2.4. Prostaglandin E2 (PGE2) Quantification

The weighed frozen brain samples (approximately 20 mg) were transferred into Eppendorf tubes. Subsequently, 80% (v/v) aqueous solution of a prechilled methanol (HPLC grade) was added with the adjusted volume based on tissue weight (400 μL of solvent per 20 mg of tissue). The tissue was ground with a small pestle grinder on dry ice. Two cycles of centrifugation 14,000× g for 10 min at 4 °C were applied. The supernatant was filtered through a 0.2-μm syringe filter (Acrodisc® CR 13 mm Syringe Filter, PALL life science, Ann Arbor, MI, USA), which was followed by LC-MS/MS analysis described previously [28]. Agilent 1200 Series Rapid Resolution LC System with Agilent 6410 triple quadrupole mass spectrometer was equipped with an electrospray ionization source (Agilent Technologies Inc., Wilmington, DE, USA). The Poroshell 120 EC-C-18 column (50 mm × 2.1 mm, 2.7 μm) was used for liquid chromatography prior to mass spectrometric analysis. The eluents were water containing 0.1% (v/v) formic acid (eluent A) and acetonitrile (eluent B). An isocratic gradient with 10% eluent B was applied. The flow rate was 0.2 mL/min, the column temperature was 40 °C, and the injection volume was 5 μL. The following mass spectrometry parameters were used: negative ion mode for drying gas (nitrogen) temperature of 300 °C, drying gas flow rate at 8 L/min, nebulizer pressure of 20 psi, and capillary voltage of 3500 V. The fragmentor voltages were 180 V and the collision energy was 6 V. Analyte detection was performed using multiple reaction monitoring with the transitions 351.4 → 315.4 and 351.4 → 271.1 (qualifier). The data was acquired using the Agilent Mass Hunter Workstation Acquisition software (Data Acquisition for Triple Quadrupole Mass Spectrometer, version B.03.01) and processed with Quantitative Analysis (B.04.00) software. The lower limit of quantification (LLOQ) for PGE2 was 1 nM. The linearity of the calibration curve (1–100 nM) as well as the selectivity, accuracy, and precision of the method were acceptable. Within-run accuracy and precision of quality control samples were ±20% of the nominal concentration.

The plasma samples were prepared in a similar manner. Plasma samples were transferred to Eppendorf tubes and 80% (v/v) aqueous solution of a pre-chilled methanol was added (1:4, v/v). The samples were gently shaked and incubated for 6 h at −80 °C, which was followed by centrifugation at 14000× g for 10 min at 4 °C. The supernatant was filtered using a 0.2 μm syringe filter (Acrodisc® CR 13 mm Syringe Filter, PALL life science, Ann Arbor, MI, USA). The samples were immediately analyzed using the LC-MS/MS method described above [28]. The statistical analyses were performed using GraphPad Prism v. 5.03 software (GraphPad Software, San Diego, CA, USA).