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Artificial sweeteners as emerging pollutants in the environment: analytical
methodologies and environmental impact
Maroula G. Kokotou,† Alexandros G. Asimakopoulos† and Nikolaos S. Thomaidis*
Received 30th December 2011, Accepted 13th June 2012
DOI: 10.1039/c2ay05950a
Published on 13 June 2012 on http://pubs.rsc.org | doi:10.1039/C2AY05950A

The primary aim of this review is to extensively demonstrate the available analytical methodologies for
the environmental determination of all eight popular artificial sweeteners: sucralose, acesulfame,
saccharin, cyclamate, alitame, aspartame, neotame, and neohesperidin dihydrochalcone. Liquid
chromatography-mass spectrometry methods play a key role in their sensitive and reliable
determination in environmental matrices. In addition, all peer reviewed literature concerning their
environmental impact (distribution and ecotoxicology) is presented. Moreover, the role of sucralose,
acesulfame, saccharin and cyclamate as pollution markers is also demonstrated. Future prospects in
terms of method developments on artificial sweeteners in environmental samples are suggested and
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discussed.

1. Introduction multitudinous ranks and uses. Artificial (high-intensity) sweeteners

Pollutants that have so far not been studied extensively enough in
terms of occurrence, environmental fate and toxicity evaluation,
and/or covered by the existing worldwide regulations, are defined
as ‘‘emerging pollutants’’ or ‘‘emerging contaminants’’.1,2 One of
the most important and interesting classes of emerging contami-
nants is artificial sweeteners. Nowadays, they are found in
Laboratory of Analytical Chemistry, Department of Chemistry, University
of Athens, Panepistimioupolis Zografou, 15771 Athens, Greece. E-mail:
ntho@chem.uoa.gr; Fax: +30 210 7274750; Tel: +30 210 7274317
† These authors contributed equally to this article.
are predominately used in the food industry for the production of
sugar-free low calorie foodstuffs.3 Their widespread use in the
human diet is mainly due to the fact that, in contrast to sugar, they
do not cause any glycemic effect/insulin response or calorie intake
once digested, and do not adversely affect the microflora of dental
plaque.3 Today, saccharin (SAC), cyclamate (CYC), aspartame
(ASP), acesulfame (ACS), sucralose (SCL), alitame (ALI), neo-
tame (NEO), and neohesperidin dihydrochalcone (NHDC) are the
most popular artificial sweeteners (Fig. 1), exhibiting countless
food chemistry applications.3–5 Use of artificial sweeteners is even
reported in drugs, and sanitary products.6,7

Maroula G. Kokotou was born Alexandros G. Asimakopoulos

Maroula G: Kokotou
in Athens, Greece in 1985. After
receiving her bachelor degree in
Chemistry and her MSc degree
in ‘‘Chemical Analysis – Quality
Control’’ from the Department
of Chemistry of the University
of Athens, Greece, she is
currently pursuing a PhD degree
under the supervision of Assis-
tant Professor N. S. Thomaidis.
She is currently working on the
development of HILIC–mass
spectrometric methods for the
determination of polar emerging
contaminants in environmental
samples.
Alexandros G: Asimakopoulos
was born in Athens, Greece in
1986. He also received his
bachelor degree in Chemistry
and MSc degree in ‘‘Chemical
Analysis – Quality Control’’
from the Department of Chem-
istry of the University of Athens,
Greece. Currently, he is pursuing
a PhD degree under the super-
vision of Assistant Professor N.
S. Thomaidis. His research is
oriented towards the develop-
ment of new mass spectrometric
methods for the determination of
pollutants in environmental
media and biomonitoring of
xenobiotics.

This journal is ª The Royal Society of Chemistry 2012 Anal. Methods, 2012, 4, 3057–3070 | 3057

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Fig. 1 Molecular structures of the most popular artificial sweeteners.
Artificial sweeteners are highly consumed, particularly in the
U.S., with increasing trends in consumption, especially after the
introduction of SCL in 1998.8 The global market for artificial
sweeteners reaches $5.1 billion, of which the U.S. and Europe
currently make up 65%.8 Production volumes of artificial
sweeteners vary between reports. ASP represents the largest
Nikolaos S. Thomaidis was born
in Mannheim, Germany, in
1968. He is currently an Assis-
tant Professor in the department
of Chemistry in the University of
Athens. He is the principal
investigator of the Trace
Analysis and Mass
Spectrometry group (http://
trams.chem.uoa.gr) in the
Laboratory of Analytical
Chemistry of the University of
Athens. His research interests
Nikolaos S: Thomaidis include, among others, the
development of mass spectro-
metric methods for the determi-
nation of emerging contaminants in environmental samples and the
investigation of their fate in the aquatic environment.
3058 | Anal. Methods, 2012, 4, 3057–3070
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artificial sweetener product segment globally, it is the most
popular artificial sweetener in the U.S., and it is used in more
than 6000 food products.9 Around 16 000 t of ASP are produced
annually in U.S. for worldwide consumption.9 The U.S. is also
currently the largest market for SCL, making use of more than
1500 t per year, followed by Europe, with around 400 t per year,
as reported by a major Chinese company that recently entered
into the SCL market.10 In the Asian Pacific market, the volume
output in total of SAC, CYC, ACS, ASP, SCL, ALI and NEO,
grew approximately 10% between 2009 and 2010, reaching
approximately 109 000 t. Among sulfamates, CYC is currently
the most produced artificial sweetener,6,11 with volumes reaching
57 800 t in 2010, followed closely by SAC.6,11 ASP and ACS are
the leading products in diet soft drinks, while SCL has become
the leader in the key tabletop sweetener market.
Following ingestion, a large percentage of SAC, CYC, ACS
and SCL pass in an unchanged form from the human body,6,12–17
whereas ASP, ALI, NEO and NHDC are eliminated to a larger
degree.18 ASP, in its dry form, is relatively stable, but below pH 3
it is unstable and is hydrolyzed to aspartylphenylalanine. Above
pH 6, it is transformed to 5-benzyl-3,6-dioxo-2-piperazine acetic
acid.19,20 ALI is soluble in water (approximately 13.1% w/v at
25
C) and is relatively stable to heat (because of its unique amide
group). ALI, when hydrolyzed, is converted to alanine amide,
aspartic acid, b-aspartic isomer (as impurity), and in the human
body the N-glucuronide is the major metabolic product.18,21 NEO
is an N-substituted aspartame derivative22 with its major
This journal is ª The Royal Society of Chemistry 2012

