Stevia rebaudiana is a plant with high sweetening capacity due to its content of glycosides, mainly stevioside and rebaudioside A. Several techniques have been used to determine the concentrations of glycosides in Stevia, although an HPLC method is recommended by the FAO/WHO-JECFA. Varieties of Stevia have been recently grown in Mexico, with no previous report of glycosides by a validated method. The aim of this study was to validate an isocratic HPLC method for content determination of main glycosides in the leaves of Stevia cultivated in Mexico. HPLC method was performed using a C18 column (250 mm × 4.6 mm, 5 μm) and UV detector set at 210 nm. The mobile phase consisted of 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. Rebaudioside A and stevioside were determined in two Stevia varieties: Morita II and Criolla, and also validation parameters were calculated. Rebaudioside A content (g/100g) in Morita II was 15.15 ± 0.02 while stevioside was 3.97 ± 0.003; in the case of Criolla they were 4.03 ± 0.01 and 8.80 ± 0.14, respectively (p < 0.001). The recoveries of fortified samples were 100% ± 10% and precision RSD was ≤6.27%. The criteria of validation showed accuracy, linearity (≥0.99), and precision; therefore, the determination of glycosides was performed with reliability.
Stevia is a genus of plants native to subtropical and tropical regions of South and Central America. Among the 407 species, Stevia rebaudiana Bertoni has the highest sweetness potential due to its high content of diterpene glycosides named stevioside, rebaudioside A, C, D, and dulcoside A. These compounds are steviol glycosides, which are formed by replacing the carboxyl hydrogen atom with glucose, xylose and rhamnose [
The variety of Stevia, weather conditions and farming are factors that contribute to the amount and type of glycosides in the plant. For example, there is one variety of Stevia with low amounts of rebaudioside A and rebaudioside C, while in another variety the main component is rebaudioside C [
Several analytical techniques have been used to determine the concentrations of glycosides in S. rebaudiana, including thin layer chromatography (HPTLC) [
In recent years some varieties of S. rebaudiana, called Criolla (from Paraguay) and Morita II, have been grown in the south of Mexico to determine their adaptation to the weather and soil of the region, with no previous report of major steviol glycosides by JECFA method [
Standards of rebaudioside A (01432) and stevioside (S3572) were purchased from Sigma-Aldrich (USA) with a purity established by manufactured of 96% and 98%, respectively; acetonitrile and water (HPLC grade) were purchased from J.T Baker (Phillipsburg, NJ). Glycosides standards were lyophilized under vacuum pressure of 133 × 10−3 Bar and a temperature of −40˚C (Labconco, Kansas City, MO), then mixed with HPLC water, filtered trough 0.45 μm and stored at −20˚C prior to use.
A high-performance liquid chromatography method was performed, according to JECFA (2010) [
Two varieties of S. rebaudiana, Criolla and Morita II was grown and collected from the Southeast of México. Five hundred milligrams of dried leaves were extracted three times with 5 mL of water each time in a boiling water bath for 30 min (100˚C). Extracts were cooled to room temperature and centrifuged at 2500 × g, 10˚C for 10 minutes. The aqueous phases were transferred to a 25 mL volumetric flask and filled to capacity. The solution was filtered through a 0.45 μm membrane filter before HPLC analysis [
In order to determine the content of rebaudioside A and stevioside with quality, reliability and consistency, validation parameters were calculated. In agreement with ICH guidelines [
Validation parameters were defined and operationalized as follows: The sensitivity is the change in response divided by the corresponding change in the stimulus (slope of the calibration function); the linearity is the ability of the method to obtain results, which are directly proportional to the analyte concentration and was obtained by a linear regression calculated by the method of least squares; limit of detection (LOD) is the lowest concentration of a analyte in a sample that can be detected which was determined by the formula: LOD = 3.3 (Standard Deviation of intercept)/slope (S); limit of quantitation (LOQ) is the lowest concentration of an analyte in a sample that can be quantitatively determined using the formula LOQ = 10 (Standard Deviation of intercept)/slope (S); accuracy is the degree of agreement between the value that is accepted as true and the value found expressed as percentage of recovery of fortified samples and calculated by the equation: Recovery (%) = S1/(S2 + S3) × 100; where S1 = amount found (μg/mL) in the spiked sample, S2 = amount present originally in the unfortified sample, and S3 = amount (μg/mL) of analyte added to the sample; finally, precision is the closeness of a series of measurements obtained from multiple samples under prescribed conditions and were expressed as relative standard deviation (%RSD) of repeatability (intra-day) and intermediate (inter-day) precision [
Statgraphics was used to evaluate the linearity of the system, whereas intercept in y, slope with their respective confidence intervals were calculated using the GraphPad Prism program version 5.00. Microsoft Office Excel was used for analysis of limit of quantification, limit of detection, accuracy and precision. Student’s t-test was performed using Statgraphics considering p < 0.05.
