- Email: [email protected]
Fast determination of seven synthetic pigments from wine and soft drinks using magnetic dispersive solid-phase extraction followed by liquid chromatography–tandem mass spectrometry
G Model
ARTICLE IN PRESS
CHROMA-355352; No. of Pages 6
Journal of Chromatography A, xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Short communication
Fast determination of seven synthetic pigments from wine and soft drinks using magnetic dispersive solid-phase extraction followed by liquid chromatography–tandem mass spectrometry夽 Xiao-Hong Chen a,b , Yong-Gang Zhao a,b , Hao-Yu Shen c , Li-Xin Zhou d , Sheng-Dong Pan a,b , Mi-Cong Jin a,b,∗ a Zhejiang Provincial Key Laboratory of Health Risk Appraisal for Trace Toxic Chemicals, Ningbo Municipal Center for Disease Control, Prevention, Ningbo, Zhejiang 315010, China b Ningbo Key Laboratory of Poison Research and Control, Ningbo Municipal Center for Disease Control and Prevention, Ningbo, Zhejiang 315010, China c Ningbo Institute of Technology, Zhejiang University, Ningbo, Zhejiang 315100, China d Medical School, Ningbo University, Ningbo, Zhejiang 315211, China
a r t i c l e
i n f o
Article history: Received 21 February 2014 Received in revised form 16 April 2014 Accepted 17 April 2014 Available online xxx Keywords: UFLC–MS/MS Amino-functionalized low degrees of cross-linking magnetic polymer (NH2 -LDC-MP) Magnetic dispersive solid-phase extraction (M-dSPE) Pigment Wine Soft drink
a b s t r a c t A novel, simple and sensitive method based on the use of magnetic dispersive solid-phase extraction (M-dSPE) procedure combined with ultra-fast liquid chromatography-tandem quadrupole mass spectrometry (UFLC–MS/MS) was developed to determine seven synthetic pigments (tartrazine, amaranth, carmine, sunset yellow, allura red, brilliant blue and erythrosine) in wines and soft drinks. An amino-functionalized low degrees of cross-linking magnetic polymer (NH2 -LDC-MP) was synthesized via suspension polymerization, and characterized by transmission electron microscopy (TEM). The NH2 LDC-MP was used as the M-dSPE sorbent to remove the matrix from the solution, and the main factors affecting the extraction were investigated in detail. The obtained results demonstrated the higher extraction capacity of NH2 -LDC-MP with recoveries between 84.0 and 116.2%. The limits of quantification (LOQs) for the seven synthetic pigments were between 1.51 and 5.0 g/L in wines and soft drinks. The developed M-dSPE UFLC–MS/MS method had been successfully applied to the real wines and soft drinks for food-safety risk monitoring in Zhejiang Province, China. The results showed that sunset yellow was in three out of thirty soft drink samples (2.95–42.6 g/L), and erythrosine in one out of fifteen dry red wine samples (3.22 g/L), respectively. It was confirmed that the NH2 -LDC-MP was a kind of highly effective M-dSPE materials for the pigments analyses. © 2014 Elsevier B.V. All rights reserved.
1. Introduction To protect consumers from health risks, many countries have established strict regulations for the allowable kinds and concentrations of synthetic pigments [1,2]. In China, the common water-soluble synthetic pigments e.g. amaranth, brilliant blue, carmine, sunset yellow, and tartrazine, are permitted in foodstuff [3,4], however, all ingredients including food pigments are required
夽 Presented at the 40th International Symposium on High Performance Liquid Phase Separations and Related Techniques (HPLC 2013 Hobart), Hobart, Tasmania, Australia, 18–21 November 2013. ∗ Corresponding author at: Ningbo Key Laboratory of Poison Research and Control, Ningbo Municipal Center for Disease Control and Prevention, Ningbo, Zhejiang 315010, China. Tel.: +86 574 87274559; fax: +86 57487277153. E-mail addresses: [email protected], [email protected] (M.-C. Jin).
to be listed on the food labels. Nowadays, more evidence indicates that the abuse of synthetic pigments may cause cancer [2,5], and some people are much sensitive to particular food pigments. Therefore, it is necessary for the determination of synthetic pigments to ensure the food safety. Today, some high-performance liquid chromatography (HPLC) methods including ultraviolet/visible (UV/vis), diode-array detectors (DAD) [6–10] and mass spectrometry (MS) [2,11,12], have been proposed for the determination of synthetic colorants. In addition, many pretreatment methods, such as liquid–liquid extraction (LLE) and solid-phase extraction (SPE), are used for the extraction of synthetic pigments from various food [2,13], however, these protocols are laborious and time consuming. Luckily, several other cleanup methods based on the traditional SPE technique, such as magnetic solid-phase extraction, dispersive solid-phase extraction (dSPE), micro-solid-phase extraction (-SPE) and dispersive
http://dx.doi.org/10.1016/j.chroma.2014.04.060 0021-9673/© 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: X.-H. Chen, et al., J. Chromatogr. A (2014), http://dx.doi.org/10.1016/j.chroma.2014.04.060
G Model CHROMA-355352; No. of Pages 6
ARTICLE IN PRESS X.-H. Chen et al. / J. Chromatogr. A xxx (2014) xxx–xxx
2
micro-solid-phase extraction (d--SPE), have been widely used for multi-residue analyses [14–26]. Although dSPE is considered as one of the most powerful cleanup technologies, to the best of our knowledge, there are no literatures by using dSPE in the analysis of synthetic pigments. In our previous work, an improved ethylenediaminefunctionalized magnetic polymer (IEDA-MP) was found to have an excellent cleanup ability for selective removal of the matrix in red wine [27]. By using IEDA-MP dSPE for cleaning up the studied synthetic pigments, a short sample preparation time and an easy cleanup procedure for the removal of various natural pigments, organic acids and sugars in wine and soft drinks were expected to be achieved, however, some analytes were also adsorbed onto IEDA-MP resulting in low recoveries. The IEDA-MP surface is generally covered with amino groups ( NH , NH2 ), which provide a special polar hook for enhanced capture of polar analytes. Therefore, we inspired that amino-functionalized low degrees of cross-linking magnetic polymer (NH2 -LDC-MP) contained less hydrophilic amino groups and more lipophilic styrene monomer would be probably become a powerful sorbent to carry out magnetic dispersive solid-phase extraction (M-dSPE) for cleaning up the studied synthetic pigments from wine and soft drinks. In this work, the effect of the usage amount of cross-linker i.e., styrene (St) used in the co-polymerization procedure, which would subsequently lead to obtain various NH2 -LDC-MP with different amount of amino groups and lipophilic styrene monomer, on the recovery of seven synthetic pigments has been investigated. In addition, an analytical procedure combined the fast M-dSPE cleanup technique with the LC–MS/MS has been proposed. It has been shown to be effective, fast and accurate in the routine analyses.
2. Experimental 2.1. Reagents and materials Ferric chloride, ferrous sulfate, oleic acid (OA), ethanol, methyl methacrylate (MMA), styrene (St), glycidylmethacrylate (GMA), ethylenediamine (EDA), polyvinyl alcohol (PVA 217) and benzoyl peroxide (BPO) of analytical grade were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Primary secondary amine (PSA) was purchased from Agilent Company (Palo Alto, USA). Tartrazine, amaranth, carmine, sunset yellow, allura red, brilliant blue and erythrosine were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). Acetonitrile and ammonium acetate (AmAc) of HPLC grade were purchased from Merck Company (Darmstadt, Germany). The wines and soft drinks were acquired on the local markets (Ningbo, China).
