Analysis of artemisinin content

Reference standard of artemisinin (98 %), n-Hexane, ethyl acetate, LC–MS grade 0.1 % formic acid in water and acetonitrile was obtained from Sigma-Aldrich (Dorset, UK). Purified water (~18 MΩ/cm) was dispensed from a Milli-Q system (Millipore, UK).

Shoots, roots and hairy roots of transformed and untransformed plants of Artemisia annua (A1, A2, A3, AC, AR1, AR2, AR3, ARC, AH1, AH2) and Artemisia dubia (D1, D2, D3, DC, DR1, DR2, DR3, DRC, DH1, DH2) were used for the analysis of artemisinin with the help of LC–MS/MS.

A standard stock solution of 1 mg mL⁻¹ of artemisinin in acetonitrile was prepared. The analytical standard was prepared as dilution from the stock in the mobile phase in the concentration range between 0.15–10 µg mL⁻¹. Beta-artemether was used as internal standard (IS) at 5 µg mL⁻¹.

Extraction of plant samples (shoots, roots, hairy roots) was based on published protocols [71, 72] with a slight modification. Briefly, 10 mL of n-hexane containing 5 % v/v ethyl acetate was used to extract 1 g of biomass in a cold (0 °C) sonication bath (PUL 125, Kerry Ultrasonics, UK) operated at 50 Hz for 30 min. The solvent from the extract was removed in vacuo and the residue re-suspended in 2 mL acetonitrile, which was then filtered through a 0.2 µm syringe filter (Fisher, UK) to remove waxes and other un-dissolved components. An aliquot of the filtrate placed in an HPLC vial was dissolved in the mobile phase and internal standard added for LC–MS/MS analysis.

The analysis was performed on an Aquity liquid chromatography unit coupled to a tandem quadruple detector (TQD) (Waters Corp., Milford, MA, USA). The liquid chromatography setup consisted of a binary pump, a cooling auto-sampler set at 10 °C with an injection loop of 10 µL. The column temperature was set at 30 °C and a Genesis^® Lightn C18 column (100 × 2.1 mm; 4 µm) (Grace, IL, USA) was used for the separation of the metabolites. The method by [73] was employed which consisting of a binary mobile phase of A: 0.1 % formic acid in water and B: 0.1 % formic acid in acetonitrile. Chromatographic separation was achieved on a linear gradient run of 0–7.0 min, 25–98 % B; 7–9.5 min, 98 % B; 9.5–10 min, 98–25 % B; 10–15 min, 25 % B; and a flow rate of 0.4 mL min⁻¹.

The mass spectrometer was operated in MRM mode with positive electrospray ionization (ESI+) using an earlier method [73]. Briefly, the cone and de-solvation gas flow rates were set at 45 L h⁻¹ and 800 L h⁻¹, respectively, while the capillary voltage, the source and de-solvation temperatures were similar for all analytes at 28 kV, 150 and 350 °C, respectively. Cone and collision voltages were set at 24 and 7 V, respectively. A MRM transition of 283 → 219 + 229 + 247 + 265 was used for identification and quantification of artemisinin. The dwell time was automatically set at 0.161 s. Data were acquired by MassLynx V4.1 software and processed for quantification with QuanLynx V4.1 (Waters Corp., Milford, MA, USA).