and coverage of the
22
urinary metabolome. In initial work, urine samples were extracted by both anion and cation
23
exchange mixed mode polymeric SPE cartridges and qualitatively compared with those using
24
conventional sample preparations using UHPLC-ESI-TOFMS analyses. Compared with neat
25
or diluted urine samples, SPE concentration of urine resulted in detection of additional
26
metabolites including bile acids, lipids, pharmaceuticals and markers of lifestyle, with little
27
loss of other components of the metabolome. Analyses of SPE preparations by nUHPLC-
28
nESI-TOFMS revealed excellent retention time repeatability with 3000 urinary metabolites have been characterised, a number that is expected to grow as
45
more sensitive analytical techniques are developed.9 These metabolites are typically 10 fold pre-concentration of sample extracts
77
which may lead to improved detection of low abundance metabolites using non-targeted LC-
78
MS techniques.19
79
Ultra-high performance LC-electrospray- ionisation-time-of-flight MS (UHPLC-ESI-
80
TOFMS) using reverse phase chromatography is a commonly used high resolution method
81
allowing separation of moderately polar and apolar metabolite classes. UHPLC technology
82
uses columns containing 1.7 µm sized particles resulting in better resolution of compounds
83
compared with traditional high-performance LC. TOFMS enables high resolution (
Article
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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 27
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
1
Solid phase extraction and nanoflow liquid chromatography-
2
nanoelectrospray ionisation mass spectrometry for improved global urine
3
metabolomics
4
5
6
Andrew J. Chetwynd, Alaa Abdul-Sada, Elizabeth M. Hill*
7
School of Life Sciences, University of Sussex, Falmer, Brighton UK BN1 9QG
8
*To whom correspondence should be addressed:
9
Tel: +44 1273678382
10
Fax: Email: [email protected]
11
12
Keywords: urine, metabolomics, SPE, nanoESI, nanoflow, nanospray, UHPLC-MS
13
Word count 7978 including figures.
1 ACS Paragon Plus Environment
Analytical Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
14
Abstract
15
Global urine metabolomics is a rapidly expanding field with the potential to discover
16
biomarkers of disease and exposure. To date most methods focus on rapid sample
17
preparation, using neat or diluted urine, together with high throughput analyses, and are
18
poorly suited for detection of low abundance metabolites present in urine samples. In this
19
study, novel methods have been developed to analyse urine by splitless nanoflowUHPLC-
20
nanoESI-TOFMS (nUHPLC-nESI-TOFMS) after pre-concentration by solid phase extraction
21
(SPE), thus enabling significant improvements in analytical sensitivity and coverage of the
22
urinary metabolome. In initial work, urine samples were extracted by both anion and cation
23
exchange mixed mode polymeric SPE cartridges and qualitatively compared with those using
24
conventional sample preparations using UHPLC-ESI-TOFMS analyses. Compared with neat
25
or diluted urine samples, SPE concentration of urine resulted in detection of additional
26
metabolites including bile acids, lipids, pharmaceuticals and markers of lifestyle, with little
27
loss of other components of the metabolome. Analyses of SPE preparations by nUHPLC-
28
nESI-TOFMS revealed excellent retention time repeatability with 3000 urinary metabolites have been characterised, a number that is expected to grow as
45
more sensitive analytical techniques are developed.9 These metabolites are typically 10 fold pre-concentration of sample extracts
77
which may lead to improved detection of low abundance metabolites using non-targeted LC-
78
MS techniques.19
79
Ultra-high performance LC-electrospray- ionisation-time-of-flight MS (UHPLC-ESI-
80
TOFMS) using reverse phase chromatography is a commonly used high resolution method
81
allowing separation of moderately polar and apolar metabolite classes. UHPLC technology
82
uses columns containing 1.7 µm sized particles resulting in better resolution of compounds
83
compared with traditional high-performance LC. TOFMS enables high resolution (
