Comparison of high pressure liquid chromatography and fluorescence polarization immunoassay methods in a theophylline...

High pressure liquid chromatography (HPLC) and fluorescence polarization immunoassay (FPIA) were compared in a theophylline pharmacokinetic study. Eight healthy subjects received single 600-mg oral doses of two different sustained-release theophylline formulations. Fourteen blood samples were collected over 57 h after each dose, and the serum was analyzed for theophylline using both HPLC and FPIA methods. In comparing the two formulations using HPLC, there was no statistical difference in the area under the curve (AUC), terminal rate constant (k), or time of peak. However, there was a 13% difference in peak theophylline concentration (p less than 0.05). The same statistical conclusions were made for all parameters when using FPIA. When comparing the kinetic parameters determined with each assay, the AUC was 12% greater and the k was 17% smaller with FPIA (p less than 0.05). Orthogonal regression of all serum theophylline concentrations showed that FPIA = 1.04 HPLC + 0.20; r = 0.987, p less than 0.001. Stratification of serum theophylline concentrations into different ranges showed that FPIA overestimated the HPLC results in each range, but the percentage of overestimation was greater at lower concentrations (p less than 0.05). The use of FPIA seems appropriate in comparative studies of theophylline pharmacokinetics; however, the calculated kinetic parameters may differ slightly from those obtained with HPLC.