Anatabine

The Agilent-1200-Series HPLC system equipped with a diode array detector (G1315D), Agilent ChemStation software and a Phenomenex Gemini-NX 5μ C18 110A 150 mm x 4.6 mm column (Phenomenex, Aschaffenburg, Germany) was used at a column temperature of 35°C and a flow rate of 1.0 ml/min. 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 nm 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 [40 ]. The reference alkaloids nicotine, anatabine, anatalline and nornicotine were purchased from Sigma aldrich (Munich, Germany), and used for sample spiking to verify the identified peaks (S1 Fig).