High-performance liquid chromatographic procedure for specificity testing of radioimmunoassays : rolipram

A high-performance liquid chromatographic (HPLC) procedure for testing the specificity of radioimmunoassays (RIA) was developed using the same method of extraction as in the RIA, followed by fractionation of the extract by HPLC and subsequent measurement of cross-reactions in all the fractions according to the normal RIA procedure. The RIA of rolipram, an antidepressant drug, was checked in plasma samples obtained from pharmacokinetic studies in rats, rabbits, cynomolgus monkeys and humans. The antibody was shown to be specific in the plasma samples from the laboratory animals, but not in human plasma. This was because in human plasma a metabolite occurred with a structure similar to that used for the hapten in the immunization process. This metabolite was not found in the plasma of the animal species investigated. The test procedure described is generally applicable, making the time-consuming development of an alternative method such as gas chromatography—mass spectrometry unnecessary.