Research article

Received: 21 February 2014, Revised: 26 June 2014, Accepted: 25 August 2014 Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/bmc.3349

Validation of HPLC-UV method for

determination of minor glycosides contained
in Stevia rebaudiana Bertoni leaves
Irma Aranda-Gonzáleza, Yolanda Moguel-Ordoñezb
and David Betancur-Anconaa*
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 cor-
relation 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. Copyright © 2014 John Wiley & Sons, Ltd.

Keywords: Stevia rebaudiana; HPLC; steviol glycosides; validation

Introduction chromatography (HPLC; Bergs et al., 2012; Gardana et al., 2010;

Hashimoto et al., 1978; Kolb et al., 2001; Tada et al., 2013;
Stevia is a genus of plants native to subtropical and tropical re- Wöelwer-Rieck et al., 2010), among others.
gions of South and Central America. Among the 407 species, Stevia However, one of simplest and most reliable methods is HPLC,
rebaudiana Bertoni has the highest potential as a sweetener and is and hence in 2010, the Food and Agriculture Organization/World
known in Guarani as ka’a he’e (‘sweet herb’). The compounds Health Organization Joint Expert Committee on Food Additives
responsible for the sweetening property of the plant are diterpene (JECFA) at the 73rd meeting recommended an HPLC method for
glycosides, named stevioside, rebaudioside A, C and D, and determination of steviol glycosides (JECFA, 2010). However the
dulcoside A. These compounds are steviol glycosides, which are existing analytical methods are mainly focused on the quantifica-
formed by replacing the carboxyl hydrogen atom with glucose, tion of either rebaudioside A or stevioside and to a lesser extent
xylose and rhamnose (Chaturvedula et al., 2011). on minor glycosides, such as rebaudioside C and B, dulcoside A
In generally cultivated varieties, the main steviol glycosides are and steviolbioside, which are also present in the extracts of the
stevioside, rebaudioside A, rebaudioside C and dulcoside A. In leaves and may contribute to sweetness or biological activity.
small quantities can be found rebaudioside C (1–2%) and Therefore, the aim of this study was to modify and validate the
dulcoside A (0.4–0.7%) along with estevolbioside, rubusoside and proposed JECFA method for the quantification of minor glycosides
rebaudioside B, D, E and F (<0.4% each) (Goyal et al., 2010; Jackson contained in S. rebaudiana Bertoni leaves grown in the southeast
et al., 2009; Wöelwer-Rieck, 2012). of Mexico.
In addition to the sweetness of S. rebaudiana, there are important
biological properties that have been identified, such as anti-
cariogenic capacity (Das et al., 1992), and antineoplastic (Mizushina
et al., 2005), antihyperglycemic (Jeppesen et al., 2002,), antihyperten-
sive (Chan et al., 2000) and anti-inflammatory activities (Yasukawa * Correspondence to: D. Betancur-Ancona, Facultad de Ingeniería Química,
et al., 2002). Universidad Autónoma de Yucatán, Periférico Norte Km. 33.5, Tablaje
Catastral 13615, Col. Chuburna de Hidalgo Inn. Mérida, Yucatán, C.P.
Various analytical techniques have been used to determine the
97203, Mexico. E‐mail: [email protected]
concentrations of glycosides in S. rebaudiana. These include high-
performance thin-layer chromatography (Chester et al., 2012; Jaitak a
Facultad de Ingeniería Química, Universidad Autónoma de Yucatán,
et al., 2008; Londhe and Nanaware, 2013), capillary electrophoresis Periférico Norte Km. 33.5, Tablaje Catastral 13615, Col. Chuburna de
(Dacome et al., 2005; Mauri et al., 1996), liquid chromatography/ Hidalgo Inn., Mérida, Yucatán, C.P. 97203, Mexico
mass spectrometry (Wang et al., 2004), electrospray ionization mass b
Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias
spectrometry (Jackson et al., 2009), infrared spectroscopy (Dacome (INIFAP)Campo experimental Mocochá, Carretera Mérida-Motul, Km. 25,
et al., 2005; Hearn and Subedi, 2009) and high-performance liquid C.P. 97454, Mocochá, Yucatán, Mexico

Biomed. Chromatogr. 2014; 9999 Copyright © 2014 John Wiley & Sons, Ltd.
 I. Aranda-González et al.

