Agilent qualitative analysis b 08

Extractions and measurements were done as previously described ^{33 (link),34 (link)}. Log-phase A. baumannii culture was incubated 0.79 or 0.38 μg/mL RBT in the presence or absence of amino acid mixture at 37 °C. Bacteria were harvested 0, 1, 8, and 24 hours and CFUs were determined by plating serial dilutions on agar media. The cell free supernatant was collected by filtration through a 0.22 μm filter. RBT were extracted by adding LC-MS grade acetonitrile:methanol:water (40:40:20) solution that was precooled to − 40 °C. Liquid chromatography mass spectrometry (LC-MS) differentiation and detection of RBT was performed using a Cogent Diamond Hydride Type C column (Microsolve Technologies) with an Agilent Accurate Mass 6230 TOF coupled with an Agilent 1290 Liquid Chromatography system as previously published.^{33 (link),35 (link)} An isocratic pump was used for continuous infusion of a reference mass solution to allow mass axis calibration. Detected RBT ion was validated based on unique accurate mass-retention time identifiers for masses. RBT level was analyzed using Agilent Qualitative Analysis B.08.00 (Agilent Technologies) with a mass tolerance of <0.005 Da. The intracellular RBT was calculated as the [RBT]_{drug only control} – [RBT]_filtrate. 3 biological replicates were tested per group.