色谱  2015, Vol. 33 Issue (1): 29-34   PDF (624 KB)    
扩展功能
加入收藏夹
复制引文信息
加入引用管理器
Email Alert
RSS
本文作者相关文章
Iman SAEIDI
Behruz BARFI
Moazameh PAYROVI
Javad FEIZY
Hojat A SHEIBANI
Mina MIRI
Farahnaz GHOLLASI MOUD
Polyamide as an efficient sorbent for simultaneous interference-free determination of three Sudan dyes in saffron and urine using high-performance liquid chromatography-ultra violet detection
Iman SAEIDI1 , Behruz BARFI2, Moazameh PAYROVI3, Javad FEIZY4, Hojat A SHEIBANI5, Mina MIRI1, Farahnaz GHOLLASI MOUD1    
1. Iranian National Standards Organization, Mashhad 91735-344, Iran;
2. Department of Chemistry, University of Semnan, Semnan 35131-19111, Iran;
3. Department of Chemistry, University of Mazandran, Babolsar 47416-95447, Iran;
4. Testa Quality Control Laboratory, Mashhad 91895/157-134, Iran;
5. Birjand University of Technology, Birjand 97198-66981, Iran
Received: 2014-08-27
Foundation item: The authors are grateful to the financial support of Institute of Standards and Industrial Research of Iran (Khorasan e RazaviProvince).
Corresponding author. Fax: +985137682074, E-mail: iman.analyst@gmail.com.
Abstract: With polyamide (PA) as an efficient sorbent for solid phase extraction (SPE) of Sudan dyes II, III and Red 7B from saffron and urine, their determination by HPLC was performed. The optimum conditions for SPE were achieved using 7 mL methanol/water (1: 9, v/v, pH 7) as the washing solvent and 3 mL tetrahydrofuran for elution. Good clean-up and high (above 90%) recoveries were observed for all the analytes. The optimized mobile phase composition for HPLC analysis of these compounds was methanol-water (70: 30, v/v). The SPE parameters, such as the maximum loading capacity and breakthrough volume, were also determined for each analyte. The limits of detection (LODs), limits of quantification (LOQs), linear ranges and recoveries for the analytes were 4.6-6.6 μg/L, 13.0-19.8 μg/L, 13.0-5000 μg/L (r2 > 0.99) and 92.5%-113.4%, respectively. The precisions (RSDs) of the overall analytical procedure, estimated by five replicate measurements for Sudan II, III and Red 7B in saffron and urine samples were 2.3%, 1.8% and 3.6%, respectively. The developed method is simple and successful in the application to the determination of Sudan dyes in saffron and urine samples with HPLC coupled with UV detection.
Key words: solid phase extraction (SPE)     high-performance liquid chromatography (HPLC)     Sudan dyes     saffron     urine     polyamide (PA)    

Azo dyes are by far the most widely used synthetic,fat-soluble,organic colorants with characteristic chromophoric azo (N=N) groups [1]. There are over 3000 azo dyes in use and they account for 65% of the commercial dye market [2, 3]. These phenyl-azoic derivatives are non-authorized and illegally used in the food industry to enhance and maintain the appearance of food products such as in chilli-,curry-,curcuma-,and palm oil-containing foodstuffs [4, 5, 6]. Besides foodstuffs,Sudan dyes are widely used as colouring agents in the products of chemical industries such as oils,fats,plastics,waxes,petrol,shoes,printing inks,shoe and floor polishing and spirit varnishing [3, 7, 8]. These dyes are categorized as Class 3 carcinogens by the International Agency for Research on Cancer (IARC). The European Commission requires products to have documentation confirming the absence of Sudan dyes.

In view of the above incidents,there is a pressing need to develop fast and sensitive methods for the simultaneous determination of Sudan dyes that contaminate foods. In general,an analytical procedure for the determination of azo dyes in sample involves three steps: extraction from the sample,separation and quantification. Regarding the complexity of matrix and the low levels of Sudan dyes contained in real samples,several sample preparation techniques such as liquid-solid extraction [9],pressurized liquid extraction [10],the molecularly imprinted polymers (MIPs) [11] and cloud point extraction [12] were developed to allow HPLC-based determination. Reversed-phase high-performance liquid chromatography combined with different detectors is the commonly used analytical method for the separation of azo dyes [4, 13, 14, 15, 16, 17, 18]. However,some of these detectors are time-consuming and require expensive instruments or laborious pretreatments.

