Ultimate 3000 pump

The digested samples were applied to MS analyses. The liquid chromatography with tandem MS (LC-MS) system comprised a PAL HTC-xt autosampler (AMR Inc., Tokyo, Japan) and Ultimate 3000 pump (Thermo Fisher Scientific) connected to a C18 tip column (10 cm length and 75 μm inner diameter, Nikkyo Technos Co., Ltd. Tokyo, Japan), and Orbitrap LTQ-Velos (Thermo Fisher Scientific). Raw data were processed with the Proteome Discoverer version 1.4.1.14 (Thermo Fisher Scientific) using the standard workflow. A database search was performed using the Mascot search engine (version 2.6.0, Matrix Science, London, UK) against SwissProt. Quantitative evaluations were conducted using an emPAI index [22 (link)].

The chemical components of LSW-ET were detected by HPLC DionexTM Ultimate 3000 system (Thermo Fischer Scientific, Inc., USA) equipped with an Ultimate 3000 pump, an Ultimate 3000 autosampler, and an Ultimate 3000 column compartment. Separation was performed using a C18 column (4.6 × 250 mm, 5 μm, Kromasil) and an optimized mobile phase composed of acetonitrile (solvent A) and 0.2% formic acid water solution (solvent B, v/v). For the combinative elution, elution was achieved using the following conditions: 0-3 min, 9% solvent A; 3-13 min, 9-12% solvent A; 13-15 min, 12-20% solvent A; 15-25 min, 20-30% solvent A; 25-30 min, 30-51% solvent A; 30-50 min, 51% solvent A (total run time 50 min). The detection wavelength was set at 296 nm and the flow rate was 0.8 mL/min. The column temperature was set at 30°C and the injection volume was 10 μL.

Animals were sacrificed by decapitation 3 h after cessation of treatment with drugs. Brains were separated, and several brain regions (striatum, frontal cortex) were dissected in anatomical borders. The tissue levels of DA, 5-HT, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) were measured using a HPLC with electrochemical detection. Briefly, tissue samples of brain structures were homogenized in ice-cold 0.1 M HClO₄ and were centrifuged at 10000×g for 10 min at 4 °C. The supernatant (3–5 μl) was injected into the HPLC system. The chromatography system consisted of an LC-4C amperometric detector with a cross-flow detector cell (BAS, IN, USA), an Ultimate 3000 pump (Thermo Scientific, USA), and a Hypersil Gold analytical column (3 μm, 100 × 3 mm, Thermo Scientific, USA). The mobile phase consisted of 0.1 M KH₂PO₄, 0.5 mM Na₂EDTA, 80 mg/l sodium 1-octanesulfonate, and a 4% methanol, adjusted to pH 3.7 with an 85% H₃PO₄. The flow rate was 1 ml/min. The potential of a 3-mm glassy carbon electrode was set at 0.7 V with sensitivity of 5 nA/V. The temperature of the column was maintained at 30 °C. The Chromax 2007 program (Pol-Lab, Warszawa, Poland) was used for data collection and analysis.

Animals were sacrificed by decapitation 3 h after intraperitoneal drugs administration. Brains were separated and several brain regions (striatum, nucleus accumbens septi, frontal cortex) were dissected in anatomical borders. The tissue levels of DA, 5-HT, DOPAC, HVA, and 5-HIAA were measured using a high-performance liquid chromatography (HPLC) with electrochemical detection. Briefly, tissue samples of brain structures were homogenized in ice-cold 0.1 M HClO₄ and were centrifuged at 10,000g for 10 min at 4 °C. The supernatant (3–5 µL) was injected into the HPLC system. The chromatography system consisted of an LC-4C amperometric detector with a cross-flow detector cell (BAS, IN, USA), a Ultimate 3000 pump (Thermo Scientific, USA), and a Hypersil Gold analytical column (3 μm, 100 × 3 mm, Thermo Scientific, USA). The mobile phase consisted of 0.1 M KH₂PO₄, 0.5 mM Na₂EDTA, 80 mg/L sodium 1-octanesulfonate, and a 4 % methanol, adjusted to pH 3.7 with an 85 % H₃PO₄. The flow rate was 1 mL/min. The potential of a 3-mm glassy carbon electrode was set at 0.7 V with sensitivity of 5 nA/V. The temperature of the column was maintained at 30 °C. The Chromax 2007 program (Pol-Lab, Warszawa, Poland) was used for data collection and analysis.

Standard stock solutions of ten different substances—quinic acid, gallic acid, chlorogenic acid, caffeic acid, catechin, quercetin, kaempferol, rutin, apingenin, and apingenin glycoside—were prepared in 50% acetonitrile (2 mg/mL). All solutions were filtered prior to analysis through a 0.45 μm syringe filter and injected three times into the HPLC. The phenolic compounds were identified by analyzing the same extracts with no added standard compounds and with added standard compounds. LC analysis was performed on a DIONEX UltiMate 3000 UHPLC+ focused system (Thermo Fisher Scientific, Waltham, MA, USA), which consists of an UltiMate 3000 pump, UltiMate 3000 autosampler, UltiMate 3000 column compartment, UltiMate 3000 variable wavelength detector, and Chromeleon software. As a stationary phase, an Ascentis Express 90 Å AQ-C18 column (15 cm × 3.0 mm, 2.7 μm, Supelco, Darmstadt, Germany) was used. The mobile phase was composed of 0.1% aqueous trichloroacetic acid (v/v) (A) and acetonitrile (B) with the following gradient elution: 0 min—95% A, 10 min—75% A, 25 min—20% A, and 30 min—95% A. Before each sample analysis, a 4 min equilibration with 5% B was performed. The flow rate was set at 0.425 mL/min, the column temperature was 40 °C, the injection volume was 1 μL, and the time of analysis was 30 min.