Shotgun Lipidomics for the Determination of Phospholipid and Eicosanoid Profiles in Atlantic Salmon (Salmo salar L.) Muscle Tissue Using Electrospray Ionization (ESI)-MS/MS Spectrometric Analysis.

Shotgun lipidomics was applied to identify and quantify phospholipids (PLs) in salmon muscle tissue by focusing on the distribution of ω-3 fatty acids (e.g., docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA)) in the form of phospholipids, as well as to identify and quantify eicosanoids, which has not yet been attempted in Atlantic salmon muscle. Shotgun lipidomics enabled the identification of 43 PL species belonging to four different classes: phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), phosphatidylserines (PSs), and phosphatidylinositols (PIs). Among others, 16:0-22:6 PtdCho m/z [M + Na]+ at 828.4 was the predominant PL species in salmon muscle tissue. The present study provided the quantification of individual phospholipid species, which has not been performed for salmon muscle tissue so far. In addition, two eicosanoids-prostaglandin E2 (PGE2) and prostaglandin F3α (PGF3α)-were identified for the first time in salmon muscle. Thus, the rapid and high-throughput shotgun lipidomics approach should shed new light on phospholipids and eicosanoids in salmon muscle tissue.

Revising the structure of a new eicosanoid from human platelets to 8,9-11,12-diepoxy-13-hydroxyeicosadienoic acid.

Eicosanoids are critical mediators of fever, pain, and inflammation generated by immune and tissue cells. We recently described a new bioactive eicosanoid generated by cyclooxygenase-1 (COX-1) turnover during platelet activation that can stimulate human neutrophil integrin expression. On the basis of mass spectrometry (MS/MS and MS3), stable isotope labeling, and GC-MS analysis, we previously proposed a structure of 8-hydroxy-9,11-dioxolane eicosatetraenoic acid (DXA3). Here, we achieved enzymatic synthesis and 1H NMR characterization of this compound with results in conflict with the previously proposed structural assignment. Accordingly, by using LC-MS, we screened autoxidation reactions of 11-hydroperoxy-eicosatetraenoic acid (11-HpETE) and thereby identified a candidate sharing the precise reverse-phase chromatographic and MS characteristics of the platelet product. We optimized these methods to increase yield, allowing full structural analysis by 1H NMR. The revised assignment is presented here as 8,9-11,12-diepoxy-13-hydroxyeicosadienoic acid, abbreviated to 8,9-11,12-DiEp-13-HEDE or DiEpHEDE, substituted for the previous name DXA3 We found that in platelets, the lipid likely forms via dioxolane ring opening with rearrangement to the diepoxy moieties followed by oxygen insertion at C13. We present its enzymatic biosynthetic pathway and MS/MS fragmentation pattern and, using the synthetic compound, demonstrate that it has bioactivity. For the platelet lipid, we estimate 16 isomers based on our current knowledge (and four isomers for the synthetic lipid). Determining the exact isomeric structure of the platelet lipid remains to be undertaken.

Co-opting oxylipin signals in microbial disease.

Oxylipins, or oxygenated lipids, are universal signalling molecules across all kingdoms of life. These molecules, either produced by microbial pathogens or their mammalian host, regulate inflammation during microbial infection. In this review, we summarise current literature on the biosynthesis pathways of microbial oxylipins and their biological activity towards mammalian cells. Collectively, these studies have illustrated how microbial pathogens can modulate immune rsponse and disease outcome via oxylipin-mediated mechanisms.

Tissue pretreatment for LC-MS/MS analysis of PUFA and eicosanoid distribution in mouse brain and liver.

