Analytical Methodologies For Determination of Artificial Sweetners in Foods - F BUN PDF

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Trends in Analytical Chemistry, Vol. 28, No. 9, 2009
Analytical methodologies for

determination of artificial
sweeteners in foodstuffs
Agata Zygler, Andrzej Wasik, Jacek Namiesnik
Artificial high-intensity sweeteners are used increasingly frequently for food production. The food industry tends to highlight
beneficial aspects of their use (e.g., tooth friendliness, increasing the quality of life of those suffering from different forms of
diabetes and the possibility of weight control without anyone sacrificing their favorite unhealthy drinks or snacks). However,
some consumers are deeply concerned about the safety of artificial sweeteners and claim that the food industry is replacing
natural beet sugar or cane sugar for purely economic reasons.
Most of these food additives have a maximum usable dose or a maximum allowable concentration specified for a given type of
food. In order to assure consumer safety, it is necessary to control the content of sweeteners in foodstuffs. Analytical methods
(including high-performance liquid chromatography, ion chromatography, thin-layer chromatography, gas chromatography,
capillary electrophoresis, flow-injection analysis, electroanalysis and spectroscopy) can determine sweeteners individually and
simultaneously in mixtures. This review focuses on the application of some popular analytical procedures for determination of
artificial sweeteners in food.
2009 Elsevier Ltd. All rights reserved.
Keywords: Artificial sweetener; Capillary electrophoresis; Electroanalysis; Flow-injection analysis; Foodstuff; Gas chromatography (GC); Highperformance liquid chromatography (HPLC); Ion chromatography (IC); Spectroscopy; Thin-layer chromatography (TLC)
1. Introduction
Agata Zygler*,
Andrzej Wasik,
Jacek Namiesnik
Gdansk University of
Technology, Department of
Analytical Chemistry, Chemical
Faculty, ul. G. Narutowicza
11/12, 80-233 Gdansk, Poland
Corresponding author.
Tel.: +485 8347 1833;
E-mail: agatazygler@wp.pl
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Artificial high-intensity sweeteners (also

called non-nutritive sweeteners) form an
important class of food additives, which
are commonly used in the food, beverage,
confectionery and pharmaceutical industries. They provide the sensation of
sweetness, but with little or no intake of
food energy. There are a large number of
known intense sweeteners, but only very
few are allowed to be used in modern food
industry. The list of authorized artificial
sweeteners varies from country to country. For example, there are six artificial
high-intensity sweeteners authorized for
use in European Union (EU) (acesulfameK, aspartame, cyclamic acid and its salts,
saccharin and its salts, sucralose and
neohesperidine dihydrochalcone) [1],
whereas, in the USA, the corresponding
list does not include cyclamates and neohesperidine dihydrochalcone, instead one
can find neotame there [2].
The food industry is heavily promoting
its artificially-sweetened products (fre-
quently called diet or light), highlighting their benefits. Low-calorie or

reduced-calorie food products and beverages can help in treatment of obesity,
maintaining body weight and management of diabetes. Last, but not least, artificial sweeteners are not fermented by the
microflora of the dental plaque, which
makes them tooth-friendly.
Sweeteners may be used separately or in
combination with other sweeteners, as socalled blends. Nowadays, the common
trend in food industry is to use sweetener
blends, because some of the sweeteners
impart side tastes and aftertastes that can
limit their applications in foods and beverages [3]. It was found that mixing such
a problematic sweetener with another
frequently yields a blend not only lacking
unwanted side or aftertastes but also
sweeter than the algebraic sum of the
components. A very well-known example
of such a mixture is saccharin-cyclamate
(1:10) blend. The bitter aftertaste of saccharin is masked by cyclamate and the
unpleasant aftertaste of cyclamate, sensed
0165-9936/$ - see front matter 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2009.06.008
by some people, is masked by saccharin. Simultaneously

(due to synergistic effect), the sweetening power of the
mixture increases. Properly formulated sweetener blends
can precisely reproduce the texture and the sweetness
profile of traditional sugar-containing products, create
new products characterized by an original sweetness
profile and improve taste stability [4].
Artificial high-intensity sweeteners, intensely promoted by the food industry are among the most controversial food additives due to suspicions of adverse
health effects [5]. These allegations include causing
dermatological problems, headaches, mood variations,
behavior changes, respiratory difficulties, seizures,
allergies and cancer.
Many experiments have been performed on the safety
of saccharin. Some associated saccharin with bladder
cancer when fed at high doses to rats. However, results
from subsequent carcinogenicity studies showed no
consistent evidence of association between saccharin
consumption and cancer in test animals. Another suspicion about saccharin is connected with the possibility
of allergic reactions in people who do not tolerate sulfa
drugs [4,6].
In the case of cyclamate, the issue is more complicated
because different people metabolize this sweetener in
different ways [5]. One study conducted in 1966 indicated that cyclamate can be metabolized by some intestinal bacteria resulting in formation of cyclohexylamine,
a compound suspected to have some chronic toxicity in
animals. Another study from 1969 linked cyclamate
consumption with increased risk of bladder cancer in
rats. In 2000, the European Food Safety Agency (EFSA)
published its opinion on safety of cyclamate, stating that
available epidemiological data revealed no indications of
harmful effects on human reproduction parameters of
cyclamate used as a food additive [7].
Aspartame is probably the most controversial artificial
high-intensity sweetener on the market, with adverse
medical effects attributed to it including brain tumors,
multiple sclerosis, systemic lupus, and methanol toxicity,
causing blindness, spasms, shooting pains, seizures,
headaches, depression, anxiety, memory loss, birth defects, leukemia and death. The European Ramazzini
Foundation (ERF) of Oncology and Environmental Sciences published, in 2006 and 2007, results of two
studies [8,9] on aspartame toxicity. Both studies linked
aspartame with cancer, lymphomas and leukemias in
tested rats. In March 2009, the EFSA discounted the
results of these studies and found no indications of any
genotoxic or carcinogenic potential of aspartame [10].
Nevertheless, people with phenylketonuria should eliminate foods containing aspartame, because excess intake
of phenylalanine (one of the aspartames metabolites)
can lead to brain damage.
Safety concerns pertaining to sucralose are mainly
caused by the presence of three chlorine atoms in its
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molecule, which make it an organochloride. Many

organochlorides are toxic or carcinogenic (e.g., pesticides
and dioxins) and this is probably the reason for the
mistrust of sucralose. However, studies in human beings
and animals have shown that this sweetener did not
pose carcinogenic, reproductive or neurological risk to
people [5].
The content of sweeteners in foodstuffs is limited by
country-specific regulations. In the EU, sweeteners are
thoroughly assessed for safety by the EFSA before they
are authorized for use. EU Directives 94/35/EC [11], 9683/EC [12], 2003/115/EC [13], 2006/52/EC [1] define,
which sweeteners have approval to be added to food
products and beverages. Considering medical and legal
aspects, the determination of these artificial sweeteners
has economic and social relevance [14].
Due to consumer safety, it is necessary to control the
content of sweeteners in foodstuffs. To obtain this
information, reliable quantitative methods of analysis
are required to measure levels of sweeteners in a broad
range of food matrices [15]. A number of analytical
methods based on different principles are available for
their determination. The aim of this review is to present
and to compare the available analytical methods for
determination of artificial sweeteners in foodstuffs.
2. Artificial sweeteners
High-intensity sweeteners can be divided into three
categories: synthetic, semi-synthetic and natural. They
comprise a wide variety of organic molecules (e.g., carbohydrate derivatives, salts of organic acids, terpenoids
and even proteins [16]). The majority of sugar substitutes approved for use in food chemistry are artificiallysynthesized compounds, so we do not consider naturally
intense sweeteners in this review. The most popular
artificial sweeteners are: acesulfame-K (ACS-K), aspartame (ASP), cyclamate (CYC), saccharin (SAC), sucralose (SCL), alitame (ALI), neotame (NEO) and
neohesperidine dihydrochalcone (NHDC), which is a
semi-synthetic sweetener. Table 1 shows the chemical
structures and the basic characteristics of aforementioned sweeteners.
3. Sample preparation
Sample preparation is an essential stage in the analytical
process, and food samples are among most difficult
matrices, due to the great variability in their composition
(e.g., preservatives, colors, thickeners, vitamins, proteins, lipids and minerals). All of the components can
interfere with the determination of sweeteners. Sample
preparation procedure must be tailored to the method of
final determination, considering the instrumentation
http://www.elsevier.com/locate/trac
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Table 1. Chemical structures and basic characteristic of artificial sweeteners

Sweetener
Structure
O
O
Full name
O
S
Acesulfame
Potassium
Basic characteristics and applications

Acronym
Molar mass
ACS-K
201.24

N K
H3C

O
-
N Na
Sodium Saccharine
SAC
205.16
S
O

O
NH
Sodium cyclamate
CYC
201.2

NH2
O
H
[5]
[6]
[17]
1,2-benzisothiazol-3(2H)-on-1,1-dioxide.
It is the oldest sweetener on the market.
The sweetening strength is fsac,g(10) = 450. It is commercially
available in three forms (i.e. acid saccharin, sodium
saccharin, and calcium saccharin).
It has high solubility and stability. However, at low pH, it can slowly
hydrolyze to 2-sulfobenzoic acid and 2-sulfoamylobenzoic acid.
It has a bitter metallic after-taste. Despite the controversy over its safety,
saccharin is allowed to be used in food and drink formulations in at
least 90 countries.
[14]
[18]
Sodium salt of cyclohexylaminosulfonic acid (cyclamic acid).