2.2. Equipment The characterizations of LDC-MP were carried out by using transmission electron microscopy (TEM) (Hitachi H-7650) (Hitachi, Japan), vibrating sample magnetometer (VSM) (Lake Shore 7410) (Westerville, USA), and elementary analyzer (EA) (ThermoFisher Flash-1112) (ThermoFisher, USA). A vortex mixer Hualida WH-866 (Taicang, China) was used during extraction. UFLC-MS/MS analyses were performed with Prominence UFLC XR system equipped with a LC-20AD pump system, a CTO-20AC column oven, a DGU20A3 degasser and a SIL-20AC autosampler (Shimadzu Corporation, Tokyo, Japan) and an AB SCIEX TRIPLE QUAD 5500 mass spectrometer (Applied Biosystems, Foster City, CA, USA). The UFLC–MS/MS system was controlled, and data were analyzed, on a computer
equipped with Analyst 1.5.1 (Applied Biosystems, Foster City, CA, USA). 2.3. Synthesis of NH2 -LDC-MP The NH2 -LDC-MP nanoparticles were prepared by the following steps based on the suspension polymerization and ring-opening reactions. 2.0 g of polyglycol was dissolved into 200 mL of hot water, followed by adding 0.04 mol of MMA, 20 mmol of St and 52 mmol of GMA. Then 2.0 g of OA-M (prepared by the reported procedure [27–30]) was added to the above system under ultrasonication. Finally, 1.0 g of BPO in 20 mL of ethanol was added dropwise under vigorously stirring. The mixture was allowed to react at 80 ◦ C for 3 h, yielding M-co-poly (MMA-St-GMA) polymer. The latter was isolated under magnetic field and washed with water and ethanol to remove redundant MMA, St and GMA. Exactly 1.25 g of the M-co-poly (MMA-St-GMA) was dispersed into 50 mL of methanol in a 100 mL flask. Then 3.5 mL (50 mmol) of EDA was added dropwise under stirring. The flask was then fitted with a water condenser and heated at 80 ◦ C for 8 h. The final improved amino-functionalized low degrees of cross-linking magnetic polymer named NH2 -LDC-MP-I was isolated under magnetic field and washed with water and methanol at pH 7.0 to remove the redundant diamine. Other NH2 -LDC-MP nanoparticles, named as NH2 -LDC-MP-II, NH2 -LDC-MP-III and NH2 -LDC-MP-IV, were synthesized in a similar way with different amounts of St (10, 5 and 0 mmol, respectively). All the NH2 -LDC-MP nanoparticles were dried in a vacuum oven at 60 ◦ C and stored in a sealed bottle for further use. 2.4. M-dSPE procedure An exactly 1.0 mL of sample was placed into an open evaporating dish and evaporated to dryness in a water bath at 80 ◦ C. Afterwards, the residue was redissolved with pure water (adjusted to pH 9.0 with ammonia 0.5 mol/L) and transferred to a polypropylene centrifuge tube (2.0 mL) containing 15.0 mg of NH2 -LDC-MP. The tube was vortexed for 1.0 min, and the adsorbed NH2 -LDC-MP was isolated under magnetic field. About 0.5 mL aliquot of the supernatant was filtered using a 0.22 m membrane prior to its injection into the UFLC–MS/MS system. 2.5. LC–QqQ-MS/MS analysis The chromatographic separation was performed on a Shimpack XR-ODS II (100 mm × 2.0 mm i.d., 2.2 m) by using 5.0 mmol/L AmAc in acetonitrile as eluent (A), and 5.0 mmol/L AmAc in water as eluent (B) as the mobile phase. The linear gradient was: 0→3.00 min, 2.0→10.0% A; 3.00→7.00 min, 10.0→80.0% A; 7.00→7.01 min, 80.0→2.0% A and 7.01→9.00 min, 2.0% A. The separation was accomplished at a constant flow of 0.40 mL/min. The column was thermostated at 40 ◦ C to increase the retention time reproducibility. The injection volume was 5.0 L. Mass spectrometry analysis was performed using an electrospray ionization source in negative mode. The operation conditions were as follows: ion spray voltage, −4500 V; curtain gas (CUR), 40 psi and interface heater was on; collision gas, medium; nebulizer gas (gas 1) and heater gas (gas 2), 50 and 50 psi; the turbo spray temperature, 500 ◦ C; entrance potential (EP), −10 V; collision cell exit potential (CXP), −10 V. Nitrogen was used in all cases. Multiple-reaction monitoring (MRM) mode was used for quantification. The results of the precursor ion, product ion are shown in Table 1. Applied Biosystems/MDS Sciex Analyst software (versions 1.5.1) was used for data acquisition and processing.
Please cite this article in press as: X.-H. Chen, et al., J. Chromatogr. A (2014), http://dx.doi.org/10.1016/j.chroma.2014.04.060
G Model CHROMA-355352; No. of Pages 6
ARTICLE IN PRESS X.-H. Chen et al. / J. Chromatogr. A xxx (2014) xxx–xxx
3
Table 1 Q1/Q3 ion pairs, linear equations, determination coefficients (r2 ), linear ranges, limits of detection (LODs) and limits of quantification (LOQs) of seven synthetic pigments. Synthetic pigment Tartrazine Amaranth Carmine Sunset yellow Allura red Brilliant blue Erythrosine a b
Precursor ion (Q1, m/z) 233.1 268.2 268.1 202.9 225.1 373.8 834.7
Product ion (Q3, m/z) a
197.9 , 117.0, 80 206.0a , 302.0 301.9a , 206.1, 80 170.9a , 207.0, 156 206.0a , 199.9 170.0a , 333.6 536.8a , 662.7
Linear equationb
r2
A = 1383C − 652 A = 2790C + 301 A = 2030C + 698 A = 5490C + 350 A = 1920C + 360 A = 2600C − 676 A = 3510C − 291
0.9993 0.9991 0.9996 0.9994 0.9990 0.9998 0.9998
Linear range (g/L) 5.0–1000.0 5.0–1000.0 5.0–1000.0 2.5–500.0 5.0–1000.0 5.0–1000.0 2.5–500.0
LOD (g/L)
LOQ (g/L)
1.25 1.51 1.00 0.45 1.22 0.71 0.50
4.16 5.00 3.33 1.51 4.02 2.38 1.66
Quantitative ion. A, peak area; C, mass concentration, g/L.
2.6. Method validation 2.6.1. Standard preparation Individual stock standard solutions were prepared at 1000 mg/L level by exact weighing and dissolution in water–methanol (1:1, v/v). The stock mixture standard solution (10.0 mg/L) was prepared by appropriate dilution of the stock solutions with water–methanol (1:1, v/v). Matrix-matched calibration standards were spiked with concentration in the range of 5.0–1000 g/L for tartrazine, amaranth, carmine, brilliant blue and allura red, 2.5–500 g/L for sunset yellow and erythrosine, respectively. The matrix-matched
calibration curves made by peak area vs concentration (g/L) were used to calibrate spike samples in recovery experiments. 2.6.2. Spiked sample Spiked recoveries were performed at concentrations of 8.0, 80, and 800 g/L for tartrazine, amaranth, carmine, brilliant blue, and allura red, 4.0, 40 and 400 g/L for sunset yellow and erythrosine in five kinds of samples, i.e., dry red wine, medium dry wine, sweet wine and medium sweet wine and soft drinks, respectively. For each spiked sample, stock mixture solution of the standards was added to 1.0 mL sample, which was free from the target compounds.
Fig. 1. TEM images of NH2 -LDC-MP-I(a), NH2 -LDC-MP-II(b), NH2 -LDC-MP-III(c), and NH2 -LDC-MP-IV(d) nanoparticles.
