24 Londhe & nanaware: JournaL of aoaC InternatIonaL V oL. 96, no. 1, 2013 Received June 13, 2011. Accepted by AP May 4, 2012. Corresponding author’s e-mail: smitaks1@rediffmail.com DOI: 10.5740/jaoacint.11-257 DIETARY SUPPLEMENTS An HPTLC method was developed and validated for simultaneous determination of stevioside (STE) and rebaudioside-A (REB-A) in Stevia rebaudiana leaves. The HPTLC separation was performed on precoated silica gel 60F 254 HPTLC plates with the mobile phase ethyl acetate–ethanol–acetone–water (15 + 3 + 6 + 6, v/v/v/v). The densitometric quantiication of steviol glycosides was carried out at λmax 580 nm in the relection-absorption mode after spraying with anisaldehyde–sulfuric acid reagent. R f values were 0.34 and 0.28 for STE and REB-A, respectively. The content of STE and REB-A in leaf extract was found to be 6.94 and 6.35%, respectively. The method was validated in terms of precision, accuracy, speciicity, and robustness. The method can be useful for routine analysis of the sweeteners. S tevia rebaudiana (Bertoni) Bertoni is a perennial shrub of the Asteraceae (Compositae) family native to certain regions of South America (Paraguay and Brazil; 1). About 150 stevia species are known; among them, S. rebaudiana is the only one with a signiicant sweet taste (2). This plant is important today because its leaves are used as a non-nutritive, high-potency sweetener, primarily in Japan, Korea, China, and South America (3). The water extract of S. rebaudiana has beneicial effects on human health, including hypoglycemic and hypotensive effects, and as a source of antioxidants (4). Its leaves contain nine sweet glycosides. They possess an ent-kaurene diterpene steviol skeleton (ent-13-hydroxy kaur- 16-en-19-oic acid). Generally dominant are stevioside (STE, 6–10%) and rebaudioside-A (REB-A, 2–4%), while other minor glycosides are present up to 1–2% in the leaves (5). The structures of stevioside and rebaudioside-A are shown in Figure 1 (6). A literature survey revealed that several HPLC methods are available for simultaneous analysis of STE and REB-A from the leaves of S. rebaudiana by using amino (NH 2 ) and C18 chemically bonded columns for single sweetener estimation (7–12). Hearn and Subedi (13) reported application of near- IR relectance spectroscopy for analysis of steviol glycosides (SGs) in S. rebaudiana leaves using HPLC as a reference method. Clos et al. (14) reported the photodegradation of either RAB-A or STE. Ni et al. (15) monitored STE in soju by HPLC and LC/MS. Dacome et al. (16) quantiied sugar and steviol derivatives by TLC-densitometry. Huang et al. (17) separated and identiied three SGs using high-speed counter current chromatography and analyzed them by HPLC. V. Jaitak et al. (4) quantiied and validated all of the SGs by HPTLC. Since STE and REB-A are the major glycosides present, a more selective method for simultaneous analysis of only these two glycosides would be useful. Therefore, the aim of present work was to develop and validate a simple, accurate, and reproducible HPTLC method that can determine STE and REB-A simultaneously in leaf extract. Experimental Chemicals, Reagents, and Solutions All chemicals and solvents used were of analytical grade (E. Merck, Ltd, Mumbai, India). Reference standards of STE and REB-A were procured from Hangzhou Dayangchem Co. Ltd, Hangzhou, China. A stock solution of STE and REB-A (1000 µg/mL) was prepared in methanol–water (8 + 2, v/v). Plant Material The leaves of S. rebaudiana were collected from Hangzhou, China. Dried material was powdered and kept in a desiccator at room temperature in the dark until the analysis. Sample specimens were authenticated at the Department of Science and Technology, Government of India, Agharkar Research Institute, Pune, India (Auth10-60). Extraction and Analysis of Samples A representative amount (5 g) of leaves was dried, powdered, and extracted at room temperature for 24 h with methanol by exhaustive maceration (5 × 20 mL). After completion of extraction, extracts were iltered through Whatmann (Florham Park, NJ) ilter paper. A 10 mL amount of extract was concentrated to 2 mL by heating on a water bath. Dilution was carried out by diluting 0.2 mL extract with 0.8 mL methanol. This solution was applied to an HPTLC plate for analysis. HPTLC Analysis A CAMAG (Wilmington, NC) HPTLC system equipped with an automatic TLC Sampler ATS4, TLC scanner 3, and integrated software winCATS version 1.4.2 was used for the analysis. HPTLC was performed on a precoated silica gel HPTLC 60F 254 (20 × 10 cm) plate (E. Merck, Darmstadt, Germany) of 250 µm layer thickness used without any pretreatment. Chromatography was carried out in a TLC chamber with 30 mL of the mobile phase ethyl acetate–ethanol–acetone–water (15 + 3 + 6 + 6, v/v/v/v). HPTLC Method for Simultaneous Analysis of Stevioside and Rebaudioside-A in Stevia rebaudiana Smita V. Londhe and Sadhana m. nanaware Sinhgad College of Pharmacy, Department of Pharmaceutical Chemistry, Vadgaon, Pune- 411041, India