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Journal acronym: TFAC
Author(s):
Mannan Hajimahmoodi, Mahdi Afsharimanesh, Ghazaleh Moghaddam, Naﬁceh Sadeghi,
Mohammad Reza Oveisi, Behrooz Jannat, Elham Pirhadi, Fatemeh Zamani Mazdeh and Hossein Kanan
Article title:
Article no:
Enclosures:
Determination of eight synthetic dyes in foodstuffs by green liquid chromatography
774465
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Surname
1
Mannan
Hajimahmoodi
2
Mahdi
Afsharimanesh
3
Ghazaleh
Moghaddam
4
Naﬁceh
Sadeghi
5
Mohammad Reza
Oveisi
6
Behrooz
Jannat
7
Elham
Pirhadi
8
Fatemeh Zamani
Mazdeh
9
Hossein
Kanan
Sufﬁx
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AQ1
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Food Additives & Contaminants: Part A, 2013
Vol. 00, No. 00, 1–6, http://dx.doi.org/10.1080/19440049.2013.774465
Determination of eight synthetic dyes in foodstuffs by green liquid chromatography
Mannan Hajimahmoodia*, Mahdi Afsharimanesha, Ghazaleh Moghaddama, Naﬁceh Sadeghia, Mohammad Reza Oveisia,
Behrooz Jannatb, Elham Pirhadic, Fatemeh Zamani Mazdehc and Hossein Kanana
a
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Department of Drug and Food Control, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran; bFood and Drug
Laboratory, Research Center, Ministry of Health and Medical Education, Tehran, Iran; cFood and Drug Administration, Tehran
University of Medical Sciences, Tehran, Iran
(Received 21 October 2012; ﬁnal version received 21 January 2013)
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Eight synthetic food colours were analysed by green liquid chromatography. Green liquid chromatography is an environmentally friendly technique which does not use organic solvents in the extraction procedure or in the chromatographic
method. Analysis was carried out for the following colours: tartrazine (E102), indigotine (E132), Quinoline Yellow (E104),
Ponceau 4R (E124), Sunset Yellow (E110), Brilliant Blue (E133), Allura Red (E129) and carmoisine (E122) in four
different foods: cookies, coloured rice, saffron and fruit juice. The method was performed on an Eurospher-100 C8 (5 μm,
4.6 × 250 mm) column with ultraviolet (UV)-VIS detection and validated by determining the calibration lines, measurement
of recovery, precision, and limits of quantiﬁcation and detection (LODs and LOQs). LOD ranged from 0.04 mg kg–1 for
E102 to 1.00 mg kg–1 for E132; LOQ ranged from 0.06 mg kg–1 for E102 to 1.12 mg kg–1 for E122. The levels of colours
in foods were compared with Iranian National Standards, but only 7.5% of cookies, 30% of coloured rice, 8% of saffron and
12% of juice samples were in compliance with these standards. Tartrazine is prohibited in Iran, but it was found as the most
prevalent food colour in the samples analysed. The results of these tests conﬁrmed that HPLC avoiding the use of organic
solvents is a suitable method and can be used for quantitative analyses or screening of food samples for synthetic food
colours.
Keywords: green chromatography; synthetic dye; HPLC analysis
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Introduction
Currently, analytical methods are used for environmental
monitoring. However, a paradoxical situation has emerged
because most of the analytical methodologies employed to
investigate environmental problems themselves generate
chemical waste, resulting in an environmental impact.
Consequently, the aim of green chemistry has been
focused on the development of methodologies less harmful to humans and the environment. Green analytical
chemistry is at an early stage of development, but there
is a clear trend towards fast and consistent growth (Vidotti
et al. 2005).
Food colours are usually added to various commercial
food products in order to make them appear more attractive
and to achieve the desired colour. Food colours can be
divided into four categories: natural, nature-identical, inorganic and synthetic (Aberoumand 2011). For safety reasons
there have been recent reductions in the number of permitted food colours but they are still being used all over the
world because of their low price, effectiveness and stability.
Moreover, the food processing industry uses all types of
food colours, but to minimise potential toxicity the amounts
of permitted synthetic colours used are strictly limited
(Huang et al. 2005; Hajimahmoodi et al. 2008).
*Corresponding author. Email: [email protected]
© 2013 Taylor & Francis
Regulations for permitted edible food colours are
applied nationally and may vary from country to country.
In Iran there are seven permitted synthetic food colours,
including: indigotine (E132), Quinoline Yellow (E104),
Ponceau 4R (E124), Sunset Yellow (E110), Brilliant
Blue (E133), Allura Red (E129) and carmoisine (E122).
