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.
authors.affiliation: Strathclyde Institute of Pharmacy and Biomedical Sciences, 161, Cathedral Street, Glasgow, Scotland, G4 0RE, UK; Institute of Clinical Pharmacology, Guangzhou University of Chinese Medicine, No. 12 Jichang Road, Guangzhou, 510405 China. Electronic address: zhangrong@gzucm.edu.cn.
authors.forename: Rong
authors.initials: R
authors.lastname: Zhang
authors.affiliation: Strathclyde Institute of Pharmacy and Biomedical Sciences, 161, Cathedral Street, Glasgow, Scotland, G4 0RE, UK. Electronic address: d.g.watson@strath.ac.uk.
authors.forename: David G
authors.initials: DG
authors.lastname: Watson
authors.affiliation: Strathclyde Institute of Pharmacy and Biomedical Sciences, 161, Cathedral Street, Glasgow, Scotland, G4 0RE, UK. Electronic address: lijie.wang@strath.ac.uk.
authors.forename: Lijie
authors.initials: L
authors.lastname: Wang
authors.affiliation: Strathclyde Institute of Pharmacy and Biomedical Sciences, 161, Cathedral Street, Glasgow, Scotland, G4 0RE, UK. Electronic address: gareth.westrop@strath.ac.uk.
authors.forename: Gareth D
authors.initials: GD
authors.lastname: Westrop
authors.affiliation: Strathclyde Institute of Pharmacy and Biomedical Sciences, 161, Cathedral Street, Glasgow, Scotland, G4 0RE, UK. Electronic address: graham.coombs@strath.ac.uk.
authors.forename: Graham H
authors.initials: GH
authors.lastname: Coombs
authors.affiliation: Strathclyde Institute of Pharmacy and Biomedical Sciences, 161, Cathedral Street, Glasgow, Scotland, G4 0RE, UK. Electronic address: tong.zhang.101@strath.ac.uk.
authors.forename: Tong
authors.initials: T
authors.lastname: Zhang
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000737:chemistry; D018320:Leishmania major / Q000737:chemistry* / Q000378:metabolism; D055432:Metabolomics / Q000379:methods*; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014867:Water / Q000737:chemistry
chemical_list: D007477:Ions; D014867:Water
keywords: HILIC-HRMS (Hydrophilic Interaction Liquid Chromatography–High Resolution Mass Spectrometry); Leishmania; Metebolomics; Untargeted metabolite profiling; Zwitterionic stationary phase