Food Chemistry 145 (2014) 956–962
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Analytical Methods
Simultaneous determination of 14 oil-soluble synthetic dyes in chilli products by high performance liquid chromatography with a gel permeation chromatography clean-up procedure Yonghong Zhu ⇑, Bo Zhao, Ruiqi Xiao, Wen Yun, Zhaojing Xiao, Dawei Tu, Shiqi Chen Center for Quality Supervision & Inspection of Food, Chongqing Academy of Metrology and Quality Inspection, Chongqing Engineering Research Center for Food Safety, Chongqing 401123, China
a r t i c l e
i n f o
Article history: Received 1 April 2013 Received in revised form 17 August 2013 Accepted 2 September 2013 Available online 11 September 2013 Keywords: High performance liquid chromatography Synthetic dyes Gel permeation chromatography Chilli products
a b s t r a c t A method was developed for the simultaneous determination of 14 fat-soluble dyes in chilli products. The samples were extracted with hexane/acetone. The cleanup was performed with gel permeation chromatography (GPC) cleanup system. A HPLC separation was performed using variable wavelength detector and a gradient elution with 0.1% formic acid and methanol–acetonitrile (1:1, v/v) as the mobile phases. Good linearity (R2 P 0.995) was observed between 0.1 and 5.0 lg/mL. Detection limits of the investigated dyes, which were evaluated at signal to noise ratio of 3, were in the ranges of 11–71 lg/kg. The recoveries of the 14 synthetic colourants in three matrices ranged from 73.4% to 103.5%. Relative standard deviations ranged from 3.7% to 12.3%. The method has been successfully used for the determination of banned dyes in real samples. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Food quality is closely associated with colour and the use of food colourants has been an age-old practice, enhancing the aesthetical appeal of foods. The use of synthetic organic dyes has been recognised as the most reliable and economical method of restoring or providing colour to a processed product. Generally, synthetic colourants can be classified into water-soluble and fatsoluble colourants based on their solubility. At present, the most synthetic colourants permitted for use in foods are water-soluble, while most fat-soluble synthetic colourants were non-edible colourants and their use in foodstuffs was prohibited. The common non-edible colourants ever reported are some azocompounds, such as Sudan orange G, Para red, Sudan I, Sudan II, Sudan III, and Sudan IV, Sudan yellow, Sudan red G, Sudan red B, and Sudan red 7B (Noguerol-Cal, López-Vilarinˇo, Fernández-Martínez, Barral-Losada, & González-Rodríguez, 2008; Pardo, Yusà, León, & Pastor, 2009; Sun, Wang, & Ai, 2007). In addition, we found an illegally added synthetic colourant in chilli products not long ago. The colourant was identified as Scarlet pigment powder, which was also a fat-soluble azo-dyes (Zhu et al., 2011). Besides above colourants, Citrus red 2, Toluidine red, and Sudan blue 2 were also commonly seen fat-soluble synthetic colourants (Liu, Hei, He, & Li, 2011; Noguerol-Cal et al., 2008; Part 74 of 21 ⇑ Corresponding author. Tel./fax: +86 23 89136317. E-mail addresses: [email protected], [email protected] (Y. Zhu). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.09.008
CFR, US). There is possibility that they could also be added into foodstuffs. Among above colourants, Citrus red 2 could be used as food dyes, but it shall be used only for colouring the skins of oranges that are not intended or used for processing (or if so used are designated in the trade as Packinghouse elimination). In US, oranges coloured with Citrus red No. 2 shall bear not more than 2.0 parts per million of such colour additive, calculated on the basis of the weight of the whole fruit (Part 74 of 21 CFR, US). The use of other fat-soluble colourants in foods is prohibited worldwide, and the prohibited colourants should not be present in foods. In EU, decisions on emergency measures concerning at Sudan I, Sudan II, Sudan III, and Sudan IV were adopted in June 2003 and May 2005 (Commission Decision, 2003, 2005). In order to prevent the possible unlawful addition of above fatsoluble synthetic colourants into foodstuffs, it is necessary to develop accurate and reliable analytical methods for those colourants in foodstuffs. Until now, there was not a method for simultaneous determination of the above 14 dyes. The chemical information of the 14 dyes is given in Table 1. Many analytical methods were reported for fat-soluble synthetic colourants, especially azo-dyes analysis in foodstuffs (Rebane, Leito, Yurchenko, & Herodes, 2010). Among the reported methods, high performance liquid chromatography (HPLC) coupled with different types of detector including mass spectrometric detector (MSD) (Calbiani et al., 2004; Liu et al., 2011; Murty, Chary, Prabhakar, Raju, & Vairamani, 2009; Pardo et al., 2009; Sun, et al., 2007), ultraviolet-visible (UV-vis) detector (Ertasß, Özer, &
Y. Zhu et al. / Food Chemistry 145 (2014) 956–962 Table 1 Chemical information about studied dyes. No.
Name
CAS number
CI number
Molecular formula
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Sudan orange G Sudan yellow Para red Citrus red 2 Sudan red G Toluidine red Sudan I Scarlet pigment powder Sudan II Sudan blue 2 Sudan III Sudan red 7B Sudan red B Sudan IV
2051-85-6 60-11-7 6410-10-2 6358-53-8 1229-55-6 2425-85-6 842-07-9 3789-75-1 3118-97-6 17354-14-2 85-86-9 6368-72-5 3176-79-2 85-83-6
11920 11020 12070 12156 12150 12120 12055 / 12140 61554 26100 26050 26110 26105
C12H10N2O2 C14H15N3 C16H11N3O3 C18H16N2O3 C17H14N2O2 C17H13N3O3 C16H12N2O C23H17N3O2 C18H16N2O C22H26N2O2 C22H16N4O C24H21N5 C24H20N4O C24H20N4O
Alasalvar, 2007; Fan et al., 2009; Long et al., 2011) and photodiode array detector (DAD) (López-Jiménez, Rubio, & Pérez-Bendito, 2010; Qi, Zeng, Wen, Liang, & Zhang, 2011; Enríquez-Gabeiras, Gallego, Garcinuño, Fernández-Hernando, & Durand, 2012) was widely used. With increasing demand for the determination of multiple dyes present in food, a high throughput method which could simultaneously determine multiple dyes was needed. Among the high throughput analytic methods, liquid chromatography tandem mass spectrometry (LC/MS/MS) based methods are more and more widely used. Because of their high sensitivity and selectivity, LC–MS based methods provide the capability of multi-component validation, quantification and microanalysis. But in ordinary labs, LC–MS are often not available. So a high throughput analytic method based on HPLC coupled with UV-vis detector or DAD could have a more practical application in routine analysis. Whichever method is used, sample pretreatment procedure was prerequisite. After solvent extraction of the analytes in foods, a further clean-up procedure was often necessary. The reported clean-up procedure included liquid–liquid extraction (Fan et al., 2009; Long et al., 2011; López-Jiménez et al., 2010), solid-phase extraction (SPE) (Qi et al., 2011), matrix solid phase dispersion (MSPD) (Enríquez-Gabeiras et al., 2012), molecularly imprinted solid-phase extraction (Yan et al., 2012), gel permeation chromatography (GPC) clean-up (Mazzetti et al., 2004; Pardo et al., 2009; Sun et al., 2007), and supercritical fluid extraction (Ávila, Zougagh, Escarpa, & Ríos, 2011), etc. Among the clean-up methods, GPC has been proven to be a universal clean-up procedure in many analytical methods, and has been used to clean co-extracted molecular interferences based on the difference in molecular size between co-extractives and target compounds (USEPA, 1994). We also found that GPC was a very useful clean-up method for oil-soluble synthetic dyes in foodstuffs (Zhu, Li, Gong, Zhou, & Li, 2007). The objective of this study was to establish a HPLC method with GPC clean-up procedure for the simultaneous determination of 14 oil-soluble synthetic colourants in pepper-containing products.
