Analytica Chimica Acta 583 (2007) 103–110

Determination of 13 synthetic food colorants in water-soluble foods

by reversed-phase high-performance liquid chromatography
coupled with diode-array detector
Katerina S. Minioti a , Christina F. Sakellariou b , Nikolaos S. Thomaidis a,∗
a Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, Panepistimioupolis Zografou, 15771 Athens, Greece
b General Chemical State Laboratory, D’ Division of Athens, Section B’, An. Tsoha 16, 11521 Athens, Greece

Received 1 August 2006; received in revised form 29 September 2006; accepted 2 October 2006
Available online 6 October 2006

Abstract
A reversed-phase high performance liquid chromatographic method for the successful separation and determination of 13 synthetic food colorants
(Tartrazine E 102, Quinoline Yellow E 104, Sunset Yellow E 110, Carmoisine E 122, Amaranth E 123, Ponceau 4R E 124, Erythrosine E 127, Red
2G E 128, Allura Red AC E 129, Patent Blue V E 131, Indigo Carmine E 132, Brilliant Blue FCF E 133 and Green S E 142) was developed. A C18
stationary phase was used and the mobile phase contained an acetonitrile–methanol (20:80 v/v) mixture and a 1% (m/v) ammonium acetate buffer
solution at pH 7.5. Successful separation was obtained for all the compounds using an optimized gradient elution within 29 min. The diode-array
detector was used to monitor the colorants between 350 and 800 nm. The method was thoroughly validated. Detection limits for all substances
varied between 1.59 (E 142) and 22.1 (E 124) ␮g L−1 . The intra-day precision (as R.S.D.r ) ranged from 0.37% (E 122 in fruit flavored drink
at a concentration of 100 mg L−1 ) to 4.8% (E 142 in icing sugar at a level of 0.9 mg kg−1 ). The inter-day precision (as R.S.D.R ) was between
0.86% for E 122 in fruit flavored drink at 100 mg L−1 and 10% for E142 in jam at a concentration of 9 mg kg−1 . Satisfactory recoveries, ranging
from 94% (E 142 in jam) to 102% (E 131 in sweets), were obtained. The method was applied to the determination of colorants in various water-
soluble foods, such as fruit flavoured drinks, alcoholic drinks, jams, sugar confectionery and sweets, with simple pre-treatment (dilution or water
extraction).
© 2006 Elsevier B.V. All rights reserved.

Keywords: Liquid chromatography; Diode-array detector; Synthetic dyes; Food analysis; Confectionary; Soft drinks

1. Introduction classes: the azo compounds (E 102, E 110, E 122, E 123, E

Synthetic colorants are a very important class of food addi-
tives. They are widely used to compensate for the loss of natural
colors of food, which are destroyed during processing and stor-
age, and to provide the desired colored appearance. However,
some of these substances pose a potential risk to human health,
especially if they are excessively consumed. For this reason,
safety data, such as the acceptable daily intake, based on tox-
icological studies on experimental animals and human clinical
studies, have been repeatedly determined and evaluated by Food
and Agricultural Organization (FAO) and World Health Orga-
nization (WHO) [1]. They are divided into five major colorant
∗ Corresponding author. Tel.: +30 210 7274317; fax: +30 210 7274750.
E-mail address: ntho@chem.uoa.gr (N.S. Thomaidis).
124, E 128 and E 129), the triarylmethane group (E 131, E 133
and E142), the chinophthalon derivative of Quinoline Yellow (E
104), xanthenes as Erythrosine (E 127) and the indigo colorants
(Indigo Carmine E 132).
The use of synthetic colorants in foods is strictly controlled
by legislation and harmonized across the European Union by
formulating the directive 94/36/EC [2]. Consequently, accu-
rate and reliable methods for the determination of synthetic
colorants are required for the assurance of food safety. Many
analytical techniques have been developed for the identification
and determination of various synthetic food colorants, such as
thin-layer chromatography [3,4], adsorptive voltammetry [5],
and differential pulse polarography [6], derivative spectrometry
[7–11] and spectrophotometric methods in combination with
chemometrics [12,13], but all of them require time-consuming
pretreatment or cannot be applied to complex colorant mixtures.

