Determination of total and free plasma carbamazepine concentrations by enzyme multiplied immunoassay: interference with the 10,11-epoxide metabolite.

The performance of the enzyme multiplied immunoassay technique (EMIT) in the measurement of total and free plasma carbamazepine (CBZ) levels was assessed in 140 clinical specimens and compared with a reference high pressure liquid chromatography (HPLC) technique. Free drug was measured in plasma filtrates obtained by the Free Level system. Both total and free CBZ levels as determined by EMIT correlated strongly with corresponding HPLC values (r = 0.88 and 0.92, respectively). Plasma CBZ concentrations, however, were higher by EMIT than by HPLC. The degree of CBZ overestimation by EMIT was relatively small (about 14%) in whole plasma but quite considerable (35% on average) in the filtrates. As a result, estimated values of free CBZ fraction were also higher for EMIT than for HPLC. Separate experiments in vitro suggested that the discrepancies between the two methods were due to cross-reaction of the EMIT reagent with the 10,11-epoxide metabolite of CBZ. The greater degree of overestimation for the free drug can be explained by the higher proportion of less protein-bound metabolite in the filtrates. These results need to be taken into account in the interpretation of free CBZ level data from laboratories using different techniques.