Engineering, 2013, 5, 154-157
http://dx.doi.org/10.4236/eng.2013.510B033 Published Online October 2013 (http://www.scirp.org/journal/eng)
Determination of Eight Sudan Dyes in Chili Powder by
UPLC-MS/MS
Lin Wang, Jie Zheng, Zhe Zhang, Tiesong Wang, Baoquan Che*
Beijing Institute for Drug Control, Beijing, 100035, China
Received April 2013
ABSTRACT
Sudan dyes are synthetic azo dyes which are widely used in industry. Although th ey are not allowed in foods tuffs, they
have been found contaminating in different food products and their presence is regularly reported. It is assumed that
these appearances are due to cross-contamination or adulteration. In this paper, we present a newly developed fast and
sensitive method for the quantification of eight Sudan dyes, using liquid-liquid extraction and UPLC-MS/MS an alysis.
The calibration curves were linear over the range of 0.1 - 25 mg/kg. Mean recovery for the eight Sudan dyes ranged
from 80.7% to 104.4%, and with the inter-day and intra-day precisions ranged from 2.24% to 12.2%. The method was
successfully applied in the determination of Sudan dyes in chili powder of 10 samples.
Keywords: Sudan Dyes; UPLC-MS/MS; Chili Powder
1. Introduction
Sudan dyes are dynthetic fat-soluble azo-compounds,
characterized by chromophoric azo groups (-N=N-) [1].
The chemical structures of Sudan I, Sudan II, Sudan III,
Sudan IV, Sudan Red G, Sudan Red 7B, Sudan Black B
and Sudan Y e llow are given in Figure 1.
These dyes are extensively used as colorants in food,
cosmetics, waxed, solvents, textiles, and so on. Lots of
them are found in various foodstuffs, like chili powder,
curry, or chili sauces fraudulently [2]. These azo dyes are
metabolized to possible carcinogenic colorless amines
that can form DNA adducts entailing mutations [3]. Su-
dan I has been found to be carcinogenic in the rat and to
produce tumors in the liver of mice. Sudan II increases
the incidence of bladder carcinomas, and Sudan IV in-
creases the risk of formation of local carcomas [4,5].
Besides, Sudan III is included in different laboratory
techniques for tissue ceroid and lipofucsin analysis [6].
Also, Sudan Black B, which is considered slightly ha-
zardous in the case of skin contact and toxic to mucus
membranes, is extensively used in diagnostic methods
[7]. Sudan I, II, and III as well as Sudan Red 7B, and
Sudan Black B reaction products have been declared
suspected carcinogens and classified group 3 compounds
by the International Agency for Research on Cancer [8,
9]. Therefore, Sudan dyes are not safe for humans, and a
sensitive and convenient method for the determination of
Sudan dyes is required.
Several methods based on liquid chromatography with
UV or MS detection [10-12] or on gas chromatography
mass spectrometry have been developed for analysis of
Sudan I-IV. Although these methods have been success-
fully applied to analysis of the dyes at trace levels in food
products, they often require complicated pretreatment,
for example, more than one solvent extraction, solid-
phase extraction, rotary evaporation, etc. The extr action
and concentration procedures are time-consuming and
the results might be interfered in matrix effect. Un til now,
the methods for the analysis of Sudan Red G, Sudan Red
7B, and Sudan Black B are very few [13], and no report
on the analysis of Sudan Yellow. To our knowledge, this
is the first research on the determination of eight Sudan
dyes.
In this study, a sensitive , robust, and fa st meth od ba sed
on UPLC-ESI-MS/MS was developed to determine eight
Sudan dyes in chili powder. The technique of extra ct io n
current of mass spectrometry was to eliminate the inter-
ference from the co-eluted substances in the complex
samp l e. The matrix effect was discussed, and the deter-
mination was accurate. The validated method was suc-
cessfully applied in the quantification of eight Sudan
dyes in 10 samples.
2. Experimental
2.1. Reagents
Sudan I, Sudan II, Sudan III, Sudan IV, Sudan Red G,
Sudan Red 7B, Sudan Black B and Sudan Yellow were
*Corresponding a uthor.

L. WANG ET AL.
155
N
N
HO
NN
HO
NNNNOH NN
CH
3
NN
HO
CH
3
N
ON
HO
NN
N
N
HN
HN NH
CH
3
H
3
C
NNNNN
NN
CH
3
CH
3
Sudan ⅠSudan Ⅱ
Sudan ⅢSudan Ⅳ
Sudan Red GSudan Red 7B
Sudan Black BSudan Yellow
Figure 1. Che m i cal structur es of Sudan dyes.
purchased from Dr. Ehrenstorfer GmbH (Augsburg,
Germany). Acetonitrile of HPLC-grade, used for the
preparation of the mobile phases in LC-MS/MS analyses
and extraction of the dyes from the chili powder, was
purchased from Dikma (Fair Lawn, NJ, USA). Ultra pure
water from a Millipor Milli-Q system (Milford, MA,
USA) was used for the preparation of mobile phases.