0003-2670/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.aca.2006.10.002
104 K.S. Minioti et al. / Analytica Chimica Acta 583 (2007) 103–110
Capillary electrophoresis [14–18] and micellar electrokinetic
capillary chromatography [19] have also been used, but they
have sensitivity problems as a result of small injection volume.
High-performance ion chromatography [20], reversed-phase liq-
uid chromatography [21–23] and ion-pair liquid chromatogra-
phy [11,19,24–29] coupled with UV or diode-array detectors
are still the most preferred methods, as they provide unrivalled
resolution, sensitivity and selectivity. Both isocratic [24,25,29]
and gradient [19–23,26–28] systems are used, and the latter
are preferred for the separation of the more complex mixtures.
However, most of the proposed methods were developed for the
determination of a small number of colorants in specific group
of foodstuffs (i.e. soft drinks) [8,23–25] or/and the analysis was
time-consuming [22,26]. Recently, analytical methods, based
on liquid chromatography–mass spectrometry (LC–MS) tech-
nique, have been developed in order to identify and quantify
artificial colorants, which are recognized to be carcinogens and
are not permitted in foodstuffs under the EU Food Regulations
for any purpose and any level (Sudan dyes) [30–33]. Selectivity
of the MS technique coupled with chromatographic separation
provided unambiguous identification and accurate determina-
tion of these dangerous compounds in complex matrices at trace
levels, without need of laborious clean up procedures.
The aim of this study was the development of a reversed-
phase high-performance liquid chromatography method for the
simultaneous determination of 13 synthetic colorants into the
minimum run time in various classes of foods. All synthetic
food colorants permitted in EU market was efficiently separated
using an optimized gradient elution in a single run within 29 min.
The developed method was validated in different food matrices,
such as fruit flavored and alcoholic drinks, jam, sweets and sugar
confectionary and was successfully applied to the analysis of real
samples of water-soluble foods with simple pretreatment that
included water extraction for solid samples and water dilution
for liquid samples.
2. Experimental
2.1. Apparatus
Chromatographic analysis was carried out with a Thermo
Finnigan Spectra System liquid chromatograph equipped with
a gradient pump Spectra System P 4000 capable for mixing
up four solvents, a SCM 1000 vacuum membrane degasser, an
Autosampler Spectra System AS 3000, a 100 ␮L loop injector
and a UV–vis Spectra System UV 6000 LP diode array detector.
The chromatographic data were collected and processed using a
personal computer running Chrom Quest 4.1 Data System soft-
ware (Thermo, San Jose, CA, USA).
A microprocessor pH-meter (HANNA Instrument, Lisbon,
Portugal) equipped with a combined glass–calomel electrode
was employed for pH measurements. The determination of
colorant purities was carried out with a double beam UV-
1601 UV/Vis spectrophotometer with 1-cm quartz cells (Shi-
madzu, Tokyo, Japan). A Transsonic T660/H ultrasonic bath
(Elma, Germany) was used for the pretreatment of solid food
samples.
2.2. Reagents
All solutions were prepared with distilled deionized water and
all chemicals were of analytical reagent grade, unless otherwise
stated.
Ultrapure water from Millipore direct-Q (Milford, MA, USA)
was used for the preparation of the aqueous mobile phase.
Ammonium acetate was from Riedel-de Haën (Seelze, Ger-
many) and sodium hydroxide (NaOH) from Panreac (Barcelona,
Spain). The HPLC grade organic solvents methanol (MeOH) and
acetonitrile (ACN) were supplied from LAB-SCAN Analytical
Sciences (Dublin, Ireland).
The colorants Tartrazine (E 102), Sunset Yellow FCF (E
110), Amaranth (E 123) and Erythrosine (E 127) were purchased
from Aldrich (Wisconsin, WI, USA). Quinoline Yellow (E 104),
Indigo Carmine (E 132) and Green S (E 142) were obtained
from Warner Jenkinson Europe (King’s Lynn, Norfolk, UK).
Carmoisine (E 122) was from Riedel-arom (Seelze, Germany)
and Ponceau 4R (E 124) was from Merck (Darmstadt, Germany).
Red 2G (E 128) and Brilliant Blue FCF (E 133) were obtained
from Sigma (Steinheim, Germany). Allura Red AC (E 129) was
from WS Simpson Ltd. (Caldicot, Monmouthshire, UK) and
Patent Blue V (E 131) was from Fluka (Buchs, Switzerland).
The chemical structures, common names, European Commu-
nity numbers (E numbers) and Color Index dominations (CI
numbers) are reported in Fig. 1.
2.3. Chromatographic conditions
A Discovery C18 (Supelco, Bellefonte, PA, USA) col-
umn (250 mm × 4.6 mm i.d.) fully-end-capped, with spherical
shaped 5-␮m particles and with carbon load 12% (3 ␮mol m−2 )
was used together with a C18 (25 mm × 4.6 mm i.d., 5 ␮m) guard
column (Supelco). A C18 column was used as the most common
column type, available in every routine laboratory.
The mobile phase consisted of an aqueous ammonium acetate
solution 1% (m/v) (0.13 M), brought to pH 7.5 by drop wise addi-
tion of a sodium hydroxide solution 10% (m/v) (mobile phase
A) and a mixture of methanol:acetonitrile (80:20 v/v) (mobile
phase B). The mobile phase A was filtered by vacuum through
a membrane filter with a pore diameter 0.45 ␮m. In order to
achieve a successful resolution of all compounds, a number of
gradient elution programs were tested, by decreasing gradually
the rate of increase of the organic solvent. Retention times (tR ),
peak widths (w), the resolution coefficient (Rs ) and the peak
purity factor (PP) of each compound were recorded in every
optimization experiment. The flow-rate of eluent was always
kept constant at 1.5 mL min−1 and the injection volume was
set at 20 ␮L. The final, optimized gradient program is given in
Table 1. All experiments were carried out at room temperature.
The diode-array detector was programmed to monitor the
colorants at a range of 350–800 nm. The detection and deter-
mination of each substance was employed at the appropriate
absorbance wavelengths.
The chromatographic system was initially conditioned by
passing the mobile phase A through the column until a stable
baseline signal was obtained (a minimum of 1 h was necessary).
 K.S. Minioti et al. / Analytica Chimica Acta 583 (2007) 103–110 105

