Automated determination of drugs in serum by columnswitching high-performance liquid chromatography II. Separation of theophylline and its metabolites

The automated determination of theophylline and related compounds in human serum by columnswitching high-performance liquid chromatography, including direct injection of serum samples, is described. TSK pre-column BSA-ODS and TSK gel ODS-80TM were used in the pre-column and analytical column, respectively. Serum samples of 20, μl were directly injected on to the pre-column. After washing out serum proteins from the pre-column with 0.1 M NaH₂PO₄ at a flow-rate of 1.0 ml/min for 3.5 min, the effluent from the pre-column was introduced on to the analytical column by a column-switching device. The analysis was performed by stepwise gradient elution using 10 and 18% methanol in 0.1 M NaH₂PO₄. Theophylline and nine derivatives could be determined simultaneously within 40 min. The recovery of these compounds from serum was 95-103%. The linearity (1.0-50, μ/ml theophylline) and reproducibility (coefficient of variation less than 2.0%) were sufficient for drug monitoring at the lower and upper limits of therapeutic concentrations of theophylline.