Comprehensive antioxidant profiling and TAC determination in Stevia rebaudiana leaves using on-line HPLC–CUPRAC: evaluation of three different post-column derivatization reactor workflows

This study presents an analytical methodology for determining the antioxidant profile and total antioxidant capacity (TAC) in Stevia rebaudiana leaves using HPLC coupled with diode array detection (DAD), and compares three novel post-column derivatization (PCD) systems. The configurations evaluated were: (i) in-column derivatization (ICD), employing a specialized column end-fitting; (ii) a novel 35 μL UHPLC gradient mixer, repurposed as a low-dispersion PCD reactor; and (iii) a blank column packed with stainless steel balls (100 μL) serving as an alternative reactor. All three systems demonstrated high efficiency in generating antioxidant fingerprints. Highly-selectivity chromatographic workflows were developed for separating 12 antioxidant standards commonly found in Stevia. The PCD process was optimized using the cupric reducing antioxidant capacity (CUPRAC) reagent to maximize derivatization efficiency and signal-to-noise ratio. Notably, ICD showed improved resolution for low-abundance peaks (6–22 % narrower) compared to the 35 μL mixer, while differences remained below 9 % for more concentrated compounds. These tools enhance compositional knowledge and add value to this natural sweetener used in foods and beverages. This is the first study to pair exhaustive extraction with high-resolution CUPRAC-HPLC-DAD, enabling the selective detection of over 48 antioxidant-related peaks in commercial Stevia samples. TAC values of 100.5 and 118.6 mg gallic acid equivalents per gram were determined. This methodology provides a robust platform for antioxidant profiling in natural products, supporting future studies on bioactive compounds in functional foods and plant-based therapeutics.