Fig. 1. Chemical structures, common names, E (European Community) and CI (Colour Index) numbers of synthetic food colorants studied.

Table 1 2.4. Preparation of colorant standards and sample

Optimized gradient program for the separation of 13 colorants by HPLC-DAD, solutions
at a flow rate of 1.5 mL min−1
t (min) A (%) B (%) Individual standard stock solutions containing each color
0.0 100 0
were prepared by dissolving 100 mg unpurified colorant in
2.0 100 0 100 mL distilled deionized water. The solutions were kept in
22.0 47.5 52.5 dark flasks. The working standard solutions of each color were
37.6 0 100 prepared by appropriate dilution of stock solutions with water
40.0 0 100 to give concentrations between 0.10 and 60 mg L−1 , taking into
41.0 100 0
43.0 100 0
consideration the purity of the colorants. The mixed standard
solutions containing all colorants at concentrations between 0.10
106 K.S. Minioti et al. / Analytica Chimica Acta 583 (2007) 103–110

and 10 mg L−1 were also prepared by mixing and dilution of 2.6. Validation of the method
appropriate aliquots from standard stock solution of each sub-
stance. All solutions were stored at 4 ◦ C in the dark and were The instrument calibration was carried out using different
stable at least for 2 months. concentrations between 0.10 and 60 mg L−1 of each compound,
All samples were obtained from market control and included with two replicates per concentration, in order to determine the
fruit flavored drinks, alcoholic drinks, jams, sweets and sugar linear region of each colorant. Calibration curves were also pre-
confectionary. A 10-mL sample of drink was diluted with water pared with the mixed standards solutions at concentration levels
in a volumetric flask of 50 mL. The samples were degassed, if 0.10, 0.20, 0.50, 0.80 and 1.0 mg L−1 to check the selectivity of
necessary, by strong stirring. The solid samples were homoge- the method. The calibration curve of each colorant was used for
nized. A portion of 10 g of jam, sweet or a confectionary product the validation experiments and quantification.
was accurately weighted and dissolved in 50 mL of water. The The limit of detection (LOD) of each compound was calcu-
sample solution was placed in ultrasonic bath for 15 min for the lated as three times the standard deviation of the response of
complete extraction of the colorants. These solutions were fil- 10 independent replicate analyses of a 0.060 mg L−1 standard
tered through a folded paper filter and the filtrate was collected solution.
in volumetric flask of 50 mL. Intra-day and inter-day precision was assessed by analyzing
All solutions were injected after filtering through 0.45-␮m six replicates of a sample during one day (n = 6, intra-day pre-
disposable syringe filters. cision) or two replicates by three analysts in two different days
(n = 2, k = 3, j = 2, inter-day precision), correspondingly. Differ-
2.5. Determination of colorant purities ent colorants (E 102, E 110, E 122, E 124, E 129, E 131 and
E 142) and food matrices (flavoured drinks, liqueurs, cherries,
Purities of colorants were determined according to the iden- jams, sweets and icing sugar) were chosen for these experiments.
tification method reported in Regulation 95/45/EC [34]. This In order to evaluate the trueness of the method, recovery
method is based on spectrophotometric absorbance measure- experiments were performed. Three different food samples (fruit
ment of a diluted standard solution (i.e. 10 mg L−1 ), for which flavoured drink, jam and flavoured sweets) were spiked with E
Beer–Lambert law is valid. The percentage amount of a colorant 122, E 142 and E 131, respectively, at fortification levels of 10,
purity is given by the equation: 50 and 100 mg kg−1 and analyzed accordingly (each sample six
times at each fortification level).
DF × A
%purity = × 105 (1)
A1%
1 cm × C 3. Results and discussion
where DF is the dilution factor of measured solution from stock
3.1. Determination of colorant purities
standard solution, A the absorbance of measured solution (A < 1)
with reference to water, the expression A1%
1 cm represents the spe- All colorant standards were analyzed to determine their col-
cific absorbance of an 1% (10 g L−1 ) aqueous solution of the
