5.2. Determination of Indigo and Indirubin by HPLC

Chromatographic determination of indigo and indirubin in Indigo Naturalis was performed using the LC-20AT HPLC system equipped with the SPD-20A UV detector and the SIL-20A autosampler (Shimadzu, Tokyo, Japan). A Kromasil C18 (250 mm × 4.6 mm, 5 µm) analytical column (Akzo Nobel, Bohus, Sweden) was used at 25 °C. The mobile phase was a mixture of acetonitrile (55%) and purified water (45%) with a flow rate of 1.0 mL/min. The detector wavelength was set at 290 nm. The injection volume was 20 µL. Duplicates of each sample were measured and the average value was used. Figure 5 shows the typical HPLC chromatograms of Indigo Naturalis and the reference compounds.

HPLC chromatograms of typical samples of Indigo Naturalis and the reference compounds.

Preparation of sample solutions for quantification: the powder (0.05 g) of each sample was dispersed in 95 mL of N,N-Dimethylformamide (DMF), sonicated for 30 min, cooled to room temperature, and the final solution volume adjusted to 100 mL with DMF.

Preparation of standard solutions: indigo (5.98 mg) or indirubin (7.66 mg) was dispersed in DMF (95 mL), sonicated for 30 min, cooled to room temperature, and the final solution volume adjusted to 100 mL with DMF.

Development of the calibration curves: the standard solutions were diluted to a series of different concentrations and tested with the above HPLC conditions. Calibration curves were developed from the chromatographic peak area relative to the weights of indigo and indirubin. The calibration curves showed good linear relationships for indigo in the range of 0.0589–0.589 µg (R = 0.9996) and indirubin in the range of 0.00766–0.0766 µg (R = 0.9995).