Hp 1100 l

The HPLC system consisted of a HP 1100 L instrument with a Diode Array Detector and managed by a HP 9000 workstation (Agilent Technologies, Palo Alto, CA, USA). The column was a Synergy max RP (150 mm × 3.0 mm) with a particle size of 4 μm (Phenomenex) maintained at 27°C. The eluents were H₂O at pH 2.0 by formic acid 5% (A) and acetonitrile (B) with a flow rate of 0.4 ml/min. The elution method involved a multistep linear solvent gradient changing from an initial concentration of 95% (A) to 78% (A) in 8 min; 5 min to 74% (A); 12 min to 65% (A) then 5 min to initial conditions. The total time of the analysis was 30 min, equilibration time 5 min. Injected volume of the samples was 5 μl solution. The UV–vis spectra were recorded between 220 and 650 nm in both cases. Chromatographic profiles were registered at 254, 280, 330, 350 and 520 nm.

The HPLC system consisted of a HP 1100 L instrument with a Diode Array Detector and was managed by a HP 9000 workstation (Agilent Techologies, Palo Alto, CA, USA). More details are provided in the Supplementary Materials.

The quantitative analyses were carried out using a HP 1100 L liquid chromatograph equipped with a DAD detector and managed by a HP 9000 workstation (Agilent Technologies). A 150 mm × 4.6 mm i.d., 5 μm Zorbax Eclipse XDB, RP18 column was used. 20 μL of each sample were injected. Chromatography was carried out in gradient mode using a flow rate of 0.6 mL/min. The mobile phase was (A) formic acid/water pH 3.2 and (B) CH₃CN. The multi-step linear solvent gradient used was: 0–5 min 15–20% B; 5–7 min 20–30% B; 7–10 min 30–40% B; 10–15 min 40–50% B; 15–20 min 50–80% B; 20–25 min 80–15% B; post time 10 min; oven temperature 30 °C, flux: 0.6 mL/min. The UV/Vis spectra were recorded in the range 200–700 nm and the chromatograms were acquired at 210, 260, 280, 350 nm.
For qualitative analysis, MS experiments were conducted using a LTQ equipped with an ESI interface (Finnigan LTQ, Thermofisher Scientific, Waltham, MA). Mass spectrometry and electrospray operating parameters were optimized for negative polarity. The following final settings were used: sheath gas flow rate (arb): 30, aux gas flow rate (arb): 5, sweep gas rate (arb): 5, capillary temp (°C): 290.00, capillary voltage (V): 16.93, tube lents (V): −99.72.