SIMULTANEOUS DETERMINATION OF PHENYTOIN, CARBAMAZEPINE, AND 10,11-CARBAMAZEPINE EPOXIDE IN HUMAN PLASMA BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH ULTRAVIOLET DETECTION

The Bioanalytical Chemistry Department at the Madison facility of Cova nce Laboratories, has developed and validated a simple and sensitive m ethod for the simultaneous determination of phenytoin (PHT), carbamaze pine (CBZ) and 10,11-carbamazepine epoxide (CBZ-E) in human plasma by high-performance liquid chromatography with 10,11 dihydrocarbamazepine as the internal standard. Acetonitrile was added to plasma samples co ntaining PHT, CBZ and CBZ-E to precipitate the plasma proteins. After centrifugation, the acetonitrile supernatant was transferred to a clea n tube and evaporated under N-2. The dried sample extract was reconsti tuted in 0.4 mi of mobile phase and injected for analysis by high-perf ormance liquid chromatography. Separation was achieved on a Spherisorb ODS2 analytical column with a mobile phase of 18:18:70 acetonitrile:m ethanol:potassium phosphate buffer. Detection was at 210 nm using an u ltraviolet detector. The mean retention times of CBZ-E, PHT and CBZ we re 5.8, 9.9 and 11.8 min, respectively. Peak height ratios were fit to a least squares linear regression algorithm with a 1/(concentration)( 2) weighting. The method produces acceptable linearity, precision and accuracy to a minimum concentration of 0.050 mu g ml(-1) in human plas ma. It is also simple and convenient, with no observable matrix interf erences. (C) 1998 Elsevier Science B.V. All rights reserved.