1260 infinity 2 lc msd iq

The extraction and detection of erythrydiol (2) follow the procedure described for β-amyrin. For the rest of the triterpenoids, 200 µl of culture was collected in a microfuge tube before it was directly extracted with 800 µl methanol with bead-beating (3,800 rpm, 1 min × 2). The mixture was centrifuged at 12,000g for 1 min to separate the pellet. Two hundred microlitres of the supernatant was transferred into an Eppendorf tube, which was then evaporated in a vacuum concentrator at room temperature and the remainders were resuspended in 200 µl methanol. Finally, samples were filtered with Amicon Ultra 0.5-ml 3-kDa filter tubes or centrifuged at 15,000g for 5 min. Products were analysed using LC–MS (1260 Infinity II LC-MSD iQ, Agilent) equipped with a reverse phase C18 column (Kinetex 2.6 µm, 250 × 4.6 mm, XB-C18, Phenomenex). A 50-min isocratic method was performed with 10:90 of water (solvent A) and acetonitrile (solvent B) using a flow rate of 0.3 ml min⁻¹. Full mass spectra were generated for metabolite identification by scanning within the m/z range of 300–600 in negative-ion mode. Data acquisition and analysis were performed using OpenLab CDS version 2.4 (Agilent).

Metabolites were analyzed as previously described with minor variations [25 (link)]. Analyses were performed on an Agilent Technologies 1260 Infinity II LC/MSD iQ (Santa Clara, CA, USA) with electrospray ionization (ESI) mass detection. Negative ion mass-to-charge ratios (m/z) were scanned from 50 to 500. The metabolite extracts were chromatographed using a Sepax Carbomix column (Newark, DE, USA; Pb-Np5:8%, 5 μm, non-porous, 4.6 × 300 mm) (mobile phase A: water; mobile phase B: acetonitrile; column temperature, 75 °C). Chromatographic separation was performed using a combination of isocratic and gradient methods, including column washing and equilibration periods at the end (0 min: 100% A, 0.12 mL/min; 60 min: 100% A, 0.12 mL/min; 70 min: 70% A, 0.5 mL/min; 145 min: 70% A, 0.5 mL/min; 150 min: 100% A, 0.5 mL/min; 164 min, 100% A, 0.5 mL/min; 165 min, 100% A, 0.12 mL/min; 170 min, 100% A, 0.12 mL/min). The metabolites were detected by the masses of their negatively charged ions (M-1 ± 0.05; 145 m/z; succinate, 115 m/z; butyrate, 87 m/z; propionate, 73 m/z; acetate, 59 m/z; lactate, 89 m/z; fructose/glucose-6-phosphate, 258 m/z; ribose-5-phosphate, 229 m/z), with their retention times and m/z verified by co-injection with authentic standards.