, Volume 78, Issue 5–6, pp 415–428 | Cite as

Development of an Online-SPE–LC–MS/MS Method for 26 Hydroxylated Polyunsaturated Fatty Acids as Rapid Targeted Metabolomics Approach for the LOX, CYP, and Autoxidation Pathways of the Arachidonic Acid Cascade

  • A. I. Ostermann
  • I. Willenberg
  • K. H. Weylandt
  • N. H. SchebbEmail author
Part of the following topical collections:
  1. Recent Developments in Clinical Omics


Hydroxylated fatty acids (OH-FAs) are formed in all branches of the arachidonic acid (AA) cascade from polyunsaturated fatty acids (PUFA). OH-FAs act as potent lipid mediators and serve as activity marker for pathways of the AA cascade, particularly the lipoxygenase branch. Current targeted metabolomics methods of the AA cascade cover several OH-FAs among other oxylipins, yet they require long runtimes and laborious sample preparation. In the present study, we developed a new rapid LC–MS method with automated sample preparation for the simultaneous quantification of 26 OH-FAs within 6.5 min. Crude biological samples are directly injected following addition of four isotopically labeled internal standards and centrifugation. The analytes are extracted from the matrix by means of online solid phase extraction on an Oasis HLB column at 3.5 mL min−1 flow rate. LC separation was carried out on a RP-18 column with fused core 1.3 µm particles. The method showed a high sensitivity with a limit of detection of 0.5–10 fmol on column and a broad linear range. Intra- and inter-batch precision and accuracy for the analytes were characterized for cell culture medium as well as human plasma and were found to be generally within 100 ± 15 %. The method was applied to the investigation of OH-FA formation in five cell lines following incubation with AA, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). The colon cancer cell lines HCA-7 and SW-480, as well as the fibroblast line Balb/c 3T3 showed significant formation of OH-FAs in the cell culture medium, with dominant formation of 15-HETE, 18-HEPE, 20-HDHA, and 8-HDHA from the precursor PUFAs.


LC–ESI/MS Polyunsaturated fatty acids Oxylipins Hydroxy-PUFA Resolvin precursor 



Arachidonic acid




Alpha-linolenic acid


Collision activated dissociation


Collision energy




Collision cell exit potential


Cytochrome P450 monooxygenase


Docosahexaenoic acid


Declustering potential


Docosapentaenoic acid


Dulbecco’s modified Eagle’s medium


Eicosapentaenoic acid


Electrospray ionization


Fetal calf serum


Full width at half maximal height


Acetic acid


Hydroxydocosahexaenoic acid, hydroxy-DHA


5-Hydroxyeicosanoid dehydrogenase


Hydroxyeicosapentaenoic acid, hydroxy-EPA


Hydroxyeicosatetraenoic acid, hydroxy-AA


Hydroperoxyeicosatetraenoic acid, hydroperoxy-AA


Hydroxyoctadecadienoic acid, hydroxy-LA


Hydroxyoctadecatrienoic acid, hydroxy-ALA


Linoleic acid


Liquid chromatography mass spectrometry


Lower limit of quantification


Limit of detection






Hydroxy fatty acid


Polyunsaturated fatty acid


Solid phase extraction



This study was supported by a Kekulé Ph.D. fellowship of the Fonds der Chemischen Industrie to IW, and a grant of the German Research Foundation (DFG) (WE 2908) to KWH as well as a Marie Curie Career Integration Grant (CIG 293536) of the European Union and a Grant of the German Research Foundation (SCHE 1801) to NHS.

Supplementary material

10337_2014_2768_MOESM1_ESM.pdf (58 kb)
Supplementary material 1 (PDF 58 kb)