degradation product being de-esterified neotame.23 NHDC is
converted by humans anoxically to 3-(3-hydroxy-4-methox-
yphenyl)propionic acid or 3-(3,4-dihydroxyphenyl)propionic
acid.24 Thus, the major metabolites of ASP, NEO, ALI and
NHDC, rather than the parent compounds, should be expected
to be present in the aquatic environment.
Excretion after human consumption is undoubtedly a major
source of artificial sweeteners in the environment, but it is surely
not the only one. For instance, in the European Union (E.U.)
SAC is authorized for use as an additive in animal feed for
piglets, pigs, bovines and calves, and it is also the major degra-
dation product of certain sulfonylurea herbicides.25–27 From
households and industries, all artificial sweeteners enter into
Published on 13 June 2012 on http://pubs.rsc.org | doi:10.1039/C2AY05950A
wastewater treatment plants and, from effluents, they eventually
reside in the receiving environmental bodies.2 In addition, direct
discharges from industry, households, animal farming and
agriculture burden surface waters with artificial sweeteners.2
From all artificial sweeteners, environmental research is espe-
cially focused on SAC, CYC, ACS and SCL, mostly for two
reasons: (i) their high determined concentrations in the aquatic
environment (at mg L1 levels) and (ii) their partial (even if high
for SAC and CYC) or limited removal in wastewater and
drinking water treatment plants.6,12,28–34 SAC is largely excreted
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by animals and can be detected in manure at high concentrations
(up to 12 mg L1), which are stable for at least 2 months of
storage.25 As a result of the application of manure in agricultural
land, SAC has a high probability of residing in significant
quantities in groundwater.25 ACS and SCL are considered
extremely persistent pollutants owing to the fact that these
compounds do not exhibit remarkable environmental degrada-
tion.31–34 Moreover, SAC, CYC, ACS and SCL are regarded as
high-priority emerging contaminants because, even though they
are detected worldwide in a variety of environmental media,
monitoring of their presence is still not required by any existing
regulations.31–35
Nevertheless, data available so far on the environmental
distribution and ecotoxicological impact of artificial sweeteners
is still limited. Therefore, there is an imperative need for multi-
analyte quantitative methods in order to determine the levels of
all popular artificial sweeteners in a broad range of environ-
mental matrices.
The basic aim of this work is to compile available information
on the latest available analytical methodologies for the deter-
mination of artificial sweeteners in environmental media, and to
present all recent findings derived from integrated environmental
studies.
2. Analytical methodologies
Solid-phase extraction (SPE) combined with liquid chromatog-
raphy (LC)-mass or tandem mass spectrometry detection (MS or
MS/MS) is the leading analytical methodology for the determi-
nation of artificial sweeteners in the environment (Table 1). SPE-
based protocols for the determination of artificial sweeteners in
environmental samples are proven to be compatible with many
chromatographic techniques, and they present the competitive
advantage of a large scale pre-concentration of samples (e.g.
greater than 100 times36) and sample clean-up. Furthermore, the
state-of-the-art LC-MS and LC-MS/MS techniques offer a
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multi-analyte determination capability and superiority in terms
of sensitivity and selectivity, even in low concentration samples
of complex matrices, such as wastewater influents, manure and
sludge. However, it is of interest to highlight the fact that, so far,
only five multi-analyte methods have been reported. The
majority of the published methods concern only the determina-
tion of SCL, mainly due to its high use in some countries, such as
the U.S., and its widespread distribution and extremely high
persistence in the aquatic environment,31–34,37 even though two
early studies have shown that SCL can be biodegraded mainly in
soils under aerobic conditions, and to a lesser extent in lake
water, wastewater and sludge.38,39
2.1. Sample preparation
The SPE sorbents employed for artificial sweeteners analysis are
polymer or silica based. They are modified with hydrophobic or/
and hydrophilic chemical moieties in order to give either
reversed-phase (e.g. StrataX-RP) or both reversed-phase and
ion-exchange functionality (mixed mode sorbents, e.g. Oasis
MAX, Oasis MCX, and Strata X-AW) (Table 1). Two
research groups, Zygler et al.47 and Scheurer et al.,12 reported
extensive work concerning a variety of parameters that affect the
recoveries of artificial sweeteners during SPE. This task was of
high importance, because artificial sweeteners belong to three
different chemical classes (sulfamates, peptides, and carbohy-
drate derivatives) and cover a wide range of polarities.47
Zygler et al. evaluated the retention behavior of all popular
artificial sweeteners from aqueous solutions on seven silica-based
cartridges (Chromabond C18ec, Bakerbond SPE Octadecyl,
Bakerbond SPE Phenyl, LiChrolut RP-18, Supelclean
LC-18, Discovery DSC-18, and Zorbax C18) and three
polymer-based cartridges (StrataX-RP, Oasis HLB, and
Bakerbond SPE SDB-1).47 The sorbent characteristics of every
tested cartridge (structure, porosity, particle diameter, carbon-
load, surface area, and the existence of end-capping or not) were
correlated with recovery. In addition, they thoroughly examined
the influence of pH and buffer type on the recoveries of the
artificial sweeteners. When Chromabond C18ec was assessed
under a variety of buffer compositions (e.g., formic acid–
ammonia buffer at pH 3.5), ACS recoveries were reported to be
better when the surface area (m2 g1) of the cartridge was 1000
instead of 500 m2 g1. Furthermore, Zorbax C18 (200 m2 g1)
and Bakerbond SPE Octadecyl (200 m2 g1) presented very
low recoveries for ACS and ASP for all tested buffer composi-
tions, apart from the case of formic acid/N,N-diisopropylethyl-
amine buffer (adjusted to pH 4.5). For Oasis HLB, quantitative
recovery of all sweeteners was obtained when the surface area of
the cartridge was 300 instead of 60 m2 g1. Low recoveries were
obtained in particular for ACS and CYC when Oasis HLB with
60 m2 g1 surface area was applied. Nevertheless, in all cases, the
best recoveries were obtained when formic acid/N,N-diisopro-
pylethylamine buffer (adjusted to pH 4.5) was applied. Finally,
Bakerbond SPE SDB-1 showed unusually low recoveries
towards ASP and ALI, exhibiting maximum recovery values of
34 and 54%, respectively, in all tested buffer compositions.47
Scheurer et al. tested ten cartridges (Isolute C18, Isolute
ENV+, StrataX-RP, Strata X-AW, Oasis HLB, Oasis
MAX, Oasis MCX, Oasis WAX, Varian PPL, and
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Table 1 Overview of analytical methodologies for environmental monitoring of artificial sweetenersa

Sample preparation
Determined artificial technique/cartridge(s)-
Analytical technique sweeteners Environmental media column(s)/Solvent(s) Column/mobile phase Analytical parameters Ref.

GC-EI-MS SCL (derivatization
(Ion trap system) with hexamethyldisilazane
and trimethylchlorosilane
in pyridine; determination of
SCL trimethylsilyl
(TMS) ether)
River surface water, SPE/Oasis HLB
oceanic water, and (600 mg and 200 mg)
wastewater column/MeOH for
conditioning and
elution, 5% v/v MeOH
95% v/v H2O for washing,
and H2O for equilibration
J & W Scientific DB-5 MS (30 LOD ¼ 0.17 nM, 40
mm  0.25 mm, 0.25 mm)/- (Rec.  S.D., n ¼ 2) %
(river surface water, oceanic water) ¼
(88.6  3) %,
(Rec.  S.D., n ¼ 2) %
(wastewater) ¼ (26.0  10) %,
linear range: 0.25–5.04 nM

TLC-UV/Vis/FL SCL Surface water, sewage SPE/extraction through TLC plate silica gel LOD ¼ 100 ng L1, 41
(Derivatization with water, and drinking Bond Elut PPL 60 F254/isopropyl acetate, (Rec.  S.D., n ¼ 3) % ¼ (84  7) %,
p-aminobenzoic water (500 mg/3 mL), and

3060 | Anal. Methods, 2012, 4, 3057–3070

MeOH and H2O working range: 100 ng L1 – 5 mg L1,
acid reagent) purification through Bond (15 : 3 : 1, % v/v/v) R2 ¼ 0.99991
Elut NH2 (200 mg/3 mL)/
MeOH followed by H2O
for conditioning,
MeOH for elution

LC-ESI-MS/MS SCL Wastewater effluents, On-line SPE/Oasis X Bridge C18 (Rec.  R.S.D., over a 15 months 42
(Triple quadrupole and source waters HLB (10 mm  2.1 mm, (150 mm  2.1 mm, period) % ¼ (91  39) %
system) with and without 25 mm)/ACN and 3.5 mm)/gradient elution with
municipal wastewater acidified H2O (pH 3) 0.2% v/v ammonium
for conditioning, and hydroxide in H2O – ACN
5% v/v ACN – 95% v/v
H2O for washing

LC-ESI()MS/MS SCL Drinking water, river SPE/Oasis HLB Restek Ultra aqueous C18 LOD ¼ 10 ng L1, 43
(Triple quadrupole and stream surface (200 mg/6 mL)/MeOH (100 mm  2.1 mm, 3 mm) or (Rec.  S.D., n ¼ 6) %
system) water, and wastewater for conditioning Hypersil Gold C18 (Drinking water) ¼ (62  9) %,
and elution, and H2O (100 mm  2.1 mm, 3 mm)/ Rec. %(Surface water) ¼ 55%,
for equilibration gradient elution with Rec. %(Wastewater) ¼ 26%,
0.1% v/v acetic acid in Signal suppression: 50%
H2O – ACN

