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Analysis of Triacylglycerols and Free Fatty Acids in Algae Using Ultra-Performance Liquid Chromatography Mass Spectrometry

  • Original Paper
  • Published:
Journal of the American Oil Chemists' Society

Abstract

To assess the suitability of microalgal strains for biodiesel production the lipid content and composition, especially individual triacylglycerols (TAG) and free fatty acids (FFA) must be determined. In this study, the compositions and concentrations of TAG and FFA were analysed in four halophytic algal species, Dunaliella salina, D. tertiolecta, D. bardawil, and D. granulata. These species were selected as part of a larger screen to identify species suitable for biofuel feedstocks. An accelerated solvent extraction instrument was used for lipids and fatty acid extraction using a dichloromethane–hexane solvent system. Ultra-performance liquid chromatography coupled with mass spectrometry (MS) detection was optimized and applied to the quantitative analysis of TAG and FFA in the different algal extracts. Individual TAG were characterized structurally using direct electrospray ionization (ESI) MS and MS/MS techniques. Cationic adducts (NH4 +) of TAG were detected and quantified in the positive ESI MS and MS/MS modes, while the negative ESI mode was used for FFA analysis. Over 20 TAG were identified and quantified in the four Dunaliella strains. Analysis of FFA compositions demonstrated that the most abundant FFA in these four algal species were palmitic, linolenic, linoleic, and oleic acids.

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Abbreviations

ASE:

Accelerated solvent extraction

ECN:

Equivalent carbon number

ESI:

Electrospray ionization

FFA:

Free fatty acid(s)

GC/MS:

Gas chromatography mass spectrometry

HPLC:

High performance liquid chromatography

LC/MS:

Liquid chromatography mass spectrometry

TAG:

Triacylglycerol(s)

UPLC:

Ultra-performance liquid chromatography

References

  1. Hu Q, Sommerfeld M, Jarvis E, Ghirardi M, Posewitz M, Seibert M, Darzins A (2008) Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. Plant J 54(4):621–639

    Article  CAS  Google Scholar 

  2. Beer LL, Boyd ES, Peters JW, Posewitz MC (2009) Engineering algae for biohydrogen and biofuel production. Curr Opin Biotechnol 20(3):264–271

    Article  CAS  Google Scholar 

  3. Chisti Y (2007) Biodiesel from microalgae. Biotechnol Adv 25(3):294–306

    Article  CAS  Google Scholar 

  4. Chisti Y (2008) Biodiesel from microalgae beats bioethanol. Trends Biotechnol 26(3):126–131

    Article  CAS  Google Scholar 

  5. Waltz E (2009) Biotechʼs green gold? Nat Biotechnol 27(1):15–18

    Article  CAS  Google Scholar 

  6. Durrett TP, Benning C, Ohlrogge J (2008) Plant triacylglycerols as feedstocks for the production of biofuels. Plant J 54(4):593–607

    Article  CAS  Google Scholar 

  7. Benning C, Pichersky E (2008) Harnessing plant biomass for biofuels and biomaterials. Plant J 54(4):533–535

    Article  CAS  Google Scholar 

  8. McGinnis KM, Dempster TA, Sommerfeld MR (1997) Characterization of the growth and lipid content of the diatom Chaetoceros muelleri. J Appl Phycol 9(1):19–24

    Article  CAS  Google Scholar 

  9. Lee SJ, Yoon BD, Oh HM (1998) Rapid method for the determination of lipid from the green alga Botryococcus braunii. Biotechnol Technol 12(7):553–556

    Article  CAS  Google Scholar 

  10. Diaz G, Melis M, Batetta B, Angius F, Falchi AM (2008) Hydrophobic characterization of intracellular lipids in situ by Nile Red red/yellow emission ratio. Micron 39(7):819–824

    Article  CAS  Google Scholar 

  11. Chen W, Zhang CW, Song LR, Sommerfeld M, Hu Q (2009) A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae. J Microbiol Methods 77(1):41–47

    Article  CAS  Google Scholar 

  12. Greenspan P, Mayer EP, Fowler SD (1985) Nile red—a selective fluorescent stain for intracellular lipid droplets. J Cell Biol 100(3):965–973

    Article  CAS  Google Scholar 

  13. Cooksey KE, Guckert JB, Williams SA, Callis PR (1987) Fluorometric-determination of the neutral lipid-content of microalgal cells using Nile Red. J Microbiol Methods 6(6):333–345

    Article  CAS  Google Scholar 

  14. Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917

    Article  CAS  Google Scholar 

  15. Inouye LS, Lotufo GR (2006) Comparison of macro-gravimetric and micro-colorimetric lipid determination methods. Talanta 70(3):584–587

    Article  CAS  Google Scholar 

  16. Brondz I (2002) Development of fatty acid analysis by high-performance liquid chromatography, gas chromatography, and related techniques. Anal Chim Acta 465(1–2):1–37

    Article  CAS  Google Scholar 

  17. Quinto Tranchida P, Donato P, Dugo P, Dugo G, Mondello L (2007) Comprehensive chromatographic methods for the analysis of lipids. TrAC, Trends Anal Chem 26(3):191–205

