AHL Quantification

All the equipment for the analyses was from Agilent technologies (Santa Clara, CA, United States) and consisted of a 6545 QTOF mass spectrometer equipped with an electrospray ionization interface (ESI) coupled to a 1260 UHPLC, a G4204A quaternary pump, G4226A ALS auto-sampler, and G1316C thermostated column compartment. UHPLC was carried out on a ZORBAX RRHD Eclipse Plus C18, 95Å, 2.1 × 50 mm, 1.8 μm column, with water (0.1% formic acid)-MeCN gradient elution, from 5 to 95% MeCN for 10 min at a flow rate of 0.5 mL/min. Twenty μL of each sample were injected into the LC-MS/MS instrument in triplicates and an average peak area of three analyses was calculated. The water-MeCN solution was injected as a blank within a sequence of samples to confirm that there was no cumulative carryover. Mass spectral parameters were optimized for 3-oxo-C6-HSL by varying the fragmentor voltage of the ion source for scan mode and collision energy for product ion mode (MS/MS), 3-oxo-C8-HSL was quantified under the same parameters. Specific parameters of the ion source were readjusted. The ESI was operated in positive mode. The source temperature was set to 300°C, Nozzle voltage 500 V and ion spray voltage was 5.5 kV.