Thermo accela uhplc system

18 mL of LB medium at different pH: 5, 7 and 9, adjusted using NaOH or HCl, was inoculated with 1 mL of bacteria (Supporting Information) and 1 mL of 0.2, 0.03 and 0.003 mg L^-1 of trimethoprim in 100 mL conical flasks. Control samples were identical except the 1 mL of trimethoprim was substituted with 1 mL of distilled water (dilution solvent). Each condition was replicated six times and each incubated for 18 h at 37°C and 200 rpm.
The overnight culture of each replicate was split for FT-IR and GC-MS to make sure that results were obtained from the same biological cultures. For FT-IR, 450 μL from each culture was collected and the biomass was washed three times with physiological saline and re-suspended in 400 μL of saline. For GC-MS, 15 mL was processed as described in Supporting Information.
In order to estimate the amount of trimethoprim inside the E. coli cells, cellular extracts were prepared, analyzed and quantified against a 20 point calibration curve constructed using a trimethoprim reference standard via LC-MS. For UHPLC-MS, a Thermo Accela UHPLC system (Thermo-Fisher Ltd.) coupled to a Thermo LTQ-Orbitrap XL MS system was employed (Thermo-Fisher). The methods used are described by Kim et al. [55 (link)]. Full details of methods are provided in S8 Fig for FT-IR and GC-MS and S9 Fig for LC-MS. In addition, the calibration curve for LC-MS is shown in Fig 2.

Sample analysis was performed using the Thermo Accela UHPLC system (Thermo Fisher Scientific, San Jose, California, United States). Chromatographic separation was performed on a maintained reverse-phase column Waters HSS T3-C18 (2.1 × 100 mm, 1.8 µm). The mobile phase was a mixture of methanol (A) and 0.1% formic acid in water (B). The following elution gradient was used: 0–5 min, 3%–10% A; 5–25 min, 10%–40% A; 25–35 min, 40%–60% A; 35–45 min, 60%–80% A; 45–50 min, 80%–95% A; 50–60 min, 95% A. The flow rate was set to 0.3 ml/min, and the injection volume was 1 µL.