Food Chemistry 157 (2014) 518523

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Fast methodology of analysing major steviol glycosides from

Stevia rebaudiana leaves
Cndida Lorenzo, Jssica Serrano-Daz, Miguel Plaza, Carmen Quintanilla, Gonzalo L. Alonso
Ctedra de Qumica Agrcola, E.T.S.I. Agrnomos, Universidad de Castilla-La Mancha, Campus Universitario, 02071 Albacete, Spain

a r t i c l e

i n f o

Article history:
Received 19 April 2013
Received in revised form 4 February 2014
Accepted 18 February 2014
Available online 28 February 2014
Keywords:
Stevioside
Rebaudioside A
Gradient elution
Ultraltration
Eco-friendly

a b s t r a c t
The aim of this work is to propose an HPLC method for analysing major steviol glycosides as well as to
optimise the extraction and clarication conditions for obtaining these compounds. Toward this aim,
standards of stevioside and rebaudioside A with purities P99.0%, commercial samples from different
companies and Stevia rebaudiana Bertoni leaves from Paraguay supplied by Insobol, S.L., were used.
The analytical method proposed is adequate in terms of selectivity, sensitivity and accuracy. Optimum
extraction conditions and adequate clarication conditions have been set. Moreover, this methodology is
safe and eco-friendly, as we use only water for extraction and do not use solid-phase extraction, which
requires solvents that are banned in the food industry to condition the cartridge and elute the steviol glycosides. In addition, this methodology consumes little time as leaves are not ground and the ltration is
faster, and the peak resolution is better as we used an HPLC method with gradient elution.
2014 Elsevier Ltd. All rights reserved.

1. Introduction
In recent years, consumers are more and more worried about
health, seeking natural and dietetic products. In this sense, the
interest in Stevia rebaudiana Bertoni has increased considerably,
as this plant has a high concentration (approximately 420%) of
sweet diterpene glycosides in its dry-leaf matter (Geuns, 2003;
Ghanta, Banerjee, Poddar, & Chattopadhyay, 2007). The high
sweetness of the steviol glycosides makes them an attractive sugar
substitute for food industries (Crammer & Ikan, 1986). Moreover,
these glycosides are non-caloric sweeteners that reduce blood glucose and protecting the organism from diseases such as diabetes
and obesity, among others (Geuns, 2003; Anton et al., 2010). Moreover, the steviol glycosides are related to other benets, such as
anti-hyperglycaemic, anti-hypertensive, anti-inammatory, antitumour, antidiarrheal, diuretic and immunomodulatory effect
(Chatsudthipong & Muanprasat, 2009). For all of these reasons,
the steviol glycosides are known as the sweeteners of the future
(Esmat, Azza, & Ferial, 2010; Brahmachari, Mandal, Rajeev, Mondal,
& Brahmachari, 2011; Lemus-Mondaca, Vega-Galvez, Zura-Bravo,
& Ah-Hen, 2012; Rao, Reddy, Ernala, Sridhar, & Ravikumar, 2012).
The main sweet components present in S. rebaudiana Bertoni
are stevioside and rebaudioside A, representing 90 wt.% of all
sweet glycosides in the leaves (Bergs, Burghoff, Joehnck, Martin,
Corresponding author. Tel.: +34 967 599310; fax: +34 967 599238.
E-mail address: Gonzalo.Alonso@uclm.es (G.L. Alonso).
http://dx.doi.org/10.1016/j.foodchem.2014.02.088
0308-8146/ 2014 Elsevier Ltd. All rights reserved.

