Drug quantitation by HPLC coupled to tandem mass spectrometry (LC/MS-MS)

Drug levels in tissue were determined by spatial quantitation in thin tissue sections by LCM followed by LC/MS-MS analysis of microdissected areas (38). The benefit of the LCM approach is the ability to obtain absolute drug levels in defined areas of Type I lung lesions such as caseum (the core of the caseous necrotic lesion) without cross-contamination. The tissue sections of 25 µm thickness were cut from infected mouse lung biopsies using a Leica CM 1860UV (Buffalo Grove, IL) and thaw-mounted onto Leica PET-Membrane FrameSlides (Buffalo Grove, IL) of 1.4 µm thickness. Approximately 10 µm thick tissue sections were thaw-mounted onto standard glass microscopy slides for H&E staining. Sequential rings of necrotic tissue proceeding from the border with the cellular rim to the core of the caseous compartment were demarcated for each tissue section by creating a mask of caseum and processing it using the Exact Euclidean Distance Transform plugin in ImageJ (National Institutes of Health, MD). Sequential rings of necrotic tissue were then dissected using a Leica LMD6 system (Buffalo Grove, IL). Dissected lesion tissues were collected into standard PCR tubes of 0.25 mL and immediately transferred to −80°C.

RIF, BDQ, INH, and verapamil (VER) were purchased from Sigma Aldrich (St. Louis, MO, USA); RIF-d8, BDQ-d6, and INH-d4 were purchased from Toronto Research Chemicals (Ontario). Drug-free lung and K₂EDTA plasma from CD-1 mice was obtained from BioIVT (Westbury, NY) for use as blank matrices to build standard curves. Neat 1 mg/mL dimethyl sulfoxide stocks were serially diluted in 50:50 acetonitrile/water to create a standard curve and quality control spiking solutions and 10 µL of neat spiking solutions were added to 2 µL of lesion homogenate prior to extraction. On the day of analysis, samples were extracted by adding 50 µL of extraction solution (acetonitrile/methanol, 1:1) containing 1, 10, 4, and 5 ng/mL of VER, INH-d4, RIF-d8, and BDQ-d6, respectively. Extracts were sonicated for 5 min and centrifuged at 10,000 rpm for 5 min, and 40 µL of supernatant was transferred to 96-well deep plates for LC-MS/MS analysis. For INH quantitation, 40 µL of 2% cinnamaldehyde in methanol was added to derivatize isoniazid prior to analysis. For RIF quantitation, 5 µl of 75 mg/mL ascorbic acid was added to extracts to stabilize RIF during analysis.

LC/MS-MS analysis was performed on a Sciex Qtrap 6500 + triple-quadrupole mass spectrometer coupled to a Shimadzu Nexera X2 UHPLC system. Chromatography was performed on an Agilent Zorbax SB-C8 column (2.1 × 30 mm; particle size 3.5 µm) using a reverse phase gradient. Deionized water with 0.1% formic acid (FA) was used for the aqueous mobile phase and 0.1% FA in acetonitrile for the organic mobile phase. Multiple-reaction monitoring (MRM) of precursor/fragment transitions in the electrospray positive-ionization mode was used to quantify the analytes. MRM transitions of 823.30/791.30, 555.00/58.00, 252.200/80.30 455.40/165.20, 831.30/799.40, 561.00/64.00, and 256.20/84.30 were used for RIF, BDQ, INH, VER, RIF-d8, BDQ-d6, and INH-d4, respectively. Data processing was performed using Analyst software version 1.6.3 (Sciex).