LC-ESI()MS/MS SCL Surface water, drinking On-line SPE/mixed-layer Atlantis T3 LOQ(Influent) ¼ 100 ng L1, 44
(Orbitrap high water, and influents and SPE cartridge containing (150 mm  3 mm, LOQ(Effluent) ¼ 50 ng L1,
resolution system) effluents of WWTP Oasis HLB, 3 mm)/gradient elution LOQ(Surface and drinking water) ¼
Strata X-CW, with 0.1% v/v formic 10 ng L1,
StrataX-AW and acid in H2O–MeOH Rec. % ¼ 80–120%,
Isolute ENV+/2.5 mM Precision at the fortification
ammonium acetate in level of 100 ng L1 (R.S.D.%): < 7%,
H2O for conditioning Linear range: 1–1000 ng L1
and MeOH for elution

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Table 1 (Contd. )

Sample preparation
Determined artificial technique/cartridge(s)-
Analytical technique sweeteners Environmental media column(s)/Solvent(s) Column/mobile phase Analytical parameters Ref.

LC-ESI()MS/MS ACS, CYC, SAC, SCL Treated wastewater, On-line SPE/two Gemini C18 Method precisionACS: 6–8%, 6
(Triple quadrupole drinking water, surface stacked Biobeads (150 mm  2 mm, Method precisionCYC: 5–6%,
system) water, and ground water SM-2 (10 mL) 5 mm)/gradient elution Method precisionSAC: 1–5%,
cartridges/- with MeOH – 1 mM Method precisionSCL: 5–8%,
ammonium acetate LODs(Surface and ground water)
1
in H2O ACS ¼ 0.01–0.03 mg L ,
LODs(Treated wastewater)ACS ¼ 0.05–0.3 mg
L1,
Ion suppression %
(untreated wastewater) ACS: 100%,
Ion suppression %
(untreated wastewater) CYC: 80%,
Ion suppression %
(untreated wastewater) SAC: 200%,
Ion suppression %

This journal is ª The Royal Society of Chemistry 2012

(untreated wastewater) SCL: 14%,
Ion suppression %
(ground water) ACS: 30%,
Ion suppression %
(ground water) CYC: 50%,
Ion suppression %
(ground water) SAC: 90%,
Ion suppression %
(ground water) SCL: 3%

LC-ESI()MS/MS ASP, NEO, ACS, Drinking water, river SPE/Bakerbond Zorbax Eclipse XDB-C8 (150 LOQNEO ¼ 1 ng L1, 12
(Triple quadrupole CYC, SAC, SCL, surface water, and SPE SDB-1 mm  4.6 mm, 5 mm)/ LOQACS, SAC ¼ 2 ng L1,
system; signal enhancement NHDC wastewater (200 mg/6 mL)/MeOH gradient elution with 20 mM LOQCYC, ASP ¼ 5 ng L1,
by post column addition of followed by H2O (pH 3) ammonium acetate in H2O – LOQSCL,NHDC ¼ 10 ng L1,
Tris(hydroxymethyl) amino for conditioning, and 20 mM ammonium acetate in Rec. % ACS ¼ 80–93%,
methane buffer) MeOH for elution MeOH Rec. % CYC ¼ 52–96%,
Rec. % SAC ¼ 23–86%,
Rec. % ASP ¼ 41–81%,
Rec. % NEO ¼ 86–111%,
Rec. % SCL ¼ 48–98%,
Rec. % NHDC ¼ 49–69%,
Calibration was linear
up to 2.25 ng on column for all
analytes, R2 > 0.995

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Table 1 (Contd. )

Sample preparation
Determined artificial technique/cartridge(s)-
Analytical technique sweeteners Environmental media column(s)/Solvent(s) Column/mobile phase Analytical parameters Ref.

LC-ESI()MS/MS ACS, CYC, Soil, feces, manure, and LLE and then on-line SPE/ Gemini C18 LOD(Soil) ACS ¼ 0.01 mg g1, 25
(Triple quadrupole SAC, SCL sewage sludge Two stacked Biobeads SM-2 (150 mm  2 mm, 5 mm)/ LOD(Soil) CYC ¼ 0.01 mg g1,
system) (10 mL) cartridges/H2O for gradient elution with LOD(Soil) SAC ¼ 0.02 mg g1,
LLE MeOH – 1 mM LOD(Soil) SCL ¼ 0.01–0.1 mg g1,
ammonium acetate LOD(Feces) SAC ¼ 10 mg g1,
in H2O LOD(Manure) SAC ¼ 0.01–0.05 mg L1,
LOD(Manure) ACS ¼ 0.001 mg L1,
Rec. % (Soils) ¼ 63–127%,
Ion suppression %
(soil, feces, manure) SCL: (1) – 2%,

3062 | Anal. Methods, 2012, 4, 3057–3070

Ion suppression %(soil, feces) ACS:
(2) – (10)%,
Ion suppression %(manure) ACS: 27%,
Ion suppression %
(soil, feces) CYC, SAC: (12) – (23)%,
Ion suppression %
(manure) CYC, SAC: (63) – (65)%

IC-ESI()MS/MS ACS, CYC, Ground water, Treated Filtered (0.22 mm PVDF Dionex IONPAC AS20 Rec. % ACS ¼ 104%, 30
SAC, SCL wastewater syringe filter) into a (250 mm  2 mm)/ Rec. % CYC ¼ 42%,
disposable container gradient with potassium Rec. % SAC ¼ 102%,
with a 5 mL hydroxide eluent Rec. % SCL ¼ 108%,
disposable syringe * Rec. % calculated in
water samples that contained
1000 mg L1 each of chloride,
sulfate and carbonate.,
R2 > 0.995

LC-ESI()MS/MS SCL Source water, Automated-SPE/HLB Restek Allure Organic Working range: 37
raw water glass cartridges/ Acids column 10–1000 ng L1, R2 $ 0.99
conditioned with 5 mL of (150 mm  3.2 mm, 5 mm)/
MTBE, 5 mL of MeOH gradient elution with
and 5 mL H2O, and methanol with 0.05% v/v
eluted with 5 mL of formic acid and 2.5 mM
MeOH followed by ammonium
5 mL of 10/90 (% v/v) acetate (A) – methanol (B)
MeOH/MTBE

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Table 1 (Contd. )

Sample preparation
Determined artificial technique/cartridge(s)-
Analytical technique sweeteners Environmental media column(s)/Solvent(s) Column/mobile phase Analytical parameters Ref.

LC-ESI()MS SCL Surface water, SPE/Extraction through Atlantis dC18 (Rec.  S.D.)% (sludge) ¼ 36
(Time-of-flight high sewage water, and Oasis HLB (200 mg), (150 mm  2.1 mm, (70  30) %,
resolution system) sewage sludge and purification through 5 mm)/gradient elution (Rec.  S.D.)% (sewage water) ¼
Isolute-MM (300 mg) with ACN – H2O (93.5  23)%
and then through Oasis
MAX (150 mg)/ACN
followed by HCl solution
(10 mM) for conditioning,
HCl solution (10 mM) for
washing, and acetone :
MeOH (5 : 1,% v/v) for
elution (*sludge samples
were suspended prior to
SPE by shaking them with

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HCl solution (10 mM))

LC-ESI(+)MS/MS SCL Surface water, SPE/Oasis HLB
(Triple quadrupole and sewage water (60 mg/3 mL)/MeOH
system) followed by H2O for
conditioning, 0.5% v/v
ammonium hydroxide in
H2O for washing, and
MeOH for elution
Hypersil BDS
C18(150 mm  2.1 mm,
5 mm) /gradient elution
with MeOH – H2O
LOQ(Sewage water) ¼ 0.2 mg L1, 45
LOQ(Surface water) ¼ 0.02 mg L1,
(Rec.  S.D., n ¼ 3) % ¼ (98  1) %,
ion suppression % (surface water):
(95) – (98)%,
Ion suppression % (sewage water):
(58) – (93)%,
working range: 22–992 mg L1,
R2 ¼ 0.999

