Lipidomic profiling of targeted oxylipins with ultra-performance liquid chromatography-tandem mass spectrometry

Abstract

Oxylipins are bioactive mediators that play diverse roles in (patho)physiology. We developed a sensitive and selective ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous profiling of 57 targeted oxylipins derived from five major n-6 and n-3 polyunsaturated fatty acids (PUFAs) that serve as oxylipin precursors, including linoleic (LA), arachidonic (AA), alpha-linolenic (ALA), eicosapentaenoic (EPA), and docosahexaenoic (DHA) acids. The targeted oxylipin panel provides broad coverage of lipid mediators and pathway markers generated from cyclooxygenases, lipoxygenases, cytochrome P450 epoxygenases/hydroxylases, and non-enzymatic oxidation pathways. The method is based on combination of protein precipitation and solid-phase extraction (SPE) for sample preparation, followed by UPLC-MS/MS. This is the first methodology to incorporate four hydroxy-epoxy-octadecenoic acids and four keto-epoxy-octadecenoic acids into an oxylipin profiling network. The novel method achieves excellent resolution and allows in-depth analysis of isomeric and isobaric species of oxylipin extracts in biological samples. The method was quantitatively characterized in human plasma with good linearity (R = 0.990-0.999), acceptable reproducibility (relative standard deviation (RSD) < 20% for the majority of analytes), accuracy (67.8 to 129.3%) for all analytes, and recovery (66.8-121.2%) for all analytes except 5,6-EET. Ion enhancement effects for 28% of the analytes in tested concentrations were observed in plasma, but were reproducible with RSD < 17.2%. Basal levels of targeted oxylipins determined in plasma and serum are in agreement with those previously reported in literature. The method has been successfully applied in clinical and preclinical studies.

Methods of the Analysis of Oxylipins in Biological Samples

“…To most effectively remove or minimize the influence of matrix effects, the following improvements can be used: modifications to the sample extraction methodology, improved chromatographic separation, and using stable isotope-labeled internal standards (IS) [154]. Deuterium-labeled standards may have disadvantages such as different retention times as compared to analytes, undesired amplification or the weakening of ionization, while 13 C-labeled standards theoretically may be better for analysis but they are not commercially available [37,156]. The choice of the optimal amount of IS is also important, since the interaction of the analyte/internal standard affects the accuracy [157,158].…”

“…Immunoassay, thin layer chromatography (TLC), HPLC with a diode array or fluorescent detector and capillary electrophoresis with a photodiode array detector were used to analyze oxylipins [13][14][15][16][17][18][19]. However, a very similar structure, limited stability and extremely low concentrations of oxylipins in the tissues impose some restrictions on these methods.…”

Plasma oxylipins and unesterified precursor fatty acids are altered by DHA supplementation in pregnancy: Can they help predict risk of preterm birth?

“…To quantify concentrations of lipid mediators in plasma, lipid extracts were purified using solid phase extraction (SPE) and quantified using LC--MS/MS as previously described [3]. Briefly, SPE of bioactive lipids from biological matrices was performed using Strata X cartridges (33 u, 200 mg/6 mL, Phenomenex, PA).…”

“…The mass spectrometer was operated in electrospray negative ionization mode using scheduled multiple reaction monitoring (sMRM) acquiring MRM data for each analyte within a retention time window of 90 s. The source parameters were set as follows: ion spray voltage, −4500 V; nebulizer gas (GS1), 65 psi; turbo-gas (GS2), 70 psi; and the turbo ion spray source temperature (TEM), 500°C. The analytes were quantified using MRM, as previously described [3].…”

Temperature and time-dependent effects of delayed blood processing on oxylipin concentrations in human plasma

“…Oxidized derivatives of 18, 20, and 22 carbon polyunsaturated fatty acids, collectively known as oxylipins, are increasingly recognized as bioactive mediators with diverse roles in human physiology and pathology [1][2][3]. Oxylipins can be divided into three general functional https://doi.org/10.1016/j.plefa.2019.09.001 Received 17 June 2019; Received in revised form 3 September 2019; Accepted 4 September 2019 T classes: (1) autacoids are labile signaling molecules that act locally and are rapidly inactivated or degraded; (2) pathway precursors are intermediates in the biosynthetic pathway leading to generation of more labile, bioactive autacoids; and (3) inactivation products are downstream metabolic derivatives that are often more stable than their autacoid precursors [4].…”