Lc solution software version 1.21 sp1

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).

HPLC analyses were performed on a Prominence LC 20A chromatographic system (Shimadzu, Kyoto, Japan) consisting of a DGU-20A3 degasser, two LC-20AD pumps, SIL-20AC autosampler, a CTO-20AC column oven, SPD-20AC detector and a CBM-20AC communication module. The data were processed by LC solution software, version 1.21 SP1 (Shimadzu).
Analysis of new chelators were performed using an Ascentis C18 chromatographic column (10×3 mm, 3 µm) protected with a guard column with the same sorbent (Sigma-Aldrich). The mobile phase was composed of 1 mM EDTA in 5 mM phosphate buffer and methanol in different ratios (Table 1). The column oven was set at 25°C and the autosampler at 5°C. A flow rate of 0.3 mL/min and injection volume of 20 µL were used. Chromatographic conditions for the determination of each chelator are given in Table 1.
The linearity, precision and accuracy of the methods were examined by the analysis of plasma samples spiked with different amounts of the chelators. Selectivity was confirmed by an analysis of blank plasma samples. All evaluated parameters reached acceptable values [40] . SIH was analyzed using a previously developed and validated method [41] (link).

Fresh and lyophilized RJ were analyzed by high-performance liquid chromatography (HPLC), using a Shimadzu apparatus equipped with a CBM-20A controller, a LC-20AT quaternary pump, a SPD-M 20A diode-array detector, and Shimadzu LC solution software, version 1.21 SP1. A Shimadzu Shim-Pack CLC-ODS (M) column (4.6 × 250 mm, particle diameter of 5 μm, pore diameter of 100 Å) was used. The mobile phase consisted of methanol in pump B and of a solution of water-phosphoric acid (0.02% v/v), pH 2.5, in pump D. The mixture was eluted using an isocratic elution with 50% B and 50% D over a period of 22 min at a flow-rate of 0.8 mL/min. Detection was set at 215 nm.
RJ was dissolved with 5 mL of methanol (HPLC grade) in 10 mL volumetric flasks, subjected to sonication for 10 min and diluted to volume with Milli-Q water. The samples were filtered through a 45 μm filter before analysis.