Simple and selective assay of 4-hydroxymephenytoin in human urine using solid-phase extraction and high-performance liquid chromatography with electrochemical detection and its preliminary application to phenotyping test.
A simple and selective HPLC method for the determination of 4-hydroxymephenytoin (4-OH-M) in human urine, using a controlled potential coulometric detector equipped with a dual working electrode cell of fully porous graphite, has been developed. After acid hydrolysis of urine, 4-OH-M and the internal standard (I.S.), 5-hydroxy-1-tetralone, were extracted from urine by means of a Bond Elut Certify LRC column. The extracts were chromatographed on a reversed-phase mu Bondapak C18 column using methanol-50 mM KH2PO4 (pH 4.0) (30:70, v/v) as the mobile phase at a flow-rate of 1.0 ml/min. Electrochemical detection at applied potential of 800 mV resulted in a limit of quantitation of 0.76 micrograms/ml. The method showed a satisfactory sensitivity, precision, accuracy, recovery and selectivity. The present method was applied to the phenotyping test in thirteen Japanese healthy volunteers who received an oral 100-mg racemic mephenytoin. The phenotypes determined by the present method were found to be in agreement with those obtained with the reported customary assay based on gas chromatography.
Tanaka
M
M
Drug Metabolism and Analytical Chemistry Research Center, Daiichi Pharmaceutical Co. Ltd, Tokyo, Japan.
Kato
K
K
Sudo
K
K
Hakusui
H
H
Ohkubo
T
T
Sugawara
K
K
Kaneko
S
S
Otani
K
K
Chiba
K
K
Ishizaki
T
T
D000328:Adult; D000818:Animals; D000927:Anticonvulsants / Q000493:pharmacokinetics; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D004563:Electrochemistry; D006801:Humans; D007202:Indicators and Reagents; D008297:Male; D008617:Mephenytoin / Q000031:analogs & derivatives* / Q000493:pharmacokinetics / Q000652:urine; D010641:Phenotype; D051381:Rats; D012996:Solutions
D000927:Anticonvulsants; D007202:Indicators and Reagents; D012996:Solutions; C025687:4-hydroxymephenytoin; D008617:Mephenytoin