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The chilli product samples including dry peppers, chilli powders, chilli oils, chilli hot pot condiments, chilli sauces, etc. were obtained from the local markets. 2.2. Standards Sudan I (purity P 97.5%), Sudan II (purity P 90.0%), Sudan III (purity P 97.0%), Sudan IV (purity P 91.0%), Sudan red G (purity P 93.5%), Sudan orange G (purity P 73.0%), Sudan red B (purity P 90.5%), Sudan red 7B (purity P 91.0%), Para red (purity P 95.5%), Butter yellow (purity P 99.0%), Sudan blue 2 (purity P 99.0%), Toluidine red (purity P 68.0%) were purchased from the Dr. Ehrenstorfer GmbH Company (Augsburg, Germany), Citrus red 2 (purity P 90.0%) was from FMC Chemicals (Philadelphia, USA), Scarlet pigment powder (purity P 95.0%) was from Chongqing standard material Science & Technology Co. Ltd. (Chongqing, China). Individual stock solutions of each compounds (0.1 mg/mL) were prepared in acetone and stored at 18 °C in the dark. Working standard solutions were prepared by mixing desired volume of individual stock standard solutions and serially diluting to different levels with acetone. These solutions were stored at 4 °C in the dark. 2.3. Instrument An automated equipment for GPC, LC-tech GPC ULTRA Automated Sample Clean-up System equipped with Autosampler and Automated Concentrator (Dorfen, Germany) was used. Common glass column (250 mm 25 mm i.d.) packed with 50 g 200–400 mesh Bio-Breads S-X3 resin (Bio-Rad Laboratories GmbH, München) was used for GPC. The HPLC system was Ultimate 3000 (ThermoFisher, USA) equipped with Ultimate 3000 pump, Ultimate 3000 autosampler, Ultimate 3000 Diode Array Detector. Chromatography was performed on an Agilent XDB C-18 column (250 mm 4.6 mm i.d., 5 lm, Agilent Technologies Com., USA) protected by a guard column (C18, 4.0 mm 3.0 mm i.d. Phenomenex, Torrance, CA, USA). 2.4. Extraction A 5 g of sample was weighed into a 50 mL centrifuge tube and 20 mL of acetone-hexane (1:3, v/v) was added, then the sample was thoroughly mixed and extracted under ultrasonic assistance for 15 min. The extracts were centrifuged at 4000 rpm for 10 min and the upper organic layer was transferred into a heart-shape flask. The pellet was re-extracted with 20 mL acetone–hexane (1:3, v/v), and the extracts were combined in the same flask and condensed to dryness using a rotary vacuum evaporator at 45 °C. The residues were dissolved in 10 mL ethyl acetate–cyclohexane (50:50, v/v), and filtered into the glass sample tubes with microfilms. Then the tubes were placed in GPC sample tray for further clean-up. For high-fat chilli oil and chilli hotpot condiment, 2.5 g of sample was weighed and extracted as the procedures above.
2. Materials and methods 2.5. Clean-up 2.1. Chemical and materials HPLC-grade methanol and acetonitrile were obtained from Merk (Darmstadt, Germany). Formic acid (purity P 88%), ethyl acetate and cyclohexane were of analytical reagent grade from Kelong (Chengdu, China). The water was purified and deionized by a water purify system (Sartorius ariumÒ pro UV, Germany). The solvents for HPLC were filtered by 0.22 lm nylon membrane (Jinteng, China) and degassed in an ultrasonic bath.
The 10 mL of sample extracts were injected into the GPC system. The elution was carried out with a mixture of ethyl acetate–cyclohexane (50:50, v/v) at a flow rate of 5 mL/min. The elution fraction in 26–36 min was collected. The collected solutions were evaporated to dryness at 45 °C. Dry residues are dissolved with 1.0 mL acetone, and mix well by a vortex shaker, the final solution was passed through 0.22 lm nylon membrane filter, and waited for HPLC analysis.
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2.6. Hplc An Agilent XDB C18 column was used. The mobile phase system consisted of solution A (0.1% formic acid aqueous solution) and B (methanol–ACN (50:50, v/v)). A gradient program was used for elution: 70–95% solution B at 0–30 min, 95–100% solution B at 30–40 min, 100–70% B at 40–41 min, 70% B at 41–46 min. The flow rate was set at 1 mL/min while the column temperature was maintained at 30 °C. The injection volume was 10 lL. The DAD was programmed to monitor the colourants at a range of 190–700 nm. The determination of each substance was conducted at the appropriate absorbance wavelengths. A total of four integration wavelengths were selected to obtain the better sensibility for each compound. The variable wavelength detector was set at 450 nm (for Sudan yellow, Sudan orange G), 490 nm (for Para red, Citrus red 2, Toluidine red, Sudan red G, Scarlet pigment powder, Sudan I, Sudan II), 520 nm (for Sudan III, Sudan IV, Sudan red B, and Sudan red 7B), and 620 nm (Sudan blue 2). The chromatographic peak Identification was performed by matching the retention time and absorption spectra with the solution of standards. The analyte shall elute at the retention time that is typical for the corresponding calibration standard under the same experimental conditions. The retention time of the analyte shall be the same as that of the calibration standard at a tolerance of ±2.5%. The absorption maxima in the spectrum of the analyte shall be at the same wavelengths as those of the calibration standard within a margin of ±2 nm. The spectrum of the analyte above 220 nm shall, for those parts of the two spectra with a relative absorbance >10%, not be visibly different from the spectrum of the calibration standard. This criterion is met when firstly the same maxima are present and secondly when the difference between the two spectra is at no point observed greater than 10% of the absorbance of the calibration standard (European Commission, 2002).
3. Results and discussion 3.1. Optimisation of HPLC separation It was often necessary to achieve a satisfactory separation of the analytes when a non-MS detector was used for HPLC method. The commonly used HPLC mobile phase for separation of fat-soluble dyes were acetonitrile or methanol containing 0.1% formic acid. We ever used methanol–acetone (9:1, v/v) containing 0.1% formic acid as mobile phase, and obtain efficient separation of 8 fat-soluble synthetic dyes, these dyes contain Para red, Sudan I–IV, Sudan red G, Sudan red B, Sudan red 7B, Sudan red G (Zhu et al., 2007). Sun et al. (2007) reported the good separation of 10 azo-dyes (Para
80.0
red, Sudan I–IV, Sudan red G, Sudan red B, Sudan red 7B, Sudan orange G, and Butter yellow) with methanol containing 0.1% formic acid and 0.1% formic acid aqueous solution as the mobile phase system under the gradient elution condition. But it could be seen from the chromatograms that the baseline separation of some compounds were not achieved, e.g. the separation between Butter yellow and Para red, Sudan red B and Sudan IV. Liu et al. (2011) reported simultaneous determination of fifteen illegal dyes in animal feeds and poultry products by UHPLC–MS/MS. Under the optimal UHPLC–MS/MS conditions, the authors stated that a satisfactory separation of the 15 dyes was achieved, but the study only involved in 11 fat-soluble dyes and some dyes were not well separated, e. g. the peaks of Sudan red 7B, Sudan red B, and Sudan IV overlapped. We achieved an ideal separation of 14 dyes with methanol–acetonitrile (1:1, v/v) and 0.1% formic acid aqueous solution as the mobile phase system under the gradient elution condition described in Section 2. The HPLC chromatogram of the 14 fat-soluble dyes are shown in Fig. 1. 3.2. The extraction of target analytes Common organic solvents (n-hexane, methanol, dichloromethane, ethyl acetate, acetonitrile, acetone, etc.) were investigated as extractant for the dyes from sample, respectively. All the organic solvents and their different mixture were found to be efficient for extraction of most fat-soluble synthetic dyes. In this study, the acetone-hexane mixture was chosen for simultaneous extraction of the 14 dyes. It was found that all the studied dyes have a relatively good solubility in acetone. For the high-fat samples, hexane was a common extraction solvent. In order to increase extraction efficiency, ultrasound-assisted extraction was introduced. We observed the extraction efficiency of the chosen extractant for the 14 dyes in representative chilli powder, chilli sauce, chilli oil, and chilli hot pot condiments. The extraction efficiency was assessed by the colour changes of extracted samples spiked with different concentrations of analytes. It could be seen with naked eyes that the colour of the extracted sample pellets was almost completely faded, which suggested an almost complete extraction of the colourants into extraction solvent. 3.3. Clean-up efficiency of GPC GPC has been proved a very useful method for clean-up of large molecular interferences from target compounds. The main interference compounds in chilli samples were large natural pigments and lipophilic materials. We found GPC could efficiently remove natural pigments and lipophilic materials in chilli samples. We injected 5 mL of mixed standard dilution (2 lg/mL for each analyte) and
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Fig. 1. HPLC chromatograph of 14-dye mixed standard (1) 1st peak of Sudan orange G; (2) Butter yellow; (3) Para red; (4) 2nd peak of Sudan orange G; (5) Citrus red 2; (6) Sudan red G; (7) Toluidine red; (8) Sudan I; (9) Scarlet pigment powder; (10) Sudan II; (11) Sudan blue 2; (12) Sudan III; (13) Sudan red 7B; (14) Sudan red B and (15) Sudan IV.
