COMPARISON OF FLUORESCENCE POLARIZATION IMMUNOASSAY AND HPLC FOR THE DETERMINATION OF THEOPHYLLINE IN SERUM

Abstract

Theophylline in serum was measured by fluorescence polarization immunoassay (FPIA) and by high-performance liquid chromatography (HPLC). Within-run precision studies using control samples in the subtherapeutic, therapeutic and toxic concentrations, resulted in coefficients of variation in the range of 2.86-3.12% (FPIA) and 2.1-3.66% (HPLC), respectively. Between-run precision ranged from 2.76-6.2% for FPIA and from 2.51-6.0% for HPLC. The mean recovery for three spiked controls was 98.9% for FPIA and 98.8% for HPLC. Comparison of 60 patients' samples, assayed with both methods, indicated an extremely good analytical correlation (r = 0.990). The FPIA method displayed a slight but consistent positive bias in relation to the concentration of theophylline present in patients sera. Caffeine was found to exhibit a positive bias to 13%, over a caffeine concentration range of 10-40 micrograms/ml. The HPLC method offers an advantage for measurements of both caffeine and theophylline simultaneously. The FPIA offers significant advantages in speed of analysis and turnover-time, while maintaining accuracy and precision compared with those of established HPLC procedures.