Evaluation of mobile phase characteristics on three zwitterionic columns in hydrophilic interaction liquid chromatography mode for liquid chromatography-high resolution mass spectrometry based untargeted metabolite profiling of Leishmania parasites

Rong Zhang, David G. Watson, Lijie Wang, Gareth D. Westrop, Graham H. Coombs, Tong Zhang

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Abstract

It has been reported that HILIC column chemistry has a great effect on the number of detected metabolites in LC-HRMS-based untargeted metabolite profiling studies. However, no systematic investigation has been carried out with regard to the optimisation of mobile phase characteristics. In this study using 223 metabolite standards, we explored the retention mechanisms on three zwitterionic columns with varied mobile phase composition, demonstrated the interference from poor chromatographic peak shapes on the output of data extraction, and assessed the quality of chromatographic signals and the separation of isomers under each LC condition. As expected, on the ZIC-cHILIC column the acidic metabolites showed improved chromatographic performance at low pH which can be attributed to the opposite arrangement of the permanently charged groups on this column in comparison with the ZIC-HILIC column. Using extracts from the protozoan parasite Leishmania, we compared the numbers of repeatedly detected LC-HRMS features under different LC conditions with putative identification of metabolites not amongst the standards being based on accurate mass (±3ppm). Besides column chemistry, the pH of the mobile phase plays a key role in not only determining the retention mechanisms of solutes but also the output of the LC-HRMS data processing. Fast evaporation of ammonium carbonate produced less ion suppression in ESI source and consequently improved the detectability of the metabolites in low abundance in comparison with other ammonium salts. Our results show that the combination of a ZIC-pHILIC column with an ammonium carbonate mobile phase, pH 9.2, at 20mM in the aqueous phase or 10mM in both aqueous and organic mobile phase components, provided the most suitable LC conditions for LC-HRMS-based untargeted metabolite profiling of Leishmania parasite extracts. The signal reliability of the mass spectrometer used in this study (Exactive Orbitrap) was also investigated.

LanguageEnglish
Pages168–179
Number of pages12
JournalJournal of Chromatography A
Volume1362
Early online date16 Aug 2014
DOIs
Publication statusPublished - 17 Aug 2014

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Leishmania
Liquid chromatography
Metabolites
Hydrophobic and Hydrophilic Interactions
Liquid Chromatography
Mass spectrometry
Mass Spectrometry
Parasites
Ammonium Compounds
Salts
Ions
Mass spectrometers
Phase composition
Isomers
Evaporation
ammonium carbonate

Keywords

  • HILIC-HRMS
  • Hydrophilic Interaction Liquid Chromatography–High Resolution Mass Spectrometry
  • zwitterionic stationary phase
  • metebolomics
  • untargeted metabolite profiling
  • Leishmania

