Development of a Flow-Based Ultrafast Immunoextraction and Reverse Displacement Immunoassay: Analysis of Free Drug Fractions

A flow-based method employing a reverse displacement immunoassay was combined with ultrafast immunoextraction and near-infrared fluorescence detection for the analysis of free drug fractions, using phenytoin as a model analyte. Factors considered in the design of this method included the sample application conditions, the design of the immobilized drug analog column, the utilization of antibodies or F-ab fragments as labeled binding agents, and the label application and column regeneration conditions. In the final method, sample injections led to the displacement of labeled binding agents from an immobilized phenytoin analog column. This displacement peak appeared within 20-30 s of sample injection and was proportional in size to the free phenytoin concentration in the sample. It was possible with this method to regenerate the column using only the application of additional label between sample injections. This method was used to measure clinically relevant concentrations of free phenytoin in serum and drug/protein mixtures and gave good correlation with ultrafiltration, while also being faster to perform and requiring significantly less sample. This technique was not limited to free phenytoin measurements but could be adapted for other drugs or analytes through the use of appropriate columns and binding agents.