The sweetening strength is fsac,g(10) = 35.
It has a bitter o-taste, but has good sweetness synergy with saccharin.
Cyclamic acid is soluble in water, and its solubility can be increased
by preparing the sodium or calcium salt.
It is stable over a wide range of pH and temperatures.
It is permitted in several countries, but, in the USA, it is banned due to
suspicions of toxicity.
[17]
[18]
(4-ethoxyphenyl)urea.
It was discovered only five years after saccharin, but it never achieved
great recognition or usage due to suspicions of its toxicity. Because of
hydrolysis to aminophenol, it may cause adverse eect during long-term usage.
Compared to saccharin, it does not have a bitter after-taste.
[18]
O Na

Potassium salt of 6-methyl-1,2,3- oxathiazine-4(3H)-one

2,2-dioxide.
Its sweetening strength is afsac,g(3) = 200.
It has a slightly bitter after-taste, especially at high concentrations.
Its water solubility is very good, whereas most synthetic sweeteners
have unsatisfactory water solubility.
It is stable at high cooking and baking temperatures.
It is approved for use in food and beverage products
in approximately 90 countries.
OCH2CH3
Dulcin
DUL
180.2

Ref.
Aspartame
ASP
294.3
O
NH2
NH
COOH
Cl
HO
Cl

HO
Sucralose
SCL
397.63
OH

Cl
OH

CH3
O
HO
[5]
[14]
[17]
4-chloro-4-deoxy-a-D-galactopyranosyl-1,6-dichloro-1,6-dideoxy-b-D-fructofuranoside.
The sweetener is derived from ordinary sugar through a multistep
manufacturing process, in which three of the hydroxyl groups on the
sugar molecule are selectively replaced with three atoms of chlorine.
A sweetening strength is fsac,g(2) = 750.
Due to its, strong sweet taste, exceptional stability under heat and over
a broad range of pH conditions, excellent solubility, and high compatibility
with commonly used food ingredients, it can be added to baked goods and
products that require a longer shelf life.
Since 1991, it has been approved for use as food additive in more than
60 countries, and recently in January 2005 in the EU.
[9]
[14]
[19]
[20]
It is composed of the amino acids L-aspartic acid and D-alanine

with a novel amid moiety (formed from 2,2,4,4-tetramethylthienanylamine).
It has a clean sweetness with no after-taste.
Like aspartame, it has a caloric value, but due to its intense sweetness,
amounts used are small enough for it to be classified as nonnutritive sweetener.
It is relatively stable to hydrolysis and to heat, because of its unique amide group.
It has been approved in some countries, for example Australia, Mexico,
New Zeeland, and China, but not in the USA or EU.
[6]
[14]
[18]
OH
O
NH
O
N-L-a -aspartyl-L-phenylalanine-1-methyl ester.

It is white, odorless, crystalline powder.
It has a caloric value of 17 kilojoules per gram like any other protein
substance, however, because its high sweetness, the amounts used
are small enough to be classified as nonnutritive sweetener.
Although it is relatively stable in its dry form, the compound undergoes
pH- and temperature- dependent degradation in solution, what makes
aspartame undesirable as a baking sweetening agent. Below pH 3 aspartame
is unstable and hydrolyzes to produce aspartylphenylalanine and above
pH 6, it changes to form 5-benzyl-3,6-dioxo-2-piperazineacetic acid.
It is permitted in more than 90 countries (the UE, the USA, Canada,
South America, Australia, Japan, etc.) for use in numerous foodstus.
NH2
NH
OH C
3
H3C
CH3
CH3
S
CH3
Alitame
ALI
331.43

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Table 1. (continued )
Sweetener
Basic characteristics and applications
Structure
Full name
OH
HO
Neohesperidine
dihydrochalcone
OH
Me
Molar mass
NHDC
612.57
HO
OH
HO
OH
HO
OMe

OH
Neotame
CH3
H3C
Acronym
NEO
378.46
NH
O
NH
HO
CH3

fsac,g(x) = Sweetness potency relative to x% sucrose solution on a weight basis.
It is a semi-synthetic sweetening agent prepared

from neohesperidine or narigin, two flavanones extracted
from citrus peel.
It has lingering menthol-liquorice after-taste.
In aqueous solutions, it has a good stability in
the pH range 2.53.5.
It has been approved for many applications in the EU,
but in the USA only for flavoring food products.
[14]
[18]
N-[N-(3,3-dimethylbutyl)-L-a-aspartyl]-L-phenylalanine
1-methyl ester, is a derivative of dipeptide, which is
made from the amino acids aspartic acid and phenylalanine.
It has a very clean sweet taste, close to sucrose, with no
undesirable bitter or metallic o-taste which occurs in
other well-known artificial sweeteners.
It has extensive shelf life in dry conditions. In aqueous
solutions, it is approximately as stable as aspartame in the
acidic pH range, but it is significantly more stable in
the neutral pH range.
It has been approved in the USA, Australia, and
New Zealand.
[5]
[20]
H3C
Ref.
used and the degree of accuracy required, whether

quantitative or qualitative.
3.1. Challenges in preparing food samples
The chemical and the physical properties of foods are
inherently variable. The variability in composition of a
given food sample can be minimized with proper sample
preparation. It is essential for the analyst to recognize the
need to utilize methods that satisfy analytical requirements.
Generally, preparation of food samples comprises
homogenization, extraction, clean up and concentration.
Analysis of liquid samples has an advantage over
analysis of solid samples that one fewer pretreatment
step is usually required, because of their liquid state.
An inherent difficulty in the preparation of food
samples is the complexity of the matrix. Some matrix
components may co-extract with analytes due to similar
solubility in the extraction solvent. The presence of
matrix interferences in a sample extract can contribute
to a multitude of problems (e.g., turbidity, generation of
emulsions and, most importantly, masking the analytical signal of the compound of interest). Such effects
usually lead to an increase in the limit of detection (LOD)
of the method. Usually, clean up of the extract resolves
this problem. Passing the extract through a column with
appropriate stationary phase, dialysis, liquid-liquid
extraction, precipitation and filtration are most commonly employed for this purpose.
The challenge for the analyst is to maximize recovery
of the analytes and to minimize the amount of interfering compounds by using appropriate extraction and
clean-up procedures. Optimal sample preparation can
reduce analysis time, eliminate sources of error, enhance
sensitivity and enable unequivocal identification, confirmation and quantification of analytes.
3.1.1. Sample pretreatment prior to the final analysis
Sweeteners are commonly used in various types of
foodstuffs (e.g., soft drinks, fruit beverages, fermented
milk drinks, cordials, instant powdered drinks, candies,
chewing gum, solid and liquid sweeteners, jams, pickles,
canned fruits, dried fruits, various sauces, dehydrated
soups, jellies, bakery products, dairy products, confectionery, and chocolates). The diversity of products and
the variability of matrices from which sweeteners are
assayed present a great challenge to analytical chemists.
The aim of their work is to come up with a compromise
between sufficient sample preparation and achieving
reliable results by using different types of analytical
techniques. Sample preparation depends on the type of
food matrix. Some samples can be analyzed directly or
after minimal pretreatment. However, extraction, clean
up and/or purification might be necessary, depending on
the complexity of the sample, in order to eliminate
interferences [21].
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There are very few known methods requiring no

