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Systematic evaluation of extraction methods for multi-platform based metabotyping: Application to the Fasciola hepatica metabolome Jasmina Saric, Elizabeth J. Want, Urs Duthaler, Matthew Lewis, Jennifer Keiser, John P Schockcor, Gordon A. Ross, Jeremy Kirk Nicholson, Elaine Holmes, and Marina Franco Maggi Tavares Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac300586m • Publication Date (Web): 12 Jul 2012 Downloaded from http://pubs.acs.org on July 21, 2012

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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

Systematic evaluation of extraction methods for multi-

2

platform based metabotyping: Application to the

3

Fasciola hepatica metabolome

4 5

Jasmina Saric,† Elizabeth J. Want,† Urs Duthaler,§ Matthew

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Lewis,† Jennifer Keiser,§ John P. Shockcor, Gordon A. Ross,#

7

Jeremy K. Nicholson,† Elaine Holmes,†,* and Marina F. M.

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Tavares, †,‡,*

9 10



11

Medicine, Imperial College London, Sir Alexander Fleming Building, South

12

Kensington, London SW7 2AZ, U.K.; §Department of Medical Parasitology

13

and Infection Biology, Swiss Tropical and Public Health Institute, CH-4002

14

Basel, Switzerland and University of Basel, CH-4003 Basel, Switzerland;

Biomolecular Medicine, Department of Surgery and Cancer, Faculty of

Pharmaceutical

15

Business

Operations,

Waters

Corporation,

Milford,

16

Massachussetts, U.S.A.; #Agilent Technologies Life Sciences and Chemical

17

Analysis, Lakeside, Cheadle Royal Business Park, Stockport, Cheshire, SK8

18

3GR, U.K.; ‡Institute of Chemistry, University of Sao Paulo, P.O. Box 26077,

19

05513-970, Sao Paulo, SP, Brazil

20 21

*

22

E-mail: [email protected]; E-mail: [email protected]

To whom correspondence should be addressed:

23

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ABSTRACT

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Combining data from multiple analytical platforms is essential for

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comprehensive study of the molecular phenotype (metabotype) of a given

27

biological sample. The metabolite profiles generated are intrinsically

28

dependent on the analytical platforms, each requiring optimization of

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instrumental parameters, separation conditions and sample extraction to

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deliver maximal biological information. An in depth evaluation of extraction

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protocols for characterizing the metabolome of the hepatobiliary fluke Fasciola

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hepatica using UPLC-MS and CE-MS is presented. The spectrometric

33

methods were characterized by performance and metrics of merit were

34

established including precision, mass accuracy, selectivity, sensitivity and

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platform stability. Although a core group of molecules was common to all

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methods, each platform contributed a unique set and 142 metabolites out of

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14,724 features were identified. A mixture design revealed that 15:59:26

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chloroform:methanol:water was globally the best composition for metabolite

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extraction across UPLC-MS and CE-MS platforms accommodating different

40

columns and ionization modes. Despite the general assumption of the

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necessity of platform-adapted protocols for achieving effective metabotype

42

characterization, we show that an appropriately designed single extraction

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procedure is able to fit the requirements of all technologies. This may

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constitute a paradigm shift in developing efficient protocols for high throughput

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metabolite profiling with more general analytical applicability.

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Analytical Chemistry

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INTRODUCTION

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Metabolic profiling using mass spectrometry (MS) hyphenated with ultra

49

performance liquid chromatography (UPLC) or gas chromatography (GC) and

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nuclear magnetic resonance (NMR) spectroscopy have been successfully

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applied to the characterization of systemic responses of organisms to

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disease, pharmaceutical intervention and dietary modulation.1,2,3 In such

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studies, the adequacy of a given analytical platform is typically dependent

54

upon the class of chemical compounds under investigation, the cost of

55

analysis, ease of sample preparation and the requirement for sensitivity,

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specificity, and robustness. No single method enables complete coverage of

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the entire metabolic information and increasingly, metabolic profiling studies

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are adopting more than one analytical platform to augment the number of

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metabolites identified and therefore enhance the extraction of biological

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information.

