analytical methodologies for determination of artificial sweetners in foods -f bun.pdf

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

Analytical methodologies fordetermination of artificialsweeteners in foodstuffsAgata 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); High-

performance liquid chromatography (HPLC); Ion chromatography (IC); Spectroscopy; Thin-layer chromatography (TLC)

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: [email protected]

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1. Introduction

Artificial high-intensity sweeteners (alsocalled non-nutritive sweeteners) form animportant class of food additives, whichare commonly used in the food, beverage,confectionery and pharmaceutical indus-tries. They provide the sensation ofsweetness, but with little or no intake offood energy. There are a large number ofknown intense sweeteners, but only veryfew are allowed to be used in modern foodindustry. The list of authorized artificialsweeteners varies from country to coun-try. For example, there are six artificialhigh-intensity sweeteners authorized foruse in European Union (EU) (acesulfame-K, aspartame, cyclamic acid and its salts,saccharin and its salts, sucralose andneohesperidine dihydrochalcone) [1],whereas, in the USA, the correspondinglist does not include cyclamates and neo-hesperidine dihydrochalcone, instead onecan find neotame there [2].

The food industry is heavily promotingits artificially-sweetened products (fre-

0165-9936/$ - see front matter 2009 Elsev

quently called diet or light), high-lighting their benefits. Low-calorie orreduced-calorie food products and bever-ages can help in treatment of obesity,maintaining body weight and manage-ment of diabetes. Last, but not least, arti-ficial sweeteners are not fermented by themicroflora of the dental plaque, whichmakes them tooth-friendly.

Sweeteners may be used separately or incombination with other sweeteners, as so-called blends. Nowadays, the commontrend in food industry is to use sweetenerblends, because some of the sweetenersimpart side tastes and aftertastes that canlimit their applications in foods and bev-erages [3]. It was found that mixing sucha problematic sweetener with anotherfrequently yields a blend not only lackingunwanted side or aftertastes but alsosweeter than the algebraic sum of thecomponents. A very well-known exampleof such a mixture is saccharin-cyclamate(1:10) blend. The bitter aftertaste of sac-charin is masked by cyclamate and theunpleasant aftertaste of cyclamate, sensed

ier Ltd. All rights reserved. doi:10.1016/j.trac.2009.06.008
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Trends in Analytical Chemistry, Vol. 28, No. 9, 2009 Trends

by some people, is masked by saccharin. Simultaneously(due to synergistic effect), the sweetening power of themixture increases. Properly formulated sweetener blendscan precisely reproduce the texture and the sweetnessprofile of traditional sugar-containing products, createnew products characterized by an original sweetnessprofile and improve taste stability [4].

Artificial high-intensity sweeteners, intensely pro-moted by the food industry are among the most con-troversial food additives due to suspicions of adversehealth effects [5]. These allegations include causingdermatological problems, headaches, mood variations,behavior changes, respiratory difficulties, seizures,allergies and cancer.

Many experiments have been performed on the safetyof saccharin. Some associated saccharin with bladdercancer when fed at high doses to rats. However, resultsfrom subsequent carcinogenicity studies showed noconsistent evidence of association between saccharinconsumption and cancer in test animals. Another sus-picion about saccharin is connected with the possibilityof allergic reactions in people who do not tolerate sulfadrugs [4,6].

In the case of cyclamate, the issue is more complicatedbecause different people metabolize this sweetener indifferent ways [5]. One study conducted in 1966 indi-cated that cyclamate can be metabolized by some intes-tinal bacteria resulting in formation of cyclohexylamine,a compound suspected to have some chronic toxicity inanimals. Another study from 1969 linked cyclamateconsumption with increased risk of bladder cancer inrats. In 2000, the European Food Safety Agency (EFSA)published its opinion on safety of cyclamate, stating thatavailable epidemiological data revealed no indications ofharmful effects on human reproduction parameters ofcyclamate used as a food additive [7].

Aspartame is probably the most controversial artificialhigh-intensity sweetener on the market, with adversemedical 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 de-fects, leukemia and death. The European RamazziniFoundation (ERF) of Oncology and Environmental Sci-ences published, in 2006 and 2007, results of twostudies [8,9] on aspartame toxicity. Both studies linkedaspartame with cancer, lymphomas and leukemias intested rats. In March 2009, the EFSA discounted theresults of these studies and found no indications of anygenotoxic or carcinogenic potential of aspartame [10].Nevertheless, people with phenylketonuria should elim-inate foods containing aspartame, because excess intakeof phenylalanine (one of the aspartames metabolites)can lead to brain damage.

Safety concerns pertaining to sucralose are mainlycaused by the presence of three chlorine atoms in its

molecule, which make it an organochloride. Manyorganochlorides are toxic or carcinogenic (e.g., pesticidesand dioxins) and this is probably the reason for themistrust of sucralose. However, studies in human beingsand animals have shown that this sweetener did notpose carcinogenic, reproductive or neurological risk topeople [5].

The content of sweeteners in foodstuffs is limited bycountry-specific regulations. In the EU, sweeteners arethoroughly assessed for safety by the EFSA before theyare authorized for use. EU Directives 94/35/EC [11], 96-83/EC [12], 2003/115/EC [13], 2006/52/EC [1] define,which sweeteners have approval to be added to foodproducts and beverages. Considering medical and legalaspects, the determination of these artificial sweetenershas economic and social relevance [14].

Due to consumer safety, it is necessary to control thecontent of sweeteners in foodstuffs. To obtain thisinformation, reliable quantitative methods of analysisare required to measure levels of sweeteners in a broadrange of food matrices [15]. A number of analyticalmethods based on different principles are available fortheir determination. The aim of this review is to presentand to compare the available analytical methods fordetermination of artificial sweeteners in foodstuffs.

2. Artificial sweeteners

High-intensity sweeteners can be divided into threecategories: synthetic, semi-synthetic and natural. Theycomprise a wide variety of organic molecules (e.g., car-bohydrate derivatives, salts of organic acids, terpenoidsand even proteins [16]). The majority of sugar substi-tutes approved for use in food chemistry are artificially-synthesized compounds, so we do not consider naturallyintense sweeteners in this review. The most popularartificial sweeteners are: acesulfame-K (ACS-K), aspar-tame (ASP), cyclamate (CYC), saccharin (SAC), sucra-lose (SCL), alitame (ALI), neotame (NEO) andneohesperidine dihydrochalcone (NHDC), which is asemi-synthetic sweetener. Table 1 shows the chemicalstructures and the basic characteristics of aforemen-tioned sweeteners.

3. Sample preparation

Sample preparation is an essential stage in the analyticalprocess, and food samples are among most difficultmatrices, due to the great variability in their composition(e.g., preservatives, colors, thickeners, vitamins, pro-teins, lipids and minerals). All of the components caninterfere with the determination of sweeteners. Samplepreparation procedure must be tailored to the method offinal determination, considering the instrumentation

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Table 1. Chemical structures and basic characteristic of artificial sweeteners

Sweetener Basic characteristics and applications Ref.

Structure Full name Acronym Molar mass

K+

CH3 O

OO

SN-

O

Acesulfame ACS-K 201.24 Potassium salt of 6-methyl-1,2,3- oxathiazine-4(3H)-one2,2-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.

[5]Potassium [6]

[17]

O

OO

N-

SNa+ Sodium Saccharine SAC 205.16 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, sodiumsaccharin, and calcium saccharin).

It has high solubility and stability. However, at low pH, it can slowlyhydrolyze 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 atleast 90 countries.

[14][18]

Na+O-O

O

SNH

Sodium cyclamate CYC 201.2 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]

H

NO

NH2

OCH2CH3Dulcin DUL 180.2 (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 ofhydrolysis to aminophenol, it may cause adverse eect during long-term usage.

Compared to saccharin, it does not have a bitter after-taste.

[18]

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COOH

NH2

O

CH3

O O

NH

Aspartame ASP 294.3 N-L-a -aspartyl-L-phenylalanine-1-methyl ester. It is white, odorless, crystalline powder. The sweetening strength is fsac,g(10) = 133. It has a caloric value of 17 kilojoules per gram like any other protein

substance, however, because its high sweetness, the amounts usedare small enough to be classified as nonnutritive sweetener.