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Fig. 2. HPLC chromatographs of chilli power sample spiked and unspiked with 14-dye mixed standard. (A) Chilli powder sample unspiked with dyes mixed standard after GPC clean-up and (B) Chilli powder sample spiked with 14-dye mixed standard after GPC clean-up. (1) Butter yellow; (2) Para red; (3) 2nd peak of Sudan orange G; (4) Citrus red 2; (5) Sudan red G; (6) Toluidine red; (7) Sudan I; (8) Scarlet pigment powder; (9) Sudan II; (10) Sudan blue 2; (11) Sudan III; (12) Sudan red 7B; (13) Sudan red B and (14) Sudan IV. The detection wavelengths were 450, 490, 520, and 600 nm.
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Fig. 3. HPLC chromatographs of Sudan orange G standard before and after GPC clean-up. (A) Sudan orange G without GPC clean-up; (B) the discarded fraction of Sudan orange G after GPC clean-up and (C) The collected fraction of Sudan orange G after GPC clean-up. (1) 1st peak of Sudan orange G; (2) 2nd peak of Sudan orange G and (3) 3rd peak of Sudan orange G.
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Table 2 Analytical quality parameters of 14 fat-soluble dyes by the proposed chromatographic method. Compound
Sudan yellow Para red Sudan orange G Citrus red 2 Sudan red G Toluidine red Sudan I Scarlet pigment powder Sudan II Sudan blue 2 Sudan III Sudan red 7B Sudan red B Sudan IV
Calibration range (mg/L)
Correlation coefficient (R2)
LOD (lg/kg) Chilli powder, chilli sauce
Chilli oil
0.2–5.0 0.1–2.5 0.2–5.0 0.1–2.5 0.1–2.5 0.1–2.5 0.1–2.5 0.1–2.5 0.1–2.5 0.1–2.5 0.1–2.5 0.1–2.5 0.1–2.5 0.1–2.5
0.998 0.999 0.995 0.999 0.999 0.998 0.999 0.998 0.999 0.997 0.999 0.999 0.996 0.998
13 11 31 27 22 15 13 19 13 36 10 15 11 11
25 22 62 55 44 30 26 38 26 71 19 30 22 22
cate the existence of Sudan orange G. Fig. 3 showed the HPLC chromatograph of Sudan orange G before and after GPC clean-up. It could be seen that the first peak mainly correspond to the discarded fraction and the second peak to the collected fraction. It was also found that an obvious peak (3rd peak) appeared in the discarded fraction, but not in collected fraction. The 3rd peak may be the transformed product of 1st peak of Sudan orange G. 3.4. Analytical performance
the extracts of chilli sample into the GPC system respectively and eluted GPC column with ethyl acetate–cyclohexane (50:50, v/v) at a flow rate of 5 mL/min. it could be seen with the naked eye that the synthetic colour band was eluted between 27 min and 32 min, while natural pigment band was completely eluted in 26 min. HPLC chromatograph (detected at 450, 490, 520, and 600 nm) for chilli power sample spiked with 14 dye mixed standard was shown in Fig. 2. No interferences were observed in corresponding retention times of target compounds by comparing chromatograms of spiked sample and blank sample. It could be seen from Fig. 2 that first peak of Sudan orange G disappeared after GPC clean-up, while the second peak was still present, which suggested that Sudan orange G was not well recovered after GPC. In previous studies, several authors observed multiple chromatographic peaks of Sudan orange G (Noguerol-Cal et al., 2008; Sun et al., 2007). Noguerol-Cal et al. (2008) further observed the UV-vis spectrum and investigated the nature of multiple chromatographic peaks of Sudan orange G using HPLC–ESI(+)-MS/MS method. It was found that the second peak’s mass spectrum do not correspond at the dye interest. The authors attributed the second peak to synthesis by-product. So when GPC was used for clean-up protocol, it was the impurity rather than the dye itself of Sudan orange G recovered. In view of the fact that presence and well separation of the impurity of Sudan arrange G from other dyes existed, we used the second peak of Sudan orange G to indi-
Under the selected conditions, some parameters such as linearity and limits of detection (LOD) of the proposed method were investigated. Linearity was studied by analysing mixed standard solution of dyes at several concentrations ranging from 0.05 to 10.0 mg/L. According to the values of the linear correlation coefficients for the calibration curves all the compounds showed good correlation (R2 P 0.995). The LOD based on signal-to-noise ratio of 3 (S/N = 3) were in the range of 11–36 lg/kg for chilli powder, chilli sauce and 22–71 lg/kg for chilli oil. Table 2 summarizes the analytical data. The LOD was believed to be much lower than the concentration of the unlawful dyes usually applied to effectively amend the colour content and colouring capacity of bad quality processed chilli products. So the LOD could meet requirement for the analysis of actual samples. EU ever announced the LOD for Sudan dyes is in the range of 0.5 and 1.0 mg/kg (0.5–1.0 ppm) using HPLC method (ASTA, 2005) and any food containing dyes above those limits must be withdrawn from the market. At present, there were not a established maximal residue level (MRL) of Sudan dyes and other banned dyes in foodstuffs worldwide. In China, the banned dyes should not be detected in foodstuffs, any foodstuff contaminated with banned dyes was thought to be unlawful. Recovery and precision were investigated on spiked samples at three levels. Dyes were spiked in chilli powder, chilli sauce at 200, 400, and 2000 lg/kg, chilli oil at 400, 800, and 4000 lg/kg. Six replicates were tested for each concentration, two replicates on each day with 3 consecutive days under repeatability conditions, with intra-day and inter-day assays combined. Spike levels, spike recoveries, and coefficients of variation were summarised in Table 3. The results showed that the recoveries ranged from 73.4% to 103.5% and coefficients of variation from 3.7% to 12.3%. The results demonstrated that the recovery and precision of the present method were basically satisfied with criterion on quality control of laboratories for chemical testing of food and could be used for routine monitoring purposes. For the determination of banned dyes in our study, the sample analysis was repeated when dyes
Table 3 Percentage recoveries and relative standard deviations of the dyes after clean-up in different matrices (n = 6). Compounds
Recovery (RSD) (%) Chilli powder
Sudan orange G Sudan yellow Para red Citrus red 2 Sudan red G Toluidine red Sudan I Scarlet pigment powder Sudan II Sudan blue 2 Sudan III Sudan red 7B Sudan red B Sudan IV
Chilli sauce
Chilli oil
200 lg/kg
400 lg/kg
2000 lg/kg
200 lg/kg
400 lg/kg
2000 lg/kg
400 lg/kg
800 lg/kg
4000 lg/kg
85.3 94.1 86.6 75.6 82.3 79.8 91.1 89.3 92.7 85.3 92.3 83.1 87.2 94.2
86.7 95.7 89.3 82.1 80.3 83.7 92.5 87.4 95.3 86.2 94.7 95.3 93.5 95.6
96.8 94.5 91.7 88.3 87.6 85.4 88.6 88.2 91.3 92.1 89.4 92.9 96.3 97.6
79.3 86.5 79.3 82.1 86.5 75.6 83.1 80.1 88.3 84.4 83.6 84.5 87.3 92.3
84.3 92.1 87.6 82.4 79.3 78.4 81.6 86.2 79.4 73.4 86.5 88.7 91.3 87.5
92.1 (7.6) 89.3 (6.5) 102.3 (7.3) 90.2 (6.5) 76.4 (4.6) 92.1 (3.7) 93.6 (7.7) 99.2 (5.4) 78.4 (4.6) 78.7 (7.7) 91.2 (8.5) 92.3 (4.6) 90.2 (6.3) 92.4 (6.1)
86.1 92.3 87.3 82.4 75.3 76.4 89.9 75.3 80.2 87.9 86.3 91.2 89.2 85.6
90.2 97.4 87.2 87.7 87.1 77.5 92.8 76.6 96.2 90.3 91.6 88.3 78.4 89.7
93.7 (7.5) 103.5 (8.4) 92.6 (7.4) 79.4 (10.3) 91.6 (6.3) 90.3 (7.6) 90.1 (8.8) 86.7 (5.6) 93.4 (7.4) 89.8 (5.5) 90.1 (7.7) 92.1 (7.5) 91.7 (6.2) 97.5 (8.8)
(10.7) (8.3) (7.7) (12.1) (9.2) (10.2) (9.8) (10.5) (10.1) (12.3) (9.4) (10.6) (7.5) (9.6)
