Rapid high-performance liquid chromatography assay for salivary and serum caffeine following an oral load. An...

A rapid isocratic reversed-phase high-performance liquid chromatography (HPLC) system for the quantitative measurement of serum and salivary caffeine is described. The best separation of caffeine from other methylxanthines was achieved by chromatography on an ODS-Hypersil column using a solvent system of 0.1 M ammonium acetate pH 4.6-acetonitrile (85:15, v/v). The effluent was monitored at 280 nm. Caffeine was extracted from diluted serum and saliva samples (10-500 microliter) by adsorption on a small Bond-Elut C18 cartridge and recovered by elution with methanol. Thermospray HPLC-mass spectrometry conditions were optimized to afford a means of directly identifying caffeine in samples. The positive-ion mass spectrum was characterized by an intense protonated molecular ion, MH+, at m/z 195 and negligible fragmentation. When the mass spectrometer was operated in selected ion monitoring mode, caffeine could be detected in less than 1 microliter of serum and saliva at a concentration of 1 microgram/ml. Caffeine (3.5 mg/kg body wt.) was administered orally to healthy adults, children, and newborn infants, and to patients with liver disease. The clearance rate and half-life were determined as a test of liver function. A prolongation in the elimination of caffeine was observed in patients with liver disease and, although there was some overlap in the values obtained for patients with noncirrhotic liver disease and healthy persons, the oral caffeine load test may usefully serve as a dynamic assessment of liver function in the serial follow-up of patients with liver disease.