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Food Chemistry 138 (2013) 17421748

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Food Chemistry
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Analytical Methods

Determination of seven synthetic dyes in animal feeds and meat by high

performance liquid chromatography with diode array and tandem mass detectors
Tingting Zou a, Pingli He a, Amangul Yasen a, Zhen Li b,

State Key Laboratory of Animal Nutrition, China Agricultural University, Beijing 100193, PR China
State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, PR China

a r t i c l e

i n f o

Article history:
Received 21 September 2012
Received in revised form 12 November 2012
Accepted 17 November 2012
Available online 29 November 2012
Synthetic food dyes
Animal feed

a b s t r a c t
An efcient method was developed for the simultaneous determination of seven commonly used synthetic sulfonate dyes (Ponceau 4RC, Sunset yellow, Allura red, Azophloxine, Ponceau xylidine, Erythrosine
and Orange II) in animal feed and meat using high performance liquid chromatography (HPLC-DAD) and
tandem mass spectrometry (HPLCMS/MS). Ethanolammoniawater (80:1:19, V/V/V) solution was used
as extract solution, which can extract target species while reducing interference from the sample matrices. The recoveries of these 7 dyes in animal feed and chicken meat were between 71% and 97% with relative standard deviations less than 14.8%. HPLCMS/MS was employed as a further means of
conrmation to assure accuracy of the results. Limits of detection for these dyes were in the range of
0.0221.83 ng mL 1. The proposed method can be applied to conrmative screening of seven commonly
used food colorants in feed and meat samples.
2012 Elsevier Ltd. All rights reserved.

1. Introduction
Food colorants are an important class of food additives. They are
widely used in drinks, juices, meat products and sweets to preserve
or restore the natural color of food products and enhance appeal
(Vidotti, Cancino, Oliveira, & Rollemberg, 2005). Natural food dyes
have been used more and more for consumer preference, however,
synthetic food dyes are still widely used in food and feed industry
because of their low cost and high stability. Most of the synthetic
dyes show good resistance against degradation and pose little
threat to human and animal health. But some of these substances
and their metabolites pose potential health risk to human beings
and may even be carcinogenic, especially when they are consumed
in excessive amounts (Robens et al., 1980). These adverse health
effects include allergy and asthmatic reaction (Ibero, Eseverri,
Barroso, & Botey, 1982; Miller, 1982; Settipane et al., 1976), DNA
damage (Combes & Haveland-Smith, 1982; Sasaki et al., 2002),
hyperactivity (McCann et al., 2007; Rowe & Rowe, 1994) and carcinogenesis (JECFA., 1975) etc. Therefore, the use of synthetic dyes in
foodstuff is strictly controlled by legislation throughout the world
(EC., 1994; GB2760-2011, 2011). In Japan, all ingredients including
food colorants are required to be listed on the package label
(Yoshioka & Ichihashi, 2008). In China, the maximum amount
allowed for most colorants is no more than 100 mg kg 1
(GB2760-2007, 2007). In Europe, the maximum level allowed for
Corresponding author. Tel./fax: +8610 62731128.
E-mail address: (Z. Li).
0308-8146/$ - see front matter 2012 Elsevier Ltd. All rights reserved.

Allura red used in luncheon meat and breakfast sausage with a

minimum cereal content of 6% is 25 mg kg 1, the maximum level
allowed for Ponceau 4RC used in jam and jellies is 100 mg kg 1
(EC, 1994). Thus, it is necessary to develop accurate and reliable
analytical methods for the conrmative determination of synthetic
food dyes in foodstuffs of various matrices to ensure food safety
and consumer health.
Various analytical methods have been reported for determining
synthetic food dyes from soft drinks, juices, fruit jellies, candies,
edible animal tissues and spices. Such methods include: capillary
electrophoresis (Huang, Shih, & Chen, 2002; Prado, Boas, Bronze,
& Godoy, 2006), thin layer chromatography (Oka, Ikaia, Kawamura,
Yamada, & Inoue, 1987; Oka et al., 1994), electrochemistry (Combeau, Chatelut, & Vittori, 2002; Ni, Bai, & Jin, 1996), spectrophotometry (Dos Santos, Demiate, & Nagata, 2010; Soylak, Unsal, &
Tuzen, 2011; Unsal, Soylak, & Tuzen, 2012) and ion-pair chromatography (Fuh & Chia, 2002). With the fast development of detection technology, high performance liquid chromatography (HPLC)
with ultraviolet/visible (UV/Vis) and diode-array detectors (DAD)
(Yoshioka & Ichihashi, 2008; Minioti, Sakellariou, & Thomaidis,
2007) and liquid chromatographymass spectrometry (LCMS)
(Feng et al., 2011) also have been applied to the detection of synthetic dyes in foodstuffs. Feng et al. established a sensitive screening method for 40 dyes in soft drinks by liquid chromatography
electrospray tandem mass spectrometry. LCMS based methods
showed signicant improvement in sensitivity and specicity compared with traditional methods, but it has strict requirements in
sample pretreatment due to interference and signal suppression

