VIETNAM NATIONAL UNIVERSITY, HA NOI

VNU UNIVERSITY OF SCIENCE

FACULTY OF CHEMISTRY

Đinh Lê Minh

Overview of analytical methods for the determination

of Steviol Glycosides

Submitted in partial fulfillment of the requirements for the course of

CHE2124 in Chemistry

( Advanced Program for talented Bachelor of Science )

Ha Noi - 2018
 VIETNAM NATIONAL UNIVERSITY, HA NOI

VNU UNIVERSITY OF SCIENCE

FACULTY OF CHEMISTRY

Đinh Lê Minh

Overview of analytical methods for the determination

of Steviol Glycosides

Submitted in partial fulfillment of the requirements for the course of

CHE2124 in Chemistry

( Advanced Program for talented Bachelor of Science )

Supervisor: Assoc. Prof. Dr. Nguyen Thi Anh Huong

Ha Noi - 2018
 Acknowledgements

I am really grateful because I have completed my study “Overview

of analytical methods for the determination of Steviol Glycosides”. I
would like to sincerely thank my supervisor, Assoc. Prof. Dr. Nguyen
Thi Anh Huong for her guidance and encouragement so that I can
complete this Overview.
 Abbreviations

CDP : Copalyl Di-photsphate

CE : Capillary Electrophoresis
CE-C4D : Capillary electrophoresis with capacitively coupled
contactless conductivity detection
HPLC : High-performance liquid chromatography
GA : Gibberellic acid
GC : Gas chromatography
GGDP : Geranyl Geranyl Di-photsphate
IPP : Iso-Pentenyl Di-photsphate
KAH : Kaurenoic acid Hydroxylase
KO : Kaurene oxidase
KS : Kaurene synthase
LC : Liquid chromatography
MEKC : Micellar electrokinetic chromatography
MEP : Methyl-Erythritol-Phosphate
PKU : Phenylketouria
SIR : Single ion reaction
TLC : Thin-layer chromatography
Contents

Preface.................................................................................................................. 1
Part 1: Overview of Steviol Glycosides .......................................................... 2
1.1. Introduction ........................................................................................... 2
1.2. Physical properties ................................................................................. 3
1.3. Stevioside Biosynthesis pathway ......................................................... 5
1.4. Phytochemical constituents of Stevia leaves ..................................... 6
1.5. Sweetness perception of Steviol glycosides ....................................... 7
1.6. Uses of Stevia plant – good agriculture practices ............................. 8
Part 2: Overview of analytical methods for the determination of Steviol
Glycosides ......................................................................................................... 10
2.1. Liquid chromatography ...................................................................... 10
2.1.1. Fundamental principle .................................................................... 10
2.1.2. Determination of Steviol Glycosides by Chromatographic
methods ....................................................................................................... 12
2.2. Capillary electrophoresis (CE) ........................................................... 15
2.2.1. Basic concepts.................................................................................. 15
2.2.2. Determination of Steviol Glycosides by CE .............................. 16
2.3. Summary of analytical methods for the determination of Steviol
Glycosides ...................................................................................................... 19

0
 Preface
The quest for sugar-replacers, driven by the obesity epidemic, is expanding.
Policy makers support the development of new energy-reduced products.
Consumers to whom “naturalness” appeals are forming an increasingly large
segment. These developments are likely to have resulted in the increased attention
for „Stevia-based‟ sweeteners. In 2007 and 2008, three applicants requested the
authorization of steviol glycosides as sweetener. In the European market the
introduction of steviol glycosides as food additives, with a purity of more than 95%
was given at the end of 2011, after EFSA issued a positive opinion on their safety
and raised the acceptable daily intake (ADI) for steviol glycosides, expressed as
steviol equivalents, to 4 mg/kg BW/day. Until then, analytical methods for the
determination of Steviol Glycosides received much concern and has been
developed by scientists all around the world.

The glycosides of plant, Stevia rebaudiana, accumulate in all the

photosynthesizing parts of the plant. A number of analytical techniques, including
liquid chromatography (LC coupled with tandem mass spectrometry, HPLC-PDA,
ultra-high-performance liquid chromatography-mass spectrometry,…), capillary
zone electrophoresis (capillary electrophoresis with micellar kinetic
chromatography, capillary zone electrophoresis with capacitively coupled
contactless conductivity detection,…) have also been applied to determine and
quantify Steviol Glycosides in plants and food samples. In order to find the most
suitable method for determination these group compounds, in these overview,
some analytical methods will be compared.

This review will provide to you some essential information about Steviol
Glycosides as well as analytical methods for the determination of Steviol
Glycosides.