Formic acid of HPLC-grade was purchased from Beijing
Chemical Reagents Company (Beijing , China).
Stock solutions of Sudan I, Sudan II, Sudan III, Sudan
IV, Sudan Red G, Sudan Red 7B, Sudan Black B and
Sudan Yellow at 100 μg/m L were prepared in acetonitrile,
and stored at 4˚C in the dark.
2.2. Sample Preparation
A 0.5 g aliquo t of th e chili pow der wa s weigh ed in to a 25
mL volumetric flask, and the flask filled with aceton itrile.
Samples were stirred with a stir bar for 1 h. Then, the
mixtures were centrifuged at 10,000 g for 10 min, and
filtered on 0.45 μm filters. Then, 2 μL of the substance
was injected into LC-MS/MS for analysis. All samples
were analyzed in duplicate (two aliquots of the same
sample individually extracted and inj e cted).
2.3. LC-MS/MS Analysis
Liquid chromatography was performed with an Acquity
UPLC system (Waters, Milford, MA, USA). Chromato-
graphic separation of eight Sudan dyes was done on a
Phenomenex XB-C18 column (2.1 mm × 100 mm, 2.6
μm particles), using acetonitrile with 0.1% formic acid
(phase A) and water with 0.1% formic acid (phase B) as
mobile phases. The flow rate was set at 0.3 mL/min and
the gradient was as follows: Isocratic elution at 70% A
for 5 min, linear gradient from 70% A to 85% A in 10
min, followed by a return to the initial condition in 1 min.
Total runtime was 20 min. The column temperature was
set at 30˚C and the sample temperature at 10˚C.
LCQ Advantage mass spectrometry (Thermo, USA)
was performed with electrospray ionization in positive-
ion mode (ESI+). Capillary column temperature was set
at 300˚C. The source voltage was set at 3500 V, and the
sampling S-lens RF amplitude and collision energy were
optimized for each molecule. Other conditions were as
listed in Table 1.
2.4. Method Validation
Blank samples (samples with amounts of Sudan below
LD) of chili powder were spiked with different amounts
of Sudan dye (0.01, 0.025, 0.05, 0.25, 0.5, 1.0, and 2.5
μg/mL in the final extract) and analyzed as described
above. Linearity was checked by calculating the correla-
tion coefficient, and the matrix effect was investigated by
comparing the standard solution at a concentration of
Table 1. MRM parameters of the analysis.
Dye parent ion product ion S-lens RF collision
(m/z ) (m/ z ) amplitude (V) energy (eV)
Sudan I 249.1 93.1 (a) 74 31
232.1 (b) 13
128.1 (b) 27
Sudan II 277.1 121.1 (a) 74 19
106.1 (b) 39
120.1 (b) 40
Sudan III 353.1 77.1 (a) 102 33
92.1 (b) 32
197.1 (b) 18
Sudan IV 381.2 91.1 (a) 110 32
224.1 (b) 21
106.1 (b) 35
Sudan Red G 279.1 108.1 (a) 74 33
123.1 (b) 19
80.1 (b) 50
Sudan Red 7B 380.2 115.0 (a) 90 50
183.1 (b) 15
169.1 (b) 32
Sudan Black B 457.2 194.1 (a) 152 33
193.1 (b) 48
246.1 (b) 26
Sudan Yellow 226.1 77.1 (a) 90 21
120.1 (b) 32
51.1 (b) 51
(a) Ion for quantification; (b) Ion fo r qu alification.

L. WANG ET AL.
156
0.05 μg/mL with the standard solution in chili powder.
Blank samples of chili powder were spiked with 0.25,
0.5, and 1.0 mg/kg of each Sudan dye, extracted, and
analyzed as described above. Precision was calculated by
dividing the standard deviation of the five determination
by the arithmetic mean of the values on three different
validation days. Recovery was determined by dividing
the measured amount of Sudan dye by the amount of
spiking.
3. Results and Discussion
3.1. Optimization of Instrumental Conditions
UPLC-ESI-MS/MS was used for qualitative and quantit-
ative analysis of the eight dyes in chili powder. Each
standard solution of 10 μg/mL for parameter tuning was
introduced directly into the electrospray ionization (ESI)
source in combination with mobile phase at a flow rate of
5 mL/min in both positive and negative ionization modes.
