Lc solution software

The HPLC apparatus consisted of a Shimadzu system (Shimadzu Corporation, Kyoto, Japan) equipped with a solvent delivery system (LC-20AD), a DGU-20A₃ on-line degasser, a SIL 20A HT autoinjector, a CTO-20A column oven, an SPD-M20A photodiode-array detector, and a CBM-20A system controller. The chromatographic system was equipped with a Rheodyne Model 7125 injector (Rheodyne Corp., Cotati, CA, USA) with a 20 μL loop. The system control and data acquisition were performed with Shimadzu LC solution software (Shimadzu Corporation).
The chromatographic separations were performed on a PhenoSphere SCX (5 μm, 150×4.6 mm) analytical column (Phenomenex Inc., Budapest, Hungary), protected by a 5 μm guard column. The column temperature was maintained constant at 35°C. Separations were performed in isocratic mode. The mobile phase used for the separation consisted of 0.5 M NaH₂PO₄ buffer (pH =3.5):acetonitrile =45:55 (v/v) pumped at a flow rate of 1 mL/min. The mobile phase was filtered by a Millipore vacuum filtration system equipped with a 0.45 μm pore size filter and degassed by ultrasonication. The detection wavelength was 200 nm.

The content of IBU was determined by the HPLC method. The HPLC instrumentation included a CBM-20Alite system controller, a Shimadzu LC-20AT solvent delivery system, a Shimadzu SPD-M20A UV/VIS photodiode array detector, a Shimadzu SIL-20A auto injector and a Shimadzu CTO-20A column oven. The chromatographic data were collected and processed by means of Shimadzu LC solution software (Shimadzu Corp). A Hypersil (4.6 × 250 mm, 5 μm) analytical column was used. The eluate was monitored at 263 nm. The mobile phase was an acetonitrile-sodium acetate buffer (pH adjusted to 2.5 with acetic acid) (60:40, v/v) with a flow speed of 1.0 mL/min. The injection volume was 20 µL, and the column temperature was 35°C. The run time was 10 minutes and the retention time of IBU was approximately 5.4 minutes. A linear correlation was acquired between peak area and concentration. The linear equation was y=447.69x-4703.6 (R²=0.9992), where x is the concentration and y is the peak area. The assay was linear in the concentration of 1.0~150 µg/mL. The limit of detection was 0.5 µg/mL.

BAT samples were dissected on a chilled plate, and immediately flash-frozen in dry ice before being stored at −80 °C until further analysis. The frozen tissue was subsequently homogenized, sonicated, and centrifugated at 14,000 g for 20 min at 4 °C. The resulting supernatant fraction was filtered and then injected into a high-performance liquid chromatography (HPLC) system (Shimadzu LC Prominence; Shimadzu Corporation) [27 ,28 (link)]. To separate norepinephrine (NE), dopamine (DA), and serotonin (5-HT), a reverse-phase analytical column (Waters Symmetry 300C18; Waters) was employed. The mobile phase consisted of a 10% MeOH solution (pH = 4) containing 70 mM KH₂PO₄, 1 mM octanesulfonic acid, and 1 mM EDTA, delivered at a flow rate of 1 ml/min. Detection of the neurotransmitters was achieved using a coulometric electrochemical detector (ESA Coulochem III; Thermo Scientific). The first and second electrodes of the analytical cell were set at +50 mV and +350 mV, respectively, while the guard cell was set at −100 mV. Data acquisition and processing were performed using the Shimadzu LC solution software (Shimadzu Corporation). The concentrations of the neurotransmitters were expressed as pg/mg of wet tissue, as shown [27 ,28 (link)].