2439
J. Sep. Sci. 2014, 37, 2439–2445
Juan Li1,2 Xiaoming Ding1 Jiaxin Zheng1,2 Dandan Liu1 Fei Guo1 Hongmin Liu1,2 ∗ Yanbing Zhang1,2 1 School
of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, China 2 New Drug Research & Development Center, Zhengzhou University, Zhengzhou, China Received March 31, 2014 Revised May 12, 2014 Accepted May 27, 2014
Research Article
Determination of synthetic dyes in bean and meat products by liquid chromatography with tandem mass spectrometry A sensitive and efficient method was developed for the simultaneous determination of eight synthetic dyes (Chrysoidin, Auramine O, Sudan(I–IV), Para Red, and Rhodamine B) in bean and meat products using high-performance liquid chromatography with tandem mass spectrometry. A simple extraction procedure using acetonitrile has been applied for the extraction of these dyes from spiked bean and meat samples. Chromatographic separation was achieved on a Waters XTerra C18 column (2.1 × 150 mm, 5 m) with a multistep gradient elution. Detection and quantification were performed using mass spectrometry in multiple reaction monitoring mode. Linear calibrations were obtained with correlation coefficients R2 > 0.99. The limits of detection and quantification for the eight dyes were in the ranges of 0.03–0.75 and 0.1–2.0 g/kg depending on matrices, respectively. The recoveries of these dyes in different food matrices were between 71.2 and 116.9% with relative standard deviations 98%. The molecular structures of the eight synthetic dyes are shown in Fig. 1.
2.2 Preparation of standard solutions Standard stock solutions (100 g/mL in acetonitrile) were separately prepared for eight synthetic dyes, and the standard solutions were stored in darkness at −20⬚C until use. Mixed standard stock solution was prepared by mixing and diluting the standard stock solution of each dye with acetonitrile to a final concentration of 10 g/mL (Sudan (I–IV), Para Red, Chrysoidin) and 1 g/mL (Rhodamine B, Auramine O). Standard working solutions were daily prepared in the determination process by acetonitrile/H2 O (50:50, v/v). C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2.3 Sample Pretreatment In order to validate the method, six different types of food samples (dried beancurd, sausage, yuba, yellow croaker, stewed chicken, and bean sauce) were purchased from a local market and stored at 4⬚C until they were processed. Dyespiked samples were prepared in a 15 mL centrifuge tube by mixing 2 g of each matrix with a series of the eight-dye mixed standard solutions at various concentrations, homogenized for 2 min, and kept at ambient temperature for 30 min before extraction. Then, sample extraction was achieved by adding 6 mL acetonitrile solution. The tube was shaken by hand for 30 s in order to allow the sample to mix thoroughly with the solvent, and the extraction continued for 30 min in an ultrasonic bath. The extraction solution was centrifuged at 12 000 rpm for 10 min to sediment the solids. Finally, the extraction supernatant was filtered through 0.22 m nylon membrane prior to LC–MS analysis. Blank samples were pretreated in the same manner.
2.4 Cleanup efficiency of SPE In order to eliminate the co-extractives, Waters Oasis HLB cartridge (hydrophilic–lipophilic balanced RP) was chosen. For RP materials, the extracted supernatant was diluted with water until the content of acetonitrile was no >5%. The supernatant was passed through the reconditioned cartridges, and then 2 mL of water was passed. The analytes were eluted with 2 mL of methanol at a rate of about 1–2 drops/s and then evaporated under a stream of nitrogen gas at 45⬚C. Finally, the residue was reconstituted with 500 L of acetonitrile and filtered through a 0.22 m nylon membrane prior to LC–MS analysis.