(5.6) (7.2) (6.3) (10.2) (11.2) (9.9) (11.5) (8.7) (7.7) (9.7) (8.3) (10.3) (5.4) (8.6)
(4.5) (6.5) (5.6) (7.3) (6.7) (8.4) (7.3) (6.5) (6.3) (7.3) (7.3) (6.3) (4.5) (8.3)
(11.5) (10.6) (8.7) (7.6) (10.9) (12.5) (8.2) (7.5) (8.7) (12.2) (9.5) (6.7) (11.6) (12.3)
(8.2) (10.2) (8.8) (5.3) (8.6) (10.3) (7.8) (8.7) (6.5) (10.3) (7.7) (6.8) (9.7) (10.3)
(11.2) (12.3) (9.7) (10.1) (7.5) (6.6) (10.3) (6.7) (8.8) (10.3) (5.7) (10.8) (7.3) (9.8)
(10.6) (10.0) (8.9) (9.2) (6.7) (10.3) (6.7) (7.9) (5.8) (9.4) (6.9) (8.1) (7.6) (6.6)
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Fig. 4. HPLC analysis of positive sample containing Scarlet pigment powder.
were detected. During the analysis of samples, the recovery was also determined for further assessing the effectiveness of the method. In addition, standard addition method or an internal standard could also be used for the quantitative determination of the target analyte in the sample when recovery was not satisfactory. 3.5. Sample analysis The more than 100 samples of different matrices have been analysed using the presented method in routine work between April 2010 and November 2012. In them the two positive chilli sauce samples were detected. Both of the samples were contaminated with Scarlet pigment powder. Fig. 4 illustrates the presence of illegal dyes in one of the positive samples. For the other chilli samples, target analytes had not been detected. Because method based on LC analysis with full-scan DAD detection could be used as confirmatory method (European Commission, 2002), the method developed in our study could be a suitable confirmatory method. For more confirmation, the measuring technique based on LC analysis with mass-spectrometric could be used as a complementary confirmation for the detected analytes. 4. Conclusions A HPLC method for the simultaneous detection of 14 fat-soluble dyes in chilli products was developed by using GPC clean-up procedure. An optimised chromatographic condition was chosen for good separation of the studied dyes. The GPC clean-up could efficiently remove large molecular lipids and natural pigments interferences from samples. The method has a good repeatability and high accuracy with satisfactory detection limits and has been successfully used for the determination of banned dyes in real samples. Acknowledgements This research was financed by the Scientific & Research Project of Scientific Committee of Chongqing under Project No. cstc2012gg-yyjs80028, and Scientific & Research Project of General Administration of Quality Supervision, Inspection and Quarantine (AQSIQ) of China under Project No. 2012QK374. References American Spice Trade Association (ASTA). (2005). Sudan red and related dyes – white paper. . Ávila, M., Zougagh, M., Escarpa, A., & Ríos, Á. (2011). Determination of Sudan dyes in food samples using supercritical fluid extraction–capillary liquid chromatography. The Journal of Supercritical Fluids, 55, 977–982.
Calbiani, F., Careri, M., Elviri, L., Mangia, A., Pistarà, L., & Zagnoni, I. (2004). Development and in-house validation of a liquid chromatography– electrospray-tandem mass spectrometry method for the simultaneous determination of Sudan I, Sudan II, Sudan III and Sudan IV in hot chilli products. Journal of Chromatography A, 1042, 123–130. Commission Decision of 20 June 2003 on emergency measures regarding hot chilli and hot chilli products, notified under document number C. (2003) 1970, 2003/ 460/EC, Official Journal of the European Union, L. 154/114, 21.6.2003. Commission decision of 23 May 2005 on emergency measures regarding chilli, chilli products, curcuma and palm oil notified under document number C. (2005) 1454, 2005/402/EC, Official Journal of the European Union, L. 135/34, 28.5.2005. Enríquez-Gabeiras, L., Gallego, A., Garcinuño, A. M., Fernández-Hernando, P., & Durand, J. S. (2012). Interference-free determination of illegal dyes in sausages and condiments by matrix solid phase dispersion (MSPD) and liquid chromatography (HPLC–DAD). Food Chemistry, 135, 193–198. Ertasß, E., Özer, H., & Alasalvar, C. (2007). A rapid HPLC method for determination of Sudan dyes and Para red in red chilli pepper. Food Chemistry, 105, 756–760. European Commission (2002). European Commission Decision 2002/657/EC. Official Journal of European Community, 221, 8–36. Fan, Y., Chen, M., Chao, S., El-Sepai, F., Wang, K., Zhu, Y., & Ye, M. (2009). Ionic liquids extraction of Para red and Sudan dyes from chilli powder, chilli oil and food additive combined with high performance liquid chromatography. Analytica Chimica Acta, 650, 65–69. Liu, R., Hei, W., He, P., & Li, Z. (2011). Simultaneous determination of fifteen illegal dyes in animal feeds and poultry products by ultra-high performance liquid chromatography-tandem mass spectrometry. Journal of Chromatography B, 879, 2416–2422. Long, C., Mai, Z., Yang, X., Zhu, B., Xu, X., Huang, X., & Zou, X. (2011). A new liquid– liquid extraction method for determination of 6 azo-dyes in chilli products by high-performance liquid chromatography. Food Chemistry, 126, 1324–1329. López-Jiménez, F. J., Rubio, S., & Pérez-Bendito, D. (2010). Supramolecular solventbased microextraction of Sudan dyes in chilli-containing foodstuffs prior to their liquid chromatography–photodiode array determination. Food Chemistry, 121, 763–769. Mazzetti, M., Fascioli, R., Mazzoncini, I., Spinelli, G., Morelli, I., & Bertoli, A. (2004). Determination of 1-phenylazo-2-naphthol (Sudan I) in chilli powder and in chilli-containing food products by GPC clean-up and HPLC with LC/MS confirmation. Food Additives & Contaminants, 21, 935–941. Murty, M. R. V. S., Sridhara Chary, N., Prabhakar, S., Prasada Raju, N., & Vairamani, M. (2009). Simultaneous quantitative determination of Sudan dyes using liquid chromatography–atmospheric pressure photoionization–tandem mass spectrometry. Food Chemistry, 115, 1556–1562. Noguerol-Cal, R., López-Vilarinˇo, J. M., Fernández-Martínez, G., Barral-Losada, L., & González-Rodríguez, M. V. (2008). High-performance liquid chromatography analysis of ten dyes for control of safety of commercial articles. Journal of Chromatography A, 1179, 152–160. Pardo, O., Yusà, V., León, N., & Pastor, A. (2009). Development of a method for the analysis of seven banned azo-dyes in chilli and hot chilli food samples by pressurised liquid extraction and liquid chromatography with electrospray ionization-tandem mass spectrometry. Talanta, 78, 178–186. Part 74 – Listing of color additives subject to certification, §74.302 Citrus red No. 2. Title 21, code of federal regulations, US. Qi, P., Zeng, T., Wen, Z., Liang, X., & Zhang, X. (2011). Interference-free simultaneous determination of Sudan dyes in chilli foods using solid phase extraction coupled with HPLC–DAD. Food Chemistry, 125, 1462–1467. Rebane, R., Leito, I., Yurchenko, S., & Herodes, K. (2010). A review of analytical techniques for determination of Sudan I–IV dyes in food matrixes. Journal of Chromatography A, 1217, 2747–2757. Sun, H., Wang, F., & Ai, L. (2007). Determination of banned 10 azo-dyes in hot chilli products by gel permeation chromatography–liquid chromatography– electrospray ionization–tandem mass spectrometry. Journal of Chromatography A, 1164, 120–128. USEPA SW-846 method 3640A, Gel-permeation clean-up. Revision 1, September 1994.
