Use of alkaline phosphatase to correct the underestimation of fosphenytoinconcentration in serum measured by phenytoin immunoassays

The pharmacologic activity of fosphenytoin, a new phosphate ester pro-drug of phenytoin, is due to in vivo conversion to phenytoin. Fosphenytoin conce ntrations cannot be accurately estimated by phenytoin immunoassays (floresc ence polarization and chemiluminescence) owing to the nonlinear relation be tween fosphenytoin concentration and the observed cross-reactivity. The pro blem of slow conversion of fosphenytoin to phenytoin in serum in vitro can be circumvented by rapidly converting fosphenytoin to phenytoin in vitro by alkaline phosphatase. Drug-free serum heparin, EDTA, or citrated plasma we re supplemented with 2 concentrations of fosphenytoin. Then to I-mt aliquot s of specimen, no enzyme (control), 10 mu L, or 25 mu L of enzyme solution was added The specimens were incubated, and phenytoin concentrations were m easured by fluorescence polarization and chemiluminescent assays. In the ab sence of enzyme, we observed little conversion of fosphenytoin to phenytoin , but in the presence of only 10 mu L of enzyme, the conversion of fospheny toin to phenytoin tvas complete in 5 minutes. We also observed complete con version of fosphenytoin to phenytoin by alkaline phosphatase in heparin, ED TA, and citrated plasma. If clinically indicated, the phenytoin concentrati on can be measured before and after addition of enzyme to roughly estimate the rate of conversion.