Analytical and Bioanalytical Chemistry

, Volume 410, Issue 14, pp 3297–3313 | Cite as

Comprehensive lipid profiling in the Mediterranean mussel (Mytilus galloprovincialis) using hyphenated and multidimensional chromatography techniques coupled to mass spectrometry detection

  • Paola Donato
  • Giuseppe Micalizzi
  • Marianna Oteri
  • Francesca Rigano
  • Danilo Sciarrone
  • Paola Dugo
  • Luigi Mondello
Research Paper
Part of the following topical collections:
  1. Euroanalysis XIX


The task of lipid analysis and profiling is taking centre stage in many research fields and as a consequence, there has been an intense effort to develop suitable methodologies to discover, identify, and quantify lipids in the systems investigated. Given the high complexity and diversity of the lipidome, researchers have been challenged to afford thorough knowledge of all the lipid species in a given sample, by gathering the data obtained by complementary analytical techniques. In this research, an “omic” approach was developed to quickly fingerprint lipids in the Mediterranean mussel (Mytilus galloprovincialis), by exploiting multidimensional and hyphenated techniques. In detail, two-dimensional comprehensive hydrophilic interaction liquid chromatography coupled to reversed-phase liquid chromatography afforded both class-type separation and lipid assignment within the total lipid species in the sample, by the coupling of a 2.1-mm I.D. partially porous stationary phase in the first dimension, to a short (50 mm) monodisperse octadecylsilica secondary column; individual molecular species were afterwards identified by means of their ion trap-time of flight mass spectra obtained by electrospray ionization. More than 200 neutral and polar lipids were identified, and among the latter, phosphatydylcholine and phosphatydylethanolamine were the most represented classes, together with their mono-acylated forms, plasmanyl and plasmenyl derivatives. Subsequently, separation of the saturated and unsaturated isomers of the fatty acids (including the saturated C16:0 and the polyunsaturated C22:6) in the offline collected phospholipid fractions was accomplished by gas chromatography analysis of the corresponding methyl esters, on a 200 m × 0.25 mm, 0.2 μm d f ionic liquid column.


Two-dimensional comprehensive LC GC-MS IT-ToF Ionic liquid column Marine organisms Lipids 



The authors gratefully acknowledge Shimadzu Corporation and Millipore Sigma/Supelco Corporation for the continuous support.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Statement of human and animal rights

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures were in accordance with guidelines for the protection of animal welfare, in compliance with the Italian National Bioethics Committee (INBC) (European Community Council Directive of November 24, 1986-86/609/EEC).

Supplementary material

216_2018_1045_MOESM1_ESM.pdf (850 kb)
ESM 1 (PDF 850 kb)