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Yan, H., Qiao, J., Pei, Y., Long, T., Ding, W., & Xie, K. (2012). Molecularly imprinted solid-phase extraction coupled to liquid chromatography for determination of Sudan dyes in preserved beancurds. Food Chemistry, 132, 649–654. Zhu, Y., Li, G., Gong, Y., Zhou, Z., & Li, L. (2007). Simultaneous determination of eight non-edible red dyes in chilli products by high performance liquid chromatography with a clean-up procedure by gel column. China Condiment, 32, 58–61.
Zhu, Y., Zhang, Q., Li, G., Li, N., Zhong, W., Wang, M., & Xiao, Z. (2011). Screening and detection of pigment scarlet powder in chilli products. China Condiment, 36, 100–104.
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Analytical Methods
Simultaneous determination of 14 oil-soluble synthetic dyes in chilli products by high performance liquid chromatography with a gel permeation chromatography clean-up procedure Yonghong Zhu ⇑, Bo Zhao, Ruiqi Xiao, Wen Yun, Zhaojing Xiao, Dawei Tu, Shiqi Chen Center for Quality Supervision & Inspection of Food, Chongqing Academy of Metrology and Quality Inspection, Chongqing Engineering Research Center for Food Safety, Chongqing 401123, China
a r t i c l e
i n f o
Article history: Received 1 April 2013 Received in revised form 17 August 2013 Accepted 2 September 2013 Available online 11 September 2013 Keywords: High performance liquid chromatography Synthetic dyes Gel permeation chromatography Chilli products
a b s t r a c t A method was developed for the simultaneous determination of 14 fat-soluble dyes in chilli products. The samples were extracted with hexane/acetone. The cleanup was performed with gel permeation chromatography (GPC) cleanup system. A HPLC separation was performed using variable wavelength detector and a gradient elution with 0.1% formic acid and methanol–acetonitrile (1:1, v/v) as the mobile phases. Good linearity (R2 P 0.995) was observed between 0.1 and 5.0 lg/mL. Detection limits of the investigated dyes, which were evaluated at signal to noise ratio of 3, were in the ranges of 11–71 lg/kg. The recoveries of the 14 synthetic colourants in three matrices ranged from 73.4% to 103.5%. Relative standard deviations ranged from 3.7% to 12.3%. The method has been successfully used for the determination of banned dyes in real samples. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Food quality is closely associated with colour and the use of food colourants has been an age-old practice, enhancing the aesthetical appeal of foods. The use of synthetic organic dyes has been recognised as the most reliable and economical method of restoring or providing colour to a processed product. Generally, synthetic colourants can be classified into water-soluble and fatsoluble colourants based on their solubility. At present, the most synthetic colourants permitted for use in foods are water-soluble, while most fat-soluble synthetic colourants were non-edible colourants and their use in foodstuffs was prohibited. The common non-edible colourants ever reported are some azocompounds, such as Sudan orange G, Para red, Sudan I, Sudan II, Sudan III, and Sudan IV, Sudan yellow, Sudan red G, Sudan red B, and Sudan red 7B (Noguerol-Cal, López-Vilarinˇo, Fernández-Martínez, Barral-Losada, & González-Rodríguez, 2008; Pardo, Yusà, León, & Pastor, 2009; Sun, Wang, & Ai, 2007). In addition, we found an illegally added synthetic colourant in chilli products not long ago. The colourant was identified as Scarlet pigment powder, which was also a fat-soluble azo-dyes (Zhu et al., 2011). Besides above colourants, Citrus red 2, Toluidine red, and Sudan blue 2 were also commonly seen fat-soluble synthetic colourants (Liu, Hei, He, & Li, 2011; Noguerol-Cal et al., 2008; Part 74 of 21 ⇑ Corresponding author. Tel./fax: +86 23 89136317. E-mail addresses: [email protected], [email protected] (Y. Zhu). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.09.008
CFR, US). There is possibility that they could also be added into foodstuffs. Among above colourants, Citrus red 2 could be used as food dyes, but it shall be used only for colouring the skins of oranges that are not intended or used for processing (or if so used are designated in the trade as Packinghouse elimination). In US, oranges coloured with Citrus red No. 2 shall bear not more than 2.0 parts per million of such colour additive, calculated on the basis of the weight of the whole fruit (Part 74 of 21 CFR, US). The use of other fat-soluble colourants in foods is prohibited worldwide, and the prohibited colourants should not be present in foods. In EU, decisions on emergency measures concerning at Sudan I, Sudan II, Sudan III, and Sudan IV were adopted in June 2003 and May 2005 (Commission Decision, 2003, 2005). In order to prevent the possible unlawful addition of above fatsoluble synthetic colourants into foodstuffs, it is necessary to develop accurate and reliable analytical methods for those colourants in foodstuffs. Until now, there was not a method for simultaneous determination of the above 14 dyes. The chemical information of the 14 dyes is given in Table 1. Many analytical methods were reported for fat-soluble synthetic colourants, especially azo-dyes analysis in foodstuffs (Rebane, Leito, Yurchenko, & Herodes, 2010). Among the reported methods, high performance liquid chromatography (HPLC) coupled with different types of detector including mass spectrometric detector (MSD) (Calbiani et al., 2004; Liu et al., 2011; Murty, Chary, Prabhakar, Raju, & Vairamani, 2009; Pardo et al., 2009; Sun, et al., 2007), ultraviolet-visible (UV-vis) detector (Ertasß, Özer, &
