Determination of Eight Sudan Dyes in Chili Powder by UPLC-MS/MS

doi:10.4236/eng.2013.510B033

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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

Email: *[email protected]

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

NNNNOH NN

HN NH

NNNNN

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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