2.5 LC–MS/MS Conditions A Waters HPLC system (Milford) including a 2695 separation module was controlled by Micromass Masslynx V4.1 software. Chromatographic separation was carried out by a Waters XTerra C18 RP column (2.1 × 150 mm, 5 m) and a binary gradient, which included 5 mM ammonium acetate buffer solution at pH 3.0 (mobile phase A) and methanol www.jss-journal.com
Other Techniques
J. Sep. Sci. 2014, 37, 2439–2445
2441
Figure 2. MRM chromatograms of the analytes obtained from dried beancurd spiked standard mix solution: (a) Sudan I (30 g/kg); (b) Sudan II (30 g/kg); (c) Sudan III (30 g/kg); (d) Sudan IV (30 g/kg); (e) Para Red (30 g/kg); (f) Chrysoidin (30 g/kg); (g) Auramine O (3 g/kg); (h) Rhodamine B (3 g/kg).
(mobile phase B). The linear gradient elution was programmed as follows: 0–10 min, 45–100% B; 10–20 min, 100% B; 20–25 min, 100–45% B. The flow rate was set at 0.2 mL/min while the column temperature was maintained at 35⬚C. The injection volume was 5 L. R , A triple quadrupole mass spectrometer (Quattro-Micro Waters, Manchester, UK) equipped with an electrospray source operating in a positive-ion mode was used for detection in MRM mode. The optimum conditions of the ESI interface for all target analytes were as follows: a capillary voltage of 3.0 kV, a source temperature of 120⬚C, and a dry temperature of 300⬚C. High purity N2 was used as both drying gas with a flow rate of 50 L/h and nebulizing gas with a flow rate of 450 L/h. The analysis was performed in just one time segment during the entire LC–MS run, which allowed obtaining sufficient scans for each peak. The average number of data points recorded across the chromatographic peaks of the analytes is 32. Each dye was analyzed using two MRM transitions in order to improve accuracy. One transition was used for quantification and qualification while the other was used as a supplemental data for qualification. For quantification, the most intense MRM transition was selected.
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
3 Results and discussion 3.1 LC–MS/MS analysis Satisfactory separation and responses for all analytes were obtained under the gradient elution conditions described above. MRM chromatograms of the analytes in spiked bean sample are shown in Fig. 2. The cone voltages and collision energies used for MRM transitions of each compound were optimized to increase sensitivity, and the results are listed in Table 1. The product-ion mass spectra of the eight dyes are shown in Fig. 3. The product ions were selected to be as specific as possible, avoiding the use of common losses to prevent false positives in the analysis of such complex matrices.
3.2 Optimization of sample pretreatment The development of an efficient sample extraction procedure is critical for accurate determination of dyes in food samples. Four extract solvents were evaluated in this experiment based on chemical properties of the eight dyes. The extract solvents include ethyl acetate, methanol, acetonitrile, and
www.jss-journal.com
2442
J. Sep. Sci. 2014, 37, 2439–2445
J. Li et al.
Table 1. Values of the instrumental settings optimized for the eight dyes tested
Compound
Retention time (min)
Molecular weight (g/mol)
Quantitative transition (m/z)
Qualitative transition (m/z)
Dwell time (s)
Cone voltage (V)
Collision energy (eV)
Sudan I
15.62
248.1
249.3/127.9
0.1
30
20
Sudan II
17.25
276.1
277.3/156.0
0.1
25
20
Sudan III
18.30
352.1
353.3/196.2
0.1
32
22
Sudan IV
20.72
380.2
381.3/224.2
0.1
32
23
Para Red
14.75
293.1
294.2/247.1
0.1
30
20
Chrysoidin
3.87
212.1
213.2/121.0
0.1
30
20
Auramine O
7.36
267.2
268.3/147.1
0.1
35
28
11.00
478.2
443.4/399.2
249.3/127.9 249.3/231.1 277.3/156.0 277.3/121.0 353.3/196.2 353.3/156.0 381.3/224.2 381.3/209.2 294.2/247.1 294.2/156.0 213.2/121.0 213.2/196.1 268.3/147.1 268.3/252.1 443.4/399.2 443.4/413.1
0.1
45
42
Rhodamine B
Table 2. Effect of different solvents on the extraction of eight synthetic dyes from dried beancurd spiked at 10 g/kg
Compound
Recovery (%) ± SD Ethyl acetate
Sudan I Sudan II Sudan III Sudan IV Para Red Chrysoidin Auramine O Rhodamine B
86.1 77.3 60.1 47.3 69.7 11.3 2.4 49.6
± ± ± ± ± ± ± ±
0.2 0.1 0.2 0.3 0.2 0.1 0.1 0.1
Acetonitrile 103.9 110.4 110.6 80.4 95.7 112.0 102.5 98.1
± ± ± ± ± ± ± ±
0.2 0.1 0.3 0.1 0.1 0.2 0.2 0.1
Acetone 70.1 44.3 9.4 9.7 64.9 5.9 35.5 79.4
± ± ± ± ± ± ± ±
0.2 0.1 0.1 0.1 0.2 0.1 0.1 0.3
Methanol 89.5 79.2 35.5 13.4 87.3 124.3 137.7 124.2
± ± ± ± ± ± ± ±
0.2 0.2 0.1 0.1 0.1 0.2 0.3 0.2
n = 5.