Please cite this article in press as: X.-H. Chen, et al., J. Chromatogr. A (2014), http://dx.doi.org/10.1016/j.chroma.2014.04.060
G Model CHROMA-355352; No. of Pages 6
ARTICLE IN PRESS X.-H. Chen et al. / J. Chromatogr. A xxx (2014) xxx–xxx
4
Table 2 Validation parameters obtained for the seven synthetic pigments at three concentration levels in dry red wine, medium dry wine, sweet wine and medium sweet wine and soft drink matrix. Synthetic pigments
Added (g/L)
Average recovery a ,% (RSD, %) Dry red wine b
c
Medium dry wine
Sweet wine
Medium sweet wine
Soft drink
Tartrazine
8.0 80.0 800.0
98.2 (2.1 , 1.5 ) 106.2 (2.6, 3.7) 97.5 (5.1, 6.8)
92.3 (3.9, 5.2) 116.2 (5.8, 6.9) 106.5 (3.5, 6.8)
90.6 (3.2, 5.7) 98.0 (4.4, 7.2) 95.6 (5.0, 6.8)
95.8 (3.5, 5.1) 91.0 (2.2, 4.7) 96.6 (4.0, 6.4)
93.9 (2.6, 5.0) 86.8 (3.0, 5.3) 89.2 (1.9, 5.2)
Amaranth
8.0 80.0 800.0
82.5 (2.4, 5.1) 90.1 (2.5, 6.6) 92.7 (3.6, 5.2)
92.8 (5.5, 7.4) 89.0 (3.3, 4.6) 95.9 (4.9, 5.2)
92.9 (2.6, 5.0) 93.2 (3.0, 5.3) 104.0 (5.2, 5.8)
82.8 (2.8, 5.0) 96.9 (3.8, 5.9) 97.5 (8.6, 9.7)
83.2 (5.1, 5.6) 97.7 (3.4, 6.2) 96.3 (5.0, 5.8)
Carmine
8.0 80.0 800.0
91.3 (2.6, 4.6) 96.6 (2.4, 4.6) 88.0 (1.9, 2.7)
92.4 (2.7, 3.7) 98.6 (3.0, 4.6) 89.1 (2.8, 5.0)
101.6 (5.7, 6.0) 99.7 (3.9, 4.7) 90.2 (1.4, 3.2)
92.8 (3.5, 7.1) 101.0 (3.3, 5.7) 95.0 (4.9, 5.6)
96.9 (3.6, 5.0) 90.8 (4.0, 4.3) 84.0 (6.2, 7.7)
Sunset yellow
4.0 40.0 400.0
86.2 (3.8, 5.7) 97.9 (8.1, 6.8) 92.6 (3.4, 7.1)
92.9 (5.8, 5.9) 86.5 (8.2, 7.8) 92.8 (5.5, 5.4)
97.0 (4.4, 7.2) 95.2 (3.0, 6.8) 96.9 (2.6, 3.0)
92.4 (1.7, 6.7) 90.6 (2.0, 3.5) 99.7 (1.6, 4.9)
92.1 (3.7, 6.0) 99.7 (2.9, 3.7) 93.3 (3.9, 6.2)
Allura red
8.0 80.0 800.0
90.3 (2.8, 3.6) 96.2 (3.4, 3.6) 92.0 (2.9, 2.8)
91.2 (2.7, 2.7) 99.2 (3.0, 3.6) 89.8 (4.8, 5.0)
102.6 (4.7, 5.0) 96.2 (2.9, 3.7) 93.2 (1.4, 3.2)
92.8 (2.5, 7.3) 100.0 (2.3, 5.2) 91.0 (2.9, 3.6)
92.9 (3.2, 4.0) 97.8 (3.0, 4.3) 89.0 (6.0, 7.2)
Brilliant blue
8.0 80.0 800.0
106.5 (5.2, 6.0) 89.2 (3.2, 4.7) 90.2 (2.4, 2.8)
92.8 (5.5, 5.4) 86.0 (6.3, 7.7) 85.3 (5.9, 6.2)
92.9 (3.6, 6.0) 90.8 (3.0, 7.3) 96.0 (2.2, 3.7)
99.2 (5.6, 5.7) 97.3 (3.1, 3.8) 96.5 (2.5, 5.8)
96.9 (3.8, 3.9) 96.5 (5.5, 6.5) 95.6 (4.0, 6.8)
Erythrosine
4.0 40.0 400.0
97.2 (4.4, 7.2) 85.3 (6.0, 6.8) 98.9 (1.6, 2.0)
93.3 (2.8, 5.7) 92.8 (4.0, 3.2) 103.7 (3.6, 6.2)
94.1 (6.7, 7.0) 89.2 (2.9, 5.8) 99.3 (3.9, 7.2)
90.8 (2.5, 5.4) 88.0 (2.3, 5.7) 97.6 (2.0, 6.4)
92.9 (2.6, 3.0) 89.3 (4.0, 4.6) 99.7 (2.9, 5.7)
a b c
The mean value was determined in one day (n = 6 replicates). Intra-day, n = 6 replicates. Inter-day, n = 3 replicates × 6 days within a 2-week period.
The spiked samples prepared were stored at 4 ◦ C for about 12 h to let the synthetic pigments permeate uniformly into the samples. 3. Results and discussion 3.1. Characterization of NH2 -LDC-MP The cross-linker used can affect the degree of crosslinking in the polymeric network. This work focused on the effect of the usage amount of cross-linker i.e., styrene (St) used in the co-polymerization procedure, which would subsequently lead to obtain various NH2 -LDC-MP nanoparticles with different amount of amino groups and lipophilic styrene monomer, on the recovery of seven synthetic pigments. Fig. 1 shows the TEM images of the NH2 LDC-MP nanoparticles with different amount of St as cross-linkers. As can be seen in Fig. 1, NH2 -LDC-MP-II prepared with 10 mmol of St was more uniform and spherical in shape than those prepared with 20, 5 and 0 mmol of St. The paramagnetic properties were verified by VSM, and the results showed that the saturation moments of NH2 -LDC-MP obtained from the hysteresis loop were in the range of 7.86–8.02 emu/g. The NH2 -LDC-MP was proved to respond to magnetic fields without any permanent magnetization. This NH2 LDC-MP characteristic could ensure to separate more quickly and easily in two-phase (solid/liquid phase) systems due to the stronger magnetism. 3.2. Optimization of UFLC–MS/MS conditions The development process of the analysis method began with the optimization of the UFLC–MS/MS data acquisition parameters. For this purpose, a compromise had to be sought between sensitivity and selectivity to choose the appropriate MS/MS multiple reaction monitoring (MRM) transitions. Suitable precursor-to-product MS/MS transitions were selected through the optimized procedure. The final MS/MS conditions were detailed in Table 1, and the MS/MS spectra of the seven synthetic pigments were shown in Fig. S1.
In order to achieve an optimal chromatographic separation, the gradient elution and the effects of AmAc and ammonia concentration on the chromatographic separation were studied. Based on the optimization, acetonitrile-AmAc (5.0 mmol/L) aqueous solution was selected as the mobile phase system for the separation of the seven studied synthetic pigments in gradient elution. 3.3. NH2 -LDC-MP M-dSPE procedure and its optimization In the study, the effect of pH value of samples and the amount of NH2 -LDC-MP on the d-SPE cleanup properties were investigated in detail. Firstly, various pH values from 2.0 to 11.0 were used to optimize the cleanup procedure by mixing 15.0 mg NH2 -LDC-MP with 1.0 mL sample spiked at a concentration of 8.0 g/L for tartrazine, amaranth, carmine, brilliant blue, and allura red, 4.0 g/L for sunset yellow and erythrosine, respectively, in a 2.0 mL polypropylene centrifuge tube. The obtained results indicated that with the increasing of pH values from 2.0 to 9.0, the recoveries of seven synthetic pigments increased significantly, as shown in Fig. S2(a). In the case of pH values above 9.0, the satisfactory recoveries of the seven synthetic pigments were consistently in the range of 90.2–103.3%. Above all, pH value 9.0 was selected for subsequent experiments. Secondly, seven dosages of NH2 -LDC-MP (5, 10, 15, 20, 30, 40 and 50 mg for 1.0 mL sample) on the cleanup properties were investigated to develop an efficient cleanup procedure for the seven synthetic pigments. The tested results showed that the combination of the least amounts of NH2 -LDC-MP adsorbents in the proportion of 10–20 mg per 1.0 mL sample would ensure efficient and robust cleanup to maintain high quantitative recoveries, as shown in Fig. S2(b). Furthermore, the results of recycling experiment showed that NH2 -LDC-MP could be reused at least twelve times without much sacrifice of the cleanup efficiency. The preparative reproducibility of the NH2 -LDC-MP used for the cleanup procedure was deeply evaluated. And the results show that the absolute deviations of the
Please cite this article in press as: X.-H. Chen, et al., J. Chromatogr. A (2014), http://dx.doi.org/10.1016/j.chroma.2014.04.060
G Model CHROMA-355352; No. of Pages 6
ARTICLE IN PRESS X.-H. Chen et al. / J. Chromatogr. A xxx (2014) xxx–xxx
5
Fig. 3. MRM chromatograms for two examined samples.
Fig. 2. MRM chromatograms for dry red wine sample spiked with seven synthetic pigments at 5.0 g/L for tartrazine, amaranth, carmine, brilliant blue and allura red, 2.5 g/L for sunset yellow and erythrosine under NH2 -LDC-MP M-dSPE cleanup procedure. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
recoveries for synthetic pigments by the three batches are less than 5.0%. This indicates that the preparation procedure of NH2 -LDC-MP has good repeatability and reproducibility.
