HPLC Analysis of Active Fractions

HPLC analyses were performed on a Shimadzu, LC-20AT system (model DGU 20A3, SHIMADZU corporation, Kyoto, Japan) equipped with LC-20AT quaternary pump, a DGU20A3/DGU-20A5 on-line degasser, an SPD-20A photodiode array detector, and a CBM-20A/20A interface. The data were acquired and processed using Lab Solution software. The active fraction was analyzed using a reverse-phase HPLC column SHIMADZU corporation, Kyoto, Japan), SunfireTM prep C18 (10 mm × 250 mm, 5 µm) column (Waters, Ireland). The mobile phase was composed of 30% H₂O with 0.1% TFA (ACROS ORGANICS) and 70% acetonitrile (LC-MS CHROMASOLVR, Fluka) with 0.1% TFA. Prior to HPLC injection, samples were filtered through a CHROMAFIL R Xtra H-PTFI filter (pore size 0.45 µm, filter 13 mm, (MACHEREY-NAGEL, Düren, Germany). Two mL of sample was injected and run for 41 min at 20 °C with a flow rate of 4 mL/min [68 (link)].