Pda 2998

For HPLC analysis, we used the HPLC system equipped with a photodiode array (PDA) detector (Waters Alliance e2695, PDA #2998, Waters Corp., Milford, MA, United States). The data were acquired and processed using Empower software (version 3; Waters Corp.). For chromatographic separation of the three compounds, a Sunfire C₁₈ analytical column (250 × 4.6 mm, 5 μm, Waters Corp.) was used, which was maintained at 35°C. The gradient conditions were 10–23% B for 30 min, 23–100% B for 10 min, and 100% B for 10 min. The mobile phases consisted of two solvents: 0.1% (v/v) TFA in water (A) and acetonitrile (B). For scanning chromatograms, PDA detection was performed at 210–400 nm. Column were used at flow rate of 1.0 ml/min and injected volume of 10 μL, respectively.

Naringenin was purified by HPLC using an Alliance 2695 HPLC with a refrigerated autosampler, a column furnace and a photodiode array detector (PDA) 2998 (Waters Corp.). Naringenin was quantified at its maximum absortion peak (290 nm). The system was equipped with a 3.5 μm particle size XBridge™ C18 (2.1 × 150 μm, Waters Corp.) column.
The culture samples were centrifuged at 13,000 rpm for 5 min and the supernatants filtered through sterile 0.2 μm polyethersulphone (PES) syringe filters (VWR, Intl.). The whole sample (100 ml) was loaded in multiple injections. HPLC conditions were as follows: column temperature 40 °C; the flow was kept at 0.32 ml/min from 0 to 2 min and then increased gradually to reach 0.40 ml/min at 10 min. A mixture of Milli Q water with 0.01 % trifluoroacetic acid (A) and acetonitrile (B) were used as solvent with the following program: 0–2 min increase from 20 to 30 % B and kept at 30 % B thereafter. The peak eluting at 6 min from repeated injections was collected and pooled and the solvent from the pooled preparation was evaporated under low pressure (SpeedVac Savant Sc110, Thermo Sci.). The concentrated solution was lyophilized and kept at −20 °C until NMR analysis and identification. Pure naringenin and genistein (Sigma Ch. Co.) were used as standards.

The quantitative analyses of the reaction mixtures were performed using an ultrahigh performance liquid chromatograph UPLC Acquity Arc Waters (Milford, MA, USA) and the products of the reaction were analyzed by two detectors, refractive index (RI 2414) and a photodiode array (PDA 2998). The reactant and the products were separated on a Shodex sugar column SH1011, heated at 30 °C. The eluent was an aqueous solution of H₂SO₄ (0.005 M) and its flow rate was set at 0.6 mL min⁻¹. The samples to be analyzed were collected at the end of the reaction: 1 mL of the reactant/products solution was diluted in 50 mL of deionized water.