The use of tartrazine (E102) is prohibited in Iran, although
in fact it was the most prevalent colour determined in food
samples in this study. Therefore, an accurate and reliable
method is needed to measure synthetic food colours to
protect consumer health.
Ion-pair extraction (Van Peteghem and Bijil 2010),
adsorption on solid materials such as alumina or polyamide (Kirschbaum et al. 2006) has been used for colorant
extraction depending on food type and the chemical structure of the colour. TLC (Lotﬁ et al. 2008), adsorptive
voltammetry (Florian et al. 2002), spectrometry (Oveisi
et al. 2003), derivative spectrometry (Gianotti et al. 2005),
and capillary electrophoresis (Huang et al. 2005; Ma et al.
2006) can be used for quantitative analysis of colorants,
but reversed-phase liquid chromatography (Zatar et al.
2007), capillary electrophoresis (Huang et al. 2005), and
ion-pair chromatography (Zatar 2007) are still the most
widely used methods.
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Techniques evaluated in this article were carried out
by green chromatography, which is a general, accurate
and safe analytical method. Smaller amounts of toxic
organic solvents were used in this method than in previous research (Khanavi et al. 2011). Analysis was carried out on food samples consisting of cookies, coloured
rice, saffron and fruit juice. Samples of cookies and juice
had the highest levels of colouring. Saffron, with its
charming fragrance and delightful taste, is one of the
most valuable herbs in Iran, which accounts for 90% of
the world’s saffron production (Moghaddasi 2010). In
Iran rice is nearly always decorated with saffron. The
main aim of this study was to evaluate uses of food
colours in terms of a market survey for identiﬁcation
and quantiﬁcation.
Materials and methods
Chemicals and reagents
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The following solid standards were donated by Iran’s
Institute of Standards and Industrial Research: E102,
E104, E124, E110, E133, E122, E129 and E132; their
purities were ≥ 98%. They were dried at 65°C for 6 h,
then individually dissolved in deionised water at a stock
concentration of 1 mg ml−1. Polyamide powder (grain size
< 0.16) and other chemicals were of analytical grade and
purchased form Merck (Darmstadt, Germany).
Samples
Samples were divided into four groups (fruit juice, saffron,
coloured rice and cookie); each group under evaluation
consisted of 25 samples. These samples had been collected from restaurants, confectioneries and groceries;
they were stored as recommended on their labels and
analysed before expiration dates.
Sample preparation
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Totals of 10 ml of each juice sample, 5 g of coloured rice
or saffron samples were taken and ﬁrst diluted with
deionised water (1:1). The solid samples (coloured rice
and cookies) were transferred to a ﬂask separately, mixed
with 30 ml NH3, centrifuged for 5 min at 1800 rpm and
then the liquid phase (supernatant) separated. Polyamide
adsorbent (2 g) was added to the liquid extract and then
the mixture was stirred vigorously to adsorb all the colorants in the solution (if the solution was still coloured,
polyamide adsorbent (1 g) should be added). pH levels of
the solutions were adjusted to 3 with HCl (10% v/v),
ﬁltered and then the adsorbent was washed three times
with 20 ml distilled deionised water. The ﬁltered adsorbent was then transferred to a 100 ml beaker, 20 ml
solution of NH3 (25% w/v) was added and ﬁltered
again. The procedure was repeated twice to remove all
the colour from the polyamide. The collected solution
was dried out on a boiling water bath and the whole
residue was transferred to a 10-ml volumetric ﬂask
using mobile phase. Then it was ﬁltered through a
0.45 μm membrane material ﬁlter before HPLC analysis
(Khanavi et al. 2011). The preparation of liquid samples
(saffron and juice) were initiated by adding HCl 10% and
then the same procedure was followed as mentioned
above for solid samples. Analysis was carried out using
calibration curves and conﬁrmed by standards for each
dyes in the HPLC.
Thin layer chromatography (TLC)
TLC is the ofﬁcial Iranian standard method, but it is not ﬁt
for determination purposes. Whatman chromatographic
paper No. 3 was used to separate colorants. The mobile
phase consisted of an equal volume of NH3 (0.25% w/v)
and NaCl (1% w/v). The paper (20 × 20 cm) was marked
with each of the standard colours allowing about a 2.0 cm
minimum distance between each different spot. The chromatography paper was placed into a 250 ml beaker. The
beaker served to hold the solvent; it was ﬁlled to a depth
of 3–7 mm (10 ml of mobile phase). Lastly, the beaker
was covered with a watch glass to prevent the solvent
vapours.
