Simultaneous determination of theophylline, tolbutamide, mephenytoin, debrisoquin, and dapsone in human plasma using high-speed gradient liquid chromatography/tandem mass spectrometry on a silica-based monolithic column

Theophylline, tolbutamide, mephenytoin, debrisoquin, and dapsone are marker substrates for CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, respectively. A silica-based monolithic column (Chromolith SpeedROD RP-18e, 50 x 4.6 mm) was used to separate these five marker substrates of cytochrome P450 within only 84 s. Linear gradient elution was from acetonitrile-water-formic acid (10 : 90 : 1, v/v/v) to acetonitrile-water-formic acid (90: 10: 1, v/v/v) in 1.4 min. The flow rate was 2.5 mL/min. The retention time was 0.52 min for theophylline, 0.67 min for debrisoquin, 0.78 min for dapsone, 0.96 min for mephenytoin, and 1.13 min for tolbutamide. Detection was by tandem mass spectrometry using a PE Sciex API 3000 mass spectrometer with a Turbo-lonspray source in positive mode. A simple protein precipitation method was used. This method was validated over the concentration range of 52000 ng/mL based on the sample volume of 0.1 mL.