1
 Part 1: Overview of Steviol Glycosides

1.1. Introduction
Steviol Rebaudiana, commonly known as Stevia, is the only one of the
Genus Stevia, family Asteraceae, with sweet tasting properties, which can be
attributed to its diterpenoid glycosides. Growing in the wild in Paraguay and
Brazil, Steviol Rebaudiana‟s leaves have been used as a natural sweetener, an
alternative ingredient for sugar by the local population for hundreds of years. The
water extract of Steviol Rebaudiana has beneficial hypoglycemic, hypotensive
effects and is a source of antioxidant compounds. In Korea, Japan, China and
South America, the leaves are used as non-nutritive, high-potency sweetener
primarily. The presence of sweet diterpenoid glycosides cause the sweetness of
leaves, and one of the most concerned are Stevioside and its derivatives.
Stevioside is a low-caloric substance and has a medical value. It has been used in
the treatment of diabetes and other disturbances of sugar metabolism. Nowadays,
Stevia Rebaudiana has became an increasing research because of the demand of
humans to find an alternative for sweet-tasting sugars.
There are many kinds of Steviol Glycosides found in leaves. The
composition of purified Stevia extracts depends on the environment,
manufacturer and genetic aspect of the original plant. The glycosides profiles of
Stevia are also subjects of consideration about geographic area, plant maturity,
environmental conditions, harvesting and processing,… The change in the plant
compositions can affect its sweetness, therapeutic, toxic activity. Strictly quality
control procedures for Stevia sweeteners must be carefully carried out to ensure
the standardization and safety requirements so that they can be used in food and
medicine. A variety of analytical methods have been reported according to the
seperation and quantification of these compounds from the Stevia rebaudiana
leaves.

2
 Compound
Steviol (ST) H H 317
Stevioside (SV) -Glc- -Glc-(2->1) 803 839
-Glc-
Rebaudioside B (Rb) H 803 839
-Glc-(2->1)-Glc(3->1)
-Glc-
Rebaudioside A (Ra) 965 1001
-Glc-(2->1)- -Glc(3->1)
Rebaudioside E (Re) -Glc- -Glc-(2->1) -Glc- -Glc-(2->1) 965 1001
Steviolbioside (Sb) H -Glc- -Glc-(2->1) 641 677
Rubusoside (Ru) 641 677
-Glc-
Rebaudioside C (Rc) 949 985
-Glc-(2->1)- -Rha(3->1)
Dulcoside A (Du) -Glc- -Rha(2->1) 787 823
Steviol glucoside H 479 515
Steviol glucoside H 479 515

Figure 1: Structure of Steviol and its glycosides found in Stevia

rebaudiana leaves from Southern Italy

1.2. Physical properties

Steviol glycosides are used as sugar substitutes for commercial purpose
because the Stevia‟ leaves accumulates several sweet tasting diterpene glycosides.
Steviol glycosides are concentrated sweeteners ranging from 50-300 times
sweeter than sugar, with high molecular weights, low water solubility and high
melting points (Table 1).
These molecules in solution seem to be highly stable at board pH and
temperatures. Heating a stevioside solution at neutral condition at 100oC for 10

3
hours causes a decrease by 16.7%. In acidic environment, it takes 4 hours to
reduce the concentration by 46.0-53.8%. Significant decrease in stevioside
concentration at 80oC was found only at highly acidic concentration (pH=1) in
solution.

Table 1: Physical properties of different steviol glycosides

Molecular Melting Solubility in RS (Relative
Compound
weight point (oC) water (%) sweetness)
Stevioside 804 196-198 0.13 143
Rebaudioside A 966 242-244 0.80 242
Rebaudioside B 804 193-195 0.10 300
Rebaudioside C 958 215-217 0.21 50
Rebaudioside D 1128 283-286 1.00 221
Rebaudioside E 966 205-207 1.70 174
Steviolbioside 642 188-192 0.03 125
Dulcoside A 788 193-195 0.58 50

The excess sugar in human bodies can cause many diseases like diabetic,
phenylketouria (PKU) and obesity. Steviosides could be a good substitute to
reduce the intake of sugar. Besides, the advantages of stevioside as a dietary
supplement for human subjects are manifold: it is stable, it is non-calorific, it
maintains good dental health. The omission of sugar added in the food is
beneficial to diabetic people by lowering the blood sugar content. No aromatic
amino acids are involved in Steviol glycosides , which is safe for PKU patients.
Futhermore, excessive sugar in food now replaced by Stevia could help obese
people lose weight. Decreasing an amount of added sucrose in food increases the
relative proportion of polymeric carbohydrates. This has a beneficial effect for
balanced food intake and for human‟s health.