Although it is relatively stable in its dry form, the compound undergoespH- and temperature- dependent degradation in solution, what makesaspartame undesirable as a baking sweetening agent. Below pH 3 aspartameis unstable and hydrolyzes to produce aspartylphenylalanine and abovepH 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.

[5][14][17]

OH

OH

OH

Cl

Cl

O

OH

Cl

OH O

O Sucralose SCL 397.63 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 thesugar 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 compatibilitywith commonly used food ingredients, it can be added to baked goods andproducts that require a longer shelf life.

Since 1991, it has been approved for use as food additive in more than60 countries, and recently in January 2005 in the EU.

[9][14][19][20]

CH3CH3

CH3CH3

SO

CH3

NH

O

NH2

OH

O

NHAlitame ALI 331.43 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. The sweetening strength is fsac,g(10) = 2000. 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]

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Table 1. (continued )

Sweetener Basic characteristics and applications Ref.

Structure Full name Acronym Molar mass

Me

OH

OH OH

OH

O O

OH

OHO

O OH

OOH

OMe

OH

Neohesperidine NHDC 612.57 It is a semi-synthetic sweetening agent preparedfrom neohesperidine or narigin, two flavanones extractedfrom citrus peel.

The sweetening strength is fsac,g(10) = 667. 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]dihydrochalcone [18]

OH

O NH

O

NH CH3O

O

CH3

CH3CH3

Neotame NEO 378.46 N-[N-(3,3-dimethylbutyl)-L-a-aspartyl]-L-phenylalanine1-methyl ester, is a derivative of dipeptide, which ismade from the amino acids aspartic acid and phenylalanine.

It has a very clean sweet taste, close to sucrose, with noundesirable bitter or metallic o-taste which occurs inother 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 theacidic pH range, but it is significantly more stable inthe neutral pH range.

It has been approved in the USA, Australia, andNew Zealand.

[5][20]

afsac,g(x) = Sweetness potency relative to x% sucrose solution on a weight basis.

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used and the degree of accuracy required, whetherquantitative or qualitative.

3.1. Challenges in preparing food samplesThe chemical and the physical properties of foods areinherently variable. The variability in composition of agiven food sample can be minimized with proper samplepreparation. It is essential for the analyst to recognize theneed to utilize methods that satisfy analytical require-ments.

Generally, preparation of food samples compriseshomogenization, extraction, clean up and concentration.Analysis of liquid samples has an advantage overanalysis of solid samples that one fewer pretreatmentstep is usually required, because of their liquid state.

An inherent difficulty in the preparation of foodsamples is the complexity of the matrix. Some matrixcomponents may co-extract with analytes due to similarsolubility in the extraction solvent. The presence ofmatrix interferences in a sample extract can contributeto a multitude of problems (e.g., turbidity, generation ofemulsions and, most importantly, masking the analyti-cal signal of the compound of interest). Such effectsusually lead to an increase in the limit of detection (LOD)of the method. Usually, clean up of the extract resolvesthis problem. Passing the extract through a column withappropriate stationary phase, dialysis, liquid-liquidextraction, precipitation and filtration are most com-monly employed for this purpose.

The challenge for the analyst is to maximize recoveryof the analytes and to minimize the amount of interfer-ing compounds by using appropriate extraction andclean-up procedures. Optimal sample preparation canreduce analysis time, eliminate sources of error, enhancesensitivity and enable unequivocal identification, con-firmation and quantification of analytes.

3.1.1. Sample pretreatment prior to the final analysisSweeteners are commonly used in various types offoodstuffs (e.g., soft drinks, fruit beverages, fermentedmilk drinks, cordials, instant powdered drinks, candies,chewing gum, solid and liquid sweeteners, jams, pickles,canned fruits, dried fruits, various sauces, dehydratedsoups, jellies, bakery products, dairy products, confec-tionery, and chocolates). The diversity of products andthe variability of matrices from which sweeteners areassayed present a great challenge to analytical chemists.The aim of their work is to come up with a compromisebetween sufficient sample preparation and achievingreliable results by using different types of analyticaltechniques. Sample preparation depends on the type offood matrix. Some samples can be analyzed directly orafter minimal pretreatment. However, extraction, cleanup and/or purification might be necessary, depending onthe complexity of the sample, in order to eliminateinterferences [21].

There are very few known methods requiring nosample preparation at all. These include electrochemicaland Fourier transform infrared (FTIR) spectrometry-based methods [121,122,135]. The biggest advantagesof direct measurement are speed and elimination ofphysico-chemical sample manipulation. Nevertheless,complicated and sample-specific calibration of FTIR-based methods limits their application to analysis ofsamples of well-defined matrices. However, electro-chemical techniques suffer from sensor instability and aneed for frequent recalibration.

Samples characterized by relatively simple matrix (i.e.table-top solid and liquid sweeteners, beverages, pow-dered drinks, syrups and juices) can simply be diluted ordissolved in deionized water, an appropriate buffer or amixture of a buffer with methanol or ethanol. In the caseof carbonated drinks, the samples have to be degassed(e.g., by sonication, or sparging with nitrogen or undervacuum) prior to analysis. Optionally, sample solutionscan be clarified using a clarifying agent (e.g., Carrez re-agent, ZnSO4/NaOH or similar). Clarifying agents areuseful for removal (by occlusion) of proteins, suspendedparticulate matter and fatty material. Clarified or not,samples are filtered before final determination. Single stepfiltration with a membrane filter is sufficient in mostcases, although some extracts need centrifugation orprefiltration with filter paper. This minimal sample prep-aration procedure is included almost universally in pub-lished methods. It is quick, cheap and simple. However,no chemical interferences with good water solubility canbe removed this way or any preconcentration of analytesachieved. Nevertheless, such a procedure is sufficient formany samples and techniques of final determination.

More sophisticated sample-preparation protocolsinvolve the use of the solid-phase extraction (SPE)technique, which is a powerful tool. It allows forfractionation of sample components based on the affinityof a compound or group of compounds to the stationaryphase. Basically, SPE can be treated as a low-resolutionliquid chromatography (LC). Among the huge number ofdifferent types of SPE cartridges available on the market,those employing different types of a non-polar C18packing material seem to be the most frequently used.Other choices include dextran, polystyrene [43] andother polymer-based fillings [53,86].

Usually, an SPE procedure comprises the followingoperations: cartridge conditioning, sample load, car-tridge wash and elution of analytes. In the most commonmode of SPE, an aliquot of the sample extract (e.g., ob-tained according to the minimal sample preparationprocedure described above) is loaded onto a previouslyconditioned SPE cartridge. The type of SPE packingmaterial, solvents, pH and the flow rates need to beproperly selected in order to retain analytes transientlywithin the cartridge. The interfering substances shouldbe retained very strongly or not retained at all. As a



result, weakly retained substances are readily removedfrom the cartridge during sample load and/or cartridgewash, analytes are eluted during the elution step andsubstances having a strong affinity to the sorbent stayadsorbed within the cartridge. The sensitivity of a finaldetermination can easily be enhanced by evaporatingthe final SPE extract to dryness and reconstituting it witha smaller amount of a solvent of choice.

Sometimes, SPE cartridges filled with polar packingmaterials (e.g., alumina) are used for decoloring samplesor removing carboxymethyl cellulose (CMC, a thickeningagent) [88]. Extracts are loaded onto preconditionedcartridges that adsorb colorants and/or CMC whileanalytes pass unretained.

Dialysis coupled with SPE was also proposed as asample preparation technique for the determination ofsucralose [40,41], aspartame, neotame and alitame [53].

SPE-based sample-preparation protocols seem to be thebest available choice. They are simple, reproducible,reasonably quick and inexpensive. They are universaland compatible with the most popular techniques used infood analysis. Other sample preparation procedures arepossible, but usually they are highly specific to the analyteunder study, the sample or the technique of final deter-mination. In such cases, details on sample preparationcan be found in the respective references given later.