sample preparation at all. These include electrochemical
and Fourier transform infrared (FTIR) spectrometrybased methods [121,122,135]. The biggest advantages
of direct measurement are speed and elimination of
physico-chemical sample manipulation. Nevertheless,
complicated and sample-specific calibration of FTIRbased methods limits their application to analysis of
samples of well-defined matrices. However, electrochemical techniques suffer from sensor instability and a
need for frequent recalibration.
Samples characterized by relatively simple matrix (i.e.
table-top solid and liquid sweeteners, beverages, powdered drinks, syrups and juices) can simply be diluted or
dissolved in deionized water, an appropriate buffer or a
mixture of a buffer with methanol or ethanol. In the case
of carbonated drinks, the samples have to be degassed
(e.g., by sonication, or sparging with nitrogen or under
vacuum) prior to analysis. Optionally, sample solutions
can be clarified using a clarifying agent (e.g., Carrez reagent, ZnSO4/NaOH or similar). Clarifying agents are
useful for removal (by occlusion) of proteins, suspended
particulate matter and fatty material. Clarified or not,
samples are filtered before final determination. Single step
filtration with a membrane filter is sufficient in most
cases, although some extracts need centrifugation or
prefiltration with filter paper. This minimal sample preparation procedure is included almost universally in published methods. It is quick, cheap and simple. However,
no chemical interferences with good water solubility can
be removed this way or any preconcentration of analytes
achieved. Nevertheless, such a procedure is sufficient for
many samples and techniques of final determination.
More sophisticated sample-preparation protocols
involve the use of the solid-phase extraction (SPE)
technique, which is a powerful tool. It allows for
fractionation of sample components based on the affinity
of a compound or group of compounds to the stationary
phase. Basically, SPE can be treated as a low-resolution
liquid chromatography (LC). Among the huge number of
different types of SPE cartridges available on the market,
those employing different types of a non-polar C18
packing material seem to be the most frequently used.
Other choices include dextran, polystyrene [43] and
other polymer-based fillings [53,86].
Usually, an SPE procedure comprises the following
operations: cartridge conditioning, sample load, cartridge wash and elution of analytes. In the most common
mode of SPE, an aliquot of the sample extract (e.g., obtained according to the minimal sample preparation
procedure described above) is loaded onto a previously
conditioned SPE cartridge. The type of SPE packing
material, solvents, pH and the flow rates need to be
properly selected in order to retain analytes transiently
within the cartridge. The interfering substances should
be retained very strongly or not retained at all. As a
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result, weakly retained substances are readily removed

from the cartridge during sample load and/or cartridge
wash, analytes are eluted during the elution step and
substances having a strong affinity to the sorbent stay
adsorbed within the cartridge. The sensitivity of a final
determination can easily be enhanced by evaporating
the final SPE extract to dryness and reconstituting it with
a smaller amount of a solvent of choice.
Sometimes, SPE cartridges filled with polar packing
materials (e.g., alumina) are used for decoloring samples
or removing carboxymethyl cellulose (CMC, a thickening
agent) [88]. Extracts are loaded onto preconditioned
cartridges that adsorb colorants and/or CMC while
analytes pass unretained.
Dialysis coupled with SPE was also proposed as a
sample preparation technique for the determination of
sucralose [40,41], aspartame, neotame and alitame [53].
SPE-based sample-preparation protocols seem to be the
best available choice. They are simple, reproducible,
reasonably quick and inexpensive. They are universal
and compatible with the most popular techniques used in
food analysis. Other sample preparation procedures are
possible, but usually they are highly specific to the analyte
under study, the sample or the technique of final determination. In such cases, details on sample preparation
can be found in the respective references given later.
4. Analytical methodology
A great variety of methods based on different principles
have been applied to the analysis of the aforementioned
compounds in food, drinks and dietary products. Most of

the methods have been developed for individual sweeteners. As can be seen in Fig. 1, commonly used sweeteners, with decades of history of usage (i.e. aspartame,
saccharin, cyclamate and acesulfame-K), can be determined by all current analytical techniques. Among other,
recently introduced or approved sweeteners, sucralose
receives the most attention, while a much smaller number of papers deals with the determination of neohesperidine dihydrochalcone, alitame and neotame.
Since there are tens of possible sweetener combinations, there is a need to develop analytical methods
capable of determining several sweeteners in one run. So
far, the majority of the published multi-sweetener
methods have focused on the determination of just a few
(34) compounds. Recently, however, some progress can
be observed with the publication of methods covering
much wider range of artificial sweeteners [15,22,23].
Table 2 provides references to recently published multianalyte methods for the determination of artificial
sweeteners in different food matrices. The reported procedures are grouped according to analytical technique.
Of the great variety of methods used for the determination of sweeteners in different food matrices, chromatographic methods have received wide recognition.
Currently, the most popular technique in this field is
high-performance LC (HPLC). The broad range of available separation columns (mechanisms) together with the
impressive portfolio of detectors makes HPLC a truly
universal technique. Reversed-phase LC (RP-LC) is a
well-known, mature technique, perfectly suited for the
separation of sweeteners.
Figure 1. The use of analytical techniques to analyze artificial sweeteners. CE, Capillary electrophoresis; ET, Electroanalytical techniques; FIA,
Flow-injection analysis; GC, Gas chromatography; HPLC, High-performance liquid chromatography; IC, Ion chromatography; TLC, Thin-layer
chromatography; ST, Spectroscopic techniques.
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Analyte
Sample
Technique
Mobile phase/Electrolyte
Column/Capillary
Analytical parametersb,c
Ref.
ASP, SAC
Dietary products
HPLC-UV
0.08 M TEA-phosphate buffer

(pH3)-MeOH-THF
Hypersil C-18 (10 lm,

250 4 mm)
Recovery 9597%
LOD not available
RSD 0.861.25%
[49]
SAC, ACS-K, Benzoic

acid, Sorbic acid
Beverages, jams
HPLC-UV
MeOH-phosphate buffer (pH 6.7)
Spherosorb ODS-1 C18

(5 lm, 250 4.6 mm)
Recovery 98.1104.2%
LOD < 0.1 mg/100 ml
[50]
ACS-K,ASP SAC,
benzoic acid, sorbic
acid, Ponceau 4R,
Sunset Yellow,
Tartrazine
Soft drinks
HPLC-UV
MeOH-phosphate buffer (pH 4)
LiChrosorb C18 (10 lm,

250 4.6 mm)
Recovery 98.6102.3%
LOD 0.13 mg/L
RSD not available
[52]
ACS-K,ASP SAC,
Vanillin, Sorbic acid
Benzoic acid
Cola drinks, instantpowder drinks
HPLC-UV
ACN-ammonium acetate buffer

(pH 4)
YMC-ODS Pack AM (5 lm,

250 4 mm)
Recovery 99101%
LOD 0.23.1 lg/g
RSD 1.02.2%
[51]
ASP, ALI, NEO
Various foods
HPLC-UV
ACN-phosphate buffer (pH 4)
Cosmosil 5C18-AR
Recovery 89104%
LOD 1lg/g
RSD not available
[53]
ACS-K, SAC, CYC, ASP,

SCL, DUL, ALI, NEO,
NHDC
Non-carbonated soft
drinks, canned
or bottled fruits and
yoghurts
HPLC-ELSD
TEA formate buffer- MeOHACTN
Zorbax Extend C18,

Purospher Star RP-18,
Nucleodur C18 Pyramid,
Nucleodur C8 Gravity;
(5 lm, 250 3 mm)
Recovery 93109%
LOD 15 lg/g
RSD 0.94.5%
[15]

SCL, DUL, GAd, STVe,
REBf
Various foods
HPLC-ESI-MS
Dibutylammonium acetate
buffer- ACN-H2O
Zorbax Eclipse XDO-C18

(150 2.1 mm)
Recovery 75.7109.2%
LOD 15 lg/g
SD 510.9%
[22]

SCL, ALI, NEO, STV
Beverages, canned fruits,

cakes
HPLC-ESI-MS
TEA formate buffer- MeOHACTN
Spherogel C18 column

(Johnson Inc., Dalian, China)
(5 lm, 250 4.5 mm)
Recovery 95.4 104.3%

LOD < 10 lg/mL
RSD not available
[23]
SAC, ACS-K, DUL,

preservatives,
antioxidants
Sugared fruits, soy

sauces, dried roast beef
Ion-paired LCUV
ACN-aqueous ahydroxyisobutyric acid solution

containing
hexadecyltrimethylammonium
bromide
Shoko stainless-steel 5C18

(5 lm, 250 4.6 mm)
Recovery 81.9103.27%
LOD 0.153 lg/g
RSD 0.35.69%
[54]
Table 2. Analytical procedures for simultaneous determination of artificial sweeteners mixtures in samples of different food products
(continued on next page)
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Table 2. (continued )
Column/Capillary
Ref.
Technique

citric acid
Drinks, powdered tabletop sweeteners
HPIC-UV-ELCD
Na2CO3
Dionex Ion Pac AS4A-SC

(254 4 mm)
Recovery 93107%
LOD 0.0190.044 mg/L
RSD 0.841.38%
[62]
ACS-K, SAC, ASP,

benzoic acid, sorbic
acid, caffeine,
theobromine,
theophyline
Drinks, juices, fermented

milk drinks,
preserved fruits, tablets
HPIC-UV
NaH2PO4 (pH 8.20)-ACN
Shim-pack IC-A3 (5 lm,

150 4.6 mm)
Recovery 85104%
LOD 430 mg/L
RSD 15%
[64]
ACS-K, SAC, CYC, ASP
Carbonated cola drinks,

fruit-juice drinks,
preserved fruits
HPICsuppressed
conductivity
detector
KOH
Dionex Ionpac AS11 (250x2

mm)
Recovery 97.96105.42%
LOD 0.0190.89 mg/L
RSD not available
[63]
ACS-K, SAC, ASP, DUL,

