C18 spe cartridge

Sample preparation for the measurements of the phenolic analytes was performed according to the method previously described (Gris et al. 2013) with slight modifications. Briefly, 10 mL of wine diluted 10 times with H₂O was applied to a C18-SPE cartridge (1 g, Waters, Milford, MA, USA), previously activated with methanol (4 mL) and conditioned with H₂O (10 mL). The cartridge was washed with 50 mL of H₂O and then eluted with 40 mL of methanol into a 100 mL flask. The elute was evaporated under reduced pressure, reconstituted in 2 mL of methanol and finally filtered through 0.22 µm polytetrafluoroethylene (PTFE) filters into a 2 mL amber LC-MS certificated vial (Waters). For analysis before the phloroglucinol reaction, right before the analysis each purified sample was diluted 5 times with H₂0/methanol 50/50. For the reaction, 100 µL of concentrated and purified wine sample reacted with 100 µL of the phloroglucinol reagent at 50 °C for 20 min and then combined with 1 mL of 40 mM aqueous sodium acetate to stop the reaction. The phloroglucinol reagent was a solution of 0.2 N HCl in methanol, containing 100 g/L of phloroglucinol and 20 g/L ascorbic acid. The final solution was filtered through a 0.22 µm PTFE filters into LC-MS certificated vials and immediately analyzed.

⁴³Sc/⁴⁷ScCl₃ was reconstituted in 0.01 M HCl and pH was adjusted to 4.0 by 1 M ammonium acetate buffer to a final buffer concentration of 0.5 M. DOTA-PSMA-617 was added and the reaction was mixed for 45 min at 95 °C on a thermomixer at 450 rpm. The resulting ⁴³Sc/⁴⁷Sc labeled ligands were then trapped on a preconditioned C18 SPE cartridge (Waters, Milford, MA, USA). The column was washed with water. The purified product was eluted with ethanol, dried and reconstituted in PBS for cellular and animal studies.

Plant extracts for bioassays were prepared by sonication (20 min) of a mixture of 1.67 g of lyophilized plant material and 50 ml H₂O/MeOH (1:1). This mixture was then centrifuged at 10,000 rpm (Beckman Avanti J-25, JA-12 rotor) for 10 min. The supernatant was separated from the sediment and evaporated to dryness using rotary evaporator (<40 °C) giving crude extracts (HC). Extract dilutions (1:1, MC and 1:10, LC) were prepared after the extracts were re-solved in the proper amount of water.
Fractions were prepared by SPE of crude extracts using C18 SPE cartridges (Waters, Milford, MA, USA) conditioned with methanol first and water after. The samples were loaded, and the cartridges were eluted first with water to obtain fraction W and then with methanol to obtain fraction M. A washing step with a 5% methanol/water solution was carried out between the two main elution steps. Each fraction was analysed by ¹H NMR.