Rapid and simultaneous measurement of midazolam, 1′-hydroxymidazolam and digoxin by liquid chromatography/tandem mass spectrometry: Application to an in vivo study to simultaneously measure P-glycoprotein and Cytochrome P450 3A activity

In order to simultaneously determine in vivo P-glycoprotein (P-gp) and Cytochrome P450 3A (CYP3A) activity, a new, rapid and sensitive liquid chromatography/tandem mass spectrometry (LC–MS/MS) method has been developed and fully validated to simultaneously determine midazolam (MDZ, as CYP3A substrate), 1′-hydroxymidazolam (1′-OHMDZ) and digoxin (DG, as P-gp substrate) in rat plasma using digitoxin as the internal standard (IS). After a single step liquid–liquid extraction with tert-butyl methyl ether/dichloromethane (75:25, v/v), analytes were subjected to LC–MS/MS analysis using positive electro-spray ionization (ESI+) under selected reaction monitoring mode (SRM). Chromatographic separation was performed on an XTerra MS C18 column (50 mm × 2.1 mm, i.d. 3.5 μm). The MS/MS detection was conducted by monitoring the fragmentation of 326.05 → 244.00 (m/z) for MDZ, 342.02 → 168.01 (m/z) for 1′-OHMDZ, 798.33 → 651.36(m/z) for DG and 782.67 → 635.24 (m/z) for IS. The method had a chromatographic running time of 3 min and linear calibration curves over the concentrations of 2–400 ng/mL for MDZ and 1′-OHMDZ and 0.5–100 ng/mL for DG. The recoveries of the method were 86.8–96.3% for MDZ, 84.6–86.4% for 1′-OH MDZ, and 81.7–85.1% for DG. The lower limit of quantification (LLOQ) of the method was 2 ng/mL for MDZ and 1′-OHMDZ and 0.5 ng/mL for DG. The intra- and inter-batch precision were less than 15% for all quality control samples at concentrations of 5, 50 and 320 ng/mL for MDZ and 1′-OHMDZ and 1, 10 and 80 ng/mL for DG. The validated LC–MS/MS method has been successfully used to analyze the concentrations of MDZ, 1′-OH MDZ and DG in rat plasma for simultaneous measurement of in vivo P-gp and CYP 3A activity.