4
1.3. Stevioside Biosynthesis pathway
The ent-kaurene backbone of Steviol Glycosides is similar to that of
Gibberellic acids (GA). In Steviol Glycosides there accumulate glycosulation at
C13 and C19. Difference between the number of saccharide units added by
various glycosyltransferases at C19 position leads to different Steviol Glycosides.
The precursor of the backbone, steviol was proved to be synthesised by the
Methyl-Erythritol-Phosphate (MEP) pathway. Firstly, the end product of the
MEP pathway, Iso-Pentenyl Di-photsphate (IPP) is converted to Geranyl Geranyl
Di-photsphate (GGDP), and then it is cyclised into Copalyl Di-photsphate
(CDP). Kaurene synthase (KS) is used to convert CDP to kaurene. Both CDP
synthase and Kaurene synthase have been characterized in stevia. Kaurene
oxidase (KO), a cyt-p450 Monooxygenase, converts kaurene to Kaurenoic acid.
Both KS and KO are duplicated in the stevia genome. Although there is an
overexpression of the KS and KO genes, stevia does not increase concentrations
of either GA or Kaurenoic acid in its tissues.
The mechanism of keeping the GA contents bases on Spatial and temporal
seperation of the key enzymes in steviol biosynthesis from the GA biosynthesis
pathway. The rate of GA concentrations in tissues was further downstream from
these enzymes, at the dioxygenase, which is the first step of GA biosynthesis.
Arabidopsis can store increasingly high concentrations of the Kaurene acid
without any bad effects.
In stevia, increased flux of Kaurenoic acid could be carried by the presence
of the sink pathway leading to steviol glycoside biosynthesis. The first committed
step to this synthesis is Kaurenoic acid -13-hydroxylase (Figure 2), where GA
precursors are hydroxylated at C7. This enzyme has immense applications in
biotechnology and is a subject of much controversy due to sequences of this
enzyme.

5
 GA biosynthesis

Figure 2: Stevioside biosynthesis pathway

1.4. Phytochemical constituents of Stevia leaves

The need of this plant for sweetening purposes has resulted in many
researches for the nutritional composition of the plant. The leaves of the plant
store diterpene glycosides, which is responsible for the sweet tastes and other
phytochemicals.
Stevia leaves have been reported to contain tannins, which are synthesised in
a wide range of plants and trees. Gallo and ellagitannins or mixtures of both,
classified as hydrolysable tannins are found in Stevia leaves too. Hydrolysable

6
tannins consist of central glucose attached to many gallic or ellagic acid units, and
their complex reaction leads to tannins in plants. To analyze tannins, it is
important to focus on sample preparation, storage and extraction techniques.
General tannin assays used for analysis of mixtures of tannin is to precipitate with
metals or protein or in the other way, by colorimetric assays for total phenols. A
sensitive method for quantifying and differentiating tannins are TCL and HPLC
methods. In Stevia leaves there accmulate high polyphenolic concentrations.
Phenolic compounds like gallic acid, ellagic acid and their derivatives, flavonoids
and even vitamins are classified as primary antioxidants (free radical scavengers,
inhibitors of lipid peroxidant and chelating agents) or chain-breakers. These
compounds act by oxidizing the free radicals which cause oxidative stress. Also,
tannins have anti-protein activity, but the major factor acting against the presence
is their sweetness.

1.5. Sweetness perception of Steviol glycosides

Complex interaction between the various constituents of the leaf leads to the
sensory attributes of Stevia extracts. Most chemical classes of sweeteners
accumulate sweet and bitter taste properties. A single compound can have both
sweet and bitter qualities. There is an interdependence between them which
comes from psychological taste experiments using mixtures containing varying
amount of sweet and bitter components. This phenomenon called mixture
supression. A possible relationship between sweet and bitter tastes can found in
many structural categories.
The two tastes are reported to be closely related with respect to their
transduction although sweet taste is usually regarded as plesant and bitter taste is
not. Biochemical studies suggest that the degree of interdependence of bitter and
sweet rates may be compound specific and rely on the transduction mechanisms
involved in inducing the taste sensations. Generally, with increasing
concentration, high-potency sweeteners containing acesulfame-K, neohesperidin

7
dihydrochalcone, sodium saccharin, rebaudioside-A and stevioside tend to
become bitter. In contrast, less bitter with increasing concentration comes from
low-potency sweeteners like fructose, sucrose, lactiol.
Taste perception is thought to begin early in our cognitive development and
relate to our aging. For example, fruits can easily be identified ripeness by color
and odour. Ripening is often associated with a transition from green to red color,
and this often parallels a major increase in the sugar concentration of fruits. If
green color in fruits shows us a preponderance of acid taste over sweetness, then
fruits with lighter green color, less acid stored will have sweeter taste. Statistically,
two properties have a stronger influence on sensory perception in adults than
children. To conclude, sweetness enhancement by odour and color associations
plays an important role in taste perception.
The sweetness of Steviol Glycosides also depends significantly on some
small variations in structure. The number of glucosyl units attached to the C13
determines magnitude of sweetness and quality of taste. Stevioside were treated
by Cyclomaltodextrin glucanotransferases (CGTastes) in the presence of starch
donor. Mono and di-glycosylated at the C13 position in products were also
concerned with major improvements.