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Although the

literature is

scattered

with platform-specific

sample

62

preparation procedures,4 there is a paucity of studies reporting the systematic

63

evaluation of sample preparation across multiple platforms.5 Despite the

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recent technological developments in the field of sample preparation of

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biofluids,

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methods,6,7,8,9

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microextractions12 to the more sophisticated use of molecularly imprinted

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polymers13 and restricted-access materials,14 sample extraction continues to

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be a crucial and time consuming step of any analytical method, with important

70

implications on the information recovered and analytical interpretation. This is

spanning

from

liquid-liquid

the or

more

traditional

solid-phase

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protein

precipitation

extractions10,11

and

3

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particularly true for comprehensive metabolic profiling, where the chemical

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complexity, sample heterogeneity, and wide concentration range of

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endogenous metabolites place a strong demand on the extraction

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procedure.15 Metabolite losses, matrix effects, artefacts and analytical

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variability are often inevitable,16 which indicate that the ultimate goal of full

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characterisation of a metabolome will be a challenging task for many

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biological samples.

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To preserve sample integrity, methods with minimal sample treatment are

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desirable and often applicable for liquid biological samples, such as urine or

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plasma, where simple dilution followed by filtration and/or centrifugation are

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the norm. However, for solid or semi-solid biological samples, such as faeces

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and tissues, more elaborate sample preparation procedures are required.

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Tissue extraction is usually performed by cooled homogenisation of tissue

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followed by stepwise addition of reagents and solvents of differing polarities

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such

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experimental approaches include the use of automated homogenizers,18,19,20

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microdialysis tissue sampling,21 and solid-phase extraction with a large variety

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of sorbents.22

as

perchloric

acid

or

methanol-chloroform

mixtures.17

Other

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Here we apply a multi-platform strategy for a more comprehensive

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assessment of the metabolic composition of tissues using the parasitic

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hepatobiliary trematode Fasciola hepatica as a model system. F. hepatica

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infects livestock and imposes a considerable economic burden across the

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globe linked to decreased productivity of the affected animals.23 Due to its

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zoonotic character, fascioliasis has emerged as human infection in the last

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two decades, whereby an estimated 91 million people are at risk. Although

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some host spots of prevalence have been identified, such as Western Europe,

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the Andes, and Egypt, human cases have been reported from as many as 51

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countries.24 Diseases management is currently suboptimal since diagnosis is

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largely based on microscopic examination of helminth eggs in stool, whereby

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detection capacity is limited in early and light infection. The first line

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therapeutic

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triclabendazole, whereby resistances have already been reported.25 Metabolic

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characterisation of the fluke may aid in deepening understanding of the

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biochemical communication between parasite and host and may provide

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leads for identifying novel diagnostic markers and drug targets at the

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metabolic level.

intervention is

limited

to

one

main

compound,

namely

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We evaluated the extraction of metabolic information from spectral profiles

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acquired across five different analytical methods. A quadrupole time-of-flight

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(Q-ToF) mass analyzer was used for UPLC-MS analysis in combination with

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two different column chemistries, i.e. reversed-phase liquid chromatography

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(RPLC) using a C18 column and hydrophilic interaction liquid chromatography

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(HILIC), in both positive and negative electrospray ionization modes (ESI+

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and ESI-, respectively). Capillary electrophoresis (CE) coupled to MS was

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applied in ESI+ mode only. Additionally, a mixture design was used to define

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the optimal solvent composition for the global platform combination, and thus

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to derive a single procedure that can be applied generically for tissue

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extraction across a range of biomedical samples and analytical platforms. In

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addition to providing an optimised and augmented metabolic screening

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capacity, such an approach would also facilitate the mathematical modelling

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across different analytical platforms since the preparation parameters are

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maintained regardless of platform, thereby reducing additional sources of