The SPE strategy comprises the isolation and preconcentration of the analyte from a complex matrix by adsorption onto an appropriate sorbent,removal of interfering impurities by washing with a suitable solvent system and selective recovery of the retained analyte with a suitable solvent. The SPE procedure used in most of the studies was based on MIPs sorbents [19, 20, 21]. Polyamide (PA) contains functional groups including acylamino,terminal amino and carboxyl groups which can reversibly form strong hydrogen bonding with substrates and eluents. It is used in the chromatography of phenols and carboxylic acids [22, 23]. So,we decided to use a PA-SPE cartridge for extraction of Sudan dyes,II,III and Red 7B in saffron and urine,which provided a simpler,less expensive and faster technique compared with the use of MIP sorbents. This investigation showed that the polyamide sorbent gave high recovery for mentioned Sudan dyes.

The aim of this work was to develop a rapid and simple analytical method for the quantitative determination of Sudan II,III and Red 7B in saffron and urine. The investigated method combined the SPE of azo dyes from saffron and urine using PA-cartridges and their separation and quantification using LC-UV.

1 Experimental conditions
1.1 Apparatus and chemicals
The standards of Sudan II,III and Red 7B were purchased from Sigma-Aldrich (St. Louis,MO,USA). Methanol and acetonitrile (HPLC grade) were from Fluka (Buches,Switzerland). PA was purchased from Fluka (particle size 50-160 μ m; bulk density 0.25 g/mL). Glacial acetic acid and HCl were from Merck (Darmstadt,Germany). Water used was double distilled deionized. The stock solutions of Sudan dyes (500 mg/L) were prepared in tetrahydrofuran (THF) and stored in the dark at 4 ℃,where they were stable at least for three months. All solutions were filtered through 0.45 μ m membrane filters (Millipore,Bedford) prior to use.

Chromatographic measurements were carried out using a KNAUER HPLC system (HPLC,KNAUER Jahre35,Germany) equipped with a K-1001 HPLC pump and a UV detector K-2800 was used for its detection. The elution was monitored at 507 nm. The other HPLC equipment included a KNAUER K-1500 solvent organizer,KNAUER K-500 degasser. Adjustment of pH of solutions was carried out by a 3030 Jenway pH meter (Leeds,UK). The column used was a C8 column (250 mm×4.5 mm,5 μ m) from Capital (Broxburn,UK). The PA-SPE cartridges (Chromabond PA,3 mL/500 mg) were obtained from Macherey-Nagel (Düren,Germany). The system was equipped with Chromgate HPLC software,Version 3.3. The sonication was done using a 50/60 kHz ultrasonic water bath (SW3,Switzerland). A Denley bench centrifuge model BS400 (Denley Instruments Ltd.,Billingshurst,UK) was used to accelerate the phase separation.

1.2 Sample preparation
The saffron samples were purchased from different local markets in Mashhad (Iran). The samples were mixed homogeneously and stored at room temperature. Five grams of saffron were weighed and mixed with 15 mL THF and then stirred for 10 min. The extract was filtered through 0.45 μ m membranes and collected for clean-up by SPE.

For the determination of Sudan dyes in human urine,different saffron samples were orally administrated by six 30-year-old healthy male volunteers. The volunteers avoided to consume possible Sudan containing foods (spice,chilli powders,paste,tea and sauce) for one week prior to the study. The urine samples (10 mL) were collected just before and 6 h after the administration.

The blank urine samples were provided by healthy volunteers in our laboratory. Actual urine samples were prepared from the same people who were suspicious to the administration of polluted saffron by Sudan dyes. The urine samples were kept frozen at -20 ℃ before clean up by SPE. The frozen urine samples were thawed at room temperature and centrifuged for 10 min at 5000 r/min. White lipidic solid was sedimentated,probably due to the co-sedimentation of the matrices (such as carbamide and uric acid) in urine. Ten milliliters of supernatants were collected and filtered through a 0.45 μ m filter for SPE.