1.6. Uses of Stevia plant – good agriculture practices

There is a strong and increasing concern among consumers and
manufacturers for products that can be used to improve health, such as natural
food supplements, herbal remedies and other products based on medical plants
and functional foods. Foods with naturally health qualities can prevent some
dangerous diseases like obesity, diabetes, cancer,… Sweetness is probably one of
the most appreciated features of the food we eat. However, not all consumers
want to (or can) consumes sugar-the most obvious source of sweetness. With an
emphasis on reducing the calorie intake, more and more individuals are focusing
on lowering their sugar consumption on food.

8
 Legally, two uses of Stevia are distinguished. The first thing is use of Stevia
in the form of plant, dried leaves and crude extracts, which are considered novel
foods and fall under EC regulation 258/97. In 2000, the placing on the market of
Stevia rabaudiana Bertoni plants and dried leaves as a novel food or novel
ingredient under EC regulation 258/97 of the European Parliament and of the
Council was refused. Second is the use of stevia in the form of purified extracts
from the plant. These extracts, steviol glycosides, exhibit enormous sweetness,
about 200-300 times more than regular sugar. The major types of Steviol
Glycosides are Stevioside and Rebaudioside A. Steviol Glycosides have been
proposed to be used as food additives and such fall under EC regulation
1333/2008.

9
 Part 2: Overview of analytical methods for the
determination of Steviol Glycosides

Nowadays, there are some methods for the determination of Steviol

Glycosides, including chromatographic methods, capillary electrophoresis,
photometric methods,… However, liquid chromatohraphy and capillary
electrophoresis are popular than other methods because of their effects for the
seperation and quantification. In this review, liquid chromatography and capillary
electrophoresis methods will be introduced.

2.1. Liquid chromatography

2.1.1. Fundamental principle
When a mixture of molecules applied onto the surface or into solid and fluid
stationary phase, there would be seperation between stable phase and mobile
phase. Different molecules have different characteristics like adsorption, partition,
affinity or the molecular weight. These differences affect seperation process, while
some components of the mixture stay longer in the stationary phase and move
slowly in chromatography system, and others pass rapidly into mobile phase, leave
the system.
Based on this approach three components form the basis of the
chromatography technique:
 Stationary phase: including a “solid” phase or “a layer of a liquid
adsorbed on the surface a solid support”
 Mobile phase: including “liquid” or “gaseous component”
 Seperated molecules
Seperation process based on type of interaction between stationary phase,
mobile phase and substances in the mixture. For seperation and identification of
small molecules as amino acids, carbohydrates, fatty acids,…, partition
chromatographies are likely to be chosen, while affinity chromatographies (for

10
 example ion-exchange chromatography) are more popular in the seperation of
macromolecules like nucleic acids, protein,… Paper chromatography is used to
study proteins and their synthesis, gas-liquid chromatography is effective for
analysis of alcohol, esther, lipid, amino groups and observation of enzymatic
interactions, while molecular-sieve chromatography is employed especially for the
determination of molecular weights of proteins. Agarose-gel chromatography is
used for the purification of ARN, ADN particles, and viruses.
Stationary phase in chromatography contained a solid phase or a liquid
phase applied on the surface of a solid. Mobile phase flowing over the stationary
phase is a gaseous phase (called gas chromatography GC, usually applied for
gases, mixtures of volatie liquids), or liquid phase (called liquid chromatography
LC, used especially for thermal unstable and non-volatie samples).