3.4. Method linearity, accuracy, LOD and LOQ The linearity of the calibration curves made by peak area vs concentration (g/L) was studied using matrix-matched calibration standards in samples. The response function was found to be linear with a determination coefficient (r2 ) higher than 0.9990 in the tested range listed in Table 1 for the seven synthetic pigments. The method accuracies were expressed as the recoveries, and the method precisions were expressed as the intra-and interday relative standard deviations (RSDs). The intra-day RSDs were obtained by repeating the three levels of spiked samples six times within a day, and the inter-day RSDs were obtained by repeating the three levels of spiked samples in triplicate on six separate days within a 2-week period. The results are summarized in Table 2. It shows that the majority of mean recoveries are in the range of 84.0–116.2% with the intra-day RSDs ranging from 1.4 to 8.6% and inter-day RSDs ranging from 1.5 to 9.7%. The limits of detection (LODs) and limits of quantification (LOQs), for the analyzed synthetic pigments are shown in Table 1. The LODs and LOQs, which were calculated on the analysis of seven synthetic pigments spiked at 5.0 g/L for tartrazine, amaranth, carmine, brilliant blue and allura red, 2.5 g/L for sunset yellow and erythrosine in blank samples that yielded a signal-to-noise
(S/N) ratio of 3 and 10 were in the range of 0.45–1.51 g/L and 1.51–5.0 g/L, respectively. 3.5. Sample analysis To further validate the feasibility of the proposed method, it was used for the analysis of sixty wine samples (including dry red wine, medium dry wine, sweet wine and medium sweet wine) and thirty soft drinks. A blank extract spiked at the low calibration level (5.0 g/L for tartrazine, amaranth, carmine, brilliant blue and allura red, 2.5 g/L for sunset yellow and erythrosine) was used to control the extraction efficiency, and UFLC–MS/MS extracted ion chromatograms were shown in Fig. 2, and no interference peak was observed in the chromatograms. The obtained results showed that sunset yellow was in three out of thirty soft drink samples (2.95–42.6 g/L), and erythrosine in one out of fifteen dry red wine sample (3.22 g/L), respectively, and other synthetic pigments were not detected in the analyzed samples with the proposed method. The typical UFLC–MS/MS chromatograms for the two examined samples were shown in Fig. 3. It indicates that a variety of illegal synthetic pigments were illegally added into market wines and soft drinks by some illicit manufacturers of Zhejiang Province, China. 4. Conclusions In this work, The M-dSPE extraction procedure using NH2 LDC-MP with rapid magnetic separation was optimized for the synthetic pigments. Acceptable recoveries for the seven studied synthetic pigments were obtained in the range of 84.0–116.2%. The results demonstrate that the accuracy and precision of the proposed M-dSPE coupled with UFLC–MS/MS method are satisfactory for analysis of the synthetic pigments in wines and soft drinks. The
Please cite this article in press as: X.-H. Chen, et al., J. Chromatogr. A (2014), http://dx.doi.org/10.1016/j.chroma.2014.04.060
G Model CHROMA-355352; No. of Pages 6
ARTICLE IN PRESS X.-H. Chen et al. / J. Chromatogr. A xxx (2014) xxx–xxx
6
present work reveals that the NH2 -LDC-MP has potential applications in the rapid effective cleanup procedure. Acknowledgements We also would like to thank the National Natural Science Foundation of China (No. 21377114), the Zhejiang Provincial Natural Science Foundation, China (No. LY12H26003, LY13B050003, LY14B040006), the Medical Health Foundation for Key Talents in Zhejiang Province, China (No. 2013KYA187), the Agriculture and Social Development Funds of Ningbo, China (No. 2011C50058), Zhejiang Provincial Analytical Foundation of China (No. 2012C37002, 2013C37089), the Advanced Key Program of Agriculture and Social Development Funds of Ningbo, China (No. 2011C11021) and Zhejiang Provincial Program for the Cultivation of High-level Innovative Health Talents for their financial support. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chroma. 2014.04.060. References [1] National Standard of the People’s Republic of China, GB/T 5009, 35-1996, Method for Determination of Synthetic Pigments in Foods, 1996. [2] F. Feng, Y. Zhao, W. Yong, L. Sun, G. Jiang, X. Chu, J. Chromatogr. B 879 (2011) 1813. [3] PRC Ministry of Health, Hygienic Standards for Uses of Food Additives, National Standard of the P.R.C, Beijing, 2007, pp. 90. [4] N. Yoshioka, K. Ichihashi, Talanta 74 (2008) 1408.
[5] IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Man, 8, IARC, Lyon, 1975, pp. 125. [6] K.S. Minioti, C.F. Sakellariou, N.S. Thomaidis, Anal. Chim. Acta 583 (2007) 103. ˜ P. Fernández-Hernando, J.S. [7] L. Enríquez-Gabeiras, A. Galleg, R.M. Garcinuno, Durand, Food Chem. 135 (2012) 193. [8] F.A.O. Olgun, B.D. Ozturk, R. Apak, Food Anal. Methods 5 (2012) 1335. [9] Y.S. Al-Degs, Food Chem. 117 (2009) 485. [10] S. Bonan, G. Fedrizzi, S. Menotta, C. Elisabetta, Dyes Pigm. 99 (2013) 36. [11] M. Holcapek, K. Volná, D. Vanˇerková, Dyes Pigm. 75 (2007) 156. [12] T. Zou, P. He, A. Yasen, Z. Li, Food Chem. 138 (2013) 1742. [13] M. Ma, X. Luo, B. Chen, S. Su, S. Yao, J. Chromatogr. A 1103 (2006) 170. [14] Q. Gao, D. Luo, J. Ding, Y.-Q. Feng, J. Chromatogr. A 1217 (2010) 5602. [15] L. Bai, B. Mei, Q.-Z. Guo, Z.-G. Shi, Y.-Q. Feng, J. Chromatogr. A 1217 (2010) 7331. [16] J. Ding, Q. Gao, D. Luo, Z.-G. Shi, Y.-Q. Feng, J. Chromatogr. A 1217 (2010) 7351. [17] Q. Gao, D. Luo, M. Bai, Z.-W. Chen, Y.-Q. Feng, J. Agric. Food Chem. 59 (2011) 8543. [18] I.S. Ibarra, J.A. Rodriguez, J.M. Miranda, M. Vega, E. Barrado, J. Chromatogr. A 1218 (2011) 2196. [19] Y.G. Zhao, H.Y. Shen, J.W. Shi, X.H. Chen, M.C. Jin, J. Chromatogr. A 1218 (2011) 5568. [20] R.H. Savant, K. Banerjee, S.C. Utture, S.H. Patil, S. Dasgupta, M.S. Ghaste, P.G. Adsule, J. Agric. Food Chem. 58 (2010) 1447. [21] J.W. Wong, K. Zhang, K. Tech, D.G. Hayward, C.M. Makovi, A.J. Krynitsky, F.J. Schenck, K. Banerjee, S. Dasgupta, D. Brown, J. Agric. Food Chem. 58 (2010) 5868. [22] S. Walorczyk, B. Gnusowski, J. Chromatogr. A 1216 (2009) 6522. [23] A. Rashid, S. Nawaz, H. Barker, I. Ahmad, M. Ashraf, J. Chromatogr. A 1217 (2010) 2933. [24] S.J. Lehotay, K.A. Son, H. Kwon, U. Koesukwiwat, W.S. Fu, K. Mastovska, E. Hoh, N. Leepipatpiboon, J. Chromatogr. A 1217 (2010) 2548. [25] C. Basheer, A.A. Alnedhary, B.S. Madhava Rao, S. Valliyaveettil, H.K. Lee, Anal. Chem. 78 (2006) 2853. [26] J.M. Jiménez-Soto, S. Cárdenas, M. Valcárcel, J. Chromatogr. A 1245 (2012) 17. [27] X.H. Chen, Y.G. Zhao, H.Y. Shen, M.C. Jin, J. Chromatogr. A 1263 (2012) 34. [28] Y.G. Zhao, M.Q. Cai, X.H. Chen, S.D. Pan, S.S. Yao, M.C. Jin, Food Res. Int. 52 (2013) 350. [29] Y.G. Zhao, X.H. Chen, S.D. Pan, H. Zhu, H.Y. Shen, M.C. Jin, J. Mater. Chem. A 1 (2013) 11648. [30] Y.G. Zhao, X.H. Chen, S.D. Pan, H. Zhu, H.Y. Shen, M.C. Jin, Talanta 115 (2013) 787.
Please cite this article in press as: X.-H. Chen, et al., J. Chromatogr. A (2014), http://dx.doi.org/10.1016/j.chroma.2014.04.060
ARTICLE IN PRESS
CHROMA-355352; No. of Pages 6
Journal of Chromatography A, xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Short communication
Fast determination of seven synthetic pigments from wine and soft drinks using magnetic dispersive solid-phase extraction followed by liquid chromatography–tandem mass spectrometry夽 Xiao-Hong Chen a,b , Yong-Gang Zhao a,b , Hao-Yu Shen c , Li-Xin Zhou d , Sheng-Dong Pan a,b , Mi-Cong Jin a,b,∗ a Zhejiang Provincial Key Laboratory of Health Risk Appraisal for Trace Toxic Chemicals, Ningbo Municipal Center for Disease Control, Prevention, Ningbo, Zhejiang 315010, China b Ningbo Key Laboratory of Poison Research and Control, Ningbo Municipal Center for Disease Control and Prevention, Ningbo, Zhejiang 315010, China c Ningbo Institute of Technology, Zhejiang University, Ningbo, Zhejiang 315100, China d Medical School, Ningbo University, Ningbo, Zhejiang 315211, China
a r t i c l e
i n f o
Article history: Received 21 February 2014 Received in revised form 16 April 2014 Accepted 17 April 2014 Available online xxx Keywords: UFLC–MS/MS Amino-functionalized low degrees of cross-linking magnetic polymer (NH2 -LDC-MP) Magnetic dispersive solid-phase extraction (M-dSPE) Pigment Wine Soft drink
a b s t r a c t A novel, simple and sensitive method based on the use of magnetic dispersive solid-phase extraction (M-dSPE) procedure combined with ultra-fast liquid chromatography-tandem quadrupole mass spectrometry (UFLC–MS/MS) was developed to determine seven synthetic pigments (tartrazine, amaranth, carmine, sunset yellow, allura red, brilliant blue and erythrosine) in wines and soft drinks. An amino-functionalized low degrees of cross-linking magnetic polymer (NH2 -LDC-MP) was synthesized via suspension polymerization, and characterized by transmission electron microscopy (TEM). The NH2 LDC-MP was used as the M-dSPE sorbent to remove the matrix from the solution, and the main factors affecting the extraction were investigated in detail. The obtained results demonstrated the higher extraction capacity of NH2 -LDC-MP with recoveries between 84.0 and 116.2%. The limits of quantification (LOQs) for the seven synthetic pigments were between 1.51 and 5.0 g/L in wines and soft drinks. The developed M-dSPE UFLC–MS/MS method had been successfully applied to the real wines and soft drinks for food-safety risk monitoring in Zhejiang Province, China. The results showed that sunset yellow was in three out of thirty soft drink samples (2.95–42.6 g/L), and erythrosine in one out of fifteen dry red wine samples (3.22 g/L), respectively. It was confirmed that the NH2 -LDC-MP was a kind of highly effective M-dSPE materials for the pigments analyses. © 2014 Elsevier B.V. All rights reserved.
1. Introduction To protect consumers from health risks, many countries have established strict regulations for the allowable kinds and concentrations of synthetic pigments [1,2]. In China, the common water-soluble synthetic pigments e.g. amaranth, brilliant blue, carmine, sunset yellow, and tartrazine, are permitted in foodstuff [3,4], however, all ingredients including food pigments are required
夽 Presented at the 40th International Symposium on High Performance Liquid Phase Separations and Related Techniques (HPLC 2013 Hobart), Hobart, Tasmania, Australia, 18–21 November 2013. ∗ Corresponding author at: Ningbo Key Laboratory of Poison Research and Control, Ningbo Municipal Center for Disease Control and Prevention, Ningbo, Zhejiang 315010, China. Tel.: +86 574 87274559; fax: +86 57487277153. E-mail addresses: [email protected], [email protected] (M.-C. Jin).
to be listed on the food labels. Nowadays, more evidence indicates that the abuse of synthetic pigments may cause cancer [2,5], and some people are much sensitive to particular food pigments. Therefore, it is necessary for the determination of synthetic pigments to ensure the food safety. Today, some high-performance liquid chromatography (HPLC) methods including ultraviolet/visible (UV/vis), diode-array detectors (DAD) [6–10] and mass spectrometry (MS) [2,11,12], have been proposed for the determination of synthetic colorants. In addition, many pretreatment methods, such as liquid–liquid extraction (LLE) and solid-phase extraction (SPE), are used for the extraction of synthetic pigments from various food [2,13], however, these protocols are laborious and time consuming. Luckily, several other cleanup methods based on the traditional SPE technique, such as magnetic solid-phase extraction, dispersive solid-phase extraction (dSPE), micro-solid-phase extraction (-SPE) and dispersive
http://dx.doi.org/10.1016/j.chroma.2014.04.060 0021-9673/© 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: X.-H. Chen, et al., J. Chromatogr. A (2014), http://dx.doi.org/10.1016/j.chroma.2014.04.060
G Model CHROMA-355352; No. of Pages 6
ARTICLE IN PRESS X.-H. Chen et al. / J. Chromatogr. A xxx (2014) xxx–xxx
2
micro-solid-phase extraction (d--SPE), have been widely used for multi-residue analyses [14–26]. Although dSPE is considered as one of the most powerful cleanup technologies, to the best of our knowledge, there are no literatures by using dSPE in the analysis of synthetic pigments. In our previous work, an improved ethylenediaminefunctionalized magnetic polymer (IEDA-MP) was found to have an excellent cleanup ability for selective removal of the matrix in red wine [27]. By using IEDA-MP dSPE for cleaning up the studied synthetic pigments, a short sample preparation time and an easy cleanup procedure for the removal of various natural pigments, organic acids and sugars in wine and soft drinks were expected to be achieved, however, some analytes were also adsorbed onto IEDA-MP resulting in low recoveries. The IEDA-MP surface is generally covered with amino groups ( NH , NH2 ), which provide a special polar hook for enhanced capture of polar analytes. Therefore, we inspired that amino-functionalized low degrees of cross-linking magnetic polymer (NH2 -LDC-MP) contained less hydrophilic amino groups and more lipophilic styrene monomer would be probably become a powerful sorbent to carry out magnetic dispersive solid-phase extraction (M-dSPE) for cleaning up the studied synthetic pigments from wine and soft drinks. In this work, the effect of the usage amount of cross-linker i.e., styrene (St) used in the co-polymerization procedure, which would subsequently lead to obtain various NH2 -LDC-MP with different amount of amino groups and lipophilic styrene monomer, on the recovery of seven synthetic pigments has been investigated. In addition, an analytical procedure combined the fast M-dSPE cleanup technique with the LC–MS/MS has been proposed. It has been shown to be effective, fast and accurate in the routine analyses.
2. Experimental 2.1. Reagents and materials Ferric chloride, ferrous sulfate, oleic acid (OA), ethanol, methyl methacrylate (MMA), styrene (St), glycidylmethacrylate (GMA), ethylenediamine (EDA), polyvinyl alcohol (PVA 217) and benzoyl peroxide (BPO) of analytical grade were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Primary secondary amine (PSA) was purchased from Agilent Company (Palo Alto, USA). Tartrazine, amaranth, carmine, sunset yellow, allura red, brilliant blue and erythrosine were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). Acetonitrile and ammonium acetate (AmAc) of HPLC grade were purchased from Merck Company (Darmstadt, Germany). The wines and soft drinks were acquired on the local markets (Ningbo, China).