1.3 Solid phase extraction
The PA-SPE cartridge was sequentially conditioned with 5 mL of n-hexane,5 mL of methanol and 10 mL of double distilled deionized water without allowing the cartridge to dry. The filtrate was passed through the cartridge,rinsed with 5 mL of water to remove polar constituents of saffron,washed by 5 mL water/methanol (9 : 1 v/v) to remove interferences and eluted with 3 mL of HPLC grade THF. The eluate was dried by blowing N2 stream and dissolved in 1 mL of THF and injected into the LC system.
1.4 Chromatographic conditions for separation and determination of Sudan dyes
LC separation of Sudan dyes was performed isocratically using a mobile phase consisting of methanol/water (70 : 30,v/v) with a flow rate of 1 mL/min at room temperature. Prior to use,all mobile phases were passed through a 0.45 μ m membrane filter and degassed under vacuum. The sample injection volume was 20 μ L and the analytes were monitored at 507 nm.
2 Results and discussion
2.1 Determination of breakthrough volume and maximum loading capacity of solid phase cartridge for Sudan dyes
Determination of breakthrough volume was performed according to the procedure presented by Hennion [24]. Standards of Sudan II,III and Red 7B (2 μ g) were dissolved in 250-3000 mL of water and passed through the cartridge. The maximum loading capacity of the analyte was determined by passing different volumes (25-450 mL) of 2 μ g/mL standard through the cartridge. In both cases,the retained analyte was eluted with 3 mL THF and dried by blowing N2 stream. The residue was dissolved in 1 mL of THF and injected into the HPLC system. Fig. 1 shows that the breakthrough volumes for Sudan II,III and Red 7B are 1000,1250 and 2250 mL,respectively. The maximum loading capacities for Sudan II,III and Red 7B were 200,250 and 350 μ g,respectively.
Fig.1 Breakthrough volume of analytes using polyamide cartridge SPE conditions: sample loaded,250-3000 mL of standard solutions containing 2 μ g of analytes; flow rate,1 mL/min.

HPLC conditions: mobile phase,methanol/water (70 : 30,v/v); flow rate,1 mL/min; column,C8 (250 mm×4.5 mm,5 μ m.); λ max,507 nm; room temperature.

2.2 Optimization of solid phase extraction
The isolation of Sudan dyes from a saffron and urine matrix is a prerequisite to any chromatographic determination and solid phase extraction based on PA cartridge which is a simple technique allowing for their extraction. The use of a suitable washing solution requires the determination of an optimum organic solvent composition in conjunction with an appropriate pH values in order to remove interfering matrix components without risking the elution of analytes. Interfering species in saffron included mineral compounds,sugar and other polar compounds.

Effect of some parameters including percentage of methanol in washing solution,pH of washing solution,type and volume of elution solvent and flow rate of sample solution through cartridge on extraction efficiency were investigated using PA cartridge as the sorbent.

To obtain a suitable solvent for elution of analytes from the cartridge,different solvents such as methanol,acetonitrile,THF,ethylacetate,n-hexane and acetone were examined. The best elution solvent for Sudan dyes was found by using 4 mL of each solvent (Fig. 2a). The best recovery of analytes was achieved by THF. For determination of a suitable volume of the elution solvent,different volumes (1,2,3,4,5 and 6 mL) of THF were used for the elution of retained analytes from the cartridge. The most suitable volume of elution was 3 mL (Fig. 2b).

Fig.2 Recovery against (a) type of organic solvent and (b) volume of THF Conditions: sample loaded,25 mL 2 mg/L standard solution; other conditions were the same as in Fig. 1.

Use of a carefully chosen washing solution in the SPE process to provide an extract free from matrix components,leads to enhanced selectivity in the separation step and more accurate determination. To achieve this purpose,the washing solution must contain the appropriate amount of organic solvent and the highest possible pH (in the recommended range for stability of the sorbent) to remove interfering matrix components without eluting the analytes.

For optimizing the washing solution,different percentages of methanol in water at different pH ranging from 2.5 to 8 were examined. According to the best recovery,the optimum conditions for washing solution were: 10% (v/v) methanol (pH 7) and 7 mL washing solution volume.

The flow rate of aqueous sample solution always has a significant impact in the SPE procedure,because the sample flow rate affected both the recoveries of analytes and loading time in an SPE system. The results indicated that the best recovery of analytes in PA cartridge was achieved at a flow rate of 2.5 mL/min.

2.3 Method validation
The limits of detection (LODs),limits of quantification (LOQs),linear ranges (LRs) for Sudan II,III and Red 7B were determined and are shown in Table 1. The results showed that the proposed method had good repeatability for the determination of the analytes in saffron and urine samples. The LOD was established using LOD=3.3×( σ /S) and the LOQ was established using LOQ=10×(σ /S),where σ is the standard deviation of the spiked sample (with 1 mg/L) and S is the slope of the calibration curve. As shown in Table 2,the intra- and inter-day precisions of the method in different spiked blank real samples were determined as relative standard deviation (RSD). The intra-day precision was assessed by five determinations per concentration in one day,while the inter-day precision was evaluated by five determinations per concentration in five different days. The accuracy of the method,in term of recovery,was calculated as the percent difference from the expected concentration. The results of the validation studies in Table 2 demonstrate that the method has the acceptable precision and accuracy.
Table 1 Analytical performance of SPE-HPLC for the determination of Sudan dyes in saffron and urine samples

Table 2 Inter- and intra-day precisions and recoveries of Sudan dyes spiked in saffron and urine samples after SPE (n=5)
2.4 Determination of Sudan dyes in actual samples
To evaluate the accuracy and applicability of the proposed method,the extraction and determination of Sudan dyes were performed in biological samples,i. e. saffron and urine. All the samples were spiked with Sudan dye standards at three levels. Subsequently,they were extracted using the PA-SPE technique. Finally,the extracts were analyzed by HPLC method.