Solvent
reservoir

Processing unit
Pump to and display
produce high
pressure

Signal to processor

Sample detector
injection
HPLC tube

waste
Figure 3: A flow scheme for HPLC

11
2.1.2. Determination of Steviol Glycosides by Chromatographic
methods

A reversed-phase liquid chromatography method coupled with tandem mass

spectrometry electrospray ionisation (HPLC-ESI-MS/MS) was developed by
Pawel Kubia, et.al. Jacek Namiesnik, Andrzej Wasik for stimultaneous
determination of eight artificial sweeteners and common Stevia rebaudiana
glycosides in soft and alcoholic beverages. First, all samples of soft and alcoholic
drinks were degassed by sonication for 15 minutes. Instant drinks were prepared
according to the manufacturer‟s directions. Samples were diluted with mobile-
phase component A in order to fall within calibration curve concentration range.
The seperation was achieved on a column. The mobile phase used for
seperation was +MeOH+acetone (75+20+5) with 0.1 %v/v of AA
(component A) and ACN+acetone (95+5) with 0.1%v/v of AA (component B).
The chromatographic seperation was performed in gradient elution mode: 0 min
(0% B), 10 min (30% B), 15 min (70%B) and 16 min (70%B). The total time of
chromatographic run was 16 min, while the column equilibration time was set to
8 min. Different concentration ranges were used for 2 different classes of
sweeteners: 5-800 ng/mL for the artificial ones and 5-1600 ng/mL for steviol
glycosides, with correlation coefficients of over 0.9987. The recoveries of the
method ranged from 97.0% to 105.7% while the relative standard deviations
(%RSD) of the results were from 0.4-4.1% (n=6). The method has been
successfully applied to the analysis of steviol glycosides in samples of drinks and
no matrix effects were observed.

A simple and sensitive thin layer chromatography method was developed by

Radoslaw J.Ekiert, et.al. Jan Krezk, Magdalena Lenartowicz, Halina Ekiert for the
analysis of stevioside and rebaudioside A in sweeteners. An 18cm x 9cm x 18cm,
Sigma Aldrich (St. Louis, USA) vertical chromatograohic chamber, Linomat 5
band applying module and Scanner 3 densitometer were used with a fluorescence

12
detector at a wavelength of 254 nm. The standard solutions and sample solutions
were applied on chromatographic plate in volumnes from 1.0 l to 50.0 l in
order to establish method‟s analytical range which was determined as 7.5-22.5 l
for stevioside and 5.0-25.0 l for rebaudioside A. The mobile phase used for
seperation was ethyl acetone-methanol-water (7:2:1, v/v/v). The development of
chromatograms took place in a closed saturated (5 min) chromatographic
chamber. The development time equaled approximately 48 min. The precision
was measured by repeatability of the results at 100% of investigated level, (RSD
for n=9 results) while accuracy was expressed by the precentage recovery of the
reference. The recovery was established for fruit methanolic extract (n=6).
Linearity was established for nine data points within the method‟s range with 5 g
intervals. The method is suitable for routine food quality testing.

Another methods to determine steviol and its glycosides in sweeteners is

using ultra-high-performance liquid chromatography with electrospray ionization
mass spectrometry (UHPLC-MS), which was developed by Claudio Gardana,
et.al. Martina Scaglianti, Paolo Simonetti. Steviol glycosides identity was
established by UV spectra comparision, molecular in and product ions
evaluation, while routine analyses were carried out in single ion reaction (SIR)
monitoring their negative chloride adducts. The seperation was achieved on
column, with added to mobile phase as source of enhance sensitivity.
Assay validation demonstrated good performances in terms of accuracy (89-
103%), precision (<4.03%), repeatability (<5.7%) and linearity (40-180mg/g). The
recovery of method ranged from 95% to 102% and the relative standard deviation
(%RSD) was 1.0-7.7%. The proposed UHPLC-MS methods can be applied for
the routine quality control of Stevia leaves and their commercial preparations.

A liquid chromatography electro-spray tandem mass spectrometry (LC-

ESI/MS/MS) method was developed by Paola Montoro, et.al. Ilaria Molferra,
Mariateresa Maldini, Lucia Ceccarini, Sonia Piacenete, Cosimo Pizza, Mario

13
Macchia for determination six steviol glycosides. Quantitative analyses of
compounds were performed in an Agilent (Paolo Alto, CA, USA) 1100 HPLC
system equipped with a Waters Atlantis RP DC18 column (150 x 2.1 mm, 5 m
i.d.) and coupled to an AB Sciex (AB Sciex, Foster city, USA) API2000 triple
quadruple instrument. The mobile phase was generated by a gradient elution
programme of phase A ( acidified with Tri-fluoroacetic acid 0.05%) and
phase B ( acidified with Tri-fluoroacetic acid 0.05%). Gradient solution
started at 35% of solvent B, to 80% of solvent B in 25 min, with a flux of 200 l
. The API2000 mass spectrometer was used in the tandem MS mode with
multiple reaction monitoring (MRM). The instrument was operated in the positive
ion mode with a de-clustering potential of 100 eV, focusing potential of 200 eV,
entrance potential of 10 eV, collision energy of 80 eV, collision cell exit potential
of 15 eV, ion spray voltage of 5000, and capillary temperature of 300 oC. The
standard deviation was no higher than 2.00%. The means recovery was
determined to be 100 2%. This technique was found to be an useful application
in the field of qualitative and quantitative analysis of foods.

