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Modified HPLC method for detection of hydroxyoctadecadienoic acid with greater sensitivity
Author(s) -
Chesmore Nathan J.,
Zhang Wenjing,
Richards Mark P.,
Shanmuganayagam Dhanansayan
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.540.9
Subject(s) - fluorophore , chemistry , conjugate , chromatography , fatty acid , high performance liquid chromatography , biochemistry , arachidonic acid , linoleic acid , fluorescence , enzyme , mathematical analysis , physics , mathematics , quantum mechanics
Hydroxyoctadecadienoic acid (HODE) is an oxidized fatty acid derivative of linoleic acid that is also a product of lipoxygenase‐mediated reactions. It has been implicated in the development of cardiovascular disease, differentiation of adipocytes, and several pro‐ and anti‐inflammatory mechanisms. HPLC, GC/MS, and LC/MS methods have been developed for the assay of HODE(s) from simple assay schemes to lipidomic approaches in fatty acid signaling, metabolism, and regulation research. Current HPLC methods used to quantify unmodified HODE(s) in fatty acid matrixes lack the required sensitivity necessary to detect levels of HODE found in tissue and serum. Sensitizing agents that bind HODE(s) or all sample fatty acids may be used to increase the signal of analytes by post‐column detectors. Here we describe the use of 2‐(2,3‐Naphthalimino)ethyl trifluoromethanesulfonate (NT) as a sensitizing fluorophore for the detection of 13(S)‐HODE in fatty acid matrixes using a c18 column for separation. NT fluorophore detection increases the sensitivity by more than 15‐fold compared to unmodified HODE conjugated diene absorbance at 234nm without modifications for fluorophore‐HODE conjugate stability. Pre‐column labeling of HODE with NT fluorophore in fatty acid matrixes also markedly increases the c18 column separation efficiency of the HODE‐fluorophore conjugate, reducing the amount of solvent and analysis time needed if shorter columns are used. Support or Funding Information The research was supported by the Biomedical & Genomic Research Group Discretionary Fund (University of Wisconsin‐Madison) and by USDA Formula Project Grant (WIS01972) National Institute of Food and Agriculture, USDA). This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .