Journal of Chromatography B, 942–943 (2013) 46–52
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Simultaneous determination of eight illegal dyes in chili products by liquid chromatography–tandem mass spectrometry Juan Li a,b , Xiao-Ming Ding a,b , Dan-Dan Liu a,b , Fei Guo a,b , Yu Chen a,b , Yan-Bing Zhang a,b,∗ , Hong-Min Liu a,b,∗ a b
School of Pharmaceutical Sciences, Zhengzhou University, 100 Science Road, Zhengzhou 450001, China New Drug Research, Development Center, Zhengzhou University, 100 Science Road, Zhengzhou 450001, China
a r t i c l e
i n f o
Article history: Received 27 July 2013 Accepted 9 October 2013 Available online 18 October 2013 Keywords: Food analysis Adulteration Illegal dyes LC–MS/MS Chili products
a b s t r a c t A sensitive and accurate method based on the use of liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed for the simultaneous determination of eight illegal synthetic dyes (Sudan (I–IV), Para Red, Rhodamine B, Chrysoidin and Auramine O) in chili products. A simple sample treatment procedure entailing the use of an extraction step with acetonitrile/H2 O (9/1) without further cleanup was developed. HPLC was performed on a C18 column using a multistep gradient elution with 5 mM ammonium acetate (pH 3.0 with formic acid) and methanol as the mobile phase. Mass spectral acquisition was done in multiple reaction monitoring (MRM) mode using positive electrospray ionization (ESI). Linear calibrations were obtained with correlation coefficients R2 > 0.99. Limit of detection (LOD) and limit of quantification (LOQ) for the studied dyes were in the ranges of 0.05–0.6 g kg−1 and 0.3–3.0 g kg−1 depending on matrices, respectively. The recoveries of the eight synthetic dyes in five matrices ranged from 70.5% to 119.2%. The intra- and inter-day precisions (RSDs) were between 2.3–15.8% and 5.7–15.6%, respectively. The applicability of the method to the determination of eight banned dyes in chili products was demonstrated. © 2013 Elsevier B.V. All rights reserved.
1. Introduction As food quality is an issue especially of aesthetical appeal, it has been an age-old practice to use food colorants for increasing the pricing level of certain commodities [1]. The use of synthetic organic dyes has been recognized as the most reliable and economical method of restoring or providing color to a processed product. According to the literature, azo compounds are by far the most widely used synthetic colorants, and they offer a wide spectrum of colors [2,3]. Sudan (I–IV), Para Red and Chrysoidin are common azo-dyes used in food and industry [4,5], characterized by chromophoric azo groups (N=N) [6]. The genetic toxicity of these azo-dyes has been confirmed, especially the Sudan group dyes and their degradation products have been recognized as carcinogens [7,8]. Therefore, the application of such dyes has been prohibited in many countries. Other dyes such as Rhodamine B and Auramine O are usually fluorescent dyes applied in industry, analytical chemistry and biological studies [9–11], which are also considered to be potentially carcinogenic [9,12]. As a result, these dyes are classified
∗ Corresponding authors. E-mail addresses: [email protected] (Y.-B. Zhang), [email protected] (H.-M. Liu). 1570-0232/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2013.10.010
into category 3 by the International Agency for Research on Cancer (IARC) [13] and are banned as food additives in the European Union [14,15]. Any level of these dyes is considered to be unsafe for human consumption. However, the synthetic dyes can still be found as a additive in food [16,17], due to their colorfastness, low cost, ready availability. For this reason, it is required to develop a sensitive and accurate method for the determination of the illegal dyes in complex matrices. Various analytical methods have been proposed for the quantitative determination of azo-dyes in foodstuffs, including liquid chromatography [18,19], partial filling micellar electrokinetic chromatography (MEKC) [20], solid-phase extraction (SPE) using single-hole hollow molecularly imprinted polymers [21], SPE using ionic liquid modified polymeric microspheres [22], and enzyme linked immunosorbent assay (ELISA) with newly developed polyclonal antibodies [23]. Due to its high sensitivity, selectivity and minimal sample treatment required, liquid chromatography coupled to mass spectrometry (LC–MS) has become the preferred method for confirmatory analysis [24–27]. A gel permeation chromatography (GPC)–liquid chromatography–tandem mass spectrometry was used for the simultaneous determination of Sudan dyes in chili products with detection limits from 0.1 to 1.8 g kg−1 [24]. A UPLC–ESI–MS/MS method was presented for the simultaneous determination of Sudan (I–IV) and Para Red in
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Fig. 1. Chemical structures of the eight synthetic dyes.
food with detection limits from 0.3 to 6.0 g kg−1 [25]. LC–TOF–MS methods have also been reported because of the high specificity provided by a TOF mass analyzer [28,29]. Prior to the detection of the synthetic colors, pretreatment of the real samples was necessary. One possible way to get rid of the matrix interferences would be sample cleanup by solid-phase extraction [30,31], molecularly imprinted solid-phase extraction [32,33], and gel permeation chromatography [34]. But such procedures increase the time and cost of the analysis, and cleanup generally leads to a loss in the number of dyes that can be analyzed in the method. Thus, a simple and low-cost pretreatment method is still required for the determination of banned dyes in foodstuffs [35,36]. Most researchers pay more attentions to red azo dyes in chili products [18,24–26]. As we know, there has been no report of the analysis of all eight banned dyes (Sudan (I–IV), Para Red, Rhodamine B, Chrysoidin and Auramine O) in chili products. In this work, we have developed a sensitive and efficient LC–MS/MS with one-step extraction procedure for the simultaneous determination of eight azo and non-azo dyes in food. The proposed method was validated by evaluating recovery, selectivity, linearity and accuracy, and has been applied for analysis of real samples. 2. Experimental 2.1. Chemicals and standards Methanol and acetonitrile (HPLC grade) were purchased from J. T. Baker (USA). Ammonium acetate and formic acid were from Sigma-Aldrich Co. Ltd. (Poole, UK). Ethyl acetate, chloroform, acetone and cyclohexane were of analytical reagent grade from Yongda (Tianjin, China). The water was purified and deionized by a water purify system (Millipore, Bedford, MA, USA). The solvents for HPLC were filtered by 0.45 m nylon membrane (Whatman, UK) and degassed in an ultrasonic bath. Standards of Sudan (I–IV), Para Red, Rhodamine B, Chrysoidin and Auramine O were purchased from Sigma (St. Louis, MO, USA). The purities of all standards were more than 98%. The molecular structures of eight synthetic dyes are shown in Fig. 1. Individual stock solutions of each compound (100 g mL−1 ) were prepared in acetonitrile for eight synthetic dyes. All stock solutions were stored at −20 ◦ C. Further dilutions were prepared in the determination process by acetonitrile/H2 O. 2.2. Sample preparation In order to achieve satisfactory results, different extraction conditions were assayed (type of extraction solvent, sample amount
and type of cleanup). The following conditions were found to be optimal for the matrices tested. The chili products were purchased from local market and were stored at room temperature until they were processed. After homogenizing the five samples (chili powder, chili sauce, nande spice, sausage and hotpot base), it was left at room temperature for 10 min prior to extraction with the solvent. A representative 2 g of solid sample (previously homogenized) was weighed into a 15 mL disposable screw-capped polypropylene tube, and 6 mL of acetonitrile/H2 O (9/1) were added. Then, the tube was shaken by hand for 30 s in order to allow the samples to mix thoroughly with the solvent, and the extraction continued for 30 min in an ultrasonic bath (100 W). Afterwards, the tube was centrifuged at 5000 rpm for 10 min to sediment the solids. An aliquot (0.5 mL) of the extraction solution was filtered through a 0.22 m polytetrafluoroethylene syringe filter before injection into the LC–MS/MS.
