Simultaneous Determination of Theophylline and Caffeine by Reversed Phase Liquid Chromatography Using Phenyl Column

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Abstract
Phenyl, C-8, and C-18 columns were evaluated for the simultaneous analysis of theophylline (TH) and caffeine (CA). The phenyl column was chosen for its capability of resolving of 1,7-dimethylxanthine from theophylline. The procedure utilized protein precipitation of either 50 or 100 ul of serum with diluted trichloroacetic acid, followed by analysis with either a manual isocratic or automated gradient elution mode. Chromatographic conditions were: column = phenyl (4.5 × 150 mm with a 4.5 × 50 mm guard column), mobile phase = phosphate or acetate/ACN, and detection wavelength = 280 nm. Peak height or area ratios of TH or CA to the internal standard, beta-hydroxyethyl theophylline (βHET) were linearly correlated to concentratiozn ranges between 2.5 to 20 or 50 mg/L. Precision studies of “quality control” samples showed that the day-to-day coefficients of variation were leas than 9% for both TH and CA. TH plasma concentrations of patients quantitated by this procedure were compared to the clinically accepted assay by FPIA using either polyclonal or monoclonal antibodies. Results were closely correlated. CA concentrations of these “patients” ranged from 0.1 to 5 mg/L. The present study showed that the phenyl column exhibited long-term stability, suitable for clinical application of drug monitoring. Further, the small sample size of 50 ul would be useful for neonatal monitoring of TH and CA.