Gas Chromatographic–Mass Spectrometric Quantitation of Theophylline and Its Metabolites in Biological Fluids

Abstract

□ 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 12min with a detection limit ranging from 500pg to 10 ng, depending on the metabolites. With this sensitivity, sample sizes of 50– 100 μl of plasma and 0.5ml of urine were sufficient for the analysis of all theophylline metabolites. The coefficient of variation of this method was <5% for the analysis of biological samples.