4. Analytical methodology

A great variety of methods based on different principleshave been applied to the analysis of the aforementioned

Figure 1. The use of analytical techniques to analyze artificial sweeteners.Flow-injection analysis; GC, Gas chromatography; HPLC, High-performanchromatography; ST, Spectroscopic techniques.

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compounds in food, drinks and dietary products. Most ofthe methods have been developed for individual sweet-eners. As can be seen in Fig. 1, commonly used sweet-eners, with decades of history of usage (i.e. aspartame,saccharin, cyclamate and acesulfame-K), can be deter-mined by all current analytical techniques. Among other,recently introduced or approved sweeteners, sucralosereceives the most attention, while a much smaller num-ber of papers deals with the determination of neohes-peridine dihydrochalcone, alitame and neotame.

Since there are tens of possible sweetener combina-tions, there is a need to develop analytical methodscapable of determining several sweeteners in one run. Sofar, the majority of the published multi-sweetenermethods have focused on the determination of just a few(34) compounds. Recently, however, some progress canbe observed with the publication of methods coveringmuch wider range of artificial sweeteners [15,22,23].Table 2 provides references to recently published multi-analyte methods for the determination of artificialsweeteners in different food matrices. The reported pro-cedures are grouped according to analytical technique.

Of the great variety of methods used for the determi-nation of sweeteners in different food matrices, chro-matographic methods have received wide recognition.Currently, the most popular technique in this field ishigh-performance LC (HPLC). The broad range of avail-able separation columns (mechanisms) together with theimpressive portfolio of detectors makes HPLC a trulyuniversal technique. Reversed-phase LC (RP-LC) is awell-known, mature technique, perfectly suited for theseparation of sweeteners.

CE, Capillary electrophoresis; ET, Electroanalytical techniques; FIA,ce liquid chromatography; IC, Ion chromatography; TLC, Thin-layer

Table 2. Analytical procedures for simultaneous determination of artificial sweeteners mixtures in samples of different food products

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% [49]LOD not availableRSD 0.861.25%

SAC, ACS-K, Benzoicacid, 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% [50]LOD < 0.1 mg/100 ml

ACS-K,ASP SAC,benzoic acid, sorbicacid, 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% [52]LOD 0.13 mg/LRSD not available

ACS-K,ASP SAC,Vanillin, Sorbic acid

Cola drinks, instant-powder drinks

HPLC-UV ACN-ammonium acetate buffer(pH 4)

YMC-ODS Pack AM (5 lm,250 4 mm)

Recovery 99101% [51]

Benzoic acidLOD 0.23.1 lg/gRSD 1.02.2%

ASP, ALI, NEO Various foods HPLC-UV ACN-phosphate buffer (pH 4) Cosmosil 5C18-AR Recovery 89104% [53]LOD 1lg/gRSD not available

ACS-K, SAC, CYC, ASP,SCL, DUL, ALI, NEO,NHDC

Non-carbonated softdrinks, cannedor bottled fruits andyoghurts

HPLC-ELSD TEA formate buffer- MeOH-ACTN

Zorbax Extend C18,Purospher Star RP-18,Nucleodur C18 Pyramid,Nucleodur C8 Gravity;(5 lm, 250 3 mm)

Recovery 93109% [15]LOD 15 lg/gRSD 0.94.5%

ACS-K, SAC, CYC, ASP,SCL, DUL, GAd, STVe,REBf

Various foods HPLC-ESI-MS Dibutylammonium acetatebuffer- ACN-H2O

Zorbax Eclipse XDO-C18(150 2.1 mm)

Recovery 75.7109.2% [22]LOD 15 lg/gSD 510.9%

ACS-K, SAC, CYC, ASP,SCL, ALI, NEO, STV

Beverages, canned fruits,cakes

HPLC-ESI-MS TEA formate buffer- MeOH-ACTN

Spherogel C18 column(Johnson Inc., Dalian, China)(5 lm, 250 4.5 mm)

Recovery 95.4 104.3% [23]LOD < 10 lg/mLRSD not available

SAC, ACS-K, DUL,preservatives,antioxidants

Sugared fruits, soysauces, dried roast beef

Ion-paired LC-UV

ACN-aqueous a-hydroxyisobutyric acid solutioncontaininghexadecyltrimethylammoniumbromide

Shoko stainless-steel 5C18(5 lm, 250 4.6 mm)

Recovery 81.9103.27% [54]LOD 0.153 lg/gRSD 0.35.69%

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Analyte Sample Technique Mobile phase/Electrolyte Column/Capillary Analytical parametersb,c Ref.

ACS-K, SAC, CYC, ASP,citric acid

Drinks, powdered table-top sweeteners

HPIC-UV-ELCD Na2CO3 Dionex Ion Pac AS4A-SC(254 4 mm)

Recovery 93107% [62]LOD 0.0190.044 mg/LRSD 0.841.38%

ACS-K, SAC, ASP,benzoic acid, sorbicacid, caffeine,theobromine,theophyline

Drinks, juices, fermentedmilk drinks,preserved fruits, tablets

HPIC-UV NaH2PO4 (pH 8.20)-ACN Shim-pack IC-A3 (5 lm,150 4.6 mm)

Recovery 85104% [64]LOD 430 mg/LRSD 15%

ACS-K, SAC, CYC, ASP Carbonated cola drinks,fruit-juice drinks,preserved fruits

HPIC-suppressedconductivitydetector

KOH Dionex Ionpac AS11 (250x2mm)

Recovery 97.96105.42% [63]LOD 0.0190.89 mg/LRSD not available

ACS-K, SAC, ASP, DUL,ALI, caffeine, benzoicacid, sorbic acid

Low-energy soft drinks,cordials, tomato sauce,marmalades, jams, table-top sweeteners

MEKC-UV Buffer consisting of Nadeoxycholate, K-dihydrogenorthophosphate, Naborate (pH 8.6)

Uncoated fused-silicacapillary (75 cm 75 lm)

Recovery 104112% [90]LOD not availableRSD 0.632.6%

ACS-K, SAC, ASP,preservatives,antioxidants

Cola beverages, low-energy jams

MEKC-UV Borate buffer with Na cholate,dodecyl sulfate, MeOH (pH 9.3)

Fused-silica capillary (52 cm 75 lm)

Recovery 98.9100.86% [91]LOD not availableRSD 0.91.5%

ACS-K, SAC, ASP,preservatives, colors

Soft drinks MEKC-UV Carbonate buffer (pH 9.5) withsodium dodecyl sulfate

Uncoated fused-silicacapillary (48.5 cm 50 lm)

Recovery [92]LOD 0.005 mg/mLRSD not available

ACS-K, SAC, ASP, MALg,SORh, XYLi, LACj

Candies, chewing gums CITP-conductivitydetector

Leading electrolyte: HCl + Tris Dionex Ion Pac AS4A-SC(254 4 mm)

Recovery 98.2102.5% [93]Terminating electrolyte: L-histidyne + Tris

LOD 0.0240.081 mMRSD 0.82.8%

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.

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Besides HPLC, other separation methods have alsofound applications in this area. Ion chromatography (IC)is a suitable tool for multi-sweetener analysis in foodproducts. Instead of organic solvent-mediated separa-tions, IC separations are performed by using innocuousand inexpensive salt solutions as the eluents.

Capillary electrophoresis (CE) is another attractiveseparation technique, useful for simultaneous determi-nation of multiple sweeteners. In some situations, CEseems to be superior to HPLC in terms of separationpower, analysis time or low solvent consumption.

When there is a need to determine just one or twosweeteners in a given sample, it is often more reasonableto apply one of the simple, rapid, reproducible analyticalmethods, specifically tailored to the particular product.So far, such rapid methods have been published fordetermination of acesulfame-K, saccharin, cyclamatesand aspartame. They utilize specific reactions or detec-tion systems rather then employing powerful but costly,universal separation-detection combos. Compared tomulti-analyte counterparts, these procedures are oftenless time consuming and less labor intensive, while beingcomparable in terms of accuracy, precision and sensi-tivity. Flow-injection analysis (FIA), electrochemical andspectroscopic methods provide ways to obtain analyticalresults in minimum time. Table 3 sets out examples ofthis kind of approach.