ALI, caffeine, benzoic
acid, sorbic acid
Low-energy soft drinks,

cordials, tomato sauce,
marmalades, jams, tabletop sweeteners
MEKC-UV
Buffer consisting of Na
deoxycholate, Kdihydrogenorthophosphate, Na
borate (pH 8.6)
Uncoated fused-silica
capillary (75 cm 75 lm)
Recovery 104112%
LOD not available
RSD 0.632.6%
[90]
ACS-K, SAC, ASP,

preservatives,
antioxidants
Cola beverages, lowenergy jams
MEKC-UV
Borate buffer with Na cholate,

dodecyl sulfate, MeOH (pH 9.3)
Fused-silica capillary (52 cm

75 lm)
LOD not available
RSD 0.91.5%
[91]
ACS-K, SAC, ASP,

preservatives, colors
Soft drinks
MEKC-UV
Carbonate buffer (pH 9.5) with

sodium dodecyl sulfate
Uncoated fused-silica
capillary (48.5 cm 50 lm)
Recovery
LOD 0.005 mg/mL
RSD not available
[92]
ACS-K, SAC, ASP, MALg,

SORh, XYLi, LACj
Candies, chewing gums
CITPconductivity
detector
Leading electrolyte: HCl + Tris

Terminating electrolyte: Lhistidyne + Tris
Dionex Ion Pac AS4A-SC

(254 4 mm)
Recovery 98.2102.5%
LOD 0.0240.081 mM
RSD 0.82.8%
[93]
Sample
b. LOD, Limit of detection.

c. RSD, Relative standard deviation.
d. GA, Glycyrrhizic acid.
e. STV, Stevioside.
f. REB, Rebaudioside.
g. MAL, Mannitol.
h. SOR, Sorbitol.
i. XYL; Xylitol.
j. LAC, Lactitol.
Mobile phase/Electrolyte
Analytical parametersb,c
Analyte
Besides HPLC, other separation methods have also

found applications in this area. Ion chromatography (IC)
is a suitable tool for multi-sweetener analysis in food
products. Instead of organic solvent-mediated separations, IC separations are performed by using innocuous
and inexpensive salt solutions as the eluents.
Capillary electrophoresis (CE) is another attractive
separation technique, useful for simultaneous determination of multiple sweeteners. In some situations, CE
seems to be superior to HPLC in terms of separation
power, analysis time or low solvent consumption.
When there is a need to determine just one or two
sweeteners in a given sample, it is often more reasonable
to apply one of the simple, rapid, reproducible analytical
methods, specifically tailored to the particular product.
So far, such rapid methods have been published for
determination of acesulfame-K, saccharin, cyclamates
and aspartame. They utilize specific reactions or detection systems rather then employing powerful but costly,
universal separation-detection combos. Compared to
multi-analyte counterparts, these procedures are often
less time consuming and less labor intensive, while being
comparable in terms of accuracy, precision and sensitivity. Flow-injection analysis (FIA), electrochemical and
spectroscopic methods provide ways to obtain analytical
results in minimum time. Table 3 sets out examples of
this kind of approach.
4.1. Techniques for final determination of analytes
This section covers nine techniques of final determination of sweeteners. We first discuss papers dealing with
the determination of a single sweetener, then multianalyte methods, if applicable.
4.1.1. High-performance liquid chromatography
LC is the most popular choice for determination of highintensity sweeteners. HPLC procedures are based on
isocratic or gradient RP chromatographic separation.
Various detection systems in conjunction with LC are
applied [e.g., ultraviolet (UV) spectrophotometry,
amperometry, coulometry, mass spectrometry (MS),
spectrofluorometry, light scattering and conductometry].
4.1.1.1. Methods for determination of individual sweeteners. Many HPLC methods have been reported for
quantitation of aspartame. RP-HPLC with spectrophotometric detection (205 nm, 214 nm and 215 nm) and
with the mobile phase containing phosphate buffer and
acetonitrile and/or methanol is the primary tool for the
determination of aspartame [2426]. Most of them were
developed on 250-mm columns, but aspartame and its
metabolites can be separated using short 30-mm
columns with UV detection. Since the column is shorter,
analysis time is reduced to less then 10 min but the
selectivity is worse than methods employing longer 250mm columns [27].
Trends
It is well known that spectrofluorometric detection

achieves better selectivity and sensitivity than spectrophotometric detection. Aspartame and its hydrolysis
products can be determined spectrofluorometrically
using pre-column derivatization. Aspartylphenylalanine,
aspartic acid and phenylalanine react with naphthalene2,3-dicarboxaldehyde in the presence of cyanide ion in a
mildly alkaline medium to give highly fluorescent 1-cyano-2-substituted benz[f]isoindole derivatives. LODs in
the sub-picomole range were reported for these compounds using HPLC with spectrofluorometric detection
(kex = 420 nm, kem = 490 nm) [28]. A similar method
with pre-column derivatization using fluorescamine was
also reported [29]. The main drawback of these methods
is the necessity of a lengthy derivatization procedure.
Native fluorescence of aspartame can be used for its
detection in HPLC.
Aspartame and phenylalanine produce native fluorescence emission with the maximum at 284 nm. An
excitation wavelength of 205 nm was found to give the
best signal-to-noise ratio. This method does not require
derivatization. Moreover, use of spectrofluorometric
detection yields much better sensitivity and slightly
better precision than spectrophotometric detection [30].
Aspartame was also determined by HPLC with electrochemical detection. Aspartame, normally electrochemically inactive, was made oxidizable by postcolumn photolytic derivatization (irradiation at
254 nm). The use of a post-column photochemical
reactor can be helpful for unequivocal identification of
aspartame using electrochemical detection [31].
The common HPLC-UV detection mode is not suitable
for determination of cyclamate, because of the lack of UV
chromophore in its molecule. Some complicated and
time-consuming procedures are necessary for the
absorbance detection of this sweetener in HPLC. This
problem has been solved by using indirect UV photometry, post-column ion-pair extraction, and pre-column
derivatization. One of the first approaches employed
post-column ion-pair extraction with absorbance detection for the LC determination of cyclamate. After chromatographic analysis the sweetener was mixed with an
appropriate dye (methyl violet or crystal violet) and detected by absorption in the visible range. In this method,
the eluted sweetener is mixed with an appropriate dye
(methyl violet or crystal violet) being detected by
absorption in the visible range [32].
Another HPLC method with UV detection at 314 nm
utilized the conversion of cyclamic acid to N,Ndichlorocyclohexylamine [33]. Cyclamate can be determined in strongly colored and protein-rich foodstuffs
after its oxidation to cyclohexylamine and derivatization
with 4-fluoro-7-nitrobenzofurane. There are two possible
ways of detection: absorbance at 485 nm and fluorescence with excitation at 485 nm; and, emission at
530 nm [34].
1091
Trends
Table 3. Simple and rapid methods for determination of artificial sweeteners in food products
Analyte
Analytical technique
Sample
Analytical parametersa,b,c,d
Ref.
ASP
FIA with spectrophotometric

detection
Table-top sweeteners
Recovery n.a.
LOD 2 lg/mL
RSD 0.2%
LR n.a.
[95]
ASP

detection
Chewing gums, custards,

sweets, jams
Recovery 95101%
LOD 0.82 lg/mL
RSD 0.55%
LR 5.0600 lg/mL
[96]
ASP, ACS-K

detection
Recovery n.a.
LODASP 5.6 lg/mL
RSDASP 3.4%
LRASP 10.0100.0 lg/mL
Recovery n.a.
LODACS-K 0.1 lg/mL
RSDASP 1.61%
LRASP 40.0100.0 lg/mL
[111]
ASP, CYC
Square-wave voltammetry in
conjunction with boron
doped diamond electrode
Powdered juice drinks,

carbonated guarana
drinks
Recovery n.a.
LODASP 4.7 10 7mol/L
LODCYC 4.2 10 6mol/L
RSD 1.11.3%
LRASP 5.0 10 64.0 10
mol/L
LRCYC 5.0 10 54.0 10
mol/L
[124]
ASP
Square-wave voltammetry in
conjunction with borondoped diamond electrode
Dietary products
Recovery n.a.
LOD 2.3 10 7 mol/L
RSD 0.2%
LR 9.9 10 6 5.2 10
[123]
mol/L
ASP
Biosensor based on carboxyl

esterase-alcohol oxidaze
Soft drinks, Table-top

sweeteners
LOD n.a.
RSD n.a.
LR 5.0 10 84.0 10 7 M
[121]
ASP
Biosensor based on graphite

