Abstract
RP-HPLC-PDA method has been developed and validated for the quantification of Steviol glycoside: Rebaudioside A in aqueous extract of Stevia rebaudiana plant leaf. Rebaudioside A was separated on a Phenomenex Luna C18 analytical column with column temperature 40 °C using an isocratic solvent system consisting (70:30 v/v) mixture of 10 mmol/L sodium phosphate buffer and acetonitrile having pH-2.6 at 1.0 mL/min flow rate with detection at 210 nm wavelength. The method has been validated for linearity, precision, accuracy, detection limit, quantitation limit and robustness. The proposed method linearity for Rebaudioside A with a regression coefficient of 0.9999 was obtained within the range of 0.0005–1.5 mg/mL. The relative standard deviation was less than 2.0% in intra-day and inter-day precision. The proposed method’s accuracy was determined by a three different level recovery study. The recovery average was between 95.20 and 96.77 percent. Rebaudioside A levels of LOD and LOQ were 0.01 and 0.05 mg/mL respectively. The leaves from eight different Indian territories were extracted by sonication extraction method. Rebaudioside A content obtained in the dried leaves was within the range of 2.39–7.53 percent w/w. The method proposed is simple, rapid and reproducible; therefore, it is appropriate for routine analysis of Rebaudioside A in plant material as well as finished products which are used commercially as high-intensity sweeteners across the globe to replace sugar.
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References
Jadhao DB, Katekhaye SD, Thorat BN (2011) Improved RP-HPLC method for quantitative estimation of Stevioside in Stevia rebaudiana Bertoni Burm. IJPP 1(2):27–34
Woelwer-Rieck U, Lankes C, Wawrzun A, Wust M (2010) Improved HPLC method for the evaluation of the major steviol glycosides in leaves of Stevia rebaudiana. Eur Food Res Technol 231:581–588
Ramesh K, Singh V, Megeji NW (2006) Cultivation of Stevia (Stevia rebaudiana Bertoni): a comprehensive review. Adv Agron 89:137–177
Kinghorn AD (2002) Overview: Stevia, the Genus of Stevia medicinal and aromatic plants-industrial profiles. Taylor and Francis, London
Genus JM (2003) Molecules of interest: Stevioside. Phytochemistry 64:913–921
Tanaka O (1982) Steviol-glycosides: new natural sweeteners. Trends Anal Chem 1(11):246–248
Brahmachari G, Mandal LC, Roy R, Mondal S, Arun KB (2011) Stevioside and related compounds-molecules of pharmaceutical promise: a critical overview. Arch Pharm Chem Life Sci 1:5–19
Supriyadi S, Siswodihardjo S, Yuwono M (2016) Method development and validation for the simultaneous determination of Stevioside, Rebaudioside A, Rebaudioside C and Dulcoside a contained in Stevia rebaudiana Bertoni using HPLC-ELSD, Int. J Pharm Pharm Sci 8(9):1–5
Benford DJ, Dinovi M, Schlatter J (2006) Steviol glycosides. Safety evaluation of certain food additives. WHO Food Additives Series, pp 117–143
Cargill (2008) GRAS Notification for Rebaudioside A, GRAS Notification FDA GRN #253
Steviol glycosides (2007) In: Combined compendium of food additives specifications, 68th meeting of the joint FAO/WHO expert committee on food additives (JECFA), FAO/JECFA monograph 4:61–65
Steviol glycosides (2008) In: Summary and conclusions of 69th meeting of the joint FAO/WHO expert committee on food additives (JECFA):1–3
Kitada Y, Sasaki M, Yamazoe Y (1989) Simultaneous determination of stevioside, rebaudioside A and C and dulcoside A in foods by high-performance liquid chromatography. J Chromatogr A 474:447–451
Kolb N, Herrera DJ, Uliana RF (2001) Analysis of sweet diterpene glycosides from Stevia rebaudiana: improved HPLC method. J Agric Food Chem 49:4538–4541
Steviol glycosides (2010) Prepared at the 73rd JECFA (2010) and published in FAO JECFA monographs 10 (INS no. 960)
Hashimoto Y, Moriyasu M, Nakamura S, Ishiguro S, Komuro M (1978) High-performance liquid chromatographic determination of stevia components on the packed hydrophilic column. J Chromatogr 161:403–405
Hutapea AM, Toskulkao C, Wilairat P, Buddhasukh D (1999) High-performance liquid chromatographic seperation and quantification of Stevioside and its metabolites. J Liq Chromatogr RT 22:1161–1170
Makapuguay HC, Nanyakkara NPD, Kinghorn AD (1984) Improved high-performance liquid chromatographic seperation of the Stevia rebaudiana sweet terpene glycosides using a linear gradient elution. J Chromatog 283:390–395
Acknowledgement
The authors Nikhil S. Kolate and Himanshu Mishra are thankful to Analytical Development Laboratory, Zydus Wellness Ltd, Ahmedabad- 380015, Gujarat, India and Department of Pharmaceutical Chemistry, Manipal College of Pharmaceutical Sciences, MAHE, Manipal- 576104, Karnataka, India for providing facilities to perform this research work.
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Kolate, N.S., Mishra, H., Kini, S.G. et al. A Validated RP-HPLC Method for Quantification of Steviol Glycoside: Rebaudioside A in Extracts of Stevia Rebaudiana Leaf. Chromatographia 84, 21–26 (2021). https://doi.org/10.1007/s10337-020-03980-2
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DOI: https://doi.org/10.1007/s10337-020-03980-2