Polyunsaturated fatty acids (PUFAs) and eicosanoids are important mediators of inflammation. The functional role of eicosanoids in metabolic-syndrome-related diseases has been extensively studied. However, their role in neuroinflammation and the development of neurodegenerative diseases is still unclear. The aim of this study was the development of a sample pretreatment protocol for the simultaneous analysis of PUFAs and eicosanoids in mouse liver and brain. Liver and brain samples of male wild-type C57BL/6J mice (11-122 mg) were used to investigate conditions for tissue rinsing, homogenization, extraction, and storage. A targeted liquid chromatography-negative electrospray ionization tandem mass spectrometry method was applied to quantify 7 PUFAs and 94 eicosanoids. The final pretreatment protocol consisted of a 5-min homogenization step by sonication in 650 µL n-hexane/2-propanol (60:40 v/v) containing 2,6-di-tert-butyl-4-methylphenol at 50 µg/mL. Homogenates representing 1 mg tissue were extracted in a single step with n-hexane/2-propanol (60:40 v/v) containing 0.1% formic acid. Autoxidation was prevented by addition of 2,6-di-tert-butyl-4-methylphenol at 50 µg/mL and keeping the samples at 4 °C during sample preparation. Extracts were dried under nitrogen and reconstituted in liquid chromatography eluent before analysis. Recovery was determined to range from 45% to 149% for both liver and brain tissue. Within-run and between-run variability ranged between 7% and 18% for PUFAs and between 1% and 24% for eicosanoids. In liver, 7 PUFAs and 15 eicosanoids were quantified; in brain, 6 PUFAs and 21 eicosanoids had significant differences within the brain substructures. In conclusion, a robust and reproducible sample preparation protocol for the multiplexed analysis of PUFAs and eicosanoids by liquid chromatography-tandem mass spectrometry in liver and discrete brain substructures was developed.

Understanding Peroxisome Proliferator-Activated Receptors: From the Structure to the Regulatory Actions on Metabolism.

Peroxisome proliferator-activated receptors (PPARs) are multi-domains proteins, belonging to the superfamily of nuclear receptors, which mainly act as ligand-activated transcription factors. A variety of lipophilic molecules, including long-chain polyunsaturated fatty acids and eicosanoids, are capable of binding to PPAR, although the nature of the physiological ligands is still under debate. PPARs regulate the expression of a set of genes involved in glucose and lipid metabolism as well as in the control of inflammatory responses. Herein we review the main molecular and cellular events associated with the activation of PPARs and their effects on metabolism.

Glycerolipid Composition of the Red Macroalga Agarophyton Chilensis and Comparison to the Closely Related Agarophyton Vermiculophyllum Producing Different Types of Eicosanoids.

The red macroalga Agarophyton chilensis is a well-known producer of eicosanoids such as hydroxyeicosatetraenoic acids, but the alga produces almost no prostaglandins, unlike the closely related A. vermiculophyllum. This indicates that the related two algae would have different enzyme systems or substrate composition. To carry out more in-depth discussions on the metabolic pathway of eicosanoids between the two algae, we investigated the characteristics of glycerolipids, which are the substrates of eicosanoids production, of A. chilensis and compared them to the reported values of A. vermiculophyllum. In A. chilensis, monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG), and phosphatidylcholine (PC) were the major lipid classes and accounted for 44.4% of the total lipid extract. The predominant fatty acids were arachidonic acid (20:4n-6), an eicosanoids precursor, and palmitic acid (16:0). The 20:4n-6 content was extremely high in MGDG and PC (>70%), and the 16:0 content was extremely high in DGDG and SQDG (>40%). A chiral-phase HPLC analysis showed that fatty acids were esterified at the sn-1 and sn-2 positions of those lipids. The glycerolipid molecular species were determined by reversed-phase HPLC⁻ESI⁻MS analysis. The main glycerolipid molecular species were 20:4n-6/20:4n-6 (sn-1/sn-2) for MGDG (63.8%) and PC (48.2%), 20:4n-6/16:0 for DGDG (71.1%) and SQDG (29.4%). These lipid characteristics of A. chilensis were almost the same as those of A. vermiculophyllum. Hence, the differences of the eicosanoids producing ability between the two algae would not be due to the difference of substrate composition but the difference of enzyme system.