T. Zou et al. / Food Chemistry 138 (2013) 17421748

from the sample matrices, also the instrument is very expensive. In

contrast, HPLC-DAD is very popular for qualitative and quantitative
determination with excellent precision, accuracy and lower cost,
which can be much more practical and economical in detecting
non-illict additives such as food colorants.
Most of the reported LC based methods focused on the determination of food dyes in liquid samples, like soft drink and fruit
drinks, or water soluble samples like fruit jelly, jam and candies.
However, these methods are not suitable for determining dyes in
solid food matrix. This is due to the high protein content in samples like meat, poultry products and animal feed. The high protein
and salt content of such samples may induce severe interference
for LCUVVis or LCMS measurements, and lead to low sensitivity
and specicity of the developed methods. In this paper, we
developed an HPLC-DAD method for the determination of seven
commonly used food dyes in high protein content matrices (feed
and chicken meat), HPLCMS was used as a conrmational step
to assure accuracy of the results.
The ethanolammonia extraction method developed here provides an efcient and convenient way of removing protein, starch
and other interferences from the sample. The extract solution can
be analyzed directly with LC-DAD and LCMS, thus, eliminate the
complicated SPE cleanup step, which is required for most LCMS
methods. Detection sensitivity in the low ppb range can be
achieved for both detection methods. With the developed
method, we analyzed dye-spiked complete feed and poultry meat
samples, recovery and accuracy of the method were also
2. Experimental
2.1. Reagents and chemicals
Orange II (94%), Ponceau 4RC (75%) and Allura red (80%) were
purchased from the Dr. Ehrenstorfer GmbH Company (Augsburg,
Germany). Azophloxine (P96%), Ponceau xylidine (P96%), Erythrosine (P96%) and Sunset yellow (P96%) were purchased from
Fluka (Buchs, Switzerland) (Table 1). HPLC grade methanol, ethanol
and acetonitrile were obtained from Fisher Scientic International
(Hampton, NH). HPLC grade formic acid and ammonium acetate
were purchased from Dikma Technology (Richmond Hill, Canada).
All other reagents were analytical grade. Ultra-pure water with a
resistance of 18.2 MX cm 1 was puried using a Milli-Q system
(Millipore, Bedford, USA). Nylon membrane lters (0.22 lm) were
obtained from Whatman (Maidstone, UK).
2.2. Apparatus
The HPLC-DAD method was developed using an Agilent 1200
HPLC system with binary gradient pump, auto-sampler, temperature controlled column oven and DAD detector (Agilent Technologies, Fermont, CA). HPLCMS/MS analysis was performed on an
Agilent 1260 HPLC system coupled with an Agilent 6460 triple
quadrupole mass spectrometer.
2.3. Preparation of standard solutions
Standard stock solutions of each dye (1 mg mL 1) were prepared
by dissolving the dyes in pure water with the exception of sunset
yellow which was dissolved in 20 mM ammonium acetatemethanol (1:1, V/V). The standard solutions were stored in darkness at
20 C until use. Mixed standard stock solution was prepared by
mixing and diluting the standard stock solution of each dye with
pure water to a nal concentration of 100 lg mL 1 and stored at
4 C. Standard working solution was prepared daily by diluting