Figure 3. The product-ion mass spectra of (a) Sudan I; (b) Sudan II; (c) Sudan III; (d) Sudan IV; (e) Para Red; (f) Chrysoidin; (g) Auramine O; (h) Rhodamine B.
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
acetone. For bean and meat samples, acetonitrile showed good extraction efficiency in terms of recovery and reproducibility for the eight dyes. Most of the interference proteins present in food samples were precipitated in the presence of acetonitrile. When acetone was used for extraction solvent, the recoveries of Sudan III, Sudan IV, and Chrysoidin from the bean sample spiked at 10 g/kg were 100% indicates signal enhancement, and a value of 0.99 in all cases. The matrix effect was also evaluated during the validation of the method, as it is known that signal suppression or enhancement as a result of matrix effect can severely compromise quantitative analysis of the compounds at trace levels as well as method reproducibility. The matrix effect was studied by the ratio between the slope of matrix-matched and solvent calibration curves multiplied by 100 (signal suppression enhancement, %). A value of >100% indicates signal enhancement, and a value of
J. Sep. Sci. 2014, 37, 2439–2445
Juan Li1,2 Xiaoming Ding1 Jiaxin Zheng1,2 Dandan Liu1 Fei Guo1 Hongmin Liu1,2 ∗ Yanbing Zhang1,2 1 School
of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, China 2 New Drug Research & Development Center, Zhengzhou University, Zhengzhou, China Received March 31, 2014 Revised May 12, 2014 Accepted May 27, 2014
Research Article
Determination of synthetic dyes in bean and meat products by liquid chromatography with tandem mass spectrometry A sensitive and efficient method was developed for the simultaneous determination of eight synthetic dyes (Chrysoidin, Auramine O, Sudan(I–IV), Para Red, and Rhodamine B) in bean and meat products using high-performance liquid chromatography with tandem mass spectrometry. A simple extraction procedure using acetonitrile has been applied for the extraction of these dyes from spiked bean and meat samples. Chromatographic separation was achieved on a Waters XTerra C18 column (2.1 × 150 mm, 5 m) with a multistep gradient elution. Detection and quantification were performed using mass spectrometry in multiple reaction monitoring mode. Linear calibrations were obtained with correlation coefficients R2 > 0.99. The limits of detection and quantification for the eight dyes were in the ranges of 0.03–0.75 and 0.1–2.0 g/kg depending on matrices, respectively. The recoveries of these dyes in different food matrices were between 71.2 and 116.9% with relative standard deviations 98%. The molecular structures of the eight synthetic dyes are shown in Fig. 1.