Y. Zhu et al. / Food Chemistry 145 (2014) 956–962 Table 1 Chemical information about studied dyes. No.
Name
CAS number
CI number
Molecular formula
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Sudan orange G Sudan yellow Para red Citrus red 2 Sudan red G Toluidine red Sudan I Scarlet pigment powder Sudan II Sudan blue 2 Sudan III Sudan red 7B Sudan red B Sudan IV
2051-85-6 60-11-7 6410-10-2 6358-53-8 1229-55-6 2425-85-6 842-07-9 3789-75-1 3118-97-6 17354-14-2 85-86-9 6368-72-5 3176-79-2 85-83-6
11920 11020 12070 12156 12150 12120 12055 / 12140 61554 26100 26050 26110 26105
C12H10N2O2 C14H15N3 C16H11N3O3 C18H16N2O3 C17H14N2O2 C17H13N3O3 C16H12N2O C23H17N3O2 C18H16N2O C22H26N2O2 C22H16N4O C24H21N5 C24H20N4O C24H20N4O
Alasalvar, 2007; Fan et al., 2009; Long et al., 2011) and photodiode array detector (DAD) (López-Jiménez, Rubio, & Pérez-Bendito, 2010; Qi, Zeng, Wen, Liang, & Zhang, 2011; Enríquez-Gabeiras, Gallego, Garcinuño, Fernández-Hernando, & Durand, 2012) was widely used. With increasing demand for the determination of multiple dyes present in food, a high throughput method which could simultaneously determine multiple dyes was needed. Among the high throughput analytic methods, liquid chromatography tandem mass spectrometry (LC/MS/MS) based methods are more and more widely used. Because of their high sensitivity and selectivity, LC–MS based methods provide the capability of multi-component validation, quantification and microanalysis. But in ordinary labs, LC–MS are often not available. So a high throughput analytic method based on HPLC coupled with UV-vis detector or DAD could have a more practical application in routine analysis. Whichever method is used, sample pretreatment procedure was prerequisite. After solvent extraction of the analytes in foods, a further clean-up procedure was often necessary. The reported clean-up procedure included liquid–liquid extraction (Fan et al., 2009; Long et al., 2011; López-Jiménez et al., 2010), solid-phase extraction (SPE) (Qi et al., 2011), matrix solid phase dispersion (MSPD) (Enríquez-Gabeiras et al., 2012), molecularly imprinted solid-phase extraction (Yan et al., 2012), gel permeation chromatography (GPC) clean-up (Mazzetti et al., 2004; Pardo et al., 2009; Sun et al., 2007), and supercritical fluid extraction (Ávila, Zougagh, Escarpa, & Ríos, 2011), etc. Among the clean-up methods, GPC has been proven to be a universal clean-up procedure in many analytical methods, and has been used to clean co-extracted molecular interferences based on the difference in molecular size between co-extractives and target compounds (USEPA, 1994). We also found that GPC was a very useful clean-up method for oil-soluble synthetic dyes in foodstuffs (Zhu, Li, Gong, Zhou, & Li, 2007). The objective of this study was to establish a HPLC method with GPC clean-up procedure for the simultaneous determination of 14 oil-soluble synthetic colourants in pepper-containing products.
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The chilli product samples including dry peppers, chilli powders, chilli oils, chilli hot pot condiments, chilli sauces, etc. were obtained from the local markets. 2.2. Standards Sudan I (purity P 97.5%), Sudan II (purity P 90.0%), Sudan III (purity P 97.0%), Sudan IV (purity P 91.0%), Sudan red G (purity P 93.5%), Sudan orange G (purity P 73.0%), Sudan red B (purity P 90.5%), Sudan red 7B (purity P 91.0%), Para red (purity P 95.5%), Butter yellow (purity P 99.0%), Sudan blue 2 (purity P 99.0%), Toluidine red (purity P 68.0%) were purchased from the Dr. Ehrenstorfer GmbH Company (Augsburg, Germany), Citrus red 2 (purity P 90.0%) was from FMC Chemicals (Philadelphia, USA), Scarlet pigment powder (purity P 95.0%) was from Chongqing standard material Science & Technology Co. Ltd. (Chongqing, China). Individual stock solutions of each compounds (0.1 mg/mL) were prepared in acetone and stored at 18 °C in the dark. Working standard solutions were prepared by mixing desired volume of individual stock standard solutions and serially diluting to different levels with acetone. These solutions were stored at 4 °C in the dark. 2.3. Instrument An automated equipment for GPC, LC-tech GPC ULTRA Automated Sample Clean-up System equipped with Autosampler and Automated Concentrator (Dorfen, Germany) was used. Common glass column (250 mm 25 mm i.d.) packed with 50 g 200–400 mesh Bio-Breads S-X3 resin (Bio-Rad Laboratories GmbH, München) was used for GPC. The HPLC system was Ultimate 3000 (ThermoFisher, USA) equipped with Ultimate 3000 pump, Ultimate 3000 autosampler, Ultimate 3000 Diode Array Detector. Chromatography was performed on an Agilent XDB C-18 column (250 mm 4.6 mm i.d., 5 lm, Agilent Technologies Com., USA) protected by a guard column (C18, 4.0 mm 3.0 mm i.d. Phenomenex, Torrance, CA, USA). 2.4. Extraction A 5 g of sample was weighed into a 50 mL centrifuge tube and 20 mL of acetone-hexane (1:3, v/v) was added, then the sample was thoroughly mixed and extracted under ultrasonic assistance for 15 min. The extracts were centrifuged at 4000 rpm for 10 min and the upper organic layer was transferred into a heart-shape flask. The pellet was re-extracted with 20 mL acetone–hexane (1:3, v/v), and the extracts were combined in the same flask and condensed to dryness using a rotary vacuum evaporator at 45 °C. The residues were dissolved in 10 mL ethyl acetate–cyclohexane (50:50, v/v), and filtered into the glass sample tubes with microfilms. Then the tubes were placed in GPC sample tray for further clean-up. For high-fat chilli oil and chilli hotpot condiment, 2.5 g of sample was weighed and extracted as the procedures above.
2. Materials and methods 2.5. Clean-up 2.1. Chemical and materials HPLC-grade methanol and acetonitrile were obtained from Merk (Darmstadt, Germany). Formic acid (purity P 88%), ethyl acetate and cyclohexane were of analytical reagent grade from Kelong (Chengdu, China). The water was purified and deionized by a water purify system (Sartorius ariumÒ pro UV, Germany). The solvents for HPLC were filtered by 0.22 lm nylon membrane (Jinteng, China) and degassed in an ultrasonic bath.
The 10 mL of sample extracts were injected into the GPC system. The elution was carried out with a mixture of ethyl acetate–cyclohexane (50:50, v/v) at a flow rate of 5 mL/min. The elution fraction in 26–36 min was collected. The collected solutions were evaporated to dryness at 45 °C. Dry residues are dissolved with 1.0 mL acetone, and mix well by a vortex shaker, the final solution was passed through 0.22 lm nylon membrane filter, and waited for HPLC analysis.
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2.6. Hplc An Agilent XDB C18 column was used. The mobile phase system consisted of solution A (0.1% formic acid aqueous solution) and B (methanol–ACN (50:50, v/v)). A gradient program was used for elution: 70–95% solution B at 0–30 min, 95–100% solution B at 30–40 min, 100–70% B at 40–41 min, 70% B at 41–46 min. The flow rate was set at 1 mL/min while the column temperature was maintained at 30 °C. The injection volume was 10 lL. The DAD was programmed to monitor the colourants at a range of 190–700 nm. The determination of each substance was conducted at the appropriate absorbance wavelengths. A total of four integration wavelengths were selected to obtain the better sensibility for each compound. The variable wavelength detector was set at 450 nm (for Sudan yellow, Sudan orange G), 490 nm (for Para red, Citrus red 2, Toluidine red, Sudan red G, Scarlet pigment powder, Sudan I, Sudan II), 520 nm (for Sudan III, Sudan IV, Sudan red B, and Sudan red 7B), and 620 nm (Sudan blue 2). The chromatographic peak Identification was performed by matching the retention time and absorption spectra with the solution of standards. The analyte shall elute at the retention time that is typical for the corresponding calibration standard under the same experimental conditions. The retention time of the analyte shall be the same as that of the calibration standard at a tolerance of ±2.5%. The absorption maxima in the spectrum of the analyte shall be at the same wavelengths as those of the calibration standard within a margin of ±2 nm. The spectrum of the analyte above 220 nm shall, for those parts of the two spectra with a relative absorbance >10%, not be visibly different from the spectrum of the calibration standard. This criterion is met when firstly the same maxima are present and secondly when the difference between the two spectra is at no point observed greater than 10% of the absorbance of the calibration standard (European Commission, 2002).