epoxy composite electrode
Diet cokes
Recovery 101.84104.08%
LOD n.a.
RSD n.a.
LR 2.5400 lM
[122]
ASP, ACS-K
FTIR
Recovery n.a.
LODASP 560 lg/g; LODACS-K 7
200 lg/g
RSD 0.170.5%
LR n.a.
[136]
ACS-K, SAC
Spectrophotometry/ in
conjunction with
chemometric analysis
Solid and liquid

sweeteners, fruit juices
Recovery 93.8105.1%
LODACS-K 0.085 lg/mL; LODSAC
0.0312 lg/mL
RSD n.a.
LR n.a.
[133]
ACS-K, CYC, SAC
Spectrophotometry in
conjunction with
chemometric analysis
Beverages, apple
vinegars, yoghurts, milk
drinks
Recovery 96.7106.0%
LODACS-K 0.08 lg/mL; LODSAC
0.2 lg/mL
LODCYC 0.019 lg/mL
RSD 14%
LRACS-K 0.24.8 lg/mL; LRCYC
0.510.0 lg/mL
LRSAC 0.85.6 lg/mL
[134]
1092
Trends
Table 3. (continued)
Analyte
Analytical technique
Sample
Analytical parametersa,b,c,d
Ref.
CYC

detection
Recovery n.a.
LOD 7.7 lmol/L
RSD 3.5%
LR < 1000 lmol/L
[106]
CYC

detection
Recovery n.a.
LOD 30 lmol/L
RSD 1.7%
LR 1003000 lmol/L
[105]
CYC
FIA with AAS detection
Soft drinks
Recovery 95.7100.5%
LOD 0.25 lg/mL
RSD 3.1%
LR 190 lg/mL
[108]
CYC, SAC
FT-Raman spectrometry in
conjunction with chemometric
analysis
Recovery n.a.
LODCYC 0.83% w/w; LODSAC
0.2% w/w
RSD 0.55%
LR n.a.
[135]
SAC
FIA with AAS detection
Recovery n.a.
LOD 3 lg/mL
RSD 2.7%
LR 575 lg/mL
[101]
SAC

detection
Soft drinks, juices, bakery products,

Recovery 98104%
LOD 0.2 lg/mL
RSD 0.78%
LR 1.0200 lg/mL
[102]
SAC
Potentiometric titration using a

silver electrode as the indicator
electrode
Solid and liquid sweeteners
Recovery 97.0102.6%
LOD 2.5 lg/mL
RSD n.a.
LR n.a.
[114]
SAC
Potentiometric membrane sensor
Dietary products
Recovery 98.2103.1%
LOD n.a.
RSD n.a.
LR 1.0 10 1 5.0 10
[117]
mol/L
SAC
Potentiometric sensor
Pt|Hg|Hg2(Sac)2|Graphite
Diet soft drinks, jams
Recovery 97.6102.0%
LOD 3.9 10 7 mol/L
RSD 1.82.3%
LR 5.0 10 71.0 10 2mol/L
[118]
SAC
Spectrophotometry
Solid and liquid sweeteners
Recovery 99.2104.3%
LOD 1.55 10 5 M
RSD 0.51.6%
LR n.a.
[128]
a
b
c
d
LOD, Limit of detection.

RSD, Relative standard deviation.
LR, Linear range.
n.a., Not available.
1093
Trends
A similar method was proposed for analysis of this

sweetener in juices and preserves. Cyclamate was oxidized to cyclohexylamine and pre-chromatographically
converted into a fluorescent derivative [35].
Indirect UV photometry has been shown to be a very
sensitive detection method useful for detection of this
sweetener [36].
Cyclamate was determined in soft drinks using RPHPLC combined with indirect visible photometry at
433 nm. The analytical signal was derived from changes
in absorbance of mobile phase with addition of chromogenic dye (Methyl Red) [37].
An HPLC-electrospray ionization (ESI)-MS method
using tris(hydroxymethyl) aminomethane as an ionpair-forming agent was employed for analysis of cyclamate in foods. Cyclamate was separated on a C8 column
in isocratic mode with 100% aqueous mobile phase. MS
was operated in negative, selected ion (m/z = 178)
recording mode. The method was found to be highly
sensitive, specific and simple [38].
An HPLC-tandem MS (HPLC-MS2) method characterized by minimal sample preparation, high sensitivity and
selectivity was developed for the determination of
cyclamate in foods. Under negative ESI conditions,
parent ions of m/z = 177.9 and product ions of m/z =
79.9 were collected and used for quantitation [39].
Since sucralose does not absorb in the usable UV/visible (UV-Vis) range, making sensitive and specific
detection by direct UV absorption difficult, a derivatization procedure is necessary. This task can be accomplished using p-nitrobenzoyl chloride (PNBCl). Sucralose
treated with PNBCl is converted into a strongly UVabsorbing derivative, having strong absorption at
260 nm, which allows for its sensitive, direct UV detection [40]. Another solution is to use refractive index (RI)
[41] or MS detector.
A sensitive LC-MS2 method has been reported for the
determination of sucralose in various foods (soft drinks,
yoghurt, chocolate and chewing gum). Sample extracts
were separated using C18 column in gradient-elution
mode. MS acquisition was done in negative selected
reaction monitoring (SRM) mode, monitoring the
395359 transition [42].
The HPLC-UV method seems to be the most popular
option for the determination of neohesperidine dihydrochalcone (NHDC). NHDC can be analyzed using a
standard C18 column in gradient mode. The optimal
analytical wavelength is 282 nm, since common food
components (e.g., sweeteners and preservatives) are
transparent or absorb very slightly at this wavelength
[4347].
4.1.1.2. Multi-analyte approaches. Despite the high
resolving power of HPLC as a separation tool, HPLCbased methods for the determination multiple sweeteners
are quite rare.
1094
Separation and quantitation of seven artificial sweeteners (aspartame, saccharin, cyclamate, acesulfame-K,
sucralose, alitame and dulcin) can be accomplished
using a C18 column working in gradient (organic solvent and pH) mode. Aspartame, acesulfame-K, saccharin, alitame and dulcin were detected using UV
absorbance at 210 nm or 200 nm. Cyclamate and
sucralose were not detected by UV absorption, but
determined by an ion-pair extraction system and RI
detection, respectively [48].
An HPLC-UV procedure that can simultaneously
determine aspartame and saccharin was found suitable
for reliable control of pharmaceutical and dietary formulations [49].
Simultaneous determination of acesulfame-K, saccharin and preservatives in beverages and jams can also
be accomplished using an HPLC-UV technique [50].
Three artificial sweeteners (acesulfame-K, aspartame
and saccharin), vanillin, benzoic and sorbic acids were
separated and quantified using a C18 column working in
isocratic mode. A diode-array detector (DAD) was used
and different analytical wavelengths were selected for
each analyte (230 nm for acesulfame-K, 220 nm for
sodium saccharin, 203 nm for a-aspartame, 280 nm for
vanillin, 225 nm for benzoic acid and 256 nm for sorbic
acid). The method was found useful to monitor the
content of sweeteners and preservatives in cola and instant-powder drinks [51].
Another HPLC-UV method was proposed for determination of the aforementioned sweeteners, preservatives and dyes present in soft drinks [52]. Good separation was obtained with a run time less than 20 min, with
a satisfactory precision. Neotame, alitame and aspartame were quantified in various foods with the aid of
HPLC-UV. Furthermore, these three sweeteners were
successfully identified by ultra-HPLC (UHPLC)-MS2.
Positive mode ESI was used, fragmentation spectra of
ions 379, 332 and 295 (m/z) were recorded for identification of neotame, alitame and aspartame, respectively
[53].
Ion-pair RP-LC with UV detection (233 nm) was employed for simultaneous determination of three sweeteners
(acesulfame-K,
saccharin
and
dulcin),
preservatives and antioxidants in food samples. The
analytes were separated using a C18 column and a
mobile phase comprising an acetonitrile - aqueous ahydroxyisobutyric acid solution with the addition of
hexadecyltrimethylammonium bromide as an ion-pairforming agent [54].
HPLC with evaporative light-scattering detection
(ELSD) was used for determination of nine sweeteners
(acesulfame-K, alitame, aspartame, cyclamic acid, dulcin, neotame, neohesperidine dihydrochalcone, saccharin and sucralose) in various food products.
Interlaboratory study showed the applicability of the
method in use by control laboratories [15,55].
Six artificial sweeteners (acesulfame K, saccharin,

