Turbovap classic lv

The extracts (2.5 mL) were transferred to a 10 mL glass vial and taken to full dryness under a gentle stream of nitrogen at 50 °C using a TurboVap Classic LV (Biotage, Charlotte, NC, USA) at constant pressure (12 psi for 1h and 16 min). Each extract was resuspended individually in 2 mL of distilled water and frozen at −20 °C for 48 h. Subsequently, the extracts were lyophilized using a Labconco freeze dryer, model 77530 (Labconco, Kansas City, MO, USA) at −56 °C for 72 h and under high vacuum conditions (0.014 mBar). Once dried, the extracts were stored under refrigeration at 4 °C protected from light.

Samples of water were defrosted at room temperature. Then, 5 mL was passed through a 0.45 μm syringe filter Filtropur S (Sarstedt, Nümbrecht, Germany) into 10 mL autosampler glass vials, and 5 ng of internal standard was added. For the tissue samples, fish were defrosted at room temperature, and then 0.1 g of muscle tissue from dorsal part of the body and whole brain were sampled into 2 mL polypropylene tubes from each fish. To extract caffeine from tissue samples, 1.5 mL of acetonitrile was added to the tubes together with 10 zirconium beads and samples were spiked with 50 ng of internal standard. Homogenization at 42000 oscillations per minute (Mini Beadbeater, BioSpec Bartlesville, USA) and centrifugation at 17500g for 10 min (Beckman Coulter Microfuge 22R Centrifuge) followed. The supernatant was pipetted into the 12-mL glass vial and whole extraction process was repeated with new 1.5 mL acetonitrile. The supernatants from both extractions were combined, evaporated to near dryness (TurboVap Classic LV, Biotage AB, Sweden), the eluent was reconstituted with 150 μL of methanol, and transferred into the 2 mL autosampler vials equipped with 200 μL inserts. The final extracts were frozen for a minimum of 24 h to ensure protein precipitation and centrifuged again directly before analysis.

Viola odorata L., collected in Bělkovice Valley (Olomouc Region, Czech Republic), was identified by Michal Hroneš, Ph.D. (Department of Botany, Palacký University Olomouc). The collection complied with all applicable laws/guidelines, both institutional and national. According to IUCN guidelines, this taxon is not threatened being qualified for Least Concern category. As a common plant species, it’s collection for scientific purpose requires no permission/license. Plants were freeze-dried and homogenized using blade grinder. 30 mg of dried plant material (DW) was extracted in a microtube using 1 mL of 0.1% HCOOH in methanol. Glass balls were added to the microtube and the plant material was homogenized for 5 min using an MM 400 oscillatory ball mill (RETSCH, Germany) operating at 27 Hz. An ultrasound-assisted extraction (10 min, 45 kHz) in a USC100T ultrasonic bath (VWR, USA) and subsequent centrifugation (21,300 g, 25 °C, 10 min) followed. The supernatant was collected and evaporated at 37 °C in a TurboVap Classic LV nitrogen evaporator (Biotage, Sweden).