Determination of total and free carbamazepine and the principal metabolites in serum by high-performance liquid chromatography with photodiode-array detection

A precise and accurate high-performance liquid chromatography (HPLC) method has been established for the simultaneous analysis of carbamazepine (CBZ), carbamazepine-10,11-epoxide (CBZ-E), and trans-10,11-dihydroxy-10,11-dihydro-CBZ (CBZ-H) in serum samples and their ultrafiltrates. CBZ and its metabolites are eluted in a 3-μM ODS-Hypersil column (250 x 2 mm) at a column temperature of 40°C. The mobile phase is a mixture containing potassium phosphate buffer-acetonitrile-methanol (110:50:30, vol/vol/vol) at a flow rate of 0.2 ml/min. Signals are monitored by a photodiodearray detector with a main sample wavelength of 215 nm and a bandwidth of 10 nm. Coefficients of variation (CVs) for within- and between-day are within 5%, with the recovery rates ranging from 98.16 to 104.64%. This method has the necessary sensitivity and linearity for routine therapeutic monitoring of both total and free CBZ and its principal metabolites. Total serum concentrations of CBZ, CBZ-E, and CBZ-H obtained from 55 epileptic children were 12.58 ± 4.42,2.45 ± 1.22, and 5.83 ± 3.17 (mean ± SD, μg/ml), respectively. Levels of free CBZ, CBZ-E, and CBZ-H were 2.59 ± 0.93, 1.05 ± 0.57 and 3.73 ± 1.87, respectively. Free fractions of CBZ, CBZ-E, and CBZ-H were 20.98 ± 4.34, 42.63 ± 8.21, and 65.41 ± 7.80%, respectively. CBZ-H and CBZ-E had larger CVs than did CBZ (54.34 and 49.75 vs 35.15%, respectively, for total levels, and 50.31 and 54.46 vs 36.22%, respectively, for free levels), as well as higher free fractions. Determination of both total and free concentrations and free fractions of CBZ and its metabolites, as well as their ratios, should provide additional needed information for therapeutic drug monitoring of CBZ.