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Oxidized fatty acid analysis by charge-switch derivatization, selected reaction monitoring, and accurate mass quantitation
- Liu, Xinping, Moon, Sung Ho, Mancuso, David J., Jenkins, Christopher M., Guan, Shaoping, Sims, Harold F., Gross, Richard W.
- Analytical biochemistry 2013 v.442 no.1 pp. 40-50
- arachidonic acid, correlation, derivatization, docosahexaenoic acid, ionization, ions, linoleic acid, liver, metabolites, mice, monitoring, stable isotopes, tandem mass spectrometry
- A highly sensitive, specific, and robust method for the analysis of oxidized metabolites of linoleic acid (LA), arachidonic acid (AA), and docosahexaenoic acid (DHA) was developed using charge-switch derivatization, liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI MS/MS) with selected reaction monitoring (SRM) and quantitation by high mass accuracy analysis of product ions, thereby minimizing interferences from contaminating ions. Charge-switch derivatization of LA, AA, and DHA metabolites with N-(4-aminomethylphenyl)-pyridinium resulted in a 10- to 30-fold increase in ionization efficiency. Improved quantitation was accompanied by decreased false positive interferences through accurate mass measurements of diagnostic product ions during SRM transitions by ratiometric comparisons with stable isotope internal standards. The limits of quantitation were between 0.05 and 6.0pg, with a dynamic range of 3 to 4 orders of magnitude (correlation coefficient r²>0.99). This approach was used to quantitate the levels of representative fatty acid metabolites from wild-type (WT) and iPLA₂γ–/– mouse liver identifying the role of iPLA₂γ in hepatic lipid second messenger production. Collectively, these results demonstrate the utility of high mass accuracy product ion analysis in conjunction with charge-switch derivatization for the highly specific quantitation of diminutive amounts of LA, AA, and DHA metabolites in biologic systems.