Kinetex 2.6 m

Frozen chemistry voucher samples of each animal were freeze-dried, then sequentially extracted with chloroform and methanol. The combined extracts were dried down and passed over a small C₁₈ plug, eluting with methanol. LC/MS data were acquired using a Bruker MaXis ESI-Q-TOF mass spectrometer coupled with a Waters Acquity UPLC system operated by Bruker Hystar software. A gradient of MeOH and H₂O (containing 0.1% formic acid) was used with a flow rate of 0.3 mL/min on a RP C18 column (Phenomenex Kinetex 2.6 µm, 2.1 mm × 100 mm). The gradient went from 10% MeOH/90% H₂O to 97% MeOH/3% H₂O in 12 mins, followed by 97% MeOH/3% H₂O held for 3.5 mins. Full scan mass spectra (m/z 150-1550) were measured in positive ESI mode. The mass spectrometer was operated using previously published parameters [56] (link). Tune mix (Agilent, ESI-L low concentration) was introduced through a divert valve at the end of each chromatographic run for automatic internal calibration.

The prepared samples were analyzed by ultra-performance liquid chromatography. The Nexera X2UPLC-MS/MS (Shimadzu Corp., Kyoto, Japan) contained two LC-30AD pumps, SiL-30AC autosampler, CTO-20AC column oven, CBM-20A communication bus module, and mass spectrometer LC-MS8050 with electro spray ionization with positive and negative ion mode (Table 1). Chromatographic separation was performed using an analytical column Kinetex 2.6 µm, Phenyl-Hexyl 100 Å 4.6 mm, 150 mm (Phenomenex, Torrance, CA, USA). The gradient program consisted of the following: 7-min sequence of linear gradient flows of solvent B (acetonitrile:methanol 1:1 v/v) balanced with solvent A (water with 0.1% formic acid) at a flow rate of 0.6 mL/min: 50–80% B over 1 min, 80–100% B over 2 min, isocratic 100% B for 3 min, and finally, 100–50% B over 1 min. The injection volume was 1 µL and column temperature was 40 °C.
The main parameters used to identify analytes were their retention times and multiple reaction monitoring (MRM) ratio, which were obtained at 0.25 µg/mL working standard solutions for most standards, and 2.5 µg/mL for AMP, IB, SPEC, STREP, SFC, SNA and TET. Chromatographic data processing was carried out using LabSolution^® software (Shimadzu Corp., Kyoto, Japan).

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).