& Schembecker, 2012). Moreover, they have the most sweetness

compared to sucrose (stevioside between 270 and 280 times more
and rebaudioside A between 350 and 450). Probably for these reasons stevioside is the most studied glycoside (Montoro et al., 2013;
Catharino & Santos, 2012) followed by rebaudioside A.
Other diterpene glycosides present in lower concentrations are
steviolbioside, rebaudioside B, C, D, F, dulcoside A and rubusoside.
The analytical determination of single diterpene glycosides by
chromatography is a difcult task because of the chemical structures of the glycosides (Fig. 1) are similar (FAO, 2010), and because
the aqueous extracts of Stevia leaf have many impurities such as
proteins, resins, organic acids, pigments and sesquiterpene lactones, among others (Kovylaeva et al., 2007).
Many and varied extraction solvents and methods for steviol
glycosides of Stevia leaves have been described in the literature:
with chloroform and methanol (Kolb, Herrera, & Uliana, 2001),
supercritical uid extraction (Pl et al., 2007), by means of microwaves (Jaitak, Singh, & Kaul, 2009) and ultrasonics and enzymatic
extraction (Jaitak et al., 2009; Liu, Li, & Tang, 2010; Puri, Sharma, &
Tiwari, 2011; Puri, Sharma, Barrow, & Tiwari, 2012). In addition,
there is abundant literature available with respect to the clarication and purication of these extracts (Zhang, Kumar, & Kutowy,
2000; Vanneste et al., 2011; Chhaya Sharma, Mondal, Majumdar,
& De, 2012; Rao et al., 2012; Li, Chen, & Di, 2012), and several
methods have also been reported for these extracts analysis:
Kovylaeva et al. (2007), Henderson and Berry (2009), Gardana,
Scaglianti, and Simonetti (2010), Woelwer-Rieck, Lankes,

C. Lorenzo et al. / Food Chemistry 157 (2014) 518523

519

Fig. 1. Molecular structure of stevioside (a) and rebaudioside-A (b).

Wawrzun, and Wst (2010), Bergs et al. (2012). However, for the
application of the sweeteners as food additives, several restrictions
and specications must be taken into account (JECFA, 2007). For
example, the use of solvents should be avoided as much as
possible.
The aim of this work was to propose a method for the analysis
of the major steviol glycosides in S. rebaudiana leaves. Towards this
goal, the optimum conditions for the extraction of these glycosides
are proposed; in addition extract clarication is accomplished by
microltration and ultraltration (UF) and quantication by
HPLC-DAD, avoiding where possible the use of dangerous solvents
that are environmentally damaging.

2. Materials and methods

2.1. Samples
Different brands of commercial samples with several steviol
glycosides concentrations, which were described on their labels,
were used: Majota pill and powder (60% and 90% steviol glycosides), Masso 60% and 98% steviol glycosides, Azelis 95% and 98%,
Cargill 98% and pure Stevia 99% steviol glycosides.
Dried leaves of S. rebaudiana Bertoni from Paraguay, supplied by
Insobol, S.L., were used. The samples were dried at room temperature to a moisture level between 5% and 6% as determined with a
halogen lamp moisture balance model XM-120T (Cobos, Barcelona,
Spain) at 105 C. When the moisture loss was less than 0.1% in
180 s, it was considered that the samples had reached a constant
mass.

Stevioside and rebaudioside A with purities P99.0% were purchased from Phytolab (Vestenbergsgreuth, Bavaria, Alemania).
The calibration solutions for the HPLC analysis of stevioside and
rebaudioside A were prepared by diluting the analytes with acetonitrile/water (8:2 v/v).
2.3. HPLC conditions
The HPLC conditions were xed with the standards cited above
and diluted with acetonitrile/water (8:2 v/v) to 50 mg/L.
The liquid chromatographer used was an Agilent 1200 HPLC
(Palo Alto, CA, USA). Two columns were tested: Develosil ODSHG 140 250 mm  4.6 mm i.d., 5 lm and Luna HILIC 150 mm
 4.6 mm i.d., 5 lm Phenomenex (Le Pecq Cedex, France). Isocratic
and gradient elution modes were tested. The eluents were water
(A) and acetonitrile (B) in both systems, trying 80% B20% A in
the isocratic elution and several ramps in the gradient, as well as
different elution times.
The oven was thermostated at 36 C. The ow rate was 1 mL/
min, and the sample injection volume was 20 ll. The DAD detector
(Hewlett Packard, Waldbronn, Germany) was set at 210, 256, 330,
360 and 450 nm.
To conrm the chemical structure of the compounds, a Mass
Spectrometer 6130 Quadrupole LC/MS, G1956 (SL) multimode
electrospray and atmospheric pressure chemical ionisation (MMESI/APCI-MS) system was used, coupled to an Agilent Chem Station
(version b.03.01) data-processing station. The parameters employed for MM-ESIMS were dry gas, N2, 10 mL/min; drying temperature, 350 C; vaporizer temperature, 200 C; nebulizer, 55
psi; capillary, 200 V (negative ionisation mode).