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variation introduced by differential extraction efficiency of metabolites.26

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MATERIALS AND METHODS

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Materials. Water, acetonitrile, methanol and chloroform (chromatography

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grade) used in the tissue extractions were obtained from Sigma (Gillingham,

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U.K.) as well as most standards used to confirm the identity of the

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chromatographic peaks. Cholic acid derivatives were acquired from Steraloids

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Inc (Newport, RI, U.S.A.) and phospholipids were purchased from Avanti

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Polar Lipids Inc (Alabaster, AL, U.S.A.) (Supplementary Table S-1). Stock

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standard solutions were prepared at 1 mg/mL in chromatography purity water.

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Working solutions used in the identification and MS/MS fragmentation studies

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were prepared at 0.010 to 0.050 mg/mL.

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Sample preparation. Fresh cattle livers were obtained from an abattoir in

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Basel and live F. hepatica worms were recovered from the bile ducts at the

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Swiss Tropical and Public Health Institute (Basel, Switzerland). Worms were

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snap frozen and forwarded on dry ice to Imperial College London and kept at -

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40°C.

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For characterization of the F. hepatica metabolome and for assessment of

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the methods performance, two batches of six individual flukes (~80 mg each)

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obtained from two different livers were thawed and weighed. Each worm was

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placed into a separate 2 mL plastic tube containing 500 mg of 1 mm diameter

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zirconia beads (Stratech Scientific Ltd, U.K.). One mL of 80% methanol, which

144

was cooled down previously to 4°C, was added to each tube. The tubes were

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processed in a tissue homogenizer (Precellys 24, Peqlab Ltd., UK) in two 30 s

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cycles of 6,000 rpm, subsequently centrifuged for 5 min at 18,894 g and the

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supernatants transferred into Eppendorf tubes placed on ice. The extraction

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procedure was repeated once by adding 500 µL cold methanol to the

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remaining pellet in each tube, followed by homogenization and centrifugation

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as described above. Both supernatants were combined and divided in

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different aliquots for spectral assessment, i.e. equal aliquots of 100 µL for

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UPLC-MS and CE-MS analysis, and the remaining was retained as a backup.

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The extracts were dried overnight in a speedvac (Eppendorf concentrator

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plus, Eppendorf UK Ltd, Cambridge, U.K.) at 45°C, under vacuum and

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rotational speed of 1,400 rpm (the g-force varies according to the position of

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the tubes in the rotors between 130 and 250 g). Dried extracts were stored at

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-40°C prior to UPLC-MS and CE-MS analysis.

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In order to evaluate the optimal solvent extraction condition to suit multiple

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analytical platforms a mixture design study was performed. A total of 13 F.

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hepatica worms (~400 mg total) were ground together in liquid nitrogen using

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a mortar and pestle. Samples of the ground tissue (~30 mg) were transferred

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into separate Eppendorf tubes and 1 mL of a given proportion of cooled

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solvent/solution (4°C) was added to the tissue as follows: (1) = 1000A, (2) =

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1000B, (3) = 100B + 900C, (4) = 500A + 500B, (5) = 500A + 500C, (6) = 500B

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+ 500C, (7) = 750A + 250B, (8) = 250A + 750B, (9) = 250B + 750C, (10) =

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700A + 150B + 150C, (11) = 150A + 700B + 150C, (12) = 150A + 150B +

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700C, and (13) = 333A + 333B + 333C, where A = 80% methanol, B = 20%

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methanol and C = pure chloroform. Samples were vortexed for 1 min,

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centrifuged (18,894 g; 5 min) and the upper aqueous phases were transferred

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into new separate Eppendorf tubes. The same amount of the solvent mixture

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was added to the remaining pelleted biomass and the extraction procedure

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repeated. The aqueous phase was again harvested and combined with the

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previous extract into a single Eppendorf tube. Final volumes were 2 mL after

174

addition of 80% methanol. The samples were vortexed again and divided into

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equal aliquots of 200 µL each for UPLC-MS, CE-MS analysis and a backup

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sample. The aqueous extracts were dried overnight at 45°C under vacuum.

