Evaluation of Caffeine Plasma Levels by an Automated Enzyme Immunoassay (EMIT) in Comparison with a High-Performance Liquid Chromatographic Method

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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 .mu.mol/l, r = 0.994 (x = HPLC, y = EMIT). Evaluation of the cross-reactivity of the 3 main human caffeine metabolites revealed no significant interference from the bromine and theophylline; however, there was significant interference (28%) by paraxanthine at a concentration range from 2.5 to 80 .mu.mol/l. At low caffeine concentrations, up to 10 .mu.mol/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. [Caffeine has been as effective as theophylline for the treatment of apnea in premature infants.].