the mixed standard stock solution with ethanolammoniawater

(80:1:19, V/V/V) to appropriate concentrations. The pH of standard
working solutions was adjusted to 5.0 with formic acid. For HPLCDAD analysis, the concentrations of standard working solution
ranged from 100 to 5000 ng mL 1; for HPLCMS/MS analysis, the
concentrations ranged from 0.1 to 2000 ng mL 1. All the working
solutions were prepared fresh before use.
2.4. Sample collection and preparation
Complete feed samples were prepared at the pilot mill of the
Ministry of Agricultural Feed Industry Center (Beijing, China). Poultry meat (chicken) was purchased from local supermarket. Before
extraction, the feed samples were pulverized by a grinder, and
sieved through a No. 60 mesh; the chicken meat samples were
homogenized in a homogenizer for 5 min (Da Kang, Tianjin, China).
2.5. Sample extraction
Dye-spiked samples were prepared in a 50 mL conical ask by
mixing 2 g of complete feed sample or homogenized chicken meat
sample with a series of the 7-dye mixed standard solutions at various concentrations. Sample extraction was achieved by adding
10 mL ethanolammoniawater (80:1:19, V/V/V) extraction solution and stirring for 30 min. The extraction solution was centrifuged at 12,000 rpm for 10 min at 4 C to remove protein. For
HPLC-DAD analysis, 2.5 mL of the supernatant was evaporated to
dryness in 50 C water bath under nitrogen stream. The residue
was reconstituted with 0.5 mL ethanolammoniawater (80:1:19,
V/V/V). The solution was adjusted to pH 5.0 with formic acid and
passed through a membrane lter (0.22 lm). Twenty micro liters
of the solution was injected into the HPLC-DAD system. By doing
so, the dyes were concentrated 5 times in the nal injected sample
compared with the extraction supernatants. For HPLCMS/MS
analysis, the extraction supernatant was adjusted to pH 5.0 with
formic acid and passed through a 0.22 lm membrane, then 10 lL
of the solution was injected for detection. Blank samples were pretreated in the same manner.
2.6. HPLC conditions
Chromatographic separation of the dyes mixture was achieved
on a Waters Atlantis T3 column (2.1 mm  150 mm, 3 lm). The
column temperature was set at 30 C. Mobile phase consisted A:
20 mM ammonium acetate and B: acetonitrile. A gradient program
was used for elution: 5% solvent B (initial), 540% solvent B (from 0
to 4 min), 4090% solvent B (from 4 to 9 min) and 90% solvent B
(from 9 to 11 min). After 11 min, the ratio was reduced to 5% solvent B. A 5-min equilibration was necessary before the next injection, so the total run time was 16 min. The mobile phase was
delivered at a ow rate of 0.2 mL min 1. The diode-array detector
was set to monitor the 7 dyes at two pre-selected wavelengths:
484 and 526 nm.
2.7. HPLCMS/MS conditions
Waters Atlantis dC18 column (2.1  150 mm, 5 lm) was used
for LCMS/MS conrmative analysis. The gradient elution program
was a little different from LC-DAD experiments: 5% solvent B (initial), 540% solvent B (from 0 to 5 min), 4090% solvent B (from 5
to 9 min) and 90% solvent B (from 9 to 11 min). After 11 min, the
ratio was reduced to 5% solvent B, the ow rate was 0.3 mL min 1.
The triple quadrupole tandem mass spectrometer operated under
multiple reaction monitor mode (MRM) for quantitative and qualitative analysis. Negatively charged ion species from the 7 dyes
were monitored. The optimized electrospray ionization conditions


Table 1
Chemical structures and tandem mass spectrometry parameters of the 7 dyes tested.

Chemical structure

Molecular weight (g/mol)

Quantitative transition m/z

Qualitative transition m/z

Fragmentor voltage (V)

Collision energy (V)

Ponceau 4RC







Sunset yellow







Allura red














T. Zou et al. / Food Chemistry 138 (2013) 17421748

Retention time (min)














Orange II







T. Zou et al. / Food Chemistry 138 (2013) 17421748




T. Zou et al. / Food Chemistry 138 (2013) 17421748

were: gas temperature 350 C, gas ow 10 L min 1, sheath gas

temperature 400 C, sheath gas ow 12 L min 1 and capillary voltage 3500 V. Detailed MRM settings were listed in Table 1.

