Chromatographia

, Volume 78, Issue 5, pp 415–428

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. Schebb
Original

DOI: 10.1007/s10337-014-2768-8

Cite this article as:
Ostermann, A.I., Willenberg, I., Weylandt, K.H. et al. Chromatographia (2015) 78: 415. doi:10.1007/s10337-014-2768-8
Part of the following topical collections:
  1. Recent Developments in Clinical Omics

Abstract

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.

Keywords

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

Abbreviations

AA

Arachidonic acid

ACN

Acetonitrile

ALA

Alpha-linolenic acid

CAD

Collision activated dissociation

CE

Collision energy

COX

Cyclooxygenase

CXP

Collision cell exit potential

CYP

Cytochrome P450 monooxygenase

DHA

Docosahexaenoic acid

DP

Declustering potential

DPA

Docosapentaenoic acid

DMEM

Dulbecco’s modified Eagle’s medium

EPA

Eicosapentaenoic acid

ESI

Electrospray ionization

FCS

Fetal calf serum

FWHM

Full width at half maximal height

HAc

Acetic acid

HDHA

Hydroxydocosahexaenoic acid, hydroxy-DHA

5-HEDH

5-Hydroxyeicosanoid dehydrogenase

HEPE

Hydroxyeicosapentaenoic acid, hydroxy-EPA

HETE

Hydroxyeicosatetraenoic acid, hydroxy-AA

HpETE

Hydroperoxyeicosatetraenoic acid, hydroperoxy-AA

HODE

Hydroxyoctadecadienoic acid, hydroxy-LA

HOTrE

Hydroxyoctadecatrienoic acid, hydroxy-ALA

LA

Linoleic acid

LC–MS

Liquid chromatography mass spectrometry

LLOQ

Lower limit of quantification

LOD

Limit of detection

LOX

Lipoxygenase

MeOH

Methanol

OH-FA

Hydroxy fatty acid

PUFA

Polyunsaturated fatty acid

SPE

Solid phase extraction

Supplementary material

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

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
  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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