Xevo g2 tof acquity uplc h class system

Drugs blood concentrations were determined by LC-MS using calibrated curves essentially as described5 (link)9 (link). Briefly, OAK and/or rifampin were given by SC or oral administration. At specified time intervals mice were euthanized (CO₂ asphyxiation) and blood samples collected from vena cava (N = 2 mice/time point). For analysis, samples were centrifuged (2 min, 6000 × g), 200 μl plasma were mixed with 0.5 ml extraction buffer (50% acetonitrile (ACN): 50% methanol) incubated (30 min on plate shaker at 200 rpm, RT), centrifuged (2 min, 14000 × g) and 300 μl supernatant diluted twofold in DDW and analyzed by LC-MS (5 μl injected to Waters Xevo G2 Tof/ACQUITY UPLC H-Class system). Flow rate, 0.5 ml/min. Run time, 5 min. Mobile phase, ACN/DDW combination containing 0.1% formic acid using an ACQUITY UPLC BEH column (C₁₈ 1.7 μm) and eluted with a 0–90% ACN gradient. Quantification was by MS detection in positive ionization mode using an identical procedure that was performed in mouse whole blood in order to establish standard calibration curves.

Electrospray ionization mass spectrometry (ESI-MS) was used to verify the FMN incorporation and determine the protein mass. Samples were applied to ESI-MS using the Waters® Xevo® G2 Tof/ACQUITY UPLC H—Class® System (Waters) coupled to a quadrupole/time-of-flight (QToF) mass spectrometer (Waters) equipped with a PDA detector. The method employed was similar as in previous work [25 ,37 (link)]. Protein samples were diluted to 1 μM and 4 μL samples were injected. The protein samples were first separated on an Acquity BEH C4 (150 × 2.1 mm, 1.7 μm, Waters) column operated at 40 °C. Protein were eluted using a gradient from solvent A (0.1% formic acid in water) to B (0.1% formic acid in acetonitrile) at a flow rate of 0.3 mL/ min using the following protocol: 2 min at 95% A, 13 min to go from 95% to 5% A. Mass spectra were obtained in the ESI-positive ion mode. The obtained charge density spectra were deconvoluted using the MagTran software.