2.3. LC–MS/MS analysis Aliquots of 5 L were analyzed on an Waters HPLC system (Milford, MA, USA), which consisted of a 2695 separation module at a flow rate of 0.2 mL min−1 , The analytical column was an XTerra C18 RP column (2.1 mm × 150 mm, 5 m, Waters). The mobile phase consisted of A (5 mM ammonium acetate buffer solution, pH 3.0 with formic acid) and B (methanol). The linear gradient elution was programmed as follows: 0–10 min, 45–100% B; 10–20 min, 100% B; 20–25 min, 100–45% B. The column temperature was maintained at 35 ◦ C. A triple quadrupole mass spectrometer (Quattro-Micro® , Waters, Manchester, UK) equipped with an electrospray source operating in positive ion mode was used for detection in MRM mode. Optimization of the measuring conditions for each compound was performed by flow injection analysis, injecting individual standard solutions directly into the source. MRM experiments were carried out to obtain the maximum sensitivity for the detection of the target compounds. High purity nitrogen (N2 ) was used as both drying gas with a flow rate of 50 L h−1 and as nebulizing gas with a flow rate of 500 L h−1 . Ultrahigh pure argon (Ar) was used as the collision gas. The main parameters were set as follows: a capillary voltage of 3.0 kV, a source temperature of 120 ◦ C, and a dry temperature of 300 ◦ C. Table 1 shows the values of the instrumental settings optimized for each compound. The analysis was performed in just one time segment, which allowed obtaining sufficient scans for each peak. For quantification, the most intense MRM transition was selected. Masslynx 4.1 software was used for data acquisition and processing.
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Table 1 Values of the instrumental settings optimized for each compound: precursor ion, cone voltage, product ions and collision energy. Compound
Retention time (min)
Molecular weight (g mol−1 )
Precursor ion (m/z)
Cone voltage (V)
Product ions (m/z)
Collision energy (eV)
Sudan I Sudan II Sudan III Sudan IV Para red Chrysoidin Auramine O Rhodamine B
15.65 17.25 18.30 20.63 14.76 3.74 7.23 10.99
248.1 276.1 352.1 380.2 293.1 212.1 267.2 478.2
249.3 277.3 353.3 381.3 294.2 213.2 268.3 443.4
30 25 32 32 30 30 35 45
127.9* 156.0* 196.2* 224.2* 247.1* 121.0* 147.1* 399.2*
20 20 22 23 20 20 28 42
*
231.1 121.0 156.0 209.2 156.0 196.1 252.1 413.1
Qualifier ions.
2.4. Method validation The specificity, linearity, sensitivity, matrix effects, as well as precision and accuracy of the method were validated by a series of experiments described below. The accuracy and precision were evaluated by recovery studies using blank matrices of the five studied samples (chili powder, chili sauce, nande spice, sausage and hotpot base) spiked at six concentration levels, 7.5, 30 and 120 g kg−1 for Sudan (I–IV), Para Red and Chrysoidin; 0.75, 3 and 12 g kg−1 for Rhodamine B, and Auramine O. Quantification of the compounds in the spiked samples was carried out comparing the peak areas of the samples with those of matrix-matched standard solutions. Linearity was evaluated both in solvent and matrix, using matrix matched calibration curves prepared as described before, in two concentration ranges of 0.2–10 ng mL−1 and 2–100 ng mL−1 (Table 2). The matrix effect was studied by comparison of the slopes of the calibration curves in solvent and in matrix. LOD and LOQ values were estimated at S/N ratio of 3:1 and 10:1, respectively, by measuring the peak height of the blank and fortified samples. The repeatability of the instrumental method was estimated by determining the inter- and intra-day relative standard deviation (RSD, %) by the repeated analysis (n = 5) of a spiked matrix extract. 3. Results and discussion 3.1. The optimization of the extracting conditions Six organic solvents (ethyl acetate, chloroform, methanol, acetonitrile, acetone and cyclohexane) were investigated as extractants for the studied dyes in chili powder. For chili powder, acetonitrile/H2 O (9/1) was found to be efficient for the simultaneous extraction of all eight studied dyes with minimum interference. The extraction efficiency of different solvents is shown in Fig. 2. With addition of 0.1% formic acid into acetonitrile/H2 O (9/1), the peak of individual analyte in the following HPLC–MS/MS detection was interfered, which may be induced by acetonitrile-soluble interferences of the samples. Consequently, one extraction with 6 mL volume of acetonitrile/H2 O (9/1), which can completely extract the analytes, was selected for chili products. This method proved to be very effective, yielding very good recoveries for most of the analytes without any further cleanup step.
The result showed that the signal of the analytes obtained with the mobile phase containing methanol/5 mM ammonium acetate (pH 3.0 with formic acid) was higher than that obtained with the mobile phase (a, c, d). Using acetonitrile containing 5 mM ammonium acetate (pH 3.0 with formic acid) (d) or 0.1% formic acid aqueous solution (c) as the mobile phase, the analysis time was relative shorter, but Chrysoidin and Auramine O were not separated under any gradient elution condition. In addition, Chrysoidin had the signal interference at retention time using the mobile phase (a, c, d). So, aqueous methanol containing 5 mM ammonium acetate (pH 3.0 with formic acid) adopted as the mobile phase was suitable for the separation of eight synthetic dyes. Using gradient elution conditions described in Section 2.3, eight synthetic dyes can be separated properly with symmetrical peak shapes (Fig. 3). The MRM chromatograms of HPLC–MS/MS are shown in Fig. 4. Furthermore, best sensitivity could be achieved when injecting a volume of 5 L per run. 3.3. Optimization of LC–MS/MS parameters In this work, ESI–MS/MS behavior of eight synthetic dyes was investigated in positive ion mode, in order to maximize the responses. The first step of the MS/MS optimization was to select the most abundant ion from the full scan spectra as the precursor ion, corresponding in all cases to the protonated molecule [M + H]+ . The full scan spectrum was performed individually for each analyte with direct infusion of 5 g mL−1 standard solution prepared in acetonitrile/H2 O at a flow rate of 10 L min−1 . Under ESI(+)–MS/MS conditions, the daughter ions of dyes from collision-induced dissociation of [M + H]+ ions were obtained and the collision energies of dyes were optimized, respectively. The [M + H]+ ions, daughter ions and collision energies are shown in Table 1, and the daughter-ion mass spectra of all the analytes are
3.2. Separation condition of eight banned dyes by HPLC The separation behaviors were investigated using different mobile phases, including (a) methanol/0.1% formic acid aqueous solution; (b) methanol/5 mM ammonium acetate (pH 3.0 with formic acid); (c) acetonitrile/0.1% formic acid aqueous solution; (d) acetonitrile/5 mM ammonium acetate (pH 3.0 with formic acid).
Fig. 2. Recoveries of individual dyes from chili powder using seven different solvents.
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Table 2 Linear regression parameters of eight banned dyes from chili powder matrix calibration curves. Compound
Concentration range (ng mL−1 )
Standard calibration curve
Correlation coefficients
Matrix-matched calibration curve
correlation coefficients
Matrix effect(%)a
Sudan I Sudan II Sudan III Sudan IV Para red Chrysoidin Auramine O Rhodamine B
2–100 2–100 2–100 2–100 2–100 2–100 0.2–10 0.2–10
Y = 30.5467x − 21.3031 Y = 62.9656x − 2.17539 Y = 39.7712x + 21.9956 Y = 23.463x + 2.71519 Y = 18.6827x + 2.2173 Y = 11.3399x − 10.0025 Y = 101.908x + 1.22843 Y = 238.39x − 2.96772
0.998402 0.998788 0.995886 0.999752 0.995883 0.992903 0.996391 0.990945
Y = 30.566x + 9.1734 Y = 60.6598x + 18.3958 Y = 37.1316x + 24.2911 Y = 24.733x − 6.77521 Y = 20.4742x − 7.74782 Y = 13.2767x − 5.48573 Y = 118.376x − 9.23422 Y = 273.241x − 4.19389
0.997612 0.993731 0.991270 0.994264 0.995819 0.994854 0.996823 0.999435
−0.06 3.7 6.6 −5.4 −9.6 −17.1 −16.2 −14.6
a Expressed as percentage of the difference between the slopes of the corresponding calibration curves in solvent and in matrix. Negative values stand for signal suppression and positive values for signal enhancement.
shown in Fig. 5. The daughter ions were selected to be as specific as possible, avoiding the use of common losses to prevent false positives in the analysis of such complex matrices. In our experiments, the daughter ions having m/z 127.9, 156.0, 196.2, 224.2, 247.1, 121.0, 147.1 and 399.2 were used for qualifier ions. 3.4. Performance of HPLC–ESI–MS/MS method 3.4.1. Specificity Specificity was evaluated by analysis of the blank sample. No interferences were observed in corresponding retention times of target compounds by comparing chromatograms of spiked samples and blank samples. From Fig. 4, it is indicated that the present method has good specificity for the studied dyes. 3.4.2. Linearity and matrix effects The matrix effects may result as positive or negative responses depending on the level of ion suppression and can greatly affect the method reproducibility and accuracy. Matrix effects must be evaluated and discussed during the validation of the method. Linearity and matrix effects were investigated using solvent and matrix-matched calibration curves. Chili powder, chili sauce, nande spice, sausage and hotpot base were examined. The calibration curves were prepared at levels of 2, 5, 10, 20, 50 and 100 ng mL−1 for Sudan (I–IV), Para Red and Chrysoidin; 0.2, 0.5, 1, 2, 5 and 10 ng mL−1 for Auramine O and Rhodamine B. The linearity of the analytical response for all studied compounds within the range of two orders of magnitude was very good, with correlation coefficients higher than 0.99 in all cases. The matrix-matched standard calibration curves were compared to normal standard solution calibration curves using the percentage of the difference between these slopes. The percentage of the difference between these slopes is positive in case of signal enhancement, whereas a negative value is indicative for signal suppression. Table 2 shows the detailed data for the chili powder studied. Most of the compounds showed soft or medium
signal suppression in the matrices investigated. In view of these results, we can confirm that these distributions depend not only on the matrix but also on the combination of the compound and the matrix. This fact highlights the importance of performing quantification with matrix-matched calibration curves.
3.4.3. Detection limit, quantification limit, recovery and precision The determined LOD and LOQ values for the studied dyes in five matrices are shown in Table 3, which were in the ranges of 0.05–0.6 g kg−1 and 0.3–3.0 g kg−1 , respectively. The LOD values are equal or lower than those from the previous reports for Sudan (I–IV) and Para Red [22,23], and lower than the LOD values for Auramine O and Rhodamine B in Refs. [4,25]. Recovery and precision were investigated on spiked samples at six levels. The data of chili powder, which are shown in Table 4, indicated that intra-day and inter-day recoveries were between 74.4–117.5% and 80.8–116.0%, respectively, intra-day and inter-day precision (RSDs) was between 2.3–15.8% and 5.7–15.6%, respectively. For chili sauce, nande spice, sausage and hotpot base, intra-day and inter-day recoveries were between 70.5–118.6% and 71.2–119.2%, respectively, and intra-day and inter-day precision was lower than 16.8% and 17.5%, respectively. The results demonstrate that the accuracy of the present HPLC–MS/MS method was acceptable for routine monitoring purposes.
3.5. Analysis of real samples Once the operating conditions had been optimized, analysis of real samples was performed. In this study, 20 chili products (curry, curcumin and chili powder), 10 chili sauces, 5 hotpot base and 5 chili meats were analyzed for their illegal dyes content. Samples were selected from local markets from May 2012 to January 2013. In them the two positive chili samples were detected, one was contaminated with Rhodamine B, the another with Auramine O. Fig. 6 illustrates the presence of illegal dyes in the positive
Fig. 3. Total ion chromatograms of the analytes obtained from chili powder spiked standard mix solution.
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Fig. 4. MRM chromatograms of the analytes obtained from chili powder spiked standard mix solution: (a) Sudan I (30 g kg−1 ); (b) Sudan II (30 g kg−1 ); (c) Sudan III (30 g kg−1 ); (d) Sudan IV (30 g kg−1 ); (e) Para red (30 g kg−1 ); (f) Chrysoidin (30 g kg−1 ); (g) Auramine O (3 g kg−1 ); (h) Rhodamine B (3 g kg−1 ). Table 3 LOD and LOQ values of the studied dyes in the different matrices. Analyte
Matrix
LOD (g kg−1 )
LOQ (g kg−1 )
Sudan I, Sudan II Sudan III, Sudan IV, Para Red Chrysoidin Auramine O, Rhodamine B
Chili powder, chili sauce, nande spice, sausage Chili powder, chili sauce, nande spice, hotpot base Chili powder, chili sauce, nande spice, sausage, hotpot base Chili powder, chili sauce, nande spice, sausage, hotpot base
0.3 0.45 0.6 0.05
0.75 1.5 3 0.3
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Fig. 5. The daughter-ion mass spectra of (a) Sudan I; (b) Sudan II; (c) Sudan III; (d) Sudan IV; (e) Para Red; (f) Chrysoidin; (g) Auramine O; (h) Rhodamine B.
Fig. 6. LC–MS/MS analysis of a positive chili sample A (containing Rhodamine B at 1.8 ± 0.1 g kg−1 ) and sample B (containing Auramine O at 2.4 ± 0.2 g kg−1 ).
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Table 4 Intra-day and inter-day precision and recovery of the HPLC–MS/MS method for chili powder sample (n = 5). Compound
Sudan I
Sudan II
Sudan III
Sudan IV
Para Red
Chrysoidin
Auramine O
Rhodamine B
Fortified concentration (g kg−1 ) 7.5 30 120 7.5 30 120 7.5 30 120 7.5 30 120 7.5 30 120 7.5 30 120 0.75 3 12 0.75 3 12
Intra-day RSD (%)
Recovery (%)
RSD (%)
74.4 84.5 97.2 76.8 84.6 108.5 102.4 84.5 117.5 102.8 84.1 109.2 99.6 91.7 99.9 112.4 90.0 97.1 92.8 96.0 101.6 96.0 97.0 104.7
12.4 5.9 5.1 6.7 2.3 11.1 11.2 8.4 10.6 14.8 9.5 12.5 13.5 5.6 7.9 15.1 11.2 14.2 15.8 6.8 8.9 9.3 12.9 5.7
80.8 92.3 102.4 86.6 93.3 106.1 102.0 106.8 115.1 104.3 103.8 110.3 105.1 96.8 110.8 116.0 95.6 97.9 97.7 97.0 96.1 97.1 98.3 106.7
14.8 11.0 14.6 6.6 5.7 9.3 9.6 14.8 14.9 16.1 13.8 14.3 14.1 12.7 14.7 15.6 10.9 12.9 15.5 11.5 8.5 7.8 13.5 12.6
samples. For the other chili samples, target analytes have not been detected. 4. Conclusions This paper developed an HPLC–MS/MS method with one-step extraction procedure for the simultaneous determination of eight illegal synthetic dyes in chili products. Selectivity of the MS/MS technique coupled with chromatographic separation proved to provide unambiguous identification and accurate determination of the compounds in complex matrices without the need of laborious cleanup procedures. And the method has a good repeatability and high accuracy with low detection limits and quantification limits. The proposed HPLC–MS/MS process is effective for detecting frauds in export chili products. Acknowledgments This work was financially supported by the Natural Science Foundation of Henan Province (no. 12A350009), the National Natural Science Foundation of China (no. 81172937). References [1] [2] [3] [4] [5] [6] [7]
Inter-day
Recovery (%)
E.C. Vidotti, J.C. Cancino, C.C. Oliveira, M.E. Rollemberg, Anal. Sci. 21 (2005) 149. M.S. Reisch, Chem. Eng. News 66 (1988) 7. F. Rafii, J.D. Hall, C.E. Cerniglia, Food Chem. Toxicol. 35 (1997) 897. H. Yan, J. Qiao, Y. Pei, T. Long, W. Ding, K. Xie, Food Chem. 132 (2012) 649. X. Hu, Q. Cai, Y. Fan, T. Ye, Y.C. Guo, J. Chromatogr. A 1219 (2012) 39. L.H. Ahlstrom, C.S. Eskilsson, E. Bjorklund, Trend. Anal. Chem. 24 (2005) 49. M.F. Boeninger, Carcinogenicity and Metabolism of Azo Dyes, Especially Those Derived from Benzidine, DHHS (NIOSH), US Department of Health and Human Services, Cincinnati, OH, 1980. [8] M. Stiborova, V. Martinek, H. Rydlova, Cancer Res. 62 (2002) 5678.
[9] M. Oplatowska, C.T. Elliott, Analyst 136 (2011) 2403. [10] N. Amdursky, D. Huppert, J. Phys. Chem. B 116 (2012) 13389. [11] T.L. Sawadogo, L.G. Savadogo, S. Diande, F. Ouedraogo, A. Mourfou, A. Gueye, I. Sawadogo, B. Nebié, L. Sangare, A.S. Ouattara, Med. Sante Trop. 22 (2012) 302. [12] E. Hmani, Y. Samet, R. Abdelhédi, Diamond Relat. Mater. 30 (2012) 1. [13] International Agency for Research on Cancer, Occupational Exposures of Hairdressers and Barbers and Personal Use of Hair Colourants: Some Hair Dyes, Cosmetic Colourants, Industrial Dyestuffs and Aromatic Amines, International Agency for Research on Cancer, Lyon, France, 1993. [14] Commission Decision 2003/460/EC, Off. J. Eur. Commun. L154 (2003) 114. [15] Commission Decision 2005/402/EC, Off. J. Eur. Commun. L135 (2005) 34. [16] C. Schummer, J. Sassel, P. Bonenberger, G. Moris, J. Agric. Food Chem. 61 (2013) 2284. [17] F. Calbiani, M. Careri, L. Elviri, A. Mangia, L. Pistarà, I. Zagnoni, J. Chromatogr. A 1042 (2004) 123. [18] C. Long, Z. Mai, X. Yang, B. Zhu, X. Xu, X. Huang, X. Zou, Food Chem. 126 (2011) 1324. [19] J. Liu, Z. Gong, Chin. J. Chromatogr. 30 (2012) 624. [20] T.S. Fukuji, M. Castro-Puyana, M.F.M. Tavares, A. Cifuentes, J. Agric. Food Chem. 59 (2011) 11903. [21] Z. Zhang, S. Xu, J. Li, H. Xiong, H. Peng, L. Chen, J. Agric. Food Chem. 60 (2012) 180. [22] H. Yan, M. Gao, J. Qiao, J. Agric. Food Chem. 60 (2012) 6907. [23] Y.H. Qi, W.S. Chong, Y.L. Zheng, Y.J. Zhang, J.P. Wang, J. Agric. Food Chem. 60 (2012) 2116. [24] H.W. Sun, F.C. Wang, L.F. Ai, J. Chromatogr. A 1164 (2007) 120. [25] C. Li, Y.L. Wu, J.Z. Shen, Food Addit. Contam. 27 (2010) 1215. [26] X. Hu, G. Xiao, W. Pan, X. Mao, P. Li, Chin. J. Chromatogr. 28 (2010) 590. [27] H. Zhang, L. Liang, Z. He, L. Shi, J. Chin. Mass Spectrom. Soc. 31 (2010) 48. [28] C. Ferrer, A.R.F. Ndez-alba, I. Ferrer, Int. J. Environ. Anal. Chem. 87 (2007) 999. [29] F. Calbiani, M. Careri, L. Elviri, A. Mangia, I. Zagnoni, J. Cromatogr. A 1058 (2004) 127. [30] Y. Uematsu, M. Ogimoto, J. Kabashima, K. Suzuki, J. AOAC Int. 90 (2007) 437. [31] Z.X. Xu, S.O. Wang, G.Z. Fang, J.J. Song, Y. Zhang, Chromatographia 71 (2010) 397. [32] C. Baggiani, L. Anfossi, C.B. Giovannoli, G. Giraudi, C. Barolo, J. Sep. Sci. 32 (2009) 3292. [33] Z.H. Zhang, H.B. Zhang, Y.F. Hu, S.Z. Yao, Anal. Chim. Acta 661 (2010) 173. [34] O. Pardo, V. Yusà, N. León, A. Pastor, Talanta 78 (2009) 178. [35] E. Ertas, H. Oezer, C. Alasalvar, Food Chem. 105 (2007) 756. [36] C. Li, T. Yang, Y. Zhang, Y.L. Wu, Chromatographia 70 (2009) 319.
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Simultaneous determination of eight illegal dyes in chili products by liquid chromatography–tandem mass spectrometry Juan Li a,b , Xiao-Ming Ding a,b , Dan-Dan Liu a,b , Fei Guo a,b , Yu Chen a,b , Yan-Bing Zhang a,b,∗ , Hong-Min Liu a,b,∗ a b
School of Pharmaceutical Sciences, Zhengzhou University, 100 Science Road, Zhengzhou 450001, China New Drug Research, Development Center, Zhengzhou University, 100 Science Road, Zhengzhou 450001, China
a r t i c l e
i n f o
Article history: Received 27 July 2013 Accepted 9 October 2013 Available online 18 October 2013 Keywords: Food analysis Adulteration Illegal dyes LC–MS/MS Chili products
a b s t r a c t A sensitive and accurate method based on the use of liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed for the simultaneous determination of eight illegal synthetic dyes (Sudan (I–IV), Para Red, Rhodamine B, Chrysoidin and Auramine O) in chili products. A simple sample treatment procedure entailing the use of an extraction step with acetonitrile/H2 O (9/1) without further cleanup was developed. HPLC was performed on a C18 column using a multistep gradient elution with 5 mM ammonium acetate (pH 3.0 with formic acid) and methanol as the mobile phase. Mass spectral acquisition was done in multiple reaction monitoring (MRM) mode using positive electrospray ionization (ESI). Linear calibrations were obtained with correlation coefficients R2 > 0.99. Limit of detection (LOD) and limit of quantification (LOQ) for the studied dyes were in the ranges of 0.05–0.6 g kg−1 and 0.3–3.0 g kg−1 depending on matrices, respectively. The recoveries of the eight synthetic dyes in five matrices ranged from 70.5% to 119.2%. The intra- and inter-day precisions (RSDs) were between 2.3–15.8% and 5.7–15.6%, respectively. The applicability of the method to the determination of eight banned dyes in chili products was demonstrated. © 2013 Elsevier B.V. All rights reserved.
1. Introduction As food quality is an issue especially of aesthetical appeal, it has been an age-old practice to use food colorants for increasing the pricing level of certain commodities [1]. The use of synthetic organic dyes has been recognized as the most reliable and economical method of restoring or providing color to a processed product. According to the literature, azo compounds are by far the most widely used synthetic colorants, and they offer a wide spectrum of colors [2,3]. Sudan (I–IV), Para Red and Chrysoidin are common azo-dyes used in food and industry [4,5], characterized by chromophoric azo groups (N=N) [6]. The genetic toxicity of these azo-dyes has been confirmed, especially the Sudan group dyes and their degradation products have been recognized as carcinogens [7,8]. Therefore, the application of such dyes has been prohibited in many countries. Other dyes such as Rhodamine B and Auramine O are usually fluorescent dyes applied in industry, analytical chemistry and biological studies [9–11], which are also considered to be potentially carcinogenic [9,12]. As a result, these dyes are classified
∗ Corresponding authors. E-mail addresses: [email protected] (Y.-B. Zhang), [email protected] (H.-M. Liu). 1570-0232/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2013.10.010
into category 3 by the International Agency for Research on Cancer (IARC) [13] and are banned as food additives in the European Union [14,15]. Any level of these dyes is considered to be unsafe for human consumption. However, the synthetic dyes can still be found as a additive in food [16,17], due to their colorfastness, low cost, ready availability. For this reason, it is required to develop a sensitive and accurate method for the determination of the illegal dyes in complex matrices. Various analytical methods have been proposed for the quantitative determination of azo-dyes in foodstuffs, including liquid chromatography [18,19], partial filling micellar electrokinetic chromatography (MEKC) [20], solid-phase extraction (SPE) using single-hole hollow molecularly imprinted polymers [21], SPE using ionic liquid modified polymeric microspheres [22], and enzyme linked immunosorbent assay (ELISA) with newly developed polyclonal antibodies [23]. Due to its high sensitivity, selectivity and minimal sample treatment required, liquid chromatography coupled to mass spectrometry (LC–MS) has become the preferred method for confirmatory analysis [24–27]. A gel permeation chromatography (GPC)–liquid chromatography–tandem mass spectrometry was used for the simultaneous determination of Sudan dyes in chili products with detection limits from 0.1 to 1.8 g kg−1 [24]. A UPLC–ESI–MS/MS method was presented for the simultaneous determination of Sudan (I–IV) and Para Red in
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Fig. 1. Chemical structures of the eight synthetic dyes.
food with detection limits from 0.3 to 6.0 g kg−1 [25]. LC–TOF–MS methods have also been reported because of the high specificity provided by a TOF mass analyzer [28,29]. Prior to the detection of the synthetic colors, pretreatment of the real samples was necessary. One possible way to get rid of the matrix interferences would be sample cleanup by solid-phase extraction [30,31], molecularly imprinted solid-phase extraction [32,33], and gel permeation chromatography [34]. But such procedures increase the time and cost of the analysis, and cleanup generally leads to a loss in the number of dyes that can be analyzed in the method. Thus, a simple and low-cost pretreatment method is still required for the determination of banned dyes in foodstuffs [35,36]. Most researchers pay more attentions to red azo dyes in chili products [18,24–26]. As we know, there has been no report of the analysis of all eight banned dyes (Sudan (I–IV), Para Red, Rhodamine B, Chrysoidin and Auramine O) in chili products. In this work, we have developed a sensitive and efficient LC–MS/MS with one-step extraction procedure for the simultaneous determination of eight azo and non-azo dyes in food. The proposed method was validated by evaluating recovery, selectivity, linearity and accuracy, and has been applied for analysis of real samples. 2. Experimental 2.1. Chemicals and standards Methanol and acetonitrile (HPLC grade) were purchased from J. T. Baker (USA). Ammonium acetate and formic acid were from Sigma-Aldrich Co. Ltd. (Poole, UK). Ethyl acetate, chloroform, acetone and cyclohexane were of analytical reagent grade from Yongda (Tianjin, China). The water was purified and deionized by a water purify system (Millipore, Bedford, MA, USA). The solvents for HPLC were filtered by 0.45 m nylon membrane (Whatman, UK) and degassed in an ultrasonic bath. Standards of Sudan (I–IV), Para Red, Rhodamine B, Chrysoidin and Auramine O were purchased from Sigma (St. Louis, MO, USA). The purities of all standards were more than 98%. The molecular structures of eight synthetic dyes are shown in Fig. 1. Individual stock solutions of each compound (100 g mL−1 ) were prepared in acetonitrile for eight synthetic dyes. All stock solutions were stored at −20 ◦ C. Further dilutions were prepared in the determination process by acetonitrile/H2 O. 2.2. Sample preparation In order to achieve satisfactory results, different extraction conditions were assayed (type of extraction solvent, sample amount
and type of cleanup). The following conditions were found to be optimal for the matrices tested. The chili products were purchased from local market and were stored at room temperature until they were processed. After homogenizing the five samples (chili powder, chili sauce, nande spice, sausage and hotpot base), it was left at room temperature for 10 min prior to extraction with the solvent. A representative 2 g of solid sample (previously homogenized) was weighed into a 15 mL disposable screw-capped polypropylene tube, and 6 mL of acetonitrile/H2 O (9/1) were added. Then, the tube was shaken by hand for 30 s in order to allow the samples to mix thoroughly with the solvent, and the extraction continued for 30 min in an ultrasonic bath (100 W). Afterwards, the tube was centrifuged at 5000 rpm for 10 min to sediment the solids. An aliquot (0.5 mL) of the extraction solution was filtered through a 0.22 m polytetrafluoroethylene syringe filter before injection into the LC–MS/MS.
2.3. LC–MS/MS analysis Aliquots of 5 L were analyzed on an Waters HPLC system (Milford, MA, USA), which consisted of a 2695 separation module at a flow rate of 0.2 mL min−1 , The analytical column was an XTerra C18 RP column (2.1 mm × 150 mm, 5 m, Waters). The mobile phase consisted of A (5 mM ammonium acetate buffer solution, pH 3.0 with formic acid) and B (methanol). The linear gradient elution was programmed as follows: 0–10 min, 45–100% B; 10–20 min, 100% B; 20–25 min, 100–45% B. The column temperature was maintained at 35 ◦ C. A triple quadrupole mass spectrometer (Quattro-Micro® , Waters, Manchester, UK) equipped with an electrospray source operating in positive ion mode was used for detection in MRM mode. Optimization of the measuring conditions for each compound was performed by flow injection analysis, injecting individual standard solutions directly into the source. MRM experiments were carried out to obtain the maximum sensitivity for the detection of the target compounds. High purity nitrogen (N2 ) was used as both drying gas with a flow rate of 50 L h−1 and as nebulizing gas with a flow rate of 500 L h−1 . Ultrahigh pure argon (Ar) was used as the collision gas. The main parameters were set as follows: a capillary voltage of 3.0 kV, a source temperature of 120 ◦ C, and a dry temperature of 300 ◦ C. Table 1 shows the values of the instrumental settings optimized for each compound. The analysis was performed in just one time segment, which allowed obtaining sufficient scans for each peak. For quantification, the most intense MRM transition was selected. Masslynx 4.1 software was used for data acquisition and processing.
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Table 1 Values of the instrumental settings optimized for each compound: precursor ion, cone voltage, product ions and collision energy. Compound
Retention time (min)
Molecular weight (g mol−1 )
Precursor ion (m/z)
Cone voltage (V)
Product ions (m/z)
Collision energy (eV)
Sudan I Sudan II Sudan III Sudan IV Para red Chrysoidin Auramine O Rhodamine B
15.65 17.25 18.30 20.63 14.76 3.74 7.23 10.99
248.1 276.1 352.1 380.2 293.1 212.1 267.2 478.2
249.3 277.3 353.3 381.3 294.2 213.2 268.3 443.4
30 25 32 32 30 30 35 45
127.9* 156.0* 196.2* 224.2* 247.1* 121.0* 147.1* 399.2*
20 20 22 23 20 20 28 42
*
231.1 121.0 156.0 209.2 156.0 196.1 252.1 413.1
Qualifier ions.
2.4. Method validation The specificity, linearity, sensitivity, matrix effects, as well as precision and accuracy of the method were validated by a series of experiments described below. The accuracy and precision were evaluated by recovery studies using blank matrices of the five studied samples (chili powder, chili sauce, nande spice, sausage and hotpot base) spiked at six concentration levels, 7.5, 30 and 120 g kg−1 for Sudan (I–IV), Para Red and Chrysoidin; 0.75, 3 and 12 g kg−1 for Rhodamine B, and Auramine O. Quantification of the compounds in the spiked samples was carried out comparing the peak areas of the samples with those of matrix-matched standard solutions. Linearity was evaluated both in solvent and matrix, using matrix matched calibration curves prepared as described before, in two concentration ranges of 0.2–10 ng mL−1 and 2–100 ng mL−1 (Table 2). The matrix effect was studied by comparison of the slopes of the calibration curves in solvent and in matrix. LOD and LOQ values were estimated at S/N ratio of 3:1 and 10:1, respectively, by measuring the peak height of the blank and fortified samples. The repeatability of the instrumental method was estimated by determining the inter- and intra-day relative standard deviation (RSD, %) by the repeated analysis (n = 5) of a spiked matrix extract. 3. Results and discussion 3.1. The optimization of the extracting conditions Six organic solvents (ethyl acetate, chloroform, methanol, acetonitrile, acetone and cyclohexane) were investigated as extractants for the studied dyes in chili powder. For chili powder, acetonitrile/H2 O (9/1) was found to be efficient for the simultaneous extraction of all eight studied dyes with minimum interference. The extraction efficiency of different solvents is shown in Fig. 2. With addition of 0.1% formic acid into acetonitrile/H2 O (9/1), the peak of individual analyte in the following HPLC–MS/MS detection was interfered, which may be induced by acetonitrile-soluble interferences of the samples. Consequently, one extraction with 6 mL volume of acetonitrile/H2 O (9/1), which can completely extract the analytes, was selected for chili products. This method proved to be very effective, yielding very good recoveries for most of the analytes without any further cleanup step.
The result showed that the signal of the analytes obtained with the mobile phase containing methanol/5 mM ammonium acetate (pH 3.0 with formic acid) was higher than that obtained with the mobile phase (a, c, d). Using acetonitrile containing 5 mM ammonium acetate (pH 3.0 with formic acid) (d) or 0.1% formic acid aqueous solution (c) as the mobile phase, the analysis time was relative shorter, but Chrysoidin and Auramine O were not separated under any gradient elution condition. In addition, Chrysoidin had the signal interference at retention time using the mobile phase (a, c, d). So, aqueous methanol containing 5 mM ammonium acetate (pH 3.0 with formic acid) adopted as the mobile phase was suitable for the separation of eight synthetic dyes. Using gradient elution conditions described in Section 2.3, eight synthetic dyes can be separated properly with symmetrical peak shapes (Fig. 3). The MRM chromatograms of HPLC–MS/MS are shown in Fig. 4. Furthermore, best sensitivity could be achieved when injecting a volume of 5 L per run. 3.3. Optimization of LC–MS/MS parameters In this work, ESI–MS/MS behavior of eight synthetic dyes was investigated in positive ion mode, in order to maximize the responses. The first step of the MS/MS optimization was to select the most abundant ion from the full scan spectra as the precursor ion, corresponding in all cases to the protonated molecule [M + H]+ . The full scan spectrum was performed individually for each analyte with direct infusion of 5 g mL−1 standard solution prepared in acetonitrile/H2 O at a flow rate of 10 L min−1 . Under ESI(+)–MS/MS conditions, the daughter ions of dyes from collision-induced dissociation of [M + H]+ ions were obtained and the collision energies of dyes were optimized, respectively. The [M + H]+ ions, daughter ions and collision energies are shown in Table 1, and the daughter-ion mass spectra of all the analytes are
3.2. Separation condition of eight banned dyes by HPLC The separation behaviors were investigated using different mobile phases, including (a) methanol/0.1% formic acid aqueous solution; (b) methanol/5 mM ammonium acetate (pH 3.0 with formic acid); (c) acetonitrile/0.1% formic acid aqueous solution; (d) acetonitrile/5 mM ammonium acetate (pH 3.0 with formic acid).
Fig. 2. Recoveries of individual dyes from chili powder using seven different solvents.
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Table 2 Linear regression parameters of eight banned dyes from chili powder matrix calibration curves. Compound
Concentration range (ng mL−1 )
Standard calibration curve
Correlation coefficients
Matrix-matched calibration curve
correlation coefficients
Matrix effect(%)a
Sudan I Sudan II Sudan III Sudan IV Para red Chrysoidin Auramine O Rhodamine B
2–100 2–100 2–100 2–100 2–100 2–100 0.2–10 0.2–10
Y = 30.5467x − 21.3031 Y = 62.9656x − 2.17539 Y = 39.7712x + 21.9956 Y = 23.463x + 2.71519 Y = 18.6827x + 2.2173 Y = 11.3399x − 10.0025 Y = 101.908x + 1.22843 Y = 238.39x − 2.96772
0.998402 0.998788 0.995886 0.999752 0.995883 0.992903 0.996391 0.990945
Y = 30.566x + 9.1734 Y = 60.6598x + 18.3958 Y = 37.1316x + 24.2911 Y = 24.733x − 6.77521 Y = 20.4742x − 7.74782 Y = 13.2767x − 5.48573 Y = 118.376x − 9.23422 Y = 273.241x − 4.19389
0.997612 0.993731 0.991270 0.994264 0.995819 0.994854 0.996823 0.999435
−0.06 3.7 6.6 −5.4 −9.6 −17.1 −16.2 −14.6
a Expressed as percentage of the difference between the slopes of the corresponding calibration curves in solvent and in matrix. Negative values stand for signal suppression and positive values for signal enhancement.
shown in Fig. 5. The daughter ions were selected to be as specific as possible, avoiding the use of common losses to prevent false positives in the analysis of such complex matrices. In our experiments, the daughter ions having m/z 127.9, 156.0, 196.2, 224.2, 247.1, 121.0, 147.1 and 399.2 were used for qualifier ions. 3.4. Performance of HPLC–ESI–MS/MS method 3.4.1. Specificity Specificity was evaluated by analysis of the blank sample. No interferences were observed in corresponding retention times of target compounds by comparing chromatograms of spiked samples and blank samples. From Fig. 4, it is indicated that the present method has good specificity for the studied dyes. 3.4.2. Linearity and matrix effects The matrix effects may result as positive or negative responses depending on the level of ion suppression and can greatly affect the method reproducibility and accuracy. Matrix effects must be evaluated and discussed during the validation of the method. Linearity and matrix effects were investigated using solvent and matrix-matched calibration curves. Chili powder, chili sauce, nande spice, sausage and hotpot base were examined. The calibration curves were prepared at levels of 2, 5, 10, 20, 50 and 100 ng mL−1 for Sudan (I–IV), Para Red and Chrysoidin; 0.2, 0.5, 1, 2, 5 and 10 ng mL−1 for Auramine O and Rhodamine B. The linearity of the analytical response for all studied compounds within the range of two orders of magnitude was very good, with correlation coefficients higher than 0.99 in all cases. The matrix-matched standard calibration curves were compared to normal standard solution calibration curves using the percentage of the difference between these slopes. The percentage of the difference between these slopes is positive in case of signal enhancement, whereas a negative value is indicative for signal suppression. Table 2 shows the detailed data for the chili powder studied. Most of the compounds showed soft or medium
signal suppression in the matrices investigated. In view of these results, we can confirm that these distributions depend not only on the matrix but also on the combination of the compound and the matrix. This fact highlights the importance of performing quantification with matrix-matched calibration curves.
3.4.3. Detection limit, quantification limit, recovery and precision The determined LOD and LOQ values for the studied dyes in five matrices are shown in Table 3, which were in the ranges of 0.05–0.6 g kg−1 and 0.3–3.0 g kg−1 , respectively. The LOD values are equal or lower than those from the previous reports for Sudan (I–IV) and Para Red [22,23], and lower than the LOD values for Auramine O and Rhodamine B in Refs. [4,25]. Recovery and precision were investigated on spiked samples at six levels. The data of chili powder, which are shown in Table 4, indicated that intra-day and inter-day recoveries were between 74.4–117.5% and 80.8–116.0%, respectively, intra-day and inter-day precision (RSDs) was between 2.3–15.8% and 5.7–15.6%, respectively. For chili sauce, nande spice, sausage and hotpot base, intra-day and inter-day recoveries were between 70.5–118.6% and 71.2–119.2%, respectively, and intra-day and inter-day precision was lower than 16.8% and 17.5%, respectively. The results demonstrate that the accuracy of the present HPLC–MS/MS method was acceptable for routine monitoring purposes.
3.5. Analysis of real samples Once the operating conditions had been optimized, analysis of real samples was performed. In this study, 20 chili products (curry, curcumin and chili powder), 10 chili sauces, 5 hotpot base and 5 chili meats were analyzed for their illegal dyes content. Samples were selected from local markets from May 2012 to January 2013. In them the two positive chili samples were detected, one was contaminated with Rhodamine B, the another with Auramine O. Fig. 6 illustrates the presence of illegal dyes in the positive
Fig. 3. Total ion chromatograms of the analytes obtained from chili powder spiked standard mix solution.
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Fig. 4. MRM chromatograms of the analytes obtained from chili powder spiked standard mix solution: (a) Sudan I (30 g kg−1 ); (b) Sudan II (30 g kg−1 ); (c) Sudan III (30 g kg−1 ); (d) Sudan IV (30 g kg−1 ); (e) Para red (30 g kg−1 ); (f) Chrysoidin (30 g kg−1 ); (g) Auramine O (3 g kg−1 ); (h) Rhodamine B (3 g kg−1 ). Table 3 LOD and LOQ values of the studied dyes in the different matrices. Analyte
Matrix
LOD (g kg−1 )
LOQ (g kg−1 )
Sudan I, Sudan II Sudan III, Sudan IV, Para Red Chrysoidin Auramine O, Rhodamine B
Chili powder, chili sauce, nande spice, sausage Chili powder, chili sauce, nande spice, hotpot base Chili powder, chili sauce, nande spice, sausage, hotpot base Chili powder, chili sauce, nande spice, sausage, hotpot base
0.3 0.45 0.6 0.05
0.75 1.5 3 0.3
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Fig. 5. The daughter-ion mass spectra of (a) Sudan I; (b) Sudan II; (c) Sudan III; (d) Sudan IV; (e) Para Red; (f) Chrysoidin; (g) Auramine O; (h) Rhodamine B.
Fig. 6. LC–MS/MS analysis of a positive chili sample A (containing Rhodamine B at 1.8 ± 0.1 g kg−1 ) and sample B (containing Auramine O at 2.4 ± 0.2 g kg−1 ).
