Reversed-Phase HPLC Analysis of Deoxyshikonin

The reversed-phase chromatographic (RP-HPLC) technique and further analysis were performed using the Dionex 3000 HPLC system (by Dionex, a brand of Thermo Scientific, Sunnyvale, CA, USA) coupled with an automated sample injector (ASI-100) and UVDAD 340S UV-Vis diode-array detector. The RP-HPLC analysis was carried out under the following conditions: eluent (gradient)-acetonitrile (60–80%, Sigma Aldrich, Burlington, MA, USA)/0.04 M ortho-phosphoric acid (40–20%, Sigma Aldrich, Burlington, MA, USA), the flow rate of eluent 1.5 cm³ min⁻¹, EC Nucleosil 120-7 ODS packed column (250 × 4.6 mm, 7 µm particles, 120 Å pores, Macherey-Nagel, Düren, Germany) according to the previously published procedure [8 (link)]. The absorbance of eluent was monitored at 215 (as reference wavelength), 237, 350, and 436 nm. According to the external standard methodology, the deoxyshikonin chromatography standards with confirmed identity were used for the peak identification.