After obtaining the values of area under the peak of the five concentrations in triplicate on two separate occasions, a plot of peak area as a function of analyte concentration was developed and the linear regression was calculated by method of least squares (
Asseen in
Since the correlation coefficients and the coefficients of determination of the standard curves were greater than 0.99, it can be concluded that the analytical methodology used was linear over the concentration range studied and therefore the model is suitable for quantifying the content of glycosides. Similar correlation coefficient and the coefficient of determination (>0.99) were obtained for rebaudioside D [
Repeatability was evaluated by analyzing relative standard deviation (RSD) for area under the peak and also for retention time through the run. Percent of RSD of all concentration were ≤1.08 and ≤5.24 for retention time and area, respectively (
Paremeters | Rebaudioside A | Stevioside |
---|---|---|
Coefficient of correlation (r) | 0.996 | 0.996 |
Coefficient of determination (R2) | 0.993 | 0.994 |
y-intercept | 12.41 | −30.54 |
CI of y-intercepta | −36.72 to 61.55 | −93.88 to 32.80 |
Slope (sensitivity) | 4.510 | 6.139 |
CI of slope | 4.263 to 4.659 | 5.949 to 6.331 |
LOD (μg/mL)b | 17.54 | 16.62 |
LOQ (μg/mL)c | 53.18 | 50.37 |
aCI: confidence interval at 95%; bLOD: limit of detection [3.3 (Standard deviation of intercept)/slope (S)]; cLOQ: limit of quantification [10 (Standard deviation of intercept)/slope (S)].
The precision for retention time was ≤1.93 %RSD in all cases, while for peak area was ≤6.27% (
After the analysis of sensibility and precision of the method, extracts of S. rebaudiana Bertoni Morita II and Criolla varieties were analyzed to identify rebaudioside A and stevioside present in both. As seen in
Once both glycosides were identified in the chromatogram of the extracts, each sample was analyzed in duplicate and the concentration calculated through the equation of linear regression constructed with the standards. In Morita II variety, the rebaudioside A content (g/100g of dry leaf) is significantly higher (p < 0.001) than that found in Criolla variety, whereas for stevioside content is exactly the opposite (
As seen in
Precision | Concentration (μg/mL) | Retention time (mean ± SD) | %RSD time | Peak area (mean ± SD) | %RSD area | |
---|---|---|---|---|---|---|
Rebaudioside A | 100 | 6.99 ± 0.05 | 0.76 | 541.58 ± 23.66 | 5.24 | |
300 | 6.97 ± 0.06 | 0.93 | 1316.0 ± 34.43 | 2.62 | ||
500 | 6.98 ± 0.08 | 1.08 | 2215.24 ± 32.19 | 1.45 | ||
Stevioside | 100 | 7.17 ± 0.02 | 0.34 | 565.63 ± 13.86 | 2.45 | |
300 | 7.18 ± 0.04 | 0.55 | 1621.16 ± 56.67 | 3.50 | ||
500 | 7.19 ± 0.07 | 0.96 | 2667.56 ± 105.02 | 3.94 | ||
%RSD: relative standard deviation.
