Quantification of mephenytoin and its metabolites 4'-hydroxymephenytoin and nirvanol in human urine using a simple sample processing method.
A reliable and easy to use liquid chromatography/tandem mass spectrometry (LC/MS/MS) method without the use of sample extraction was developed for the simultaneous quantification of urinary concentrations of mephenytoin, a standard phenotyping substrate for the cytochrome P450 enzyme CYP2C19, and its phase I metabolites 4'-hydroxymephenytoin and nirvanol. Fifty microL of urine were diluted with a buffered beta-glucuronidase solution and incubated at 37 degrees C for 6 h followed by addition of methanol, containing the internal standard 4'-methoxymephenytoin. The chromatographic separation was achieved using a 100 x 3 mm, 5 micro Thermo Electron Aquasil C18 column with a gradient flow, increasing the organic fraction (acetonitrile/methanol 50:50) of the mobile phase from 10 to 90%. Quantification by triple-stage mass spectrometry (TSQ Quantum, Thermo Electron) was accomplished by negative electrospray ionization in the selected reaction monitoring mode. Linearity was observed for all substances in the concentration range 15-10 000 ng/mL. The lower limit of quantification (LLOQ) was 20 ng/mL for 4'-hydroxymephenytoin and 30 ng/mL for nirvanol and mephenytoin, respectively. Intra- and inter-day inaccuracy did not exceed 9.5% for all substances from LLOQ to 10 000 ng/mL. Intra- and inter-day precision were in the range of 0.8-10.5%. The method was validated according to international ICH and FDA guidelines and successfully applied for phenotyping of Caucasian male volunteers who received an oral dose of 50 mg mephenytoin.
Department of Pharmacology, Clinical Pharmacology, University of Cologne, Gleueler Str. 24, 50931 Köln, Germany. Tobias.Klaassen@medizin.uni-koeln.de
Tobias
T
Klaassen
Dirk
D
Kasel
Stefanie
S
Harlfinger
Uwe
U
Fuhr
D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics; D002138:Calibration; D002853:Chromatography, Liquid; D065731:Cytochrome P-450 CYP2C19; D006801:Humans; D008297:Male; D013058:Mass Spectrometry; D008617:Mephenytoin / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000652:urine*; D006899:Mixed Function Oxygenases / Q000235:genetics; D015394:Molecular Structure; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
C014544:ethylphenylhydantoin; C025687:4-hydroxymephenytoin; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C045793:CYP2C19 protein, human; D065731:Cytochrome P-450 CYP2C19; D008617:Mephenytoin