Identification of biologically active δ-lactone eicosanoids as paraoxonase substrates.

The mammalian paraoxonases (PONs 1, 2 and 3) are a family of esterases that are highly conserved within and between species. They exhibit antioxidant and anti-inflammatory activities. However, their physiological function(s) and native substrates are uncertain. Previous structure-activity relationship studies demonstrate that PONs have a high specificity for lipophilic lactones, suggesting that such compounds may be representative of native substrates. This report describes the ability of PONs to hydrolyze two bioactive δ-lactones derived from arachidonic acid, 5,6-dihydroxy-eicosatrienoic acid lactone (5,6-DHTL) and cyclo-epoxycyclopentenone (cyclo-EC). Both lactones were very efficiently hydrolyzed by purified PON3. PON1 efficiently hydrolyzed 5,6-DHTL, but with a specific activity about 15-fold lower than PON3. 5,6-DHTL was a poor substrate for PON2. Cyclo-EC was a poor substrate for PON1 and not hydrolyzed by PON2. Studies with the PON inhibitor EDTA and a serine esterase inhibitor indicated that the PONs are the main contributors to hydrolysis of the lactones in human and mouse liver homogenates. Studies with homogenates from PON3 knockout mouse livers indicated that >80% of the 5,6-DHTL and cyclo-EC lactonase activities were attributed to PON3. The findings provide further insight into the structural requirements for PONs substrates and support the hypothesis that PONs, particularly PON1 and PON3, evolved to hydrolyze and regulate a class of lactone lipid mediators derived from polyunsaturated fatty acids.

Molecular modelling insights into a physiologically favourable approach to eicosanoid biosynthesis inhibition through novel thieno[2,3-b]pyridine derivatives.

In this research, we exploited derivatives of thieno[2,3-b]pyridine as dual inhibitors of the key enzymes in eicosanoid biosynthesis, cyclooxygenase (COX, subtypes 1 and 2) and 5-lipoxygensase (5-LOX). Testing these compounds in a rat paw oedema model revealed potency higher than ibuprofen. The most active compounds 7a, 7b, 8b, and 8c were screened against COX-1/2 and 5-LOX enzymes. Compound 7a was the most powerful inhibitor of 5-LOX with IC50 = 0.15 µM, while its p-chloro analogue 7b was more active against COX-2 (IC50 = 7.5 µM). The less desirable target COX-1 was inhibited more potently by 8c with IC50 = 7.7 µM. Surflex docking programme predicted that the more stable anti- conformer of compound (7a) formed a favourable complex with the active site of 5-LOX but not COX-1. This is in contrast to the binding mode of 8c, which resembles the syn-conformer of series 7 and binds favourably to COX-1.

Long-chain acyl-CoA synthetase isoforms differ in preferences for eicosanoid species and long-chain fatty acids.

Because the signaling eicosanoids, epoxyeicosatrienoic acids (EETs) and HETEs, are esterified to membrane phospholipids, we asked which long-chain acyl-CoA synthetase (ACSL) isoforms would activate these molecules and whether the apparent FA substrate preferences of each ACSL isoform might differ depending on whether it was assayed in mammalian cell membranes or as a purified bacterial recombinant protein. We found that all five ACSL isoforms were able to use EETs and HETEs as substrates and showed by LC-MS/MS that ACSLs produce EET-CoAs. We found differences in substrate preference between ACS assays performed in COS7 cell membranes and recombinant purified proteins. Similarly, preferences and Michaelis-Menten kinetics for long-chain FAs were distinctive. Substrate preferences identified for the purified ACSLs did not correspond to those observed in ACSL-deficient mouse models. Taken together, these data support the concept that each ACSL isoform exhibits a distinct substrate preference, but apparent substrate specificities depend upon multiple factors including membrane character, coactivators, inhibitors, protein interactions, and posttranslational modification.

Mass Spectrometry-Based Chemical Cartography of a Cardiac Parasitic Infection.