  1. 1.
    Teslovich TM, Musunuru K, …, Kathiresan S. Biological, clinical and population relevance of 95 loci for blood lipids. Nature. 2010;466:707–13.Google Scholar
  2. 2.
    Akoh CC. Food lipids: chemistry, nutrition, and biotechnology. 4th ed. Boca Raton: CRC Press; 2017.CrossRefGoogle Scholar
  3. 3.
    Hyötyläinen T, Orešič M. Optimizing the lipidomics workflow for clinical studies—practical considerations. Anal Bioanal Chem. 2015;407:4973–93.CrossRefPubMedGoogle Scholar
  4. 4.
    Donato P, Cacciola F, Beccaria M, Dugo P, Mondello L. Lipidomics. In: Picò Y, editor. Advanced mass spectrometry for food safety and quality. Amsterdam: Elsevier; 2015. p. 395–439.CrossRefGoogle Scholar
  5. 5.
    Donato P, Inferrera I, Sciarrone D, Mondello L. Supercritical fluid chromatography for lipid analysis in foodstuffs. J Sep Sci. 2017;40:361–82.CrossRefPubMedGoogle Scholar
  6. 6.
    Beccaria M, Sullini G, Cacciola F, Donato P, Dugo P, Mondello L. High performance characterization of triacylglycerols in milk and milk-related samples by liquid chromatography and mass spectrometry. J Chromatogr A. 2014;1360:172–87.CrossRefPubMedGoogle Scholar
  7. 7.
    Lísa M, Holčapek M. 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. 2008;1198:115–30.CrossRefPubMedGoogle Scholar
  8. 8.
    Hutchins PM, Barkley RM, Murphy RC. Separation of cellular non polar neutral lipids by normal-phase chromatography and analysis by electrospray ionization mass spectrometry. J Lipid Res. 2008;49:804–13.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Donato P, Cacciola F, Cichello F, Russo M, Dugo P, Mondello L. Determination of phospholipids in milk samples by means of hydrophilic interaction liquid chromatography coupled to evaporative light scattering and mass spectrometry detection. J Chromatogr A. 2011;1218:6476–82.CrossRefPubMedGoogle Scholar
  10. 10.
    Tranchida PQ, Donato P, Dugo P, Dugo G, Mondello L. Comprehensive chromatographic methods for the analysis of lipids. TrAC Trends Anal Chem. 2007;26:191–205.CrossRefGoogle Scholar
  11. 11.
    Jandera P. Column selectivity for two-dimensional liquid chromatography. J Sep Sci. 2006;29:1763–83.CrossRefPubMedGoogle Scholar
  12. 12.
    Jandera P. Comprehensive two-dimensional liquid chromatography—practical impacts of theoretical considerations. A review. Cent Eur J Chem. 2012;10:844–75.Google Scholar
  13. 13.
    Dugo P, Fawzy N, Cichello F, Cacciola F, Donato P, Mondello L. Stop-flow comprehensive two-dimensional liquid chromatography combined with mass spectrometric detection for phospholipid analysis. J Chromatogr A. 2013;1278:46–53.CrossRefPubMedGoogle Scholar
  14. 14.
    Lísa M, Cífková E, Holčapek M. Lipidomic profiling of biological tissues using off-line two-dimensional high-performance liquid chromatography-mass spectrometry. J Chromatogr A. 2011;1218:5146–56.CrossRefPubMedGoogle Scholar
  15. 15.
    Li M, Tong X, Lv P, Feng B, Yang L, Wu Z, et al. A not-stop-flow online normal-/reversed-phase two-dimensional liquid chromatography–quadrupole time-of-flight mass spectrometry method for comprehensive lipid profiling of human plasma from atherosclerosis patients. J Chromatogr A. 2014;1372:110–9.CrossRefGoogle Scholar
  16. 16.
    Donato P, Cacciola F, Tranchida PQ, Dugo P, Mondello L. Mass spectrometry detection in comprehensive liquid chromatography: basic concepts, instrumental aspects, applications and trends. Mass Spectrom Rev. 2012;31:523–59.CrossRefPubMedGoogle Scholar
  17. 17.
