Prominence lc 20ad system

Quantitative analysis of the PMX and DFOA was performed using ion-exchange HPLC using a Prominence LC-20AD system (Shimadzu, Kyoto, Japan) equipped with a diode matrix detector and an ultra-short monolithic analytical CIM-SO₃ column (5 × 5 mm) (BIA Separations, Ljubljana, Slovenia). Buffer solutions were used as the mobile phases: A—0.005 M sodium phosphate buffer solution, pH 7.0; B—2 M NaCl. Analysis was performed under binary gradient conditions: 0–2 min—100% A, 2–7 min—0 to 100% B, 7–12 min—100% B. The mobile phase flow rate was 0.5 mL/min. Detection was performed at 215 nm. DFOA and PMX B retention times were 3.5 and 7.5–7.9 min, respectively. The quantification was carried out regarding a calibration plot pre-built for PMX and DFOA in the range of concentration 0.05–5.00 mg/mL and 0.05 to 2.00 mg/mL, respectively.

Phenolic compounds were purified using a column Xbridge^® BEH C18 (4.6 mm × 250 mm, 130 Å, 5µm particle size, Waters, Milford, MA, USA). The column was connected to the Shimadzu Prominence LC-20AD system, including a CBM-20A controller, two LC-20 AD XR pumps, and a DGU-20As online degasser, equipped with an SPD-M20A UV detector, and an autocollector FRC-10A (Shimadzu) was employed. Data acquisition was performed by the LabSolution version 5.53 software (Shimadzu, Kyoto, Japan). The detector was set at 280 nm. The sample was eluted at a flow rate of 0.6 mL min⁻¹ using ddH₂O with 10 mmol L⁻¹ ammonium formate at pH 10 as phase A and MeOH:ddH₂O (90:10, v/v) with 10 mmol L⁻¹ ammonium formate at pH 10 as phase B. The gradient started at 5% of B, then increased to 50% in 44 min; then, the column was equilibrated for 10 min. Six fractions were collected as follows: F1 (1–11 min), F2 (12–14 min), F3 (15–16 min), F4 (16–19 min), F5 (20–25 min), and F6 (26–40 min). The number of collected fractions from chromatographic separation is shown in Figure 1A.
Each collected fraction (F1–6) was subjected to a PLpro bioactivity test to identify the most active ones.