molecules Review Methods of the Analysis of Oxylipins in Biological Samples Ivan Liakh 1, Alicja Pakiet 2, Tomasz Sledzinski 1 and Adriana Mika 1,2,* 1 Department of Pharmaceutical Biochemistry, Medical University of Gdansk, Debinki 1, 80-211 Gdansk, Poland; [email protected] (I.L.); [email protected] (T.S.) 2 Department of Environmental Analysis, Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308 Gdansk, Poland; [email protected] * Correspondence: [email protected]; Tel.: +48-585235190 Received: 13 December 2019; Accepted: 13 January 2020; Published: 15 January 2020 Abstract: Oxylipins are derivatives of polyunsaturated fatty acids and due to their important and diverse functions in the body, they have become a popular subject of studies. The main challenge for researchers is their low stability and often very low concentration in samples. Therefore, in recent years there have been developments in the extraction and analysis methods of oxylipins. New approaches in extraction methods were described in our previous review. In turn, the old analysis methods have been replaced by new approaches based on mass spectrometry (MS) coupled with liquid chromatography (LC) and gas chromatography (GC), and the best of these methods allow hundreds of oxylipins to be quantitatively identified. This review presents comparative and comprehensive information on the progress of various methods used by various authors to achieve the best results in the analysis of oxylipins in biological samples. Keywords: oxylipins; biological samples; HPLC; UHPLC; GC–MS; LC–MS 1. Introduction Oxylipins are important lipid mediators that are formed from polyunsaturated fatty acids (PUFAs) such as arachidonic acid (ARA), linoleic acid (LA), α-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) [1–3] in reactions catalyzed by cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP 450) enzymes, and non-enzymatic oxidation pathways [1,2]. Oxylipins are involved in various biological processes but, primarily, are important for the regulation of inflammation [4–8]. The direction of oxylipins influence on inflammation depends on their PUFA precursor, usually n-3 PUFA derived oxylipins are anti-inflammatory and pro-resolving [1], while n-6 PUFA metabolites can promote inflammation [1,3]. The ability of oxylipins to act as molecular mediator arises from their binding to peroxisome proliferator-activated receptors (PPARs) and G protein-coupled receptors (GPCRs) [9,10]. In epithelial-derived cancers altered ARA metabolism by COX and LOX leads to production of pro-inflammatory factors that promote tumor growth and facilitate formation of tumor microenvironment conductive to angiogenesis and immunosuppression [6,10]. Altered PUFA metabolism was also shown in obesity [4], where the interaction with PPAR and GPCR can modulate adipogenesis [9]. Plasma oxylipin levels have also been correlated with outcome of cardiovascular disease, metabolic syndrome, preeclampsia, due to vasoconstrictive effects of some oxylipins, or cardiac arrythmias [11]. The presence of most oxylipins in low concentrations, as well as their enormous heterogeneity and the emergence of many structurally similar oxylipins, makes their qualitative and quantitative determination difficult due to the low sensitivity of traditional methods. In the previous review [12], we described the current methods of sample preparation from various biological materials preceding the analysis of oxylipins. This paper included a description of the stages of sample collection and Molecules 2020, 25, 349; doi:10.3390/molecules25020349 www.mdpi.com/journal/molecules Molecules 2020, 25, 349 2 of 33 Molecules 2020, 25, x FOR PEER REVIEW 2 of 38 a storagesummary and of the a summary used pre-extraction of the used additives pre-extraction and standards. additives These and are standards.especially important These are to especiallyconsider inimportant order to tominimize consider oxidation, in order to minimizephotodegradation oxidation, or photodegradation heat destruction orof heat oxylipins destruction during of oxylipinssample handling.during sample Furthermore, handling. the main Furthermore, extraction the methods mainextraction including protein methods precipitation including protein(PPT), liquid-liquid precipitation extraction(PPT), liquid-liquid (LLE), solid-phase extraction extrac (LLE),tion solid-phase(SPE), and the extraction derivatization (SPE), andprocess the derivatizationwere described. process Choosing were appropriatedescribed. extraction Choosing method appropriate and extractionsolvents is methodessential and in obtaining solvents isgood essential target inanalyte obtaining recoveries good targetand reproducibilityanalyte recoveries needed and for reproducibility