Elsevier

Journal of Chromatography A

Volume 1220, 13 January 2012, Pages 26-34
Journal of Chromatography A

An efficient hydrophilic interaction liquid chromatography separation of 7 phospholipid classes based on a diol column

https://doi.org/10.1016/j.chroma.2011.11.034Get rights and content

Abstract

A hydrophilic interaction liquid chromatography (HILIC) – ion trap mass spectrometry method was developed for separation of a wide range of phospholipids. A diol column which is often used with normal phase chromatography was adapted to separate different phospholipid classes in HILIC mode using a mobile phase system consisting of acetonitrile, water, ammonium formate and formic acid. An efficient between-class separation of seven phospholipid classes including phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinostol, phosphatidylcholine, phosphatidylserine, sphingomyelin and lysophosphatidylcholine was successfully achieved within 14 min using a gradient elution which starts with 90% of organic solvent and ends with 70% of organic solvent. 53 mM formic acid (in both organic phase and aqueous phase) and 60 mM ammonium formate (only in aqueous phase) were used as mobile phase modifier. The relatively high amount of ammonium formate was essential to obtain well-shaped peaks of each phospholipid class, especially phosphatidylserines; actually, no negative effect due to ammonium formate was observed for electrospray-mass spectrometry detection in real-life samples. Good chromatographic separation between different lipid classes was obtained (Rs, from 0.73 to 4.97) and well-shaped peaks (tailing factor, from 0.98 to 1.20) were obtained. The developed method was fully validated and the satisfactory performance characteristics such as linearity (R2, 0.990–0.999), retention time stability (RSD < 1%), within day repeatability (RSD, 5–13%), between day variation (RSD, 7–14%) and recoveries (99.6–115.5%) indicated the gradient HILIC method was appropriate for profiling of plasma phospholipids. The method was successfully applied to separate phospholipids extracts from human plasma, mouse plasma and rat plasma.

Highlights

► An efficient HILIC separation of 7 phospholipid classes. ► The relatively high amount of ammonium formate is beneficial to separation of PLs. ► The possible mechanism of HILIC separation of phospholipids on diol-bonded column. ► IPA (isopropanol) method for extracting phospholipids from plasma.

Introduction

Phospholipids (PLs) are the primary structural constituents of biological membranes. Phospholipid (PL) molecules consist of a polar phosphoryl head group and one or two non-polar fatty acid tails with varying numbers of carbons and double bonds [1] (Fig. 1). PLs can be divided into two major families glycerophospholipids (GPs) and sphingomyelins (SMs). The GPs have in common a glycerol backbone and they differ in their polar head group, i.e., ethanolamine, choline, glycerol, inositol, and serine. Sphingomyelins (SMs) differ from GPs as they have a sphingosine base as the backbone instead of a glycerol.

In addition to their membrane function PLs are important in the emulsification of neutral fat and cholesterol deposits in blood vessels [2], [3], intelligence improvement [4] and cell activation [5], [6], [7]. Next, PL metabolism is very closely associated with many different diseases. Furthermore, PLs have already been recognized to be important signaling molecules [8], [9], [10], [11], [12], [13], [14], [15], [16] and potential biomarkers for ovarian cancer [17], [18], [19], [20], diabetes mellitus [21], [22] and many other diseases [23], [24], [25], [26], [27]. Due to this apparent important role of PLs in biology, identification and quantification of PLs in biological samples are very important.

HPLC separation of PL classes is widely reported using either reversed phase liquid chromatography (RPLC) or normal phase liquid chromatography (NPLC) [28], [29], [30], [31]. Mainly two basic solvent systems, viz. acetonitrile (ACN)-based solvent systems [30] and isopropanol (IPA)-based solvent systems [31], have been reported as mobile phase systems in the past 30 years. Some limited work though is reported on the use of chloroform (CHCl3) as important mobile phase constituent [32], but due to the effects on (public) health, these systems have not been further evaluated in this study and the use of either ACN or IPA based solvents are the preferred choice. Some of the PLs are charged molecules and may require a counter ion in solution for efficient solution by RPLC system. Whereas sulphuric and phosphoric acid have been used for PLs as pH modifiers in liquid chromatography using ultraviolet (UV) detection, they are incompatible with evaporative light scattering detection and mass spectrometry (MS) detection [33], [34], [35]. Organic pH modifiers/buffers such as triethylamine, acetic acid, formic acid (FA), ammonia, ammonium formate (AmFm) and ammonium acetate (AmAc) are commonly applied [36], [37], [38]. Various modifications to silica like diol, nitrile, nitro, methyl, phenylcyano or phenylsulphonate substituent groups have been reported for PL separation. Each of these stationary phases offer specific selectivity.

Both RPLC and NPLC systems, with a variety of detectors, have been reported in the literature for separations both between PL classes and within PL class. Still, both have their limitations for separation of PLs. With NPLC, PL class separation was achieved but retention time shifting has been reported; this may be due to solvent mixing effects, slow equilibration of the stationary phase or may be due to phase separation since the mobile phase mixtures used often contain water and a water-insoluble solvent [36], [39], [40]. With RPLC, different compounds within the various PL classes were separated successfully but between-class separation was not obtained completely [41], [42]. As a result, ion suppression of lipids of a certain class can occur due to presence of lipids of another class [43]. Therefore, the aim of the current study is to develop a separation method which can provide better class separation.