A high performance liquid chromatography method was developed by

Wilhad Reuter for qualitative and quantitative analysis of Steviol Glycosides in
drinks. The HPLC sysem used photodiodiode array detector and a 50 mm flow
cell. Following the JECFA protocol, 210 nm was chosen as the analytical wave
length since Steviol glycosides adequately absorb only in this region. The
seperation was achieved on column. The mobile phase used in this case was
32% acetonitrile, 68% 10 mM buffer; pH 2.7 with phosphoric acid.
The analysis time was 25 min, with flow rate 1.0 mL/min. All solvents and
diluents used were HPLC grade and filtered via 0.45 m filters to remove small
particles. All standard dilutions were made using 30:70 acetonitrile/ water. The
limit of quantitation were calculated to be 0.5-0.7 ppm. Good linearities between

14
 response (peak area) and concentration of Reb A and Stevioside were found over
a concentration range of 1.0/5.0 ppm to 200/100 ppm.

A method based on LC-MS/MS by using a triple quadrupole detector was

developed by M. Molina-Calle, et.al. V. Sánchez de Medina, M. P. Delgado de la
Torre, F. Priego-Capote, M. D. Luque de Castro for quantitation of 8 steviol
glycosides in extracts from Stevia leaves. A column was used for the
seperation together with deionized water with 0.1% of formic acid (A) and
methanol with 0.1% of formic acid (B) as mobile phases. The gradient was as
follows: start with 60% B, change from 60% to 100% B in 15 min and constant
100% B for 5 min more. A post-run of 5 min was programmed to equilibrate the
column between analyses. The flow rate was constant at 0.7 mL/min and the
injected volume 10 µL. The column temperature was set at 40 ºC. The ionization
and fragmentation parameters for selected reaction monitoring were optimized.
The ionization operating conditions were studied by direct injection of individual
standard solutions of each analyte at 5 µg/mL using the positive and negative
ionization modes. Detection and quantitation limits ranging from 0.1 to 0.5
ng/mL and from 0.5 to 1 ng/mL, respectively. The method provided a high
sensitivity, thanks to the optimization of all the parameters involved in both steps,
ionization and fragmentation.

2.2. Capillary electrophoresis (CE)

2.2.1. Basic concepts
Capillary electrophoresis is an analytical technique that seperates ions based
on their electrophoretic mobility with the use of an applied voltage. The
electrophoretic mobility is dependent upon the charge of the molecule, the
viscosity, and the atom‟s radius. The rate at which the particle moves is directly
proportional to the applied electric field--the greater the field strength, the faster
the mobility. Neutral species are not affected, only ions move with the electric

15
 Figure 4: Instrumental setup

field. If two ions are the same size, the one with greater charge will move the
fastest. For ions of the same charge, the smaller particle has less friction and
overall faster migration rate. Capillary electrophoresis is used most predominately
because it gives faster results and provides high resolution separation. It is a
useful technique because there is a large range of detection methods available.

2.2.2. Determination of Steviol Glycosides by CE

A simple and sensitive capillary electrophoresis method was developed by
Antonio S. Dacome, et.al. Cleuza C. da Silva, Cecılia E.M. da Costa, Jose D.
Fontana, Juliana Adelmann, Silvio Claudio da Costa for the isolation and
quantitative of diterpenic glycosides in Stevia plants. Capillary electrophoreses
(CE) were run in a CE Aligent apparatus using a 70cm length and 50 i.d.
fused-silica capillary tube at 28 kV/70 for 15 min and at 250C after a 50 mbar
hydrostatic injection for 2s. 30 mM, pH 10.3 sodium tetraborate containing 3 mM
-cyclodextrin was used as buffer, 22.5% of acetonitrile and 15% of methanol as

16
solvent polarity modifiers. The rebaudioside-A:stevioside peaks ratio when
considering the peak heights as proportional to the effective concentrations from
2.09:1.95 to 5.63:5.15 mg/mL. The regression coefficients for both of them were
0.9986 and 0.9974 respectively. It was found that a more alkaline borate buffer
pH 10.3 could increase the capillary silanol group‟s ionization and the resulting
electroendosmotic flow, proved beneficious for resolution improvement, and run
time shortening.

A simple and efficient capillary electrophoretic method was developed by J.

