μSPE followed by HPLC–MS/MS for the determination of series D and E resolvins in biological matrices

The critical role of acute inflammatory processes is recognized in many chronic diseases; a key point in molecular mechanisms of acute inflammation resolution is represented by a new group of pro-resolving lipid mediators that include distinct families of molecules: lipoxins, resolvins, protectins and maresins, collectively termed “specialized pro-resolving mediators” (SPMs). In particular, resolvins are active in the picogram to nanogram dose range, whereby they can directly modulate a plethora of anti-inflammatory responses. The presented method proposes an analytical protocol able to extract and to quantify 6 different resolvins from 3 different matrices (plasma, cells and exudates). The method, validated according to the EMA guideline for bioanalysis, exhibited good precision (1%–20%) and accuracy (2%–20%). In particular, the combination of two different sample preparation techniques, Liquid-Liquid Extraction (LLE) and micro-Solid Phase Extraction (μSPE), applied for the first on this class of molecules, used for the extraction and clean-up respectively, led to high enrichment factor (20 fold) and consequently a high sensitivity (LOQ between 1 and 38 pg mL⁻¹); moreover the validation data proved the versatility of μSPE as clean-up tool as it was capable to manage huge enrichment factor without negatively affect accuracy and precision of analysis.