Development and Validation of a Bioanalytical Method for the Quantification of Nitrated Fatty Acids in Plasma Using LC-MS/MS: Application to Cardiovascular Patients

Abstract

Nitrated fatty acids (NO2-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 NO2-FAs identified in vivo so far are nitro oleic acid (NO2-OA), nitro linoleic acid (NO2-LA) and its structural isomer nitro-conjugated linoleic acid (NO2-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 NO2-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 NO2-OA and NO2-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 m/z 326 > 46 and m/z 324 > 46 for NO2-OA and NO2-LA, respectively, and m/z 269 > 250 for the internal standard heptadecanoic acid. Linear responses were observed for both analytes over the studied range (R2 = 0.9805 and 0.9644 for NO2-OA and NO2-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 NO2-FAs) were below basal endogenous levels, thereby providing a good tool to accurately measure these NO2-FAs in plasma. We applied the validated method to compare NO2-OA and NO2-LA levels in the plasma of 28 ischemic heart disease (IHD) patients and 18 healthy controls. The levels of NO2-OA were found to be significantly higher in the plasma of patients (21.7 ± 9.8 nM) versus healthy controls (12.6 ± 6 nM) (p-value < 0.01). Whereas the levels of NO2-LA were comparable in both groups (3 ± 1 nM in patients, 3.2 ± 1.7 nM in controls, p-value = 0.87288). The early elevation of NO2-OA in plasma samples, which were collected 2–3 h post myocardial injury, implies the potential use of NO2-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 NO2-FAs in humans with and without IHD.