Simultaneous separation and determination (in serum) of phenytoin and carbamazepine and their deuterated analogues by high-performance liquid chromatography--ultraviolet detection for tracer studies.

The use of stable isotope-labeled tracer compounds is the safest and most effective method to perform many steady state pharmacokinetic and drug interaction studies. We describe a method by which the heavily deuterated 2H10 analogues of carbamazepine (2H10 CBZ) and phenytoin (2H10 PHT) can be chromatographically separated by high-performance liquid chromatography from unlabeled CBZ and PHT. All compounds are quantitated against an internal standard (IS) (10,11-dihydrocarbamazepine) and measured using conventional UV detection rather than mass spectrometry. Baseline resolution of extracted serum containing 2H10 CBZ, CBZ, 2H10 PHT, PHT and IS is achieved on a heated (55 degrees C) 25 cm x 4.6 mm BioAnalytical Systems Phase II 5 microns ODS column with an isocratic mobile phase consisting of water-acetonitrile-tetrahydrofuran (80:16:4, v/v/v) at 1.2 ml/min. Eluting compounds were monitored at a UV wavelength of 214 nm. Calculated resolution of 2H10 CBZ from CBZ and of 2H10 PHT from PHT were 1.3. Serum standard curves were linear (R greater than or equal to 0.999) over a range of 0.5-14 micrograms/ml for 2H10 CBZ, 0.5-20 micrograms/ml for CBZ, 0.5-20 micrograms/ml for 2H10 PHT, and 0.5-30 micrograms/ml for PHT. Within-day percent relative standard deviations (precision) were less than 6% in all cases.

Simultaneous determination of p-hydroxylated and dihydrodiol metabolites of phenytoin in urine by high-performance liquid chromatography.

Accurate urinary measurements of the two major metabolites of phenytoin, 5-(p-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) and 5-(3,4-dihydroxy-cyclohexa-1,5-dienyl)-5-phenylhydantoin (dihydrodiol, DHD), are necessary for pharmacokinetic and drug-interaction studies of this commonly used antiepileptic drug. We describe a simple, rapid, acid hydrolysis, with liquid-liquid extraction and simultaneous isocratic reversed-phase high-performance liquid chromatography of p-HPPH and 5-(m-hydroxyphenyl)-5-phenylhydantoin (m-HPPH) (hydrolytic end product of DHD). p-HPPH and m-HPPH were quantitated against their separate respective internal standards of alphenal and tolylbarb. The mobile phase consisted of water-dioxane-tetrahydrofuran (80:15:5, v/v/v) at 2 ml/min and at 50 degrees C, with detection at 225 nm. Baseline separation was achieved by use of a 16 cm x 3.9 mm Nova-Pak C18 column and total analysis time of 12 min. p-HPPH and m-HPPH concentrations ranged from 10 to 200 and from 2 to 30 micrograms/ml, respectively, with between-day coefficients of variations of 3.3-4.5% and 2.2-5.1% for controls. All standard curves were linear with r values greater than 0.993. The DHD concentration was determined by multiplying m-HPPH concentrations by 2.3.