2.2. Reagents and standards

2.4. Method evaluation

HPLC-grade acetonitrile was used from Panreac Qumica, SAU

(Castellar del Valls, Barcelona, Espaa). Ultra-high-purity water
was produced using a Milli-Q System from Millipore (Bedford,
MA), and PVDF lters (13 mm, 0.45 lm) were also purchased from
Millipore.

Steviol glycosides were tentatively identied with the DAD

detector by comparison with the corresponding UVVis spectra,
on the basis of their molecular ion by electrospray ionisation mass
spectrometry (MM-ESIMS) (Fig. 1), and comparing with retention
time of their pure standards in the chromatogram.

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C. Lorenzo et al. / Food Chemistry 157 (2014) 518523

The selectivity was determined by comparing the chromatograms obtained from the leaf samples with those of the standards
solutions. For the linearity study, calibration graphs were performed by injecting standard solutions of stevioside and rebaudioside A with six different concentrations of each analyte. Each level
of concentration was analysed in triplicate. The concentration
ranges were from 25 to 500 mg/L. Qualitative determination was
achieved by comparing the retention times and the UVVis spectrum of the standard solution with those of the samples. Quantication was possible by applying the calibration plot equations
calculated by the least-squares method.
The sensitivity of the method was determined with regard to
the limit of detection (LOD) and the limit of quantication (LOQ),
comparing the height of a sample peak and the height of a noise
peak. The LOD is reached at a signal-to-noise ratio greater than
three, and the LOQ is reached at a signal-to-noise ratio greater than
10 (IHC, 2005).
The accuracy of the method was calculated with eight commercial samples spiked with different amounts (from 10% to 50% concentration increase) of the two steviol glycosides, which were
diluted to different volumes with a mixture of acetonitrile/water
(8:2, v/v) according to the glycoside concentration declared on
the samples labels. The standard deviation for each compound
(square root of the arithmetic mean of the variances) was calculated to obtain the repeatability (%RSD). The standard deviation
of the three values for each compound multiplied by the square
root of 3 was taken as the reproducibility value (if this value was
higher than the repeatability; if not, this last value was also taken
as the reproducibility) (Ortega, Lpez, Cacho, & Ferreira, 2001).
2.5. Extraction conditions
To choose the best extraction conditions for the steviol glycosides, we tried both milled (to 0.5 lm) and unmilled dry leaves.
Other factors such as the temperature, agitation and extraction
time were considered. On the basis of other authors (Pl et al.,
2007; Vanek, Nepovm, & Valcek, 2001; Woelwer-Rieck et al.,
2010) and our own experiences, the temperatures chosen for testing were 37, 60 and 100 C with and without agitation for 20 min.
After setting the temperature, the extraction time was tested by
analysing the sample extracts every 10 min until no increase in
the steviol glycosides concentration was observed.
Finally, 50 g of unmilled dry leaves were extracted with 2 L of
distilled water. The extractor used was a Thermomix TM-31 de Vorwerk & Co. KG (Wuppertal, Germany). One millilitre of the extract
was diluted to 10 ml with acetonitrile/water (8:2 v/v). This solution was ltered through a membrane lter (0.45 lm) before the
HPLC analysis. All of the samples were analysing in triplicate.
2.6. Centrifugal UF treatment
The extract was centrifugated in a Centronic BL-II centrifuge
from JP Selecta S.A. (Abrera, Barcelona, Spain) at 4400 rpm for
10 min. The supernatant was centrifugated at 12,000 rpm for
5 min, and the new supernatant was subjected to successive ltrations in a vacuum with membranes of 10, 2.5 and 1 lm. The last
extract was subjected to different centrifugal UF conditions: a
5000 Da (5 KDa) Centricon Plus-20 ultraltration membrane by
centrifugation at 4400 rpm for 1 h; a 3000 Da (3 KDa) Centriplus
YM-3 ultraltration membrane by centrifugation at 4400 rpm for
1 h; a 3000 Da Centriplus YM-3 ultraltration membrane by centrifugation at 4400 rpm for 30 min; and nally, a 3000 Da Centriplus
YM-3 ultraltration membrane by centrifugation at 12,000 rpm for
5 min. All of these membranes were obtained from Merck Millipore Headquarters (Billerica, MA, USA).

