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Updated: Jun 26, 2026
Tracing de novo Lipids using Stable Isotope Labeling LC-TIMS-TOF MS/MS
Published on: August 23, 2024
Specific GC-MS/MS stable-isotope dilution methodology for free 9- and 10-nitro-oleic acid in human plasma challenges
Dimitrios Tsikas1, Alexander Zoerner, Anja Mitschke
1Institute of Clinical Pharmacology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30623 Hannover, Germany. tsikas.dimitros@mh-hannover.de
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.
