Development and Validation of a Rapid High-Performance Liquid Chromatography Method for Simultaneous Determination of...

Cocoa husk tea has gained attention as a value-added beverage from cocoa processing by-products, due to its potential content of bioactive compounds associated with health benefits. However, rapid and reliable analytical methods for the simultaneous determination of methylxanthines and flavanols in this matrix remain limited. This study aimed to develop and validate a rapid HPLC-PDA method for the simultaneous determination of methylxanthines and selected flavanols in cocoa husk tea. Separation was performed using a Zorbax 300SB-C18 with a gradient system of acetic acid, water and acetonitrile, and detection at 280 nm. The method enabled separation of theobromine, caffeine, catechin, epicatechin, procyanidin B1, and procyanidin B2 within 15 min. Validation followed ICH Q2(R2) guidelines, demonstrating satisfactory linearity, sensitivity, accuracy, and precision. The method was applied to ten commercial cocoa husk tea products from Thailand. Theobromine was the predominant methylxanthine (10.483-16.027 mg g^-1), whereas caffeine was lower (0.923-1.909 mg g^-1), while flavanol contents varied among samples. These findings demonstrate that the developed method provides a rapid and reliable approach for the analysis and quality assessment of cocoa husk tea products and may support the further utilization of cocoa by-products in the functional beverage industry.