HPLC-MS/MS Analysis of Quorum Sensing Signals

The HPLC-MS/MS analysis was performed as described in Purohit et al. (2013) (link) and Hansen et al. (2015) (link). Briefly HPLC-MS/MS was performed using an Ascentis Express C18 reversed-phase column (50 × 2.1 mm, 2.7 µm particle size; Sigma). A sample of 20 µl was injected into the column and eluted using 0.1% formic acid in water and 0.1% formic acid in acetonitrile at a flow rate of 200 µl/min. The elution profile obtained was 5% acetonitrile in 30 s, 90% in 300 s and 5% in the next 60 s. The separated compounds were detected by Linear Ion Trap Quadrapole (LTQ) part of the LTQ-Orbitrap (Thermo Fisher Scientific). The LTQ was used in selected reaction monitoring (SRM) mode, and the SRM was divided into two segments. Segment 1 scanned 3OHC10-HSL and segment 2 scanned the internal standard 3O-C12-HSL with a retention time of 0–3.15 min and 3.15–6.00 min, respectively. The ion trap parameters chosen for MS/MS were maximum injection time 50 ms, isowidth 1.0 m/z, collision energy 35, act Q 0.25 and act time 30 ms. The measured AHLs are presented in ng/ml/OD₆₀₀. The AHL measurements at different temperatures were performed twice.