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The organic phase was collected and stored at -40°C but was not analyzed

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further here, since the aqueous phase gave information relating to both the

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low molecular weight components and lipids.

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The dried aliquots for UPLC-MS and CE-MS were dissolved in 200 µL of

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50% methanol and 20% methanol, respectively, vortexed for 20 s and

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subsequently sonicated for 5 min. A volume of 120 µL was transferred either

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into the wells of a 96-well plate (Waters, Hertfordshire, U.K.) for UPLC-MS or

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to the CE-MS sample vials (Agilent Technologies UK Ltd, Edinburgh, U.K.). A

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quality control (QC) sample pool was prepared by mixing 5 µL of each

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aqueous extract sample in a separate Eppendorf tube. At the beginning of

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each of the chromatographic/electrophoretic runs, 5 injections from the QC

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sample pool were made; further QC injections were made after every 5

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samples throughout the run, in order to assess repeatability and platform

190

stability.

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Instrumentation and data acquisition. All extracts were analyzed on a

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UPLC system (UPLC Acquity, Waters Ltd., Elstree, U.K.) coupled online via

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electrospray ionization to a Q-ToF Premier mass spectrometer (Waters MS

194

Technologies Ltd., Manchester, U.K.) using both a Waters Acquity UPLC BEH

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C18 column (1.8 µm, 2.1 x 100 mm) at 50°C and a Waters Acquity HILIC BEH

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column (1.7 µm, 2.1 x 100 mm) at 40°C, operated under gradient elution, as

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follows. For C18: A = 0.1% formic acid in water, B = 0.1% formic acid in

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methanol, under flow rate of 0.4 mL/min. Gradient elution: 0-2 min, 99.9%

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A:0.10% B; 6 min, 75% A:25% B; 10 min, 20% A:80% B, 12 min, 10% A:90%

200

B, 21-23 min, 0.10% A:99.9% B, 24-26 min: 99.9% A:0.10% B. For HILIC: A

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= 95% acetonitrile: 5% 200 mmol/L ammonium acetate, containing a total of

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0.1% formic acid; B = 50% acetonitrile: 50% 20 mmol/L ammonium acetate,

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containing a total of 0.1% formic acid, under flow rate of 1.4 mL/min. Strong

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wash solvent was 5% acetonitrile whereas the weak wash solvent was 95%

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acetonitrile. Gradient elution: 0-1 min, 99% A:1% B; 12 min, 100% B; 12.1-15

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min, 99% A:1% B.

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Other chromatographic conditions common to both modes include the

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temperature of the autosampler compartment (4°C), the injection volume (5

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µL) and the injection loop option (partial loop with needle overfill). Capillary

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voltage was 3,200 V (positive ionization) and 2,400 V (negative ionization),

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and the sample cone voltage was maintained at 35 V. The desolvation

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temperature was set to 350°C and the source temperature to 120°C, and

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cone gas flow and desolvation gas flow were maintained at 25 L/h and 900

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L/h, respectively. The Q-ToF Premier was operated in V optics mode, with a

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data acquisition rate of 0.2 s and a 0.01 s interscan delay. Leucine enkephalin

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(m/z 556.2771) was used as lockmass whereby a solution of 200 pg/µL in

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50% acetonitrile was infused into the instrument at a rate of 3 µL/min via an

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auxiliary sprayer. Data were collected in centroid mode with a scan range of

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50 – 1000 m/z, with lockmass scans collected every 15 s and averaged over 3

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scans to perform mass correction.