2.2. Equipment The characterizations of LDC-MP were carried out by using transmission electron microscopy (TEM) (Hitachi H-7650) (Hitachi, Japan), vibrating sample magnetometer (VSM) (Lake Shore 7410) (Westerville, USA), and elementary analyzer (EA) (ThermoFisher Flash-1112) (ThermoFisher, USA). A vortex mixer Hualida WH-866 (Taicang, China) was used during extraction. UFLC-MS/MS analyses were performed with Prominence UFLC XR system equipped with a LC-20AD pump system, a CTO-20AC column oven, a DGU20A3 degasser and a SIL-20AC autosampler (Shimadzu Corporation, Tokyo, Japan) and an AB SCIEX TRIPLE QUAD 5500 mass spectrometer (Applied Biosystems, Foster City, CA, USA). The UFLC–MS/MS system was controlled, and data were analyzed, on a computer
equipped with Analyst 1.5.1 (Applied Biosystems, Foster City, CA, USA). 2.3. Synthesis of NH2 -LDC-MP The NH2 -LDC-MP nanoparticles were prepared by the following steps based on the suspension polymerization and ring-opening reactions. 2.0 g of polyglycol was dissolved into 200 mL of hot water, followed by adding 0.04 mol of MMA, 20 mmol of St and 52 mmol of GMA. Then 2.0 g of OA-M (prepared by the reported procedure [27–30]) was added to the above system under ultrasonication. Finally, 1.0 g of BPO in 20 mL of ethanol was added dropwise under vigorously stirring. The mixture was allowed to react at 80 ◦ C for 3 h, yielding M-co-poly (MMA-St-GMA) polymer. The latter was isolated under magnetic field and washed with water and ethanol to remove redundant MMA, St and GMA. Exactly 1.25 g of the M-co-poly (MMA-St-GMA) was dispersed into 50 mL of methanol in a 100 mL flask. Then 3.5 mL (50 mmol) of EDA was added dropwise under stirring. The flask was then fitted with a water condenser and heated at 80 ◦ C for 8 h. The final improved amino-functionalized low degrees of cross-linking magnetic polymer named NH2 -LDC-MP-I was isolated under magnetic field and washed with water and methanol at pH 7.0 to remove the redundant diamine. Other NH2 -LDC-MP nanoparticles, named as NH2 -LDC-MP-II, NH2 -LDC-MP-III and NH2 -LDC-MP-IV, were synthesized in a similar way with different amounts of St (10, 5 and 0 mmol, respectively). All the NH2 -LDC-MP nanoparticles were dried in a vacuum oven at 60 ◦ C and stored in a sealed bottle for further use. 2.4. M-dSPE procedure An exactly 1.0 mL of sample was placed into an open evaporating dish and evaporated to dryness in a water bath at 80 ◦ C. Afterwards, the residue was redissolved with pure water (adjusted to pH 9.0 with ammonia 0.5 mol/L) and transferred to a polypropylene centrifuge tube (2.0 mL) containing 15.0 mg of NH2 -LDC-MP. The tube was vortexed for 1.0 min, and the adsorbed NH2 -LDC-MP was isolated under magnetic field. About 0.5 mL aliquot of the supernatant was filtered using a 0.22 m membrane prior to its injection into the UFLC–MS/MS system. 2.5. LC–QqQ-MS/MS analysis The chromatographic separation was performed on a Shimpack XR-ODS II (100 mm × 2.0 mm i.d., 2.2 m) by using 5.0 mmol/L AmAc in acetonitrile as eluent (A), and 5.0 mmol/L AmAc in water as eluent (B) as the mobile phase. The linear gradient was: 0→3.00 min, 2.0→10.0% A; 3.00→7.00 min, 10.0→80.0% A; 7.00→7.01 min, 80.0→2.0% A and 7.01→9.00 min, 2.0% A. The separation was accomplished at a constant flow of 0.40 mL/min. The column was thermostated at 40 ◦ C to increase the retention time reproducibility. The injection volume was 5.0 L. Mass spectrometry analysis was performed using an electrospray ionization source in negative mode. The operation conditions were as follows: ion spray voltage, −4500 V; curtain gas (CUR), 40 psi and interface heater was on; collision gas, medium; nebulizer gas (gas 1) and heater gas (gas 2), 50 and 50 psi; the turbo spray temperature, 500 ◦ C; entrance potential (EP), −10 V; collision cell exit potential (CXP), −10 V. Nitrogen was used in all cases. Multiple-reaction monitoring (MRM) mode was used for quantification. The results of the precursor ion, product ion are shown in Table 1. Applied Biosystems/MDS Sciex Analyst software (versions 1.5.1) was used for data acquisition and processing.
Please cite this article in press as: X.-H. Chen, et al., J. Chromatogr. A (2014), http://dx.doi.org/10.1016/j.chroma.2014.04.060
G Model CHROMA-355352; No. of Pages 6
ARTICLE IN PRESS X.-H. Chen et al. / J. Chromatogr. A xxx (2014) xxx–xxx
3
Table 1 Q1/Q3 ion pairs, linear equations, determination coefficients (r2 ), linear ranges, limits of detection (LODs) and limits of quantification (LOQs) of seven synthetic pigments. Synthetic pigment Tartrazine Amaranth Carmine Sunset yellow Allura red Brilliant blue Erythrosine a b
Precursor ion (Q1, m/z) 233.1 268.2 268.1 202.9 225.1 373.8 834.7
Product ion (Q3, m/z) a
197.9 , 117.0, 80 206.0a , 302.0 301.9a , 206.1, 80 170.9a , 207.0, 156 206.0a , 199.9 170.0a , 333.6 536.8a , 662.7
Linear equationb
r2
A = 1383C − 652 A = 2790C + 301 A = 2030C + 698 A = 5490C + 350 A = 1920C + 360 A = 2600C − 676 A = 3510C − 291
0.9993 0.9991 0.9996 0.9994 0.9990 0.9998 0.9998
Linear range (g/L) 5.0–1000.0 5.0–1000.0 5.0–1000.0 2.5–500.0 5.0–1000.0 5.0–1000.0 2.5–500.0
LOD (g/L)
LOQ (g/L)
1.25 1.51 1.00 0.45 1.22 0.71 0.50
4.16 5.00 3.33 1.51 4.02 2.38 1.66
Quantitative ion. A, peak area; C, mass concentration, g/L.
2.6. Method validation 2.6.1. Standard preparation Individual stock standard solutions were prepared at 1000 mg/L level by exact weighing and dissolution in water–methanol (1:1, v/v). The stock mixture standard solution (10.0 mg/L) was prepared by appropriate dilution of the stock solutions with water–methanol (1:1, v/v). Matrix-matched calibration standards were spiked with concentration in the range of 5.0–1000 g/L for tartrazine, amaranth, carmine, brilliant blue and allura red, 2.5–500 g/L for sunset yellow and erythrosine, respectively. The matrix-matched
calibration curves made by peak area vs concentration (g/L) were used to calibrate spike samples in recovery experiments. 2.6.2. Spiked sample Spiked recoveries were performed at concentrations of 8.0, 80, and 800 g/L for tartrazine, amaranth, carmine, brilliant blue, and allura red, 4.0, 40 and 400 g/L for sunset yellow and erythrosine in five kinds of samples, i.e., dry red wine, medium dry wine, sweet wine and medium sweet wine and soft drinks, respectively. For each spiked sample, stock mixture solution of the standards was added to 1.0 mL sample, which was free from the target compounds.
Fig. 1. TEM images of NH2 -LDC-MP-I(a), NH2 -LDC-MP-II(b), NH2 -LDC-MP-III(c), and NH2 -LDC-MP-IV(d) nanoparticles.
