Analytical Method Development and Validation with Greenness Assessment of HPLC-DAD Method to Determine Methylxanthines

In this study, a simple, and eco-friendly high-performance liquid chromatography-diode array detection (HPLC-DAD) method was developed and validated for analyzing methylxanthines, including theobromine (TB), theophylline (TF), and caffeine (CF). This method demonstrated excellent performance using a mobile phase of water and ethanol in a core-shell Kinetex® C18 column. The method was linear (1-100 µg mL-1), exhibiting high precision and accuracy with relative standard deviations below 2.67% and recoveries ranging from 98.17% to 108.23%. The detection and quantification limits ranged from 0.19-0.26 µg mL-1 and 0.64-0.87 µg mL-1, respectively. We applied this method to analyze powdered lemon-flavored beverages enriched with black or green tea extracts. CF was detected exceeding 1.62 g kg-1. The proposed method showed excellent greenness, evidenced by the Analytical Eco-Scale, Analytical GREEnness metric (AGREE) calculator, and ComplexGAPI.