sucralose, cyclamate, aspartame, dulcin) and three
natural sweeteners (glycyrrhizic acid, stevioside, rebaudioside A) were quantified in different foods by HPLC-MS.
Under negative ESI conditions, quantitation was
achieved by monitoring the selected ions for each
sweetener [22].
Recently, an HPLC-MS method was published for the
determination of seven artificial sweeteners (aspartame,
saccharin, acesulfame-K, neotame, sucralose, cyclamate
and alitame) and one natural sweetener (stevioside).
Unlike many other methods, this one does not include
any clean-up step. Food samples were extracted with
methanol-water mixture and, after filtration, injected
into the HPLC-MS system. The target compounds were
quantified using selective ionization recording (SIR) at
m/z 178, 397, 377, 293, 641, 312, 162 and 182 to
cyclamate, sucralose, neotame, aspartame, stevioside,
alitame, acesulfame-K and saccharin, respectively, with
warfarin sodium (m/z = 307) being used as an internal
standard [23].
4.1.2. Ion chromatography
High-performance IC (HPIC) offers an attractive alternative to the aforementioned HPLC methods. There are
few reported methods for the determination of individual
sweeteners.
4.1.2.1. Methods for determination of individual sweeteners. Acesulfame-K was determined in various foodstuffs
by anion-exchange chromatography coupled to a conductivity detector. The separation was achieved by using
a Dionex AS5 anion-separation column and sodium
carbonate as a mobile phase [56]. Quantitation of
sodium saccharin in shrimps has also utilized highperformance anion-exchange chromatography (HPAEC)
[57].
An IC method with integrated amperometric detection
was proposed for determination of aspartame in drink
and powder forms. A Dionex AS4A-SC separation
column was applied to separate this sweetener from
other food additives. Moreover, commonly added
sweeteners (e.g., sodium saccharin, acesulfame-K and
cyclamate) give no electrochemical signal at all, and
could be eliminated as interference [58].
A few procedures for determination of sucralose in
various food and beverage products were reported. In all
cases, HPAEC with pulsed amperometric detection (PAD)
was applied. The high resolving power of HPAEC and the
specificity of PAD allow the determination of sucralose
with little interference from other ingredients. High
precision, method ruggedness, and high spike recovery
are possible for these complex sample matrices [5961].
4.1.2.2. Multi-analyte approaches. There are few reported methods using IC with different detection modes
Trends
for simultaneous determination of multiple sweeteners.

Four artificial sweeteners (acesulfame-K, aspartame,
saccharin and cyclamate) and citric acid were separated
by AEC and quantitated using UV detection in combination with conductivity detection [62].
A suppressed conductivity detector alone was found
sensitive enough for simultaneous determination of four
sweeteners (aspartame, acesulfame-K, sodium saccharin
and sodium cyclamate) in foods by IC when KOH eluent
generator was used [63]. Good agreement of results
obtained by AEC with those provided by HPLC was observed in the case of simultaneous determination of three
artificial sweeteners (saccharin, acesulfame-K and
aspartame), two preservatives, caffeine, theophylline and
theobromine in foods. Analytes were separated in isocratic mode and detected by UV detector [64].
4.1.3. Thin-layer chromatography
Thin-layer chromatography (TLC) has been used for
identification and/or determination of artificial sweeteners, because the equipment needed is simple, inexpensive and flexible.
4.1.3.1. Methods for determination of individual sweeteners. The content of saccharin in soft drinks can be
measured using TLC and UV spectrophotometry. After
chromatographic separation on a silica gel GF254 plate,
the saccharin band is scraped off, extracted and, finally,
absorbance is measured at 235 nm and 244 nm [65].
A simple, fast TLC method was proposed for the
quantification of sucralose in various food matrices. The
method requires little or no sample preparation to isolate
or to concentrate the analyte. The separation of sucralose
was performed on amino-bonded silica-gel HPTLC-plate.
The use of the DAD resulted in excellent LODs [66].
4.1.3.2. Multi-analyte approaches. Saccharin, cyclamate
and dulcin can be extracted from foods with ethyl acetate and separated and quantified by TLC analysis using
polyamide plates [67]. The same combination of sweeteners was determined in soft drinks using silica-gel H
plates [68]. Polyamide plates were also employed for
identification of acesulfame-K, saccharin and cyclamate
in foodstuffs and cosmetics. Analytes were separated
using a mixture of xylene, n-propanol and formic acid
[69]. Aspartame, acesulfame-K cyclamate were
determined in sparkling and non-sparkling drinks using
silica-gel G plates and an ethanol-isopropanol-aqueous
ammonia system [70].
4.1.4. Gas chromatography
As an analytical tool, gas chromatography (GC) is not
used very often for determination of sweeteners because
of the low volatility of these compounds. Before analysis,
sweeteners must be converted into volatile compounds,
and that is main drawback of using GC for this purpose.
1095
Trends
The derivatization step is not only labor-intensive and

time-consuming but also a possible source of erratic
results.
4.1.4.1. Methods for determination of individual
sweeteners. Several methods were developed for
GC determination of saccharin. Esterification with
N,O-bis(trimethylsilyl)acetamide [71], N-methylation
using diazomethane [72] and methylation with trimethylsilyldiazomethane [73] are commonly used techniques.
Various GC methods for the determination of cyclamate have been developed. This sweetener can be
determined in soft drinks by GC detection of the cyclohexene produced during pre-column derivatization with
nitrous acid [74]. However, it was noted that formation
of other products (e.g., monochlorocyclohexane, cyclohexanone and cyclohexanol) can occur and hinder the
analysis. A modified method employed acidification of
the sample with sulfuric acid instead of hydrochloric
acid, prior to reaction with sodium nitrite. This precluded the formation of monochlorocyclohexane [75].
These procedures were improved by replacing benzene,
as an internal standard, with decane. This change increased the sensitivity and the precision of results [76].
Aspartame and its decomposition products were analyzed by GC-MS after derivatization with BSA-N,Obis(trimethylsilyl)acetamide [77].
Pyrolysis GC-MS was applied for the determination of
aspartame in table-top sweeteners and soft drinks. No
preliminary sample extraction or derivatization was
necessary [78].
Sucralose can be analyzed by GC after silylation.
Treating sucralose dissolved in pyridine with the mixture
of hexamethyldisilazane (HMDS) and trimethylchlorosilane (TMS-Cl) yields volatile sucraloseTMS ether.
Identification and quantification of sucralose was done
by means of GC-MS and GC-flame-ionization detection
(FID), respectively [79].
4.1.4.2. Multi-analyte approaches. Saccharin, cyclamate
and dulcin were determined together by GC. Saccharin
and cyclamate were derivatized with diazomethane.
Dulcin does not undergo methylation with diazomethane, but it is volatile enough to be analyzed by GC
without derivatization [80].
Simultaneous determination of saccharin and acesulfame-K in foods can be achieved by means of GC-nitrogen phosphorus detection (NPD). Both sweeteners were
methylated with diazomethane prior to analysis [81].
4.1.5. Capillary electrophoresis
CE is a powerful tool in food analysis. Due to its high
resolving power, low solvent consumption and ease of
automation, CE is a viable alternative to HPLC for
determination of high-intensity sweeteners in various
1096
foodstuffs. Different modes of CE have been used in the

analysis of individual sweeteners or sweetener mixtures.
Capillary-zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC), and capillary isotachophoresis (CITP) are the modes of CE used most. The
electrolytes used to fill the silica capillary are generally
buffers based on phosphate, borate, benzoate or glycinate with pH values in the range (69). Several papers
have demonstrated the utility of CE for the analysis of
single sweeteners.
4.1.5.1. Methods for determination of individual sweeteners. A CZE method was employed for determination of
aspartame in foods using a 30 mM phosphate-19 mM
TRIS buffer with detection at 211 nm. The analysis time
was significantly shorter than that reported for HPLC
and no interferences were observed in the samples tested.
The main drawback of this method is the low pH value
(2.14) of the buffer used, because of the instability of
aspartame at pH below 3 [82].
Simultaneous separation of aspartame, caffeine and
benzoic acid in soft drinks can be achieved at pH 11
using 25 mM phosphate buffer. Separation of these three
compounds was completed within 10 min. However,
achievement of good reproducibility was difficult due to
problems with keeping the capillary walls in the same
conditions between days of analysis [83].
Aspartame, caffeine and benzoic acid can be determined in soft drinks using a glycine buffer which provide
much shorter migration times than borate buffers. Separation of analytes was achieved in 2 min using a
20 mM glycine buffer at pH 9 and direct spectrophotometric detection at 215 nm. Excellent reproducibility of
the method was also reported [84].
Cyclamate has also been successfully determined by
CE methods. Indirect UV detection at 254 nm was used
for the determination of cyclamate in beverages and
jams. Sample preparation was minimal. Beverages were
diluted with deionized water, and jams blended with
deionized water and filtered. Uncoated fused-silica capillary column with an electrolyte consisting of 1 mM
hexadecyltrimethylammonium hydroxide in 10 mM sodium benzoate was employed. The analysis time was less
than 5 min [85].
This method has been modified for use with the
broader range of foods by addition of an SPE clean-up
step. Sample extracts were passed through the Oasis HLB
SPE cartridges in order to remove interfering compounds. The running buffer containing 1 mM hexadecyltrimethylammonium bromide in 10 mM potassium
sorbate was used for separation. Detection and reference
wavelengths of cyclamate were 300 nm and 254 nm,
respectively [86].
CE with indirect absorption measurement is also a
suitable tool for monitoring the content of sucralose in
various foodstuffs. Sucralose determination in low-calo-
rie soft drinks can be achieved without any sample clean

