SIMULTANEOUS SEPARATION AND DETERMINATION (IN SERUM) OF PHENYTOIN AND CARBAMAZEPINE AND THEIR DEUTERATED ANALOGS 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 duterated H-2(10) analogues of carbamazepine (H-2(10) CBZ) and phenytoin (H-2 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 H-2(10) CBZ, CBZ, H-2(10) PHT, PHT and IS is achieved on a heated (55-degrees-C) 25 cm x 4.6 mm BioAnalytical Systems Phase II 5-mu-m 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 H-2(10) CBZ from CBZ and H-2(10) 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-mu-g/ml for H-2(10) CBZ, 0.5-20 mu-g/ml for CBZ, 0.5-20-mu-g/ml for H-2(10) PHT, and 0.5-30-mug/ml for PHT. Within-day percent relative standard deviations (precision) were less than 6% in all cases.