HPLC-MS/MS setup consisted of an Agilent ZORBAX HPLC system (SB-C18 column) coupled with an Agilent G6410 triple quadrupole mass spectrometer and an ESI interface. The adjustments of the HPLC setup and the protocol were as follows: a sonicated, degassed, and filtered mobile phase of Acetonitrile:Water (0.1% Formic Acid) consists of 20 min 45% acetonitrile, 90% acetonitrile for 3 min, 7 min 100% acetonitrile, 10 min 45% acetonitrile with a flow rate of 0.6 mL/min. The column temperature was set at 25°C, and 20 μL of the sample was subjected to the instrument via an auto-sampler. Mass spectrometer adjustments were as follows: capillary voltage: 4500 V, mass range: 120–450 amu, Dwell time: 500 ms, fragmentor voltage: 120 V, collision energy: 7. Nine ultra-pure AHLs, including N-butanoyl-L-homoserine lactone (C4-HSL), N-hexanoyl-L-homoserine lactone (C6-HSL), N-(3-oxo-hexanoyl)-L-homoserine lactone (Oxo-C6-HSL), N-heptanoyl-L-homoserine lactone (C7-HSL), N-octanoyl-L-homoserine lactone (C8-HSL), N-(3-oxo-octanoyl)-L-homoserine lactone (Oxo-C8-HSL), N-decanoyl-L-homoserine lactone (C10-HSL), N-dodecanoyl-L-homoserine lactone (C12-HSL), and N-(3-oxo-dodecanoyl)-L-homoserine lactone (Oxo-C12-HSL), purchased from Sigma-Aldrich, were used as standards to determine the characteristic daughter ions, which were found to be at m/z 102, reflecting the lactone ring, similar to previous reports (Morin et al., 2003 (link); Fekete et al., 2007 (link); Bose et al., 2017 (link)). Accordingly, each sample was evaluated by the precursor ion method screening for AHL-characteristic daughter ion at m/z 102. Afterward, the total ion chromatogram (TIC) of each bacterial medium extract was screened for major masses, and each mass was depicted independently through an extraction chromatogram (EIC). To further confirm each mass to be an AHL, the concurrent existence of at least one other daughter ion at m/z 56, 74, or 84 was evaluated via the production method (Rodrigues et al., 2022 (link)). All the results were analyzed using Agilent MassHunter Qualitative Analysis.
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