3. Results and discussion

3.1. HPLC conditions
To choose the best HPLC method, two columns together with
two elution types were tested, as it was described in Materials
and methods Fig. 2(a and b) show the different chromatograms obtained with the column Luna HILIC (150  4.6 mm i.d., 5 lm) and
Fig. 2(c and d) show those obtained with Develosil ODS-HG
(140 250  4.6 mm i.d., 5 lm). HPLC method proposed is based
on Woelwer-Rieck et al. (2010) procedure and, as well, the best results were obtained for Luna HILIC column. In terms of elution gradient, several modications were carried out to get a better peak
resolution, as shown in Fig. 2. This Figure shows that using isocratic elution in both columns (b and d), the compounds stevioside
and rebaudioside A are coeluted. However, they are clearly resolved when gradient elution was assayed in both columns
(Fig. 2a and c), showing Luna column the best results. For these
reasons, Luna column and gradient elution were chosen for the
method. This nding is in accordance with the results shown by
Hutapea, Toskulkao, Wilairat, and Buddhasukh (1999), who report
that this type of column shows poor selectivity, which can be
solved using gradient elution. After testing different elution
gradients with water (A) and acetonitrile (B) as eluents, we
obtained the best results with the following: 80% B, 010 min;
8050% B, 1012 min; 500% B, 1214 min; 080% B, 1416 min;
80% B, 1618 min. The ow rate was 1 mL/min, and the sample
injection volume was 20 ll. The wavelength used for quantication was 210 nm as this wavelength was the most adequate for
our objectives.
The evaluation of the method, performed by means of its selectivity, sensitivity and accuracy (Tables 1 and 2), showed that the
method was adequate for our objectives. In Table 1, we can see
the linearity of the proposed method by means of calibration
graphs for stevioside (y = 0.2091x) and rebaudioside A
(y = 0.2092x) in a wide concentration range from 25 to 500 mg/L.
Note that the correlation coefcients in both steviol glycosides
(stevioside and rebaudioside A) were higher than 0.99, which demonstrates an excellent linearity of the method.
The sensitivity was similar in both compounds, which showed a
LOD of 1.07 mg/L and LOQs of 3.55 and 3.56 mg/L for stevioside
and rebaudioside A, respectively. We have not found information
about the LOD and LQD in the method described by Woelver-Rieck
et al. (2010), which we relied on, but these limits are similar to
those obtained in the method proposed by Bergs et al. (2012).
The accuracy of the experimental procedure was also evaluated by studying the reproducibility and repeatability. For the
reproducibility of a method (%RSD) to be considered acceptable,
its value should be less than 20%. As seen in the results summarised in Table 2, this parameter ranged from 3.33% to 5.39%. The
same limit (20%) was taken to represent good repeatability; in
this case, this value ranged from 5.76% to 9.32%. Therefore, the
method proposed shows very good repeatability and reproducibility parameters.
3.2. Extraction conditions
To choose the best extraction conditions, we studied the concentrations of the steviol glycosides in the different situations.
The rst major decision we made was not to grind the sample.
This choice was because, in the extraction conditions used
(50 g dry leaves, 2 L water, 60 C for 1 h), grinding the sample
did not signicantly improve the extraction, whereas the ltration was much more tedious. That is to say, the increase in
the efciency of the extraction does not compensate for the
higher cost of ltration.

C. Lorenzo et al. / Food Chemistry 157 (2014) 518523

521

Fig. 2. Comparison between chromatograms of same Stevia leaf extract using Luna HILIC column (a: gradient elution; b: isocratic elution) and Develosil column (c: gradient
elution; d: isocratic elution). (1) Stevioside, (2) rebaudioside A.

