Simultaneous determination of carbamazepine, phenytoin, phenobarbital, primidone and their principal metabolites by high-performance liquid chromatography with photodiode-array detection

We have established a precise and accurate high-performance liquid chromatographic method for the simultaneous assay of carbamazepine, phenytoin, phenobarbital, primidone and their principal metabolites. This method has been used for the analysis of these drugs and the metabolites in serum, saliva and urine samples. Acetonitrile is used for the deproteinization of serum and saliva samples while solid-phase extraction is utilized for urine sample pretreatment. Samples of 2 μl are injected onto a 3-μm ODS-Hypersil column (250 mm × 2 mm I.D.) with a column temperature of 40°C. The drugs and metabolites are eluted with a mobile phase containing potassium phosphate buffer-acetonitrile-methanol (110:50:30, v/v/v) at a flow-rate of 0.2 ml/min. Signals are monitored by a photodiode-array detector at a sample wavelength of 200 nm with a bandwidth of 10 nm. These four commonly used antiepileptic drugs and their six metabolites are well separated from one another within 15 min. Within-day coefficients of variation (C.V.) are within 5% in most cases and between-day C.V. are from 2.32 to 4.75%. The recovery rates range from 95.12 to 104.42%. This method has the necessary sensitivity and linearity for routine therapeutic monitoring of both total and free drug levels and may be employed for pharmacokinetics studies of drug interactions and metabolism as well.