Liquid chromatographic analysis
Samples were analysed by a Knauer HPLC (Germany)
system which consisted of a binary pump, a degasser, an
automated injector, a column oven and an ultraviolet (UV)
detector. The system was controlled by Eurochrom 2000
software. Since all eight colours tested in the study are not
usually present in foodstuff simultaneously, a different
strategy and approach that involved two different HPLC
conditions for the separation of these dyes were used
(Khanavi et al. 2011).
Chromatographic conditions were evaluated and optimised using a Eurospher-100 C8 (5 μm, 4.6 × 250 mm)
column. Before analysis, the temperature of the column
was set at 35°C and was conditioned making the mobile
phase ﬂow through the system for 30 min at 1.0 ml min−1.
The mobile phase was prepared by dissolving 0.25 ml
of Triton X-100 up to 100 ml with 50 mmol phosphate
buffer solution adjusted to pH 7 and the solution was
ﬁltered through a 0.45 µm membrane ﬁlter.
Due to polarity of the analytes, two different solvent
gradient systems (A and B) were employed to accomplish
a quick separation of the colours under analysis (Table 1).
After identifying the colour present in each sample by
TLC the most suitable method for each sample was
selected.
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Food Activities & Contaminants: Part A
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Table 1. Methods of the colour standard solutions.
Method A
Method B
Colour
E number
Detection wavelength (nm)
Retention time (min)
Flow rate (ml min–1)
Tartrazine
Quinoline Yellow
Ponceau 4R
Brilliant Blue
Allura Red
Carmoisine
Indigotine
Ponceau 4R
Sunset Yellow
Allura Red
Carmoisine
E102
E104
E124
E133
E129
E122
E132
E124
E110
E129
E122
450
450
630
630
515
515
600
450
450
515
515
2.103
5.634
7.818
12.409
20.120
33.256
4.017
7.761
10.436
20.120
33.256
1.5
1.5
1.5
1.5
2.25
2.25
1.5
1.5
1.5
2.25
2.25
Results and discussion
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Iranian National Standards have some stringent rules for
regulating the use of food colouring additives and only
seven synthetic colours are permitted under these regulations. However, methods to evaluate levels of colouring in
food for quality control are currently made by the TLC
method. Therefore, based on the achieved results, it seems
that approved standards need to be revised both in terms
of colour type and related quantitative limitations made
regarding further risk assessments.
Figure 1 shows the separation of a mixture of edible
colours which was actually found in one randomly
selected juice sample, including the prevalent Iranian
edible colours.
Some important method validation parameters are presented in Table 2. Results for correlation coefﬁcients were
always > 0.995 and closer to unity showing a good relationship between peak areas and concentrations. Detection
limits of colours in the samples were also found to be
satisfactory.
The ADI of a food additive is determined from an
estimated amount of that food additive expressed on a
body weight basis that can be ingested daily over a
lifetime without any appreciable health risk. Table 2
presents corresponding ADIs as set by the FAO/WHO
(1999), the European Food Safety Authority (EFSA
2009) and it also shows the percentages of each colour
in juice, coloured rice, saffron and cookie. Tartrazine is
commonly understood to contribute to several health
problems; it is therefore a prohibited food colour in
Iran Standards (Beseler 1999; Iran-Standard 2012).
However, according to these tests tartrazine was the
most commonly used colour in all four studied groups,
with a prevalence of 92% in saffron solution, 88% in
juice, 60% in coloured rice and 59% in cookie samples
(Table 2), but there were low percentages of indigotine
and Allura Red detected in the studied samples. Lok
et al. (2010) found that tartrazine and Sunset Yellow
were also the most common synthetic colourings used
in Hong Kong.
To check the accuracy of the method, different kinds
of matrices were spiked with the analyte of interest at
intermediate concentrations of the each calibration curves.
The concentrations were recalculated from the corresponding calibration straight line (experimental concentration)
and were compared with the theoretical concentrations.
Recovery was estimated as the relationship between the
experimental and the theoretical concentration expressed
as a percentage: (Cexp/Ctheo) × 100. Table 3 presents the
recoveries level of four groups of samples. The recoveries
(n = 6) ranged from 93.5 ± 0.9% to 99.7 ± 1.2%. Precision
accuracy showed good standard deviations and recovery
values.
