title: Specific GC-MS/MS stable-isotope dilution methodology for free 9- and 10-nitro-oleic acid in human plasma challenges previous LC-MS/MS reports.
abstract: Nitrated unsaturated fatty acids including nitro-oleic acid (NO(2)-OA) have been measured in human blood samples in their free and esterified forms. Plasma concentrations in healthy subjects have been reported to be of the order of 600 nM for free NO(2)-OA and 300 nM for esterified NO(2)-OA, as measured by LC-MS/MS. In the present article we report a GC-MS/MS method for the specific and accurate quantification of two NO(2)-OA isomers, i.e., 9-NO(2)-OA and 10-NO(2)-OA, in human plasma using newly prepared, isolated, characterized and standardized (15)N-labeled analogs. This method involves SPE extraction of fatty acids from slightly acidified plasma samples (pH 5), conversion to their pentafluorobenzyl (PFB) esters, isolation by HPLC, solvent extraction from a single HPLC fraction and GC-MS/MS analysis in the electron capture negative-ion chemical ionization (ECNICI) mode. Quantification was performed by selected-reaction monitoring (SRM) of m/z 46 ([NO(2)](-)) and m/z 47 ([(15)NO(2)](-)) produced by collision-induced dissociation (CID) from the parent ions [M-PFB](-) at m/z 326 for endogenous 9-NO(2)-OA and 10-NO(2)-OA and m/z 327 for the internal standards 9-(15)NO(2)-OA and 10-(15)NO(2)-OA. We partially validated the GC-MS/MS method for 9-NO(2)-OA and 10-NO(2)-OA in human plasma and quantified these nitro-oleic species in plasma of 15 healthy volunteers. We identified two isomers, i.e., 9-NO(2)-OA and 10-NO(2)-OA, in human plasma under physiological conditions and found these nitrated fatty acids at a mean concentration of 1 nM each. This concentration is about 600 times lower than that reported by others using LC-MS/MS. Our GC-MS/MS studies on nitro-oleic acid and 3-nitrotyrosine suggest that the extent of nitration of biomolecules such as unsaturated fatty acids and tyrosine is very low in health. In this article we discuss analytical and biological ramifications potentially associated with measurement of nitrated biomolecules in biological systems.
authors.affiliation: Institute of Clinical Pharmacology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30623 Hannover, Germany. tsikas.dimitros@mh-hannover.de
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Tsikas
authors.forename: Alexander
authors.initials: A
authors.lastname: Zoerner
authors.forename: Anja
authors.initials: A
authors.lastname: Mitschke
authors.forename: Yahya
authors.initials: Y
authors.lastname: Homsi
authors.forename: Frank-Mathias
authors.initials: FM
authors.lastname: Gutzki
authors.forename: Jens
authors.initials: J
authors.lastname: Jordan
mesh_terms: D000328:Adult; D002853:Chromatography, Liquid; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D008297:Male; D008875:Middle Aged; D009587:Nitrogen Isotopes / Q000737:chemistry*; D009829:Oleic Acids / Q000097:blood / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: C521487:10-nitro-oleic acid; D009587:Nitrogen Isotopes; D009829:Oleic Acids