Table 1
Linearity of the method.
Compound

Retention time
(min)

Slope

Correlation coef. (r2)

Stevioside
Rebaudioside A

5.15
6.15

0.2091
0.2092

0.999
0.996

Table 2
Precision of the method.
Compound

LOD
(mg/L)

LOQ
(mg/L)

Reproducibility
(%)

Repeatability
(%)

Stevioside
Rebaudioside A

1.07
1.07

3.55
3.56

3.33
5.39

5.76
9.32

The results obtained by comparing the concentrations we extracted at different temperatures over 20 min (37, 60 and 100 C,
with and without agitation) led us to set the extraction temperature at 100 C, which was the same temperature used by other
authors (Vanek et al., 2001). Once the temperature was reached
in the equipment (with water boiling), sample extracts were taken
every 10 min and analysed to evaluate the inuence of the

extraction time (Fig. 3). The maximum stevioside concentration

was at 20 min, with a concentration of 2714.95 mg/L, representing
10.86% in plant weight. The highest quantity of rebaudioside A was
reached at 30 min (683.72 mg/L), representing 2.73% in plant
weight. These values are within the range of concentrations described by several authors for sweetening compounds (Geuns,
2003; Puri et al., 2011). Taking into account these results, we chose
30 min as the best extraction time for the major steviol glycosides
(stevioside and rebaudioside A).
We chose not to use solid-phase extraction to purify the sample
as this technique requires solvents that are banned in the food
industry to condition the cartridge and elute the steviol glycosides.
For this reason, we tried to clarify the samples only by micro- and
ultraltration.
After removing solids by means of centrifugation, the clarication of the extracts was performed using different membranes
(Fig. 4) for microltration (successive ltrations in vacuum: 10,
2.5 and 1 lm). After passing through the nal membrane (1 lm),
the steviol glycosides concentrations showed by the extracts were
as follows: 3409.83 mg/L for stevioside and 1853.73 mg/L for
rebaudioside A, representing 9.09% and 4.94%, respectively, of the
total mass of the starting plant material. Therefore, these sweeteners represent approximately 14% of the plant mass, which is

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C. Lorenzo et al. / Food Chemistry 157 (2014) 518523

2800
2700
mg/L

2600
2500
2400
2300
2200
10

20

30

40

50

60

Extracon me (min)

700

mg/L

680

because most of the water passes through the membranes, diluting

the nal concentrations. This fact is a problem because there are
losses of these compounds; we could therefore improve the efciency by, for example, introducing wash steps of rejections in
the nal method. Other authors (Chhaya et al., 2012) tested different UF membranes to clarify Stevia extract and obtained approximately 45% recovery of stevioside as the most suitable choice.
For all of these reasons, we can say that the use of the proposed
membranes in this study is an adequate rst step for the clarication of Stevia leaf extracts, although the process must be completed with nanoltration, as is proposed by other authors
(Vanneste et al., 2011; Rao et al., 2012).
4. Conclusions

660
640
620
600
10

20

30

40

50

60

Extracon me (min)

Fig. 3. Inuence of the extraction time in the concentration of stevioside (a) and
rebaudioside A (b).

100%
90%
80%
70%
60%

The HPLC method proposed allows the major steviol glycosides

to be quantied in an easy way with good selectivity, sensitivity
and accuracy. We propose the best extraction conditions as follows: 100 C for 30 min without agitation and with no grinding
of the leaves. The clarication of the extracts by means of microand ultraltration allows an acceptable percentage of the major
steviol glycosides to be extracted.
The proposed methodology is adequate for determining, in a
quick and easy procedure, that the major steviol glycosides (stevioside and rebaudioside A) are safer for health and have a lower
environmental impact than other methodologies described in literature. This is of great relevance in the food industry, which are
seeking substitutes for the articial sweeteners widely used up
to now, but whose consumption starts to be rejected by consumers
increasingly concerned about their health.

50%

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40%
30%
20%
10%
0%
5KDa 1h
4400 rpm

3KDa 1h
4400 rpm

3KDa 1/2h
4400 rpm

3KDa 5min
12000 rpm

Fig. 4. Percentages of stevioside rejection (above, light grey) and recovery (down,
dark grey) when extract is passed through different membranes.

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To perform the ultraltration, the resultant extract was subjected to different centrifugal UF conditions to nd the conditions
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at 4400 rpm both for 1 h and for 30 min, whereas in the case of
centrifugation at 12,000 rpm for 5 min, the percentage of rejection
is 75%. The higher percentage of compounds in the rejections is

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