Liu & S. F. Y. Li for the determination of Steviol Glycosides in sweeteners. CE
was carried out on a commercial and a laboratory-built CE system. A fused-silica
capillary tube of 50 cm effective length and 50 m I.D. was used as the separation
column. The peaks were detected by a Micro-UVis2O detector with wavelength
set at 210 nm. The window for the on-column detection cell was made by
removing a small section of the polyimide coating on the fused silica capillary.
Samples were injected into the capillary by gravity feed with injection time of 20
seconds and injection height of 10 cm. The buffer solution contained acetonitrile
and sodium tetraborate solution. Good linearity was from 0.01 to 20.0 mg/mL.
The four steviol glycosides were neutral compounds and usually could not be
seperated electrophoretically. But in borate buffer systems adding acetonitrile,
these compounds were transformed into negatively charged borate complexes
that could be partially separated in CE. An applied voltage of 16.5 kV was chosen
for subsequent experiments since it gave satisfactory separation and short
migration times for all the peaks. The detection limit of the CE system was 0.010
mg/ml. The correlation coefficients was r = 0.9936. The method has been
sucessfully used as an alternative analytical procedure to HPLC when the amount
of sample available was small, or in an orthogonal manner to provide additional
information.

17
 Two electrophoretic methods with contactless conductivity detection have
been developed by Václav Pavlícˇek, et.al. Petr Tuma for determination of the
content of rebaudioside A and stevioside in samples of sweeteners and beverages
prepared from extracts of the plant Stevia rebaudiana Bertoni. The total content
of rebaudioside A and stevioside can be determined in a fused silica capillary tube
with an inner diameter of 10 m and total length of 31.5 cm in optimised
background electrolyte with the composition 170 mM /LiOH (pH 9.0).
The combined peak of the two glucosides is characterised by a migration time of
54s, which completely separates it from EOF. INST coating solution in an
amount of 0.5% v/v, which effectively suppresses the electroosmotic flow, was
added to the background electrolyte for mutual separation of rebaudioside A and
stevioside. The CE method with suppression of EOF is characterised by
complete separation of rebaudioside A and stevioside, LOD is 0.3 mg/L (0.1
M). The calibration dependence for the area of the rebaudioside A peak in the
concentration range 10–100 mg/L. The accuracy at low and high level is 10.0 ±
0.5 mg/L and 100.0 ± 4.0 mg/L for rebaudioside A; 10.0 ± 1.0 mg/L and 100.0
± 5.1 mg/L for stevioside. The repeatability of the migration time for 20
consecutive analyses of a combined sample of rebaudioside A and stevioside with
a concentration of 50 mg/L is 0.6% (RSD). This method was a simple and rapid
treatment for analysis foods and beverages.

The determination of diterpene glycosides from Stevia rebaudiana leaves

using capillary electrophoresis was described by Pierluigi Mauri, et.al. Giovanna
Catalano, Claudio Gardana, Piergiorgio Pietta. Analysises was performed on a
fused silica capillaries with 20 nM sodium tetraborate buffer, pH 8.3, and 30 mM
sodium dodecyl sulfate. Stevioside standard solution was obtained by dissolving
10 mg in 20% methanol (2 mL). Replicate injections (n = 6), were made at 50
mbar x 2s. The purity of these compounds was assessed by CE, and their identity
was confirmed by electrospray mass spectrometry (ESI). The detector response
for each glycoside was linear over the range 0.2-5 mg/mL and the linear

18
 regression coefficient was 0.995-0.997 (RSD= 1.1-1.3 %). The contents of
stevioside was 56.7mg/100mg extract, rebaudioside A was 26.1 mg/100mg
extract while the concentration of steviolbioside was smallest.

A newly developed electrokinetic chromatographic method for the

simultaneous separation and determination of steviol glycosides in real stevia
samples by capillary electrophoresis and supported by molecular docking studies
was described by Bathinapatla Ayyappa, et.al. Suvardhan Kanchi, Parvesh Singh,
Myalowenkosi I. Sabela, Martin Dovey , Krishna Bisetty. The results obtained
using 30-mM heptakis-(2,3,6-tri-o-methyl betacyclodextrin) as seperating agents.
The detection limits of rebaudioside A and stevioside were 0.02 mM and 0.073
mM respectively. In addition, the molecular docking studies explained to a certain
extent why the seperation was successful. The calculated binding free energy
results for the rebaudioside-A and stevioside complexes formed with the
separating agent showed that although both ligands penetrated deeply into the
hydrophobic cavity of the separating agent, the presence of additional hydrogen
bonding in the case of stevioside is probably responsible for its stronger binding
affinity than that of rebaudioside A.

