Purospher rp 18e 250 4 mm 5 mm column

HPLC analysis was performed to determine the content of metabolites in the methanol extracts from biomass (2 h, at the solvent boiling point of 64.7 °C) and in hydrolysates (2 M HCl, 30 min, at the solvent boiling point of 100 °C) obtained from the agitated shoot cultures. RP-HPLC analysis was carried out as previously described [41 (link)] on Merck-Hitachi liquid chromatograph (LaChrom Elite, Hitachi, Tokyo, Japan) equipped with a DAD detector L-2455 and Purospher^® RP-18e (250 × 4 mm/5 mm) column (Merck, Darmstadt, Germany). Analysis was carried out at 25 °C. The mobile phase used for the analysis consisted of methanol (A), and methanol:0.5% acetic acid (1:4, v/v) (B). The flow rate was 1 mL/min, and the gradient was as follows: 100% B for 0–20 min; 100–80% B for 20–35 min; 80–60% B for 35–55 min; 60–0% B for 55–70 min; 0% B for 70–75 min; 0–100% B for 75–80 min; and 100% B for 80–90 min. Quantification was done by measuring the peak area with reference to a standard curve derived from five concentrations (0.03125–0.5 mg/mL). Exemplary chromatograms showing the content of the analyzed compounds in an extract from R. graveolens cultures and chromatograms of the standards are included in the Supplementary Files (Figures S1–S4).

Lyophilised and powdered samples (about 250 mg) were extracted with 2 mL of methanol using sonication (10 min at 25 ± 2 °C) and centrifuged for 10 min at 15,000 rpm. The supernatant was filtered through 0.22 μm filter (Millex^®GP, Millipore, Merck, Darmstadt, Germany). RP-HPLC analyses was carried out according to Simlat et al.^{34 (link)} using a Merck–Hitachi liquid chromatograph (LaChrom Elite, Hitachi, Tokyo, Japan) equipped with a DAD detector L-2455 and Purospher^®RP-18e (250 × 4 mm/5 mm) column (Merck, Darmstadt, Germany). Quantification was carried out at λ = 254 nm for phenolic acids and λ = 370 nm for flavonoids. Identification was performed through a comparison of the retention times of the peaks with authentic reference compounds and co-chromatography with standards. Quantification consisted of measurement of the peak area with reference to the standard curve derived from five concentrations (0.03125–0.5 mg ml⁻¹). Commercially available standards of the phenolic acids: chlorogenic, neochlorogenic (Sigma–Aldrich, St Louis, MO, USA), isochlorogenic A (ChromaDex, Irvine, CA, USA), and rosmarinic (Sigma–Aldrich, St Louis, MO, USA), as well as the flavonoids isoquercetin, and quercitrin (Sigma–Aldrich, St Louis, MO, USA), were used to generate the calibration curves. The analysis was repeated three times.