Trypanosoma cruzi parasites are the causative agents of Chagas disease, a leading infectious form of heart failure whose pathogenesis is still not fully characterized. In this work, we applied untargeted liquid chromatography-tandem mass spectrometry to heart sections from T. cruzi-infected and uninfected mice. We combined molecular networking and three-dimensional modeling to generate chemical cartographical heart models. This approach revealed for the first time preferential parasite localization to the base of the heart and regiospecific distributions of nucleoside derivatives and eicosanoids, which we correlated to tissue-damaging immune responses. We further detected novel cardiac chemical signatures related to the severity and ultimate outcome of the infection. These signatures included differential representation of higher- vs lower-molecular-weight carnitine and phosphatidylcholine family members in specific cardiac regions of mice infected with lethal or nonlethal T. cruzi strains and doses. Overall, this work provides new insights into Chagas disease pathogenesis and presents an analytical chemistry approach that can be broadly applied to the study of host-microbe interactions.

Biomimetic synthesis of hemiketal eicosanoids for biological testing.

The hemiketal (HK) eicosanoids HKE2 and HKD2 are the major products resulting from the biosynthetic cross-over of the 5-lipoxygenase and cyclooxygenase-2 pathways. They are formed by activated human leukocytes ex vivo, and, therefore, may be involved in regulation of the inflammatory response as autocrine or paracrine mediators. HKE2 and HKD2 are not commercially available and, so far, no method for their total chemical synthesis has been reported. The limited availability has impeded the characterization of their biological effects. Here, we describe a method for biomimetic preparation of HKE2 and HKD2 by reaction of recombinant human cyclooxygenase-2 with chemically synthesized 5S-HETE. We found that HKE2 did not induce or inhibit the release of TNFα and IL-1ß by human THP-1 monocytes and phorbol ester treatment-derived macrophages.

The Bibenzyl Canniprene Inhibits the Production of Pro-Inflammatory Eicosanoids and Selectively Accumulates in Some Cannabis sativa Strains.

Canniprene (1), an isoprenylated bibenzyl unique to Cannabis sativa, can be vaporized and therefore potentially inhaled from marijuana. Canniprene (1) potently inhibited the production of inflammatory eicosanoids via the 5-lipoxygenase pathway (IC50 0.4 µM) and also affected the generation of prostaglandins via the cyclooxygenase/microsomal prostaglandin E2 synthase pathway (IC50 10 µM), while the related spiranoid bibenzyls cannabispiranol (2) and cannabispirenone (3) were almost inactive in these bioassays. The concentration of canniprene (1) was investigated in the leaves of 160 strains of C. sativa, showing wide variations, from traces to >0.2%, but no correlation was found between its accumulation and a specific phytocannabinoid profile.

Structural analogs of pulmonary surfactant phosphatidylglycerol inhibit toll-like receptor 2 and 4 signaling.

The pulmonary surfactant phospholipid, 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG), potently inhibits toll-like receptor (TLR)2 and TLR4 signaling from the cell surface of macrophages. Analogs of POPG that vary in polar head group length, hydroxylation, and alkyl branching were synthesized using a phospholipase D-catalyzed transphosphatidylation reaction and a 1-palmitoyl-2-oleoyl phosphatidylcholine substrate. Lipid analogs with C3 and C4 alkyl head group length (POP-propanol and POP-butanol) are less effective than POPG as TLR2 and TLR4 antagonists. However, adding a hydroxyl group at the alkyl chain 3- or 4-position (POP-propanediols or POP-butanediols) greatly increased their inhibitory effects against TLR2 and TLR4. POP-2',2'-dimethylpropanediol is a weak inhibitor of TLR2 and TLR4 activation that results in arachidonic acid release, but an effective inhibitor of TLR4 activation that results in TNF-α production. Addition of an amino group at the alkyl-2 position (POP-2'-aminopropanediol) completely abolished the antagonism of TLRs 2 and 4. Multiple analogs strongly bind to the TLR4 coreceptors, cluster of differentiation 14 (CD14) and myeloid differentiation 2, but competition for di[3-deoxy-D-manno-octulosonyl]-lipid A binding to CD14 is the best predictor of biological activity at the cellular level. Collectively, these findings identify new compounds for antagonizing TLR2 and TLR4 activation and define structural properties of POPG analogs for discriminating between two TLR systems.