Positive mode resulted in better sensitivity. The quanti-
fication was accomplished by calculating the detector’s
responses of daugh ter ion in multiple rea ction monito ring
(MRM) mode. The parameters of eight Sudan dyes were
showed in Table 1.
3.2. Me thod Validation
The possibility of matrix effects was investigated by
comparing responses obtained from the standard solution
with or without matrix. The results were shown in Fig-
ure 2. Significant matrix effects were observed, includ-
ing ion suppression and ion promotion. Because of the
matrix effect, calibration plots were prepared using a
series of calibration solutions prepared in blank extract
instead of standard solutions in the analysis.
A typical chromatogram of the analyses was shown in
Figure 3. The extracted ions current technique in the
mass spectrometry can significantly enhance the selectiv-
ity and sensitivity. The linearity of the calib ration curves
was obtained between the concentrations and the area of
each Sudan dye. The correlation coefficients for the cali-
bration plots were better than 0.99 for all eight Sudan
Figure 2. Matrix effects on LC-MS/MS anal ysis.
Figure 3. Extracted ion chromatogram of eight Sudan dyes.
Table 2. LOD and LOQ of LC- MS/MS analysis.
Dye LOD LOQ
(mg/kg) (mg/kg)
Sudan I 0.03 0.10
Sudan II 0.01 0.05
Sudan III 0.003 0.01
Sudan IV 0.01 0.05
Sudan Red G 0.01 0.05
Sudan Red 7B 0.003 0.01
Sudan Black B 0.002 0.005
Sudan Yellow 0.001 0.002
dyes, showing the linearity of the method over the entire
calibration range. The LODs (S/N = 3) and LOQs (S/N =
10) of the method for eight Sudan dyes in chili powder
were in the ranges of 0.001 - 0.03 and 0.002 - 0.1 mg/kg,
respectively (Table 2). This showed that the method was
specific and should not cause false positive results.
As shown in Table 3, the intra-day precision ranged
from 2.49% to 10.16% and inter-day precision ranged
from 2.24% to 12.20% for each amount spiked at 0.25,
0.50, and 1.00 mg/kg. Mean recovery was 80.7% - 103.1%
with intra-day a nd 85.6% - 104.4% with in ter-day. The se
results demonstrate that the method was repeatable and
no noteworthy losses occur during the extraction proce-
dure.
3.3. Analysis of Real Samples
The method was successfully applied in the determina-
tion of eight Sudan dyes in chili powder. A total of 10
samples of chili powder were analyzed in duplicate. No
Sudan dyes were detected in these samples.

L. WANG ET AL.
157
Table 3. Precision and recovery of LC- MS/MS analysis (n =
5).
Dye
amount
spiked Intra-day Inter-day
(mg/kg) Recovery Precision Recovery Precision
Sudan I 0.25 87.7% 4.75% 90.7% 5.27%
0.50 90.0% 3.97% 93.2% 3.16%
1.00 94.0% 7.05% 90.4% 5.80%
Sudan II 0.25 86.1% 4.59% 91.9% 3.43%
0.50 88.5% 2.49% 91.7% 2.24%
1.00 91.1% 5.27% 89.7% 4.64%
Sudan III 0.25 80.7% 7.60% 86.3% 5.50%
0.50 82.5% 2.91% 89.3% 3.12%
1.00 89.2% 5.80% 88.9% 5.06%
Sudan IV 0.25 81.1% 6.27% 85.6% 7.58%
0.50 101.6% 3.97% 104.4% 4.76%
1.00 103.1% 4.49% 100.1% 12.20%
Sudan Red G 0.25 90.8% 7.31% 93.4% 5.06%
0.50 90.5% 3.83% 93.5% 2.88%
1.00 96.2% 5.42% 93.3% 4.14%
Sudan Red 7B 0.25 92.4% 10.16% 99.7% 6.24%
0.50 101.8% 3.21% 100.5% 3.15%
1.00 91.7% 5.97% 95.9% 7.68%
Sudan Black B 0.25 88.3% 4.10% 89.2% 4.40%
0.50 89.6% 4.23% 89.1% 3.25%
1.00 93.7% 7.20% 91.4% 4.07%
Sudan Yellow 0.25 98.8% 5.77% 94.2% 4.34%
0.50 98.7% 3.37% 94.8% 2.42%
1.00 100.7% 5.88% 93.0% 3.60%
4. Conclusion
A sensitive UPLC-ESI-MS/MS assay was developed to
determine eight Sudan dyes in chili powder. The method
had simple pretreatment, and offered high selectivity.
Sensitivity, precision and rec ov ery of variation were ac-
ceptable. The method could be used for routine analysis.
5. Acknowledgements
This work was supported by the Plan of Science and
Technology of Beijing (Z111100074211021).
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