Nitro‐fatty Acids Occur in Human Plasma in the Picomolar Range: a Targeted Nitro‐lipidomics GC–MS/MS Study

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Abstract
First studies on the occurrence of nitrated fatty acids in plasma of healthy subjects revealed basal concentrations of 600 nM for free/nonesterified nitro‐oleic acid (NO2‐OA) as measured by liquid chromatography tandem mass spectrometry (LC–MS/MS). We recently showed by a gas chromatography tandem mass spectrometry (GC–MS/MS) method the physiological occurrence of two isomers, i.e., 9‐NO2‐OA and 10‐NO2‐OA, at mean basal plasma concentrations of 880 and 940 pM, respectively. In consideration of this large discrepancy we modified our originally reported method by replacing solid‐phase extraction (SPE) by solvent extraction with ethyl acetate and by omitting the high‐performance liquid chromatography (HPLC) step for a more direct detection and with the potential for lipidomics studies. Intra‐assay imprecision and accuracy of the modified method in human plasma were 1–34% and 91–221%, respectively, for added NO2‐OA concentrations in the range 0–3,000 pM. This method provided basal plasma concentrations of 306 ± 44 pM for 9‐NO2‐OA and 316 ± 33 pM for 10‐NO2‐OA in 15 healthy subjects. Nitro‐arachidonic acid and nitro‐linolenic acid were not detectable in the plasma samples. In summary, our studies show 9‐NO2‐OA and 10‐NO2‐OA as endogenous nitrated fatty acids in human plasma in the pM range; HPLC is recommendable as a sample clean‐up step for reliable quantification of nitro‐oleic acids by GC–MS/MS.

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