HPLC was achieved on an Agilent 1260 Series (Agilent Technologies) equipped with a 1260 Quat pump VL quaternary pump, 1260 ALS autosampler, 1260 TCC column thermostat, and 1260 DAD VL diode array detector. The separation was done on a Hypersil BDS C18 column (4.6 × 100 mm i.d., 3.5 μm) with a C18 guard column. The mobile phases were (A) 0.2% formic acid in water and (B) methanol using gradient elution: 75% B in A to 90% B in A for 10 min; 90% B in A to 100% B for 5 min; 100% B for 10 min. This column was re-equilibrated with 75% B in A for 10 min prior to each analysis and the flow rate was set at 1.0 ml/min with controlled temperature at 25 °C. DAD detector was set at the wavelength of 245 nm and injection volume was 5 μl for every sample and standard.
Stock solutions of standard compounds (1)–(4) were prepared by accurately weighing and dissolving the compounds in methanol to obtain the final concentration of 1000 μg/ml. Working solutions of standard compounds were obtained by diluting the stock standard solutions with methanol to achieve the desired concentrations.