Postcolumn Immunodetection Following Conditioning of the HPLC Mobile Phase by On. Line Ion-Exchange Extraction

摘要

Postcolumn immunodetection can provide enhancement of selectivity and sensitivity when used in combination with HPLC. Typical reversed-phase HPLC employs a variety of organic modifiers and buffers, and immunoassays generally perform best in a low ionic strength aqueous matrix with a pH between 7 and 8. There is therefore a need for removal of an analyte from a typical mobile phase into an environment compatible with an immunoreactor. An on-line system utilizing an ion-exchange column along with a switching valve to sequester an analyte from a HPLC mobile phase and apply a counterion eluent solution was designed to provide the proper matrix for immunoassay. An on-line HPLC-ion-exchange system was developed for the model analyte procainamide hydrochloride. The eluents from the online HPLC and ion-exchange system were collected and analyzed off-line using fluorescence polarization immunoassay. The fluorescence polarization immunoassay was calibrated using standards in a buffer matrix, and the system was validated using controls at 2.0, 7.0, and 15.0 μg/mL in mobile phase. Procainamide and its metabolite were extracted from spiked plasma using liquid-liquid ex, action, and validation of the system with plasma samples demonstrated 15.60, 15.63 and 15.70% (n = 6) RSD for the three controls and accuracies of 6.42, 10.30, and 17.70% (n = 6) difference from spiked, respectively.