Analytical and Bioanalytical Chemistry

, Volume 407, Issue 17, pp 5033–5043 | Cite as

Continuous comprehensive two-dimensional liquid chromatography–electrospray ionization mass spectrometry of complex lipidomic samples

  • Michal HolčapekEmail author
  • Magdaléna Ovčačíková
  • Miroslav Lísa
  • Eva Cífková
  • Tomáš Hájek
Paper in Forefront
Part of the following topical collections:
  1. Lipidomics


A new continuous comprehensive two-dimensional liquid chromatography–electrospray ionization mass spectrometry method has been developed for the lipidomic characterization of complex biological samples. The reversed-phase ultra-high-performance liquid chromatography with a C18 column (150 mm × 1 mm, 1.7 μm) used in the first dimension makes the separation of numerous lipid species differing in their hydrophobic part of the molecule, mainly fatty acyl chain lengths and the number and positions of double bonds, possible. Coeluted lipid species in the first dimension are resolved by the fast hydrophilic interaction liquid chromatography separation (50 mm × 3 mm, 2.7 μm, core–shell particles) of lipid classes according to their different polarities in the second dimension. Retention times in both dimensions, accurate m/z values, and tandem mass spectra provide high confidence in the identification of lipid species. The retention behavior of individual lipids in reversed-phase mode follows the equivalent carbon number pattern, which provides an additional tool for unambiguous identification. This analytical method is applied for the lipidomic characterization of total lipid extracts of human plasma and porcine brain samples, which resulted in the identification of 143 lipid species from four lipid categories and ten lipid classes.

Graphical Abstract

2D-LC/ESI-MS of porcine brain lipid extract


Lipids Lipidomics Comprehensive 2D liquid chromatography Mass spectrometry Plasma Brain 



This work was supported by ERC CZ project no. LL1302 sponsored by the Ministry of Education, Youth, and Sports of the Czech Republic. E.C. acknowledges the support of project no. CZ.1.07/2.3.00/30.0021 sponsored by the Ministry of Education, Youth, and Sports of the Czech Republic.

Supplementary material

216_2015_8528_MOESM1_ESM.pdf (461 kb)
ESM 1 (PDF 461 kb)


