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

Nitrated unsaturated fatty acids including nitro-oleic acid (NO2-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 NO2-OA and 300 nM for esterified NO2-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 NO2-OA isomers, i.e., 9-NO2-OA and 10-NO2-OA, in human plasma using newly prepared, isolated, characterized and standardized 15N-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 ([NO2]−) and m/z 47 ([15NO2]−) produced by collision-induced dissociation (CID) from the parent ions [M−PFB]− at m/z 326 for endogenous 9-NO2-OA and 10-NO2-OA and m/z 327 for the internal standards 9-15NO2-OA and 10-15NO2-OA. We partially validated the GC–MS/MS method for 9-NO2-OA and 10-NO2-OA in human plasma and quantified these nitro-oleic species in plasma of 15 healthy volunteers. We identified two isomers, i.e., 9-NO2-OA and 10-NO2-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.