Analytical and Bioanalytical Chemistry

, Volume 406, Issue 30, pp 7937–7948 | Cite as

LC/MS lipid profiling from human serum: a new method for global lipid extraction

  • Roberto Maria Pellegrino
  • Alessandra Di Veroli
  • Aurora Valeri
  • Laura Goracci
  • Gabriele CrucianiEmail author
Research Paper


Over the last decade, technological advances have improved the sensitivity and selectivity of LC/MS analyzers, providing very efficient tools for lipidomics research. In particular, the nine lipid classes that constitute 99 % of the human serum lipidome (sterols, cholesteryl esters, phosphocholines, phosphoethanolamines, sphingomyelins, triacylglycerols, fatty acids, lysophosphocholines, and diacylglycerols) can be easily detected. However, until today there has not been a unique technique for sample preparation that provides a satisfactory recovery for all of these nine classes together. In this work, we have developed and validated a new one-phase extraction (OPE) method that overcomes this limitation. This method was also compared with the gold standard lipid extraction methods such as Folch, Bligh & Dyer, and recently developed methods with methanol and methyl-tert-butyl ether. Results demonstrate that the mixture of methanol/chloroform/MTBE (MMC) provides a recovery very close to 100 % for all nine lipid classes of the human serum investigated. For this extraction method, 100 μL of human serum is incubated with 2 mL of the solvents mixture, then vortexed and centrifuged. For its simplicity of execution, rapidity, reproducibility, and the reduced volume of sample required, this method opens the door to the use of human serum lipid profiling for large-scale applications in scientific research and clinical trials.


Lipidomics Mass spectrometry Clinical trials Diagnostic tools Dyslipidemias 



Bligh & Dyer




Methanol:acetone method


Methanol:chloroform method


Methanol method


Methanol:isopropanol method


Methanol:methyl-tert-butyl ether:chloroform method


One-phase extraction


Standard deviation


Two-phase extraction



A.D.V. thanks Regione Umbria, this research project was partly supported by 2007–2013 ESF “Competitiveness and Employment objective” Umbrian Regional Operational  Programme (ROP), Avviso pubblico aiuti individuali per la realizzazione di progetti di ricerca, project “Correlazione tra impronta lipidica del sangue e incidenza delle malattie neurodegenerative. Sviluppo di un metodo non invasivo e poco costoso per abbattere i costi della sanità pubblica regionale”.

The authors thank the Centro Ospedaliero di Riabilitazione Intensiva (C.O.R.I.), Passignano sul Trasimeno (Perugia, Italy), for providing serum samples, and Molecular Discovery Ltd. (UK) for free software and financial support.


