Simultaneous analysis of cytochrome P450 probes—dextromethorphan, flurbiprofen and midazolam and their major metabolites by HPLC-mass-spectrometry/fluorescence after single-step extraction from plasma

Cytochrome P450 enzymes catalyze oxidative metabolism of most pharmaceutical compounds. Consequently dextromethorphan, flurbiprofen, midazolam and other compounds are commonly used as probe substrates to evaluate cytochrome P450 function in humans. A “cocktail” approach employing simultaneous administration of two or more of the probe substrates has been used by various investigators in recent years. An analytical strategy to simultaneously extract and analyze dextromethorphan, flurbiprofen and midazolam and their major metabolites (dextrorphan, 4′-hydroxy-flurbiprofen and 1′-hydroxy-midazolam) by HPLC-MS/fluorescence was developed and is described here. The three probe substrates and their major metabolites were extracted simultaneously by means of a solid-phase (Bond Elut Certify ® cartridges) extraction procedure from 200 μl of pig plasma. The extraction efficiency was more than 79.5% for each of the six analytes. The extracted compounds were chromatographically separated on a Luna C8(II) column (50 mm L × 3 mm ID) in a single run of 20 min and analyzed by either fluorescence (flurbiprofen and 4′-hydroxy-flurbiprofen) or selective ion monitoring (dextromethorphan, dextrorphan, midazolam and 1′-hydroxy-midazolam) with positive electrospray ionization. The limit of quantification was 2.5 ng/ml for midazolam and 5 ng/ml for the other five analytes. The assay was precise and accurate (error: −9.1 to 12.1) with total CVs of 13.9% or better for each of the 6 analytes. This method was used to analyze concentrations of the three probes and their metabolites in plasma after intravenous administration to a healthy pig.