3. Results and discussion

3.1. Optimization of sample pretreatment
The development of an efcient sample extraction procedure is
critical for accurate determination of dyes in animal feed and
meat samples. The complex matrices of the samples pose challenges for the sample extraction procedure, which has to be effective in removing interfering matrices while retaining the highest
sensitivity and specicity. Four extract solvents were evaluated
in this experiment based on chemical properties of the 7 dyes
(Table 1). The extract solvents include: ethanolammoniawater
80:1:19, (V/V/V), 80:5:15, (V/V/V), 80:10:10, (V/V/V) and methanolammoniawater 80:1:19, (V/V/V). Ethanolammoniawater
80:1:19, (V/V/V) and 80:10:10, (V/V/V) showed similar extraction
efciency in terms of recovery and reproducibility for the 7 dyes
(Fig. 1). Most of the interference proteins present in feed and meat
samples were precipitated in the presence of high percentage ethanol, and they can be removed efciently by the subsequent
refrigerated centrifugation procedure. Afterwards, pH of the
supernatant solutions was adjusted to approximate 5.0 with formic acid, so that it is compatible with the HPLC mobile phase. Ethanolammoniawater 80:1:19 (V/V/V) was chosen as the nal
extract solvent for the 7 dyes because its much easier to adjust
the pH of supernatant to pH 5.0 with less ammonia in the
In this experiment, further purication of the extract solution
with solid-phase extraction was also considered, Waters Oasis
3 cc,
60 mg) and Waters Oasis MAX (mixed-mode anion exchange sorbent, 3 cc, 60 mg) cartridge were tested. With the extra SPE purication step, the chromatograms showed little improvement,
impurities observed during the rst 2 min of LC elution cannot
be removed with SPE. Since all analytes of interest eluted after
4 min, the existence of these low retention interferences did not affect the detection. Moreover, these 7 dyes possess different chemical characteristics, especially ER, which has distinct chemical
structure from the other dyes, no single SPE column can achieve
satisfactory retention and recovery performance for each single
dye. For these reasons, in the developed method, SPE was not
adopted as it is not necessary, and without SPE, the whole pretreatment process is very convenient and effective.

Fig. 1. Recoveries of each dye from complete feed using four different extract
solutions: (a) ethanolammoniawater (80:1:19, V/V/V); (b) ethanolammonia
water (80:5:15, V/V/V); (c) ethanolammoniawater (80:10:10, V/V/V); (d) methanolammoniawater (80:1:19, V/V/V). 4RC, Ponceau 4RC; SY, Sunset yellow; AR,
Allura red; AZ, Azophloxine; PX, Ponceau xylidine; ER, Erythrosine; OG, Orange II.

3.2. HPLC-DAD method development

The chemical structures of the 7 dyes studied are shown in
Table 1. Here we chose Atlantis T3 column due to its excellent
resolving power for middle to high polar compounds. The ow rate
was set at 0.2 mL/min, as a higher ow rate resulted in poor separation between Ponceau 4RC and Sunset yellow. Optimal UVVis
signals were obtained using scan mode of the DAD detector, most
of the dyes showed good sensitivity at 526 nm, but for Sunset yellow, the wavelength was set at 484 nm for enhancing signal intensity. The chromatogram of mixed standard solution showed clear
baseline separation of the 7 dyes in a single 16-min LC run
(Fig. 2A).
Linearity, limit of detection, precision and recovery were determined to evaluate the validity of the method. Linearity was studied
by analyzing mixed standard working solutions of the 7 dyes at
several concentrations ranging from 100 to 5000 ng mL 1 in HPLC.
Most of the 7 dyes showed satisfactory linearity within the concentration range of 5005000 ng mL 1 (R2 > 0.996). Limit of detection
(LOD), which was dened as the concentration at 3 times the signal
intensity of noise, was determined by examining HPLC-DAD chromatograms of the extract solution of the dye-spiked complete feed
samples. The LOD values were in the range of 6.4374.81 ng mL 1
(Table 2).
The precision of the method was evaluated for intra- and interday precision. The intra-day RSD for 7 dyes in spiked feed sample
ranged from 3.0% to 19.0%, and the inter-day RSD ranged from
0.7% to 8.6%. The intra-day RSD was higher than inter-day RSD because some of the dyes showed degradation at room temperature
during the extended period of analysis of 5 replicates. The overall
precision and accuracy of the method were sufcient for the quantication of the 7 dyes in real samples.
The established method was applied to recovery analysis of
dye-spiked blank feed and meat samples to validate this method
for routine monitoring of animal feed and meat samples. Dyes
were spiked in complete feed at 1 and 5 mg kg 1, and poultry meat
at 0.5 and 1 mg kg 1. Six replicates were tested for each concentration. Chromatograms for complete feed sample spiked with 7 dye
mixture (1 mg kg 1) and a blank feed sample were shown in
Fig. 2B. The clean baseline of the chromatogram indicates the
developed sample extraction method can extract dyes from the
sample effectively while bringing in minimum interference from
the sample matrix. Calibration curves obtained from external standards were used to calculate recovery of the developed method.
The recoveries for spiked feed samples ranged from 71% to 94%