LC-ESI(+)MS ASP, SAC, SCL Wastewater effluents, Automated-SPE /Oasis Zorbax Eclipse XDB-C8 LODASP ¼ 0.02 mg L1, 46
(Time of flight surface waters from HLB (500 mg /6 mL) /MeOH column (150 mm  LODSAC ¼ 0.5 mg L1,
system) rivers and reservoirs, followed by H2O for 4.6 mm, 5 mm) /gradient LODSCL ¼ 0.05 mg L1,
and groundwater conditioning, and MeOH for elution with ACN – 0.1% v/v LOQASP ¼ 0.2 mg L1,
elution formic acid in H2O LOQSAC ¼ 5 mg L1,
LOQSCL ¼ 0.5 mg L1,
(Rec.  R.S.D.) % SAC ¼ (53  8) %,
(Rec.  R.S.D.) % ASP ¼ (90  6) %,
(Rec.  R.S.D.) % SCL ¼ (73  5) %,
inter-day precision (R.S.D.%, n ¼ 5): 3–
8%,
Linear range ASP: 0.05–5 mg mL1,
linear range SAC: 1–10 mg mL1,
linear range SCL: 0.1–5 mg mL1,
R2 > 0.99
a
GC, gas chromatography; LC, liquid chromatography; TLC, thin-layer chromatography; IC, Ion chromatography; ESI, electrospray ionization; EI, electron impact ionization; MS, mass
spectrometry detection; MS/MS, tandem mass spectrometry detection; SPE, solid phase extraction; LLE, liquid–liquid extraction; MTBE, Methyl-t-Butyl-ether; ACN, acetonitrile; MeOH,
methanol; H2O, water; ASP, aspartame; NEO, neotame; ACS, acesulfame; SAC, saccharin; CYC, cyclamate; SCL, sucralose; ALI, alitame; NHDC, neohesperidin dihydrochalcone; LOD, limit of
detection; LOQ, limit of quantification; Rec. %, recovery %; R.S.D. %, relative standard deviation %; S.D. %, standard deviation %.

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Bakerbond SPE SDB-1) and examined the effect of pH on the
recoveries from water samples of all popular artificial sweet-
eners.12 Optimum recoveries were obtained by using Bakerbond
SPE SDB-1 (200 m2 g1) cartridge and by adjusting the sample
at pH 3 prior to loading. Moreover, recoveries of ACS, CYC,
SAC and SCL seemed to decrease in the order of: drinking water
> surface water > wastewater.12 A correlation of large amounts
of dissolved organic matter with low recovery was stated for SCL
by Mead et al.40
Artificial sweeteners contain nitrogen and/or oxygen in their
molecular structure (Fig. 1). Thus, when the sorbent possesses
hydrophilic chemical moieties, retention is theoretically favored
and the recovery values increase. StrataX-RP and Oasis HLB
Published on 13 June 2012 on http://pubs.rsc.org | doi:10.1039/C2AY05950A
sorbents consist of poly(divinylbenzene-N-vinylpyrrolidone) and
therefore maintain the ability to apply hydrophobic, p–p and
even hydrophilic intermolecular forces to the artificial sweet-
eners. On the other hand, Bakerbond SPE SDB-1 sorbent
consists of poly(divinylbenzene-ethylvinylbenzene) and therefore
maintains the ability to apply only hydrophobic and p–p inter-
molecular forces to the artificial sweeteners. For this reason, the
conclusions reached by Zygler et al.47 and Scheurer et al.12 seem
contradictory on this aspect. Furthermore, Zygler et al.47 have
observed better recoveries of all the analytes using a silica-based
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cartridge, while Scheurer et al.12 did so using a polymeric-based
cartridge. Keeping in mind the differences that can be derived
from the buffers and pH that were used, further studies are
needed in this direction.
High pre-concentration factors for the determination of
artificial sweeteners were generally achieved by loading a high
volume of sample onto the SPE cartridge, generally ranging
from 50 mL to 1 L, and then applying eluent volumes ranging
from 3 to 7 mL.6,12,36,43 The SPE eluent volume could be further
decreased to a smaller volume through solvent evapora-
tion.6,12,25,40 However, the pre-concentrated sample extract not
only contains the target analytes, but also contains pre-
concentrated matrix components that co-elute with the analytes.
Matrix components can have a particularly negative effect on
LC-ESI-MS (or MS/MS) analysis. This effect, mostly known as
the ‘‘matrix effect’’, is mainly presented as a false decrease
(suppression) or increase (enhancement) of signal
response.6,12,25,42,45 Consequently, the matrix effect is more
severe when samples are pre-concentrated, highlighting the fact
that the matrix effect is entirely matrix-dependent. For instance,
wastewater samples cannot be pre-concentrated as extensively
as drinking water samples, since apart from the obvious diffi-
culty of the task, severe matrix effects will be induced by elec-
trospray ionization (ESI) due to the high concentration of
interfering compounds in the wastewater matrix.6,25,42,45 The
factor by which a sample will be pre-concentrated is a parameter
that needs to be carefully assessed and optimized during LC-
ESI-MS and LC-ESI-MS/MS method development in order to
achieve optimal and accurate detection, taking into account the
demands of the matrix analyzed (fit for purpose). It is noted that
the matrix effect is an important analytical parameter in mass
spectrometry based methods and should be reported in all cases.
It should be quantitatively assessed through the calculation of
relative suppression % (Table 1). The matrix effect (%) should be
always estimated in accordance with absolute recoveries
through SPE.
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At this point, it should be noted that little effort has been
focused towards sample stability. The need for quality assurance
from sampling to analysis is imperative. In literature, two studies
by Van Stempvoort et al. assessed the impact of storage condi-
tions on SAC, CYC, ACS and SCL concentrations in wastewater
and groundwater samples.30,48 Van Stempvoort et al.30,48 stated
that no perceptible differences were observed in the concentra-
tions of ACS and SCL in groundwater samples impacted by a
septic system, after 13.5 months of storing in the refrigerator
(5
C) and in the freezer, whereas losses were observed for SAC
and CYC in the refrigerated samples compared to the frozen
samples. In treated wastewater and groundwater samples, this
study30 also showed that CYC decreased in refrigerated samples
over 3 weeks, whereas ACS and SAC appeared to be stable over
this time period. These studies concluded that saccharin and
cyclamate concentrations were strongly decreased in samples in
the refrigerator, probably by microbial degradation, and that
long-term storage under refrigerated conditions is not suitable
for the determination of these two sweeteners even though the
samples had been filtered.48 The authors’ view is that stability
studies like those of Van Stempvoort et al. should be extended to
other environmental matrices and to include other artificial
sweeteners (ALI, ASP, NEO and NHDC), where no reports
concerning stability are available. Moreover, Mawhinney et al.37
described in detail the protocol that was followed from sampling
to analysis for SCL in drinking water. Mawhinney et al.37 made
use of sodium azide and ascorbic acid during sampling and
shipping, in order to quench any residual oxidant. Stability
factors reported by Van Stempvoort et al.,30,48 and recommen-
dations or guidelines, such as those presented by Mawhinney
et al.37 for SCL, should be incorporated into any future quality
assurance protocols and adopted practice.
2.2. Instrumental analysis
To the best of our knowledge, only one gas chromatographic
(GC)40 and one thin-layer chromatographic (TLC) method41
were reported for the determination of SCL in environmental
media. Both methods employed a derivatization step prior to
instrumental analysis. In the GC method, the derivatization step
was needed in order to convert SCL to a volatile substance that
could be analyzed by GC.40,49 In the TLC method, the derivati-
zation step was needed in order to achieve greater selectivity and
sensitivity by converting SCL to both a strong UV absorbing and
fluorescent compound.41 In particular, that study proposed
p-aminobenzoic acid as a new selective derivatization reagent for
SCL, leading to blue fluorescent zones under UV irradiation at
366 nm.41 On the other hand, LC-MS and LC-MS/MS methods
are established as the best choice for the environmental analysis
of artificial sweeteners, since low LODs can be achieved without
the need of the time-consuming derivatization step (Table 1).
In all published LC-MS and LC-MS/MS methods, the
instruments used were equipped with an electrospray interface
(ESI) for the ionization of the artificial sweeteners. For quanti-
fication purposes, the internal standardization method was
mainly used due to the need to adequately compensate for matrix
effects.6,25,42,43 Mostly, the deuterated internal standard (IS) of
SCL (SCL-d6) has been applied so far.6,25,42–45 Apart from the
internal standardization method, quantification by external
This journal is ª The Royal Society of Chemistry 2012