3. Results and discussion 3.1. Optimisation of HPLC separation It was often necessary to achieve a satisfactory separation of the analytes when a non-MS detector was used for HPLC method. The commonly used HPLC mobile phase for separation of fat-soluble dyes were acetonitrile or methanol containing 0.1% formic acid. We ever used methanol–acetone (9:1, v/v) containing 0.1% formic acid as mobile phase, and obtain efficient separation of 8 fat-soluble synthetic dyes, these dyes contain Para red, Sudan I–IV, Sudan red G, Sudan red B, Sudan red 7B, Sudan red G (Zhu et al., 2007). Sun et al. (2007) reported the good separation of 10 azo-dyes (Para
80.0
red, Sudan I–IV, Sudan red G, Sudan red B, Sudan red 7B, Sudan orange G, and Butter yellow) with methanol containing 0.1% formic acid and 0.1% formic acid aqueous solution as the mobile phase system under the gradient elution condition. But it could be seen from the chromatograms that the baseline separation of some compounds were not achieved, e.g. the separation between Butter yellow and Para red, Sudan red B and Sudan IV. Liu et al. (2011) reported simultaneous determination of fifteen illegal dyes in animal feeds and poultry products by UHPLC–MS/MS. Under the optimal UHPLC–MS/MS conditions, the authors stated that a satisfactory separation of the 15 dyes was achieved, but the study only involved in 11 fat-soluble dyes and some dyes were not well separated, e. g. the peaks of Sudan red 7B, Sudan red B, and Sudan IV overlapped. We achieved an ideal separation of 14 dyes with methanol–acetonitrile (1:1, v/v) and 0.1% formic acid aqueous solution as the mobile phase system under the gradient elution condition described in Section 2. The HPLC chromatogram of the 14 fat-soluble dyes are shown in Fig. 1. 3.2. The extraction of target analytes Common organic solvents (n-hexane, methanol, dichloromethane, ethyl acetate, acetonitrile, acetone, etc.) were investigated as extractant for the dyes from sample, respectively. All the organic solvents and their different mixture were found to be efficient for extraction of most fat-soluble synthetic dyes. In this study, the acetone-hexane mixture was chosen for simultaneous extraction of the 14 dyes. It was found that all the studied dyes have a relatively good solubility in acetone. For the high-fat samples, hexane was a common extraction solvent. In order to increase extraction efficiency, ultrasound-assisted extraction was introduced. We observed the extraction efficiency of the chosen extractant for the 14 dyes in representative chilli powder, chilli sauce, chilli oil, and chilli hot pot condiments. The extraction efficiency was assessed by the colour changes of extracted samples spiked with different concentrations of analytes. It could be seen with naked eyes that the colour of the extracted sample pellets was almost completely faded, which suggested an almost complete extraction of the colourants into extraction solvent. 3.3. Clean-up efficiency of GPC GPC has been proved a very useful method for clean-up of large molecular interferences from target compounds. The main interference compounds in chilli samples were large natural pigments and lipophilic materials. We found GPC could efficiently remove natural pigments and lipophilic materials in chilli samples. We injected 5 mL of mixed standard dilution (2 lg/mL for each analyte) and
1 2 3 4 mAU
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0.0
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Fig. 1. HPLC chromatograph of 14-dye mixed standard (1) 1st peak of Sudan orange G; (2) Butter yellow; (3) Para red; (4) 2nd peak of Sudan orange G; (5) Citrus red 2; (6) Sudan red G; (7) Toluidine red; (8) Sudan I; (9) Scarlet pigment powder; (10) Sudan II; (11) Sudan blue 2; (12) Sudan III; (13) Sudan red 7B; (14) Sudan red B and (15) Sudan IV.
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Fig. 2. HPLC chromatographs of chilli power sample spiked and unspiked with 14-dye mixed standard. (A) Chilli powder sample unspiked with dyes mixed standard after GPC clean-up and (B) Chilli powder sample spiked with 14-dye mixed standard after GPC clean-up. (1) Butter yellow; (2) Para red; (3) 2nd peak of Sudan orange G; (4) Citrus red 2; (5) Sudan red G; (6) Toluidine red; (7) Sudan I; (8) Scarlet pigment powder; (9) Sudan II; (10) Sudan blue 2; (11) Sudan III; (12) Sudan red 7B; (13) Sudan red B and (14) Sudan IV. The detection wavelengths were 450, 490, 520, and 600 nm.
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Fig. 3. HPLC chromatographs of Sudan orange G standard before and after GPC clean-up. (A) Sudan orange G without GPC clean-up; (B) the discarded fraction of Sudan orange G after GPC clean-up and (C) The collected fraction of Sudan orange G after GPC clean-up. (1) 1st peak of Sudan orange G; (2) 2nd peak of Sudan orange G and (3) 3rd peak of Sudan orange G.
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Table 2 Analytical quality parameters of 14 fat-soluble dyes by the proposed chromatographic method. Compound
Sudan yellow Para red Sudan orange G Citrus red 2 Sudan red G Toluidine red Sudan I Scarlet pigment powder Sudan II Sudan blue 2 Sudan III Sudan red 7B Sudan red B Sudan IV
Calibration range (mg/L)
Correlation coefficient (R2)
LOD (lg/kg) Chilli powder, chilli sauce
Chilli oil
0.2–5.0 0.1–2.5 0.2–5.0 0.1–2.5 0.1–2.5 0.1–2.5 0.1–2.5 0.1–2.5 0.1–2.5 0.1–2.5 0.1–2.5 0.1–2.5 0.1–2.5 0.1–2.5
0.998 0.999 0.995 0.999 0.999 0.998 0.999 0.998 0.999 0.997 0.999 0.999 0.996 0.998
13 11 31 27 22 15 13 19 13 36 10 15 11 11
25 22 62 55 44 30 26 38 26 71 19 30 22 22
cate the existence of Sudan orange G. Fig. 3 showed the HPLC chromatograph of Sudan orange G before and after GPC clean-up. It could be seen that the first peak mainly correspond to the discarded fraction and the second peak to the collected fraction. It was also found that an obvious peak (3rd peak) appeared in the discarded fraction, but not in collected fraction. The 3rd peak may be the transformed product of 1st peak of Sudan orange G. 3.4. Analytical performance
the extracts of chilli sample into the GPC system respectively and eluted GPC column with ethyl acetate–cyclohexane (50:50, v/v) at a flow rate of 5 mL/min. it could be seen with the naked eye that the synthetic colour band was eluted between 27 min and 32 min, while natural pigment band was completely eluted in 26 min. HPLC chromatograph (detected at 450, 490, 520, and 600 nm) for chilli power sample spiked with 14 dye mixed standard was shown in Fig. 2. No interferences were observed in corresponding retention times of target compounds by comparing chromatograms of spiked sample and blank sample. It could be seen from Fig. 2 that first peak of Sudan orange G disappeared after GPC clean-up, while the second peak was still present, which suggested that Sudan orange G was not well recovered after GPC. In previous studies, several authors observed multiple chromatographic peaks of Sudan orange G (Noguerol-Cal et al., 2008; Sun et al., 2007). Noguerol-Cal et al. (2008) further observed the UV-vis spectrum and investigated the nature of multiple chromatographic peaks of Sudan orange G using HPLC–ESI(+)-MS/MS method. It was found that the second peak’s mass spectrum do not correspond at the dye interest. The authors attributed the second peak to synthesis by-product. So when GPC was used for clean-up protocol, it was the impurity rather than the dye itself of Sudan orange G recovered. In view of the fact that presence and well separation of the impurity of Sudan arrange G from other dyes existed, we used the second peak of Sudan orange G to indi-
