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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
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
Keywords:
Synthetic food dyes
Animal feed
Meat
HPLC-DAD
HPLCMS/MS
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: lizhenchem@gmail.com (Z. Li).
0308-8146/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2012.11.084
1743
1744
Table 1
Chemical structures and tandem mass spectrometry parameters of the 7 dyes tested.
Compound
Chemical structure
Ponceau 4RC
4.9
604.5
536.7/301.9
536.7/301.9
536.7/428.9
130
130
10
20
Sunset yellow
5.2
452.0
406.8/206.9
406.8/206.9
406.8/326.9
170
170
30
25
Allura red
5.6
496.4
472.8/194.8
472.8/194.8
472.8/199.8
190
190
35
35
Azophloxine
5.7
509.0
463.9/343.8
463.9/344.1
463.9/359.1
120
120
30
25
6.3
480.4
434.9/355.0
434.9/355.0
434.9/274.1
120
120
30
25
Erythrosine
7.1
879.6
834.1/662.8
834.1/662.8.
834.1/536.8
150
150
35
35
Orange II
7.6
350.3
327.0/171.1
327.0/171.7
327.0/155.8
150
150
25
35
Ponceau
xylidine
1745
1746
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.
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.
1747
Ponceau 4RC
Sunset yellow
Allura red
Azophloxine
Ponceau xylidine
Erythrosine
Orange II
HPLC-DAD
HPLCMS/MS
2
Linear Range
(ng mL 1)
Linear equation
5005000
1001000
5005000
5005000
5005000
5005000
5005000
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
LOD
(ng mL
0.993
0.999
0.998
0.996
0.998
0.999
0.999
22.01
74.81
68.02
19.97
14.74
6.43
8.57
R2
Linear range
(ng mL 1)
Linear equation
5.01000
1.01000
2.0100
3.01500
2.02000
0.2200
0.1100
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
LOD
(ng mL
0.997
0.999
0.999
0.999
0.980
0.999
0.999
6.49
2.18
14.49
19.87
21.83
0.10
0.02
Table 3
Percentage recoveries (%) and relative standard deviations (%) of the 7 dyes in different matrices (n = 6).
Compound
HPLC-DAD
HPLCMS/MS
Feed
1 mg kg
Ponceau 4RC
Sunset yellow
Allura red
Azophloxine
Ponceau xylidine
Erythrosine
Orange II
83(7.8)
90(2.5)
94(3.2)
88(2.5)
87(2.2)
84(4.1)
90(3.4)
Poultry meat
1
5 mg kg
91(10.6)
72(7.0)
90(4.9)
81(4.4)
71(1.6)
83(3.8)
86(2.7)
0.5 mg kg
87(10.5)
94(6.3)
97(4.7)
94(3.1)
91(1.8)
84(4.0)
93(0.9)
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).
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.
Feed
1 mg kg
92(14.8)
94(2.0)
85(1.2)
84(0.7)
83(0.8)
78(4.0)
89(0.2)
1 mg kg
53(9.4)
57(4.3)
106(9.5)
86(14.5)
102(9.7)
73(13.8)
103(9.1)
Poultry meat
1
5 mg kg
63(10.8)
51(3.6)
85(9.4)
83(5.8)
65(4.0)
88(8.9)
87(4.7)
0.5 mg kg
108(8.8)
94(15.8)
113(19.3)
92(13.9)
83(6.4)
81(11.5)
100(7.3)
1 mg kg
59(3.6)
74(7.2)
76(9.9)
78(6.9)
82(6.8)
79(5.6)
89(7.0)
1748
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.
Acknowledgments
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.
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