Hplc dad

The esterification reaction was monitored by HPLC-DAD (Merck) in a reverse-phase C18 column (Agilent) with 250 × 4.6 mm i.d., 2.7 μm at 25 °C. The eluents used were (A) 1% (v/v) formic acid in water and (B) 1% (v/v) formic acid in acetonitrile, and the elution gradient was performed from 50 to 100% B during 51 min at a flow rate of 0.4 mL/min. After 51 min, the column was washed with 100% B for 15 min and, then, it was stabilized with the initial conditions for more 10 min.
Mass spectrometry (MS) analysis of the pyranoflavylium-cinnamic derivative esters was performed using a Finnigan Surveyor series liquid chromatograph equipped with a reversed-phase C18 column (Agilent) with 250 × 4.6 mm i.d., 2.7 μm thermostatted at 25 °C). The mass detection was carried out in the positive ion mode in a Finnigan LCQ DECA XP MAX (Finnigan Corp., San José, CA, USA) mass detector with an API (Atmospheric Pressure Ionization) source of ionization and an ESI (ElectroSpray Ionization) interface. The solvents and HPLC gradient were used as the same reported above for the HPLC analysis. Spectra were recorded in the positive ion mode between m/z 300 and 1500.

One gram of ground dried leaves (knife mill after oven at 60 °C with forced air circulation) was extracted by maceration in ultrasonic bath (Quimis, Diadema, Brazil) at room temperature in 10 mL ethanol:water (7:3, v/v) for 30 min. The obtained ACHE was filtered in a 0.45 μm PTFE filter (Merck Millipore, Darmstadt, Germany) before analyzed in a High-Performance Liquid Chromatograph coupled to Diode-Array Detector (HPLC-DAD). Carajurin was isolated and characterized [16 (link)]. The determined purity was 98% by HPLC-DAD [16 (link)].

Compounds purity and stability were checked by HPLC-DAD (Merck) in a reversed-phase C18 column (Agilent) with 250 × 4.6 mm i.d., particle size 2.7 μm and at 25 °C (Fig. S1). The eluents used were (A) 1% (v/v) formic acid in water and (B) 0.5% (v/v) formic acid in 80% (v/v) acetonitrile and the elution gradient consisted in 40–85% B during 50 min at a flow rate of 0.4 mL/min. After 50 min, the column was washed with 100% B during 10 min and then it was stabilized with the initial conditions for more 10 min.