*4.4. Alkaloid Identification*

In order to identify the steroidal alkaloids in *V. californicum* leaf, stem and root/rhizome extracts, samples were analyzed by HPLC-MS, where the mass spectrometer was an ultra-high resolution Quadrupole Time of Flight (QTOF) instrument (Bruker maXis, Billerica, MA, USA). The electrospray ionization (ESI) source was operated under the following conditions: positive ion mode, 1.2 bar nebulizer pressure, 8 L/min flow of N2 drying gas heated to a temperature of 200 ◦C, 3000 V to −500 V voltage between HV capillary and HV end-plate offset, mass range set from 80 to 800 *m*/*z*, and the quadrupole ion energy at 4.0 eV. Sodium formate was used to calibrate the system in this mass range of 80 to 800 *m*/*z*. HPLC separation was achieved using a XTerra MS C18 column, 3.5 μm, 2.1 × 150 mm

(Waters, Milford, MA, USA). The flow rate was 250 μL/min. The mobile phases were 5% acetonitrile and 0.1% formic acid in water (Buffer A) and acetonitrile and 0.1% formic acid (Buffer B). The linear gradient method was used to separate analytes starting at 5% Buffer B and increasing to 60% Buffer B over 25 min. A 1 μL sample injection was used. Data were analyzed with the Compass Data Analysis software package (Bruker Corporation).