  1. 1.
    LIPID MAPS (2014) LIPID MAPS Lipidomics Gateway. Accessed 2 Dec 2014
  2. 2.
    Fahy E, Subramaniam S, Brown HA, Glass CK, Merrill AH, Murphy RC, Raetz CRH, Russell DW, Seyama Y, Shaw W, Shimizu T, Spener F, van Meer G, VanNieuwenhze MS, White SH, Witztum JL, Dennis EA (2005) A comprehensive classification system for lipids. Eur J Lipid Sci Technol 107:337–364CrossRefGoogle Scholar
  3. 3.
    Fahy E, Subramaniam S, Murphy RC, Nishijima M, Raetz CRH, Shimizu T, Spener F, van Meer G, Wakelam MJO, Dennis EA (2009) Update of the LIPID MAPS comprehensive classification system for lipids. J Lipid Res 50:S9–S14CrossRefGoogle Scholar
  4. 4.
    Santos CR, Schulze A (2012) Lipid metabolism in cancer. FEBS J 279:2610–2623CrossRefGoogle Scholar
  5. 5.
    van Meer G, Voelker DR, Feigenson GW (2008) Membrane lipids: where they are and how they behave. Nat Rev Mol Cell Biol 9:112–124CrossRefGoogle Scholar
  6. 6.
    Oresič M, Hanninen VA, Vidal-Puig A (2008) Lipidomics: a new window to biomedical frontiers. Trends Biotechnol 26:647–652CrossRefGoogle Scholar
  7. 7.
    Li M, Yang L, Bai Y, Liu HW (2014) Analytical methods in lipidomics and their applications. Anal Chem 86:161–175CrossRefGoogle Scholar
  8. 8.
    Han XL, Gross RW (2005) Shotgun lipidomics: electrospray ionization mass spectrometric analysis and quantitation of cellular lipidomes directly from crude extracts of biological samples. Mass Spectrom Rev 24:367–412CrossRefGoogle Scholar
  9. 9.
    Han X, Yang K, Gross RW (2012) Multi-dimensional mass spectrometry-based shotgun lipidomics and novel strategies for lipidomic analyses. Mass Spectrom Rev 31:134–178CrossRefGoogle Scholar
  10. 10.
    Papan C, Penkov S, Herzog R, Thiele C, Kurzchalia T, Shevchenko A (2014) Systematic screening for novel lipids by shotgun lipidomics. Anal Chem 86:2703–2710CrossRefGoogle Scholar
  11. 11.
    Sandra K, Pereira AD, Vanhoenacker G, David F, Sandra P (2010) Comprehensive blood plasma lipidomics by liquid chromatography/quadrupole time-of-flight mass spectrometry. J Chromatogr A 1217:4087–4099CrossRefGoogle Scholar
  12. 12.
    Holčapek M, Cífková E, Červená B, Lísa M, Vostálová J, Galuszka J (2015) Determination of nonpolar and polar lipid classes in human plasma, erythrocytes and plasma lipoprotein fractions using ultrahigh-performance liquid chromatography-mass spectrometry. J Chromatogr A 1377:85–91CrossRefGoogle Scholar
  13. 13.
    Sokol E, Almeida R, Hannibal-Bach HK, Kotowska D, Vogt J, Baumgart J, Kristiansen K, Nitsch R, Knudsen J, Ejsing CS (2013) Profiling of lipid species by normal-phase liquid chromatography, nanoelectrospray ionization, and ion trap–orbitrap mass spectrometry. Anal Biochem 443:88–96CrossRefGoogle Scholar
  14. 14.
    Cífková E, Holčapek M, Lísa M, Ovčačíková M, Lyčka A, Lynen F, Sandra P (2012) Nontargeted quantitation of lipid classes using hydrophilic interaction liquid chromatography-electrospray ionization mass spectrometry with single internal standard and response factor approach. Anal Chem 84:10064–10070CrossRefGoogle Scholar
  15. 15.
    Cífková E, Holčapek M, Lísa M (2013) Nontargeted lipidomic characterization of porcine organs using hydrophilic interaction liquid chromatography and off-line two-dimensional liquid chromatography-electrospray ionization mass spectrometry. Lipids 48:915–928CrossRefGoogle Scholar
  16. 16.
    Lísa M, Netušilová K, Franěk L, Dvořáková H, Vrkoslav V, Holčapek M (2011) Characterization of fatty acid and triacylglycerol composition in animal fats using silver-ion and non-aqueous reversed-phase high-performance liquid chromatography/mass spectrometry and gas chromatography/flame ionization detection. J Chromatogr A 1218:7499–7510CrossRefGoogle Scholar
  17. 17.
    Adlof RO (1997) Normal-phase separation effects with lipids on a silver ion high-performance liquid chromatography column. J Chromatogr A 764:337–340CrossRefGoogle Scholar
  18. 18.
    Holčapek M, Dvořáková H, Lísa M, Girón AJ, Sandra P, Cvačka J (2010) Regioisomeric analysis of triacylglycerols using silver-ion liquid chromatography–atmospheric pressure chemical ionization mass spectrometry: comparison of five different mass analyzers. J Chromatogr A 1217:8186–8194CrossRefGoogle Scholar
  19. 19.
    Lísa M, Holčapek M (2013) Characterization of triacylglycerol enantiomers using chiral HPLC/APCI-MS and synthesis of enantiomeric triacylglycerols. Anal Chem 85:1852–1859CrossRefGoogle Scholar
  20. 20.
    Murphy SA, Nicolaou A (2013) Lipidomics applications in health, disease and nutrition research. Mol Nutr Food Res 57:1336–1346CrossRefGoogle Scholar
  21. 21.
    Römpp A, Spengler B (2013) Mass spectrometry imaging with high resolution in mass and space. Histochem Cell Biol 139:759–783CrossRefGoogle Scholar
  22. 22.
    Dugo P, Cacciola F, Kumm T, Dugo G, Mondello L (2008) Comprehensive multidimensional liquid chromatography: theory and applications. J Chromatogr A 1184:353–368CrossRefGoogle Scholar
  23. 23.
    François I, Sandra K, Sandra P (2009) Comprehensive liquid chromatography: Fundamental aspects and practical considerations—a review. Anal Chim Acta 641:14–31CrossRefGoogle Scholar