Soluble epoxide hydrolase inhibitor 1-trifluoromethoxyphenyl-3- (1-propionylpiperidin-4-yl) urea attenuates bleomycin-induced pulmonary fibrosis in mice.

Epoxyeicosatrienoic acids (EETs), the metabolites of arachidonic acid derived from the cytochrome P450 (CYP450) epoxygenases, are mainly metabolized by soluble epoxide hydrolase (sEH) to their corresponding diols. EETs but not their diols, have anti-inflammatory properties and inhibition of sEH might provide protective effects against inflammatory fibrosis. We test the effects of a selected sEH inhibitor, 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), on bleomycin-induced pulmonary fibrosis (PF) in mice. A mouse model of PF was established by intratracheal injection of bleomycin and TPPU was administered for 21 days after bleomycin injection. We found TPPU treatment improved the body weight loss and survival rate of bleomycin-stimulated mice. Histological examination showed that TPPU treatment alleviated bleomycin-induced inflammation and maintained the alveolar structure of the pulmonary tissues. TPPU also decreased the bleomycin-induced deposition of collagen and the expression of procollagen I mRNA in lung tissues of mice. TPPU decreased the transforming growth factor-ß1 (TGF-ß1), interleukin-1ß (IL-1ß) and IL-6 levels in the serum of bleomycin-stimulated mice. Furthermore, TPPU inhibited the proliferation and collagen synthesis of mouse fibroblasts and partially reversed TGF-ß1-induced α-smooth muscle actin expression. Our results indicate that the inhibition of sEH attenuates bleomycin-induced inflammation and collagen deposition and therefore prevents bleomycin-induced PF in a mouse model.

Proinflammatory Pathways: The Modulation by Flavonoids.

Inflammation is a natural, carefully orchestrated response of the organism to tissue damage, involving various signaling systems and the recruitment of inflammatory cells. These cells are stimulated to release a myriad of mediators that amplify the inflammatory response and recruit additional cells. These mediators present numerous redundancies of functions, allowing a broad and effective inflammatory response, but simultaneously make the understanding of inflammation pathways much difficult. The extent of the inflammatory response is usually self-limited, although it depends on the balance between the pro- and anti-inflammatory signals. When that equilibrium is dislocated, a more widespread inflammatory response may take place. Flavonoids have been shown to be possible alternatives to the traditionally molecules used as anti-inflammatory agents. In fact, the biological activities of flavonoids include the modulation of the diverse phases of inflammatory processes, from the gene transcription and expression to the inhibition of the enzymatic activities and the scavenging of the reactive species. In the present review, the inflammatory network is widely revised and the flavonoids' broad spectrum of action in many of the analyzed inflammatory pathways is revised. This kind of integrated revision is original in the field, providing the reader the simultaneous comprehension of the inflammatory process and the potential beneficial activities of flavonoids.

Simultaneous Determination Method of Epoxyeicosatrienoic Acids and Dihydroxyeicosatrienoic Acids by LC-MS/MS System.

