Extraction of lipids and oxylipins from plasma for quantification of total oxylipins – Challenges and strategies

Several eicosanoids and other oxylipins are potent lipid mediators which are involved in theregulation of physiological functions such as inflammation. It is believed that they act predominantlyin their free, i.e. non-esterified, form. However, a major portion of oxylipins is found as esters, e.g. inpolar lipids.Only little information is available on the biological activity of these esterified oxylipins. An importantstep towards a better understanding of their biological role is a comprehensive comparison of thechanges in the pattern of free vs. esterified mediators induced by pharmacological intervention ordietary supplementation as well as during onset and progression of diseases.While several LC-MS based methods for the detection of free oxylipins have been developed,esterified oxylipins are commonly quantified as a sum of free and esterified oxylipins following basehydrolysis. However, different approaches have been described for preparation of samples beforehydrolysis and for the solid phase extraction. Here, we present a three-step strategy for thequantification of total oxylipins including extraction of total lipids, saponification to liberate esterifiedoxylipins and solid phase extraction of free oxylipins. We thoroughly investigated different liquidliquidextraction procedures and protein precipitation in terms of extraction efficiency for variouslipid classes from plasma. Moreover, to optimize sample throughout we compared extraction of freeoxylipins via solid phase extraction on cartridges with 96-well plates.In conclusion, our results emphasis the challenges related to the extraction procedures and providedifferent strategies for reliable oxylipin quantification with a focus on efficient and reproducibleextraction