Validation of HPLC‐UV method for determination of minor glycosides contained in Stevia rebaudiana Bertoni leaves

Abstract

Leaves of Stevia rebaudiana contain glycosides with sweetness and biological activity. However besides the major glycosides, there are other glycosides within extracts that may contribute to its activity, and therefore it is important to quantify them. In this work, an isocratic HPLC method was validated for determination of dulcoside A, steviolbioside, rebaudioside C and rebaudioside B. An HPLC method was performed using a C18 column (250 × 4.6 mm, particle size 5 µm) and a UV detector set at 210 nm. The mobile phase consisted of a 32:68 (v/v) mixture of acetonitrile and sodium phosphate buffer (10 mmol/L, pH 2.6), set to a flow rate of 1.0 mL/min. The calculated parameters were: sensitivity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy and precision. The calibration curves were linear over the working range 25-150 µg/mL, with coefficient of correlation of ≥0.99 and coefficient of determination of ≥0.98. The LOD was 5.68-8.81 µg/mL, while the LOQ was 17.21-26.69 µg/mL. The percentage recoveries of fortified samples were 100 ± 10% and precision, relative standard deviation, was <10%. The method validation showed accuracy, linearity and precision; therefore this method can be applied for quantitative analysis of minor steviol glycosides in S. rebaudiana leaves.

Effect of different drying methods on the composition of steviol glycosides in Stevia rebaudiana Bertoni leaves

“…The linearity response and linear regression equation for each glycoside is presented in Table 1. A good correlation coefficient and coefficient of determination was obtained as expected, given that the method used was previously validated (Aranda-González et al, 2014); however, they are presented to establish the conditions of quantification.…”

“…Although there were no significant differences in the content, it was possible to quantify dulcoside A, rebaudioside D, and steviolbioside in the four drying methods, as previously published (Aranda-González et al, 2014).…”

“…The high-performance liquid chromatography method (Aranda-González et al, 2014;, previously validated in the laboratory, was performed according to JECFA (2010). The chromatographic analysis was carried out on a Luna C18 (2) (length: 250 mm; inner diameter: 4.6 mm, particle size: 5 μm) column (Phenomenex Co., Ltd., CA, USA) without temperature control.…”

Evaluation of the euglycemic effect of oral administration of S. rebaudiana B. cultivated in Mexico in normoglycemic and induced-diabetic rats

“…It is noteworthy that the quantification of glycosides was obtained from a previously validated HPLC method, which chromatographic data and validation parameters can be consulted elsewhere. [22,23] In this regard, all steviol glycosides are at lower concentration than previously reported, [22,23] possibly attributed to a different batch. Comparing the varieties Morita II and Criolla, there are no differences in the content of minor glycosides, except for Rebaudioside B and Rebaudioside D; however, the most important difference are the content of Stevioside and Rebaudioside A. Morita II variety had higher content of total glycosides as compared to the Criolla variety (16.74 vs. 9.65 g/100 g) with a higher content of Rebaudioside A (4.5 times more).…”

“…Quantification of the steviol glycosides content was based on peak area, from linear regression curves of the standards. [22,23] …”