  1. 1.
    Wolf C, Quinn PJ (2008) Lipidomics: practical aspects and applications. Prog Lipid Res 47:15–36CrossRefGoogle Scholar
  2. 2.
    Wenk MR (2005) The emerging field of lipidomics. Nat Rev Drug Discov 4:594–610CrossRefGoogle Scholar
  3. 3.
    Feng L, Prestwich GD (2005) Functional lipidomics. Dekker-CRC, New York 1–329Google Scholar
  4. 4.
    Yang K, Han X (2011) Accurate quantification of lipid species by electrospray ionization mass spectrometry meets a key challenge in lipidomics. Metabolites 1:21–40CrossRefGoogle Scholar
  5. 5.
    Han X, Holtzman DM, McKeel DW Jr, Kelley J, Morris JC (2002) Substantial sulfatide deficiency and ceramide elevation in very early Alzheimer’s disease: potential role in disease pathogenesis. J Neurochem 82:809–818CrossRefGoogle Scholar
  6. 6.
    Mills GB, Moolenaar WH (2003) The emerging role of lysophosphatidic acid in cancer. Nat Rev Cancer 3:582–591CrossRefGoogle Scholar
  7. 7.
    Zhao Z, Xiao Y, Elson P, Tan H, Plummer SJ, Berk M, Aung PP, Lavery IC, Achkar JP, Li L, Casey G, Xu Y (2007) Plasma lysophosphatidylcholine levels: potential biomarkers for colorectal cancer. J Clin Oncol 25:2696–2701CrossRefGoogle Scholar
  8. 8.
    Postle AD, Wilton DC, Hunt AN, Attard GS (2007) Probing phospholipid dynamics by electrospray ionization mass spectrometry. Prog Lipid Res 46:200–224CrossRefGoogle Scholar
  9. 9.
    Graessler J, Schwudke D, Schwarz PEH, Herzog R, Shevchenko A, Bornstein SR (2009) Top-down lipidomics reveals ether lipid deficiency in blood plasma of hypertensive patients. PLoS ONE 4:e6261CrossRefGoogle Scholar
  10. 10.
    Fernandez C, Sandin M, Sampaio JL, Almgren P, Narkiewicz K, Hoffman M, Hedner T, Wahlstrand B, Simons K, Shevchenko A, James P, Melander O (2013) Plasma lipid composition and risk of developing cardiovascular disease. PLoS ONE 8:e71846CrossRefGoogle Scholar
  11. 11.
    Rossouw JE (1994) The effects of lowering serum cholesterol on coronary heart disease risk. Med Clin N Am 78:181–195Google Scholar
  12. 12.
    Han X, Gross RW (2003) Global analyses of cellular lipidomes directly from crude extracts of biological samples by ESI mass spectrometry: a bridge to lipidomics. J Lipid Res 44:1071–1079CrossRefGoogle Scholar
  13. 13.
    Breuer HW (2001) Hypertriglyceridemia: a review of clinical relevance and treatment options: focus on cerivastatin. Curr Med Res Opin 17:60–73CrossRefGoogle Scholar
  14. 14.
    Pulfer M, Murphy RC (2003) Electrospray mass spectrometry of phospholipids. Mass Spectrom Rev 22:332–364CrossRefGoogle Scholar
  15. 15.
    Folch J, Lees M, Sloane Stanley GH (1957) A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 226:497–509Google Scholar
  16. 16.
    Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917CrossRefGoogle Scholar
  17. 17.
    Retra K, Bleijerveld OB, van Gestel RA, Tielens AGM, van Hellemond JJ, Brouwers JF (2008) A simple and universal method for the separation and identification of phospholipid molecular species. Rapid Commun Mass Spectrom 22:1853–1862CrossRefGoogle Scholar
  18. 18.
    Reis A, Rudnitskaya A, Blackburn GJ, Fauzi NM, Pitt AR, Spickett CM (2013) A comparison of five lipid extraction solvent systems for lipidomic studies of human LDL. J Lipid Res 54:1812–1824CrossRefGoogle Scholar
  19. 19.
    Matyash V, Liebisch G, Kurzchalia TV, Shevchenko A, Schwudke D (2008) Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics. J Lipid Res 49:1137–1146CrossRefGoogle Scholar
  20. 20.
    Löfgren L, Ståhlman M, Forsberg GB, Saarinen S, Nilsson R, Hansson GI (2012) The BUME method: a novel automated chloroform-free 96-well total lipid extraction method for blood plasma. J Lipid Res 53:1690–1700CrossRefGoogle Scholar
  21. 21.
    Quehenberger O, Armando AM, Brown AH, Milne SB, Myers DS, Merrill AH, Bandyopadhyay S, Jones KN, Kelly S, Shaner RL, Sullards CM, Wang E, Murphy RC, Barkley RM, Leiker TJ, Raetz CRH, Guan Z, Laird GM, Six DA, Russell DW, McDonald JG, Subramaniam S, Fahy E, Dennis EA (2010) Lipidomics reveals a remarkable diversity of lipids in human plasma. J Lipid Res 51:3299–3305CrossRefGoogle Scholar
  22. 22.
    Zhao Z, Xu Y (2010) An extremely simple method for extraction of lysophospholipids and phospholipids from blood samples. J Lipid Res 51:652–659CrossRefGoogle Scholar
  23. 23.
    Thompson M, Ellison SLR, Wood R (2002) Harmonized guidelines for single laboratory validation of methods of analysis. (IUPAC Technical Report). Pure Appl Chem 74:835–855CrossRefGoogle Scholar
  24. 24.
    Bird SS, Marur VR, Sniatynski MJ, Greenberg HK, Kristal BS (2011) Serum lipidomics profiling using LC-MS and high-energy collisional dissociation fragmentation: focus on triglyceride detection and characterization. Anal Chem 83:6648–6657CrossRefGoogle Scholar
  25. 25.
    Sud M, Fahy E, Cotter D, Brown A, Dennis EA, Glass CK, Merrill AH, Murphy RC, Raetz CR, Russell DW, Subramaniam S (2007) LMSD: LIPID MAPS structure database. Nucleic Acids Res 35:D527–D532CrossRefGoogle Scholar
  26. 26.
    Taguchi R, Houjou T, Nakanishi H, Yamazaki T, Ishida M, Imagawa M, Shimizu T (2005) Focused lipidomics by tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 823:26–36CrossRefGoogle Scholar
  27. 27.
    Camera E, Ludovici M, Galante M, Sinagra J, Picardo M (2010) Comprehensive analysis of the major lipid classes in sebum by rapid resolution high-performance liquid chromatography and electrospray mass spectrometry. J Lipid Res 51:3377–3388CrossRefGoogle Scholar
  28. 28.
    Cruciani G, Crivori P, Carrupt PA, Testa B (2000) Molecular fields in quantitative structure-permeation relationships: the VolSurf approach. J Mol Struct THEOCHEM 503:17–30CrossRefGoogle Scholar
  29. 29.
    Milne S, Ivanova P, Forrester J, Brown HA (2006) Lipidomics: an analysis of cellular lipids by ESI-MS. Methods 39:92–103CrossRefGoogle Scholar
  30. 30.
    Liebisch G, Lieser B, Rathenberg J, Drobnik W, Schmitz G (2004) High-throughput quantification of phosphatidylcholine and sphingomyelin by electrospray ionization tandem mass spectrometry coupled with isotope correction algorithm. Biochim Biophys Acta 1686:108–117CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Roberto Maria Pellegrino
    • 1
  • Alessandra Di Veroli
    • 1
  • Aurora Valeri
    • 1
  • Laura Goracci
    • 1
  • Gabriele Cruciani
    • 1
    Email author
  1. 1.Department of Chemistry, Biology, and BiotechnologiesUniversity of PerugiaPerugiaItaly

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