Fig. 2. Chromatograms of HPLC-DAD: (A) the 7-dye mixed standard; (B) complete
feed sample spiked with 7-dye mixed standard (1 mg kg 1) and blank feed sample.
(1) Ponceau 4RC; (2) Sunset yellow; (3) Allura red; (4) Azophloxine; (5) Ponceau
xylidine; (6) Erythrosine; (7) Orange II.


T. Zou et al. / Food Chemistry 138 (2013) 17421748

Table 2
Linear relationships, sensitivities and LODs for the detection of 7 dyes using HPLC-DAD and HPLCMS/MS.

Ponceau 4RC
Sunset yellow
Allura red
Ponceau xylidine
Orange II



Linear Range
(ng mL 1)

Linear equation


Y = 76.018x + 35.811
Y = 13.146x + 1.67
Y = 68.8x 12.9
Y = 128.12x + 21.238
Y = 222.85x 32.005
Y = 482.67x 9.8918
Y = 353.62x 2.8836

(ng mL




Linear range
(ng mL 1)

Linear equation


y = 49.073x 537.22
y = 78.37x 400.03
y = 95.469x + 114.81
y = 72.931x + 740.79
y = 105.49x + 4900.3
y = 936.78 + 364.14
y = 1550.7x + 1391.9

(ng mL



Table 3
Percentage recoveries (%) and relative standard deviations (%) of the 7 dyes in different matrices (n = 6).



1 mg kg
Ponceau 4RC
Sunset yellow
Allura red
Ponceau xylidine
Orange II


Poultry meat

5 mg kg

0.5 mg kg


and RSDs were less than 10.6%. The recoveries for spiked chicken
samples ranged from 78% to 97%, and RSDs were less than 14.8%
(Table 3).

3.3. HPLCMS/MS analysis

3.3.1. Optimization of HPLCMS/MS conditions
The complexity of the sample matrix may induce interference in
HPLC-DAD measurements, so HPLC-DAD alone is not adequate for
conrmative determination of low-level additives in food and feed
samples. HPLCMS/MS was used as a conrmative step to further
conrm the existence of the dyes in the sample matrices.

Fig. 3. Chromatograms of HPLCMS: (A) the 7-dye mixed standard; (B) chicken
sample spiked with 7-dye mixed standard (0.5 mg kg 1) and blank chicken meat
sample; (Inset) quantitative product ion chromatograms of the 7 dyes. (1) Ponceau
4RC; (2) Sunset yellow; (3) Allura red; (4) Azophloxine; (5) Ponceau xylidine; (6)
Erythrosine; (7) Orange II.

