Evaluation of caffeine plasma levels by an automated enzyme immunoassay (EMIT) in comparison with a high-performance liquid chromatographic method

Abstract

A new enzyme immunoassay (EMIT) for the measurement of levels of caffeine in plasma was adapted to an automated centrifugal analyzer (Cobas Bio) and compared with a high-performance liquid chromatographic (HPLC) method. Precision of the EMIT test was similar to that of the HPLC method with intraassay coefficients of variation in the range of 2.0-4.1% (EMIT) and 1.5-3.3% (HPLC), respectively, depending on the concentration range tested. Day-to-day precision ranged from 2.7 to 5.6% for EMIT and was 3% for HPLC. Comparison of 69 patient samples assayed with both methods yielded the following equation: y = 1.06x + 1.25 mumol/L, r = 0.994 (X = HPLC, y = EMIT). Evaluation of the cross-reactivity of the three main human caffeine metabolites revealed no significant interference from theobromine and theophylline; however, there was significant interference (28%) by paraxanthine at a concentration range from 2.5 to 80 mumol/L. At low caffeine concentrations, up to 10 mumol/L, the level of this metabolite may be more than twice the corresponding caffeine concentration; therefore, the latter may be falsely elevated in the EMIT test. Despite this cross-reactivity, the new EMIT test proved to be suitable for use in a drug assay laboratory, as well as in the routine screening of outpatients for liver disease.