Acrodisc

A freshly prepared solution (1 ml) containing 100 mM Bis–Tris–HCl (pH 7.2), 5 mM 2-oxoglutrate, 10 mM sodium ascorbate, 0.2 mM FeSO₄, 25 μM α-solamarine, was introduced into 10 ml round bottomed flask with a stir bar. Then, the flask was capped by a three-way cock which was connected to a vacuum pump line and a balloon filled with ¹⁸O₂, respectively. The reaction mixture was degassed in vacuo, and then ¹⁸O₂ was incorporated into the reaction mixture by purging once and stirring with a stirrer bar. An aliquot of the reaction mixture was quickly transferred to 1.5 ml microtube and mixed with the purified enzyme. The reaction was carried out at 30 °C for 30 min. The reaction was then stopped by incubation for 2 min at 90 °C. After centrifugation, 20 μl of supernatant was diluted with 180 μl methanol and filtered through 0.2-μm nylon membrane filters (Acrodisc, Waters). An aliquot (2 μl) was analyzed using LC–MS, as described in “Feeding experiments” above.

HRESIMS data were collected and in positive and negative ionization modes using a Thermo QExactive Plus mass spectrometer (ThermoFisher) equipped an electrospray ionization (ESI) source and via an Acquity UPLC system (Waters Corp). The higher-energy collisional dissociation (HCD) cell used a normalized collision energy of 30 eV for all the compounds to obtain MS/MS data. The UPLC separation was performed using an Acquity BEH C₁₈ column equilibrated at 40 °C and a flow rate set at 0.3 mL/min. The mobile phase consisted of 15% CH₃CN–H₂O (0.1% formic acid) for 0.5 min, and then a linear gradient from 15% CH₃CN to 100% CH₃CN over 6 min, and 1 min holding 100% CH₃CN before returning to the starting conditions. Samples were dissolved in MS grade methanol and filtered through a 0.2 μm Acrodisc (Waters) filter. Tentative metabolite identification was performed by comparison of HRMS data, UV maxima and fragmentation patterns (MS/MS data) with those contained in the Dictionary of Natural Products [20 ] reference compounds refined for Thevetia plant metabolites.