Please cite this article in press as: X.-H. Chen, et al., J. Chromatogr. A (2014), http://dx.doi.org/10.1016/j.chroma.2014.04.060
G Model CHROMA-355352; No. of Pages 6
ARTICLE IN PRESS X.-H. Chen et al. / J. Chromatogr. A xxx (2014) xxx–xxx
4
Table 2 Validation parameters obtained for the seven synthetic pigments at three concentration levels in dry red wine, medium dry wine, sweet wine and medium sweet wine and soft drink matrix. Synthetic pigments
Added (g/L)
Average recovery a ,% (RSD, %) Dry red wine b
c
Medium dry wine
Sweet wine
Medium sweet wine
Soft drink
Tartrazine
8.0 80.0 800.0
98.2 (2.1 , 1.5 ) 106.2 (2.6, 3.7) 97.5 (5.1, 6.8)
92.3 (3.9, 5.2) 116.2 (5.8, 6.9) 106.5 (3.5, 6.8)
90.6 (3.2, 5.7) 98.0 (4.4, 7.2) 95.6 (5.0, 6.8)
95.8 (3.5, 5.1) 91.0 (2.2, 4.7) 96.6 (4.0, 6.4)
93.9 (2.6, 5.0) 86.8 (3.0, 5.3) 89.2 (1.9, 5.2)
Amaranth
8.0 80.0 800.0
82.5 (2.4, 5.1) 90.1 (2.5, 6.6) 92.7 (3.6, 5.2)
92.8 (5.5, 7.4) 89.0 (3.3, 4.6) 95.9 (4.9, 5.2)
92.9 (2.6, 5.0) 93.2 (3.0, 5.3) 104.0 (5.2, 5.8)
82.8 (2.8, 5.0) 96.9 (3.8, 5.9) 97.5 (8.6, 9.7)
83.2 (5.1, 5.6) 97.7 (3.4, 6.2) 96.3 (5.0, 5.8)
Carmine
8.0 80.0 800.0
91.3 (2.6, 4.6) 96.6 (2.4, 4.6) 88.0 (1.9, 2.7)
92.4 (2.7, 3.7) 98.6 (3.0, 4.6) 89.1 (2.8, 5.0)
101.6 (5.7, 6.0) 99.7 (3.9, 4.7) 90.2 (1.4, 3.2)
92.8 (3.5, 7.1) 101.0 (3.3, 5.7) 95.0 (4.9, 5.6)
96.9 (3.6, 5.0) 90.8 (4.0, 4.3) 84.0 (6.2, 7.7)
Sunset yellow
4.0 40.0 400.0
86.2 (3.8, 5.7) 97.9 (8.1, 6.8) 92.6 (3.4, 7.1)
92.9 (5.8, 5.9) 86.5 (8.2, 7.8) 92.8 (5.5, 5.4)
97.0 (4.4, 7.2) 95.2 (3.0, 6.8) 96.9 (2.6, 3.0)
92.4 (1.7, 6.7) 90.6 (2.0, 3.5) 99.7 (1.6, 4.9)
92.1 (3.7, 6.0) 99.7 (2.9, 3.7) 93.3 (3.9, 6.2)
Allura red
8.0 80.0 800.0
90.3 (2.8, 3.6) 96.2 (3.4, 3.6) 92.0 (2.9, 2.8)
91.2 (2.7, 2.7) 99.2 (3.0, 3.6) 89.8 (4.8, 5.0)
102.6 (4.7, 5.0) 96.2 (2.9, 3.7) 93.2 (1.4, 3.2)
92.8 (2.5, 7.3) 100.0 (2.3, 5.2) 91.0 (2.9, 3.6)
92.9 (3.2, 4.0) 97.8 (3.0, 4.3) 89.0 (6.0, 7.2)
Brilliant blue
8.0 80.0 800.0
106.5 (5.2, 6.0) 89.2 (3.2, 4.7) 90.2 (2.4, 2.8)
92.8 (5.5, 5.4) 86.0 (6.3, 7.7) 85.3 (5.9, 6.2)
92.9 (3.6, 6.0) 90.8 (3.0, 7.3) 96.0 (2.2, 3.7)
99.2 (5.6, 5.7) 97.3 (3.1, 3.8) 96.5 (2.5, 5.8)
96.9 (3.8, 3.9) 96.5 (5.5, 6.5) 95.6 (4.0, 6.8)
Erythrosine
4.0 40.0 400.0
97.2 (4.4, 7.2) 85.3 (6.0, 6.8) 98.9 (1.6, 2.0)
93.3 (2.8, 5.7) 92.8 (4.0, 3.2) 103.7 (3.6, 6.2)
94.1 (6.7, 7.0) 89.2 (2.9, 5.8) 99.3 (3.9, 7.2)
90.8 (2.5, 5.4) 88.0 (2.3, 5.7) 97.6 (2.0, 6.4)
92.9 (2.6, 3.0) 89.3 (4.0, 4.6) 99.7 (2.9, 5.7)
a b c
The mean value was determined in one day (n = 6 replicates). Intra-day, n = 6 replicates. Inter-day, n = 3 replicates × 6 days within a 2-week period.
The spiked samples prepared were stored at 4 ◦ C for about 12 h to let the synthetic pigments permeate uniformly into the samples. 3. Results and discussion 3.1. Characterization of NH2 -LDC-MP The cross-linker used can affect the degree of crosslinking in the polymeric network. This work focused on the effect of the usage amount of cross-linker i.e., styrene (St) used in the co-polymerization procedure, which would subsequently lead to obtain various NH2 -LDC-MP nanoparticles with different amount of amino groups and lipophilic styrene monomer, on the recovery of seven synthetic pigments. Fig. 1 shows the TEM images of the NH2 LDC-MP nanoparticles with different amount of St as cross-linkers. As can be seen in Fig. 1, NH2 -LDC-MP-II prepared with 10 mmol of St was more uniform and spherical in shape than those prepared with 20, 5 and 0 mmol of St. The paramagnetic properties were verified by VSM, and the results showed that the saturation moments of NH2 -LDC-MP obtained from the hysteresis loop were in the range of 7.86–8.02 emu/g. The NH2 -LDC-MP was proved to respond to magnetic fields without any permanent magnetization. This NH2 LDC-MP characteristic could ensure to separate more quickly and easily in two-phase (solid/liquid phase) systems due to the stronger magnetism. 3.2. Optimization of UFLC–MS/MS conditions The development process of the analysis method began with the optimization of the UFLC–MS/MS data acquisition parameters. For this purpose, a compromise had to be sought between sensitivity and selectivity to choose the appropriate MS/MS multiple reaction monitoring (MRM) transitions. Suitable precursor-to-product MS/MS transitions were selected through the optimized procedure. The final MS/MS conditions were detailed in Table 1, and the MS/MS spectra of the seven synthetic pigments were shown in Fig. S1.
In order to achieve an optimal chromatographic separation, the gradient elution and the effects of AmAc and ammonia concentration on the chromatographic separation were studied. Based on the optimization, acetonitrile-AmAc (5.0 mmol/L) aqueous solution was selected as the mobile phase system for the separation of the seven studied synthetic pigments in gradient elution. 3.3. NH2 -LDC-MP M-dSPE procedure and its optimization In the study, the effect of pH value of samples and the amount of NH2 -LDC-MP on the d-SPE cleanup properties were investigated in detail. Firstly, various pH values from 2.0 to 11.0 were used to optimize the cleanup procedure by mixing 15.0 mg NH2 -LDC-MP with 1.0 mL sample spiked at a concentration of 8.0 g/L for tartrazine, amaranth, carmine, brilliant blue, and allura red, 4.0 g/L for sunset yellow and erythrosine, respectively, in a 2.0 mL polypropylene centrifuge tube. The obtained results indicated that with the increasing of pH values from 2.0 to 9.0, the recoveries of seven synthetic pigments increased significantly, as shown in Fig. S2(a). In the case of pH values above 9.0, the satisfactory recoveries of the seven synthetic pigments were consistently in the range of 90.2–103.3%. Above all, pH value 9.0 was selected for subsequent experiments. Secondly, seven dosages of NH2 -LDC-MP (5, 10, 15, 20, 30, 40 and 50 mg for 1.0 mL sample) on the cleanup properties were investigated to develop an efficient cleanup procedure for the seven synthetic pigments. The tested results showed that the combination of the least amounts of NH2 -LDC-MP adsorbents in the proportion of 10–20 mg per 1.0 mL sample would ensure efficient and robust cleanup to maintain high quantitative recoveries, as shown in Fig. S2(b). Furthermore, the results of recycling experiment showed that NH2 -LDC-MP could be reused at least twelve times without much sacrifice of the cleanup efficiency. The preparative reproducibility of the NH2 -LDC-MP used for the cleanup procedure was deeply evaluated. And the results show that the absolute deviations of the
Please cite this article in press as: X.-H. Chen, et al., J. Chromatogr. A (2014), http://dx.doi.org/10.1016/j.chroma.2014.04.060
G Model CHROMA-355352; No. of Pages 6
ARTICLE IN PRESS X.-H. Chen et al. / J. Chromatogr. A xxx (2014) xxx–xxx
5
Fig. 3. MRM chromatograms for two examined samples.
Fig. 2. MRM chromatograms for dry red wine sample spiked with seven synthetic pigments at 5.0 g/L for tartrazine, amaranth, carmine, brilliant blue and allura red, 2.5 g/L for sunset yellow and erythrosine under NH2 -LDC-MP M-dSPE cleanup procedure. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
recoveries for synthetic pigments by the three batches are less than 5.0%. This indicates that the preparation procedure of NH2 -LDC-MP has good repeatability and reproducibility.