orant content. The procedure using a spectrophotometer enabled
colorant at the prescribed wavelength using a path length of
easy calculation of colorant purities, presented in Table 2. The
1 cm at 20 ± 1 ◦ C, and C is the concentration of stock solution
purity of the individual colorant standards ranged from 52.5% (E
expressed as the mg of the unpurified colorant in 100 mL dis-
102) to 97.0% (E 123). The low colorant content is often a conse-
tilled deionized water. The A1%1 cm value for each compound is quence of the specific manufacturing process. The by-products
presented in Table 2 [34].
are usually inorganic salts, such as NaCl [22].
Table 2
Dissociation constants, specific absorbance, A1% 3.2. Optimization of the separation
1 cm , and calculated purities of
synthetic colorants studied
A simple method for the determination of all permitted syn-
Colorant pKa ± S.D.a A1%
1 cm Purity (%)
thetic food colorants is advantageous for practical application.
E 102 9.40 ± 0.01 530 52.5 Reversed-phase liquid chromatography (RP-LC) is suitable for
E 104 n.a.b 865 69.9 the analysis of these compounds, if conditions are chosen in
E 110 10.36 ± 0.01 555 89.2
E 122 n.a. 510 83.6
which the ionized analytes are able to form neutral molecules,
E 123 10.36 ± 0.02 440 97.0 taking into account their available dissociation constants, shown
E 124 11.24 ± 0.01 430 56.9 in Table 2 [35]. Hence, the addition of an ion-pairing reagent,
E 127 n.a. 1100 84.2 which makes the resolution more complicate and causes a vari-
E 128 n.a. 620 67.5 ety of problems [36], is not always necessary.
E 129 11.35 ± 0.01 540 84.6
E 131 7.67 ± 0.02 2000 88.1
The main characteristics of the colorants that should be taken
E 132 n.a. 480 62.3 into account for an efficient separation are their hydrophobic-
E 133 n.a. 1630 60.3 ity and the presence of acidic and alkaline groups (Fig. 1). The
E 142 n.a. 1720 78.5 hydrophobicity of non-azo compounds is weaker than those of
a Dissociation constants ± standard deviations. azo colorants. In addition, the hydrophobicity of colorants with
b n.a.: not available. naphthalene ring is stronger than those with benzene ring [37].
 K.S. Minioti et al. / Analytica Chimica Acta 583 (2007) 103–110
Under a RP-LC separation, the more polar compounds elute
first. However, since the chromatographic determination of the
colorants is performed, normally, at pH around 7, the acidic
and alkaline groups present in the molecules can change the
elution sequence [37]. Thus, under the conditions of the cur-
rent experiments, colorants are carried along with the organic
solvent (mixture of methanol:acetonitrile). The addition of ace-
tonitrile was found to improve significantly the asymmetry of
the peaks, which has also been noticed when used in methanol
[38]. However, the addition of an inorganic electrolyte to the
mobile phase, as a modifier, is necessary in order to improve
the separation of ionizable species and to obtain the separation
in reasonably short analysis time. Ammonium acetate buffer
has been used as a modifier for the purification and separa-
tion of seven azo colorants by RP-HPLC and preparative liquid
chromatography [39]. It was also used (at a pH 5) for the sep-
aration and determination of two (E 102 and E 110) [38] or
five azo food dyes (E 102, E 104, E 110, E 122 and E 124)
in soft drinks by RP-HPLC [23]. A gradient elution prepared
by a mixture of sodium acetate buffer (0.1 M and pH 7) with
acetonitrile was used for the successful separation of 14 syn-
thetic food colorants in standard solutions [22]. It was also used
for the successful determination of eight azo-colorants in food-
stuff by liquid chromatography–electrospray-mass spectrometry
(LC–ESI-MS), where it was found that excellent separation,
with symmetrical peak shapes, was achieved only in the pres-
ence of ammonium acetate and acetic acid [40]. The optimum
concentration of ammonium acetate in terms of plate number
and peak symmetry was reported to be around 0.1 M [39]. At
higher concentrations of ammonium acetate, retention times are
increased, likely due to an enhanced “salting-out” effect and so
enhance the interaction of the analyte with the stationary phase.
