Neat 500 µM micafungin solution was serially diluted in 50/50 acetonitrile (ACN)/water to create neat standard curves and quality control spiking solutions. 10 µL of neat spiking solutions were added to 90 µL of drug-free macrophage lysate to create standard and QC samples. 10 µL of control, standard, or study sample lysate were added to 100 µL of a 50:50 acetonitrile: methanol protein precipitation solvent mix containing 10 ng/mL of the internal standard verapamil to extract micafungin. Extracts were vortexed for 5 min and centrifuged at 3700 × g for 5 min. 75 µL of supernatant was transferred for LC-MS/MS analysis and diluted with 75 µL of Milli-Q deionized water. LC-MS/MS analysis was performed on a Sciex Applied Biosystems Qtrap 6500+ triple-quadrupole mass spectrometer coupled to a Shimadzu Nexera X2 UHPLC system to quantify each drug concentrations in each sample. Chromatography was performed on an Agilent SB-C8 (2.1 × 30 mm; particle size, 3.5 µm) using a reverse phase gradient. Milli-Q deionized water with 0.1% formic acid was used for the aqueous mobile phase and 0.1% formic acid in acetonitrile for the organic mobile phase. Multiple-reaction monitoring of parent/daughter transitions in electrospray positive-ionization mode was used to quantify all analytes. The MRM transitions of 455.40/165.20, 1270.40/1190.40, 823.5/791.6, 402.2/358.0, 473.2/431.2 were used for verapamil, micafungin, rifampicin, moxifloxacin, and clofazimine, respectively. Data processing was performed using Analyst software (version 1.6.3; Applied Biosystems Sciex).
Free full text: Click here
