Shim pack vp ods

HPLC was chosen to detect the concentration of PB. The first step was to configure the standard solution. PB standards were accurately weighed and dissolved in methanol to obtain a 500 μg/mL PB stock solution. A certain volume of the PB stock solution was precisely measured in a volumetric flask and diluted with methanol to obtain a series of standard solutions with different concentrations (0.1, 0.2, 0.5, 1, 2.5, 10, 20, and 30 μg/mL). Then, 50 μL of ultrapure water and 50 µL of the PB solution from the series were mixed to obtain a series of samples with concentrations of 0, 0.05, 0.1, 0.25, 0.5, 1.25, 5, 10, and 15 μg/mL. Then, 300 µL of methanol were added to the sample, and the sample was analyzed after sufficient mixing. The ordinate represents the peak area of PB (A), and the abscissa represents the concentration of PB (ρ, μg/mL). The standard curve was drawn, and the minimum detection limit was calculated. Finally, the sample was processed and tested. Fifty microlitres of ultrapure water, 50 µL of sample and 300 µL of methanol were mixed and injected for determination.
Chromatography conditions: The column type was Shim-pack VP-ODs (4.6 × 150 L, SHIMADZU), and methanol-water (95:5, V/V) was used as the mobile phase. The injection volume was 20 μL, and the flow rate was 1.0 mL/min. Chromatographic information was acquired at a wavelength of 242 nm at room temperature.

ALE was dissolved in 80% methanol and centrifuged for 5 min at 3,000 rpm. The supernatant was used as the analytical sample for the determination. The components of ALE were determined with the Prominence LC-20A (Shimadzu, Kyoto, Japan). The HPLC system was equipped with a CMA-20A communications bus module, LC 20AD liquid chromatograph, SPD-M20A diode array detector, SIL-20AC autosampler, DGU-20A3 degasser, and CTO-20AC column oven. HPLC analysis was conducted using a Shim-pack VP-ODS (3 × 75 mm, 2.2 μm, Shimadzu, Japan) The flow rate of the mobile phase was 0.15 ml/min. The chromatogram was monitored at 330 nm. The injection volume was 10 μL. The column temperature was maintained at 30°C. The mobile phase, consisting of 100% acetonitrile (A) and water containing 0.1% formic acid (B), was run with the gradient programs shown in Table 1. The LC/MS-IT-TOF was run with the gradient programs shown in Table 1. The LC/MS-IT-TOF was operated with a nebulizer gas flow rate of 1.5 ml/min, detector voltage of 1.53 kV, and probe voltage of 450 kV. The mass range (m/z) was 100–1500 amu.

Serotonin and 5-HIAA (5 mg) were dissolved in 100 mL of eluting solution (50 ml MilliQ water, 0.43 ml HClO₄ 70% [0.2 N], 10 mg EDTA, 9.5 mg sodium metabissulfite) and frozen at −20°C, to later be used as a standard.
The HPLC system consisted of a delivery pump (LC20-AT, Shimadzu), a 20 µL sample injector (Rheodyne), a degasser (DGA-20A5), and an analytical column (Shimadzu Shim-Pack VP-ODS, 250×4.6 mm internal diameter). The integrating recorder was a Shimadzu CBM-20A (Shimadzu, Kyoto, Japan). An electrochemical detector (Model L-ECD-6A) with glassy carbon was be used at a voltage setting of +0.83 V, with a sensitivity set at 8 nA full deflection. The mobile phase consisted of a solution of 70 mM phosphate buffer (pH 2.9), 0.2 mM EDTA, 5% methanol and 20% sodium metabissulfite as a conservative. The column temperature was set at 17°C, and the isocratic flow rate was 1.8 ml/min. 0.5 mL of extracellular fluid (ECF) were extracted by quickly removing one brain from the skull and incubating it in 2 mL of 50 mM TBS, pH 7.4, containing 90 mM NaCl, 2.5 mM CaCl2, 1 mM glutathione for 30 min at 4°C (7). This fluid was then mixed with 0.5 mL of eluting solution, filtered through a 0.22 µm syringe filter, and then injected into the HPLC system.

The PTA, BHET, and MHET released in the fermented broth were obtained using liquid–liquid extraction (adapted from Kim and Lee [44 (link)]) (Figure 3). The chromatographic separations were performed on a Shimadzu equipped with an octadecylsilane (C18) column Shim-pack VP-ODS (4.6 mm × 250 mm, 5 µm) coupled to a pre-column of the same material (Shimadzu, Santa Clara, CA, USA), with a manual injector and using the following mobile phases: Solvent A: 0.5% (v/v) formic acid:acetonitrile, HPLC grade 90:10, and Solvent B: 0.5% (v/v) formic acid:acetonitrile, HPLC grade 60:40. These mobile phases were followed by an elution gradient, according to Table S1 (supplementary material), in a flow of 0.5 mL·min⁻¹. The analytes were detected at a wavelength of 254 nm. Benzoic acid (retention time = 23.4 min) was used as an internal standard. The method was the following: linearity from 0.15 ppm to 300.0 ppm for PTA, BHET, and MHET, with a correlation coefficient of 0.999 for the three analytes (6 calibration solutions n = 3); precision determined by the relative standard deviation (variation coefficient) between 1.1% and 31.1%. The latter was the lowest concentration of the curve. The chromatogram in Figure 4 shows the separation efficiency of the methodology used.

Phenolic analysis of total grape pomace (skins, seeds and stalks) was performed with a methanol solution with 10 mg mL⁻¹ crude extract that was solubilized with ultrasound, filtered through a 0.45 µm nylon filter and injected into the loop (20 µL) at a flow rate of 0.7 mL min⁻¹ at ambient temperature [16 (link)]. The polyphenols were separated in a C18 reversed-phase column, Shim-pack VP-ODS (250 × 4.6 mm; 5 µm particle size) with a system controller CBM-20A (Shimadzu Co., Kioto, Japan) connected to binary pumps LC-20AD (Shimadzu^®) that had a UV/VIS SPD-20A detector (Shimadzu^®), CTO-20A (Shimadzu^®) and self-injector SIL 20AC (Shimadzu^®). The solvent system used to elute the compounds was: A—0.1% acetic acid water solution, and B—methanol. The following gradient conditions were used: 0–10 min (95% A/5% B); 10–60 min (50% A/50% B); 60–80 min (30% A/70% B); and 80–90 min (95% A/5% B). A chromatographic profile of the extracts was obtained at a wavelength of 254 nm. The identification of the compounds in grape pomace was performed in an AmaZon ETD (Bruker Co., Billerica, MA, USA) instrument with positive ion mode mass spectrometry using 27 psi and a capillary voltage of 4500 V.