4.1. Techniques for final determination of analytesThis section covers nine techniques of final determina-tion of sweeteners. We first discuss papers dealing withthe determination of a single sweetener, then multi-analyte methods, if applicable.

4.1.1. High-performance liquid chromatographyLC is the most popular choice for determination of high-intensity sweeteners. HPLC procedures are based onisocratic or gradient RP chromatographic separation.Various detection systems in conjunction with LC areapplied [e.g., ultraviolet (UV) spectrophotometry,amperometry, coulometry, mass spectrometry (MS),spectrofluorometry, light scattering and conductometry].

4.1.1.1. Methods for determination of individual sweeten-ers. Many HPLC methods have been reported forquantitation of aspartame. RP-HPLC with spectropho-tometric detection (205 nm, 214 nm and 215 nm) andwith the mobile phase containing phosphate buffer andacetonitrile and/or methanol is the primary tool for thedetermination of aspartame [2426]. Most of them weredeveloped on 250-mm columns, but aspartame and itsmetabolites can be separated using short 30-mmcolumns with UV detection. Since the column is shorter,analysis time is reduced to less then 10 min but theselectivity is worse than methods employing longer 250-mm columns [27].

It is well known that spectrofluorometric detectionachieves better selectivity and sensitivity than spectro-photometric detection. Aspartame and its hydrolysisproducts can be determined spectrofluorometricallyusing pre-column derivatization. Aspartylphenylalanine,aspartic acid and phenylalanine react with naphthalene-2,3-dicarboxaldehyde in the presence of cyanide ion in amildly alkaline medium to give highly fluorescent 1-cy-ano-2-substituted benz[f]isoindole derivatives. LODs inthe sub-picomole range were reported for these com-pounds using HPLC with spectrofluorometric detection(kex = 420 nm, kem = 490 nm) [28]. A similar methodwith pre-column derivatization using fluorescamine wasalso reported [29]. The main drawback of these methodsis the necessity of a lengthy derivatization procedure.Native fluorescence of aspartame can be used for itsdetection in HPLC.

Aspartame and phenylalanine produce native fluo-rescence emission with the maximum at 284 nm. Anexcitation wavelength of 205 nm was found to give thebest signal-to-noise ratio. This method does not requirederivatization. Moreover, use of spectrofluorometricdetection yields much better sensitivity and slightlybetter precision than spectrophotometric detection [30].

Aspartame was also determined by HPLC with elec-trochemical detection. Aspartame, normally electro-chemically inactive, was made oxidizable by post-column photolytic derivatization (irradiation at254 nm). The use of a post-column photochemicalreactor can be helpful for unequivocal identification ofaspartame using electrochemical detection [31].

The common HPLC-UV detection mode is not suitablefor determination of cyclamate, because of the lack of UVchromophore in its molecule. Some complicated andtime-consuming procedures are necessary for theabsorbance detection of this sweetener in HPLC. Thisproblem has been solved by using indirect UV photom-etry, post-column ion-pair extraction, and pre-columnderivatization. One of the first approaches employedpost-column ion-pair extraction with absorbance detec-tion for the LC determination of cyclamate. After chro-matographic analysis the sweetener was mixed with anappropriate dye (methyl violet or crystal violet) and de-tected 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 byabsorption in the visible range [32].

Another HPLC method with UV detection at 314 nmutilized the conversion of cyclamic acid to N,N-dichlorocyclohexylamine [33]. Cyclamate can be deter-mined in strongly colored and protein-rich foodstuffsafter its oxidation to cyclohexylamine and derivatizationwith 4-fluoro-7-nitrobenzofurane. There are two possibleways of detection: absorbance at 485 nm and fluores-cence with excitation at 485 nm; and, emission at530 nm [34].


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 spectrophotometricdetection

Table-top sweeteners Recovery n.a. [95]LOD 2 lg/mLRSD 0.2%LR n.a.

ASP FIA with spectrophotometricdetection

Chewing gums, custards,sweets, jams

Recovery 95101% [96]LOD 0.82 lg/mLRSD 0.55%LR 5.0600 lg/mL

ASP, ACS-K FIA with spectrophotometricdetection

Table-top sweeteners Recovery n.a. [111]LODASP 5.6 lg/mLRSDASP 3.4%LRASP 10.0100.0 lg/mLRecovery n.a.LODACS-K 0.1 lg/mLRSDASP 1.61%LRASP 40.0100.0 lg/mL

ASP, CYC Square-wave voltammetry inconjunction with borondoped diamond electrode

Powdered juice drinks,carbonated guaranadrinks

Recovery n.a. [124]LODASP 4.7 107mol/LLODCYC 4.2 106mol/LRSD 1.11.3%LRASP 5.0 1064.0 105

mol/LLRCYC 5.0 1054.0 104

mol/L

ASP Square-wave voltammetry inconjunction with boron-doped diamond electrode

Dietary products Recovery n.a. [123]LOD 2.3 107 mol/LRSD 0.2%LR 9.9 106 5.2 105 mol/L

ASP Biosensor based on carboxylesterase-alcohol oxidaze

Soft drinks, Table-topsweeteners

Recovery 102.5106.3% [121]LOD n.a.RSD n.a.LR 5.0 1084.0 107 M

ASP Biosensor based on graphiteepoxy composite electrode

Diet cokes Recovery 101.84104.08% [122]LOD n.a.RSD n.a.LR 2.5400 lM

ASP, ACS-K FTIR Table-top sweeteners Recovery n.a. [136]LODASP 560 lg/g; LODACS-K 7200 lg/gRSD 0.170.5%LR n.a.

ACS-K, SAC Spectrophotometry/ inconjunction withchemometric analysis

Solid and liquidsweeteners, fruit juices

Recovery 93.8105.1% [133]LODACS-K 0.085 lg/mL; LODSAC0.0312 lg/mLRSD n.a.LR n.a.

ACS-K, CYC, SAC Spectrophotometry inconjunction withchemometric analysis

Beverages, applevinegars, yoghurts, milkdrinks

Recovery 96.7106.0% [134]LODACS-K 0.08 lg/mL; LODSAC0.2 lg/mLLODCYC 0.019 lg/mLRSD 14%LRACS-K 0.24.8 lg/mL; LRCYC0.510.0 lg/mLLRSAC 0.85.6 lg/mL




Analyte Analytical technique Sample Analytical parametersa,b,c,d Ref.

CYC FIA with spectrophotometricdetection

Table-top sweeteners Recovery n.a. [106]LOD 7.7 lmol/LRSD 3.5%LR < 1000 lmol/L

CYC FIA with spectrophotometricdetection

Table-top sweeteners Recovery n.a. [105]LOD 30 lmol/LRSD 1.7%LR 1003000 lmol/L

CYC FIA with AAS detection Soft drinks Recovery 95.7100.5% [108]LOD 0.25 lg/mLRSD 3.1%LR 190 lg/mL

CYC, SAC FT-Raman spectrometry inconjunction with chemometricanalysis

Table-top sweeteners Recovery n.a. [135]LODCYC 0.83% w/w; LODSAC0.2% w/wRSD 0.55%LR n.a.

SAC FIA with AAS detection Table-top sweeteners Recovery n.a. [101]LOD 3 lg/mLRSD 2.7%LR 575 lg/mL

SAC FIA with spectrophotometricdetection

Soft drinks, juices, bakery products,Table-top sweeteners

Recovery 98104% [102]LOD 0.2 lg/mLRSD 0.78%LR 1.0200 lg/mL

SAC Potentiometric titration using asilver electrode as the indicatorelectrode

Solid and liquid sweeteners Recovery 97.0102.6% [114]LOD 2.5 lg/mLRSD n.a.LR n.a.

