HPLC Analysis of Metabolites

A 900 µL aliquot from the elicited culture medium was extracted with an equal amount of ethyl acetate in a 2 mL microcentrifuge tube by vortexing for 30 s. Then, 500 µL of the upper organic phase was removed, transferred to an amber HPLC vial, and dried under a nitrogen stream using a Reacti-Vap III apparatus (Thermo Fisher Scientific, Waltham, MA, USA). HPLC analyses were performed in an Ultimate 3000 UHPLC system (Thermo Fisher Scientific, Waltham, MA, USA). The extract was resuspended in 500 µL of MeOH and analyzed by reversed-phase HPLC. Briefly, the chromatography was performed in a SunFire^TM C18 column (5 µm, 4.6 × 250 mm, UV detection at 340 nm) (Waters, Milford, MA, USA) at 40 °C and a flow rate of 1.0 mL/min. The mobile phase was composed of MeOH (A) and 0.5% HCO₂H (v/v) (B). The column was initially calibrated with B for 1 min. Then, a linear gradient was performed using the following program: 60% A to 65% A for 1–20 min, 65% A and 35% B to 100% B for 20–25 min, and 100% B for 25–30 min. Calibration curves for reference compounds were established previously [36 (link)]. Similarly, re-elicited culture medium was extracted and analyzed as described above.