Kinetex c18 100 lc column

Phenomenex Kinetex® C18 100 Å LC column (50 × 2.1 mm, 2.6 μm) coupled with a Phenomenex SecurityGuard ULTRA cartridge UPLC Evo C18 (both from Phenomenex, Torrance, CA, USA) was used in this assay. The mobile phase used for analysis was a mixture of water with 0.1 % formic acid (solvent A) and acetonitrile with 0.1 % formic acid (solvent B), which were combined in a gradient as follows: 2 % B (0–0.3 min), 40 % B (0.3–2.8 min), and held at 2 % B until the end of the run. The mobile phase was delivered at a total flow rate of 0.25 mL/min. The total run time was 7.0 minutes. Under these conditions, piperacillin and tazobactam had typical retention times of 3.45 and 2.75 minutes, respectively. ²H₅-piperacillin, IS for piperacillin and tazobactam, eluted with typical retention times of 3.42 minutes. The MRM transitions for the antibiotics and their respective IS with the corresponding compound specific parameters are shown in Table 2.

E. coli BAP1 containing pEXT06 or pEXT07 was cultivated on LB plates supplemented with 1% glucose and 50 μg/mL chloramphenicol at 37 °C. The following day, a loop of E. coli cells was transferred for precultures grown at 37 °C in 10 mL of LB medium supplemented with 1% glucose and 50 μg/mL chloramphenicol for 4–5 h. One microliter of each preculture was transferred to 50 mL of fresh LB with the same supplements and grown at 37 °C to an OD₆₀₀ of 0.4 to 0.6. Cultures were induced with 200 μM IPTG and incubated for an additional 12–14 h at 30 °C with shaking (220 rpm). Cultures were harvested, and 1 mL of H₂O supplemented with 0.5 mg/mL lysozyme was added to the pellets. Cells were disrupted by sonication at room temperature. The lysates were acidified with acetic acid (1% final concentration) and extracted twice with an equal volume of EtOAc. The organic phase was evaporated, resuspended in MeOH (0.2 mL), and filtered through Acrodisc MS PTFE Syringe filters (Pall Inc., Ann Arbor, MI, USA) prior to HPLC analysis. Each extract was monitored at 280 nm during separation by HPLC using a Kinetex C18 100 Å, LC Column (5 μm, 150 × 2.1 mm; Phenomenex, US) as follows: 0–10 min, 30% B; 10–11 min, 30%−100% B; 11–25 min, 100% B; 26–27 min, 100%−30% B; 28–35 min, 30% B (solvent A: H₂O/TFA (999:1, v/v); solvent B: CH₃CN/TFA (999:1)).