221

CE-MS

analysis

was

performed

on

an

Agilent

7100

capillary

222

electrophoresis system coupled to an Agilent 6224 Accurate-Mass time-of-

223

flight mass spectrometer (Agilent Technologies, Waldbronn, Germany) by

224

means of a coaxial spray needle configured to an electrospray ionization

225

source. A fused-silica capillary (Composite Metal Services, Hallow, U.K.) with

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dimensions 50 µm internal diameter, 375 µm outer diameter and 65 cm total

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length was used as separation device for the positive ionization mode. New

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fused-silica capillaries were conditioned for 30 min with 1 mol/L NaOH at 1

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bar pressure followed by 10 min deionized water and 30 min background

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electrolyte (BGE) composed of 0.80 mol/L formic acid at pH 1.8 containing

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20% methanol. Before analysis, the capillary was conditioned by pressure

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flushes (925 bar) of 1 mol/L NaOH (10 min), 0.10 mol/L NaOH (10 min),

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deionized water (10 min) and BGE (30 min) at 25 °C. In both cases the

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capillary was conditioned with the distal end outside of the MS source.

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Between runs the BGE vials were replenished to a height of 1.5 cm and the

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capillary was flushed with BGE for 5 min. At the end of the day the capillary

237

was pressure flushed with deionized water (10 min), methanol (10 min) and

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air (10 min). Samples were injected hydrodynamically (50 mbar for 10 s)

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followed by injection of BGE (50 mbar, 5 s). The CE system was operated

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under constant voltage of +30 kV and the cartridge was thermostatized at

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25°C. A sheath liquid (SHL) composed of 70% methanol containing 0.5%

242

formic acid was delivered at a flow rate of 4 µL/min via a 1:100 splitter

243

connected to an isocratic pump (1260 Infinity Series, Agilent Technologies)

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running at 400 µL/min. The nebulizer was set to 10 psig at 0.5 min after

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injection and a flow of heated dry nitrogen gas (150 °C) was maintained at 10

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L/min. Transfer capillary voltage was 4500 V, the fragmentor was set to 120

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V, skimmer to 65 V and ion guide octapole to 750 V. The CE unit was

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operated by the 3D-CE ChemStation Rev B.04.03 software and MS data were

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acquired by the MassHunter WorkStation Acquisition B.02.01 in centroid

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mode with a data acquisition rate of 5 spectra per second. Purine

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(C5H4N4; [M+H]+ 121.05087) and HP921 (hexakis (2,2,3,3,-tetrafluoropropoxy)

252

phosphazine; CAS No.: 58943-98-9; C18H18O6N3P3F24; [M+H]+ 922.00980)

253

were used as reference masses whereby a solution containing 10 mL water,

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90 mL methanol, 100 µL formic acid, 1600 µL of 5 mmol/L purine and 600 µL

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of 2.5 mmol/L phosphazine derivative listed above (HP 0921) were infused

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directly into the ion source. The reference mass spectra were collected

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simultaneously with the analytical data and used for accurate mass correction.

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Data processing and peak identification. The raw data files derived from

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UPLC-MS and CE-MS acquisition were preprocessed using the publically

260

available XCMS software (version 1.24.1).27 Isotope peaks, fragments and

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adducts were treated as separate features. For the dataset containing the

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extracts from individual flukes, the samples were grouped according to the

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liver they were extracted from, whereby QC samples were treated as a

264

separate group. Default settings were employed in XCMS with the exception

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of width of overlapping m/z slices used for creating peak density

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chromatograms and grouping peaks across samples (mzwid=0.025),

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bandwidth for the grouping performed after retention time correction (bw=10

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s) and the degree of smoothing for local polynomial regression fitting

269

(span=0.3). A table of time aligned detected features containing the retention

270

times, m/z ratio and intensities of each sample was then obtained. Median

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normalization was performed using an in-house developed R script (Dr. K.