SAC Potentiometric membrane sensor Dietary products Recovery 98.2103.1% [117]LOD n.a.RSD n.a.LR 1.0 101 5.0 105 mol/L

SAC Potentiometric sensorPt|Hg|Hg2(Sac)2|Graphite

Diet soft drinks, jams Recovery 97.6102.0% [118]LOD 3.9 107 mol/LRSD 1.82.3%LR 5.0 1071.0 102mol/L

SAC Spectrophotometry Solid and liquid sweeteners Recovery 99.2104.3% [128]LOD 1.55 105 MRSD 0.51.6%LR n.a.

a LOD, Limit of detection.b RSD, Relative standard deviation.c LR, Linear range.d n.a., Not available.




A similar method was proposed for analysis of thissweetener in juices and preserves. Cyclamate was oxi-dized to cyclohexylamine and pre-chromatographicallyconverted into a fluorescent derivative [35].

Indirect UV photometry has been shown to be a verysensitive detection method useful for detection of thissweetener [36].

Cyclamate was determined in soft drinks using RP-HPLC combined with indirect visible photometry at433 nm. The analytical signal was derived from changesin absorbance of mobile phase with addition of chro-mogenic dye (Methyl Red) [37].

An HPLC-electrospray ionization (ESI)-MS methodusing tris(hydroxymethyl) aminomethane as an ion-pair-forming agent was employed for analysis of cycla-mate in foods. Cyclamate was separated on a C8 columnin isocratic mode with 100% aqueous mobile phase. MSwas operated in negative, selected ion (m/z = 178)recording mode. The method was found to be highlysensitive, specific and simple [38].

An HPLC-tandem MS (HPLC-MS2) method character-ized by minimal sample preparation, high sensitivity andselectivity was developed for the determination ofcyclamate 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/vis-ible (UV-Vis) range, making sensitive and specificdetection by direct UV absorption difficult, a derivatiza-tion procedure is necessary. This task can be accom-plished using p-nitrobenzoyl chloride (PNBCl). Sucralosetreated with PNBCl is converted into a strongly UV-absorbing derivative, having strong absorption at260 nm, which allows for its sensitive, direct UV detec-tion [40]. Another solution is to use refractive index (RI)[41] or MS detector.

A sensitive LC-MS2 method has been reported for thedetermination of sucralose in various foods (soft drinks,yoghurt, chocolate and chewing gum). Sample extractswere separated using C18 column in gradient-elutionmode. MS acquisition was done in negative selectedreaction monitoring (SRM) mode, monitoring the395fi359 transition [42].

The HPLC-UV method seems to be the most popularoption for the determination of neohesperidine dihydro-chalcone (NHDC). NHDC can be analyzed using astandard C18 column in gradient mode. The optimalanalytical wavelength is 282 nm, since common foodcomponents (e.g., sweeteners and preservatives) aretransparent or absorb very slightly at this wavelength[4347].

4.1.1.2. Multi-analyte approaches. Despite the highresolving power of HPLC as a separation tool, HPLC-based methods for the determination multiple sweetenersare quite rare.


Separation and quantitation of seven artificial sweet-eners (aspartame, saccharin, cyclamate, acesulfame-K,sucralose, alitame and dulcin) can be accomplishedusing a C18 column working in gradient (organic sol-vent and pH) mode. Aspartame, acesulfame-K, saccha-rin, alitame and dulcin were detected using UVabsorbance at 210 nm or 200 nm. Cyclamate andsucralose were not detected by UV absorption, butdetermined by an ion-pair extraction system and RIdetection, respectively [48].

An HPLC-UV procedure that can simultaneouslydetermine aspartame and saccharin was found suitablefor reliable control of pharmaceutical and dietary for-mulations [49].

Simultaneous determination of acesulfame-K, sac-charin and preservatives in beverages and jams can alsobe accomplished using an HPLC-UV technique [50].Three artificial sweeteners (acesulfame-K, aspartameand saccharin), vanillin, benzoic and sorbic acids wereseparated and quantified using a C18 column working inisocratic mode. A diode-array detector (DAD) was usedand different analytical wavelengths were selected foreach analyte (230 nm for acesulfame-K, 220 nm forsodium saccharin, 203 nm for a-aspartame, 280 nm forvanillin, 225 nm for benzoic acid and 256 nm for sorbicacid). The method was found useful to monitor thecontent of sweeteners and preservatives in cola and in-stant-powder drinks [51].

Another HPLC-UV method was proposed for deter-mination of the aforementioned sweeteners, preserva-tives and dyes present in soft drinks [52]. Good separa-tion was obtained with a run time less than 20 min, witha satisfactory precision. Neotame, alitame and aspar-tame were quantified in various foods with the aid ofHPLC-UV. Furthermore, these three sweeteners weresuccessfully identified by ultra-HPLC (UHPLC)-MS2.Positive mode ESI was used, fragmentation spectra ofions 379, 332 and 295 (m/z) were recorded for identi-fication of neotame, alitame and aspartame, respectively[53].

Ion-pair RP-LC with UV detection (233 nm) was em-ployed for simultaneous determination of three sweet-eners (acesulfame-K, saccharin and dulcin),preservatives and antioxidants in food samples. Theanalytes were separated using a C18 column and amobile phase comprising an acetonitrile - aqueous a-hydroxyisobutyric acid solution with the addition ofhexadecyltrimethylammonium bromide as an ion-pair-forming agent [54].

HPLC with evaporative light-scattering detection(ELSD) was used for determination of nine sweeteners(acesulfame-K, alitame, aspartame, cyclamic acid, dul-cin, neotame, neohesperidine dihydrochalcone, saccha-rin and sucralose) in various food products.Interlaboratory study showed the applicability of themethod in use by control laboratories [15,55].


Six artificial sweeteners (acesulfame K, saccharin,sucralose, cyclamate, aspartame, dulcin) and threenatural sweeteners (glycyrrhizic acid, stevioside, rebau-dioside A) were quantified in different foods by HPLC-MS.Under negative ESI conditions, quantitation wasachieved by monitoring the selected ions for eachsweetener [22].

Recently, an HPLC-MS method was published for thedetermination of seven artificial sweeteners (aspartame,saccharin, acesulfame-K, neotame, sucralose, cyclamateand alitame) and one natural sweetener (stevioside).Unlike many other methods, this one does not includeany clean-up step. Food samples were extracted withmethanol-water mixture and, after filtration, injectedinto the HPLC-MS system. The target compounds werequantified using selective ionization recording (SIR) atm/z 178, 397, 377, 293, 641, 312, 162 and 182 tocyclamate, sucralose, neotame, aspartame, stevioside,alitame, acesulfame-K and saccharin, respectively, withwarfarin sodium (m/z = 307) being used as an internalstandard [23].

4.1.2. Ion chromatographyHigh-performance IC (HPIC) offers an attractive alter-native to the aforementioned HPLC methods. There arefew reported methods for the determination of individualsweeteners.

4.1.2.1. Methods for determination of individual sweeten-ers. Acesulfame-K was determined in various foodstuffsby anion-exchange chromatography coupled to a con-ductivity detector. The separation was achieved by usinga Dionex AS5 anion-separation column and sodiumcarbonate as a mobile phase [56]. Quantitation ofsodium saccharin in shrimps has also utilized high-performance anion-exchange chromatography (HPAEC)[57].

An IC method with integrated amperometric detectionwas proposed for determination of aspartame in drinkand powder forms. A Dionex AS4A-SC separationcolumn was applied to separate this sweetener fromother food additives. Moreover, commonly addedsweeteners (e.g., sodium saccharin, acesulfame-K andcyclamate) give no electrochemical signal at all, andcould be eliminated as interference [58].

A few procedures for determination of sucralose invarious food and beverage products were reported. In allcases, HPAEC with pulsed amperometric detection (PAD)was applied. The high resolving power of HPAEC and thespecificity of PAD allow the determination of sucralosewith little interference from other ingredients. Highprecision, method ruggedness, and high spike recoveryare possible for these complex sample matrices [5961].

4.1.2.2. Multi-analyte approaches. There are few re-ported methods using IC with different detection modes

for simultaneous determination of multiple sweeteners.Four artificial sweeteners (acesulfame-K, aspartame,saccharin and cyclamate) and citric acid were separatedby AEC and quantitated using UV detection in combi-nation with conductivity detection [62].