calibration12,43 has also been performed. Loos et al. proved that
between the two methods of quantification, better quantification
of SCL was performed with the internal standardization method
(SCL-d6 applied), in view of the lower uncertainty that was
achieved.43 Scheurer et al. quantified SCL with the internal
standardization method (SCL-d6 applied), whereas the rest of
artificial sweeteners (ASP, NEO, ACS, CYC, SAC, and NHDC)
were quantified by external calibration.12 On the contrary,
Buerge et al. quantified four artificial sweeteners (ACS, CYC,
SAC, and SCL) only by the internal standardization method
(SCL-d6 applied).6,25 Van Stempvoort et al.48 reported the use of
SCL-d6, acesulfame-d4 (ACS-d4), saccharin-13C6 (SAC-13C6)
and cyclamic acid-d11 (CYC acid-d11) for quantification with the
Published on 13 June 2012 on http://pubs.rsc.org | doi:10.1039/C2AY05950A
internal standardization method for SCL, ACS, SAC and CYC,
respectively. SCL-d6 and ACS-d4 were also used by Scheurer
et al.28 To the best of our knowledge, SAC-13C6 is the only 13C6-
labeled artificial sweetener that has been reported for the envi-
ronmental analysis of artificial sweeteners, and it was obtained
from Toronto Research Chemicals Inc. (North York, ON,
Canada). Furthermore, other newly released ISs, such as aspar-
tame-d5 (ASP-d5), neotame-d3 (NEO-d3), neotame-d5 (NEO-d5),
and neohesperidin dihydrocalcone-d3 (NHDC-d3) can also be
obtained from the same company. Similarly, both deuterated and
Downloaded on 05 October 2012
13
C-labeled artificial sweeteners can be obtained from Santa Cruz
Biotechnology Inc (California, USA). These ISs should be
evaluated in the near future in terms of compensating sufficiently
for matrix effects, losses during sample preparation and insta-
bility of the MS or MS/MS detector. It should be noted that 13C
labeled-compounds are preferred theoretically, since 1H and 2H
(D) have greater differences in their physical properties than 12C
and 13C,50 however, they are considerably more expensive.
The published LC-MS/MS data on precursor and product ions
under negative and positive ionization mode are presented in
Table 2. ASP, SAC, and SCL are the only artificial sweeteners
that can also be determined (individually or as sodium adducts)
under positive ionization mode.45,46 Moreover, their fragmenta-
tion pathways were extensively studied under positive ESI
polarity with a LC/TOF-MS system by Ferrer and Thurman.46
However, fragmentation pathways were not demonstrated for
negative ESI-MS/MS methods. Neset et al. were the only ones to
mention that SCL and SCL-d6 form significant amounts of
adducts with formic acid in the negative ESI mode, and they used
these adducts to quantify SCL.44
Throughout literature, it is clear that the only possible way to
achieve simultaneous determination of all popular artificial
sweeteners by LC-MS and LC-MS/MS techniques is by running
analysis under negative ionization mode (Table 1). Under those
ionization conditions, Scheurer et al. achieved high levels of
sensitivity for all determined artificial sweeteners by post column
addition of tris(hydroxymethyl)aminomethane buffer.12 The
signal was augmented from 30% for NHDC to 290% for SAC.12
According to Scheurer et al., the addition of a basic buffer
facilitated the deprotonation of the target-analytes, but buffer
concentrations greater than 20 mM were avoided in order to
prevent unnecessary contamination of the mass spectrometer
interface.12 Furthermore, Loos et al. characteristically reported
that SCL under negative ionization mode presented a poor
ionization efficiency such as that of nonylphenol (molecule
containing a hydroxyl group),43 a fact that was also confirmed
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from the authors’ experience.51–53 Finally, ASP, SAC, and SCL
could be analyzed with both ionization polarities, although
higher sensitivity and more fragments were obtained only
through the positive ionization mode.45,46
In general, throughout literature, the methods developed for
the determination of artificial sweeteners in environmental liquid
samples involved pre-concentration factors of 100,6,12,28 400,46
800,43 and even 1000,36 in order to achieve the demanded very
low LODs in the ng L1 range (Table 1). A minimum volume
water sample (5 mL) was used in on-line SPE systems,6 whereas
typical volumes for off-line SPE are 200–1000 mL for water
analysis36,43,46 and 50–200 mL for wastewater analysis.12,36
As already mentioned, it is apparent from Table 1 that the
majority of methods referred to the determination of SCL. Only
the method of Scheurer et al.12 included seven out of the 8 most
popular artificial sweeteners. Polymeric-based cartridges should
be the best SPE materials for successful multi-analyte extraction;
however, it is apparent from the analytical data presented in
Table 1 that recoveries ranged considerably between the pub-
lished studies. Taking into account the large differences in
physicochemical properties between the compounds, one suitable
SPE material giving efficient recoveries for all the compounds
simultaneously could be difficult to find. On the contrary,
LC-MS/MS with negative ESI seems to be the best choice for
multi-analyte determination, using appropriate isotope labeled
analogs for internal standards. It is interesting to observe from
Table 1 that the available analytical methods did not present all
the necessary analytical parameters for method performance
(LOD, LOQ, at least intra-day precision, linear range, correla-
tion coefficients, recoveries, and matrix effects/matrix factors for
LC-MS and LC-MS/MS methods). Therefore, there is a need for
a multi-analyte, fully validated method in environmental
samples, due to the extremely interesting environmental chem-
istry of these compounds, as described in the following section.
3. Environmental studies
The central pillar of environmental studies on artificial sweet-
eners is admittedly the distribution in the environment and, by
extension, the ecotoxicological impact. Since 2009, a constantly
rising number of environmental studies have focused on the role
of artificial sweeteners as pollution tracing compounds.
3.1. Distribution in the environment
All reported concentrations of artificial sweeteners in the envi-
ronment from the peer reviewed literature were compiled in
Table 3.
Scheurer et al. detected SCL, ACS, SAC and CYC in waste-
water from German sewage treatment plants (STPs).12 Concen-
trations in wastewater influents ranged between 34 and 50 mg L1
for ACS and SAC, up to 190 mg L1 for CYC, and below 1 mg
L1 for SCL. ACS was removed in quantities up to 41% and SCL
was removed by about 20%, whereas SAC and CYC were
removed by greater than 90%.12 Furthermore, Scheurer et al.
detected all four artificial sweeteners in German surface waters:
ACS was found in concentrations exceeding 2 mg L1, SAC and
CYC were found at levels between 50 and 150 ng L1, and SCL
was mainly found from 60 to 80 ng L1.12 Then, Scheurer et al.
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Table 2 Selected reaction monitoring (SRM) data published for artificial sweetenersb

Selected reaction monitoring

Artificial sweeteners ESI mode (SRM) (presented with their nominal mass) Ref.

