HPLC ANALYTICAL VALIDATION FOR STEVIOL GLYCOSIDES IN COMMERCIAL STEVIA POWDER PRODUCTS

Stevia (Stevia rebaudiana Bertoni) is widely used as a natural sweetener due to the high glycoside content of steviol. These compounds have similar chemical structures, varying by the number and position of glycosidic residues, making chromatographic separation difficult and requiring robust analytical methodologies for quality control. The aim of this study was to validate a high-performance liquid chromatography (HPLC) method for the determination of Rebaudiosides D (RD), M (RM), C (RC) and Rubusoside (R) in commercial stevia powder products. The analyses were conducted in a Shimadzu system with quaternary pump, UV/DAD and RID detectors, C18 column (250 × 4.6 mm, 5 μm) and mobile phase in water gradient and acetonitrile acidified with 0.1% H₃PO₄, operating at 1.0 mL/min. The samples were prepared by extraction in aqueous solvent, filtered in 0.45 μm membranes and injected (20 μL). Quantification was based on calibration curves obtained with commercial purity standards ≥99.9%. The compounds showed consistent retention times of 3,770 min (RD), 4,085 min (RM), 9,245 min (RC), and 12,387 min (R). The method demonstrated linearity (R² > 0.99), satisfactory accuracy (RSD < 5%) and adequate recoveries, meeting the validation criteria. For stability evaluation, three points of the calibration curve (0.8, 1.0, and 2.0 ppm) were stored under freezing conditions for 28 days and reanalyzed; statistical comparison by ANOVA (α = 0.05) indicated no significant differences (p > 0.05), confirming the stability of both the method and the glycosides under storage conditions. Application of the methodology to three commercial products (A, A2, and AA-801) revealed no significant difference in RD content among the samples, whereas significant differences were observed for RM and RC: stevia A2 presented higher concentrations (RM = 7.140 ± 0.317 mg/L; RC = 17.057 ± 0.318 mg/L), while A (RM = 1.717 ± 0.292 mg/L; RC = 3.034 ± 0.041 mg/L) and AA-801 (RM = 5.030 ± 1.160 mg/L; RC = 2.942 ± 1.607 mg/L) did not differ from each other for RC. It is concluded that the validated methodology is efficient for determining steviol glycosides in powdered stevia, showing robustness, reproducibility, and applicability in the quality control of commercial products, establishing itself as a reliable tool for industry and research in food science and technology.