Shim pack clc ods column

Cells were harvested after 2-hour incubation with 5 mM _L-ascorbic acid and washed with PBS. The cells were resuspended in 1 mL of PBS with 10% meta-phosphoric acid (MPA) solution and lysed three times by freeze-thaw cycles in a −80 °C deep freezer. The lysate was centrifuged at 16000 rpm at 4 °C for 5 minutes and the supernatant was harvested. Next, 100 μL of sample was mixed with 100 μL of precipitation reagents of vitamin C diagnostics kits (Chromosystems, Gräfelfing, Germany) and incubated for 10 minutes at 4 °C. The mixture was centrifuged at 13000 rpm for 5 minutes and the supernatant was analyzed using a high-performance liquid chromatography (HPLC) system (Shimadzu Corporation, Tokyo, Japan) equipped with Shim-pack CLC-ODS column (6 mm × 15 cm) connected to a Shim-pack G-ODS guard column (4 mm × 1 cm) (Shimadzu). The mobile phase was provided by Chromsystems and the experiment was performed according to the instruction manual. The concentration of _L-ascorbic acid in cells was determined by manual calculation $C_{Analyte,Sample}(mg/l)=\frac{A_{Sample}\times I{S}_{Standard}}{A_{Standard}\times I{S}_{Sample}}\times C_{Standard}$ . The following instrument settings were used: injection volume 20 μL, run time 10 min, flow rate 1 mL/min, column temperature 25 °C, and UV detector wavelength 245 nm.

HPLC was used to identify and quantify the flavonoid and phenolic contents of PAAPEE. The sample which is prepared by dissolving 10 mg of PAAPEE in 5 mL distilled water and 12 mL ethanol and after that the solution was vortexed to ensure homogeneity. The reaction mixture was then placed in an oven at 90°C for 2 h, after which distilled water (6 mL) was added, followed by 10 mL of 15 M of HCl. The reaction mixture was put in an oven at 90°C for 2 h (Sultana and Anwar, 2008 (link)). The samples were filtered using syringe filters. The flavonoids and phenolic metabolites were separated using a Shim-Pack CLC-ODS column (Shimadzu, Japan) where its stationary phase is octadecyl group; its particle size is 5 μm; its dimensions are 6.0 mm i. d X 15 cm with reversed phase as the separation mode and catalog number of 228-00808-91. The flow rate was 1 mL/min; the column length was 10 cm. Gradients A (H₂O: AA-94:6, pH = 2.27), B (CAN 100%), 0–15 min = 15% V, 15–30 min = 45% B, and 30–45 min = 45% B were used in the mobile phase. A 280-nm UV-visible detector (SPD-10AV) and an LC-10AV pump were used (Hamed et al., 2021 (link)).

Brazilian green propolis (EPP-AF^®) extract and gel were evaluated using an HPLC/DAD system (Shimadzu apparatus equipped with a CBM-20 A controller, a LC-20AT quaternary pump, an SPD-M 20 A diode-array detector, and Shimadzu LC solution software, version 1.21 SP1) coupled to a Shimadzu Shim-Pack CLC-ODS column (4.6 mm × 250 mm, 5 µm particle diameter, 100 Å pore diameter). The mobile phase consisted of methanol (HPLC grade) and a water-formic acid solution (0.1% v/v), pH 2.7 (A). The method consisted of a linear gradient of 20%–95% methanol over a period of 77 min at a flow rate of 0.8 mL/min. Detection was set at 275 nm, in accordance with a previously published protocol (Berretta et al., 2012 (link)). Samples were diluted in 5 mL of methanol in 10 mL volumetric flasks, subjected to sonication for 10 min and filled to volume with Milli-Q water. All samples were filtered through a 0.45 µm filter before analysis. The chemical references used were caffeic acid (Sigma-Aldrich, L: SLBZ6416), p-coumaric acid (Sigma-Aldrich, L: 091M119V), 3,5 dicaffeoylquinic acid (Phytolab, L. 3215), 4,5–dicaffeoylquinic acid (Phytolab, L. 9943), galangin (Sigma-Aldrich: BCCG2648), artepillin C (Phytolab, L: 111674647), as well as aromadendrin-4′-O-methyl ether, drupanin and baccharin previously isolated by De Sousa et al. (2007) (link).