    Article  Google Scholar 

  18. Basconcillo LS, McCarry BE (2008) Comparison of three GC/MS methodologies for the analysis of fatty acids in Sinorhizobium meliloti: development of a micro-scale, one-vial method. J Chromatogr B 871(1):22–31

    Article  CAS  Google Scholar 

  19. Vosough M, Salemi A (2007) Second-order standard addition for deconvolution and quantification of fatty acids of fish oil using GC-MS. Talanta 73(1):30–36

    Article  CAS  Google Scholar 

  20. Wang G, Wang T (2012) Characterization of lipid components in two microalgae for biofuel application. J Am Oil Chem Soc 89(1):135–143

    Article  CAS  Google Scholar 

  21. Guedes AC, Meireles LA, Amaro HM, Malcata FX (2010) Changes in lipid class and fatty acid composition of cultures of Pavlova lutheri, in response to light intensity. J Am Oil Chem Soc 87(7):791–801

    Article  CAS  Google Scholar 

  22. Harrabi S, Herchi W, Kallel H, Mayer PM, Boukhchina S (2009) Liquid chromatographic-mass spectrometric analysis of glycerophospholipids in corn oil. Food Chem 114(2):712–716

    Article  CAS  Google Scholar 

  23. Sommer U, Herscovitz H, Welty FK, Costello CE (2006) LC-MS-based method for the qualitative and quantitative analysis of complex lipid mixtures. J Lipid Res 47(4):804–814

    Article  CAS  Google Scholar 

  24. Malone M, Evans JJ (2004) Determining the relative amounts of positional isomers in complex mixtures of triglycerides using reversed-phase high-performance liquid chromatography-tandem mass spectrometry. Lipids 39(3):273–284

    Article  CAS  Google Scholar 

  25. Dorschel CA (2002) Characterization of the TAG of peanut oil by electrospray LC-MS-MS. J Am Oil Chem Soc 79(8):749–753

    Article  CAS  Google Scholar 

  26. Byrdwell WC (2005) The bottom-up solution to the triacylglycerol lipidome using atmospheric pressure chemical ionization mass spectrometry. Lipids 40(4):383–417

    Article  CAS  Google Scholar 

  27. Sandra P, Medvedovici A, Zhao Y, David F (2002) Characterization of triglycerides in vegetable oils by silver-ion packed-column supercritical fluid chromatography coupled to mass spectroscopy with atmospheric pressure chemical ionization and coordination ion spray. J Chromatogr A 974(1–2):231–241

    CAS  Google Scholar 

  28. Leskinen HM, Suomela JP, Kallio HP (2010) Quantification of triacylglycerol regioisomers by ultra-high-performance liquid chromatography and ammonia negative ion atmospheric pressure chemical ionization tandem mass spectrometry. Rapid Commun Mass Spectrom 24(1):1–5

    Article  CAS  Google Scholar 

  29. Cai SS, Short LC, Syage JA, Potvin M, Curtis JM (2007) Liquid chromatography-atmospheric pressure photoionization-mass spectrometry analysis of triacylglycerol lipids—effects of mobile phases on sensitivity. J Chromatogr A 1173(1–2):88–97

    CAS  Google Scholar 

  30. Zehethofer N, Pinto DM (2008) Recent developments in tandem mass spectrometry for lipidomic analysis. Anal Chim Acta 627(1):62–70

    Article  CAS  Google Scholar 

  31. Zehethofer N, Pinto DM, Volmer DA (2008) Plasma free fatty acid profiling in a fish oil human intervention study using ultra-performance liquid chromatography/electrospray ionization tandem mass spectrometry. Rapid Commun Mass Spectrom 22(13):2125–2133

    Article  CAS  Google Scholar 

  32. Jones J, Manning S, Montoya M, Keller K, Poenie M (2012) Extraction of algal lipids and their analysis by HPLC and mass spectrometry. J Am Oil Chem Soc 89(8):1371–1381

    Google Scholar 

  33. Kind T, Meissen JK, Yang DW, Nocito F, Vaniya A, Cheng YS, VanderGheynst JS, Fiehn O (2012) Qualitative analysis of algal secretions with multiple mass spectrometric platforms. J Chromatogr A 1244:139–147

    Article  CAS  Google Scholar 

  34. MacDougall KM, McNichol J, McGinn PJ, O’Leary SJB, Melanson JE (2011) Triacylglycerol profiling of microalgae strains for biofuel feedstock by liquid chromatography-high-resolution mass spectrometry. Anal Bioanal Chem 401(8):2609–2616

    Article  CAS  Google Scholar 

  35. Cheng CF, Gross ML (1998) Complete structural elucidation of triacylglycerols by tandem sector mass spectrometry. Anal Chem 70(20):4417–4426

    Article  CAS  Google Scholar 

  36. Marzilli LA, Fay LB, Dionisi F, Vouros P (2003) Structural characterization of triacylglycerols using electrospray ionization-MSn ion-trap MS. J Am Oil Chem Soc 80(3):195–202