Precision | Concentration (μg/mL) | Retention time (mean ± SD) | %RSD time | Peak area (mean ± SD) | %RSD area | ||
---|---|---|---|---|---|---|---|
Rebaudioside A | 100 | 6.94 ± 0.10 | 1.51 | 445.21 ± 19.31 | 4.34 | ||
300 | 6.91 ± 0.13 | 1.89 | 1338.36 ± 17.93 | 1.34 | |||
500 | 6.92 ± 0.13 | 1.93 | 2206.31 ± 53.04 | 2.40 | |||
Stevioside | 100 | 7.24 ± 0.05 | 0.74 | 584.92 ± 36.67 | 6.27 | ||
300 | 7.25 ± 0.04 | 0.52 | 1745.14 ± 85.62 | 4.91 | |||
500 | 7.22 ± 0.04 | 0.52 | 2852.71 ± 127.10 | 4.46 | |||
%RSD: relative standard deviation.
Glycosides | Morita II (mean ± S.D.) | Criolla (mean ± S.D.) |
---|---|---|
Rebaudioside A | 15.15 ± 0.02a | 4.03 ± 0.01b |
Stevioside | 3.97 ± 0.003a | 8.80 ± 0.14b |
Values are expressed as mean ± standard deviation. Different letter in the same row denote significant differences at p < 0.001 (Student’s t-test).
recommended. In this context it is also possible to use a pre-analytical method such as SPE extraction [
The high content of rebaudioside A found in the variety Morita II is somewhat expected since this variety was bred to be used as a sweetener [
the mobile phase than the method described for amino bonded columns. Taking these data together, the content of major glycosides found here are from a validated method, which involves less time and use of moderately toxic substance as it is the acetonitrile.
Finally, accuracy was evaluated by performing recovery studies of fortified samples; One hundred μg/mL of rebaudioside A or stevioside was added to three samples of S. rebaudiana extract previously quantified and analyzed trough the HPLC method. The analysis of spiked samples, conducted in triplicate, demonstrated a good recovery of rebaudioside A of 99.5% - 92.29%, whereas the recovery of stevioside was in the range of 97.91% - 104.49% in both varieties of Stevia (
The recovery rate found for rebaudioside A and stevioside are in the range that has been reported previously for rebaudioside A (93% - 108%) or stevioside (95.7% - 106%) [
Taking in consideration that using the same HPLC method it is possible to quantify minor glycosides such as rebaudioside D [
Variety | Glycoside (% of recovery) | ||
---|---|---|---|
Rebaudioside A | Stevioside | ||
S. rebaudiana Criolla | 99.50 | 97.91 | |
S. rebaudiana Morita II | 92.29 | 104.49 |
Rebaudioside A and stevioside were determined in Stevia rebaudiana leaves grown in Mexico; rebaudioside A content (g/100g) in Morita II was 15.15 ± 0.02 while stevioside was 3.97 ± 0.003; in the case of Criolla they were 4.03 ± 0.01 and 8.80 ± 0.14, respectively (p < 0.001). This was performed by a validated HPLC method that showed sensitivity of 4.15 and 6.139, with a LOD of 17.54 μg/mL and 16.62 μg/mL, a LOQ of 53.18 μg/mL and 50.37 μg/mL, for Rebaudioside A and Stevioside, respectively; the method was accurate (100% ± 10%), linear (R = 0.99), and precise (≤6.27 %RSD), and therefore it could be routinely applied to quantify these glycosides in S. rebaudiana grown elsewhere.
This work was supported by Programa de Mejoramiento al Profesorado-PROMEP-SEP and Fondos Fiscales- INIFAP, Also, I.A.G. received a CONACYT’s scholarship during postgraduate studies.
IrmaAranda-González,YolandaMoguel-Ordoñez,DavidBetancur-Ancona, (2015) Determination of Rebaudioside A and Stevioside in Leaves of S. rebaudiana Bertoni Grown in México by a Validated HPLC Method. American Journal of Analytical Chemistry,06,878-885. doi: 10.4236/ajac.2015.611083