Rapid absolute quantification of caffeine, theophylline, and theobromine in beverages by MALDI mass spectrometry

A rapid method utilizing matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been developed and validated for the quantification of methylxanthines in beverages. Beverage samples were directly analyzed by MS after dilution, with the entire procedure completed within two minutes. Caffeine-D₃ and 1-methylxanthine were used as internal standards for the quantitative analysis of caffeine, theophylline, and theobromine, which exhibited strong linear correlations (R² ≥ 0.9978). By integrating the concentration ratios of the isomeric compounds theophylline and theobromine derived from two diagnostic ions generated through MALDI tandem mass spectrometry, this approach enables the absolute quantification of both analytes. The method demonstrated satisfactory accuracy, with values from the spiked beverage samples ranging from 84.7% to 119.5%. Furthermore, real beverage samples were analyzed using both the developed method and the conventional high-performance liquid chromatography (HPLC) approach, yielding comparable results. The proposed method provides a rapid, accurate, and high-throughput analysis of methylxanthines in beverages.