Experimental squares method. Also, the correlation coefficient and the intercept on y
were calculated. Limit of detection was determined using the formula:
Reagents and samples LOD = 3.3 (standard deviation of intercept)/slope (S). Limit of quantification
was determined using the formula LOQ = 10 (standard deviation of inter-
Standards of steviolbioside (ASB-00019349), dulcoside A (ASB-00004949), cept)/S. Accuracy was evaluated by performing recovery studies of fortified
rebaudioside C (ASB-00018228) and rebaudioside B (ASB-00018227) samples with steviolbioside, dulcoside A, rebaudioside C or rebaudioside B;
were purchased from Chromadex (Irvine, CA, USA) whereas acetonitrile recoveries were determined by adding 25 μg/mL of each glycoside to three
and water (HPLC grade) were purchased from J. T. Baker (Phillipsburg, samples of S. rebaudiana extract previously quantified; the recovery of each
NJ, USA). Prior to use, glycoside standards were lyophilized under analyte was expressed as a percentage and calculated by the equation:
3
vacuum pressure of 133 × 10 bar and a temperature of 40°C recovery (%) = S1/(S2 + S3) × 100, where S1 = amount found (μg/mL) in
(Labconco, Kansas City, MO, USA), then mixed with HPLC water, filtered the spiked sample, S2 = amount present originally in the unfortified
trough 0.45 μm and stored at -20°C. sample, and S3 = amount (μg/mL) of analyte added to the sample. Repeat-
ability and intermediate precision were evaluated and expressed as relative
standard deviation (RSD); for this purpose, three samples of each glycoside
HPLC conditions were analyzed using the chromatographic conditions described on the
Liquid chromatography under isocratic conditions was performed using same day (intraday precision) or on different days (interday precision).
an Agilent 100 HPLC system with a UV–vis detector set to a wavelength
of 210 nm. Separation was carried out on a Luna C18(2) (length, 250 mm;
inner diameter, 4.6 mm; particle size, 5 μm) column (Phenomenex Co. Statistical analysis
Ltd, CA, USA) without temperature control. The mobile phase consisted Statistical analyses were performed using Statgraphics to evaluate the line-
of a 32:68 mixture of acetonitrile and 10 mmol/L sodium phosphate arity of the system; the confidence intervals of the intercept in y and confi-
buffer (pH 2.6) used at a constant flow rate of 1 mL/min. Chromato- dence intervals of the slope were calculated using the GraphPad Prism
graphic analysis was performed with Clarity software, version 2.7.3.498 program version 5.00. Microsoft Office Excel was used for analysis of limit
(2009). The sample injection volume was 20 μL. of quantification, limit of detection, accuracy and precision.

Stevia rebaudiana extract Results and discussion

The leaves of two varieties of S. rebaudiana, Morita and Criolla II, were
collected from the southeast of Mexico. Dried material was powdered and
Method validation
kept in the dark until analysis. For preparing extracts, 0.5 g of powdered After obtaining the values of area under the peak of the six con-
leaves was weighed and extracted three times with water with 5 mL each centrations in triplicate on two separate occasions, a plot of peak
time in a boiling water bath at 100°C for 30 min. Extracts were cooled to room area as a function of analyte concentration was developed and
temperature and centrifuged for 10 min (2500g, 10°C) to facilitate separation
the linear regression was calculated by the least squares method
of the aqueous phases of the leaves. The aqueous phases were transferred to
a 25 mL volumetric flask and filled to capacity after the last extraction. The
(Fig. 1). As seen in Table 1, the parameters of simple linear
solution was filtered through a membrane filter (0.45 μm) to remove any regression were calculated for each of the four standards of
solid residue before HPLC analysis (Wöelwer-Rieck et al., 2010). steviol glycosides at six concentrations of 25–150 μg/mL.
Since the correlation coefficients of the standard curves were
>0.99, while the coefficients of determination were ≥98%, it can
Method validation be concluded that the analytical methodology used was linear
In agreement with International Conference on Harmonization (ICH) over the concentration range studied and therefore the model
guidelines (ICH, 2005), the quality, reliability and consistency of the vali- is suitable for quantifying the content of glycosides.
dated method were determined based on the sensitivity, linearity, limit When tested with a similar method but at concentrations of
of detection (LOD), limit of quantification (LOQ), accuracy and precision 0.01–0.5 mg/mL, limits of detection of 4 μg/mL for dulcoside
of each of the four glycosides. For this purpose, the standard solutions A, rebaudioside C and B and steviolbioside were reported
were prepared by dilution of the stock solution with HPLC water to reach (Tada et al., 2013). Therefore, this method has the advantage
concentration ranges of 25–150 μg/mL for each glycoside. Each concen-
tration was injected a total of six times onto the HPLC equipment.
According to ICH guidelines (ICH, 2005), each parameter was defined as
follows: sensitivity is the change in the response of a measuring instrument
divided by the corresponding change in the stimulus; linearity is the ability
of the method (within a given range) to obtain results that are directly
proportional to the concentration (amount) of analyte; LOD is the lowest
concentration of an analyte in a sample that can be detected but not
necessarily quantitated as an exact value; LOQ is the lowest concentration
of an analyte contained in a sample that can be quantitatively determined;
accuracy is the closeness of agreement between the value that is accepted
either as a conventional true value or an accepted reference value and the
value found; and precision is the closeness of agreement between a series
of measurements obtained from multiple samples of a homogeneous
sample under the prescribed conditions.
Also, in agreement with ICH guidelines (ICH, 2005) the parameters were
operationalized as follows: sensitivity was calculated as the slope of the cal-
ibration function; linearity was calculated as the evaluation of six concentra-
tions of the standards in a concentration range of 25–150 μg/mL; to Figure 1. Linear regression of steviol glycosides standards curves under
evaluate the linearity, a graph was constructed of the signal produced as chromatographic conditions. Data are means ± SD of individual steviol
a function of analyte concentration and the linear regression by the least glycosides absorbance (solid line) and linear regression (dotted line).

