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.

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.

Figure 1

Chemical structures of prostanoid metabolites.

Figure 2

Chromatographic profiles, corresponding to 25 pg on column, depicting the separation of all prostanoids using a UPLC tandem MS/MS triple quadrupole mass spectrometer. Separation of metabolites was performed on a reversed-phase Acquity BEH C-18 column (2.1 × 150 mm; 1.7 μm i.d.). Metabolites were eluted at a flow rate of 0.4 ml/min over 12 min run time with a gradient from 35% acetonitrile containing 0.005% acetic acid to 98% acetonitrile containing 0.005% acetic acid.

Figure 3

Concentrations of prostanoid metabolites detected in brain cortical tissue of pediatric rats at 5 min post resuscitation after being subjected to 12 min of asphyxial cardiac arrest. Quantitative amounts of PGD₂, PGJ₂, PGE₂, 15-d-PGD₂, PGA₂, 15-d-PGJ₂, PGF_2α, PGF_1α, 11β-PGF_2α were measured in these tissue samples.

Figure 4

Concentrations of eicosanoid metabolites detected in brain cortical tissue of pediatric rats at 5 min post resuscitation after being subjected to 12 min of asphyxial cardiac arrest. Quantitative amounts of 12-, 15-, 20-HETE, 8,9-, 11,12-, 14,15-EET, and 11,12-, 14,15-DiHETE were measured in these tissue samples. These eicosanoids were analyzed from same tissue extracts used for prostanoids measurement.