Epoxyeicosatrienoic acids (EETs) are produced primarily by CYPs from arachidonic acid (AA) and then further metabolized to the corresponding dihydroxyeicosatrienoic acids (DHETs). EETs play important roles in physiological processes such as regulating vasodilation and inflammation. Thus, the drug inhibition of CYP-mediated AA metabolism could reduce production of EETs, potentially resulting in adverse cardiovascular events. The aim of this study was to develop a simple method to simultaneously determine the concentrations of both EETs and DHETs using a conventional LC-MS/MS system to evaluate drug-endogenous substance interactions, including eicosanoids. Eight eicosanoids (5,6-EET, 8,9-EET, 11,12-EET, 14,15-EET, 5,6-DHET, 8,9-DHET, 11,12-DHET, and 14,15-DHET) were detected with their corresponding deuterium-labeled eicosanoids as internal standards. The samples were purified by solid-phase extraction columns. Liquid chromatographic separation was achieved on a C18 column. DHETs and EETs were eluted at 4-7 and 18-26 min, respectively. The weighted (1/y(2)) calibration curves were linear over a range of 5-2000 nmol/L for EETs and 2-2000 nmol/L for DHETs. In quality control (QC) samples, the recoveries of eicosanoids were 95.2-118%. The intra-day precisions were within 6% in all three QC samples, and the inter-day precisions were <16.7% at 50 nmol/L, <8.6% at 200 nmol/L, and <9.8% at 1000 nmol/L. We have applied this method for the determination of the eicosanoid levels in samples from incubation studies of AA by using human recombinant CYP enzyme (rCYP), and confirmed that the method has sensitivity sufficient for assessment of rCYP incubation study.

Lipoxygenase-catalyzed transformation of epoxy fatty acids to hydroxy-endoperoxides: a potential P450 and lipoxygenase interaction.

Herein, we characterize a generally applicable transformation of fatty acid epoxides by lipoxygenase (LOX) enzymes that results in the formation of a five-membered endoperoxide ring in the end product. We demonstrated this transformation using soybean LOX-1 in the metabolism of 15,16-epoxy-α-linolenic acid, and murine platelet-type 12-LOX and human 15-LOX-1 in the metabolism of 14,15-epoxyeicosatrienoic acid (14,15-EET). A detailed examination of the transformation of the two enantiomers of 15,16-epoxy-α-linolenic acid by soybean LOX-1 revealed that the expected primary product, a 13S-hydroperoxy-15,16-epoxide, underwent a nonenzymatic transformation in buffer into a new derivative that was purified by HPLC and identified by UV, LC-MS, and ¹H-NMR as a 13,15-endoperoxy-16-hydroxy-octadeca-9,11-dienoic acid. The configuration of the endoperoxide (cis or trans side chains) depended on the steric relationship of the new hydroperoxy moiety to the enantiomeric configuration of the fatty acid epoxide. The reaction mechanism involves intramolecular nucleophilic substitution (SNi) between the hydroperoxy (nucleophile) and epoxy group (electrophile). Equivalent transformations were documented in metabolism of the enantiomers of 14,15-EET by the two mammalian LOX enzymes, 15-LOX-1 and platelet-type 12-LOX. We conclude that this type of transformation could occur naturally with the co-occurrence of LOX and cytochrome P450 or peroxygenase enzymes, and it could also contribute to the complexity of products formed in the autoxidation reactions of polyunsaturated fatty acids.

New families of bioactive oxidized phospholipids generated by immune cells: identification and signaling actions.

Phospholipids are of critical importance in mammalian cell biology, both through providing a permeability barrier and acting as substrates for synthesis of lipid mediators. Recently, several new families of bioactive lipids were identified that form through the enzymatic oxidation of membrane phospholipids in circulating innate immune cells and platelets. These comprise eicosanoids attached to phosphatidylethanolamine and phosphatidylcholine and form within 2-5 minutes of cell activation by pathophysiologic agonists, via the coordinated action of receptors and enzymes. In this review, we summarize what is currently known regarding their structures, mechanisms of formation, cell biology, and signaling actions. We show that phospholipid oxidation by acutely activated immune cells is a controlled event, and we propose a central role in regulating membrane biology and innate immune function during health and disease. We also review the mass spectrometry methods used for identification of the lipids and describe how these approaches can be used for discovery of new lipid mediators in complex biologic samples.

Antagonizing arachidonic acid-derived eicosanoids reduces inflammatory Th17 and Th1 cell-mediated inflammation and colitis severity.

