Electrochemical immunoassay combined with column liquid chromatography: determination of phenytoin in human serum.

A new electrochemical immunoassay combined with column liquid chromatography has been developed for the determination of phenytoin in human serum. Phenytoin was labelled with the electrochemically active nitroxide, and the separation of the free labelled antigen from other electrochemically active compounds in serum was accomplished by the use of gel chromatography. Serum samples were mixed with the antibody and the labelled antigen, incubated for 90 min, and then a 100-microliter aliquot of the mixture was directly injected to the column, which was equipped with an electrochemical detector. With 10 microliter of serum, the smallest detectable concentration of phenytoin was 2 micrograms/ml.