The mean concentration of colours in each sample
group is presented in Table 4. All studied colours were
present in the juice group, but carmoisine (208 ±
65.3 mg kg–1) and indigotine (5.00 ± 0.83 mg kg–1)
recorded the highest and lowest amounts for colour additives, respectively. Research by Ma et al. (2006), carried
out in China, reported that tartrazine, Ponceau 4R and
Sunset Yellow contents in soft drinks were 10.52 ± 0.91,
2.44 ± 0.41 and 11.71 ± 0.93 mg kg–1, respectively,
records which were lower than the colour contents in
juice samples tested in this study (Table 4). Other research
in Taiwan showed that tartrazine was the most prevalent
colour used in soft drinks; Brilliant Blue at a level of
0.6 mg kg–1 and Allura Red at a level of 67.1 mg kg–1
had the highest and lowest concentrations (Huang et al.
2005). According to Brazilian legislation, maximum
allowable concentrations of Sunset Yellow, Allura Red
and tartrazine are set at 100 mg kg–1 in soft drinks
(Pereira Alves et al. 2008). Therefore, according to these
Brazilian Standards, these three dyes in the juice samples
tested in this study could be approved, but the amounts of
carmoisine were higher than the permitted level.
Saffron has been used as a spice and colouring agent
for many centuries, especially in Iran, and it is known to
have numerous medicinal properties (Moghaddasi 2010).
Crocin (C44 H64 O24) is the most signiﬁcant substances
providing the colouring power of saffron. It is a rare
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M. Hajimahmoodi et al.
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Figure 1. HPLC chromatogram of mixed colours which was actually found in one randomly selected juice sample by: 1. Method A:
(tartrazine, Quinoline Yellow; Ponceau; Brilliant Blue; Allura Red; carmoisine); and 2. Method B: (indigotine; Ponceau; Sunset Yellow;
Allura Red; carmoisine).
Table 2. Linear range, coefﬁcient of determination (R2), ADI permitted colours (mg kg–1 of body) and percentages of each dyes in
studied sample (%).
Colour
Tartrazine
Ponceau 4R
Brilliant Blue
Carmoisine
Sunset Yellow
Quinoline Yellow
Indigotine
Allura Red
250
Linear range (ppm)
R2
ADI (mg kg–1 bw–1)
Juice (%)
Coloured rice (%)
Saffron (%)
Cookie (%)
0.5–2.5
1–20
2–25
2–25
1–10
2–15
1–10
1–15
0.9923
0.9971
0.9996
0.9908
0.9984
0.9974
0.9952
0.9980
0–7.5
0–4.0
0–12.5
0–4.0
0–1
0–0.5
0–5.0
0–7.0
88
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40
52
40
24
8
8
92
4
−
−
20
12
−
−
60%
−
−
−
20%
10%
−
−
59
−
7
7
29
55
−
−
carotenoid found in nature which can easily dissolve in
water. In comparison with other carotenoids, crocin has a
wider application as a colour in food and medicine, mainly
because of its high solubility. As saffron is the world’s
most expensive spice, a dishonest producer may adulterate
saffron by adding a similar looking material or a yellow
dye to give the saffron the appearance of good quality.
Pure saffron contains only the stigma of the crocus ﬂower
Food Activities & Contaminants: Part A
5
Table 3. LOD, LOQ and recoveries of the edible dyes fortiﬁed to cookie, coloured rice, saffron and juice samples.
Dyes
Allura Red Indigotine Quinoline Yellow Sunset Yellow Carmoisine Brilliant Blue Ponceau
Matrices
Juice
Juice
Cookie
Spiked level
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5
8
Recovery (n = 6) 99.0 ± 0.5 98.9 ± 0.8
96.6 ± 0.6
0.45
0.06
0.40
LOD (mg kg–1)
LOQ (mg kg–1)
0.92
0.11
0.46
Table 4.
Saffron
5
93.5 ± 0.9
0.15
0.22
Juice
13
99.7 ± 1.2
1.00
1.12
Juice
13
98.3 ± 0.2
0.60
0.65
Tartrazine
Juice
Coloured rice
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1
97.0 ±0.4 98.1 ± 0.4
0.23
0.04
0.29
0.06
Mean and range of dye concentrations (mg kg–1) in studied samples.
Colour:
Sample
Tartrazine
Ponceau
Brilliant
Blue
Carmoisine
Sunset
Yellow
Quinoline
Yellow
Indigotine Allura Red
Juice
Mean 24.42
22.89
24.85
208.57
25.49
5.24
Range 0.06–121.82 0.37–187.72 2.75–71.44 11.9–603.14 0.48–215.14 0.8–13.79
2.46
6.46
1.93–3.00 5.77–7.15
Saffron
Mean 56.55
0.60
Range 0.04–129.69 0.60
n.d.
