Determination of phenobarbital, phenytoin and carbamazepine in human serum: comparison of fluorescence polarization immunoassay with high performance liquid chromatography

ABSTRACT

A simple high performance-liquid chromatography [HPLC] method for simultaneous determination of antiepileptic drugs i.e., carbamazepine, phenobaebital and phenytion in human serum is described. The procedure involves deproteinization of serum with acetonitrile containing phenacetin as an internal standard prior to injection onto the chromatograph. The drugs were eluted from a resolve 5 micro spherical c18 column with a mobile phase consisting of 0.01 m potassium dihydrogen phosphate methanol [5554%, v/v] adjusted to PH 6.0 with potassium hydroxide. The drugs and the internal standard were eluted at a flow rate of 1.5 ml per minute, and the optimum detector wavelength was 220 nm. The chromatography time was ten minutes and the resolution between the drugs and internal standard was excellent. Quantitation was achieved by the measurement of peak height ratios and the minimum detectable concentration was less than 0.1 mg/l. the calibration curve of the assay was linear over the range of 0.1-25 mg/l, 0.5-120mg/L and 0.1-80 mg/L for carbamazepine, Phenobarbital and phenytoin respectively respectively. The Abbott TDX FLXTM fluorescence polarization immunoassay [FPIA] has been evaluated and compared with the developed HPLC method for precision, calibration curve stability, specificity and accuracy. Using control samples in the subtherapeutic, therapeutic and toxic concentrations resulted in mean analytical recoveries which varied from 95.3 to 99.8% for HPLC and 96.8 to 101.2 for FPIA. Within run coefficient of variation was in the range of 1.66-4.47% for HPLC and 1.50-3.75% for FPIA. Between run precision ranged from 2.10-4.71% for HPLC and 1.21-4.28% for FPIA. Serum specimens from 270 patients taking carnamazepine, 191 patients taking Phenobarbital and 225 patients taking phenytion were analyzed by both methods, with good correlation. The HPLC method offered reproducibility and accuracy when compared with an established method for routine analysis of carbamazepine, Phenobarbital and phenytoin