3.4. Method linearity, accuracy, LOD and LOQ The linearity of the calibration curves made by peak area vs concentration (g/L) was studied using matrix-matched calibration standards in samples. The response function was found to be linear with a determination coefficient (r2 ) higher than 0.9990 in the tested range listed in Table 1 for the seven synthetic pigments. The method accuracies were expressed as the recoveries, and the method precisions were expressed as the intra-and interday relative standard deviations (RSDs). The intra-day RSDs were obtained by repeating the three levels of spiked samples six times within a day, and the inter-day RSDs were obtained by repeating the three levels of spiked samples in triplicate on six separate days within a 2-week period. The results are summarized in Table 2. It shows that the majority of mean recoveries are in the range of 84.0–116.2% with the intra-day RSDs ranging from 1.4 to 8.6% and inter-day RSDs ranging from 1.5 to 9.7%. The limits of detection (LODs) and limits of quantification (LOQs), for the analyzed synthetic pigments are shown in Table 1. The LODs and LOQs, which were calculated on the analysis of seven synthetic pigments spiked at 5.0 g/L for tartrazine, amaranth, carmine, brilliant blue and allura red, 2.5 g/L for sunset yellow and erythrosine in blank samples that yielded a signal-to-noise
(S/N) ratio of 3 and 10 were in the range of 0.45–1.51 g/L and 1.51–5.0 g/L, respectively. 3.5. Sample analysis To further validate the feasibility of the proposed method, it was used for the analysis of sixty wine samples (including dry red wine, medium dry wine, sweet wine and medium sweet wine) and thirty soft drinks. A blank extract spiked at the low calibration level (5.0 g/L for tartrazine, amaranth, carmine, brilliant blue and allura red, 2.5 g/L for sunset yellow and erythrosine) was used to control the extraction efficiency, and UFLC–MS/MS extracted ion chromatograms were shown in Fig. 2, and no interference peak was observed in the chromatograms. The obtained results showed that sunset yellow was in three out of thirty soft drink samples (2.95–42.6 g/L), and erythrosine in one out of fifteen dry red wine sample (3.22 g/L), respectively, and other synthetic pigments were not detected in the analyzed samples with the proposed method. The typical UFLC–MS/MS chromatograms for the two examined samples were shown in Fig. 3. It indicates that a variety of illegal synthetic pigments were illegally added into market wines and soft drinks by some illicit manufacturers of Zhejiang Province, China. 4. Conclusions In this work, The M-dSPE extraction procedure using NH2 LDC-MP with rapid magnetic separation was optimized for the synthetic pigments. Acceptable recoveries for the seven studied synthetic pigments were obtained in the range of 84.0–116.2%. The results demonstrate that the accuracy and precision of the proposed M-dSPE coupled with UFLC–MS/MS method are satisfactory for analysis of the synthetic pigments in wines and soft drinks. The
Please cite this article in press as: X.-H. Chen, et al., J. Chromatogr. A (2014), http://dx.doi.org/10.1016/j.chroma.2014.04.060
G Model CHROMA-355352; No. of Pages 6
ARTICLE IN PRESS X.-H. Chen et al. / J. Chromatogr. A xxx (2014) xxx–xxx
6
present work reveals that the NH2 -LDC-MP has potential applications in the rapid effective cleanup procedure. Acknowledgements We also would like to thank the National Natural Science Foundation of China (No. 21377114), the Zhejiang Provincial Natural Science Foundation, China (No. LY12H26003, LY13B050003, LY14B040006), the Medical Health Foundation for Key Talents in Zhejiang Province, China (No. 2013KYA187), the Agriculture and Social Development Funds of Ningbo, China (No. 2011C50058), Zhejiang Provincial Analytical Foundation of China (No. 2012C37002, 2013C37089), the Advanced Key Program of Agriculture and Social Development Funds of Ningbo, China (No. 2011C11021) and Zhejiang Provincial Program for the Cultivation of High-level Innovative Health Talents for their financial support. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chroma. 2014.04.060. References [1] National Standard of the People’s Republic of China, GB/T 5009, 35-1996, Method for Determination of Synthetic Pigments in Foods, 1996. [2] F. Feng, Y. Zhao, W. Yong, L. Sun, G. Jiang, X. Chu, J. Chromatogr. B 879 (2011) 1813. [3] PRC Ministry of Health, Hygienic Standards for Uses of Food Additives, National Standard of the P.R.C, Beijing, 2007, pp. 90. [4] N. Yoshioka, K. Ichihashi, Talanta 74 (2008) 1408.
[5] IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Man, 8, IARC, Lyon, 1975, pp. 125. [6] K.S. Minioti, C.F. Sakellariou, N.S. Thomaidis, Anal. Chim. Acta 583 (2007) 103. ˜ P. Fernández-Hernando, J.S. [7] L. Enríquez-Gabeiras, A. Galleg, R.M. Garcinuno, Durand, Food Chem. 135 (2012) 193. [8] F.A.O. Olgun, B.D. Ozturk, R. Apak, Food Anal. Methods 5 (2012) 1335. [9] Y.S. Al-Degs, Food Chem. 117 (2009) 485. [10] S. Bonan, G. Fedrizzi, S. Menotta, C. Elisabetta, Dyes Pigm. 99 (2013) 36. [11] M. Holcapek, K. Volná, D. Vanˇerková, Dyes Pigm. 75 (2007) 156. [12] T. Zou, P. He, A. Yasen, Z. Li, Food Chem. 138 (2013) 1742. [13] M. Ma, X. Luo, B. Chen, S. Su, S. Yao, J. Chromatogr. A 1103 (2006) 170. [14] Q. Gao, D. Luo, J. Ding, Y.-Q. Feng, J. Chromatogr. A 1217 (2010) 5602. [15] L. Bai, B. Mei, Q.-Z. Guo, Z.-G. Shi, Y.-Q. Feng, J. Chromatogr. A 1217 (2010) 7331. [16] J. Ding, Q. Gao, D. Luo, Z.-G. Shi, Y.-Q. Feng, J. Chromatogr. A 1217 (2010) 7351. [17] Q. Gao, D. Luo, M. Bai, Z.-W. Chen, Y.-Q. Feng, J. Agric. Food Chem. 59 (2011) 8543. [18] I.S. Ibarra, J.A. Rodriguez, J.M. Miranda, M. Vega, E. Barrado, J. Chromatogr. A 1218 (2011) 2196. [19] Y.G. Zhao, H.Y. Shen, J.W. Shi, X.H. Chen, M.C. Jin, J. Chromatogr. A 1218 (2011) 5568. [20] R.H. Savant, K. Banerjee, S.C. Utture, S.H. Patil, S. Dasgupta, M.S. Ghaste, P.G. Adsule, J. Agric. Food Chem. 58 (2010) 1447. [21] J.W. Wong, K. Zhang, K. Tech, D.G. Hayward, C.M. Makovi, A.J. Krynitsky, F.J. Schenck, K. Banerjee, S. Dasgupta, D. Brown, J. Agric. Food Chem. 58 (2010) 5868. [22] S. Walorczyk, B. Gnusowski, J. Chromatogr. A 1216 (2009) 6522. [23] A. Rashid, S. Nawaz, H. Barker, I. Ahmad, M. Ashraf, J. Chromatogr. A 1217 (2010) 2933. [24] S.J. Lehotay, K.A. Son, H. Kwon, U. Koesukwiwat, W.S. Fu, K. Mastovska, E. Hoh, N. Leepipatpiboon, J. Chromatogr. A 1217 (2010) 2548. [25] C. Basheer, A.A. Alnedhary, B.S. Madhava Rao, S. Valliyaveettil, H.K. Lee, Anal. Chem. 78 (2006) 2853. [26] J.M. Jiménez-Soto, S. Cárdenas, M. Valcárcel, J. Chromatogr. A 1245 (2012) 17. [27] X.H. Chen, Y.G. Zhao, H.Y. Shen, M.C. Jin, J. Chromatogr. A 1263 (2012) 34. [28] Y.G. Zhao, M.Q. Cai, X.H. Chen, S.D. Pan, S.S. Yao, M.C. Jin, Food Res. Int. 52 (2013) 350. [29] Y.G. Zhao, X.H. Chen, S.D. Pan, H. Zhu, H.Y. Shen, M.C. Jin, J. Mater. Chem. A 1 (2013) 11648. [30] Y.G. Zhao, X.H. Chen, S.D. Pan, H. Zhu, H.Y. Shen, M.C. Jin, Talanta 115 (2013) 787.
Please cite this article in press as: X.-H. Chen, et al., J. Chromatogr. A (2014), http://dx.doi.org/10.1016/j.chroma.2014.04.060