4.2.2. Lipid Extraction and LC–MS Measurement of Oxylipins
REGRESSION
All samples were stored at −80 °C, thawed once, and immediately used for free fatty acid and oxylipin isolation as previously described [Oxylipins were quantified by the stable isotope dilution method. Briefly, identical amounts of deuterated internal standards were added to each sample and to all the primary standards used to generate standard curves. To calculate the amount of oxylipins and free fatty acids in a sample, ratios of peak areas between endogenous metabolite and matching deuterated internal standards were calculated. Ratios were converted to absolute amounts by linear regression analysis of standard curves generated under identical conditions. Oxylipin levels are expressed in picomole/milliliter (pmol/mL). To account for batch effects, quality control samples were run in each batch; the average coefficient of variance for the quantified oxylipins was 4% (standard deviation 0.01).