    Holčapek M, Ovčačíková M, Lísa M, Cífková E, Hájek T. Continuous comprehensive two-dimensional liquid chromatography–electrospray ionization mass spectrometry of complex lipidomic samples. Anal Bioanal Chem. 2015;407:5033–43.CrossRefPubMedGoogle Scholar
  18. 18.
    Baglai A, Gargano AFG, Jordens J, Mengerink Y, Honing M, van der Wal S, et al. Comprehensive lipidomic analysis of human plasma using multidimensional liquid- and gas-phase separations: two-dimensional liquid chromatography-mass spectrometry vs. liquid chromatography-trapped-ion-mobility-mass spectrometry. J Chromatogr A. 2017;1530:90–103.CrossRefPubMedGoogle Scholar
  19. 19.
    Cajka T, Fiehn P. Comprehensive analysis of lipids in biological systems by liquid chromatography-mass spectrometry. TrAC Trends Anal Chem. 2014;61:192–206.CrossRefGoogle Scholar
  20. 20.
    Zoccali M, Schug KA, Walsh P, Smuts J, Mondello L. Flow-modulated comprehensive two-dimensional gas chromatography combined with a vacuum ultraviolet detector for the analysis of complex mixtures. J Chromatogr A. 2017;1497:135–43.CrossRefPubMedGoogle Scholar
  21. 21.
    Delmonte P, Fardin-Kia AR, Rader JI. Separation of fatty acid methyl esters by GC-online hydrogenation×GC. Anal Chem. 2013;85:1517–24.CrossRefPubMedGoogle Scholar
  22. 22.
    Anderson JL, Armstrong DW. High-stability ionic liquids. A new class of stationary phases for gas chromatography. Anal Chem. 2003;75:4851–8.CrossRefPubMedGoogle Scholar
  23. 23.
    Amaral MSS, Marriott PJ, Bizzo HR, et al. Ionic liquid capillary columns for analysis of multi-component volatiles by gas chromatography-mass spectrometry: performance, selectivity, activity and retention indices. Anal Bioanal Chem. 2017;
  24. 24.
    Delmonte P, Fardin-Kia AR, Kramer JKG, Mossoba MM, Sidisky L, Rader J. Separation characteristics of fatty acid methyl esters using SLB-IL111, a new ionic liquid coated capillary gas chromatographic column. J Chromatogr A. 2011;1218:545–54.CrossRefPubMedGoogle Scholar
  25. 25.
    Zapadlo M, Krupcik J, Májek P, Armstrong DW, Sandra P. Use of a polar ionic liquid as second column for the comprehensive two-dimensional GC separation of PCBs. J Chromatogr A. 2010;1217:5859–67.CrossRefPubMedGoogle Scholar
  26. 26.
    Seeley JV, Seeley SK, Libby EK, Breitbach ZS, Armstrong DW. Comprehensive two-dimensional gas chromatography using a high-temperature phosphonium ionic liquid column. Anal Bioanal Chem. 2008;390:323–32.CrossRefPubMedGoogle Scholar
  27. 27.
    Fanali C, Micalizzi G, Dugo P, Mondello L. Ionic liquids as stationary phases for fatty acid analysis by gas chromatography. Analyst. 2017;
  28. 28.
    Albergamo A, Rigano F, Purcaro G, Mauceri A, Fasulo S, Mondello L. Free fatty acid profiling of marine sentinels by nanoLC-EI-MS for the assessment of environmental pollution effects. Sci Total Environ. 2016;571:955–62.CrossRefPubMedGoogle Scholar
  29. 29.
    Martínez-Pita I, Sánchez-Lazo C, Ruíz-Jarabo R, Herrera M, Mancera JM. Biochemical composition, lipid classes, fatty acids and sexual hormones in the mussel Mytilus galloprovincialis from cultivated populations in south Spain. Aquaculture. 2012;358-359:274–83.CrossRefGoogle Scholar
  30. 30.
    Rigano F, Albergamo A, Sciarrone D, Beccaria M, Purcaro G, Mondello L. Nano liquid chromatography directly coupled to electron ionization mass spectrometry for free fatty acid elucidation in mussel. Anal Chem. 2016;88:4021–8.CrossRefPubMedGoogle Scholar
  31. 31.
    Bligh EG, Dyer WG. A rapid method of total lipid extraction and purification. Can J Biochem Phys. 1959;37:911–7.CrossRefGoogle Scholar
  32. 32.
    OECD. Test no. 305: bioaccumulation in fish: aqueous and dietary exposure. Paris: OECD Publishing; 2012. Scholar
  33. 33.