up using 3,5-dinitrobenzoate buffer at pH 12.1 and
indirect UV detection at 238 nm [87].
The scope of the method has been extended to include
the possibility of analysis of yoghurts and candies by
introducing a sample clean-up step (centrifugation, filtration, and SPE on Alumina A cartridges) [88].
Neohesperidine dihydrochalcone (NHDC) is a minor
component of sweetener blends. It was separated from
other commonly-added sweeteners using a 100 mM
borate buffer (pH 8.3). Direct UV detection was applied,
and a wavelength of 282 nm was found optimal to avoid
interferences from other sweeteners and sugars. The
method was successfully applied to the determination of
low levels of NHDC in soft drinks, fruit juices and yoghurts [89].
4.1.5.2. Multi-analyte approaches. Different modes of CE
were applied for simultaneous determination of several
artificial sweeteners. MEKC is the most common mode.
Aspartame, acesulfame-K, saccharin, alitame, dulcin,
caffeine, benzoic acid and sorbic acid were separated in a
single run by MEKC. The complete separation was
achieved in less than 12 min using uncoated fused-silica
capillary column and 10 mM phosphate-10 mM borate
buffer at pH 8.6 with 50 mM sodium deoxycholate as the
micellar phase. The analytes were detected by direct UV
spectrophotometry at 220 nm. The method was applied
to a range of foods including low-energy soft drinks,
tomato sauce, marmalade and table-top sweeteners [90].
A more sophisticated buffer system was developed for
MEKC-UV (214 nm) determination of aspartame, saccharin and acesulfame-K together with several antioxidants and preservatives in cola beverages and lowenergy jams. The separation of the mixture was successfully accomplished utilizing a 20 mM borate buffer
with 35 mM sodium cholate, 15 mM sodium dodecyl
sulfate and 10% methanol added at pH 9.3 [91].
A rapid CE method for the analysis of aspartame,
acesulfame-K and saccharin in the presence of preservatives and food colors used as food additives in soft
drink was also presented. The mixture of such additives
was successfully separated using MEKC under optimized
conditions using a 20 mM carbonate buffer at pH 9.5
with 62 mM sodium dodecyl sulfate as the micellar
phase. Sample components were identified by a DAD in
the UV-Vis range (190600 nm) [92]. Four artificial
high-intensity sweeteners (acesulfame-K, saccharin,
cyclamate and aspartame) and four bulk sweeteners
(lactitol, sorbitol, mannitol and xylitol) were determined
in candies and chewing gums by means of CITP with
conductivity detection. The separation was achieved in
about 20 min using a capillary filled with electrolyte
system consisting of 10 mM HCl + 14 mM TRIS, pH 7.7
(leading electrolyte) and 5 mM L-histidine + 5 mM TRIS,
pH 8.3 (terminating electrolyte) [93].
Trends
4.1.6. Flow-injection analysis

FIA is a powerful technique for automated, continuous
determination of artificial sweeteners, especially when
only one or two analytes need to be determined in a
large number of samples. The use of FIA methodologies
presents advantages (e.g., high sample throughput, low
consumption of sample and reagents, high
reproducibility, speed of analysis, and simple and automated operation).
4.1.6.1. Methods for determination of individual sweeteners. Aspartame in table-top sweetener, pudding, gelatin, and powdered drink was determined by a
spectrophotometric FIA method using ninhydrin as a
colorimetric reagent. Aspartame reacted with ninhydrin
in a methanol-isopropanol medium containing potassium hydroxide. The absorbance measurements were
made at 603 nm [94].
Another spectrophotometric FIA method employed a
solid-phase reactor with copper (II) phosphate. Aspartame reacts with immobilized copper (II) ions giving
Cu(II)(aspartame)2 complex. Release of Cu (II) occurs
when the complex is mixed with alizarin red S solution,
which forms another colored complex. The absorbance
of Cu(II)-alizarin complex is measured at 550 nm. The
method was used for the determination of aspartame in
table-top sweeteners [95].
A very simple, sensitive flow-through spectrophotometric sensor was developed for determination of
aspartame in low-calorie and dietary products. The
sensor was implemented in a monochannel flow-injection (FI) system with UV spectrophotometric detection
using Sephadex CM-C25 cationic exchanger packed in a
flow cell. Aspartame was determined by measuring its
intrinsic absorbance at 219 nm at its residence time (pH
5.0) without any derivatization [96].
Biosensors have also been used for the measurement
of aspartame in foodstuffs. An FIA-biosensor system
comprised two enzyme columns, containing purified
peptidase and aspartate aminotransferase, respectively,
immobilized on activated aminopropyl glass beads and
an enzyme electrode connected in series [97].
The same authors proposed a system comprising an
enzyme column of pronase immobilized on activated
arylamine glass beads and 1-amino acid oxidase electrode connected in series [98].
An amperometric enzyme electrode for the determination of aspartame was developed by covalent immobilization of alcohol oxidase and a-chymotrypsin on the
surface of platinum-based hydrogen peroxide electrode.
The electrode was used in FI mode for the determination of aspartame in seven different food matrices, with
good recoveries. Sensitivity was significantly better than
those of previously constructed aspartame electrodes
[99].
1097
Trends
Saccharin in dietary product was analyzed using a

precipitation FIA method. Saccharin was precipitated as
mercurous saccharinate and the mercury-cation excess
was measured potentiometrically using silver wire
coated with a mercury film as the working electrode.
High sample throughput (60 samples/h) could be
achieved with this method [100].
Another precipitation FIA method, which utilized
atomic absorption spectrometry (AAS) as a detection
technique, permitted indirect determination of saccharin
in the presence of other sweeteners. It was based on the
continuous precipitation of saccharin with AgNO3 in a
flow manifold, followed by filtration, washing, dissolution in ammonia and measurement of dissolved silver by
flame atomic absorption spectrometry (FAAS) [101].
A flow-through spectrophotometric sensor for monitoring the content of saccharin in low-calorie products
utilized the transient adsorption of the sweetener on solid
phase packed in a flow cell, followed by absorbance
measurement at 217 nm [102].
FIA procedures have also been proposed for determination of cyclamate in foodstuffs, involving detection by
chemiluminescence, spectrophotometry, FAAS or
amperometry. An FIA method based on the sensitizing
effect of sodium cyclamate on the chemiluminogenic
oxidation of sulfite by cerium (IV) has been described
[103].
Cyclamates in table-top sweeteners and some low
calorie soft drinks were determined by an FIA spectrophotometric method. The procedure utilized the reaction
between nitrite and cyclamate in medium containing
phosphoric acid. The excess of nitrite was determined by
the measurement of absorbance of Griess reaction
product at 535 nm [104].
Hydrolysis of cyclamate to cyclohexylamine has been
employed for the analysis of table-top sweeteners. The
hydrolysis step was performed batch wise by treating
cyclamate with hydrogen peroxide and hydrochloric
acid. The cyclohexylamine was derivatized with 1,2naphthoquione-4-sulfonate (NQS) in an FI system. The
NQS derivative was monitored at 480 nm [105].
An interesting FIA system for the determination of
cyclamate in table-top sweeteners has been proposed.
The procedure is based on the reaction of cyclamate with
nitrite in acidic medium. The excess nitrite reacts with
iodide to form the triiodide anion, which is determined
spectrophotometrically at 350 nm. No toxic reagents are
employed and the amounts of chemicals consumed and
wastes generated are minimized by replacing a traditional continuous-flow system with a set of sequentiallyoperated solenoid micro-pumps [106].
Automated turbidimetric determination of cyclamate
in low-calorie soft drinks and sweeteners (without pretreatment) employed oxidation to sulfate by nitrite, followed by precipitation with barium and measurement of
analytical signal at 420 nm [107].
1098
Cyclamate was also determined by an indirect AAS

