Comparison of total and free phenytoin serum concentrations measured by high-performance liquid chromatography and standard TDx assay: implications for the prediction of free phenytoin serum concentrations

Ther Drug Monit. 1998 Dec;20(6):619-23. doi: 10.1097/00007691-199812000-00006.


The free fraction of phenytoin (PHT) in serum increases considerably in combination with valproic acid (VPA), depending on the VPA concentration. Equations to predict the free PHT concentration (PHTf) from the total PHT concentration (PHTt) and from the VPA concentration were developed by Haidukewych and colleagues. (equation 1: PHTf = 0.095 x PHTt + 0.001 x VPA x PHTt) and May and colleagues (equation 2: PHTf = 0.0792 x PHTt + 0.000636 x VPA x PHTt]; in both equations, PHTf, PHTt, and VPA are given in microg/ml. Obviously, the equations give different predictions. The aim of this study was to investigate whether different methods for the determination of PHTt and PHTf were responsible for the differences; equation 1 was calculated from standard TDx measurements and equation 2 from high-performance liquid chromatography (HPLC) values. A total of 52 samples from patients with VPA (n=26) or without VPA (n=26) were analyzed using HPLC and TDx. The concentrations measured by HPLC and TDx were highly correlated but TDx yields significantly higher PHTt (Y = 0.98 x X + 2.46; X = HPLC, Y = TDx, r2 = 0.957) and, in particular, higher PHTf concentrations (Y = 1.03 x X + 0.30; X = HPLC, Y = TDx, r2 = 0.919), compared with our HPLC method. The accuracy of the predictive equations depends on the method used for the determination of PHTt and PHTf. The best predictions of PHTf were obtained if equation 2 and HPLC measurements were used. However, the differences in the predicted PHTf could only partly be explained by the different methods of determination.

Publication types

  • Comparative Study

MeSH terms

  • Chromatography, High Pressure Liquid
  • Dose-Response Relationship, Drug
  • Drug Interactions
  • Fluorescence Polarization
  • Humans
  • In Vitro Techniques
  • Models, Theoretical
  • Phenytoin / blood*
  • Protein Binding
  • Valproic Acid / blood*


  • Valproic Acid
  • Phenytoin