wileyonlinelibrary.com/journal/bmc Copyright © 2014 John Wiley & Sons, Ltd. Biomed. Chromatogr. 2014; 9999
HPLC-UV method for S. rebaudiana minor glycosydes

Table 1. Parameters calculated from linear regression model peak area vs concentration (y = a + b*x) of minor steviol glycosides

Parameters Rebaudioside C Dulcoside A Rebaudioside B Steviolbioside

r 0.995 0.997 0.996 0.994
R2 99.14 99.50 99.34 98.80
y-Intercept 5.142 30.135 6.544 2.404
CI of y- intercept 26.36 to 16.08 52.20 to 8.07 15.35 to 2.257 8.035 to 3.228
Slope (sensitivity) 4.425 6.048 2.104 0.993
CI of slope 4.20 a 4.64 5.822 a 6.275 2.01 a 2.19 0.93 a 1.05
LOD (μg/mL) 7.47 5.68 6.51 8.81
LOQ (μg/mL) 22.62 17.21 19.72 26.69
r, Correlation coefficient; R2, coefficient of determination; CI, confidence interval at 95%; LOD, limit of detection [3.3 (standard
deviation of intercept)/slope (S)]; LOQ, limit of quantification [10 (standard deviation of intercept)/S].

that it can be used to quantify small or large quantities of Intraday precision. Three concentrations of each glycoside
steviol glycosides. standard were prepared in triplicate and analyzed according to
the HPLC method. The integrated peak areas are shown in
Table 3. The RSD values for all three concentrations were <8%,
Accuracy. The recovery experiment with the spiked samples,
ranging from 2.13 to 7.98%. Intraday precision was also evaluated
conducted in triplicate, demonstrated a good recovery in the
by analyzing retention time through the run. In all cases, the RSD
range of 92.2–104.4% (Table 2). To date, no studies evaluating
for retention time was <1%, except for the highest concentration
the accuracy in quantifying minority glycosides with the same
of rebaudioside C and for all three concentrations of dulcoside A.
method have been reported, since most of the published studies
The average retention time was 9.5 min for rebaudioside C,
are about rebaudioside A or stevioside. However, the recovery
10.28 min for dulcoside A, 20.73 min for rebaudioside B and
rates found for dulcoside A, rebaudioside C, rebaudioside B
22.29 min for steviolbioside.
and steviolbioside are in the range that has been reported for
rebaudioside A (93–108%) (Chester et al., 2012; Jaworska et al., Interday precision. Intermediate or interday precision was
2012; Wöelwer-Rieck et al., 2010) or stevioside (95.7–106%) found by repeating the same procedure, although 1–4 weeks later.
(Chester et al., 2012; Ni et al., 2007; Wöelwer-Rieck et al., 2010) The precision for retention time was <2.5% RSD in all cases except
in different chromatographic methods and are therefore consid- for dulcoside A, whereas the relative standard deviation for peak
ered acceptable. area was <9% (Table 4). Although the values of RSD of peak area

Table 2. Accuracy of the analythical method expressed as a recovery percentage of spiked samples

Variety Glycoside (percentage recovery)

Rebaudioside C Dulcoside A Rebaudioside B Steviolbioside
Stevia rebaudiana criolla 99.50 97.91 92.63 102.62
Stevia rebaudiana Morita II 92.29 104.49 101.24 100.71