  24. 24.
    Česla P, Hájek T, Jandera P (2009) Optimization of two-dimensional gradient liquid chromatography separations. J Chromatogr A 1216:3443–3457CrossRefGoogle Scholar
  25. 25.
    D'Attoma A, Grivel C, Heinisch S (2012) On-line comprehensive two-dimensional separations of charged compounds using reversed-phase high performance liquid chromatography and hydrophilic interaction chromatography. Part I: orthogonality and practical peak capacity considerations. J Chromatogr A 1262:148–159CrossRefGoogle Scholar
  26. 26.
    Jandera P, Hájek T, Staňková M, Vyňuchalová K, Česla P (2012) Optimization of comprehensive two-dimensional gradient chromatography coupling in-line hydrophilic interaction and reversed phase liquid chromatography. J Chromatogr A 1268:91–101CrossRefGoogle Scholar
  27. 27.
    Lísa M, Cífková E, Holčapek M (2011) Lipidomic profiling of biological tissues using off-line two-dimensional high-performance liquid chromatography mass spectrometry. J Chromatogr A 1218:5146–5156CrossRefGoogle Scholar
  28. 28.
    Holčapek M, Velínská H, Lísa M, Česla P (2009) Orthogonality of silver-ion and non-aqueous reversed-phase HPLC/MS in the analysis of complex natural mixtures of triacylglycerols. J Sep Sci 32:3672–3680CrossRefGoogle Scholar
  29. 29.
    Dugo P, Kumm T, Crupi ML, Cotroneo A, Mondello L (2006) Comprehensive two-dimensional liquid chromatography combined with mass spectrometric detection in the analyses of triacylglycerols in natural lipidic matrixes. J Chromatogr A 1112:269–275CrossRefGoogle Scholar
  30. 30.
    Yang Q, Shi X, Gu Q, Zhao S, Shan Y, Xu G (2012) On-line two dimensional liquid chromatography/mass spectrometry for the analysis of triacylglycerides in peanut oil and mouse tissue. J Chromatogr B 895–896:48–55CrossRefGoogle Scholar
  31. 31.
    Ling YS, Liang HJ, Lin MH, Tang CH, Wu KY, Kuo ML, Lin CY (2014) Two-dimensional LC-MS/MS to enhance ceramide and phosphatidylcholine species profiling in mouse liver. Biomed Chromatogr 28:1284–1293CrossRefGoogle Scholar
  32. 32.
    Sun C, Zhao Y-Y, Curtis JM (2014) Elucidation of phosphatidylcholine isomers using two dimensional liquid chromatography coupled in-line with ozonolysis mass spectrometry. J Chromatogr A 1351:37–45CrossRefGoogle Scholar
  33. 33.
    Dugo P, Fawzy N, Cichello F, Cacciola F, Donato P, Mondello L (2013) Stop-flow comprehensive two-dimensional liquid chromatography combined with mass spectrometric detection for phospholipid analysis. J Chromatogr A 1278:46–53CrossRefGoogle Scholar
  34. 34.
    Wang SY, Li J, Shi XZ, Qiao LZ, Lu X, Xu GW (2013) A novel stop-flow two-dimensional liquid chromatography-mass spectrometry method for lipid analysis. J Chromatogr A 1321:65–72CrossRefGoogle Scholar
  35. 35.
    Li M, Feng BS, Liang Y, Zhang W, Bai Y, Tang W, Wang T, Liu HW (2013) Lipid profiling of human plasma from peritoneal dialysis patients using an improved 2D (NP/RP) LC-QToF MS method. Anal Bioanal Chem 405:6629–6638CrossRefGoogle Scholar
  36. 36.
    Bang DY, Moon MH (2013) On-line two-dimensional capillary strong anion exchange/reversed phase liquid chromatography-tandem mass spectrometry for comprehensive lipid analysis. J Chromatogr A 1310:82–90CrossRefGoogle Scholar
  37. 37.
    Liebisch G, Vizcaino JA, Kofeler H, Trotzmuller M, Griffiths WJ, Schmitz G, Spener F, Wakelam MJO (2013) Shorthand notation for lipid structures derived from mass spectrometry. J Lipid Res 54:1523–1530CrossRefGoogle Scholar
  38. 38.
    Folch J, Lees M, Stanley GHS (1957) A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 226:497–509Google Scholar
  39. 39.
    Holčapek M, Jandera P, Fischer J, Prokeš B (1999) Analytical monitoring of the production of biodiesel by high-performance liquid chromatography with various detection methods. J Chromatogr A 858:13–31CrossRefGoogle Scholar
  40. 40.
    Holčapek M, Jandera P, Zderadička P, Hrubá L (2003) Characterization of triacylglycerol and diacylglycerol composition of plant oils using high-performance liquid chromatography–atmospheric pressure chemical ionization mass spectrometry. J Chromatogr A 1010:195–215CrossRefGoogle Scholar
  41. 41.
    Lísa M, Holčapek M (2008) Triacylglycerols profiling in plant oils important in food industry, dietetics and cosmetics using high-performance liquid chromatography–atmospheric pressure chemical ionization mass spectrometry. J Chromatogr A 1198–1199:115–130CrossRefGoogle Scholar
  42. 42.
    Berdeaux O, Juaneda P, Martine L, Cabaret S, Bretillon L, Acar N (2010) Identification and quantification of phosphatidylcholines containing very-long-chain polyunsaturated fatty acid in bovine and human retina using liquid chromatography/tandem mass spectrometry. J Chromatogr A 1217:7738–7748CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Michal Holčapek
    • 1
    Email author
  • Magdaléna Ovčačíková
    • 1
  • Miroslav Lísa
    • 1
  • Eva Cífková
    • 1
  • Tomáš Hájek
    • 1
  1. 1.Department of Analytical Chemistry, Faculty of Chemical TechnologyUniversity of PardubicePardubiceCzech Republic

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