  1. 1.
    Buczynski MW, Dumlao DS, Dennis EA (2009) J Lipid Res 50:1015–1038CrossRefGoogle Scholar
  2. 2.
    Fischer R, Konkel A, Mehling H, Blossey K, Gapelyuk A, Wessel N, von Schacky C, Dechend R, Muller DN, Rothe M, Luft FC, Weylandt K, Schunck WH (2014) J Lipid Res 55:1150–1164CrossRefGoogle Scholar
  3. 3.
    Mulugeta S, Suzuki T, Hernandez NT, Griesser M, Boeglin WE, Schneider C (2010) J Lipid Res 51:575–585CrossRefGoogle Scholar
  4. 4.
    Serhan CN (2014) Nature 510:92–101CrossRefGoogle Scholar
  5. 5.
    Weylandt KH, Chiu CY, Gomolka B, Waechter SF, Wiedenmann B (2012) Prostagladins Other Lipid Mediat 97:73–82CrossRefGoogle Scholar
  6. 6.
    Schuchardt JP, Schmidt S, Kressel G, Dong H, Willenberg I, Hammock BD, Hahn A, Schebb NH (2013) Prostagladins Leukot Essent Fat Acids 89:19–29CrossRefGoogle Scholar
  7. 7.
    Dangi B, Obeng M, Nauroth JM, Teymourlouei M, Needham M, Raman K, Arterburn LM (2009) J Biol Chem 284:14744–14759CrossRefGoogle Scholar
  8. 8.
    Chiu CY, Gomolka B, Dierkes C, Huang NR, Schroeder M, Purschke M, Manstein D, Dangi B, Weylandt KH (2012) Inflamm Res 61:967–976CrossRefGoogle Scholar
  9. 9.
    Porter NA, Lehman LS, Weber BA, Smith KJ (1981) J Am Chem Soc 103:6447–6455CrossRefGoogle Scholar
  10. 10.
    Yin H, Brooks JD, Gao L, Porter NA, Morrow JD (2007) J Biol Chem 282:29890–29901CrossRefGoogle Scholar
  11. 11.
    Konkel A, Schunck WH (2011) Biochim Biophys Acta 1814:210–222CrossRefGoogle Scholar
  12. 12.
    Inceoglu B, Wagner K, Schebb NH, Morisseau C, Jinks SL, Ulu A, Hegedus C, Rose T, Brosnan R, Hammock BD (2011) Proc Natl Acad Sci USA 108:5093–5097CrossRefGoogle Scholar
  13. 13.
    Miyata N, Roman RJ (2005) J Smooth Muscle Res 41:175–193CrossRefGoogle Scholar
  14. 14.
    Bylund J, Kunz T, Valmsen K, Oliw EH (1998) J Pharmacol Exp Ther 284:51–60Google Scholar
  15. 15.
    Buckley CD, Gilroy DW, Serhan CN (2014) Immunity 40:315–327CrossRefGoogle Scholar
  16. 16.
    Bento AF, Claudino RF, Dutra RC, Marcon R, Calixto JB (2011) J Immunol 187:1957–1969CrossRefGoogle Scholar
  17. 17.
    Gonzalez-Periz A, Planaguma A, Gronert K, Miquel R, Lopez-Parra M, Titos E, Horrillo R, Ferre N, Deulofeu R, Arroyo V, Rodes J, Claria J (2006) Faseb J 20:2537–2539CrossRefGoogle Scholar
  18. 18.
    Lima-Garcia JF, Dutra RC, da Silva KABS, Motta EM, Campos MM, Calixto JB (2011) Br J Pharmacol 164:278–293CrossRefGoogle Scholar
  19. 19.
    Neuhofer A, Zeyda M, Mascher D, Itariu BK, Murano I, Leitner L, Hochbrugger EE, Fraisl P, Cinti S, Serhan CN, Stulnig TM (2013) Diabetes 62:1945–1956CrossRefGoogle Scholar
  20. 20.
    Wilson R, Lyall K (2002) Lipids 37:917–924CrossRefGoogle Scholar
  21. 21.
    Tsikas D, Zoerner AA (2014) J Chromatogr B 964:79–88CrossRefGoogle Scholar
  22. 22.
    Dumlao DS, Buczynski MW, Norris PC, Harkewicz R, Dennis EA (2011) Biochim Biophys Acta 1811:724–736CrossRefGoogle Scholar
  23. 23.
    Gomolka B, Siegert E, Blossey K, Schunck WH, Rothe M, Weylandt KH (2011) Prostagladins Other Lipid Mediat 94:81–87CrossRefGoogle Scholar
  24. 24.
    Inceoglu B, Wagner KM, Yang J, Bettaieb A, Schebb NH, Hwang SH, Morisseau C, Haj FG, Hammock BD (2012) Proc Natl Acad Sci USA 109:11390–11395CrossRefGoogle Scholar
  25. 25.
    Yang J, Schmelzer K, Georgi K, Hammock BD (2009) Anal Chem 81:8085–8093CrossRefGoogle Scholar
  26. 26.
    Strassburg K, Huijbrechts AM, Kortekaas KA, Lindeman JH, Pedersen TL, Dane A, Berger R, Brenkman A, Hankemeier T, van Duynhoven J, Kalkhoven E, Newman JW, Vreeken RJ (2012) Anal Bioanal Chem 404:1413–1426CrossRefGoogle Scholar
  27. 27.
    Willenberg I, Elsner L, Steinberg P, Schebb NH (2015) Food Chem 166:537–543CrossRefGoogle Scholar
  28. 28.
    Schebb NH, Huby M, Morisseau C, Hwang SH, Hammock BD (2011) Anal Bioanal Chem 400:1359–1366CrossRefGoogle Scholar
  29. 29.