This effect was also observed in the separation of Tartrazine (E
102), Brilliant Blue (E 133) and Sunset Yellow (E 110) by a C18
column with the use of phosphate as buffer in the mobile phase
[37]. The acetate buffer solution is also stable; it does not inter-
act with the HPLC system and is adequate for UV absorbance
measurements. All colorants are present as neutral compounds
at pH 7.5, in order a reversed phase mechanism to take place.
Decreasing the pH of the aqueous portion of the mobile phase
resulted in poor resolution of the LC separation of the azo dyes
and the data could not be used for quantitative analysis [37,39].
Therefore, no further pH optimization of the mobile phase A
was attempted.
A 10 mg L−1 mixed standard solution containing the 13 col-
orants was used to study the optimum conditions of separation.
The initial gradient elution program used was as follows. For
the initial 2 min, the mobile phase A (ammonium acetate buffer)
goes through the column for elution of inorganic compounds.
Between 2 and 25 min, a gradient is carried out and for the last
2 min (25–27 min) the mobile phase B passes through the col-
umn for the elution of possible organic impurities. Finally, a
2-min equilibrium phase of the column is taking place for the
next run.
The elution of the colorants was observed in a chromatogram
that was obtained by scanning the wavelength range from 350
to 800 nm, continuously. From software, the retention time (tR ),
107
the width (w) and the peak purity factor (PP) of each peak were
calculated. The peak purity factor is produced from the compar-
ison of the absorbance spectrum of a compound in mixture with
the corresponding spectrum in its individual standard solution.
In addition, the resolution coefficient (Rs ) for two successive
peaks was determined.
Two main resolution problems were observed when the first
gradient elution program was applied. The colorants E 128 and
E 142 were co-eluted and, although they were determined in
different wavelengths, a mutual interference appeared. The res-
olution of colorants E 122 and E 133 was also unsatisfactory.
However, in this case, only E 133 interfered with the determi-
nation of E 122. These conclusions were also verified from the
low peak purities values of E 128, E 142 and E 122, the high
peak purity value of E 133 and the unsatisfactory Rs values for
these two couples of colorants (0.30 and 0.49, respectively).
The resolution problems were solved by decreasing grad-
ually the amount of the organic solvent (mobile phase B) in
gradient programs. Consequently, the time of analysis increased.
Seven experiments were performed for which the rate of increase
of solvent B gradually decreased from 4.3% B per min in the
first experiment to 2.6% B per min in the last experiment (data
not shown). Finally, a combination of two experiments resulted
in the final optimized gradient program, which is presented in
Table 2. Use of this program resulted in an efficient separa-
tion with no interferences for all colorants. The chromatogram
and the chromatographic characteristics obtained from the opti-
mized gradient program are presented in Fig. 2 and Table 3,
respectively. The peak purity (PP) factor is greater than 0.999
for all compounds. The simultaneous determination of the col-
orants E 128 and E 142 have been achieved and E 133 no longer
interferes with the determination of E 122. The elution order of
E 133 and E 104III has been changed. These compounds seem
to be co-eluted (small Rs value), but from their high PP values
and the fact that they have different absorption spectra and they
are determined in different wavelengths, obviously, they do not
interfere each other. Along with the PP factor (the purity of the
absorption spectra), retention times could also be used for qual-
itative analysis for all the compounds since their repeatability is
very good. The R.S.D. of tR (n = 10) was always less than 0.32%
(E 102) (Table 3).
Fig. 2. Chromatogram of a mixed standard solution using the optimized gra-
dient program of Table 2. The concentrations of all colorants were 10 mg L−1 .
Identification of the peaks and their corresponding tR are given in Table 3.
108 K.S. Minioti et al. / Analytica Chimica Acta 583 (2007) 103–110

Table 3
Chromatographic data of the colorants with the optimized gradient program of Table 1
Peak Colorant λmax (nm) tR ± S.D. (min) (n = 10) w (min) Rs Peak purity

1 E 102 427 10.90 ± 0.035 0.45 1.0000

2 E 123 520 12.20 ± 0.036 0.47 2.83 0.9990
3 E 132 608 12.62 ± 0.030 0.48 0.88 0.9999
4 E 104I 417 15.35 ± 0.040 0.48 5.69 1.0000
5 E 124 508 16.22 ± 0.037 0.43 1.91 0.9999
6 E 110 482 16.87 ± 0.039 0.43 1.51 0.9999
7 E 104II 417 17.55 ± 0.042 0.48 1.5 0.9995
8 E 129 507 19.07 ± 0.059 0.4 3.45 0.9999
9 E 128 530 19.93 ± 0.052 0.5 1.92 0.9999
10 E 142 630 20.38 ± 0.047 0.62 0.8 1.0000
11 E 122 516 24.02 ± 0.059 0.5 6.49 0.9999
13 E 104III 417 24.55 ± 0.045 0.4 1.18 0.9999
12 E 133 624 24.68 ± 0.032 0.52 0.29 0.9999
14 E 131 631 27.48 ± 0.011 0.6 5 0.9999
15 E 127 528 28.95 ± 0.018 0.48 2.72 0.9999

Table 4
Limits of detection, linear range, calibration equations and coefficients of determination (R2 ) of all colorants
Colorant LOD (␮g L−1 ) Linear range (mg L−1 ) Calibration equation R2

E 102 1.87 0.006–21 y = 261 + (30.3 × 104 )x 0.9999

E 104 4.72 0.014–49 y = 459 + (26.9 × 104 )x 0.9999
E 110 4.41 0.013–55 y = (4.46 × 103 ) + (19.1 × 104 )x 0.9995
E 122 4.35 0.013–54 y = −87.0 + (18.3 × 104 )x 1.0000
E 123 10.2 0.031–58 y = 213 + (12.0 × 104 )x 1.0000
E 124 22.1 0.067–46 y = (3.22 × 103 ) + (16.4 × 104 )x 0.9999
E 127 6.68 0.020–34 y = −145 + (38.8 × 104 )x 1.0000
E 128 6.26 0.019–48 y = 590 + (22.1 × 104 )x 0.9999
E 129 7.46 0.023–51 y = 609 + (20.3 × 104 )x 1.0000
E 131 10.5 0.032–27 y = 584 + (75.9 × 104 )x 1.0000
E 132 8.09 0.025–38 y = 920 + (10.7 × 104 )x 0.9999
E 133 2.72 0.008–19 y = (1.46 × 103 ) + (57.1 × 104 )x 0.9999
E 142 1.59 0.005–32 y = (5.09 × 103 ) + (60.9 × 104 )x 0.9999

Many of colorants have isomers that appear in the chro- E 127, E 131, E 133 and E 142, have been characterized from
matogram as small peaks. The colorant Quinoline Yellow (E shorter linear ranges. Limits of detection for all substances var-
104) exhibits three peaks corresponding to isomers, probably ied between 1.59 ␮g L−1 for E 142 and 22.1 ␮g L−1 for E 124.
disulfo- and monosulfo-derivatives (E 104I, peak 4, E 104II, In case of Quinoline Yellow (E 104) only the E 104I isomer was
peak 7 and E 104III, peak 13 in Fig. 2) [22]. The unspec-
ified peaks shown in chromatogram were identified by their
absorbance spectra and were found to correspond to isomers Table 5
of E 132 (15.8 min), E 142 (19.4 min), E 133 (25.8 min), E 127 Precision data (intra-day, as R.S.D.r and inter-day, as R.S.D.R ) and concentration
levels (C) of seven colorants in various food matrices
(26.1 min) and E 131 (26.3 min) (Fig. 2).
Colorant Matrix Concentration R.S.D.r R.S.D.R
level (C) (%) (%)
3.3. Validation of the method
13.6 1.2 3.9
Sour cherry flavoured
Calibration equations of mixed standard solutions, coeffi- E 122 47.5 0.53 1.1
drink (mg L−1 )
97.6 0.37 0.86
cients of determination (R2 ), linear range and the limits of
detection of all colorants are presented in Table 4. Calibration E 102 Icing sugar (mg kg−1 ) 12.4 1.4 2.2
equations were calculated using the peak area of the substances. E 110 Icing sugar (mg kg−1 ) 10.1 1.4 3.9
E 142 Icing sugar (mg kg−1 ) 0.94 4.8 9.3
In case of E 104, the sum of the peak areas of the three iso- E 124 Liqueur (mg kg−1 ) 14.4 2.4 3.5
mers was used. The slopes of calibration equations of mixed E 129 Cherries (mg kg−1 ) 73.6 1.9 6.5
solutions were compared with t-test with those calculated from
4.7 2.7 6.2
the measurement of individual standard solutions (concentra- E 131 Sweets (mg kg−1 )
72.2 1.9 3.6
tion levels 0.10–60 mg L−1 ) and it was found that they do not
E Jam 9 1.9 10
differ significantly. The linear range is different between the ana- (mg kg−1 ) 84.5 0.90 3.4
142
lytes. In general, colorants that have high A1% 1 cm value, such as
 K.S. Minioti et al. / Analytica Chimica Acta 583 (2007) 103–110 109

Table 6
Mean recoveries and R.S.D.s (n = 6) of three colorants from spiked food matrices at various fortification levels
Colorant Food matrices Concentration level

100 50 10

Recovery (%) R.S.D. (%) Recovery (%) R.S.D. (%) Recovery (%) R.S.D. (%)

E 122 (mg L−1 ) Fruit flavoured drink 97.9 0.44 99.6 0.73 97.7 1.19
E 142 (mg kg−1 ) Jam 94.4 0.90 95.1 1.87
E 131 (mg kg−1 ) Mint flavoured sweets 101.8 1.98

used for the determination of the LOD. The LODs of the col- Table 7
orants obtained in this study are in the same range as or slightly Determination of colorants in water-soluble foodstuffs collected from the Greek
market
better than those reported in other studies using ion-pair HPLC
methods [27–29]. Sample Colorant found Mean content of colorant R.S.D. (%)
The precision experiments resulted in good R.S.D.s for both A (mg L−1 ) E 110 0.35 3.3
intra-day and inter-day precision, for the colorants and food E 122 0.63 7.3
matrices chosen. Precision was determined for colorants with E 124 0.89 3.9
different polarity, which eluted at the beginning (E 102), in the B (mg L−1 ) E 110 2.7 2.7
middle (E 110, E 124 and E 129) and near the end of the chro- E 122 125 1.9
matogram (E 142, E 122 and E 131). Six different food matrices, C (mg kg−1 ) E 110 12.2 1.5
in which the addition of these colorants is frequent, were cho- E 122 23.2 1.3
sen (Table 5). The intra-day precision (as R.S.D.r ) ranged from E 124 15.3 1.5
0.37% for E 122 in fruit flavored drink at a concentration of E 132 44.9 1.2
100 mg L−1 , to 4.8% for E 142 in icing sugar at a level of D (mg kg−1 ) E 131 4.7 2.7
0.9 mg kg−1 . The inter-day precision (as R.S.D.R ) was between E (mg kg−1 ) E 104 15.8 1.6
0.86% for E 122 in fruit flavored drink at 100 mg L−1 and 10% E 110 5.9 2.1
for E142 in jam at a concentration of 9 mg kg−1 (Table 5). The E 122 0.97 2.5
results obtained from this work are in general agreement with E 129 18.1 1.8
E 133 0.5 3.1
those obtained from other studies, where maximum R.S.D.s
ranged between 2.1% [23] and 6.4% [40]. F (mg kg−1 ) E 102 0.5 3.2
E 104 10.8 1.6
The results from recovery experiments in three different
E 110 10.8 1.2
matrices at levels ranged from 10 to 100 mg kg−1 are shown in E 122 2.3 2.0
Table 6. The high level (100 mg kg−1 ) was chosen in accordance E 129 27.8 1.9
to the legislation limits [2]. E 122 and E 142, which had shown E 133 4.8 1.7
poor resolution, and E 131, a compound with low polarity (thus, G (mg kg−1 ) E 124 52 1.6
with a possibly low affinity to water extraction), were chosen for
recovery experiments. The developed method resulted in satis-
factory recoveries for all the tested compounds, ranging from
94% for E 142 in jam to 102% for E 131 in sweets. The recov- at low concentrations. In general, colorants were determined in
eries are more than adequate for the developed application and foods collected from the Greek market in a more wide concen-
slightly better than those reported in literature [26–28]. tration range than those reported in other studies [23,26–29].

3.4. Application to real samples

The method developed was applied to food samples from
market control. The samples were prepared as described in Sec-
tion 2.4. A fruit flavored drink (A), a fruit flavored liqueur (B),
a sample of icing sugar (C), three samples of sweets (D, E and
F) and a blackberry flavored jam (G) were analyzed, at least, in
duplicate and the results are summarized in Table 7. Data from
Tables 5 and 7 show that 10 out of the 13 colorants were detected,
at least once. The concentrations of colorants in analyzed food-
stuffs ranged from 0.35 mg L−1 (E 110 in fruit flavoured drink) to
125 mg kg−1 (E 122 in fruit flavoured liqueur). The low detection
limits allowed the accurate determination of colorants in foods
4. Conclusions
An efficient and accurate analytical method for the simultane-
ous determination of 13 permitted in EU market food colorants
in a single run by liquid chromatography-diode array detection
was developed and optimized. The proposed method includes
a simple pretreatment procedure for the extraction of colorants
from food and offers a combination of sensitivity and selectiv-
ity, simplicity and relatively short time of analysis. This method
permits the detection of colorants at very low concentrations
(␮g kg−1 range). The applicability was verified by the determi-
nation of colorants present in various water-soluble foodstuffs.
110 K.S. Minioti et al. / Analytica Chimica Acta 583 (2007) 103–110
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