A suppressed conductivity detector alone was foundsensitive enough for simultaneous determination of foursweeteners (aspartame, acesulfame-K, sodium saccharinand sodium cyclamate) in foods by IC when KOH eluentgenerator was used [63]. Good agreement of resultsobtained by AEC with those provided by HPLC was ob-served in the case of simultaneous determination of threeartificial sweeteners (saccharin, acesulfame-K andaspartame), two preservatives, caffeine, theophylline andtheobromine in foods. Analytes were separated in iso-cratic mode and detected by UV detector [64].

4.1.3. Thin-layer chromatographyThin-layer chromatography (TLC) has been used foridentification and/or determination of artificial sweet-eners, because the equipment needed is simple, inex-pensive and flexible.

4.1.3.1. Methods for determination of individual sweeten-ers. The content of saccharin in soft drinks can bemeasured using TLC and UV spectrophotometry. Afterchromatographic 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 thequantification of sucralose in various food matrices. Themethod requires little or no sample preparation to isolateor to concentrate the analyte. The separation of sucralosewas 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, cyclamateand dulcin can be extracted from foods with ethyl ace-tate and separated and quantified by TLC analysis usingpolyamide plates [67]. The same combination of sweet-eners was determined in soft drinks using silica-gel Hplates [68]. Polyamide plates were also employed foridentification of acesulfame-K, saccharin and cyclamatein foodstuffs and cosmetics. Analytes were separatedusing a mixture of xylene, n-propanol and formic acid[69]. Aspartame, acesulfame-K cyclamate weredetermined in sparkling and non-sparkling drinks usingsilica-gel G plates and an ethanol-isopropanol-aqueousammonia system [70].

4.1.4. Gas chromatographyAs an analytical tool, gas chromatography (GC) is notused very often for determination of sweeteners becauseof 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.



The derivatization step is not only labor-intensive andtime-consuming but also a possible source of erraticresults.

4.1.4.1. Methods for determination of individualsweeteners. Several methods were developed forGC determination of saccharin. Esterification withN,O-bis(trimethylsilyl)acetamide [71], N-methylationusing diazomethane [72] and methylation with trim-ethylsilyldiazomethane [73] are commonly used tech-niques.

Various GC methods for the determination of cycla-mate have been developed. This sweetener can bedetermined in soft drinks by GC detection of the cyclo-hexene produced during pre-column derivatization withnitrous acid [74]. However, it was noted that formationof other products (e.g., monochlorocyclohexane, cyclo-hexanone and cyclohexanol) can occur and hinder theanalysis. A modified method employed acidification ofthe sample with sulfuric acid instead of hydrochloricacid, prior to reaction with sodium nitrite. This pre-cluded the formation of monochlorocyclohexane [75].These procedures were improved by replacing benzene,as an internal standard, with decane. This change in-creased the sensitivity and the precision of results [76].

Aspartame and its decomposition products were ana-lyzed by GC-MS after derivatization with BSA-N,O-bis(trimethylsilyl)acetamide [77].

Pyrolysis GC-MS was applied for the determination ofaspartame in table-top sweeteners and soft drinks. Nopreliminary sample extraction or derivatization wasnecessary [78].

Sucralose can be analyzed by GC after silylation.Treating sucralose dissolved in pyridine with the mixtureof hexamethyldisilazane (HMDS) and trimethylchlorosi-lane (TMS-Cl) yields volatile sucraloseTMS ether.Identification and quantification of sucralose was doneby means of GC-MS and GC-flame-ionization detection(FID), respectively [79].

4.1.4.2. Multi-analyte approaches. Saccharin, cyclamateand dulcin were determined together by GC. Saccharinand cyclamate were derivatized with diazomethane.Dulcin does not undergo methylation with diazometh-ane, but it is volatile enough to be analyzed by GCwithout derivatization [80].

Simultaneous determination of saccharin and acesul-fame-K in foods can be achieved by means of GC-nitro-gen phosphorus detection (NPD). Both sweeteners weremethylated with diazomethane prior to analysis [81].

4.1.5. Capillary electrophoresisCE is a powerful tool in food analysis. Due to its highresolving power, low solvent consumption and ease ofautomation, CE is a viable alternative to HPLC fordetermination of high-intensity sweeteners in various


foodstuffs. Different modes of CE have been used in theanalysis of individual sweeteners or sweetener mixtures.Capillary-zone electrophoresis (CZE), micellar electroki-netic chromatography (MEKC), and capillary isotacho-phoresis (CITP) are the modes of CE used most. Theelectrolytes used to fill the silica capillary are generallybuffers based on phosphate, borate, benzoate or glycin-ate with pH values in the range (69). Several papershave demonstrated the utility of CE for the analysis ofsingle sweeteners.

4.1.5.1. Methods for determination of individual sweeten-ers. A CZE method was employed for determination ofaspartame in foods using a 30 mM phosphate-19 mMTRIS buffer with detection at 211 nm. The analysis timewas significantly shorter than that reported for HPLCand 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 ofaspartame at pH below 3 [82].

Simultaneous separation of aspartame, caffeine andbenzoic acid in soft drinks can be achieved at pH 11using 25 mM phosphate buffer. Separation of these threecompounds was completed within 10 min. However,achievement of good reproducibility was difficult due toproblems with keeping the capillary walls in the sameconditions between days of analysis [83].

Aspartame, caffeine and benzoic acid can be deter-mined in soft drinks using a glycine buffer which providemuch shorter migration times than borate buffers. Sep-aration of analytes was achieved in 2 min using a20 mM glycine buffer at pH 9 and direct spectrophoto-metric detection at 215 nm. Excellent reproducibility ofthe method was also reported [84].

Cyclamate has also been successfully determined byCE methods. Indirect UV detection at 254 nm was usedfor the determination of cyclamate in beverages andjams. Sample preparation was minimal. Beverages werediluted with deionized water, and jams blended withdeionized water and filtered. Uncoated fused-silica cap-illary column with an electrolyte consisting of 1 mMhexadecyltrimethylammonium hydroxide in 10 mM so-dium benzoate was employed. The analysis time was lessthan 5 min [85].

This method has been modified for use with thebroader range of foods by addition of an SPE clean-upstep. Sample extracts were passed through the Oasis HLBSPE cartridges in order to remove interfering com-pounds. The running buffer containing 1 mM hexade-cyltrimethylammonium bromide in 10 mM potassiumsorbate was used for separation. Detection and referencewavelengths of cyclamate were 300 nm and 254 nm,respectively [86].

CE with indirect absorption measurement is also asuitable tool for monitoring the content of sucralose invarious foodstuffs. Sucralose determination in low-calo-


rie soft drinks can be achieved without any sample cleanup using 3,5-dinitrobenzoate buffer at pH 12.1 andindirect UV detection at 238 nm [87].

The scope of the method has been extended to includethe possibility of analysis of yoghurts and candies byintroducing a sample clean-up step (centrifugation, fil-tration, and SPE on Alumina A cartridges) [88].

Neohesperidine dihydrochalcone (NHDC) is a minorcomponent of sweetener blends. It was separated fromother commonly-added sweeteners using a 100 mMborate buffer (pH 8.3). Direct UV detection was applied,and a wavelength of 282 nm was found optimal to avoidinterferences from other sweeteners and sugars. Themethod was successfully applied to the determination oflow levels of NHDC in soft drinks, fruit juices and yo-ghurts [89].

4.1.5.2. Multi-analyte approaches. Different modes of CEwere applied for simultaneous determination of severalartificial sweeteners. MEKC is the most common mode.Aspartame, acesulfame-K, saccharin, alitame, dulcin,caffeine, benzoic acid and sorbic acid were separated in asingle run by MEKC. The complete separation wasachieved in less than 12 min using uncoated fused-silicacapillary column and 10 mM phosphate-10 mM boratebuffer at pH 8.6 with 50 mM sodium deoxycholate as themicellar phase. The analytes were detected by direct UVspectrophotometry at 220 nm. The method was appliedto a range of foods including low-energy soft drinks,tomato sauce, marmalade and table-top sweeteners [90].

