Chemstation software

HPLC-UV analysis: Equipment consisted of an Agilent 1200 series HPLC system with a UV-VIS PDA detector (Agilent Technologies, United States), Agilent ChemStation software, Phenomenex^® C18 column (250 mm × 4.6 mm id, 5 μm). Solvent A (H₂O + acetic acid 0.2% v/v) and B (methanol + acetic acid 0.2% v/v) were mixed in gradient mode as follows: 0 min 90% A, 0–5 min 80% A, 5–65 min 50% A, 65–75 min 20% A; flow rate 0.8 ml/min. The injection volume and column temperature were set at 10 μl and 40°C, respectively.

The Agilent-1200-Series HPLC system equipped with a diode array detector (G1315D), the Agilent ChemStation software and a Phenomenex Gemini-NX 5 μ C18 110A 150 mm × 4.6 mm column (Phenomenex, http://www.phenomenex.com/) was used at a column temperature of 35 °C and a flow rate of 1.0 ml·min⁻¹. The injection volume was 20 μl for cell extracts and 30 μl for medium extracts; peaks were quantified at 260 nm. UV spectra were collected over the wavelength range from 200 to 700 nm. Eluent A contained 10% acetonitrile in 20 mM ammonium formate, adjusted to pH 8.7, and eluent B consisted of 100% acetonitrile. A gradient program was employed, composed of a sequence of linear gradients with an initial step of 100% A to 80% A and 20% B over the first 10 min, followed by a second gradient to 10% A and 90% B over the next 10 min, and a final step to 100% B from 21 min after injection till the end of the run at 30 min after injection (Trehy et al. 2011 (link)). The reference alkaloids nicotine and nornicotine were purchased from Sigma-Aldrich (Munich, Germany) and used for sample spiking to verify the identified peaks (Online Resource 1). Data represent mean values and standard errors from three independent experimental series. The significance of the observed differences was probed with the non-parametrical Friedman test.