CYC Negative 178 m/z > 80 m/za 6, 30

178 m/z > 96 m/z
178 m/z > 80 m/z 12
ACS Negative 162 m/z > 82 m/za 6, 12, 41
162 m/z > 78 m/z
NHDC Negative 611 m/z > 303 m/za 12
293 m/z > 125 m/z
NEO Negative 377 m/z > 195 m/za 12
377 m/z > 345 m/z
ASP Negative 293 m/z > 261 m/za 12
293 m/z > 200 m/z
Positive 295 m/z > 120 m/z 46
Published on 13 June 2012 on http://pubs.rsc.org | doi:10.1039/C2AY05950A

295 m/z > 235 m/z

295 m/z > 260 m/z
295 m/z > 180 m/z
SAC Negative 182 m/z > 106 m/za 6, 30
182 m/z > 62 m/z
182 m/z > 42 m/za 12
182 m/z > 106 m/z
Positive 184 m/z > 166 m/z 46
SCL Negative 395 m/z > 359 m/za 6, 12, 30
397 m/z > 361 m/z
395 m/z > 359 m/za 43
397 m/z > 359 m/z
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395 m/z > 35 m/za 37

397 m/z > 35 m/z
441 m/z (formic acid adduct) > 395 m/z 44
Positive 397 m/z > 145 m/z 46
397 m/z > 163 m/z
397 m/z > 181m/z
397 m/z > 199 m/z
419 m/z (sodium adduct) > 239 m/z 46
SCL-d6 Negative 401 m/z > 365 m/z 6, 30
401 m/z > 365 m/za 43
403 m/z > 367 m/z
403 m/z > 367 m/z 37
447 m/z (formic acid adduct) > 401 m/z 44
ACS-d4 Negative 166 m/z > 82 m/z 30
CYC acid-d11 Negative 189 m/z > 80 m/z 30
SAC-13 C6 Negative 188 m/z > 106 m/z 30
a
Stated as primary product. b Abbreviations: CYC, cyclamate; ACS, acesulfame; NHDC, neohesperidin dihydrochalcone; NEO, neotame; ASP,
aspartame; SAC, saccharin; SCL, sucralose; SCL-d6, sucralose-d6; acesulfame-d4, ACS-d4; saccharin-13C6., SAC-13C6; cyclamic acid-d11, CYC acid-d11.

investigated the effectiveness of drinking water treatment in
removing these four artificial sweeteners.28 They concluded that
only SAC and CYC could be completely removed by applying
the treatments of river bank filtration and artificial groundwater
recharge.28
Buerge et al. investigated the distribution of SCL, ACS, SAC
and CYC in wastewater, surface waters (from lakes and rivers),
ground waters, and drinking water of Switzerland.6 Concentra-
tions ranging between 12–43 mg L1, 10–65 mg L1, 3.9–18 mg
L1, and 2.0–9.1 mg L1 were found in influent wastewater
samples for ACS, CYC, SAC, and SCL, respectively, whereas the
respective concentrations in effluent wastewater samples were
between 14 and 46 mg L1, <LOD–0.82 mg L1, <LOD–3.2 mg
L1, and 2.0–88 mg L1 for ACS, CYC, SAC, and SCL,
respectively.6 Furthermore, all four compounds were found in
surface waters (lakes and rivers), whereas only ACS was detected
in ground waters and drinking water. ACS concentrations
ranged from <0.01 mg L1 (LOD) up to 2.8 mg L1 in surface
water samples. It was detected in 65 out of 100 analyzed
groundwater samples at concentrations up to 4.7 mg L1. ACS
concentrations up to 2.6 mg L1 were measured in tap water
samples that were obtained from groundwater resources, and
links between concentrations in groundwater samples and
samples from the infiltrating river upstream of the pumping
stations were indicated. This study particularly denoted the
extensive distribution of ACS in the Swiss aquatic environment.6
The authors concluded that elevated concentrations of ACS in
groundwater may result primarily from infiltration of waste-
water-polluted surface water through stream beds.6,25 Concen-
trations of SAC in groundwater samples reaching up to 0.26 mg
L1 were also reported by Buerge et al., likely due to manure
application in agricultural soils.25
An extensive study on the distribution of SCL in the aquatic
environment of Europe was performed by Loos et al., screening
stream and river waters from 27 countries.43 Data obtained from
the UK, Belgium, France, Switzerland, Italy, Spain, The Neth-
erlands, Norway and Sweden showed that SCL concentrations in
these countries were as high as 1 mg L1.43 On the contrary, data

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 View Online

Table 3 Reported concentrations of artificial sweeteners in environmental samplesa

Environmental sample Reported concentrations of artificial sweeteners Ref.

Untreated wastewater, ground
water, surface water, and drinking
water from Switzerland (Canton of
Zurich)
Wastewater and surface waters
from Germany
Ground water and digested sewage
sludge from Switzerland
Published on 13 June 2012 on http://pubs.rsc.org | doi:10.1039/C2AY05950A
12–43 mg L1 for ACS in influents, 10–65 mg L1 for CYC in influents, 3.9–18 mg L1 for 6
SAC in influents, 2.0–9.1 mg L1 for SCL in influents, 14–46 mg L1 for ACS in effluents,
<0.1 (LOD)–0.82 mg L1 for CYC in effluents, <0.1 (LOD)–3.2 mg L1 for SAC in
effluents, 2.0–8.8 mg L1 for SCL in effluents, <0.01 (LOD)–2.8 mg L1 ACS in surface
water, 65 out of 100 analyzed groundwater samples reached up to 4.7 mg L1 only for
ACS, up to 2.6 mg L1 only of ACS was found in tap water
34–50 mg L1 for ACS and SAC in influents, up to 190 mg L1 for CYC in influents, below 12
1 mg L1 for SCL in influents, removal in the STPs was limited for ACS and SCL and
>94% for SAC and CYC, 0.27–2.7 mg L1 for ACS in German rivers, 0.01–0.35 mg L1 for
SAC in German rivers, 0.03–0.32 mg L1 for CYC in German rivers, 0.01–0.11 mg L1 for
SCL in German rivers
Up to 0.26 mg L1 of SAC in ground water, 23–43 mg L1 for ACS in digested sludge, 25
0.6–5.5 mg L1 for CYC in digested sludge, 10–16 mg L1 for SAC in digested sludge,

(Canton of Zurich) 5.4–8.6 mg L1 for SCL in digested sludge

Drinking water from drinking
water treatment plants (DWTPs)
in the U.S.
Open ocean waters in the U.S.
European surface waters
(stream and river waters)
Wastewater, surface water
and groundwater samples
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SCL in source water: 47–2900 ng L1 in 15 out of 19 DWTPs, SCL in finished water: 37
49–2400 ng L1 in 13 out of 17 DWTPs, SCL in distribution system water:
48–2400 ng L1 in 8 out of 12 DWTPs
Up to 67 ng L1 of SCL from the Gulf Stream, up to 147 ng L1 of SCL from Northern 40
Florida Keys, up to 372 ng L1 of SCL from Cape Fear River Estuary of North Carolina,
up to 392 ng L1 of SCL from Middle Florida Keys
Up to 1 mg L1 of SCL in U.K., Belgium, France, Switzerland, Italy, Spain, The 43
Netherlands, Norway and Sweden, lower than 100 ng L1 of SCL in Germany and
Eastern Europe
<0.5 (LOD)–5 mg L1 of SAC in wastewater, <0.5 (LOD) for SAC in surface and ground 46
water, 0.8–1.8 mg L1 of SCL in wastewater, <0.05 (LOD)–1.8 mg L1 of SCL in surface

from the U.S. water, 0.6–2.4 mg L1 of SCL in ground water

a
Abbreviations: CYC, cyclamate; ACS, acesulfame; NHDC, neohesperidin dihydrocalcone; NEO, neotame; ASP, aspartame; SAC, saccharin; SCL,
sucralose; DWTPs, drinking water treatment plants; LOD, limit of detection.

