Development and validation of a sensitive LC–MS/MS method for the simultaneous quantitation of theophylline and its metabolites in rat plasma

A rapid, specific, and reliable LC–MS/MS-based bioanalytical method was developed and validated in rat plasma for the simultaneous quantitation of theophylline and its four metabolites: 1,3-dimethyluric acid (1,3-DMU), 3-methylxanthine (3-MX), 1-methylxanthine (1-MX), and 1-methyluric acid (1-MU). Chromatographic separation of these analytes was achieved on a Gemini C18 column (50 mm × 4.60 mm, 5 μm) using reversed phase chromatography. The analytes were monitored by electrospray ionization in negative ion multiple reaction monitoring mode. Modification of collision energies was performed in parallel with chromatographic separation to further eliminate interference peaks. The method was validated from 0.05 to 30 μg/mL for 1-MX, 1,3-DMU, 1-MU, and theophylline and from 0.1 to 30 μg/mL for 3-MX using 0.2 mL of plasma sample. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels exhibited relative standard deviations (RSD) of less than 13% and with relative error (RE) values of −8.8% to 9.7%. The method was successfully applied for the quantitation of theophylline and its metabolite in rat plasma samples.