Cbm 20a controller

The HPLC setup was based on a Shimadzu system and consisted of a CBM-20A controller, a SIL-HTA auto sampler, two 10ADVP pumps, a DGU-14A degasser, a CTO-10AVP column oven and an SPD-10AVP variable wavelength detector. The setup was coupled to a micrOTOF time of flight mass analyzer (Bruker) and controlled by Compass HyStar (Bruker) version 3.2 and microTOFControl (Bruker) version 3.0. Measurement data was analyzed using Compass DataAnalysis version 5.0 R1 (Bruker).
The column oven was set to 40 °C. A SeQunant ZIC-HILIC (Merk KGaA, 150 × 2.1 mm, particle size 3.5 µM, pore size 200 Å) with a SeQunant ZIC-HILIC (Merk KGaA, 20 × 2.1 mm, particle size 5 µM, pore size 200 Å) precolumn was used as stationary phase. The mobile phase was a mixture of water (0.1 % NH₄FA, pH 3.2) and acetonitrile. The injection volume was 5 µL. The following gradient was used with a flow rate of 150 µL/min (Table 2).

The quantitative and qualitative analysis of polyphenolic compounds was performed using the high-performance liquid chromatography (HPLC) method described earlier by Król et al. [47 (link)]. Briefly, a 3 mL sample of coffee brew was combined with 5 mL of 80% methanol. The mixture was then mechanically shaken and subjected to extraction in an ultrasonic bath (30 °C, 10 min). Following extraction, the sample was centrifuged (5 °C, 3780× g, 10 min) and was subsequently subjected to chromatographic analysis (HPLC-DAD using two LC-20AD pumps, a CBM-20A controller, an SIL-20AC column oven, and UV/Vis SPD-20 AV, and SPD-M20A spectrometers; Shimadzu, Kyoto, Japan).
Chromatographic separation was carried out on a Phenomenex Fusion-RP 80A column (Torrance, CA, USA, 250 mm × 4.60 mm). The mobile phase consisted of two components: (A) 90% water and 10% acetonitrile, and (B) 45% water and 55% acetonitrile, both with a pH of 3.0. The applied gradient program is detailed in Table 3. The injection volume was set at 100 μL, with the sample temperature maintained at 30 °C, and the column temperature also held at 30 °C. UV detection was performed at wavelengths: λ = 250 nm for flavonoids (quercetin-3-O-rutinoside, kaempferol-3-O-glucoside, quercetin, quercetin-3-O-glucoside, kaempferol, epigallocatechin) and λ = 370 nm for phenolic acids (gallic, chlorogenic, caffeic, salicylic).

We used a developed LC‐HRAM method for the determination of peptide concentrations in mouse tissues (heart, kidney, liver, lung and spleen) and plasma. The linear range of the method was 0.200–50.0 µg ml⁻¹. The heart, kidney, liver, lung and spleen tissue samples of mice were degraded into 20% tissue homogenate (tissue: homogenate solution = 1:4) with protease K. Then, 50 µl samples (standard curve samples, quality control samples, blank samples and unknown samples) were added to 10 µl of internal standard solution (500 µg ml⁻¹ peptide in water) in addition to the blank samples. After vortex mixing, 100 µL of 5 mg mL⁻¹ Protein K in water was added. After 1500 rpm for 1 min, multitube vortex mixing and tape winding were subsequently performed at 1000 rpm in a 65 °C constant temperature incubator overnight. For serum testing, a 10 µl sample (500 µg mL⁻¹ peptide in water) of internal standard solution was added to the L sample (standard curve sample, quality control sample and blank sample). The next day, 1% FA in water and 200 µl of acetonitrile were added for protein precipitation, followed by mixing (1000 rpm, 5 min) and centrifuging (4000 rpm, 10 min). Then, 200 µl of supernatant was centrifuged at 1000 rpm for 1 min and then tested on a Shimadzu SIL‐30ACMP Autosampler with an LC‐30AD pump and a CBM‐20A controller (Shimadzu, Columbia, MD).

Fractions of crude extracts H and E were obtained through vacuum liquid chromatography (VLC) on silica gel (0.040–0.063 mm, Merck, Darmstadt, Germany) with solvent mixtures of increasing polarity (hexane, ethyl acetate, methanol, all Roth, Germany). Fractions were controlled by thin-layer chromatography (silica gel 60 F₂₅₄, Merck, Darmstadt, Germany) and those of similar substance patterns combined to yield five fractions for H (H1–H5) and 9 fractions for E (E1–E9).
Four compounds were isolated by semi-preparative HPLC using a Shimadzu CBM-20A controller, LC-20AT solvent delivery module, SIL-10AF autosampler, CTO-20AC column oven, SPD-M20A diode array detector, and FRC-10A fraction collector (all Shimadzu, Kyoto, Japan).
All separations were performed on a Luna C18(2) column, 250 × 10 mm, 10 μm (Phenomenex, Torrance, CA, USA) via isocratic elution with a flow rate of 4 mL/min, 25 °C column temperature; 200 μL injection volume.
Compounds 1 and 7 were obtained from E8 (VLC fraction with hexane:ethyl acetate:methanol = 4:80:16) by isocratic elution with a solvent composition of acetonitrile:water = 70:30 (v/v). Compound 4 was isolated with acetonitrile:water = 63:37 (v/v) from H4 (hexane:ethyl acetate:methanol = 36:60:4) and compound 6 with acetonitrile:water = 75:25 (v/v) from E2 (hexane:ethyl acetate = 70:30).