During colitis, activation of two inflammatory T cell subsets, Th17 and Th1 cells, promotes ongoing intestinal inflammatory responses. n-6 polyunsaturated fatty acid- (PUFA-) derived eicosanoids, such as prostaglandin E2 (PGE2), promote Th17 cell-mediated inflammation, while n-3 PUFA antagonize both Th17 and Th1 cells and suppress PGE2 levels. We utilized two genetic mouse models, which differentially antagonize PGE2 levels, to examine the effect on Th17 cells and disease outcomes in trinitrobenzene sulfonic acid- (TNBS-) induced colitis. Fat-1 mice contain the ω3 desaturase gene from C. elegans and synthesize n-3 PUFA de novo, thereby reducing the biosynthesis of n-6 PUFA-derived eicosanoids. In contrast, Fads1 Null mice contain a disrupted Δ5 desaturase gene and produce lower levels of n-6 PUFA-derived eicosanoids. Compared to Wt littermates, Fat-1 and Fads1 Null mice exhibited a similar colitic phenotype characterized by reduced colonic mucosal inflammatory eicosanoid levels and mRNA expression of Th17 cell markers (IL-17A, RORγτ, and IL-23), decreased percentages of Th17 cells and, improved colon injury scores (P ≤ 0.05). Thus, during colitis, similar outcomes were obtained in two genetically distinct models, both of which antagonize PGE2 levels via different mechanisms. Our data highlight the critical impact of n-6 PUFA-derived eicosanoids in the promotion of Th17 cell-mediated colonic inflammation.

Development and Validation of Methodologies for the Identification of Specialized Pro-Resolving Lipid Mediators and Classic Eicosanoids in Biological Matrices.

Lipid mediators, which include specialized pro-resolving mediators and classic eicosanoids, are pivotal in both initiating and resolving inflammation. The regulation of these molecules determines whether inflammation resolves naturally or persists. However, our understanding of how these mediators are regulated over time in various inflammatory contexts is limited. This gap hinders our grasp of the mechanisms underlying the disease onset and progression. Due to their localized action and low endogenous levels in many tissues, developing robust and highly sensitive methodologies is imperative for assessing their endogenous regulation in diverse inflammatory settings. These methodologies will help us gain insight into their physiological roles. Here, we establish methodologies for extracting, identifying, and quantifying these mediators. Using our methods, we identified a total of 37 lipid mediators. Additionally, by employing a reverse-phase HPLC method, we successfully separated both double-bond and chiral isomers of select lipid mediators, including Lipoxin (LX) A4, 15-epi-LXA4, Protectin (PD) D1, PDX, and 17R-PD1. Validation of the method was performed in both solvent and surrogate matrix for linearity of the standard curves, lower limits of quantitation (LLOQ), accuracy, and precision. Results from these studies demonstrated that linearity was good with r2 values > 0.98, and LLOQ for the mediators ranged from 0.01 to 0.9 pg in phase and from 0.1 to 8.5 pg in surrogate matrix. The relative standard deviation (RSD) for inter- and intraday precision in solvent ranged from 5% to 12% at low, intermediate, and high concentrations, whereas the RSD for the inter- and intraday variability in the accuracy ranged from 95% to 87% at low to high concentrations. The recovery in biological matrices (plasma and serum) for the internal standards used ranged from 60% to 118%. We observed a marked ion suppression for molecules evaluated in negative ionization mode, while there was an ion enhancement effect by the matrix for molecules evaluated in positive ionization mode. Comparison of the integration algorithms, namely, AutoPeak and MQ4, and approaches for calculating signal-to-noise ratios (i.e., US Pharmacopeia, relative noise, peak to peak, and standard deviation) demonstrated that different integration algorithms tested had little influence on signal-to-noise ratio calculations. In contrast, the method used to calculate the signal-to-noise ratio had a more significant effect on the results, with the relative noise approach proving to be the most robust. The methods described herein provide a platform to study the SPM and classic eicosanoids in biological tissues that will help further our understanding of disease mechanisms.