The mass concentrations of Sudan dyes in saffron and urine samples were determined by five replicate measurements using standard addition method (n=5). Table 3 presents the mass concentrations of the Sudan dyes found in saffron and urine samples. The analytes were identified by spiking the samples with the standards and comparing their retention times with those of the standard Sudan II,III and Red 7B. Representative chromatograms of saffron and urine samples acquired at optimum mobile phase conditions are presented in Fig. 3.

Table 3 Contents of Sudan dyes in saffron and urine samples

Fig.3 Representative chromatograms of spiked

(a) saffron and (b) urine samples Experimental details are described in the text.

In a comparative study with other techniques used for determination of Sudan dyes in different samples,shown in Table 4,one can see that comparable results were achieved for this method versus other conventional methods and without the use of expensive and advanced instruments.

Table 4 Comparison of the presented method with other methods used for the determination of the interested Sudan dyes
3 Conclusions
In this work,determination of Sudan dyes in saffron and urine was performed using SPE with a polyamide cartridge and isocratic LC. SPE parameters (selection of a suitable solvent and its volume for elution of analytes,maximum loading capacity of polyamide cartridge and breakthrough volume) were optimized. The use of polyamide cartridge provided a fast sample treatment with low solvent consumption,good clean up and high (above 90% ) recovery in saffron and urine samples. Good resolution,sensitivity,linearity and repeatability as well as the simplicity of HPLC and low-cost sample preparation made this method a useful tool for quality control,routine analysis of adulterated saffron samples,and can be applied to standard institutes and laboratories,and also bioanalytical determinations.
参考文献
[1]Reisch M S. Chem Eng News, 1992, 70: 16  
[2]Ahlstrom L H, Eskilsson C S, Bjorklund E. TrAC-Trend Anal Chem, 2005, 24: 49  
[3]Rafii F, Hall J D, Cerniglia C E. Food Chem Toxicol, 1997, 35: 897  
[4]Calbiani F, Careri M, Elviri L, et al. J Chromatogr A, 2004, 1042: 123  
[5] Commission Decision 2005/402/EC of 23 May 2005 on Emergency Measures Regarding Chilli, Chilli Products, Curcuma and Palm Oil
[6]Cornet V, Govaert Y, Moens G, et al. J Agric Food Chem, 2006, 54: 639  
[7]Dillion D, Combes R, Zeiger E. Mutagen, 1994, 9: 295  
[8]Society of Dyers and Colourists. Colour Index. 3rd ed. Bradford: Society of Dyers and Colourists, 1971: 3
[9]European Commission, NEWS notification: 03/99. Analysis and Dosage of the Colorants Sudan and Bixin in Chilli Powder and Pepper-Based Products
[10]Anibal C V D, Odena M, Ruisánchez I, et al. Talanta, 2009, 79: 887  
[11]Long C, Mai Z, Yang Y, et al. J Chromatogr A, 2009, 1216: 8379  
[12]Liu W, Zhao W, Chen J, et al. Anal Chim Acta, 2007, 605: 41  
[13]Zhang Y, Zhang Z, Sun Y. J Chromatogr A, 2006, 1129: 34  
[14]Liu S, Zhang X, Lin X, et al. Electrophoresis, 2007, 28: 1696  
[15]Mejia E, Ding Y, Mora M F, et al. Food Chem, 2007, 102: 1027  
[16]Liu R, Hei W, He P, et al. J Chromatogr B, 2011, 879: 2416  
[17]Chailapakul O, Wonsawat W, Siangproh W, et al. Food Chem, 2008, 109: 876  
[18]Qi P, Zeng T, Wen Z, et al. Food Chem, 2011, 125: 1462  
[19]Qiao F, Geng Y, He C, et al. J Chromatogr B, 2011, 879: 2891  
[20]Zhao C, Zhao T, Liu X, et al. J Chromatogr A, 2010, 1217: 6995  
[21]Yan H, Wang H, Qiao H, et al. J Chromatogr A, 2011, 1218: 2182  
[22]Dueñas M, Sun B, Hernández T, et al. J Agric Food Chem, 2003, 51: 7999  
[23]Monagas M, Gómez-Cordovés C, Bartolomé B, et al. J Agric Food Chem, 2003, 51: 6475  
[24]Hennion M C. J Chromatogr A, 1999, 856: 3  
[25]Calbiani F, Careri M, Elviri L, et al. J Chromatogr A, 2004, 1058: 127