2.2 Preparation of standard solutions Standard stock solutions (100 g/mL in acetonitrile) were separately prepared for eight synthetic dyes, and the standard solutions were stored in darkness at −20⬚C until use. Mixed standard stock solution was prepared by mixing and diluting the standard stock solution of each dye with acetonitrile to a final concentration of 10 g/mL (Sudan (I–IV), Para Red, Chrysoidin) and 1 g/mL (Rhodamine B, Auramine O). Standard working solutions were daily prepared in the determination process by acetonitrile/H2 O (50:50, v/v). C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2.3 Sample Pretreatment In order to validate the method, six different types of food samples (dried beancurd, sausage, yuba, yellow croaker, stewed chicken, and bean sauce) were purchased from a local market and stored at 4⬚C until they were processed. Dyespiked samples were prepared in a 15 mL centrifuge tube by mixing 2 g of each matrix with a series of the eight-dye mixed standard solutions at various concentrations, homogenized for 2 min, and kept at ambient temperature for 30 min before extraction. Then, sample extraction was achieved by adding 6 mL acetonitrile solution. The tube was shaken by hand for 30 s in order to allow the sample to mix thoroughly with the solvent, and the extraction continued for 30 min in an ultrasonic bath. The extraction solution was centrifuged at 12 000 rpm for 10 min to sediment the solids. Finally, the extraction supernatant was filtered through 0.22 m nylon membrane prior to LC–MS analysis. Blank samples were pretreated in the same manner.
2.4 Cleanup efficiency of SPE In order to eliminate the co-extractives, Waters Oasis HLB cartridge (hydrophilic–lipophilic balanced RP) was chosen. For RP materials, the extracted supernatant was diluted with water until the content of acetonitrile was no >5%. The supernatant was passed through the reconditioned cartridges, and then 2 mL of water was passed. The analytes were eluted with 2 mL of methanol at a rate of about 1–2 drops/s and then evaporated under a stream of nitrogen gas at 45⬚C. Finally, the residue was reconstituted with 500 L of acetonitrile and filtered through a 0.22 m nylon membrane prior to LC–MS analysis.
2.5 LC–MS/MS Conditions A Waters HPLC system (Milford) including a 2695 separation module was controlled by Micromass Masslynx V4.1 software. Chromatographic separation was carried out by a Waters XTerra C18 RP column (2.1 × 150 mm, 5 m) and a binary gradient, which included 5 mM ammonium acetate buffer solution at pH 3.0 (mobile phase A) and methanol www.jss-journal.com
Other Techniques
J. Sep. Sci. 2014, 37, 2439–2445
2441
Figure 2. MRM chromatograms of the analytes obtained from dried beancurd spiked standard mix solution: (a) Sudan I (30 g/kg); (b) Sudan II (30 g/kg); (c) Sudan III (30 g/kg); (d) Sudan IV (30 g/kg); (e) Para Red (30 g/kg); (f) Chrysoidin (30 g/kg); (g) Auramine O (3 g/kg); (h) Rhodamine B (3 g/kg).
(mobile phase B). The linear gradient elution was programmed as follows: 0–10 min, 45–100% B; 10–20 min, 100% B; 20–25 min, 100–45% B. The flow rate was set at 0.2 mL/min while the column temperature was maintained at 35⬚C. The injection volume was 5 L. R , A triple quadrupole mass spectrometer (Quattro-Micro Waters, Manchester, UK) equipped with an electrospray source operating in a positive-ion mode was used for detection in MRM mode. The optimum conditions of the ESI interface for all target analytes were as follows: a capillary voltage of 3.0 kV, a source temperature of 120⬚C, and a dry temperature of 300⬚C. High purity N2 was used as both drying gas with a flow rate of 50 L/h and nebulizing gas with a flow rate of 450 L/h. The analysis was performed in just one time segment during the entire LC–MS run, which allowed obtaining sufficient scans for each peak. The average number of data points recorded across the chromatographic peaks of the analytes is 32. Each dye was analyzed using two MRM transitions in order to improve accuracy. One transition was used for quantification and qualification while the other was used as a supplemental data for qualification. For quantification, the most intense MRM transition was selected.
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
3 Results and discussion 3.1 LC–MS/MS analysis Satisfactory separation and responses for all analytes were obtained under the gradient elution conditions described above. MRM chromatograms of the analytes in spiked bean sample are shown in Fig. 2. The cone voltages and collision energies used for MRM transitions of each compound were optimized to increase sensitivity, and the results are listed in Table 1. The product-ion mass spectra of the eight dyes are shown in Fig. 3. The product ions were selected to be as specific as possible, avoiding the use of common losses to prevent false positives in the analysis of such complex matrices.