Under the selected conditions, some parameters such as linearity and limits of detection (LOD) of the proposed method were investigated. Linearity was studied by analysing mixed standard solution of dyes at several concentrations ranging from 0.05 to 10.0 mg/L. According to the values of the linear correlation coefficients for the calibration curves all the compounds showed good correlation (R2 P 0.995). The LOD based on signal-to-noise ratio of 3 (S/N = 3) were in the range of 11–36 lg/kg for chilli powder, chilli sauce and 22–71 lg/kg for chilli oil. Table 2 summarizes the analytical data. The LOD was believed to be much lower than the concentration of the unlawful dyes usually applied to effectively amend the colour content and colouring capacity of bad quality processed chilli products. So the LOD could meet requirement for the analysis of actual samples. EU ever announced the LOD for Sudan dyes is in the range of 0.5 and 1.0 mg/kg (0.5–1.0 ppm) using HPLC method (ASTA, 2005) and any food containing dyes above those limits must be withdrawn from the market. At present, there were not a established maximal residue level (MRL) of Sudan dyes and other banned dyes in foodstuffs worldwide. In China, the banned dyes should not be detected in foodstuffs, any foodstuff contaminated with banned dyes was thought to be unlawful. Recovery and precision were investigated on spiked samples at three levels. Dyes were spiked in chilli powder, chilli sauce at 200, 400, and 2000 lg/kg, chilli oil at 400, 800, and 4000 lg/kg. Six replicates were tested for each concentration, two replicates on each day with 3 consecutive days under repeatability conditions, with intra-day and inter-day assays combined. Spike levels, spike recoveries, and coefficients of variation were summarised in Table 3. The results showed that the recoveries ranged from 73.4% to 103.5% and coefficients of variation from 3.7% to 12.3%. The results demonstrated that the recovery and precision of the present method were basically satisfied with criterion on quality control of laboratories for chemical testing of food and could be used for routine monitoring purposes. For the determination of banned dyes in our study, the sample analysis was repeated when dyes
Table 3 Percentage recoveries and relative standard deviations of the dyes after clean-up in different matrices (n = 6). Compounds
Recovery (RSD) (%) Chilli powder
Sudan orange G Sudan yellow Para red Citrus red 2 Sudan red G Toluidine red Sudan I Scarlet pigment powder Sudan II Sudan blue 2 Sudan III Sudan red 7B Sudan red B Sudan IV
Chilli sauce
Chilli oil
200 lg/kg
400 lg/kg
2000 lg/kg
200 lg/kg
400 lg/kg
2000 lg/kg
400 lg/kg
800 lg/kg
4000 lg/kg
85.3 94.1 86.6 75.6 82.3 79.8 91.1 89.3 92.7 85.3 92.3 83.1 87.2 94.2
86.7 95.7 89.3 82.1 80.3 83.7 92.5 87.4 95.3 86.2 94.7 95.3 93.5 95.6
96.8 94.5 91.7 88.3 87.6 85.4 88.6 88.2 91.3 92.1 89.4 92.9 96.3 97.6
79.3 86.5 79.3 82.1 86.5 75.6 83.1 80.1 88.3 84.4 83.6 84.5 87.3 92.3
84.3 92.1 87.6 82.4 79.3 78.4 81.6 86.2 79.4 73.4 86.5 88.7 91.3 87.5
92.1 (7.6) 89.3 (6.5) 102.3 (7.3) 90.2 (6.5) 76.4 (4.6) 92.1 (3.7) 93.6 (7.7) 99.2 (5.4) 78.4 (4.6) 78.7 (7.7) 91.2 (8.5) 92.3 (4.6) 90.2 (6.3) 92.4 (6.1)
86.1 92.3 87.3 82.4 75.3 76.4 89.9 75.3 80.2 87.9 86.3 91.2 89.2 85.6
90.2 97.4 87.2 87.7 87.1 77.5 92.8 76.6 96.2 90.3 91.6 88.3 78.4 89.7
93.7 (7.5) 103.5 (8.4) 92.6 (7.4) 79.4 (10.3) 91.6 (6.3) 90.3 (7.6) 90.1 (8.8) 86.7 (5.6) 93.4 (7.4) 89.8 (5.5) 90.1 (7.7) 92.1 (7.5) 91.7 (6.2) 97.5 (8.8)
(10.7) (8.3) (7.7) (12.1) (9.2) (10.2) (9.8) (10.5) (10.1) (12.3) (9.4) (10.6) (7.5) (9.6)
(5.6) (7.2) (6.3) (10.2) (11.2) (9.9) (11.5) (8.7) (7.7) (9.7) (8.3) (10.3) (5.4) (8.6)
(4.5) (6.5) (5.6) (7.3) (6.7) (8.4) (7.3) (6.5) (6.3) (7.3) (7.3) (6.3) (4.5) (8.3)
(11.5) (10.6) (8.7) (7.6) (10.9) (12.5) (8.2) (7.5) (8.7) (12.2) (9.5) (6.7) (11.6) (12.3)
(8.2) (10.2) (8.8) (5.3) (8.6) (10.3) (7.8) (8.7) (6.5) (10.3) (7.7) (6.8) (9.7) (10.3)
(11.2) (12.3) (9.7) (10.1) (7.5) (6.6) (10.3) (6.7) (8.8) (10.3) (5.7) (10.8) (7.3) (9.8)
(10.6) (10.0) (8.9) (9.2) (6.7) (10.3) (6.7) (7.9) (5.8) (9.4) (6.9) (8.1) (7.6) (6.6)
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Absorbance [mAU]
25.0
1 2 3 4 mAU
60.0
Peak #5
%
100% at 26.97 min
WVL:450 nm
50% at 26.88 min: 999.99 -50% at 27.06 min: 1000.00
50.0
20.0
40.0 30.0 20.0 10.0
10.0
0.0 -10.0 200
0.0 -5.0
nm
250
300
350
400
450
500
550
600
650
700
1 2 3 4
min
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
45.0
Retention Time [min]
Fig. 4. HPLC analysis of positive sample containing Scarlet pigment powder.
were detected. During the analysis of samples, the recovery was also determined for further assessing the effectiveness of the method. In addition, standard addition method or an internal standard could also be used for the quantitative determination of the target analyte in the sample when recovery was not satisfactory. 3.5. Sample analysis The more than 100 samples of different matrices have been analysed using the presented method in routine work between April 2010 and November 2012. In them the two positive chilli sauce samples were detected. Both of the samples were contaminated with Scarlet pigment powder. Fig. 4 illustrates the presence of illegal dyes in one of the positive samples. For the other chilli samples, target analytes had not been detected. Because method based on LC analysis with full-scan DAD detection could be used as confirmatory method (European Commission, 2002), the method developed in our study could be a suitable confirmatory method. For more confirmation, the measuring technique based on LC analysis with mass-spectrometric could be used as a complementary confirmation for the detected analytes. 4. Conclusions A HPLC method for the simultaneous detection of 14 fat-soluble dyes in chilli products was developed by using GPC clean-up procedure. An optimised chromatographic condition was chosen for good separation of the studied dyes. The GPC clean-up could efficiently remove large molecular lipids and natural pigments interferences from samples. The method has a good repeatability and high accuracy with satisfactory detection limits and has been successfully used for the determination of banned dyes in real samples. Acknowledgements This research was financed by the Scientific & Research Project of Scientific Committee of Chongqing under Project No. cstc2012gg-yyjs80028, and Scientific & Research Project of General Administration of Quality Supervision, Inspection and Quarantine (AQSIQ) of China under Project No. 2012QK374. References American Spice Trade Association (ASTA). (2005). Sudan red and related dyes – white paper. . Ávila, M., Zougagh, M., Escarpa, A., & Ríos, Á. (2011). Determination of Sudan dyes in food samples using supercritical fluid extraction–capillary liquid chromatography. The Journal of Supercritical Fluids, 55, 977–982.
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