Table 3. Repeatibility or intraday precision of minor steviol glycosides

Glycoside Concentration Retention time (mean ± SD) RSD time (%) Peak area (mean ± SD) RSD Area (%)
(μg/mL)
Rebaudioside C 25 9.52 ± 0.07 0.70 106.02 ± 8.46 7.98
75 9.47 ± 0.08 0.85 371.80 ± 12.26 3.30
150 9.51 ± 0.14 1.42 718.74 ± 36.17 5.03
Dulcoside A 25 10.30 ± 0.20 1.95 155.28 ± 3.30 2.13
75 10.27 ± 0.23 2.22 449.18 ± 27.25 6.07
150 10.29 ± 0.29 2.80 902.97 ± 43.56 4.82
Rebaudioside B 25 20.76 ± 0.09 0.41 38.29 ± 2.15 5.61
75 20.75 ± 0.08 0.38 126.49 ± 6.08 4.81
150 20.68 ± 0.18 0.88 295.31 ± 16.01 5.42
Steviolbioside 25 22. 29 ± 0.03 0.15 15.94 ± 1.04 6.55
75 22.22 ± 0.04 0.18 67.21 ± 4.99 7.42
150 22.36 ± 0.13 0.57 151.83 ± 4.83 3.18
RSD, Relative standard deviation.

Biomed. Chromatogr. 2014; 9999 Copyright © 2014 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/bmc
 I. Aranda-González et al.

Table 4. Intermediate precision of minor steviol glycosides

Glycoside Concentration Retention time (mean ± SD) RSD time (%) Peak area (mean ± SD) RSD Area (%)
(μg/mL)
Rebaudioside C 25 9.49 ± 0.09 0.97 112.99 ± 1.86 1.64
75 9.43 ± 0.12 1.26 361.28 ± 3.75 1.04
150 9.47 ± 0.18 1.95 714.64 ± 32.30 4.52
Dulcoside A 25 10.26 ± 0.25 2.41 150.84 ± 3.25 2.15
75 10.09 ± 0.45 4.48 439.14 ± 20.43 4.65
150 10.29 ± 0.28 2.73 879.56 ± 21.80 2.48
Rebaudioside B 25 20.73 ± 0.11 0.52 36.79 ± 1.00 2.73
75 20.55 ± 0.30 1.47 128.21 ± 4.17 3.25
150 20.37 ± 0.50 2.47 298.67 ± 17.85 5.98
Steviolbioside 25 22.34 ± 0.06 0.28 16.04 ± 1.30 8.08
75 22.31 ± 0.11 0.51 61.08 ± 4.84 7.92
150 22.30 ± 0.19 0.83 161.36 ± 14.38 8.91
RSD, Relative standard deviation.

Figure 2. Chromatogram of a mixture of seven steviol glycosides standards under chromatography conditions: (a) rebaudioside D; (b) rebaudioside A;
(c) stevioside; (d) rebaudioside C; (e) dulcoside A; (f) rebaudioside B; and (g) steviolbioside.

wileyonlinelibrary.com/journal/bmc Copyright © 2014 John Wiley & Sons, Ltd. Biomed. Chromatogr. 2014; 9999
HPLC-UV method for S. rebaudiana minor glycosydes

are slightly higher than those reported for rebaudioside A and in S. rebaudiana Bertoni leaf (Jarma-Orozco et al., 2011), so
stevioside (0.70 and 0.41, respectively) using the same chromato- these factors could explain these slight differences.
graphic method (Tada et al., 2013), RSD <10% is generally
regarded as appropriate. The chromatogram with the proposed
conditions is shown in Fig. 2(A), where in addition to the four Conclusion
glycosides validated in the current work it is possible to identify An HPLC method was validated for determination and quan-
rebaudioside D, stevioside and rebaudioside A. tification of rebaudioside C, dulcoside A, rebaudioside B and
steviolbioside. The method validation showed accuracy, linearity
Glycoside identification and quantification in S. rebaudiana and precision; therefore it can be applied for quantitative analy-
extracts sis of minor steviol glycosides in S. rebaudiana leaves grown in
southeast Mexico.
After the validation of the method, extracts from Morita and
Criolla II varieties were analyzed to identify steviol glycosides.
As shown in Fig. 2(B and C), it is possible to identify steviol
Acknowledgments
glycosides in both varieties. There is a high matrix load visible This work was financially supported by Programa de Mejoramiento
at the beginning of the extract chromatograms (Fig. 2B and C) al Profesorado-PROMEP-SEP and Fondos Fiscales-INIFAP through
within 5 min, which may be caused by proteins, resins, organic projects named ‘Evaluación del efecto hipoglucemiante de las hojas
acids, pigments, etc. (Bergs et al., 2012). These components de Stevia rebaudiana’ and ‘Desarrollo de productos alimenticios
may decrease the life of the column and therefore the use of a elaborados con hoja de Stevia rebaudiana Bertoni’, respectively.
Also, Aranda-González received a CONACYT scholarship during
pre-column in the HPLC equipment has been described, or a
postgraduate studies.
previous analytical method such as solid-phase extraction
(Wöelwer-Rieck, 2012), to remove these impurities. However,
although the pre-treatment is useful and improves the chro- References
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