    Schebb NH, Flores I, Kurobe T, Franze B, Ranganathan A, Hammock BD, Teh SJ (2011) Aquat Toxicol 105:448–454CrossRefGoogle Scholar
  30. 30.
    Schebb NH, Inceoglu B, Rose T, Wagner K, Hammock BD (2011) Anal Methods 3:420–428CrossRefGoogle Scholar
  31. 31.
    Hsieh Y, Duncan CJG, Brisson JM (2007) Anal Chem 79:5668–5673CrossRefGoogle Scholar
  32. 32.
    Bollinger JG, Thompson W, Lai Y, Oslund RC, Hallstrand TS, Sadilek M, Turecek F, Gelb MH (2010) Anal Chem 82:6790–6796CrossRefGoogle Scholar
  33. 33.
    Masoodi M, Mir AA, Petasis NA, Serhan CN, Nicolaou A (2008) Rapid Commun Mass Spectrom 22:75–83CrossRefGoogle Scholar
  34. 34.
    Miller TM, Donnelly MK, Crago EA, Roman DM, Sherwood PR, Horowitz MB, Poloyac SM (2009) J Chromatogr B 877:3991–4000CrossRefGoogle Scholar
  35. 35.
    Harkewicz R, Fahy E, Andreyev A, Dennis EA (2007) J Biol Chem 282:2899–2910CrossRefGoogle Scholar
  36. 36.
    Masoodi M, Eiden M, Koulman A, Spaner D, Volmer DA (2010) Anal Chem 82:8176–8185CrossRefGoogle Scholar
  37. 37.
    Schebb NH, Ostermann AI, Yang J, Hammock BD, Hahn A, Schuchardt JP (2014) Prostagladins Other Lipid Mediat. doi: 10.1016/j.prostaglandins.2014.05.002 Google Scholar
  38. 38.
    Norris PC, Dennis EA (2012) Proc Natl Acad Sci USA 109:8517–8522CrossRefGoogle Scholar
  39. 39.
    Norris PC, Reichart D, Dumlao DS, Glass CK, Dennis EA (2011) J Leukoc Biol 90:563–574CrossRefGoogle Scholar
  40. 40.
    Glasgow WC, Eling TE (1990) Mol Pharmacol 38:503–510Google Scholar
  41. 41.
    Little JM, Kurkela M, Sonka J, Jantti S, Ketola R, Bratton S, Finel M, Radominska-Pandya A (2004) J Lipid Res 45:1694–1703CrossRefGoogle Scholar
  42. 42.
    Turgeon D, Chouinard S, Belanger P, Picard S, Labbe JF, Borgeat P, Belanger A (2003) J Lipid Res 44:1182–1191CrossRefGoogle Scholar
  43. 43.
    Grant GE, Rokach J, Powell WS (2009) Prostagladins Other Lipid Mediat 89:98–104CrossRefGoogle Scholar
  44. 44.
    Green FA (1990) Lipids 25:618–623CrossRefGoogle Scholar
  45. 45.
    Powell WS, Gravel S, Gravelle F (1995) J Lipid Res 36:2590–2598Google Scholar
  46. 46.
    Sharma RA, Gescher A, Plastaras JP, Leuratti C, Singh R, Gallacher-Horley B, Offord E, Marnett LJ, Steward WP, Plummer SM (2001) Carcinogenesis 22:1557–1560CrossRefGoogle Scholar
  47. 47.
    Sheng H, Shao J, Kirkland SC, Isakson P, Coffey RJ, Morrow J, Beauchamp RD, DuBois RN (1997) J Clin Invest 99:2254–2259CrossRefGoogle Scholar
  48. 48.
    Yoshinaga M, Buchanan FG, DuBois RN (2004) Prostagladins Other Lipid Mediat 73:111–122CrossRefGoogle Scholar
  49. 49.
    Bednar W, Holzmann K, Marian B (2007) Food Chem Toxicol 45:508–514CrossRefGoogle Scholar
  50. 50.
    Melstrom LG, Bentrem DJ, Salabat MR, Kennedy TJ, Ding XZ, Strouch M, Rao SM, Witt RC, Ternent CA, Talamonti MS, Bell RH, Adrian TA (2008) Clin Cancer Res 14:6525–6530CrossRefGoogle Scholar
  51. 51.
    Tavolari S, Bonafe M, Marini M, Ferreri C, Bartolini G, Brighenti E, Manara S, Tomasi V, Laufer S, Guarnieri T (2008) Carcinogenesis 29:371–380CrossRefGoogle Scholar
  52. 52.
    Lepage C, Liagre B, Cook-Moreau J, Pinon A, Beneytout JL (2010) Int J Oncol 36:1183–1191Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • A. I. Ostermann
    • 1
  • I. Willenberg
    • 1
  • K. H. Weylandt
    • 2
    • 3
  • N. H. Schebb
    • 1
    • 4
    Email author
  1. 1.Institute for Food Toxicology and Analytical ChemistryUniversity of Veterinary Medicine HannoverHannoverGermany
  2. 2.Department of Hepatology, Gastroenterology and Metabolism, Rudolf-Virchow-HospitalCharité University MedicineBerlinGermany
  3. 3.Lipid Clinic, Experimental and Clinical Research Center (ECRC), Max-Delbrück-Center for Molecular MedicineCharité University MedicineBerlinGermany
  4. 4.Institute of Food Chemistry, University of WuppertalWuppertalGermany

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