–
n.d.
–
2.14
0.94–4.81
44.8
0.84–73.10
n.d.
–
n.d.
–
Coloured Rice Mean 9.66
Range 0.20–38.01
n.d.
–
n.d.
–
n.d.
–
3.297
0.24–7.03
27.027
n.d.
10.23–43.82 –
n.d.
–
Cookie
n.d.
–
0.95
0.90–0.99
3.75
2.21–5.28
6.30
0.08–28.29
19.77
0.61–96.36
n.d.
–
Mean 4.66
Range 0.13–17.36
n.d.
–
Note: n.d., Not detected.
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with nothing else added. Therefore, related yellow synthetic dyes in saffron must be considered in evaluations of
purity in saffron. Adulteration of saffron, especially by
synthetic dyes, has not only an economic impact, but
also may have direct pharmacological and toxicological
consequences (Alonso et al. 1998; Fernandez 2004). In
view of the diversity of fraudulent methods, the purity of
saffron from the ﬁeld down to the consumer is important.
Chromatographic analyses showed evidence of the
colours tartrazine, Ponceau, Sunset Yellow and Quinoline
Yellow in saffron samples tested in this study and that
tartrazine with a mean concentration of 56 ± 39.9 mg kg–1
was the most predominantly used colorant. In Iran rice is
usually decorated with saffron and its high price contributes to the use of synthetic colour for dying rice.
Additives of synthetic dyes that were found in samples
of coloured rice were tartrazine, Quinoline Yellow and
Sunset Yellow, of which Quinoline Yellow had the record
for the maximum amount with a mean concentration of
27 ± 18.25 mg kg–1.
Concentrations of evaluated colours in cookies were
lower than those in other sample groups, but in spite of the
low amount of detected colours they had diverse colour
types after the juice samples. Quinoline Yellow and
Brilliant Blue with mean concentrations of 19 ± 9.5 and
0.95 ± 0.31 ppm were the lowest and highest records of
colour in cookie samples, respectively (Table 4).
In Greece, food colour contents were determined by
reversed-phase HPLC. Apart from Quinoline Yellow and
carmoisine as exceptions, all sweets had higher colour
concentrations than cookie samples in this study (Minioti
et al. 2007). Rao and Bhat (2003) studied 218 cookie
samples taken from the city and the countryside. In this
research only 38% of samples from the city and 39% of
samples from the countryside met the Indian Standard,
which is 100 ppm (maximum) for all determined food
colorants. In a study by Guler (2005) in which synthetic
colours were evaluated in confectionary and powder
drinks, 45% of whole samples had more than the approved
amounts of colorant under Turkish standards.
The TLC technique is the approved standard method
for food colour evaluation in Iran, but it does not provide
an accurate quantiﬁcation method (Iran-Standard 2012).
Iranian Standard numbers for different materials (saffron
solution A-1-259, orange juice 507, Cherry juice 5528,
Barberry juice 2736, multi-fruit juice 507, coloured rice
259, cookie 3493) recommend that there must be no trace
of synthetic colours in current unbranded products.
According to these above-mentioned standards, most of
the analysed samples in this study were rejected and just
7.5% of cookies, 30% of coloured rice, 8% of saffron and
12% of juice were approved.
Compared with other countries, Ponceau 4R is prohibited in the United States and Norway. Brilliant Blue is
prohibited in many countries including Belgium, France,
Germany, Switzerland, Sweden, Austria and Norway.
Tartrazine is also forbidden in the United States and
Austria (Vachirapatama et al. 2008). However, countries
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have different restrictions. Regarding Japanese standards
for food additives, Brilliant Blue, indigo carmine, amaranth, erythrosine, Allura Red, Sunset Yellow and tartrazine are permitted without any restrictions (Speciﬁcations
and food standards, food additives 2011). Synthetic colours are used in Hong Kong; they follow the Codex
Alimentarius Commission of the FAO/WHO, which is
currently being updated (Lok et al. 2010).
Conclusion
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There have been problems with Iranian National Standards
for regulating the use of food colorants in terms of determining food colouring types and quantitative limitations.
Therefore, it is necessary to revise standards and introduce
a reliable quantitative method to control food quality.
Quantitative analysis of colorants resulting from this
study has demonstrated that some manufacturers have
used synthetic dyes to improve the appearance of their
product against available standards.
References
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