obtained from Germany and Eastern Europe showed that SCL
concentrations were lower than 100 ng L1.43 It should be noted
that the advantage of presenting capita values, in contrast to
concentration values (e.g. see Loos et al.43), is that these values
are independent of the factors of discharge and population
density.44 Thus, for the year 2009, the estimated average daily
load per capita of SCL to WWTPs of Sweden was reported to be
0.76 mg per cap d.44 Neset et al. also compared available data of
estimated daily load to WWTP for artificial sweeteners for
several countries (Switzerland,6 Germany,12 and Sweden36) and
found that the load is considerably lower in Germany (daily load
per capita 0.14–0.23 mg per cap d) than in Switzerland (1.5  0.6
mg per cap d) and Sweden (1.7–2.1 mg per cap d in 200736),
concluding that this difference could be attributed to the fact that
the German food industry prefers other artificial sweeteners.44
There were no significant differences in the loads of ACS, CYC
and SAC to the WWTP of Switzerland and Germany.44
The first study that presented concentration data of SCL for
North American coastal and open ocean waters was by Mead
et al.40 Marine waters from the Gulf Stream, Northern Florida
Keys, Cape Fear River Estuary of North Carolina, and Middle
Florida Keys reached levels of up to 67, 147, 372, and 392 ng L1,
respectively.40 Furthermore, that study highlighted the potentially
widespread distribution of SCL from United States (U.S.) waters
due to the major oceanographic current of the Gulf Stream.40
According to Mawhinney et al.,37 SCL was found to be present in
source water of 15 out of 19 DWTPs (drinking water treatment
plants) in the U.S. at concentrations between 47 and 2900 ng L1,
in finished water of 13 out of 17 DWTPs at concentrations
between 49 and 2400 ng L1, and in distribution system water of
8 out of 12 DWTPs in a concentration range of 48–2400 ng L1.
Moreover, Ferrer and Thurman46 detected SCL in wastewater,
surface water and groundwater samples, collected from different
locations and states around the U.S., in concentrations that
ranged between 0.8–1.8 mg L1, <0.05–1.8 mg L1 and 0.6–2.4 mg
L1, respectively. Additionally, Ferrer and Thurman46 detected
SAC in only one wastewater sample at 5 mg L1.
3.2. Ecotoxicological studies on sucralose
The worldwide environmental distribution of SCL at high
concentrations, as demonstrated in the previous section, has
channeled research exclusively in the last three years towards the
assessment of the ecotoxicological impact of this particular
artificial sweetener. SCL is considered safe for human
consumption (the acceptable daily intake for SCL was set at 5 mg
kg1 of body weight per day),54–56 but its effects in the ecosystem
have not yet been studied in depth, since limited ecotoxicological
data are available in the peer reviewed literature. So far, SCL has
low bioaccumulation potential and negligible acute/chronic
toxicity in aquatic organisms57,58 and does not appear toxic to
plant growth.59
In 2010, an important study was conducted by Hjorth et al. in
order to investigate the short-term effects (96 h) of SCL in Arctic
aquatic ecosystems.60 The factors that were assessed were the egg
production, hatching rate, food intake and mortality of two
species of Arctic copepods of the genus Calanus, C. glacialis and
C. finmarchicus. The copepods were exposed to six different
concentrations (0–50 mg L1) of SCL, which simulates the range
of concentrations found in screening studies. The results showed

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that both species responded weakly to SCL, but with C. glacialis
being possibly slightly more sensitive than C. finmarchicus. 60 In
2011, a study was conducted by Huggett and Stoddard to assess
the effects of SCL on the survival, growth and reproduction of
Daphnia magna and Americamysis bahia (mysid shrimp).61 The
survival or reproduction of D. magna was not reduced even at
concentrations as high as 61.8 g L1. The no observable effect
concentration (NOEC) and lowest observable effect concentra-
tions (LOEC) for D. magna were 1800 and >1800 mg L1,
respectively. The survival, growth and reproduction of the mysid
shrimp were not affected by concentrations of 693 mg L1 of
SCL. The NOEC and LOEC for the mysid shrimp were 93 and
>93 mg L1, respectively. Thus, the study concluded that the
Published on 13 June 2012 on http://pubs.rsc.org | doi:10.1039/C2AY05950A
concentrations of SCL detected in the environment are well
below those required to elicit chronic effects in freshwater or
marine water bodies.61 Recently, a study on crustaceans showed
for the first time that physiology and locomotive behavior can be
affected by exposure to SCL. The behavioral response of D.
magna manifested as altered swimming height and increased
swimming speed, whereas in gammarids the time to reach food
and shelter was prolonged.58 Research on the ecotoxicology of
artificial sweeteners is expected to increase in subsequent years,
especially for SCL and ACS, since the long-term effects resulting
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from chronic exposure to low levels of these compounds are
largely unknown.
3.3. Artificial sweeteners as pollution markers
When the concentration of a contaminant is measured at
different spatial locations, it often exhibits concentration values
that differ between the locations. This phenomenon is known as
‘‘spatial variability of the contaminant’’ and this is due to the
existence of complex sources of contamination within an
urbanized watershed. Basically, the water bodies of surface
water, wastewater, groundwater, and tap water mix through the
urban infrastructure. Thus, it is particularly difficult to trace the
fate of a pollutant in a given urban setting.30,42,62,63 For water
quality control purposes, the development of pollution indicators
or pollution markers specific to wastewater effluents aid in
determining the source contributions in recreational waters or
potable supply sources.42 The characteristics of an ideal waste-
water marker are perfectly depicted by Oppenheimer et al.,42 and
recently ACS and SCL have proven to be reliable pollution
markers.6,37,42,59,62,63
Oppenheimer et al.42 compared the suitability of a variety of
anthropogenic pollutants as wastewater indicators to SCL, by
examining occurrence data for 85 organic compounds in samples
of effluents and surface waters with or without known wastewater
influence. The results demonstrated the superior performance of
SCL as a potential indicator of domestic wastewater input in the
U.S. While many compounds were detected in all of the effluent
samples, only SCL was consistently detected in the surface waters
with known wastewater discharges and it was absent in the
samples without wastewater influence.42 Mawhinney et al.37
confirmed that SCL will function well as an indicator compound
of anthropogenic influence on source, finished drinking and
distribution system water, as well as a marker for the presence of
other recalcitrant compounds in finished drinking water.37
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In Swiss lakes from densely populated catchment areas, ACS,
CYC and SAC were found to present good linear correlations
(R 2 $ 0.88) between their concentrations and the ratio of pop-
ulation (P)/water outflow (Q), a ratio that expresses the actual
anthropogenic burden in a quantitative sense to a lake water
body by wastewater.6 Additionally, ACS proved to be suffi-
ciently hydrophilic to reach groundwater (high frequency of
detection, 65%) and therefore the widespread occurrence of ACS
confirmed that many groundwater aquifers were substantially
influenced by infiltration of river water, where the infiltrating
water received considerable discharge from WWTPs.6
At the Rhine and main rivers in Germany, data from a seven-
month monitoring program revealed high correlation between
ACS and carbamazepine (R 2 ¼ 0.94) and also a good correlation
with the emerging pollutants 1H-benzotriazole and 4-methyl-
benzotriazole.63 The concentration decrease of SCL was found to
be correlated to prolonged residence time of wastewater in the
subsurface.63 A higher consumption of SCL in Switzerland
compared to Germany was assumed, since the ACS/SCL ratio
was observed to steadily increase along the river Rhine.63 Besides
this fact, the persistent behavior of SCL according to Soh et al.
suggests that it may be used as a long-term aquatic environ-
mental marker of anthropogenic activity, when compared to
ACS. In addition, SCL sorption onto soil was reported by Soh
et al. as significantly lower than that of ACS, indicating that SCL
is a better aquatic marker of anthropogenic activity.59 On the
contrary, there is evidence from Scheurer et al.63 that a concen-
tration decrease was observed for SCL at long-term residence
time in the subsurface, whereas again according to Scheurer
et al.,63 ACS seemed to present the strongest prediction power if
the influence of point sources and regional differences is limited.
Therefore, the ranking of ACS and SCL as long-term aquatic
environmental markers needs further consideration.
Also in Switzerland, correlation analysis showed that some
perfluoroalkyl acids (PFAAs) concentrations correlated well
with population and less so with catchment area. The correlation
with ACS confirmed this observation, but also highlighted a few
elevated PFAA levels, some of which could be attributed to
industrial emissions.62
In a municipal wastewater plume at Jasper (Alberta, Canada),
ACS was strongly correlated with chloride and was positively
correlated with other wastewater-related contaminants, indi-
cating that this sweetener has the potential to be a good marker of
‘‘young’’ wastewater (<20 years residence time).30 In the same
study, the concentration of SAC was found to be higher than that
of ACS in many samples in an old landfill in Ontario (Canada).30
These observations proved that simultaneous determination of
multiple sweeteners may provide useful information on contam-
inant sources and groundwater conditions in an urban setting, and
the authors expect future developments towards this direction.
3.4. Advanced treatment processes for the removal of artificial
sweeteners
Different technologies have been used to minimize the discharge
of artificial sweeteners in the aquatic environment and for their
removal during drinking water production. Upgrading WWTPs
with advanced oxidation processes like ozonation as tertiary
treatment seems to be a promising and expanding option. So far,
This journal is ª The Royal Society of Chemistry 2012

