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Hu Jinsheng Zhu Taoyu.
Introduction. Synthetic colorants are synthetic substances that color food and improve food color [1], which have the characteristics of bright color, stable properties, low cost and strong coloring strength, and are widely used in various types of food, such as candy, pastries, candied fruits, beverages, canned food, etc. Synthetic colorants play an indispensable role in the modern food industry, imparting a rich color to food and presenting consumers with a variety of choices. However, with people's increasing concern about food quality and safety, a series of potential hazards caused by the abuse of synthetic colorants in the production process and excessive intake in the consumption process have also aroused widespread concern and discussion from all walks of life.
Recently, the National Food Safety Standards Review Committee has reviewed and issued 85 national food safety standards, including the testing standard for synthetic colorants - GB 500935-2023。
gb 5009.35-2023 New National Standard Changes[2][3].
1. gb 5009.35-2023 was officially released on 2023-09-06 and officially implemented on 2024-03-06;
2. gb 5009.35-2023 instead of GB 500935-2016、gb 5009.141-2016、gb/t 9695.6-2008、gb/t 21916-2008；
3.Compared to GB500935-2016，gb 5009.35-2023 Four synthetic colorants were added: indigo, ** red, acid red and quinoline yellow, the types of applicable matrices were increased and clarified, the sample preparation method and instrument conditions were modified, and the detection and quantification limits were modified.
Highlights of Thermo Scientific's new national standard solution:
1.The Thermo Scientific laboratory fully reproduced the method with standard gradient elution procedures, and only increased the column temperature to 35 for further optimization of sensitivity and resolution
2.Tartrazine and new red, quinoline yellow-5 and amaranth red, quinoline yellow-6 and carmine, sunset yellow and quinoline yellow-2, and acid red and quinoline yellow-3 can all be separated at baseline for accurate qualitative and quantitative separation
3.The acidic red peak is located after Brilliant Blue-1 and does not interfere with the qualitative quantification of Brilliant Blue-3, and the separation and retention are better than the reference spectrum provided by the standard
4.Based on Chromeleon 73 Data processing software, using the compound group mode to accurately quantify multiple isomers of quinoline yellow and brilliant blue, improve work efficiency.
gb 5009.35-2023 Thermo Scientific Application Solutions.
01 Instrument configuration and chromatographic conditions.
Liquid chromatograph:Vanquish Core HPLC LC System with Chameleon Data Processing Software.
Column(s):
acclaim 120 c18, 5 μm, 4.6×250 mm (p/n: 059149)
betasil c18, 5 μm, 4.6×250 mm (p/n: 70105-254630)
Quaternary pump pump:vc-p20-a
Mobile phase:A: 20 mM ammonium acetate B: Methanol.
Flow rate:1.0 ml/min
Gradient Elution Program:
Autosampler Module: VC-A12-A
Injection volume: 10 l;Tray temperature: 15
Column Comp: VC-C10-A
Column temperature:
Detector DAD: VC-D11-A (UV Vis).
Acquisition wavelengths: 415 nm, 520 nm, 610 nm;
Acquisition frequency: 5 Hz;
Spectral acquisition: 400-800 nm
02 Spectra and data.
2.1 Acclaim 120 C18 is applied to the separation of 11 synthetic colorants.
Figure 1 Separation overlay of 11 synthetic colorants at 415 520 610 nm (200 μg/ml, 10 μl)
Fig. 2 Separation spectra and data of tartrazine and quinoline yellow at 415 nm (20.)0 μg/ml, 10 μl)
Fig. 3 Separation spectra and data of 7 synthetic colorants including Xinhong at 520 nm (20.)0 μg/ml, 10 μl)
Figure 4 Separation spectra and data of indigo and brilliant blue at 610 nm (20.)0 μg/ml, 10 μl)
2.2 Separation of quinoline yellow and brilliant blue isomers.
2.2.1 Isolation of quinoline xanthagorene.
The food additive quinoline yellow is prepared by sulfonation of 2-(2-quinolino)-1,3-nindione (type) or two-thirds of 2-(2-quinolinyl)-1,3-nindione and one-third of 2-[(6-methylquinoline)]-1,3-nindione. Among them, the main component of the type process product is 2-(2-quinolinyl)-indengino-1,3-dione disulfonic acid disodium salt (80.).0%), 2-(2-quinolinyl)-indenhydrin-1,3-dione monosulfonate sodium salt (15.)0%), 2-(2-quinolinyl)-indengino-1,3-diketotrisulfonic acid trisodium salt (7.)0%), the paradyes 2-(2-quinolinyl)-1,3-nindione and 2-[(6-methylquinoline)]-1,3-nindione (4.)0%)；The main component of the process product is 2-(2-quinolinyl)-indengino-1,3-dione disulfonic acid disodium salt (70.).0%), the paradyes 2-(2-quinolinyl)-1,3-nindione and 2-[(6-methylquinoline)]-1,3-nindione (4.)0%)[4]。There are two isomers of 2-(2-quinolino)-indanyl-1,3-dione disulfonic acid disodium salt qyna2 due to the different substitution sites during the sulfonation reaction, and 2-(2-quinolinyl)-indanyl-1,3-dione monosulfonate sodium salt qyna and qyna [5].