1 mg kg

1 mg kg

Poultry meat

5 mg kg

0.5 mg kg

1 mg kg


Most of the dyes contain a sulfonate group in their molecular

structure, and the most abundant precursor ions of the dyes were
their negatively charged sodium adduct ions [M Na] or
[M 2Na]2 , these ions were chosen as the precursor ions for
MRM. Characteristic product ions were obtained by ramping the
collision energy; the two most abundant product ions of the species were selected for MRM conrmation with the most abundant
product ion used for quantitation. Optimal MS parameters were
listed in Table 1 and quantitative product ion chromatograms of
the 7 dyes were shown in Fig. 3 (Inset). Since, the colorants showed
degradation at room temperature, in order to obtain comparable
data from LC-DAD and LCMS/MS experiments, samples must be
run simultaneously on both instruments. A Waters dC18 column
was used here in addition to the T3 column used in LC-DAD analysis. The relatively old dC18 column showed slightly lower separation efciency, so Allura red and Azophloxine were co-eluted from
the column, but with different MRM transition, these two compounds could still be distinguished. Chromatograms for the 7-dye
mixed standard was shown in Fig. 3A, extract ion chromatogram
for chicken meat spiked with the 7-dye mixture (0.5 mg kg 1)
and a blank chicken sample were shown in Fig. 3B.
3.3.2. HPLCMS/MS method validation
LCMS based method showed better sensitivity, most of the 7
dyes showed satisfactory linearity within the concentration range
of 0.11000 ng mL 1 (R2 > 0.996), and limit of quantitation (LOD)
was in the range of 0.0221.83 ng mL 1 (Table 2). Intra-day RSDs
for 7-dye mixed standard ranged from 2.0% to 16.2%, and the inter-day RSDs ranged from 0.1% to 2.6%. The intra-day RSDs for seven colorants in spiked samples ranged from 5.7% to 17.7%, and the
inter-day precision ranged from 4.3% to 14.5%.
The actual spiked quantity and the measured concentration in
feed and meat matrices showed good consistency (Table 3). The
recoveries for spiked feed samples ranged from 51% to 106% and
relative standard deviations were less than 14.5%. The recoveries
for spiked chicken samples ranged from 59% to 113% and relative
standard deviations were less than 15.8%. The HPLCMS/MS results


T. Zou et al. / Food Chemistry 138 (2013) 17421748

were in good accordance with HPLC-DAD, which proved the accuracy of the method developed.
4. Conclusion
In summary, the very simple and effective HPLC-DAD method
with ammoniaethanol extraction provides satisfactory specicity
and sensitivity for the determination of seven commonly used sulfonate dyes in feed and meat samples. Meanwhile, HPLCMS/MS,
which served as a further conrmative step, showed good sensitivity under complex sample matrix situation and assured accuracy of
the method developed. Analysis of dye-spiked samples with the
simple extraction proctol developed here showed that the recoveries ranged from 71% to 97%, which can meet regular analytical
requirements. With miniature and portability of new LC instruments, this method could provide a way for onsite monitoring of
colorants in feed and animal products.
Financial supports from the Key Projects in The National Science & Technology Pillar Program during the eleventh ve-year
plan (2009BADB7B07), Agro scientic Research in the Public Interest (201203088), and the National Natural Science Foundation of
China (31170343) are gratefully acknowledged.
Combeau, S., Chatelut, M., & Vittori, O. (2002). Identication and simultaneous
determination of Azorubine, Allura red and Ponceau 4R by differential pulse
polarography: Application to soft drinks. Talanta, 56, 115122.
Combes, R. D., & Haveland-Smith, R. B. (1982). A review of the genotoxicity of food,
drug and cosmetic colours and other azo, triphenylmethane and xanthene dyes.
Mutation Research, 98, 101243.
Dos Santos, M. E., Demiate, I. M., & Nagata, N. (2010). Simultaneous determination
of tartrazine and sunset yellow in food by spectrophotometry UVVIS and
multivariate calibration methodology. Ciencia E Tecnologia De Alimentos, 30,
EC. (1994). Directive of the European Parliament and of the council 94/36/EC of June
30, 1994 on colours for use in foodstuffs. Ofcial Journal, L237, 13 (10/9/1994).
Feng, F., Zhao, Y. S., Yong, W., Sun, L., Jiang, G. B., & Chu, X. G. (2011). Highly sensitive
and accurate screening of 40 dyes in soft drinks by liquid chromatography
electrospray tandem mass spectrometry. Journal of Chromatography B, 879,
Fuh, M. R., & Chia, K. J. (2002). Determination of sulphonated azo dyes in food by
ion-pair liquid chromatography with photodiode array and electrospray mass
spectrometry detection. Talanta, 56, 663671.
GB2760-2007 (2007). Hygienic standards for the use of food additives. Ministry of
Health of the Peoples Republic of China.
GB2760-2011 (2011). Hygienic standards for the use of food additives. Ministry of
Health of the Peoples Republic of China.