only one study have been performed to identify oxidation
products of artificial sweeteners.
Torres et al. studied three chemical oxidation processes (UV
radiation, chlorination and ozonation) in order to remove or
transform SCL under potential WWTP operation scenarios.29
Prolonged exposure to UV radiation did not oxidize SCL
significantly, and chlorine and ozone showed only a slow
oxidation.29 SCL would not be expected to degrade by free
chlorine or ozone under typical WWTP operational conditions.
Soh et al.59 also systematically evaluated the extent of degrada-
tion or mineralization of SCL through the same advanced
oxidation processes and reached the same conclusions as Torres
et al.29 for the chlorination and UV radiation. However, signifi-
Published on 13 June 2012 on http://pubs.rsc.org | doi:10.1039/C2AY05950A
cant degradation of SCL was found after one hour at an ozone
dose of 100 mM. Oxidation by ozone was achieved through the
radical mediated oxidation mechanism. Though SCL was
significantly degraded with hydroxyl radicals produced from
ozone, the amount of degradation that will actually occur during
ozonation as part of the water treatment process will likely be
minimal as other organic compounds will effectively act as
radical scavengers.29 This would agree with the findings of
Hollender et al.,64 showing only 31% removal in a water treat-
ment plant upgraded with ozonation.
Downloaded on 05 October 2012
Scheurer et al. also investigated the removal of SCL, ACS,
SAC and CYC by ozonation and chlorine disinfection.28 The
compounds were not transformed in chlorination experiments.
SCL proved to be persistent against ozone (removal of 8–15% in
waterworks and <20% after 30 min in the batch test). ACS
reacted readily with ozone, but ozone levels typically used in
drinking water treatment would only remove 18–60%. Only
<10% of SAC and 30–50% of CYC was transformed within 30
min, with ozone concentrations of 5 mg L1.28 It is known that
ozonation can lead to the formation of undesired oxidation by-
products, so research on the identification of ozonation products
(OPs) should be prioritized. Thus, Scheurer et al. in a subsequent
study identified the main oxidation products of CYC and ACS.65
Detailed reaction pathways were proposed for both compounds.
Amidosulfonic acid and cyclohexanone are formed as main OPs
of CYC, whereas acetic acid, and two anionic compounds with
m/z 170 (ACS OP170) and m/z 168 (ACS OP168), were identified
as the main OPs for ACS.65 ACS OP170 was identified as
(dihydroxyacetyl) sulfamate, which should form a fairly stable
aldehyde hydrate in aqueous solutions. ACS OP168 is the direct
oxidation product of ACS OP170 ((carboxycarbonyl)
sulfamate).65
Finally, the oxidation of SCL, along with common carbohy-
drates (sucrose, maltose, glucose and fructose), by ferrate(VI)
(Fe(VI)) was studied as a function of pH (6.5–10.1) at 25
C.66
Interestingly, SCL and sucrose reacted slower than the other
reducing sugars. Rate constants decreased with an increase in
pH. Comparison of the reactivity of Fe(VI) with other oxidants
suggests that free radical species, such as hydroxyl radicals, have
much higher reactivity than Fe(VI) towards SCL.66
4. Conclusions and prospects
Recently, a hydrophilic interaction liquid chromatographic
(HILIC) method was developed for the determination of all
popular artificial sweeteners in environmental samples51 and it
This journal is ª The Royal Society of Chemistry 2012
View Online
proved to be a promising alternative to reversed-phase chroma-
tography. Kokotou and Thomaidis51 included ALI for the first
time in a method for the determination of artificial sweeteners in
wastewater. So far, only Scheurer et al.12 had tried to separate
seven sweeteners (ASP, NEO, ACS, CYC, SAC, NHDC and
SCL) on a HILIC column (Hypercarb, 150  2.1 mm; 5 mm), but
NHDC was eventually not eluted and they preferred to use a
reversed-phase separation (Zorbax Eclipse XDB-C8 column,
150  4.6 mm; 5 mm).
Since artificial sweeteners are polar compounds, more studies
and methods based on their HILIC separation are expected in
the near future. Moreover, the development of small column
particles67 and the constant increase in the availability of ultra-
high pressure LC systems make ultra-fast sample profiling for
sweeteners in environmental samples a task that can be easily be
witnessed in the near future. Ultra-fast sample profiling implies a
high-throughput analysis and, consequently, involves a higher
performance when conducting studies that assess environmental
distribution.
When artificial sweeteners are applied in foodstuffs or
eventually enter the environment, transformation or/and
degradation may follow, by forming toxic substances. For
instance, SCL in the presence of glycerol may generate toxic
chloropropanols.68 Since SCL proved to be abundant, the co-
existence of chloropropanols cannot be excluded. In a recent
study, dehydrohalogenation of SCL was proposed after
hydrolysis at pH 10.59 As already mentioned in the introduc-
tion, transformation and biodegradation products of mainly
peptides, sweeteners and NHDC are expected to occur in the
environment. The identification of unknown transformation
products, such as microbial degradation products of SAC and
CYC, which are removed effectively during wastewater treat-
ment, should be investigated. Therefore, the scope of environ-
mental monitoring of artificial sweeteners should include their
potentially toxic transformation and degradation by-products.
As an extension of this, much is still needed to be done in order
to accurately assess the biogeochemical implications of artificial
sweetener usage on ecosystems worldwide.
It is worthwhile mentioning that the analytical performance of
a method often shows poor validation data (Table 1). Sole
demonstration of the instrumental LOD, without reference to the
method LOQ is useless, especially for monitoring application of
the method. The confirmed method LOQ is a very important
analytical parameter, since it determines the suitability of the
analytical approaches intended for monitoring purposes and
other environmental applications. In addition, validation data
such as the working range and linearity of the method, the
absolute and relative recoveries of the target analytes from at
least six replicates, and inter- and/or intra-day precision should
always be presented in order to demonstrate the fitness for
purpose and the applicability of an analytical method. Internal
standardization by isotope-labelled compounds for sulfamates
and dipeptides should be used in order to correct systematic
errors in the determination, and compounds such as acesulfame-
d4 (ACS-d4) and aspartame-d3 (ASP-d3) definitely improve the
analytical performance of the developed methods.51 Clearly
described and validated methods for the simultaneous determi-
nation of all artificial sweeteners in the environment are highly
required and expected.
Anal. Methods, 2012, 4, 3057–3070 | 3069
 View Online

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3070 | Anal. Methods, 2012, 4, 3057–3070 This journal is ª The Royal Society of Chemistry 2012