Determination of Caffeine and Dimethylxanthines in Biological Fluids and Tissues by High Performance Liquid Chromatography

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Abstract
Simple, rapid and sensitive methods of separation and quantification of caffeine and/or dimethylxanthines in biological fluids and tissues were developed by high performance liquid chromatography. A 0.1-ml of the serum was denatured and precipitated with 0.1 ml of acetonitrile. A 0.01-ml of the supernatant was injected into the chromatograph with a reversed-phase Zorbax ODS column and ultraviolet (UV) detector set at 273 nm and 0.01 a.u.f.s. The flow rate of the mobile phase of 0.2 M acetate buffer-acetonitrile (85:15, v/v) was 0.5 ml/min. The limit of detection was 0.2 .mu.g ml for caffeine. On analyses of biological fluids, caffeine and its metabolites were extracted from 0.2 ml of the plasma or saliva by using 2.5 ml of chloroform-isopropanol solution (75:25, v/v). On analyses of tissues, the liver, placenta or fetal cerebrum was homogenated by saline, adjusted to 10% solution, and caffeine and its metabolites were extracted from 0.5 ml of 10% tissue homogenates by using 5.0 ml of chloroform-isopropanol solution. The reversed-phase .mu.Bondapak C18 column was used, connected with UV detector set at 280 nm and 0.01 a.u.f.s. The flow rate of mobile phase of 0.005 M acetate buffer-methanol-acetonitrile-tetrahydrofuran (92.5:3.0:2.8:1.7,v/v) was 1.5 ml/min. The limits of detection of caffeine and dimethylxanthines on analyses of biological fluids and tissues were 0.1-0.2 .mu.g/ml and 1.0-2.0 .mu.g/g wet weight, respectively. The practicability and utility of the method were demonstrated in human and rat studies.

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