High-performance liquid chromatography–quadrupole time of flight mass spectrometry (HPLC–Q-TOF–MS)

Aliquots of 50 μL cell extracts and PBQC samples were transferred into 2 mL autosampler vials with glass inserts. A mixture of 550 authentic standards (in 13 separate mixes), covering a range of metabolic pathways was run with each batch of bacterial species to aid metabolite identification (MSI level 1). Analysis was performed on the Agilent 1200 series HPLC system coupled with a 6545 Q-TOF MS using Agilent MassHunter Workstation LC/MS Data Acquisition for 6200 series TOF/6500 series Q-TOF (version 10.1) as follows⁶⁶: Samples were maintained at 4 °C in the autosampler and metabolite separation was performed by injecting 7 µL of sample into a SeQuant® ZIC-pHILIC PEEK coated column (150 mm × 4.6 mm, 5 µm polymer, Merck) maintained at 25 °C, with a binary gradient made up of solvent A (20 mM ammonium carbonate, pH 9.0, Sigma-Aldrich) and solvent B (100% MeCN, Hypergrade for LCMS LiChrosolv, Merck) at a flow rate of 300 µL/min. A 33.0 min gradient was set up with time (t) = 0 min, 80% B; t = 0.5 min, 80% B; t = 15.5 min, 50% B; t = 17.5 min, 30% B; t = 18.5 min, 5% B; t = 21.0 min, 5% B; t = 23.0 min, 80% B.

Metabolites were ionised in an electrospray ionisation source (ESI) with a capillary voltage of 2.5 kV, drying nitrogen gas flow at 10 L/min, with the temperature and nebuliser pressure of 225 °C and 20 psi, respectively. The voltages were set at −125 V for fragmentor and at −45 V for skimmer. The acquisition was conducted with a scan range of 85–1200m/z at a rate of 1.5 spectra/s in negative all-ion fragmentation (AIF) mode, which included three collision energies (0, 10, 20 V). A QTOF reference mass solution with 2.5 µM Hexakis(1H, 1H, 3H-tetrafluoropropoxy)phosphazine and 5 µM purine in 95:5 acetonitrile: water (API-TOF Reference Mass Solution Kit, Agilent Technologies) was continuously infused into the ESI source for internal mass calibration during data acquisition. In accordance with the metabolomics standards initiative (MSI), metabolite identification (MSI level 1) was based on the retention time and molecular masses matching to an authentic standard⁶⁷. Peak area integration was performed using MassHunter TOF Quantitative Analysis software (version B.07.00, Agilent Technologies) using the Metabolomics Australia In-house Data processing pipeline.