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Table 4 Intra-day and inter-day precision and recovery of the HPLC–MS/MS method for chili powder sample (n = 5). Compound
Sudan I
Sudan II
Sudan III
Sudan IV
Para Red
Chrysoidin
Auramine O
Rhodamine B
Fortified concentration (g kg−1 ) 7.5 30 120 7.5 30 120 7.5 30 120 7.5 30 120 7.5 30 120 7.5 30 120 0.75 3 12 0.75 3 12
Intra-day RSD (%)
Recovery (%)
RSD (%)
74.4 84.5 97.2 76.8 84.6 108.5 102.4 84.5 117.5 102.8 84.1 109.2 99.6 91.7 99.9 112.4 90.0 97.1 92.8 96.0 101.6 96.0 97.0 104.7
12.4 5.9 5.1 6.7 2.3 11.1 11.2 8.4 10.6 14.8 9.5 12.5 13.5 5.6 7.9 15.1 11.2 14.2 15.8 6.8 8.9 9.3 12.9 5.7
80.8 92.3 102.4 86.6 93.3 106.1 102.0 106.8 115.1 104.3 103.8 110.3 105.1 96.8 110.8 116.0 95.6 97.9 97.7 97.0 96.1 97.1 98.3 106.7
14.8 11.0 14.6 6.6 5.7 9.3 9.6 14.8 14.9 16.1 13.8 14.3 14.1 12.7 14.7 15.6 10.9 12.9 15.5 11.5 8.5 7.8 13.5 12.6
samples. For the other chili samples, target analytes have not been detected. 4. Conclusions This paper developed an HPLC–MS/MS method with one-step extraction procedure for the simultaneous determination of eight illegal synthetic dyes in chili products. Selectivity of the MS/MS technique coupled with chromatographic separation proved to provide unambiguous identification and accurate determination of the compounds in complex matrices without the need of laborious cleanup procedures. And the method has a good repeatability and high accuracy with low detection limits and quantification limits. The proposed HPLC–MS/MS process is effective for detecting frauds in export chili products. Acknowledgments This work was financially supported by the Natural Science Foundation of Henan Province (no. 12A350009), the National Natural Science Foundation of China (no. 81172937). References [1] [2] [3] [4] [5] [6] [7]
Inter-day
Recovery (%)
E.C. Vidotti, J.C. Cancino, C.C. Oliveira, M.E. Rollemberg, Anal. Sci. 21 (2005) 149. M.S. Reisch, Chem. Eng. News 66 (1988) 7. F. Rafii, J.D. Hall, C.E. Cerniglia, Food Chem. Toxicol. 35 (1997) 897. H. Yan, J. Qiao, Y. Pei, T. Long, W. Ding, K. Xie, Food Chem. 132 (2012) 649. X. Hu, Q. Cai, Y. Fan, T. Ye, Y.C. Guo, J. Chromatogr. A 1219 (2012) 39. L.H. Ahlstrom, C.S. Eskilsson, E. Bjorklund, Trend. Anal. Chem. 24 (2005) 49. M.F. Boeninger, Carcinogenicity and Metabolism of Azo Dyes, Especially Those Derived from Benzidine, DHHS (NIOSH), US Department of Health and Human Services, Cincinnati, OH, 1980. [8] M. Stiborova, V. Martinek, H. Rydlova, Cancer Res. 62 (2002) 5678.
[9] M. Oplatowska, C.T. Elliott, Analyst 136 (2011) 2403. [10] N. Amdursky, D. Huppert, J. Phys. Chem. B 116 (2012) 13389. [11] T.L. Sawadogo, L.G. Savadogo, S. Diande, F. Ouedraogo, A. Mourfou, A. Gueye, I. Sawadogo, B. Nebié, L. Sangare, A.S. Ouattara, Med. Sante Trop. 22 (2012) 302. [12] E. Hmani, Y. Samet, R. Abdelhédi, Diamond Relat. Mater. 30 (2012) 1. [13] International Agency for Research on Cancer, Occupational Exposures of Hairdressers and Barbers and Personal Use of Hair Colourants: Some Hair Dyes, Cosmetic Colourants, Industrial Dyestuffs and Aromatic Amines, International Agency for Research on Cancer, Lyon, France, 1993. [14] Commission Decision 2003/460/EC, Off. J. Eur. Commun. L154 (2003) 114. [15] Commission Decision 2005/402/EC, Off. J. Eur. Commun. L135 (2005) 34. [16] C. Schummer, J. Sassel, P. Bonenberger, G. Moris, J. Agric. Food Chem. 61 (2013) 2284. [17] F. Calbiani, M. Careri, L. Elviri, A. Mangia, L. Pistarà, I. Zagnoni, J. Chromatogr. A 1042 (2004) 123. [18] C. Long, Z. Mai, X. Yang, B. Zhu, X. Xu, X. Huang, X. Zou, Food Chem. 126 (2011) 1324. [19] J. Liu, Z. Gong, Chin. J. Chromatogr. 30 (2012) 624. [20] T.S. Fukuji, M. Castro-Puyana, M.F.M. Tavares, A. Cifuentes, J. Agric. Food Chem. 59 (2011) 11903. [21] Z. Zhang, S. Xu, J. Li, H. Xiong, H. Peng, L. Chen, J. Agric. Food Chem. 60 (2012) 180. [22] H. Yan, M. Gao, J. Qiao, J. Agric. Food Chem. 60 (2012) 6907. [23] Y.H. Qi, W.S. Chong, Y.L. Zheng, Y.J. Zhang, J.P. Wang, J. Agric. Food Chem. 60 (2012) 2116. [24] H.W. Sun, F.C. Wang, L.F. Ai, J. Chromatogr. A 1164 (2007) 120. [25] C. Li, Y.L. Wu, J.Z. Shen, Food Addit. Contam. 27 (2010) 1215. [26] X. Hu, G. Xiao, W. Pan, X. Mao, P. Li, Chin. J. Chromatogr. 28 (2010) 590. [27] H. Zhang, L. Liang, Z. He, L. Shi, J. Chin. Mass Spectrom. Soc. 31 (2010) 48. [28] C. Ferrer, A.R.F. Ndez-alba, I. Ferrer, Int. J. Environ. Anal. Chem. 87 (2007) 999. [29] F. Calbiani, M. Careri, L. Elviri, A. Mangia, I. Zagnoni, J. Cromatogr. A 1058 (2004) 127. [30] Y. Uematsu, M. Ogimoto, J. Kabashima, K. Suzuki, J. AOAC Int. 90 (2007) 437. [31] Z.X. Xu, S.O. Wang, G.Z. Fang, J.J. Song, Y. Zhang, Chromatographia 71 (2010) 397. [32] C. Baggiani, L. Anfossi, C.B. Giovannoli, G. Giraudi, C. Barolo, J. Sep. Sci. 32 (2009) 3292. [33] Z.H. Zhang, H.B. Zhang, Y.F. Hu, S.Z. Yao, Anal. Chim. Acta 661 (2010) 173. [34] O. Pardo, V. Yusà, N. León, A. Pastor, Talanta 78 (2009) 178. [35] E. Ertas, H. Oezer, C. Alasalvar, Food Chem. 105 (2007) 756. [36] C. Li, T. Yang, Y. Zhang, Y.L. Wu, Chromatographia 70 (2009) 319.
