title: Chiral chromatography-tandem mass spectrometry applied to the determination of pro-resolving lipid mediators.
abstract: Pro-resolving lipid mediators are a class of endogenously synthesized molecules derived from different fatty acids, such as arachidonic, docosahexaenoic or eicosapentaenoic acid, which are derived into four different product families: lipoxins, resolvins, maresins and protectins. For quantitation of these compounds, a sensitive, selective and robust liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous quantitation of lipoxin A4, 6-epi-lipoxin A4, lipoxin B4 and lipoxin A5, the D-series resolvins D1 and D2 as well as aspirin-triggered lipoxin A4 and resolvin D1, maresin and protectin and the pathway markers 17(S)-hydroxy-docosahexaenoic acid and 17(R)-hydroxy-docosahexaenoic acid in cell culture supernatants. For this purpose, a chiral column was connected in series with a reversed-phase column to achieve efficient analyte separation and high sensitivity. Sample pre-treatment included a fast and simple liquid-liquid extraction procedure. Limits of quantitation in the range of 0.1-0.5ng/mL cell culture media, absolute recoveries between 90 and 115%, intra- and interday precision of less than 13% and an accuracy of less than 11% were obtained. Stability of the samples after 60 days storage at -80°C, three freeze/thaw cycles and 4h at room temperature has been demonstrated for all analytes. Sample extracts can be stored at 7°C for 24h without degradation of the analytes. Deviations of less than 13% in the accuracy, evaluated in terms of relative error, were obtained. The suitability of the method has been demonstrated in cell culture supernatants of human polymorphonuclear leukocytes, stimulated with 15R-hydroxy-eicosatetraenoic acid and in cell culture media of human polymorphonuclear leukocytes co-incubated with human platelets. From all studied analytes, lipoxin A4 and 6-epi-lipoxin A4 were found in cell culture media under both incubation conditions, while 15-epi-lipoxin A4 was additionally detected in cell culture supernatants of polymorphonuclear leukocytes stimulated with 15R-hydroxy-eicosatetraenoic acid.
authors.affiliation: pharmazentrum frankfurt/ZAFES, Institute of Clinical Pharmacology, Goethe-University, Frankfurt, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Homann
authors.affiliation: Fraunhofer Institute for Molecular Biology & Applied Ecology, IME - Project Group Translational Medicine & Pharmacology TMP, Frankfurt, Germany; Goethe University, Institute of Pharmaceutical Chemistry, ZAFES, Frankfurt, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Lehmann
authors.affiliation: Goethe University, Institute of Pharmaceutical Chemistry, ZAFES, Frankfurt, Germany.
authors.forename: Astrid S
authors.initials: AS
authors.lastname: Kahnt
authors.affiliation: Goethe University, Institute of Pharmaceutical Chemistry, ZAFES, Frankfurt, Germany.
authors.forename: Dieter
authors.initials: D
authors.lastname: Steinhilber
authors.affiliation: Fraunhofer Institute for Molecular Biology & Applied Ecology, IME - Project Group Translational Medicine & Pharmacology TMP, Frankfurt, Germany.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Parnham
authors.affiliation: pharmazentrum frankfurt/ZAFES, Institute of Clinical Pharmacology, Goethe-University, Frankfurt, Germany; Fraunhofer Institute for Molecular Biology & Applied Ecology, IME - Project Group Translational Medicine & Pharmacology TMP, Frankfurt, Germany.
authors.forename: Gerd
authors.initials: G
authors.lastname: Geisslinger
authors.affiliation: pharmazentrum frankfurt/ZAFES, Institute of Clinical Pharmacology, Goethe-University, Frankfurt, Germany. Electronic address: ferreirosbouzas@em.uni-frankfurt.de.
authors.forename: Nerea
authors.initials: N
authors.lastname: Ferreirós
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D057230:Limit of Detection; D044045:Lipoxins / Q000032:analysis / Q000737:chemistry; D059625:Liquid-Liquid Extraction; D009504:Neutrophils / Q000737:chemistry; D013237:Stereoisomerism; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D044045:Lipoxins
keywords: Chiral chromatography–tandem mass spectrometry; Lipoxin; Maresin; Pro-resolving lipid mediators; Protectin; Resolvin