    Article  CAS  Google Scholar 

  37. Mekhedov S, de Ilarduya OM, Ohlrogge J (2000) Toward a functional catalog of the plant genome. A survey of genes for lipid biosynthesis. Plant Physiol 122(2):389–401

    Article  CAS  Google Scholar 

  38. Duffin KL, Henion JD, Shieh JJ (1991) Electrospray and tandem mass-spectrometric characterization of acylglycerol mixtures that are dissolved in nonpolar-solvents. Anal Chem 63(17):1781–1788

    Article  CAS  Google Scholar 

  39. Chu XP, Zhao T, Zhang YY, Zhao AH, Zhou MM, Zheng XJ, Dan M, Jia W (2009) Determination of 13 free fatty acids in Pheretima using ultra-performance LC-ESI-MS. Chromatographia 69(7–8):645–652

    Article  CAS  Google Scholar 

  40. Rogatsky E, Stein D (2005) Evaluation of matrix effect and chromatography efficiency: new parameters for validation of method development. J Am Soc Mass Spectrom 16(11):1757–1759

    Article  CAS  Google Scholar 

  41. Belmans D, Vanlaere A (1988) Effect of ionophores on the ATP-pool and glycerol content in cells of the halotolerant green-alga Dunaliella-tertiolecta. J Gen Microbiol 134:2261–2268

    CAS  Google Scholar 

  42. Gouveia L, Oliveira AC (2009) Microalgae as a raw material for biofuels production. J Ind Microbiol Biotechnol 36(2):269–274

    Article  CAS  Google Scholar 

  43. Ben-Amotz A, Polle JEW, Rao DVS (2009) The alga Dunaliella: biodiversity, physiology, genomics and biotechnology. Science Publishers, Enfield

    Google Scholar 

  44. Avron M, Ben-Amotz A (1980) Production of glycerol, catoenes and algae meal. US Patent 4,199,895

  45. Cooney M, Young G, Nagle N (2009) Extraction of bio-oils from microalgae. Sep Purif Rev 38(4):291–325

    Article  CAS  Google Scholar 

  46. Denery JR, Dragull K, Tang CS, Li QX (2004) Pressurized fluid extraction of carotenoids from Haematococcus pluvialis and Dunaliella salina and kavalactones from Piper methysticum. Anal Chim Acta 501(2):175–181

    Article  CAS  Google Scholar 

  47. Herrero M, Martin-Alvarez PJ, Senorans FJ, Cifuentes A, Ibanez E (2005) Optimization of accelerated solvent extraction of antioxidants from Spirulina platensis microalga. Food Chem 93(3):417–423

    Article  CAS  Google Scholar 

  48. Wolk CP (1973) Physiology and cytological chemistry of blue-green algae. Bacteriol Rev 37(1):32–101

    CAS  Google Scholar 

  49. Otles S, Pire R (2001) Fatty acid composition of Chlorella and Spirulina microalgae species. J AOAC Int 84(6):1708–1714

    CAS  Google Scholar 

  50. Volkman JK, Jeffrey SW, Nichols PD, Rogers GI, Garland CD (1989) Fatty-acid and lipid-composition of 10 species of microalgae used in mariculture. J Exp Mar Biol Ecol 128(3):219–240

    Article  CAS  Google Scholar 

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Acknowledgments

Funding support from the U.S. Department of Energy under award DE-EE0008272, SunGrant Initiative (U.S. Department of Transportation), and University of Nevada Agricultural Experiment Station (NEV-00342) to JCC is gratefully acknowledged. The authors thank Rebecca Albion for invaluable technical support. We would also like to thank Mary Ann Cushman for her critical reading of the manuscript. This publication was also made possible by NIH Grant Number P20 RR-016464 from the INBRE Program of the National Center for Research Resources through its support of the Nevada Genomics, Proteomics and Bioinformatics Centers.

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Authors and Affiliations

  1. Division of Atmospheric Sciences, Desert Research Institute, Reno, NV, 89512-1095, USA

    Vera Samburova, S. Kent Hoekman & Barbara Zielinska

  2. Department of Chemical Engineering and Materials Science, University of California, Davis, CA, 95616-5294, USA

    Mark S. Lemos

  3. Department of Biochemistry and Molecular Biology, University of Nevada, Reno, NV, 89557-0330, USA

    Sage Hiibel & John C. Cushman

Authors
  1. Vera Samburova
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  2. Mark S. Lemos
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  3. Sage Hiibel
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  4. S. Kent Hoekman
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  5. John C. Cushman
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  6. Barbara Zielinska
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Correspondence to Vera Samburova.

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Samburova, V., Lemos, M.S., Hiibel, S. et al. Analysis of Triacylglycerols and Free Fatty Acids in Algae Using Ultra-Performance Liquid Chromatography Mass Spectrometry. J Am Oil Chem Soc 90, 53–64 (2013). https://doi.org/10.1007/s11746-012-2138-3

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  • DOI: https://doi.org/10.1007/s11746-012-2138-3

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