A Validated RP-HPLC Method for Quantification of Steviol Glycoside: Rebaudioside A in Extracts of Stevia Rebaudiana Leaf
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ORIGINAL
A Validated RP‑HPLC Method for Quantification of Steviol Glycoside: Rebaudioside A in Extracts of Stevia Rebaudiana Leaf Nikhil S. Kolate1 · Himanshu Mishra1 · Suvarna G. Kini1 · Govindarajan Raghavan2 · Tejas B. Vyas2 Received: 8 May 2020 / Revised: 7 October 2020 / Accepted: 14 October 2020 © Springer-Verlag GmbH Germany, part of Springer Nature 2020
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. Keywords Stevia rebaudiana · Rebaudioside A · Quantitation · RP-HPLC method
Introduction Stevia rebaudiana, a medicinal plant in the family Asteraceae [1], has considerable economic value due to its high content of valuable natural and dietary sweeteners in the leaves [2–4]. It is a rich source of diterpene glycosides [2], namely Rebaudiosides (Rebaudioside A, B, D, M, X) and Stevioside. A native of South America [1], nevertheless, because of the increasing economic value, stevia farming has been growing in many other parts of the world, including Japan, China, Argentina, Colombia, India, Vietnam and Brazil over the past few decades, meaning the health-related benefits of using plant and natural materials [1, 2, 5, 6]. The * Suvarna G. Kini [email protected] 1
Department of Pharmaceutical Chemistry, Manipal College of Pharmaceutical Sciences, MAHE, Manipal, Karnataka, India 576104
Analytical Development Laboratory, Zydus Wellness Ltd, Ahmedabad, Gujarat, India 380015
2
use of stevia leaves is as a sugar substitute, especially for diabetics and health-conscious, as it a non-caloric sweetener which helps in obesity and helps maintain the normal level of blood glucose an
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