272

Veselkov,

273

normalization. Output tables containing information on the average retention

274

time and average m/z were also prepared as data input of an in-house

275

developed MATLAB script (Dr. P. Masson, Imperial College London) that

276

allowed searching of candidate metabolites in an in-house built UPLC-MS

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database within specified errors; in this work 0.05 Da for m/z and 1 min for

278

retention time were used to start with. A set of candidate compounds was

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then generated as a first pass and errors on the m/z and retention time

280

assignments were provided for each compound. Compounds with m/z and

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retention time differing by more than 20 ppm and 0.5 min, respectively, were

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disregarded. Eventually, a mass chromatogram of the corresponding

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authentic standard and the sample were acquired and the peak submitted to

284

fragmentation in a MS/MS experiment. If the fragmentation pattern of both

285

standard and sample peak matched, the peak was then assigned to the

286

alleged compound with improved reliability.

Imperial

College

London),28

followed

by

tissue

weight

287

288

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Analytical Chemistry

289

RESULTS AND DISCUSSION

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Analytical Platform Selectivity for F. hepatica Metabolites. MS has the

291

necessary sensitivity to assess low abundance molecular species in biological

292

matrices. When combined with appropriately designed extraction methods

293

and any gas or liquid based separation technique, MS is capable of analyzing

294

a plethora of metabolites of different chemical classes. Moreover, the variety

295

of separation modes, ionization schemes and mass analyzers qualifies the

296

use of hyphenated MS platforms as a resourceful approach to metabolic

297

profiling.29,30,31,32

298

We compared individual F. hepatica extracts at 80% methanol prepared

299

from flukes obtained from 2 different cow livers using analytical conditions and

300

instrumental parameters provided in previous work for UPLC-MS,18,33 and a

301

method developed specifically for CE-MS.

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Typical base peak mass chromatograms acquired using ESI+ and ESI-,

303

respectively, on the C18 column are depicted in Figures 1A and S-1A. As

304

expected, many polar components exhibited poor retention, co-eluting close

305

to the column dead volume. Therefore, assignments for peaks eluting by less

306

than 0.94 min (k22). A polyethylene glycol peak envelope (~8 min) was observed in

329

the mass chromatogram but was also detected in the pure water and

330

therefore attributed to contamination.

331

Similar features were observed in the negative ionization C18 mass

332

chromatogram of Figure S-1A. In addition to phenylalanine and tryptophan, a

333

few carboxylic acids (methylmalonic and pantothenic acids), adenosine

334

monophosphate (AMP) and inosine compose the early eluting polar

335

compounds, whereas in the cholic acid region, deoxycholic acid was further

336

detected. Similar compounds were identified in the phospholipid region for

337

both positive and negative ionization modes, although the monosubstituted

338

glycerophosphocholine derivatives exhibited much higher ionization yields in

339

negative ion mode when compared to the disubstituted phosphatidylcholine

340

homologs, in direct contrast to the observation made from the positive

341

ionization mode data.

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As the results of Figures 1A and S-1A testify, the choice of C18 columns for

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UPLC-MS studies allows immediate visualisation of nonpolar and moderately

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polar compounds. However, its use discriminates instantly against highly polar

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compounds. Recently, HILIC has been offered as a powerful alternative to

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compensate for the poor RPLC retention of highly polar compounds,

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ubiquitous components of many biological fluids.34,35,36 HILIC is a form of

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normal-phase liquid chromatography (NPLC) in the sense that uses polar

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stationary phases, usually water-rich layers immobilized onto silica particles.

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However, unlike NPLC, HILIC employs aqueous mobile phases; during

ACS Paragon Plus Environment

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Analytical Chemistry

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gradient elution, the polarity is increased from a low organic content to a high

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water content mobile-phase in order to promote the elution of polar

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compounds. HILIC columns have found application in numerous areas,

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including metabolic profiling of various pathologies, but as yet a systematic

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evaluation of their performance as a metabolic profiling tool is lacking.37,38

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Typical base peak mass chromatograms are shown in Figures 1B and S-

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1B, acquired under positive and negative ionization modes, respectively, on

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the HILIC column. In the positive ionization mode (Figure 1B), prominent

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peaks from a few quaternary ammonium compounds (choline and betaine)

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and proline (a cyclic aminoacid) were bracketed by moderately retained

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phospholipids (4