Empower 3

Ferroverdin extraction with ethyl acetate, drying, and resuspension in acetonitrile were performed as described previously [19 (link)]. Samples were analyzed by HPLC and Ferroverdin A was detected at 440 nm and quantified by peak area integration, as described previously [19 (link)]. Data were analyzed using Empower 3 (Waters, Milford, MA, USA). The HPLC-based protocol for ferroverdin A semi-quantitative analysis is detailed in Appendix A.

The buffer of the nanobody was changed to 1 × PBS (pH = 7.4) by ultrafiltration discs (Merck, PLBC07610). 50 μL nanobody (1 mg/mL) was filtered through a 0.2 μm filter. After that, filtered samples were prepared for HPLC analysis. An HPLC instrument (Waters, ACQUITY UPLC H-Clas) was used for purity analysis of proteins. PBS and a gel permeation chromatography column (TSKgel G3000SW_XL, Tosoh Bioscience, 0008541) were used as the mobile phase and the stationary phase, respectively. The pump was running for 5 min, mainly to eliminate air bubbles in the system, and then all exhaust valves were closed. The mobile phase was run at a fixed rate (1 mL/min) until the baseline was stable. Then, the operating parameters (flow rate: 1 mL/min, pressure: 3.3 MPa, temperature: 25 °C, detection: UV 280 nm) of the sample were set in the software (Waters, Empower3). After that, the sample was injected into the sampling valve, and the line was monitored by software. The sample was allowed to run for 20 min (more than 1.5✕column volume) before stopping.

The hydroxyl and carboxyl contents of tannins were determined according to Granata and Argyropoulos [33 (link)] from freshly phosphitylated tannins by ³¹P NMR on a Bruker 500 MHz NMR spectrometer at room temperature using a previously published method. Nitrogen contents were determined as described by Nordlund [34 (link)]. Carbohydrates were determined by HPAEC-PAD from acid hydrolysed samples [35 (link)]. Molar mass measurements of tannins, dissolved in 0.1 M NaOH and filtered (0.45 μm), were performed by size exclusion chromatography using 0.1 M NaOH as the eluent (pH 13, 0.5 mL/min, T = 25 °C) on PSS MCX 1000 and 100,000 Å columns. The elution curves were detected using a Waters 2998 Photodiode Array detector at 280 nm. The weight (M_w)- and number (M_n)-average molar masses were calculated against polystyrene sulfonate standards (eight standards with a range of 3420−148,500 g/mol) using Waters Empower 3 (Milford, MA, USA) software.

The quantification of DCF was performed by an Alliance 2690 (Waters Corp, Milford, MA) HPLC system equipped with a photodiode array detector and a computer software (Empower 3, Waters). DCF was determined at 280 nm using a SunFire C18 column (3.5 µm, 4.6 mm × 100 mm, Waters, Milan, Italy), and a mixture of 40.75% water, 59.225% acetonitrile, and 0.025% acetic acid (v/v) as a mobile phase, delivered at a flow rate of 0.5 mL/min. A calibration curve was built by using standard solutions (0.01–0.5 mg/mL) prepared by the dilution of a stock standard solution. The limit of detection was 1 ng, while the limit of quantification was 2 ng.

Quantitative determination of BDP and CUR was performed by HPLC using a liquid chromatograph Alliance 2690 (Waters Corp, Milford, MA, USA) equipped with a photodiode array detector and a computer integrating apparatus (Empower 3). Analyses were performed with a Sunfire C18 column (3.5 µm, 4.6 mm × 150 mm, Waters). The mobile phase was a mixture of acetonitrile, water, and acetic acid (95:4.84:0.16 v/v), delivered at a flow rate of 0.5 mL/min. Samples (10 µL) were injected using an auto sampler. CUR was revealed at 421 nm, whereas BDP was at 240 nm. The stock standard solutions of CUR and BDP were prepared by dissolving the drug in methanol and stored at 4 °C. A standard calibration curve (peak area of CUR/BDP vs. known drug concentration) was built up by using standard solutions prepared by dilution of the stock standard solution with the mobile phase. Calibration graphs were plotted according to the linear regression analysis, which gave a correlation coefficient value (R²) of 0.999. The described HPLC method was also used to quantify the drug in the obtained nanosuspensions and to evaluate the presence of any degradation products. The limit of quantification was 1 ng/µL for CUR and 0.5 ng/µL for BDP, while the limit of detection was 0.2 ng/µL for both compounds. Sample preparation and analyses were performed at room temperature.