Simultaneous determination of caffeine and theophylline in human plasma with a weak cation monolithic SPE-column

A simple reversed-phase high performance liquid chromatography (RP-HPLC) method was developed to determine the level of caffeine and theophylline in human plasma samples. The sample clean-up step involved the on-line solid-phase extraction (SPE) of the analytes from plasma samples into a weak cation monolithic column using a column switching system. Separation was performed on a C ₁₈ column (5 μm, 150 mm ×4.6 mm) with ultraviolet detection at 274 nm. The mobile phase consisted of methanol-water (32/68, V/V) under isocratic conditions at a flow rate of 0.6 mL ^•min ¹. The measured concentration of caffeine and theophylline showed a good linear relationship over the concentrations range, 0.1-80.0 μg ^•mL ¹. The absolute recoveries ranged from 77.10% to 85.39%, and the inter-day and intra-day relative standard deviations (RSD) were all less than 5%. This method avoids a tedious pretreatment and provides an economic, repeatable and effective method for assaying trace drugs in biological samples.