further quantitative needed analysis for further and depends quantitative on the analysis oxylipin and group depends of interest. on the SPE,oxylipin due to group the availability of interest. of SPE, various due types to the of availability sorbents and of varioussolvents, types is the of most sorbents widely and used solvents, extraction is the methodmost widely in the usedanalysis extraction of oxylipins. method For in LLE the analysisand SPE, of the oxylipins. extraction For methods LLE and have SPE, been the extractiongrouped accordingmethods to have the been characteristics grouped according of the studied to the biological characteristics material of the (biofluids, studied biologicalsolid tissues, material cell cultures). (biofluids, Finally,solid tissues,new approaches cell cultures). and trends Finally, in material new approaches collection (dried and trends blood in spot), material precipitation collection (ferromagnetic (dried blood particlespot), precipitationenhanced deproteination), (ferromagnetic and particle extraction enhanced (microextraction, deproteination), online and SPE, extraction mixed-mode (microextraction, extraction withonline a spin SPE, column) mixed-mode for the extraction analysis of with oxylipins a spin we column)re described. for the Owing analysis to of th oxylipinse fact that were oxylipins described. are usuallyOwing present to the factat very that low oxylipins concentrations are usually in biological present atsamples, very low the concentrations quality of sample in biologicalcollection, samples,storage andthe extraction quality of methods sample collection,is paramount storage for achieving and extraction accurate methods quantification. is paramount The present for achieving review, being accurate an extensionquantification. of the previous The present paper, review, describes being further an extension stages of ofanalysis the previous and the paper, quantitative describes determination further stages of oxylipinof analysis levels and in thethe quantitativeprepared samples determination using existing of oxylipin analytical levels techniques. in the prepared samples using existing analyticalImmunoassay, techniques. thin layer chromatography (TLC), HPLC with a diode array or fluorescent detectorImmunoassay, and capillary thin electrophoresis layer chromatography with a (TLC),photodiode HPLC witharray a diodedetector array were or fluorescentused to analyze detector oxylipinsand capillary [13–19]. electrophoresis However, witha very a photodiode similar arraystructure, detector limited were usedstability to analyze and oxylipinsextremely [ 13low–19 ]. concentrationsHowever, a very of similaroxylipins structure, in the tissues limited impose stability some and extremelyrestrictions low on concentrations these methods. of oxylipinsTherefore, in recently,the tissues gas impose chromatography–mass some restrictions on spectrometry these methods. (GC–MS) Therefore, and recently, liquid gas chromatography–mass chromatography–mass spectrometryspectrometry (LC–MS) (GC–MS) have and most liquid often chromatography–mass been used to determine spectrometry oxylipin (LC–MS) levels in have biological most oftensamples been [20–24].used to A determine summary oxylipinof the most levels frequently in biological used samples analytical [20 methods–24]. A summary in the analysis of the of most oxylipins frequently as wellused as analyticaltheir advantages methods and in limitations the analysis is presented of oxylipins in Table as well 1. asAlso, their the advantages comparison and of mass limitations spectra is ofpresented PGE2 and in PGD2 Table 1obtained. Also, the by comparison two most popular of mass techniques spectra of PGE2 for lipids and PGD2identification: obtained GC–MS by two mostand LC–MSpopular is techniquespresented on for Figure lipids identification:1 [25]. GC–MS and LC–MS is presented on Figure1[25]. Figure 1. Representative mass spectra of prostaglandin E2 (PGE2) and prostaglandin D2 (PGD2) Figureobtained 1. Representative by using hard (gasmass chromatography–mass spectra of prostaglandi spectrometryn E2 (PGE2) (GC–MS)) and prostaglandin and soft ionization D2 (PGD2) (liquid obtainedchromatography–mass by using hard spectrometry(gas chromatography–m (LC–MS)/MS)ass in spectrometry mass.(GC–MS)) and soft ionization (liquid chromatography–mass spectrometry (LC–MS)/MS) in spectrometry mass. Molecules 2020, 25, 349 3 of 33 Table 1. Advantages and limitations of the most frequently used analytical methods in the analysis of oxylipins. Analytical Technique Application Advantages Limitations Enzyme immunoassay (EIA) and dioimmunoassay (RIA) are easy to use, sensitive and do not require expensive Unspecificity due to cross-reactivity [27] instrumentation