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

The free fraction of phenytoin (PHT) in serum increases considerably i n combination with valproic acid (VPA), depending on the VPA concentra tion. Equations to predict the free PHT concentration (PHTf) from the total PHT concentration (PHTt) and from the VPA concentration were dev eloped by Haidukewych and colleagues. (equation 1: PHTf = 0.095 x PHTt + 0.001 x VPA x PHTt) and May and colleagues (equation 2: PHTf = 0.07 92 x PHTt + 0.000636 x VPA x PHTt); in both equations, PHTf, PHTt, and VPA are given in mu g/ml. Obviously, the equations give different pre dictions. The aim of this study was to investigate whether different m ethods 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) val ues. A total of 52 samples from patients with VPA (n = 26) or without VPA (n = 26) were analyzed using HPLC and TDx. The concentrations meas ured by HPLC and TDx were highly correlated but TDx yields significant ly higher PHTt (Y = 0.98 x X + 2.46; X = HPLC, Y = TDx, r(2) = 0.957) and, in particular, higher PHTf concentrations (Y = 1.03 x X + 0.30; X = HPLC, Y = TDx, r(2) = 0.919), compared with our HPLC method. The ac curacy of the predictive equations depends on the method used for the determination of PHTt and PHTf. The best predictions of PHTf were obta ined if equation 2 and HPLC measurements were used. However, the diffe rences in the predicted PHTf could only partly be explained by the dif ferent methods of determination.