Analysis of lipoxygenase‐derived fatty acid hydroperoxides by electrospray ionization tandem mass spectrometry

Abstract

A rapid method is described for the identification of fatty acid hydroperoxides by electrospray ionization-tandem mass spectrometry coupled to liquid chromatography without any derivatization required prior to analysis. Localization of fatty acid hydroperoxides in complex mixtures was achieved by monitoring the loss of hydrogen peroxide using constant neutral loss scanning. In the presence of 5 mM NH4OAc in methanol-water, adduct ions [M + NH4]+ were formed almost exclusively, directly revealing the molecular mass of the thermolabile hydroperoxides. In addition, low energy collision-induced dissociation of precursor ions [M + NH4]+ led to characteristic product ions from both the 9- and 13-regioisomers. Thus, electrospray ionization-tandem mass spectrometry provides a straightforward approach for the study of the regioselectivity of lipoxygenase catalysis without any derivatization step required prior to analysis.

LOXPsa1, the first recombinant lipoxygenase from a basidiomycete fungus

“…Regiospecific analysis of linoleic hydroperoxides was carried out according to [32] using a Nucleodur C18 Pyramid column (250 mm × 4 mm, 5 m; Macherey-Nagel, Düren, Germany). Eluent A consisted of (v/v) THF/MeOH/water/acetic acid (25:30:44.9:0.1) and B was MeOH/water (9:1).…”

A Dual Function α-Dioxygenase-Peroxidase and NAD+Oxidoreductase Active Enzyme from Germinating Pea Rationalizing α-Oxidation of Fatty Acids in Plants,

“…The peaks are: 1, 2-hydroperoxypalmitic acid; 2, 2-hydroxypalmitic acid; 3, 1-pentadecanal and pentadecanoic acid; and 4, unconverted palmitic acid. (Schneider et al, 1997).…”

“…Injection volume was 1 L both for reference compounds and biological samples. LC-ESI-MS/MS analyses were performed according to our previously described conditions (Schneider et al, 1997) using a triple-quadrupole TSQ 7000 apparatus with electrospray interface (Finnigan-MAT, Bremen, Germany). For pneumatically assisted electrospray ionization the spray capillary voltage was set to 4 kV and the temperature of the heated inlet capillary serving simultaneously as repeller electrode (8.3 V) was 170°C.…”

Simultaneous determination by GC‐MS of epoxy and hydroxy FA as their methoxy derivatives

“…We therefore used hydroxy FA derived from linoleic acid as a surrogate measure of free radical oxidative damage (23,28). Hydroxy FA have been used as a surrogate measurement of hydroperoxy FA in biological systems in many studies (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). Hydrogenation is an essential step in many methods because it not only stabilizes the hydroperoxy group early in the analytical procedure (as a hydroxy group) but through elimination of double bonds also prevents further oxidation during sample workup.…”

“…The use of surrogate markers of lipid peroxidation (5) and their association with disease are now well documented (1). GC-MS provides unequivocal identification of metabolites and has become increasingly popular for the quantitative analysis of markers of lipid peroxidation in biological material (5)(6)(7)(8)(9)(10). Epoxy FA are also formed during normal metabolic processes in the body (11)(12)(13) and, like hydroperoxy FA or their hydroxy derivatives, epoxy FA exhibit biological and toxicological behavior (1)(2)(3)5,(11)(12)(13).…”