HPLC Analysis of Medicinal Plant Compounds

HPLC analysis of ANM and selected plant fractions (ANE and ANA) was performed using HPLC-DAD (Agilent 1200, Germany) equipped with Zorbex RXC8 (Agilent, USA) analytical column with 5 μm particle size and 25 ml capacity using previously reported method [28 (link)]. Each sample was diluted with HPLC grade methanol. Mobile phase was consisted of eluent A, (acetonitrile- methanol--water- acetic acid-/5: 10: 85: 1)-and eluent B (acetonitrile-methanol-acetic acid/40: 60:-1). The gradient (A: B) utilized was the following: 0–20 min (0 to 50 % B), 21–25 min (50 to 100 % B), 26–30 min (100 % B) and 31–40 (100 to 0 % B) at flow rate ofL1 ml/min. The standards and samples were prepared in HPLC grade methanol (1 mg/ml), filtered through 0.45 μm-membrane filter andL20 μl was injected for the analysis. Among the standards rutin was investigated at 257 nm, catechin and gallic acid at 279 nm, caffeic acid and apigenin at 325 nm while quercetin, myricetin and kampferol were analyzed atL368 nm [29 (link)]. The analysis was performed in triplicate and the column was reconditioned for 10 min after each run. Quantification was done by the integration of the peak by using the external standard method.