hydrophilic interaction liquid chromatography (HILIC) is highly capable of separating polar and hydrophilic compounds. In HILIC mode, the retention of solutes is increased if the percentage of organic solvent is increased. The high composition of organic solvent in the mobile phase which is required to retain polar compounds by HILIC has a positive effect on the ionization efficiency in ESI [44]. Therefore, HILIC coupled with ESI-MS has been widely used to measure polar compounds such as peptides, amino acids, sugars, proteins, nucleic acids and monosaccharides [45], [46], [47], [48], [49], [50], [51]. Up to now, HILIC applications to separation of PLs were sparsely reported. The separation of a mixture of PCs, PEs and PIs on an amino column by gradient elution was reported [52]. Using a silica gel column and IPA–ACN mobile phase system, Zheng et al. [53] successfully separated lipid extracts from Leishmania donovani. Schwalbe-Herrmann et al. [54] separated at least four classes of PLs with isocratic elution in HILIC mode using a silica-based HILIC method. However, gradient elution usually gives a faster overall analysis, narrower peaks and better resolution without loss in linearity and repeatability compared with isocratic elution [55]. Therefore, a gradient elution HILIC method might be more suitable and efficient for PLs separation.

This paper describes the development, validation and application of a gradient HILIC method using a diol column for separating PL classes. In this paper, a diol column and an ACN–water mobile phase system containing ammonium formate (AmFm) and formic acid (FA) were explored to separate PL classes for, to our best knowledge, the first time. The method was validated to determine several performance characteristics (retention time stability, linearity, intermediate precision and recovery) and subsequently the applicability of the method for the analysis of PLs in various (blood) plasma samples.

Section snippets

Chemicals and materials

ULC-MS grade IPA, CHCl3, water and HPLC-S grade ACN, methanol (MeOH) were purchased from Biosolve (Valkenswaard, The Netherlands). FA (99+%) was purchased from ACROS OGANICS (New Jersey, NJ, USA). AmFm (≥99.995%) was obtained from Sigma Aldrich (St. Louis, MO, USA). PLs standards of 1,2-Dimyristoyl -sn-Glycero-3-[phospho-rac-(1-glycerol)](sodium salt) PG (C14:0/14:0), 1,2-Diheptadecanoyl -sn-Glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) PG (C17:0/17:0), 1,2-Dipentadecanoyl-sn-Glycero-3

Method optimization

Two methods for extraction of lipids from plasma were compared, extraction by MeOH and extraction by IPA. The IPA method was a modification of the MeOH method by Zhao and Xu [56] and was compared with that MeOH extraction as reference. Since IPA has a lower polarity than MeOH, theoretically, IPA should have higher extraction efficiency for more apolar PLs. The peak areas of two selected compounds in each class were compared to evaluate the relative difference on extraction efficiency between

Conclusions

HILIC is very promising for separation of PLs. A gradient HILIC method for plasma PL class separation was developed successfully. An efficient between-class separation of seven PL classes as well as within-class separation of PL individuals was achieved in HILIC mode by using a diol column and an ACN–water mobile phase system with AmFm as additive in the aqueous phase. The relatively high amount of AmFm in aqueous mobile phase is beneficial to well-shaped peaks of PLs, especially PSs, without

Acknowledgements

C. Zhu is a joint Ph.D. student educated by East China University of Science and Technology (ECUST) and Leiden University (LU). This study was performed with the help of staffs of the Division of Analytical Bioscience, Leiden University. We would like to thank all of members related to this work.

This study was supported by the research programme of the Netherlands Metabolomics Centre and the Sino Dutch Centre for Personalised and Preventive Medicine, which are part of The Netherlands Genomics

References (59)

  • S. Sengupta et al.

    Semin. Cell Dev. Biol.

    (2004)
  • H.R. Yoon et al.

    J. Chromatogr. B

    (2003)
  • T.E. Merchant et al.

    Cancer Lett.

    (2002)
  • C.Y. Lee et al.

    J. Lipid Res.

    (2005)
  • W.M.A. Hax et al.

    J. Chromatogr.

    (1977)
  • S. Uran et al.

    J. Chromatogr. B

    (2001)
  • S.S.H. Chen et al.

    J. Chromatogr.

    (1982)
  • T.L. Kaduce et al.

    J. Lipid Res.

    (1983)
  • L.Q. Pang et al.

    J. Chromatogr. B

    (2008)
  • S.L. Abidi

    J. Chromatogr.

    (1991)
  • B. Sas et al.

    J. Chromatogr. A

    (1999)
  • P. Fagan et al.

    J. Chromatogr. A

    (2004)
  • D.Y. Bang et al.

    J. Chromatogr. B

    (2007)
  • H. Tanaka et al.

    J. Chromatogr. A

    (2003)
  • T. Yoshida

    J. Biochem. Biophys. Methods

    (2004)
  • A.J. Alpert

    J. Chromatogr.

    (1990)
  • G. Karlsson et al.

    J. Chromatogr. A

    (2005)
  • M. Schwalbe-Herrmann et al.

    J. Chromatogr. A

    (2010)
  • A.P. Schellinger et al.

    J. Chromatogr. A

    (2006)
  • Z.W. Zhao et al.

    J. Lipid Res.

    (2010)
  • ...
  • B.R. Gordon et al.

    J. Infect. Dis.

    (2005)
  • ...
  • ...
  • M. Mueller et al.

    J. Immunol.

    (2005)
  • A. Nishikimi et al.

    Science

    (2009)
  • A. Zimman et al.

    J. Proteome Res.

    (2010)
  • W.H. Moolenaar

    N. Y. Ann. Acad. Sci.

    (2000)
  • G.B. Mills et al.

    Nat. Rev. Cancer

    (2003)
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