Dionex 3000 hplc system

Before analytical procedures, dried extracts from biomass, xerogels, and culture medium were mixed with methanol (HPLC-grade) (Merck, Poznań, Poland). Redissolved samples of extracts were chromatographically analyzed by a reversed-phase HPLC technique supported by the DIONEX 3000 HPLC system (Thermo Scientific, Waltham, MA, USA) equipped with an EC Nucleosil 120-7 ODS packed column (250 × 4.6 mm) filled by 7 μm particles with 120 Å pores (Macherey-Nagel, Allentown, PA, USA). A UVD 340S diode-array detector (Thermo Scientific, Waltham, MA, USA) was applied for qualitative and quantitative detection of chemical compounds. The chromatographic separation was performed using a gradient elution of acetonitrile (60–80%) and 0.04 M orthophosphoric acid (40–20%) at a flow rate of 1.5 mL min⁻¹. Eluent absorbance was measured at four wavelengths: 215, 237, 350, and 436 nm. The concentrations of naphthoquinones, i.e., deoxyshikonin and rinderol, in extracts were quantitatively determined by the analysis of specified peaks at 215 nm (deoxyshikonin) and 237 nm (rinderol) wavelengths on chromatograms following the standard external method. Referenced deoxyshikonin and rinderol standards were used for qualitative identification of naphthoquinones peaks and calibration of the curve for quantitative analysis.

The HPLC analysis was performed by Dionex 3000 HPLC system (Thermo Fisher Scientific Inc., Waltham, MA, USA). Chromatograms were detected at 450 nm wavelength; data acquisition was performed by Chromeleon 7.20 software. The separation was carried out on an endcapped C₃₀ column (250 × 4.6 mm i.d.; YMC C₃₀, 3 µm, YMC Europe GmbH, Dinslaken, Germany). Eluents: (A) MeOH: MTBE (methyl-t-butylether): H₂O = 81:15:4 v/v%; (B) MeOH: MTBE: H₂O = 6:90:4 v/v%. The chromatography was performed in a linear gradient from 100% A eluent to 50% B mixture in 45 min, with 1.00 mL/min flow rate, at 22 °C.