Therefore, due to the differences in synthesis and purification processes, the food additive quinoline yellow will have multiple chromatographic peaks during chromatographic separation, and the same chromophore may have up to 7 chromatographic peaks at 415 nmThe standard quinoline yellow is usually purified by chromatographic preparation to remove the paradye, and up to 5 chromatographic peaks may be separated. As shown in Figure 5, the spectra of the 7 components of quinoline yellow are highly similar, and the peak order and retention time of the 4 components of quinoline yellow marked with the new national standard are consistent, and the quinolino-yellow-5 6 7 may be qyna3 or paradye components, which are often embedded by other adjacent peaks in the standard mixture due to insufficient chromatographic separation, and then ignored.
Figure 5 Isomer separation spectra of quinoline xanthamides at 415 nm (1000 μg/ml, 5 μl)
2.2.2 Separation of brilliant blue isomers.
The synthesis route of the food additive Brilliant Blue is prepared by condensation and oxidation of one part benzaldehyde o-sulfonic acid and two parts N-ethyl-N-(3-sulfobenzyl)-aniline as raw materials [6]. Among them, N-ethyl-N-(3-sulfobenzyl)-aniline is obtained by the sulfonation of N-ethyl-N-benzylaniline, and the sulfonation position is mainly in the meta-position on its benzyl group, followed by para-position, and ortho-sulfonation is more difficult. Because it is difficult to separate, the industrial product of n-ethyl-n-(3-sulfobenzyl)-aniline is actually a mixture of three isomers, which is generally roughly composed of meta (m): para (p) : ortho (o) = 75 85 : 15 20 : 0 8. Therefore, the main component of the prepared brilliant blue commercial pigment should also be a mixture of 6 isomers, m-m, m-p, m-o, p-p, p-o, and o-o[7]. While the proportion of O-O and O-P isomers is very low, commercially available liquid chromatograms of pure brilliant blue typically show 4 chromatographic peaks. As shown in Figure 6, the spectra of the four components of Brilliant Blue are highly similar, and the peak order and retention time of the two Brilliant Blue components marked by Brilliant Blue-1 2 are consistent with those of the new national standard, and Brilliant Blue-3 4 is omitted in the standard, where Brilliant Blue-3 may be disturbed by Acid Red.
Figure 6 Separation spectra of brilliant blue isomers at 610 nm (1000 μg/ml, 5 μl)
2.3 Calibration curves for 11 synthetic colorants standard series working fluids.
Fig. 7 Linear overlay spectra of tartrazine and quinoline yellow (0.)20-10.0 μg/ml, 10 μl)
Fig. 8 Linear overlay spectra of 7 synthetic colorants including Xinhong (020-10.0 μg/ml, 10 μl)
Fig. 9 Indigo and brilliant blue linear overlay spectra (0.)20-10.0 μg/ml, 10 μl)
2.4 Samples and sample spike spectra.
Fig. 10 Samples of vitamin functional drinks and their spike spectra (with detection of tartrazine and carmine).
Figure 11 Jasmine tea samples and sample spike spectra.
2.5 Quantification of quinoline yellow and brilliant blue isomers.
Figure 12 Schematic diagram of quinoline yellow and brilliant blue isomers quantified using a compound group.
tips:Want to learn more about the operation details of the chameleon software for compound group quantitation?Speak with the Thermo Scientific LC Applications team.
2.6 Betasil C18 is applied to the separation of 11 synthetic colorants (complementary protocol).
Figure 13 Separation spectra of BETASIL C18 applied to 11 synthetic colorants (20.)0 μg/ml, 10 μl)
03 gb 5009.35-2023 Related Chromatography Consumables Recommendations.
References: 1] GB 500935-2023 National Food Safety Standard Determination of Synthetic Colorants in Food.
2] gb 5009.35-2016 National Food Safety Standard Determination of Synthetic Colorants in Food.
3] GB 2760-2014 National Food Safety Standard Standard for the Use of Food Additives.
4] gb 1886.104-2015 National Food Safety Standard Food Additive Quinoline Yellow.
5] DBS32 012-2016 Local Standard for Food Safety Detection of Quinoline Yellow in Food High Performance Liquid Chromatography, Liquid Chromatography-Mass Spectrometry Mass Spectrometry.
6] gb 1886.217-2016 National Food Safety Standard Food Additives Bright Blue.
7] Ding Qiulong, Wang Libin, Chen Jianxin. Isomeric studies of edible bright blue pigments.
Liquid chromatograph: