Analytica Chimica Acta 716 (2012) 128132

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Analytica Chimica Acta

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Applicability of accelerated solvent extraction for synthetic colorants analysis in

meat products with ultrahigh performance liquid chromatographyphotodiode
array detection
Qie Gen Liao, Wei Hong Li, Lin Guang Luo
Jiangxi Academy of Agricultural Sciences Key Laboratory of Quality and Safety of Agricultural Products, Agricultural Product Quality Safety and Standards Institute, Jiangxi Academy
of Agricultural Sciences, Nanchang, 330200, PR China

a r t i c l e

i n f o

Article history:
Received 8 September 2011
Received in revised form
14 December 2011
Accepted 15 December 2011
Available online 24 December 2011
Keywords:
Ultrahigh performance liquid
chromatography (UHPLC)
Photodiode array detection (PDA)
Synthetic colorants
Accelerated solvent extraction (ASE)
Meat products

a b s t r a c t
Accelerated solvent extraction (ASE) coupled with ultrahigh performance liquid chromatography
(UHPLC) with photodiode array detection (PDA) has been used for the quantitative determination of
synthetic colorants in meat products. Samples were extracted with ethanolwaterammonia with a
ratio of 75:24:1 (v/v/v) using ASE instrument at 85 C. As a result, all the colorants in meat products were
separated using an optimized gradient elution within 3.5 min. Detection and quantication limits of synthetic colorants were in the ranges of 0.010.02 mg kg1 and 0.05 mg kg1 , respectively. The intra-day
and inter-day precision of the synthetic colorants were ranged between 1.7% (E123) to 5.2% (E124) and
3.2% (E124) to 6.0% (E129), respectively. The intra-day and inter-day recoveries of the synthetic colorants
were ranged between 76.9% (E124) to 84.9% (E102) and 76.3% (E124) to 84.3% (E127), respectively. The
method has been applied for the determination of seven synthetic colorants in meat products.
2011 Elsevier B.V. All rights reserved.

1. Introduction
Food colorants are very interesting classes of food additives.
Frequently color of a product determines its attractiveness to consumers. Incentive hues are associated with freshness, good taste,
and nutritional value. However, natural colorants are unstable and
easily undergo degradation during the food processing and storage.
Moreover, in poultry farming, because of -carotene or carotenoid
deciency or short feeding periods, poultry products may be of
lighter colors that do not meet the requirements of consumers. Synthetic colorants are a very important class of food additives, which
are widely used to compensate for the lack of natural colors. However, some researchers have conrmed their negative inuence on
human organism. For instance, azodyes are decomposed by natural intestinal ora into aromatic amines [1], which cause frequent
headaches in adults and cause children to become distractible and
hyperactive [2]. Thus, content of synthetic dyes in food must be
strictly controlled. In China, the use of synthetic colorants in meat
products is strictly controlled by Directive GB 2760-2011 of the
Ministry of Health. Consequently, accurate and reliable methods

Corresponding author. Tel.: +86 791 87090293; fax: +86 791 87090293..
E-mail address: luolinguang@hotmail.com (L.G. Luo).
0003-2670/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.aca.2011.12.033

for the determination of synthetic colorants are required for the

safety assurance of meat products.
Several analytical techniques have been developed for the
identication and determination of various synthetic colorants,
including adsorptive voltammetry [3], differential pulse polarography [4], derivative spectrometry [5] and spectrophotometric
methods in combination with chemometrics [6,7]. Unfortunately,
these methods cannot be applied to complex colorant mixtures or
simultaneous determination of various synthetic colorants. Capillary electrophoresis [810] and micellar electrokinetic capillary
chromatography [11] have also been used, however, these methods have sensitivity issues due to their small injection volume.
High-performance ion chromatography [12], reversed-phase liquid chromatography [1316] and ion-pair liquid chromatography
[1723] coupled with UV or diode-array detectors, continue to be
the most preferred methods due to unrivalled resolution, sensitivity, and selectivity; however, analysis speeds need to be improved.
The use of ultrahigh-performance liquid chromatography (UHPLC)
could improve analysis speed [24]. Recently, analytical methods, based on liquid chromatographymass spectrometry (LCMS)
technique [2527], have been developed in order to identify and
quantify synthetic colorants or their degradation [28].
The preparation of samples is also a very important problem
in the analysis of synthetic colorants. Extracting them from different types of matrices is not an easy task. Most of the above

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129

Fig. 1. Representative UHPLC chromatograms of grilled chicken wings spiked at 0.5 mg kg1 (A) and ham sausage (B) of each colorant extracted by ASE. The detection
wavelengths were as follows: 0 min1.38 min, 429 nm; 1.38 min1.70 min, 522 nm; 1.70 min2.00 min, 509 nm; 2.00 min2.10 min, 483 nm; 2.10 min2.50 min, 508 nm;
2.50 min2.90 min, 625 nm; and 2.80 min3.50 min, 529 nm.

mentioned methods have been developed for the determination

of synthetic colorants in specic and simple foodstuffs (i.e., soft
drinks, solid juice powders, and candy) [1223,2527]. These methods do not apply to pretreatment of samples with high protein
content due to strong adsorption between the colorant and protein [29]. In our preparatory experiments, ethanol containing
1% (v/v) ammonia, acetonitrile containing 1% (v/v) ammonia, ammoniawaterethanol, ammoniawateracetonitrile, and
ultrasonic extraction were explored as reagent or extraction
method. Unfortunately, the measures mentioned above were
unsatisfactory in the extraction of the low concentration colorants.
Accelerated solvent extraction (ASE) is a recent advancement in
sample preparation for trace analytes. ASE uses conventional solvents at elevated pressures and temperatures to extract samples
quickly [30,31]. Thus, ASE may be a potential chance to reduce the
adsorption.
The aim of the current study is the development of a rapid
UHPLC method for the simultaneous determination of seven synthetic colorants in meat products using an optimized gradient
elution. The ASE experimental parameters (i.e., extraction solvent,
temperature, static time and cycles) are optimized to achieve the
maximum extraction efciencies for synthetic colorants. The proposed method has been applied to the analysis of meat products
from local commercial and farmers markets.

2. Experimental
2.1. Reagents
Ammonium acetate was obtained from Riedel-de Han (Seelze,
Germany). The HPLC grade organic solvents methanol (MeOH) and
acetonitrile (ACN) were supplied by Merck (Darmstadt, Germany).

Ethanol, ammonia and hexane were obtained from Shanghai chemicals company (Shanghai, China).
Tartrazine (E102), Sunset Yellow FCF (E110), Amaranth (E123),
Ponceau 4R (E124), Brilliant Blue FCF (E133), Erythrosine (E127)
and Allura Red (E129) containing 0.5 mg mL1 were purchased from
National Standard Material Center (Beijing, China). Working standards of individual colorants were prepared by dilution of the stock
solutions with deionized water. All chemicals were of analytical
reagent grade, unless otherwise stated. Deionized water from Millipore (Milford, MA, USA) was used throughout the study.
2.2. Samples
The meat products were purchased from local commercial and
farmers markets. After being homogenized in a high-speed food
blender, the samples were stored below 20 C. Sub-samples of
approximately 5 g were spiked with the right amount of the mixed
standard solutions. The spiked samples were mixed 1 min by a
vortex mixer, and were set aside for a minimum of 1 h before extraction.
2.3. ASE extraction
Samples were extracted with a Dionex accelerated solvent
extractor 350 (Dionex, Sunnyvale, California, USA). Approximately
5 g of the spiked blank/sample material mixed with 10 g of hydromatrix was packed in a 34 mL stainless steel extraction cell. The
free space in the cell was lled up with hydromatrix. Each cell
was locked with stainless steel screw caps equipped with Teon
O-ring seals. Circular glass microber lters of 3.0 cm diameter
were placed above and below the packing. The solvent selected
was ethanolwaterammonia (75:24:1, v/v/v). Conditions of the
extraction were as follows: time heating cell, 5 min; time of solvent

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Q.G. Liao et al. / Analytica Chimica Acta 716 (2012) 128132

in contact with the sample, 10 min (static time); pressure, 1500 psi;
temperature, 85 C; time purging with nitrogen to expulse rest of
solvent in the cell, 120 s; ushing volume respect to the cell size in
percentage, 60%; cycles, 2.
After extraction, each resulting extract was evaporated to dryness under vacuum distillation at 75 C. The residue was redissolved
with 5.0 mL of 0.02 mol L1 ammonium acetate and 5.0 mL hexane,
and then transferred to a 50 mL centrifuge tube. After liquidliquid
extraction and high-speed centrifugation at 16,000 rpm for 5 min,
the lower solutions were used for UHPLC analysis.
2.4. Chromatographic analysis
Samples were analyzed by UHPLC. The UHPLC system consisted
of a binary pump, a degasser, an automated injector, a column
oven and a photodiode array (PDA) detector (Waters, USA). The
system was controlled by an Empower ChemStation. Chromatographic conditions (mobile phase composition and ow-rate) were
evaluated and optimized in an acquity UPLC BEH C18 (1.7 m,
2.1 mm 100 mm) column.
Solvent A was 0.02 mol L1 of ammonium acetate aqueous
solution, whereas solvent B was acetonitrile. In gradient-elution
analysis, the initial mobile phase was 95% A with 5% B, which then
linearly increased as follows: up to 30% B at 1.8 min, and holding
at 30% B until 1.9 min; up to 70% B at 2.3 min; and up to 98% B
at 2.8 min, and holding at 98% B until 3.1 min. A return to the initial conditions was carried out at 3.2 min and held at 5% B until
3.5 min. The ow rate was set at 0.3 mL min1 , whereas the column
temperature was maintained at 30 C. The injection volume was
5.0 L.
The PDA detector was programmed to monitor the colorants at
a range of 220700 nm. The determination of each substance was
conducted at the appropriate absorbance wavelengths. Identication was performed by matching the retention time and absorption
spectra of standards.
2.5. Validation procedure
To check the sensitivity of the proposed method, calibration
curves were also prepared with the mixed standards solutions spiking blank samples at concentration levels of 0.05, 0.1, 0.5, 1.0, 5.0
and 10.0 mg kg1 . The matrix-match calibration curve of each colorant was used for the validation experiments and quantication.
The limit of detection (LOD) and limit of quantication (LOQ)
were determined by considering 3 and 10 times the standard deviation of the signals from the blank matrix, respectively.
Intra-day and inter-day reproducibility of the method were
assessed by performing replicate analyses. Intra-day precision was
determined by analyzing six fortied samples on the same day.
Inter-day repeatability was determined by analyzing the fortied
sample on six different days.
In order to evaluate the trueness of the method, recovery experiments were performed. Absolute recoveries of the extraction
were measured by spiking different concentrations of the studied colorants to the blank samples. The spiked samples were then
extracted and quantied, as described above.
3. Results and discussion
3.1. Optimum separation conditions of synthetic dyes by UHPLC
The separation behaviors using the following mobile phases
were investigated: aqueous phases containing 0.02 mol L1 ammonium acetate or 0.02 mol L1 ammonium acetate and 0.1% acetic
acid, organic phases containing acetonitrile or methanol, respectively. The peaks of E102, E110, E123, E124, E133, E127, and

Fig. 2. Effects of solvent proportion (A) and extraction temperature (B) on recovery
[constant pressure (1500 psi) and static time (10 min) were maintained].

E129 were broadened, partially overlapped, or disappeared with

either methanol or aqueous phase containing 0.02 mol L1 ammonium acetate and 0.1% acetic acid as the mobile phase. However,
the excellent separation for all of compounds explored could
be achieved with acetonitrile containing 0.02 mol L1 ammonium
acetate as the mobile phase system. Therefore, it was adopted as
the mobile phase in subsequent studies.
The separation was also investigated under different gradient
elution conditions. Under the optimal gradient elution conditions,
the extraction UHPLC chromatograms of grilled chicken wings
spiked at 0.5 mg kg1 of each colorant and ham sausage showed
that seven synthetic colorants properly separated with sharp symmetrical peak shapes (Fig. 1 A and B respectively). Rapid analysis
was achieved within 3.5 min.
3.2. Optimization ASE procedure
The inuences of solvent proportion, temperature, static cycles,
and static time were investigated to acquire the most effective conditions. Ammonia-containing organic solvents, such as
ethanol containing 1% (v/v) ammonia, acetonitrile containing 1%
(v/v) ammonia, can be used to extract colorants with low extract
recoveries. When a certain percentage of water was added to
the ethanol containing 1% (v/v) ammonia, the recovery signicantly improved. The best ratio of ethanolwaterammonia was
75:24:1(v/v/v) (Fig. 2A). The presence of 1% (v/v) ammonia could

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131

Table 1
The detection wavelength, retention time, linear range, linear equation, limit of detection (LOD) and coefcients of determination (R2 ) of all colorants.
Colorant
E102
E123
E124
E110
E129
E133
E127

tR (min)
1.313
1.458
1.953
2.032
2.227
2.879
3.036

 (nm)
429
522
509
483
508
625
529

Linear range (mg kg1 )

Linear equation

0.0510.0
0.0510.0
0.0510.0
0.0510.0
0.0510.0
0.0510.0
0.0510.0

y = 6.94 10 + (1.50 10 )x
y = 6.01 102 + (1.25 105 )x
y = 1.18 103 + (1.21 105 )x
y = 1.63 103 + (1.87 105 )x
y = 5.29 103 + (1.62 105 )x
y = 4.96 103 + (4.85 105 )x
y = 1.33 103 + (2.10 105 )x
2

reduce the electrostatic adsorption between the colorants and protein, for the colorants and protein are negatively charged in the
alkaline medium. On the other hand, the presence of ethanol could
reduce hydrophobic force between the colorants and protein, and
causing protein denaturation. However, a percentage higher than
25% (v/v) of water in the extraction solvent also resulted in lower
recoveries. Due to increased viscosity by water, the ushing and
purging steps did not completely expel the extraction solvent from
the extraction cell. The remaining solvent could contain colorants,
which probably led to the lower recoveries [32]. On the other hand,
the extraction efciencies were affected by extraction temperature.
Recovery of the colorants increased with increasing extraction temperature from 55 C to 85 C, indicating the acceleration of protein
denaturation, and then slightly decreased at higher temperatures
(Fig. 2B). A small amount of aggregation from meat products was
observed when heated at higher than 85 C, and the aggregation
could wrap or adsorb on the synthetic colorants. Consequently,
85 C was adopted as the extraction temperature.
Static cycles introduce fresh solvent during the extraction process and are useful for matrices that are difcult to penetrate. The
number of the extraction cycles was tested to assure a rapid extraction, as well as high recovery. The number of extraction cycle was

LOD (mg kg1 )

LOQ (mg kg1 )

R2

0.02
0.02
0.02
0.01
0.01
0.01
0.02

0.05
0.05
0.05
0.05
0.05
0.05
0.05

0.9997
0.9998
0.9998
0.9998
0.9999
0.9994
0.9992

varied between one and three. After two cycles of extraction of the
same matrix, the recovery could no longer increase.
To evaluate if extraction time could inuence extraction efciency, different extraction times (5, 7, 10, 12, 15 min) were used.
Increasing the static extraction time from 5 min to 10 min resulted
in a recovery improvement at about 10%. Consequently, static time
of 10 min was selected in order to minimize the time of analysis.
Flush percentage refers to the amount of solvent ushed
through the cell following the static heating step, expressed as a
percentage of the cell volume. Increasing the ush volume allowed
more solvent to pass through the sample, but also increased the
nal volume of the extract. Different ush volumes (40%, 60%, 80%,
100%) did not signicantly affect the extraction efciencies of the
analytes. Therefore, the ush volume was set at their default values
(60%).
3.3. Validation of the method
Each colorant has a different wavelength of maximum absorption. The maximum absorption wavelengths were monitored as
detection wavelengths. Matrix-matched calibration curves were
prepared daily by spiking blank control samples of meat products

Table 2
Recoveries and precision of each colorant spiked in the grilled chicken wings.
Colorant

Spiked level (mg kg1 )

Intra-day recovery (n = 6)

Inter-day recovery (n = 6)

Mean (%)

R.S.D. (%)

Mean (%)

R.S.D. (%)

E102

0.05
0.5
2.0
5.0

78.3
80.5
82.3
84.9

4.4
2.3
2.9
3.3

77.9
79.3
80.4
83.9

5.2
3.8
5.3
4.0

E123

0.05
0.5
2.0
5.0

78.3
78.5
84.5
84.5

4.5
1.7
2.6
3.3

77.0
77.4
83.7
83.9

5.7
4.3
4.5
4.0

E124

0.05
0.5
2.0
5.0

76.9
77.5
81.9
83.6

5.2
2.5
3.5
2.6

763
76.6
80.4
81.9

5.5
3.8
4.2
3.2

E110

0.05
0.5
2.0
5.0

80.4
81.3
83.3
84.7

4.6
2.5
2.9
3.4

79.7
80.3
82.0
83.8

5.5
3.8
4.3
4.1

E129

0.05
0.5
2.0
5.0

80.9
81.5
84.3
84.6

5.1
3.2
2.9
3.5

79.0
79.5
83.3
84.3

6.0
4.0
4.5
3.7

E133

0.05
0.5
2.0
5.0

83.7
84.1
83.3
84.7

3.9
2.5
2.9
3.4

82.4
82.3
82.7
83.5

4.6
3.8
4.3
4.1

E127

0.05
0.5
2.0
5.0

80.9
81.5
83.3
84.6

4.2
3.2
2.9
2.5

80.1
79.5
82.3
84.3

4.9
4.0
4.5
3.7

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Q.G. Liao et al. / Analytica Chimica Acta 716 (2012) 128132

with mixtures colorants. Calibration equations of mixed standard

solutions, coefcients of determination (R2 ), detection wavelength,
linear range, limit of detection and limit of quantication of all
colorants are presented in Table 1. Calibration equations were
calculated using the peak area of the colorants, and calculated
from the measurement of six standard solutions (concentration
levels of 0.0510.0 mg kg1 ). Correlation coefcients (R2 ) were
better than 0.99. Detection and quantication limits of synthetic colorants were in the ranges of 0.010.02 mg kg1 and
0.05 mg kg1 , respectively. To reduce matrix effects, matrix-match
calibrations curves were chosen as reference curves throughout the
study.
Precision and recovery were investigated in order to research
the inuence of the matrix. The results from recovery experiments in the same matrices at levels ranging from 0.05 mg kg1
to 5.0 mg kg1 were shown in Table 2. The results of precision indicated that intra-day and inter-day repeatability (R.S.D. of synthetic
colorants were between 1.7% (E123) to 5.2% (E124) and 3.2% (E124)
to 6.0% (E129), respectively. The results of recovery indicated that
the intra-day and inter-day recoveries of synthetic colorants were
between 76.9% (E124) to 84.9% (E102) and 76.3% (E124) to 84.3%
(E127), respectively. The results demonstrate that the precision and
accuracy of the present method are acceptable for routine monitoring purposes.
3.4. Application to real samples
The proposed method was applied to meat products from
local commercial and farmers market. In the eighty samples,
only a few samples tested positive for E102, E123, and/or
E124; their concentrations were found to be in the range of
2.510.9 mg kg1 . The results also indicate that the method is suitable for determining low amounts of synthetic colorants in meat
products.

the meat products. The developed method could also be potentially

applied to other products with high protein content.
Acknowledgements
This work was supported by the fund from nonprot industryspecic agricultural research projects (no. 200903055) and
innovation fund of Jiangxi Academy of Agricultural Sciences (no.
2010CJJ012).
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In the current study, an efcient and accurate analytical method
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