Cite this

@article{7326b7a4e9e2496ea2722bb7235492e8,
title = "Evaluation of mobile phase characteristics on three zwitterionic columns in hydrophilic interaction liquid chromatography mode for liquid chromatography-high resolution mass spectrometry based untargeted metabolite profiling of Leishmania parasites",
abstract = "It has been reported that HILIC column chemistry has a great effect on the number of detected metabolites in LC-HRMS-based untargeted metabolite profiling studies. However, no systematic investigation has been carried out with regard to the optimisation of mobile phase characteristics. In this study using 223 metabolite standards, we explored the retention mechanisms on three zwitterionic columns with varied mobile phase composition, demonstrated the interference from poor chromatographic peak shapes on the output of data extraction, and assessed the quality of chromatographic signals and the separation of isomers under each LC condition. As expected, on the ZIC-cHILIC column the acidic metabolites showed improved chromatographic performance at low pH which can be attributed to the opposite arrangement of the permanently charged groups on this column in comparison with the ZIC-HILIC column. Using extracts from the protozoan parasite Leishmania, we compared the numbers of repeatedly detected LC-HRMS features under different LC conditions with putative identification of metabolites not amongst the standards being based on accurate mass (±3ppm). Besides column chemistry, the pH of the mobile phase plays a key role in not only determining the retention mechanisms of solutes but also the output of the LC-HRMS data processing. Fast evaporation of ammonium carbonate produced less ion suppression in ESI source and consequently improved the detectability of the metabolites in low abundance in comparison with other ammonium salts. Our results show that the combination of a ZIC-pHILIC column with an ammonium carbonate mobile phase, pH 9.2, at 20mM in the aqueous phase or 10mM in both aqueous and organic mobile phase components, provided the most suitable LC conditions for LC-HRMS-based untargeted metabolite profiling of Leishmania parasite extracts. The signal reliability of the mass spectrometer used in this study (Exactive Orbitrap) was also investigated.",
keywords = "HILIC-HRMS, Hydrophilic Interaction Liquid Chromatography–High Resolution Mass Spectrometry, zwitterionic stationary phase, metebolomics, untargeted metabolite profiling, Leishmania",
author = "Rong Zhang and Watson, {David G.} and Lijie Wang and Westrop, {Gareth D.} and Coombs, {Graham H.} and Tong Zhang",
year = "2014",
month = "8",
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doi = "10.1016/j.chroma.2014.08.039",
language = "English",
volume = "1362",
pages = "168–179",
journal = "Journal of Chromatography A",
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T1 - Evaluation of mobile phase characteristics on three zwitterionic columns in hydrophilic interaction liquid chromatography mode for liquid chromatography-high resolution mass spectrometry based untargeted metabolite profiling of Leishmania parasites

AU - Zhang, Rong

AU - Watson, David G.

AU - Wang, Lijie

AU - Westrop, Gareth D.

AU - Coombs, Graham H.

AU - Zhang, Tong

PY - 2014/8/17

Y1 - 2014/8/17

N2 - It has been reported that HILIC column chemistry has a great effect on the number of detected metabolites in LC-HRMS-based untargeted metabolite profiling studies. However, no systematic investigation has been carried out with regard to the optimisation of mobile phase characteristics. In this study using 223 metabolite standards, we explored the retention mechanisms on three zwitterionic columns with varied mobile phase composition, demonstrated the interference from poor chromatographic peak shapes on the output of data extraction, and assessed the quality of chromatographic signals and the separation of isomers under each LC condition. As expected, on the ZIC-cHILIC column the acidic metabolites showed improved chromatographic performance at low pH which can be attributed to the opposite arrangement of the permanently charged groups on this column in comparison with the ZIC-HILIC column. Using extracts from the protozoan parasite Leishmania, we compared the numbers of repeatedly detected LC-HRMS features under different LC conditions with putative identification of metabolites not amongst the standards being based on accurate mass (±3ppm). Besides column chemistry, the pH of the mobile phase plays a key role in not only determining the retention mechanisms of solutes but also the output of the LC-HRMS data processing. Fast evaporation of ammonium carbonate produced less ion suppression in ESI source and consequently improved the detectability of the metabolites in low abundance in comparison with other ammonium salts. Our results show that the combination of a ZIC-pHILIC column with an ammonium carbonate mobile phase, pH 9.2, at 20mM in the aqueous phase or 10mM in both aqueous and organic mobile phase components, provided the most suitable LC conditions for LC-HRMS-based untargeted metabolite profiling of Leishmania parasite extracts. The signal reliability of the mass spectrometer used in this study (Exactive Orbitrap) was also investigated.

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KW - HILIC-HRMS

KW - Hydrophilic Interaction Liquid Chromatography–High Resolution Mass Spectrometry

KW - zwitterionic stationary phase

KW - metebolomics

KW - untargeted metabolite profiling

KW - Leishmania

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M3 - Article

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EP - 179

JO - Journal of Chromatography A

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JF - Journal of Chromatography A

SN - 0021-9673

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