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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


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.

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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


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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.

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Correspondence to N. H. Schebb.

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Published in the topical collection Recent Developments in Clinical Omics with guest editors Martin Giera and Manfred Wuhrer.

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Ostermann, A.I., Willenberg, I., Weylandt, K.H. et al. 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. Chromatographia 78, 415–428 (2015).

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