Splash lipidomix

Plasma samples were divided into four batches in such a way that there were no differences among the batches in terms of the number of samples from each study group or other variables such as age, sex, and presence of comorbidities. Each sample batch was prepared simultaneously and run sequentially in order to stabilize the quality control of the experiment with regard to the instrumental setup and pH of chromatographic buffers. The plasma sample (25 μL) was transferred into a borosilicate glass tube followed by addition of internal standards (Splash Lipidomix, Avanti Polar, Alabaster, AL) at a 1:1 ratio of internal standard (IS):plasma. The complete list of IS and their corresponding concentrations has been described previously.^{1 (link)} Water, methanol, and chloroform at a 1:2:0.9 ratio was added to the glass tube. The mixture was vortexed and left to sit at room temperature for 30 min. Next, water and chloroform were added at a 1:0.9 ratio and the sample tube was inverted several times. The tubes were centrifuged at 3500 rpm for 30 min. The bottom organic layer containing the lipids was carefully collected, and 2 mL of chloroform was added to the aqueous phase (upper layer) to re-extract the lipids. Tubes were vortexed and centrifuged again. The subsequent bottom layer was combined with the previously collected lipids and dried down using a nitrogen stream.