title: Rapid and simultaneous quantitation of prostanoids by UPLC-MS/MS in rat brain.
abstract: The metabolites of arachidonic acid (AA) produced from the cyclooxygenase (COX) pathway, collectively termed as prostanoids, and from the CYP 450 pathway, eicosanoids, have been implicated in various neuro-degenerative and neuroinflammatory diseases. This study developed a quantitative UPLC-MS/MS method to simultaneously measure 11 prostanoids including prostaglandins and cyclopentenone metabolites in the rat brain cortical tissue. Linear calibration curves ranging from 0.104 to 33.3ng/ml were validated. The inter-day and intra-day variance for all metabolites was less than 15%. The extraction recovery efficiency and matrix (deionized water) effects measured at 12.5ng/ml (750pg on column) ranged from 88 to 100% and 3 to 14%, respectively, with CV% values below 20%. Additionally, applying the processing and extraction conditions of this method to our previous CYP450 eicosanoids method resulted in overall improvement in extraction recovery and reduction in matrix effects at low (0.417ng/ml) and high (8.33ng/ml) concentrations. In rat brain cortical tissue samples, concentrations of prostanoids ranged from 10.2 to 937pmol/g wet tissue and concentration of eicosanoids ranged from 2.23 to 793pmol/g wet tissue. These data demonstrate that the successive measurement of prostanoids and eicosanoids from a single extracted sample of rat brain tissue can be achieved with a UPLC-MS/MS system and that this method is necessary for evaluation of these metabolites to delineate their role in various neuroinflammatory and cerebrovascular disorders.
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261, USA.
authors.forename: Jafar Sadik B
authors.initials: JS
authors.lastname: Shaik
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261, USA.
authors.forename: Tricia M
authors.initials: TM
authors.lastname: Miller
authors.affiliation: Geriatric Research Educational and Clinical Center, V.A. Pittsburgh Healthcare System, Pittsburgh, PA 15261, USA; Department of Neurology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15261, USA.
authors.forename: Steven H
authors.initials: SH
authors.lastname: Graham
authors.affiliation: Division of Pediatric Emergency Medicine, Department of Pediatrics, Children's Hospital of Pittsburgh, Pittsburgh, PA 15261, USA; Safar Center for Resuscitation Research, University of Pittsburgh, Pittsburgh, PA 15261, USA.
authors.forename: Mioara D
authors.initials: MD
authors.lastname: Manole
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261, USA. Electronic address: poloyac@pitt.edu.
authors.forename: Samuel M
authors.initials: SM
authors.lastname: Poloyac
mesh_terms: D000818:Animals; D001923:Brain Chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D057230:Limit of Detection; D008297:Male; D011453:Prostaglandins / Q000032:analysis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D011453:Prostaglandins
keywords: AA; Arachidonic acid; COX; CV; CYP450; Cyclooxygenase; DiHETE; EET; Eicosanoids; HETE; Ischemic stroke; PG; Prostanoids; QC; SPE; UPLC–MS/MS; arachidonic acid; coefficient of variation; cyclooxygenase; cytochrome P450; dihydroxyeicosatrienoic acid; epoxyeicosatrienoic acid; hydroxyeicosatetraenoic acid; prostaglandins; quality control; solid phase extraction; ultra performance liquid chromatography tandem mass spectrometry