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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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0165-9936/$ - see front matter 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2009.06.008
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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
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Full name
O
S
Acesulfame
Potassium
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]
[17]
[18]
(4-ethoxyphenyl)urea.
The sweetening strength is fsac,g(5) = 109.
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
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
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[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]
[6]
[14]
[18]
OH
O
NH
O
NH2
NH
OH C
3
H3C
CH3
CH3
S
CH3
Alitame
ALI
331.43
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Table 1. (continued )
Sweetener
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
[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.
The sweetening strength is fsac,g(10) = 6000.
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.
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4. Analytical methodology
A great variety of methods based on different principles
have been applied to the analysis of the aforementioned
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.
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ASP, SAC
Dietary products
HPLC-UV
Recovery 9597%
LOD not available
RSD 0.861.25%
[49]
Beverages, jams
HPLC-UV
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
Recovery 98.6102.3%
LOD 0.13 mg/L
RSD not available
[52]
ACS-K,ASP SAC,
Vanillin, Sorbic acid
Benzoic acid
HPLC-UV
Recovery 99101%
LOD 0.23.1 lg/g
RSD 1.02.2%
[51]
Various foods
HPLC-UV
Cosmosil 5C18-AR
Recovery 89104%
LOD 1lg/g
RSD not available
[53]
Non-carbonated soft
drinks, canned
or bottled fruits and
yoghurts
HPLC-ELSD
Recovery 93109%
LOD 15 lg/g
RSD 0.94.5%
[15]
Various foods
HPLC-ESI-MS
Dibutylammonium acetate
buffer- ACN-H2O
Recovery 75.7109.2%
LOD 15 lg/g
SD 510.9%
[22]
HPLC-ESI-MS
[23]
Ion-paired LCUV
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
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Table 2. (continued )
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Column/Capillary
Ref.
Technique
HPIC-UV-ELCD
Na2CO3
Recovery 93107%
LOD 0.0190.044 mg/L
RSD 0.841.38%
[62]
HPIC-UV
Recovery 85104%
LOD 430 mg/L
RSD 15%
[64]
HPICsuppressed
conductivity
detector
KOH
Recovery 97.96105.42%
LOD 0.0190.89 mg/L
RSD not available
[63]
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]
MEKC-UV
Recovery 98.9100.86%
LOD not available
RSD 0.91.5%
[91]
Soft drinks
MEKC-UV
Uncoated fused-silica
capillary (48.5 cm 50 lm)
Recovery
LOD 0.005 mg/mL
RSD not available
[92]
CITPconductivity
detector
Recovery 98.2102.5%
LOD 0.0240.081 mM
RSD 0.82.8%
[93]
Sample
Mobile phase/Electrolyte
Analytical parametersb,c
Analyte
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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
Table-top sweeteners
Recovery n.a.
LOD 2 lg/mL
RSD 0.2%
LR n.a.
[95]
ASP
Recovery 95101%
LOD 0.82 lg/mL
RSD 0.55%
LR 5.0600 lg/mL
[96]
ASP, ACS-K
Table-top sweeteners
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
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
Recovery 102.5106.3%
LOD n.a.
RSD n.a.
LR 5.0 10 84.0 10 7 M
[121]
ASP
Diet cokes
Recovery 101.84104.08%
LOD n.a.
RSD n.a.
LR 2.5400 lM
[122]
ASP, ACS-K
FTIR
Table-top sweeteners
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
Recovery 93.8105.1%
LODACS-K 0.085 lg/mL; LODSAC
0.0312 lg/mL
RSD n.a.
LR n.a.
[133]
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]
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Table 3. (continued)
Analyte
Analytical technique
Sample
Analytical parametersa,b,c,d
Ref.
CYC
Table-top sweeteners
Recovery n.a.
LOD 7.7 lmol/L
RSD 3.5%
LR < 1000 lmol/L
[106]
CYC
Table-top sweeteners
Recovery n.a.
LOD 30 lmol/L
RSD 1.7%
LR 1003000 lmol/L
[105]
CYC
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
Table-top sweeteners
Recovery n.a.
LODCYC 0.83% w/w; LODSAC
0.2% w/w
RSD 0.55%
LR n.a.
[135]
SAC
Table-top sweeteners
Recovery n.a.
LOD 3 lg/mL
RSD 2.7%
LR 575 lg/mL
[101]
SAC
Recovery 98104%
LOD 0.2 lg/mL
RSD 0.78%
LR 1.0200 lg/mL
[102]
SAC
Recovery 97.0102.6%
LOD 2.5 lg/mL
RSD n.a.
LR n.a.
[114]
SAC
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
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
Recovery 99.2104.3%
LOD 1.55 10 5 M
RSD 0.51.6%
LR n.a.
[128]
a
b
c
d
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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].
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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].
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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
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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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