A more sophisticated buffer system was developed forMEKC-UV (214 nm) determination of aspartame, sac-charin and acesulfame-K together with several antioxi-dants and preservatives in cola beverages and low-energy jams. The separation of the mixture was suc-cessfully accomplished utilizing a 20 mM borate bufferwith 35 mM sodium cholate, 15 mM sodium dodecylsulfate 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 preser-vatives and food colors used as food additives in softdrink was also presented. The mixture of such additiveswas successfully separated using MEKC under optimizedconditions using a 20 mM carbonate buffer at pH 9.5with 62 mM sodium dodecyl sulfate as the micellarphase. Sample components were identified by a DAD inthe UV-Vis range (190600 nm) [92]. Four artificialhigh-intensity sweeteners (acesulfame-K, saccharin,cyclamate and aspartame) and four bulk sweeteners(lactitol, sorbitol, mannitol and xylitol) were determinedin candies and chewing gums by means of CITP withconductivity detection. The separation was achieved inabout 20 min using a capillary filled with electrolytesystem 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].

4.1.6. Flow-injection analysisFIA is a powerful technique for automated, continuousdetermination of artificial sweeteners, especially whenonly one or two analytes need to be determined in alarge number of samples. The use of FIA methodologiespresents advantages (e.g., high sample throughput, lowconsumption of sample and reagents, highreproducibility, speed of analysis, and simple and auto-mated operation).

4.1.6.1. Methods for determination of individual sweeten-ers. Aspartame in table-top sweetener, pudding, gela-tin, and powdered drink was determined by aspectrophotometric FIA method using ninhydrin as acolorimetric reagent. Aspartame reacted with ninhydrinin a methanol-isopropanol medium containing potas-sium hydroxide. The absorbance measurements weremade at 603 nm [94].

Another spectrophotometric FIA method employed asolid-phase reactor with copper (II) phosphate. Aspar-tame reacts with immobilized copper (II) ions givingCu(II)(aspartame)2 complex. Release of Cu (II) occurswhen the complex is mixed with alizarin red S solution,which forms another colored complex. The absorbanceof Cu(II)-alizarin complex is measured at 550 nm. Themethod was used for the determination of aspartame intable-top sweeteners [95].

A very simple, sensitive flow-through spectrophoto-metric sensor was developed for determination ofaspartame in low-calorie and dietary products. Thesensor was implemented in a monochannel flow-injec-tion (FI) system with UV spectrophotometric detectionusing Sephadex CM-C25 cationic exchanger packed in aflow cell. Aspartame was determined by measuring itsintrinsic absorbance at 219 nm at its residence time (pH5.0) without any derivatization [96].

Biosensors have also been used for the measurementof aspartame in foodstuffs. An FIA-biosensor systemcomprised two enzyme columns, containing purifiedpeptidase and aspartate aminotransferase, respectively,immobilized on activated aminopropyl glass beads andan enzyme electrode connected in series [97].

The same authors proposed a system comprising anenzyme column of pronase immobilized on activatedarylamine glass beads and 1-amino acid oxidase elec-trode connected in series [98].

An amperometric enzyme electrode for the determi-nation of aspartame was developed by covalent immo-bilization of alcohol oxidase and a-chymotrypsin on thesurface of platinum-based hydrogen peroxide electrode.The electrode was used in FI mode for the determina-tion of aspartame in seven different food matrices, withgood recoveries. Sensitivity was significantly better thanthose of previously constructed aspartame electrodes[99].



Saccharin in dietary product was analyzed using aprecipitation FIA method. Saccharin was precipitated asmercurous saccharinate and the mercury-cation excesswas measured potentiometrically using silver wirecoated with a mercury film as the working electrode.High sample throughput (60 samples/h) could beachieved with this method [100].

Another precipitation FIA method, which utilizedatomic absorption spectrometry (AAS) as a detectiontechnique, permitted indirect determination of saccharinin the presence of other sweeteners. It was based on thecontinuous precipitation of saccharin with AgNO3 in aflow manifold, followed by filtration, washing, dissolu-tion in ammonia and measurement of dissolved silver byflame atomic absorption spectrometry (FAAS) [101].

A flow-through spectrophotometric sensor for moni-toring the content of saccharin in low-calorie productsutilized the transient adsorption of the sweetener on solidphase packed in a flow cell, followed by absorbancemeasurement at 217 nm [102].

FIA procedures have also been proposed for determi-nation of cyclamate in foodstuffs, involving detection bychemiluminescence, spectrophotometry, FAAS oramperometry. An FIA method based on the sensitizingeffect of sodium cyclamate on the chemiluminogenicoxidation of sulfite by cerium (IV) has been described[103].

Cyclamates in table-top sweeteners and some lowcalorie soft drinks were determined by an FIA spectro-photometric method. The procedure utilized the reactionbetween nitrite and cyclamate in medium containingphosphoric acid. The excess of nitrite was determined bythe measurement of absorbance of Griess reactionproduct at 535 nm [104].

Hydrolysis of cyclamate to cyclohexylamine has beenemployed for the analysis of table-top sweeteners. Thehydrolysis step was performed batch wise by treatingcyclamate with hydrogen peroxide and hydrochloricacid. The cyclohexylamine was derivatized with 1,2-naphthoquione-4-sulfonate (NQS) in an FI system. TheNQS derivative was monitored at 480 nm [105].

An interesting FIA system for the determination ofcyclamate in table-top sweeteners has been proposed.The procedure is based on the reaction of cyclamate withnitrite in acidic medium. The excess nitrite reacts withiodide to form the triiodide anion, which is determinedspectrophotometrically at 350 nm. No toxic reagents areemployed and the amounts of chemicals consumed andwastes generated are minimized by replacing a tradi-tional continuous-flow system with a set of sequentially-operated solenoid micro-pumps [106].

Automated turbidimetric determination of cyclamatein low-calorie soft drinks and sweeteners (without pre-treatment) employed oxidation to sulfate by nitrite, fol-lowed by precipitation with barium and measurement ofanalytical signal at 420 nm [107].


Cyclamate was also determined by an indirect AASmethod using a flow system with precipitate dissolution.It was based on oxidation of the sulfamic group to sulfateand continuous precipitation with lead ion in a flowmanifold, followed by filtration, washing, dissolution inammonium acetate and measurement of lead by FAAS[108].

A bioamperometric titration procedure was foundsuitable for the determination of cyclamate in variousdietary products. After the reaction of cyclamate withthe nitrite, the excess of nitrite was detected in a poly-urethane cell containing two platinum wires polarized at0.7 V [109].

4.1.6.2. Multi-analyte approaches. There are only veryfew reported FIA procedures suitable for simultaneousdetermination of artificial sweeteners.

Cyclamate, acesulfame-K and saccharin were deter-mined in table-top sweeteners, yoghurts, soft diet drinksand wines by means of an FIA system containing filter-supported bilayer membrane (BLM) formed usinglyophilized egg phosphatidycholine. The detection wasperformed using two Ag/AgCl reference electrodes biasedby an external power supply at the level of +25 mV(sensing electrode). Transient electrical signals with dif-ferent time delays were observed after injection of themixture of sweeteners into the stream of the carrierelectrolyte [110].

Simultaneous determination of aspartame and ace-sulfame-K in table-top sweeteners was demonstratedusing a multi-analyte flow-through sensor. Theprocedure was based on the transient retention ofacesulfame-K in the ion exchanger Sephadex DEAE A-25placed in the flow-through cell of a monochannel FIAsystem using pH 2.70 ortophosphoric acid/sodiumdihydrogen phosphate buffer 0.06 M as carrier. In theseconditions aspartame is very weakly retained, and thatmakes it possible to measure the intrinsic UV absorbanceof first aspartame and then acesulfame-K after desorp-tion by the carrier itself [111].