3.2 Optimization of sample pretreatment The development of an efficient sample extraction procedure is critical for accurate determination of dyes in food samples. Four extract solvents were evaluated in this experiment based on chemical properties of the eight dyes. The extract solvents include ethyl acetate, methanol, acetonitrile, and
www.jss-journal.com
2442
J. Sep. Sci. 2014, 37, 2439–2445
J. Li et al.
Table 1. Values of the instrumental settings optimized for the eight dyes tested
Compound
Retention time (min)
Molecular weight (g/mol)
Quantitative transition (m/z)
Qualitative transition (m/z)
Dwell time (s)
Cone voltage (V)
Collision energy (eV)
Sudan I
15.62
248.1
249.3/127.9
0.1
30
20
Sudan II
17.25
276.1
277.3/156.0
0.1
25
20
Sudan III
18.30
352.1
353.3/196.2
0.1
32
22
Sudan IV
20.72
380.2
381.3/224.2
0.1
32
23
Para Red
14.75
293.1
294.2/247.1
0.1
30
20
Chrysoidin
3.87
212.1
213.2/121.0
0.1
30
20
Auramine O
7.36
267.2
268.3/147.1
0.1
35
28
11.00
478.2
443.4/399.2
249.3/127.9 249.3/231.1 277.3/156.0 277.3/121.0 353.3/196.2 353.3/156.0 381.3/224.2 381.3/209.2 294.2/247.1 294.2/156.0 213.2/121.0 213.2/196.1 268.3/147.1 268.3/252.1 443.4/399.2 443.4/413.1
0.1
45
42
Rhodamine B
Table 2. Effect of different solvents on the extraction of eight synthetic dyes from dried beancurd spiked at 10 g/kg
Compound
Recovery (%) ± SD Ethyl acetate
Sudan I Sudan II Sudan III Sudan IV Para Red Chrysoidin Auramine O Rhodamine B
86.1 77.3 60.1 47.3 69.7 11.3 2.4 49.6
± ± ± ± ± ± ± ±
0.2 0.1 0.2 0.3 0.2 0.1 0.1 0.1
Acetonitrile 103.9 110.4 110.6 80.4 95.7 112.0 102.5 98.1
± ± ± ± ± ± ± ±
0.2 0.1 0.3 0.1 0.1 0.2 0.2 0.1
Acetone 70.1 44.3 9.4 9.7 64.9 5.9 35.5 79.4
± ± ± ± ± ± ± ±
0.2 0.1 0.1 0.1 0.2 0.1 0.1 0.3
Methanol 89.5 79.2 35.5 13.4 87.3 124.3 137.7 124.2
± ± ± ± ± ± ± ±
0.2 0.2 0.1 0.1 0.1 0.2 0.3 0.2
n = 5.
Figure 3. The product-ion mass spectra of (a) Sudan I; (b) Sudan II; (c) Sudan III; (d) Sudan IV; (e) Para Red; (f) Chrysoidin; (g) Auramine O; (h) Rhodamine B.
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
acetone. For bean and meat samples, acetonitrile showed good extraction efficiency in terms of recovery and reproducibility for the eight dyes. Most of the interference proteins present in food samples were precipitated in the presence of acetonitrile. When acetone was used for extraction solvent, the recoveries of Sudan III, Sudan IV, and Chrysoidin from the bean sample spiked at 10 g/kg were 100% indicates signal enhancement, and a value of 0.99 in all cases. The matrix effect was also evaluated during the validation of the method, as it is known that signal suppression or enhancement as a result of matrix effect can severely compromise quantitative analysis of the compounds at trace levels as well as method reproducibility. The matrix effect was studied by the ratio between the slope of matrix-matched and solvent calibration curves multiplied by 100 (signal suppression enhancement, %). A value of >100% indicates signal enhancement, and a value of