    Zhu C, Dane A, Spijksma G, Wang M, van der Greef J, Luo G, et al. An efficient hydrophilic interaction liquid chromatography separation of 7 phospholipid classes based on a diol column. J Chromatogr A. 2012;1220:26–34.CrossRefPubMedGoogle Scholar
  34. 34.
    Jandera P. Programmed elution in comprehensive two-dimensional liquid chromatography. J Chromatogr A. 2012;1255:112–29.CrossRefPubMedGoogle Scholar
  35. 35.
    Facchini L, Losito I, Cataldi TR, Palmisano F. Seasonal variations in the profile of main phospholipids in Mytilus galloprovincialis mussels: a study by hydrophilic interaction liquid chromatography-electrospray ionization Fourier transform mass spectrometry. J Mass Spectrom. 2017;
  36. 36.
    Facchini L, Losito I, Cataldi TR, Palmisano F. Ceramide lipids in alive and thermally stressed mussels: an investigation by hydrophilic interaction liquid chromatography-electrospray ionization Fourier transform mass spectrometry. J Mass Spectrom. 2016;51:768–81.CrossRefPubMedGoogle Scholar
  37. 37.
    Jandera P, Hájek T, Cesla P. Comparison of various second-dimension gradient types in comprehensive two-dimensional liquid chromatography. J Sep Sci. 2010;33:1382–97.CrossRefPubMedGoogle Scholar
  38. 38.
    Pirok BWJ, Gargano AFG, Schoenmakers PJ. Optimizing separations in online comprehensive two-dimensional liquid chromatography. J Sep Sci. 2017:1–30.
  39. 39.
    Donato P, Rigano F, Cacciola F, Schure M, Farnetti S, Russo M, et al. Comprehensive two-dimensional liquid chromatography–tandem mass spectrometry for the simultaneous determination of wine polyphenols and target contaminants. J Chromatogr A. 2016;1458:54–62.CrossRefPubMedGoogle Scholar
  40. 40.
    Murphy KJ, Mooney BD, Mann NJ, Nichols PD, Sinclair AJ. Lipid, FA, and sterol composition of New Zealand green lipped mussel (Pernacanaliculus) and Tasmanian blue mussel (Mytilusedulis). Lipids. 2002;37:587–95.CrossRefPubMedGoogle Scholar
  41. 41.
    Gorinstein S, Moncheva S, Katrich E, Toledo F, Arancibia P, Goshev I, et al. Antioxidants in the black mussel (Mytilus galloprovincialis) as an indicator of black sea coastal pollution. Mar Pollut Bull. 2003;46:1317–25.CrossRefPubMedGoogle Scholar
  42. 42.
    Delmonte P, Fardin-Kia AR, Kramer JKG, Mossoba MM, Sidisky L, Tyburczy C, et al. Evaluation of highly polar ionic liquid gas chromatographic column for the determination of the fatty acids in milk fat. J Chromatogr A. 2012;1233:137–46.CrossRefPubMedGoogle Scholar
  43. 43.
    Christie WW. Preparation of ester derivatives of fatty acids for chromatographic analysis. In: Christie WW, editor. Advances in lipid methodology–two. Dundee: Oily Press; 1993. p. 69–111.Google Scholar
  44. 44.
    Marinetti GV. Hydrolysis of lecithin with sodium methoxide. Biochemistry. 1962;1:350–3.CrossRefPubMedGoogle Scholar
  45. 45.
    Marinetti GV. Low temperature partial alcoholysis of triglycerides. J Lipid Res. 1966;7:786–8.PubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Paola Donato
    • 1
  • Giuseppe Micalizzi
    • 2
  • Marianna Oteri
    • 2
  • Francesca Rigano
    • 3
  • Danilo Sciarrone
    • 2
  • Paola Dugo
    • 2
    • 3
    • 4
  • Luigi Mondello
    • 2
    • 3
    • 4
  1. 1.Dipartimento di Scienze Biomediche, Odontoiatriche e delle Immagini Morfologiche e FunzionaliUniversity of MessinaMessinaItaly
  2. 2.Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed AmbientaliUniversity of Messina - Polo AnnunziataMessinaItaly
  3. 3.Chromaleont S.r.l.MessinaItaly
  4. 4.Centro Integrato di Ricerca (C.I.R.)University Campus Bio-Medico of RomeRomeItaly

Personalised recommendations