Aspartame, acesulfame-K, saccharin and a few anti-oxidants and preservatives were simultaneously deter-mined in selected foods using a single-channel FIAsystem incorporating a short monolithic C18 pre-col-umn. The separation of analytes was based on theirretention time and quantification was done by means ofa DAD. This system should be treated as a simple, low-resolution liquid chromatograph [112].

4.1.7. Electrochemical techniquesElectrochemical techniques are powerful and versatiletool for determination of artificial sweeteners, becausethey are simple, fast and low cost. Moreover, they givethe opportunity to determine analytes directly even inturbid and colored solutions. Several electrochemicaltechniques have been developed for the analysis of these


compounds separately or in mixtures in dietary prod-ucts.

4.1.7.1. Methods for determination of individual sweeten-ers. There are two basic approaches concerning apotentiometric determination of saccharin. The first isbased on precipitation titration of saccharin with eithermercurous [113] or silver [114] ions. The other way isto use an ion-selective electrode (ISE) sensitive to sac-charinate ions. Most constructions of such electrodescomprise a graphite rod coated with a dopedpoly(vinylchloride) membrane. Usually, the dopant is anion pair formed between saccharinate anion and acounter cation. Basic dyes [115], silesquioxane 3-n-propylpirydinium chloride [116] and Aliquat 336S[117] were employed as ion-pair forming agents.

An interesting saccharinate-sensitive ISE, based on asolid, polycrystalline membrane has also been proposed.The membrane was prepared by compressing a mixtureof mercury saccharinate, graphite and a small amount ofmetallic mercury. A wide linear range (102106 M)and relatively long lifetime have been reported for thissensor [118].

Various electrochemical methods have been proposedfor aspartame. Most of them utilized biosensors. Abienzymatic electrode, which utilized the chemical co-immobilization of carboxypeptidaze and L-aspartase onan ammonia-gas-sensing electrode, has been employedfor the determination of aspartame in several dietaryproducts. This electrode suffered from a very narrowlinear range and short lifetime [119].

Another method used an amperometric biosensorcomprising a-chymotrypsin and alcohol oxidase, whichwere co-immobilized on a dissolved oxygen electrode[120]. The bienzyme system comprising carboxyl ester-ase and alcohol oxidase, immobilized in gelatin mem-brane, was another way of monitoring of the aspartamecontent.

In order to determine the concentration of aspartame,oxygen consumption during the enzymatic reaction wasmeasured using an oxygen meter [121].

The same bienzyme system was integrated withgraphite epoxy composite electrode (GECE). Greaterlinear range and a longer lifetime were claimed for thisset-up [122].

Determination of aspartame by square-wave voltam-metry was also reported [123].

4.1.7.2. Multi-analyte approaches. There are only a fewreported electrochemical methods suitable for simulta-neous determination of several artificial sweeteners infoodstuffs. One of them described a simultaneous square-wave (SW) voltammetric method for determination ofaspartame and cyclamate in beverages. The use of SWvoltammetry in conjunction with boron-doped diamondelectrode gives an opportunity to separate the oxidation-

peak potentials of aspartame and cyclamate by 400 mV.The results showed that the proposed method wassimple, inexpensive and very sensitive [124].

4.1.8. Spectroscopic techniquesVarious types of spectroscopic techniques have beenapplied for analysis of sweeteners in foodstuffs. UV-spectrophotometric methods are the most widely used,but Raman and infrared spectroscopy have also beenused for this purpose.

4.1.8.1. Methods for determination of individual sweeten-ers. A lot of spectrophotometric methods have beenreported for determination of saccharin.

The first spectrophotometric methods of saccharindetermination based on reaction with azure B [125],Nile-blue [126] or astrazone pink FG [127] were oftentime-consuming and tedious.

A simple, rapid and sensitive spectrophotometricmethod was proposed for routine analysis of saccharin inliquid and solid sweeteners. Saccharin was reacted withp-chloranil in the presence of hydrogen peroxide, andthat resulted in formation of a violet-red compound,exhibiting an absorption maximum at 550 nm. Themain drawback of this method was a strong interferencefrom cyclamate, which had to be eliminated duringsample preparation by precipitation in ethanol [128].

A spectrophotometric method involving ninhydrin asa colorimetric reagent was proposed for the determina-tion of aspartame. To increase the selectivity of thedetermination, extraction with propylene carbonate wasnecessary [129].

Another spectrophotometric method for the determi-nation of aspartame in beverages was based on theenzymatic conversion of aspartame into formaldehydeby the a-chymotrypsinalcohol oxidase system. Subse-quently, formaldehyde was exposed to derivatizationwith Fluoral-P [130].

A rapid method for the determination of aspartame insoft drinks utilized Fourier transform infrared spectros-copy with an attenuated total-reflectance accessory andpartial least-squares regression. The method gives goodresults for the samples with well-defined matrices, so it issuitable for routine quality-control analysis of aspartamein the beverage-manufacturing sector [131].

Aspartame, saccharin and acesulfame-K can bedetermined (but not simultaneously) in various foodproducts by an extractive spectrophotometric techniqueusing an oxazine dye, Sevron Blue 5G, as the reagent.Those sweeteners form chloroform-extractable ion-asso-ciation complexes with the dye. The content of asweetener can be determined by measuring the absor-bance of a chloroform extract at 655 nm [132].

4.1.8.2. Multi-analyte approaches. An analyticalprocedure for simultaneous determination of aspartame,



saccharin and acesulfame-K was based on the oxidativereaction of the analytes with KMnO4 in alkaline solution.The green potassium-magnate reaction product fromthree sweeteners was formed at different kinetic rates,which allowed for selective determination of each ana-lyte. To improve the accuracy and the precision ofanalysis, chemometric multivariate-calibration methodswere applied [133].

A simple, rapid method was used to determine ace-sulfame-K and saccharin in liquid and solid sweetenersand powdered fruit juice. The application of UV-Visspectrophotometry coupled to chemometric analysis[partial least squares (PLS)] allowed the determination ofthese compounds in real samples. Strong interferencefrom aspartame was taken into account during calibra-tion and validation [134].

A PLS Fourier-transform Raman spectrometry proce-dure was suitable for simultaneous determination ofsaccharin and cyclamate in the formulation of table-topsweeteners. This procedure offered the possibility ofcarrying out direct and reagent-free measurements onsolid samples [135].

Fourier-transform mid-infrared spectrometry was usedto determine aspartame and acesulfame-K in table-topsweeteners. Off-line and on-line FTIR procedures weredeveloped to provide results statistically comparable withthose obtained by the HPLC reference method [136].

5. Conclusion

Due to medical and legal aspects, many researchers havefocused their efforts on developing analytical methods forthe determination of artificial sweeteners individually orsimultaneously in mixtures. They have applied a widevariety of instrumental techniques.

The method of choice for the determination of artificialsweeteners in different food matrices is HPLC because ofits multi-analyte capability, compatibility with thephysico-chemical properties of sweeteners, high sensi-tivity and robustness.

CE and IC are both interesting alternatives to HPLC.The resolving power of these techniques is in many casescomparable with that of HPLC and, frequently, theirrunning costs are lower. However, it seems that due tolimited robustness, in the case of CE methods, and themodest choice of separation mechanisms, in the case ofIC, these methods are less popular.

TLC and GC have been applied occasionally to analysisof artificial sweeteners. TLC methods are characterizedby poor separation efficiency and GC methods requirederivatization that is time consuming and labor inten-sive. Due to a demand for simple, rapid and low-costalternative methods for determination of sweeteners, inmany instances chromatographic methods can be re-placed by electroanalytical, spectroscopic or FI proce-


dures. Some of them are even more sensitive andselective and require very little sample preparation.Unfortunately, their applications are limited to one ortwo sweeteners only.

However, there still remains the challenge of devel-oping stable, reliable and robust methods for the deter-mination of artificial sweeteners in difficult foodmatrices. Robust and reliable analytical methods areessential to meet the needs of growing markets in qualitycontrol and consumer safety.

AcknowledgementsThis work was partly financed within the framework of aresearch project funded by the Polish Committee for theScientific Research (Research project # N N404027535).

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