Huang, H. Y., Shih, Y. C., & Chen, Y. C. (2002). Determining eight colorants in milk
beverages by capillary Electrophoresis. Journal of Chromatography A, 959,
Ibero, M., Eseverri, J. L., Barroso, C., & Botey, J. (1982). Dyes, preservatives and
salicylates in the induction of food intolerance and/or hypersensitivity in
children. Allergologia et immunopathologia (Madrid), 10, 263268.
JECFA (1975). Amaranth. In WHO food additives series (vol. 8). Geneva: World Health
McCann, D., Barrett, A., Cooper, A., Crumpler, D., Dalen, L., Grimshaw, K., et al.
(2007). Food additives and hyperactive behaviour in 3-year-old and 8/9-yearold children in the community: A randomised, doubleblinded, placebocontrolled trial. Lancet, 370, 15601567.
Miller, K. (1982). Sensitivity to Tartrazine. British Medical Journal, 285, 15971598.
Minioti, K. S., Sakellariou, C. F., & Thomaidis, N. S. (2007). Determination of 13
synthetic food colorants in water-soluble foods by reversed-phase highperformance liquid chromatography coupled with diode-array detector.
Analytica Chimica Acta, 583, 103110.
Ni, Y., Bai, J., & Jin, L. (1996). Simultaneous adsorptive voltammetric analysis of
mixed colorants by multivariate calibration approach. Analytica Chimica Acta,
329, 6572.
Oka, H., Ikaia, Y., Kawamura, N., Yamada, M., & Inoue, H. (1987). Simple method for
the analysis of food dyes on reversed-phase thin-layer plates. Journal of
Chromatography, 411, 437444.
Oka, H., Ikaia, Y., Ohno, T., Kawamura, N., Hayakawa, J., Harada, K., et al. (1994).
Identication of unlawful food dyes by thin-layer chromatography-fast atom
bombardment mass spectrometry. Journal of Chromatography A, 674, 301307.
Prado, M. A., Boas, L. F. V., Bronze, M. R., & Godoy, H. T. (2006). Validation of
methodology for simultaneous determination of synthetic dyes in alcoholic
beverages by capillary electrophoresis. Journal of Chromatography A, 1136,
Robens, J. F., Dill, G. S., Ward, J. M., Joiner, J. R., Griesemer, R. A., & Douglas, J. F.
(1980). Thirteen-week subchronic toxicity studies of Direct Blue 6, Direct Black
38, and Direct Brown 95 dyes. Toxicology and Applied Pharmacology, 54,
Rowe, K. S., & Rowe, K. J. (1994). Synthetic food coloring and behavior: A dose
response effect in a double-blind, placebo controlled, repeated-measures study.
Journal of Pediatrics, 125, 691698.
Sasaki, Y. F., Kawaguchi, S., Kamaya, A., Ohshita, M., Kabasawa, K., Iwama, K., et al.
(2002). The comet assay with 8 mouse organs: results with 39 currently used
food additives. Mutation Research, 519, 103119.
Settipane, G. A., Chafee, F. H., Postman, I. M., Levine, M. I., Saker, J. H., Barrick, R. H.,
et al. (1976). Signicance of Tartrazine sensitivity in chronic urticaria of
unknown etiology. Journal of Allergy and Clinical Immunology, 57, 541546.
Soylak, M., Unsal, Y. E., & Tuzen, M. (2011). Spectrophotometric determination of
trace levels of allura red in water samples after separation and
preconcentration. Food and Chemical Toxicology, 49, 11831187.
Unsal, Y. E., Soylak, M., & Tuzen, M. (2012). Column solid-phase extraction of sunset
yellow and spectrophotometric determination of its use in powdered beverage
and confectionery products. International Journal of Food Science & Technology,
47, 12531258.
Vidotti, E. C., Cancino, J. C., Oliveira, C. C., & Rollemberg, M. C. E. (2005).
spectrophotometry with sorption onto polyurethane foam. Analytical Sciences,
21, 149153.
Yoshioka, N., & Ichihashi, K. (2008). Determination of 40 synthetic food colors in
drinks and candies by high-performance liquid chromatography using a short
column with photodiode array detection. Talanta, 74, 14081413.