Gas chromatographic-mass spectrometric quantitation of theophylline and its metabolites in biological fluids.

In premature infants, theophylline is converted to caffeine, and the biological half-life is prolonged. To assess the metabolic alterations of theophylline during development of premature infants, a sensitive and simple method was developed which quantitated all theophylline metabolites in plasma, urine, and red blood cells. Theophylline and its metabolites in the sample were converted to the N-propyl derivative using n-propyl iodide in dimethylformamide with potassium carbonate catalysis and were analyzed under isothermal conditions on a gas chromatograph-mass spectrometer with a 3% methylsilicone-phenylsilicone column. Deuterated caffeine (caffeine-d3) was used as the internal standard. A selected ion-monitoring technique, together with 70-eV electron impact ionization mode, was used. The ion current ratios between caffeine-d3 (m/z 197) and caffeine (m/z 194), theophylline (m/z 222), 3-methylxanthine (m/z 250), 1,3-dimethyluric acid (m/z 280), and 1-methyluric acid (m/z 308) were monitored. The total analysis time was 12 min with a detection limit ranging from 500 pg to 10 ng, depending on the metabolites. With this sensitivity, sample sizes of 50-100 microliters of plasma and 0.5 ml of urine were sufficient for the analysis of all theophylline metabolites. The coefficient of variation of this method was less than 5% for the analysis of biological samples.