| Summary: | Nitrated fatty acids (NO<sub>2</sub>-FAs) are a newly discovered class of biologically active compounds with distinct biochemical features that induce physiologically beneficial alterations in transcriptional regulatory protein function, leading to a variety of modulatory and protective actions. The most common NO<sub>2</sub>-FAs identified in vivo so far are nitro oleic acid (NO<sub>2</sub>-OA), nitro linoleic acid (NO<sub>2</sub>-LA) and its structural isomer nitro-conjugated linoleic acid (NO<sub>2</sub>-cLA). Analytical limitations that compromise accurate quantitation of these endogenous compounds are their low concentrations, compromised stability and different distribution profiles in tissues and biofluids. As a result, reliable analytical methods for the quantitative determination of their endogenous levels are rare. Only NO<sub>2</sub>-OA was quantified by GC-MS while LC-MS methods are still scarce. In this work, an LC-MS/MS bioanalytical method was developed and validated for the quantification of NO<sub>2</sub>-OA and NO<sub>2</sub>-LA in human plasma via a standard addition protocol after protein precipitation, liquid extraction and LC-MS/MS analysis in the negative ion mode. Quantification was performed via multiple reaction monitoring of the transitions <i>m/z</i> 326 > 46 and <i>m/z</i> 324 > 46 for NO<sub>2</sub>-OA and NO<sub>2</sub>-LA, respectively, and <i>m/z</i> 269 > 250 for the internal standard heptadecanoic acid. Linear responses were observed for both analytes over the studied range (R<sup>2</sup> = 0.9805 and 0.9644 for NO<sub>2</sub>-OA and NO<sub>2</sub>-LA, respectively). Sufficient accuracy and precision were also achieved at low, medium and high levels within the linearity range. The limits of quantification of our method (2 nM for both NO<sub>2</sub>-FAs) were below basal endogenous levels, thereby providing a good tool to accurately measure these NO<sub>2</sub>-FAs in plasma. We applied the validated method to compare NO<sub>2</sub>-OA and NO<sub>2</sub>-LA levels in the plasma of 28 ischemic heart disease (IHD) patients and 18 healthy controls. The levels of NO<sub>2</sub>-OA were found to be significantly higher in the plasma of patients (21.7 ± 9.8 nM) <i>versus</i> healthy controls (12.6 ± 6 nM) (<i>p</i>-value < 0.01). Whereas the levels of NO<sub>2</sub>-LA were comparable in both groups (3 ± 1 nM in patients, 3.2 ± 1.7 nM in controls, <i>p</i>-value = 0.87288). The early elevation of NO<sub>2</sub>-OA in plasma samples, which were collected 2–3 h post myocardial injury, implies the potential use of NO<sub>2</sub>-OA levels as a biomarker for IHD after further investigation with a larger number of IHD patients. To our knowledge, this is the first comparative study on the levels of NO<sub>2</sub>-FAs in humans with and without IHD.
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