Novel type of flow injection immunoassay for the determination of phenytoin in serum Original Research Article

A novel flow injection immunoassay using fluorimetric detection successfully measured an antiepileptic drug, phenytoin, from a serum background. The assay was based on the competitive binding of fluorescein labelled and unlabelled phenytoin followed by the separation of bound and unbound antigens with gel filtration chromatography. The immunoreaction and subsequent detection of products was performed in a newly designed stopped flow injection system employing a simple two injector system for reagent injection, mixing and rigorous timing of the incubation. Two experimental set-ups were tested. The optimal parameters and configurations of the systems were established and the results were verified against an independent method. The calibration curves were linear showing good sensitivity and reproducibility in the concentration range required for clinical analyses suggesting that this technique could be a viable alternative for the measurement of the phenytoin in serum samples.