method using a flow system with precipitate dissolution.
It was based on oxidation of the sulfamic group to sulfate
and continuous precipitation with lead ion in a flow
manifold, followed by filtration, washing, dissolution in
ammonium acetate and measurement of lead by FAAS
[108].
A bioamperometric titration procedure was found
suitable for the determination of cyclamate in various
dietary products. After the reaction of cyclamate with
the nitrite, the excess of nitrite was detected in a polyurethane cell containing two platinum wires polarized at
0.7 V [109].
4.1.6.2. Multi-analyte approaches. There are only very
few reported FIA procedures suitable for simultaneous
determination of artificial sweeteners.
Cyclamate, acesulfame-K and saccharin were determined in table-top sweeteners, yoghurts, soft diet drinks
and wines by means of an FIA system containing filtersupported bilayer membrane (BLM) formed using
lyophilized egg phosphatidycholine. The detection was
performed using two Ag/AgCl reference electrodes biased
by an external power supply at the level of +25 mV
(sensing electrode). Transient electrical signals with different time delays were observed after injection of the
mixture of sweeteners into the stream of the carrier
electrolyte [110].
Simultaneous determination of aspartame and acesulfame-K in table-top sweeteners was demonstrated
using a multi-analyte flow-through sensor. The
procedure was based on the transient retention of
acesulfame-K in the ion exchanger Sephadex DEAE A-25
placed in the flow-through cell of a monochannel FIA
system using pH 2.70 ortophosphoric acid/sodium
dihydrogen phosphate buffer 0.06 M as carrier. In these
conditions aspartame is very weakly retained, and that
makes it possible to measure the intrinsic UV absorbance
of first aspartame and then acesulfame-K after desorption by the carrier itself [111].
Aspartame, acesulfame-K, saccharin and a few antioxidants and preservatives were simultaneously determined in selected foods using a single-channel FIA
system incorporating a short monolithic C18 pre-column. The separation of analytes was based on their
retention time and quantification was done by means of
a DAD. This system should be treated as a simple, lowresolution liquid chromatograph [112].
4.1.7. Electrochemical techniques
Electrochemical techniques are powerful and versatile
tool for determination of artificial sweeteners, because
they are simple, fast and low cost. Moreover, they give
the opportunity to determine analytes directly even in
turbid and colored solutions. Several electrochemical
techniques have been developed for the analysis of these
compounds separately or in mixtures in dietary products.

4.1.7.1. Methods for determination of individual sweeteners. There are two basic approaches concerning a
potentiometric determination of saccharin. The first is
based on precipitation titration of saccharin with either
mercurous [113] or silver [114] ions. The other way is
to use an ion-selective electrode (ISE) sensitive to saccharinate ions. Most constructions of such electrodes
comprise a graphite rod coated with a doped
poly(vinylchloride) membrane. Usually, the dopant is an
ion pair formed between saccharinate anion and a
counter cation. Basic dyes [115], silesquioxane 3-npropylpirydinium chloride [116] and Aliquat 336S
[117] were employed as ion-pair forming agents.
An interesting saccharinate-sensitive ISE, based on a
solid, polycrystalline membrane has also been proposed.
The membrane was prepared by compressing a mixture
of mercury saccharinate, graphite and a small amount of
metallic mercury. A wide linear range (10 210 6 M)
and relatively long lifetime have been reported for this
sensor [118].
Various electrochemical methods have been proposed
for aspartame. Most of them utilized biosensors. A
bienzymatic electrode, which utilized the chemical coimmobilization of carboxypeptidaze and L-aspartase on
an ammonia-gas-sensing electrode, has been employed
for the determination of aspartame in several dietary
products. This electrode suffered from a very narrow
linear range and short lifetime [119].
Another method used an amperometric biosensor
comprising a-chymotrypsin and alcohol oxidase, which
were co-immobilized on a dissolved oxygen electrode
[120]. The bienzyme system comprising carboxyl esterase and alcohol oxidase, immobilized in gelatin membrane, was another way of monitoring of the aspartame
content.
In order to determine the concentration of aspartame,
oxygen consumption during the enzymatic reaction was
measured using an oxygen meter [121].
The same bienzyme system was integrated with
graphite epoxy composite electrode (GECE). Greater
linear range and a longer lifetime were claimed for this
set-up [122].
Determination of aspartame by square-wave voltammetry was also reported [123].
4.1.7.2. Multi-analyte approaches. There are only a few
reported electrochemical methods suitable for simultaneous determination of several artificial sweeteners in
foodstuffs. One of them described a simultaneous squarewave (SW) voltammetric method for determination of
aspartame and cyclamate in beverages. The use of SW
voltammetry in conjunction with boron-doped diamond
electrode gives an opportunity to separate the oxidation-
Trends
peak potentials of aspartame and cyclamate by 400 mV.

The results showed that the proposed method was
simple, inexpensive and very sensitive [124].
4.1.8. Spectroscopic techniques
Various types of spectroscopic techniques have been
applied for analysis of sweeteners in foodstuffs. UVspectrophotometric methods are the most widely used,
but Raman and infrared spectroscopy have also been
used for this purpose.
4.1.8.1. Methods for determination of individual sweeteners. A lot of spectrophotometric methods have been
reported for determination of saccharin.
The first spectrophotometric methods of saccharin
determination based on reaction with azure B [125],
Nile-blue [126] or astrazone pink FG [127] were often
time-consuming and tedious.
A simple, rapid and sensitive spectrophotometric
method was proposed for routine analysis of saccharin in
liquid and solid sweeteners. Saccharin was reacted with
p-chloranil in the presence of hydrogen peroxide, and
that resulted in formation of a violet-red compound,
exhibiting an absorption maximum at 550 nm. The
main drawback of this method was a strong interference
from cyclamate, which had to be eliminated during
sample preparation by precipitation in ethanol [128].
A spectrophotometric method involving ninhydrin as
a colorimetric reagent was proposed for the determination of aspartame. To increase the selectivity of the
determination, extraction with propylene carbonate was
necessary [129].
Another spectrophotometric method for the determination of aspartame in beverages was based on the
enzymatic conversion of aspartame into formaldehyde
by the a-chymotrypsinalcohol oxidase system. Subsequently, formaldehyde was exposed to derivatization
with Fluoral-P [130].
A rapid method for the determination of aspartame in
soft drinks utilized Fourier transform infrared spectroscopy with an attenuated total-reflectance accessory and
partial least-squares regression. The method gives good
results for the samples with well-defined matrices, so it is
suitable for routine quality-control analysis of aspartame
in the beverage-manufacturing sector [131].
Aspartame, saccharin and acesulfame-K can be
determined (but not simultaneously) in various food
products by an extractive spectrophotometric technique
using an oxazine dye, Sevron Blue 5G, as the reagent.
Those sweeteners form chloroform-extractable ion-association complexes with the dye. The content of a
sweetener can be determined by measuring the absorbance of a chloroform extract at 655 nm [132].
4.1.8.2. Multi-analyte approaches. An analytical
procedure for simultaneous determination of aspartame,
1099
Trends
saccharin and acesulfame-K was based on the oxidative

reaction of the analytes with KMnO4 in alkaline solution.
The green potassium-magnate reaction product from
three sweeteners was formed at different kinetic rates,
which allowed for selective determination of each analyte. To improve the accuracy and the precision of
analysis, chemometric multivariate-calibration methods
were applied [133].
A simple, rapid method was used to determine acesulfame-K and saccharin in liquid and solid sweeteners
and powdered fruit juice. The application of UV-Vis
spectrophotometry coupled to chemometric analysis
[partial least squares (PLS)] allowed the determination of
these compounds in real samples. Strong interference
from aspartame was taken into account during calibration and validation [134].
A PLS Fourier-transform Raman spectrometry procedure was suitable for simultaneous determination of
saccharin and cyclamate in the formulation of table-top
sweeteners. This procedure offered the possibility of
carrying out direct and reagent-free measurements on
solid samples [135].
Fourier-transform mid-infrared spectrometry was used
to determine aspartame and acesulfame-K in table-top
sweeteners. Off-line and on-line FTIR procedures were
developed to provide results statistically comparable with
those obtained by the HPLC reference method [136].
5. Conclusion
Due to medical and legal aspects, many researchers have
focused their efforts on developing analytical methods for
the determination of artificial sweeteners individually or
simultaneously in mixtures. They have applied a wide
variety of instrumental techniques.
The method of choice for the determination of artificial
sweeteners in different food matrices is HPLC because of
its multi-analyte capability, compatibility with the
physico-chemical properties of sweeteners, high sensitivity and robustness.
CE and IC are both interesting alternatives to HPLC.
The resolving power of these techniques is in many cases
comparable with that of HPLC and, frequently, their
running costs are lower. However, it seems that due to
limited robustness, in the case of CE methods, and the
modest choice of separation mechanisms, in the case of
IC, these methods are less popular.
TLC and GC have been applied occasionally to analysis
of artificial sweeteners. TLC methods are characterized
by poor separation efficiency and GC methods require
derivatization that is time consuming and labor intensive. Due to a demand for simple, rapid and low-cost
alternative methods for determination of sweeteners, in
many instances chromatographic methods can be replaced by electroanalytical, spectroscopic or FI proce1100
dures. Some of them are even more sensitive and

selective and require very little sample preparation.
Unfortunately, their applications are limited to one or
two sweeteners only.
However, there still remains the challenge of developing stable, reliable and robust methods for the determination of artificial sweeteners in difficult food
matrices. Robust and reliable analytical methods are
essential to meet the needs of growing markets in quality
control and consumer safety.
Acknowledgements
This work was partly financed within the framework of a
research project funded by the Polish Committee for the
Scientific Research (Research project # N N404
027535).
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Trends in Analytical Chemistry, Vol. 28, No. 9, 2009