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Quantification of mephenytoin and its metabolites 4′‐hydroxymephenytoin and nirvanol in human urine using a simple sample processing method
Author(s) -
Klaassen Tobias,
Kasel Dirk,
Harlfinger Stefanie,
Fuhr Uwe
Publication year - 2004
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.1539
Subject(s) - chemistry , chromatography , mephenytoin , electrospray ionization , mass spectrometry , selected reaction monitoring , formic acid , isotope dilution , detection limit , urine , tandem mass spectrometry , cyp2c19 , enzyme , cytochrome p450 , biochemistry
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 μL of urine were diluted with a buffered β ‐glucuronidase solution and incubated at 37°C for 6 h followed by addition of methanol, containing the internal standard 4′‐methoxymephenytoin. The chromatographic separation was achieved using a 100 × 3 mm, 5 μ 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. Copyright © 2004 John Wiley & Sons, Ltd.