Empower data system

Standard solutions containing 1 µg/ml concentrations of indigo and indirubin in DMSO were prepared and diluted with 1:1 DMSO:acetonitrile mixtures to concentrations of 1, 5 and 10 ng/ml. Standard stock solutions and working solutions were stored at 4°C. For preparation of the sample, samples of PR extract were weighed, and dissolved in 1:1 DMSO and acetonitrile mixtures. The samples were then sonicated for 1 h at room temperature. Prior to high performance liquid chromatographic system (HPLC) analysis, the sample preparations were filtered through a 0.45-µm membrane filter. A Waters Alliance HPLC system (Waters Corporation, Milford, MA, USA), equipped with a Waters 2489 UV/Visible detector was used for analysis. An Empower Data System and YMC-Pack Pro C-18 column (1.7 µm, 2.1×100 mm; both Waters Corporation) were used for recording the output signal of the detector and for separation, respectively. The mobile phase consisted of 0.1% aqueous formic acid and 0.1% formic acid in acetonitrile. The flow rate in the gradient elution system was 1.0 ml/min, the injection volume was 10 µl, the UV detection wavelength was 540 nm and the column was maintained at room temperature.