2.3. Summary of analytical methods for the determination

of Steviol Glycosides
In order to get an overview of analytical methods for determination of
Steviol Glycosides, the information mentioned above have been summarized in
table 2.
Table 2: Determination of Steviol Glycosides by some mentioned methods
Or- Objects of LOD/
Method Condition Linear range Result
der study LOQ
Soft and +MeOH+ LOQ: Recovery
HPLC-ESI- 5 - 1600
1 alcoholic acetone as 0.323-1.36 from 97.0%
MS/MS ng/mL
beverages mobile phase ng/mL to 105.7%

19
 Stevioside: Recovery of
Using ethyl s/n=3 for
15.0-55.0 Stevioside:
acetone – LOD;
2 TLC Sweeteners Rebaudioside 97.4%;
methanol – water s/n =9 for
A : 10.0-50.0 Rebaudioside
as mobile phase LOQ
( l/band) A: 96.8%
LOD for
Stevia leaves SV: 15;
and added to Ra: 50; Recovery:
3 UHPLC-MS 40-180 mg/g
commercial mobile phase Sb: 10; 95-102%
sweeteners ST: 1
(ng/ml)
Phase A: O Stevioside: LOD: 0.1-
Stevia Means
LC- and phase B: 6-10%; 2.8 ng/mL
4 rebaudiana recovery:
ESI/MS/MS as mobile Rebaudioside LOQ: 2.1-
samples 100 2%
phases A: 2-4% 8.9 ng/mL
ACN +
Energy/ Regression
1.0/5.0 ppm to LOQ: 0.5-
5 HPLC-PDA Vitamins buffer; pH 2.7, coefficient:
200/100 ppm 0.7 ppm
drinks phosphoric acid =0.99995
as mobile phase
Phase A:
LOD: 0.1-
deionized water Regression
0.5 ng/mL
6 LC- MS/MS Stevia leaves and phase B: <1% coefficient:
LOQ: 0.5-1
methanol as =0.98
ng/mL
mobile phases
Rebaudioside
Regression
Using fused- A: 2.09-5.63; LOD:
coefficient:
7 CE-MEKC Stevia plants silica capillary Stevioside: 1.95-5.63
=0.9986
tube 1.95-5.15 mg/ml
and 0.9974
(mg/mL)
Fused-silica
LOD: Correlation
capillary tube; 0.01-20.0
8 CE-UV/Vis Sweeteners 0.010 coefficients:
Micro UVis mg/mL
mg/ml r =0.9936
detector

20
 Sweeteners 170 mM Regression
LOD: 0.3
9 CE- and /LiOH (pH 9.0) 10-100 mg/mL coefficient:
mg/L
beverages electrolyte =0.999
Stevioside:
5-8%; Regression
Using fused silica Rebaudioside 0.2-5 coefficient:
10 CE-MEKC Stevia leaves
capillary tube A: 1%; mg/mL =0.995-
Others: 0.997
<0.04%
30-mM heptakis-
(2,3,6-tri-o- Stevioside:
methyl LOD: 0.02- 94.57%;
11 CE-EKC Stevia leaves 2-10%
betacyclodex-trin 0.073 mM Rebaudioside
as seperating A: 99.08%
agent

It is quite clear that Liquid chromatography is usually used for analyze

Steviol Glycosides in food and berverages while CE is usually used for analyze
them in Stevia leaves. CE-EKC has the smallest value of LOD so that this
method is suitable for the analyse if Steviol Glycosides present a very little of
concentration. Otherwise, with small value of LOD, high recoveries, short time
analyse, HPLC is the most effective method for the determination of Steviol
Glycosides, especially in food and drinks.

21
 Conclusions

The analyses of Steviol Glycosides in various samples of differing matrix

have been carried out by a variety of methods, including liquid chromatography,
capillary electrophoresis. It can be clearly seen that High performance liquid
chromatography is the most preferable for the determination of Steviol glycosides.

Like other forms of chromatography, HPLC allows the separation of

chemical constituents through the use of a mobile phase and a stationary phase.
The mobile phase is liquid and the stationary phase is solid. Because the different
components move at different speeds they separate from each other. Compared to
other chromatographic techniques, such as TLC, HPLC is extremely quick and
efficient. It uses a pump, rather than gravity, to force a liquid solvent through a
solid adsorbent material, with different chemical components separating out as
they move at different speeds. It is accurate and highly reproducible, despite its
costly and the requirements for large quantities of expensive organics.

In general, High-performance liquid chromatography is versatile and

extremely precise method when it comes to identifying and quantifying chemical
components with high molecular weights, so that it is suitable for steviol
glycosides.

22
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