QuEChERS Determination Method for 5 Common Synthetic Food Colorants in Tea

Abstract

QuEChERS Determination Method for 5 Common Synthetic Food Colorants in Tea Leaves, belonging to the technical field of chemical analysis. This method includes the preparation of standard solutions; the preparation of test samples; extraction and purification; chromatographic analysis; qualitative and quantitative analysis. The present invention has established a QuEChERS determination method for 5 common synthetic food colorants in tea leaves, which uses matrix solid-phase dispersion extraction for purification, combined with pH value adjustment and selective wavelength detection, effectively reducing the interference of the complex matrix of tea leaves. While maintaining the characteristics of the original technology being simple, fast and easy to operate, it significantly reduces matrix interference and optimizes the method accuracy and sensitivity.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical analysis, and particularly relates to a QuEChERS determination method for five commonly used food synthetic colorants in tea. Background Art

[0002] According to GB 2760-2014 National Food Safety Standard - Food Additives Usage Standard, it is clearly stated that no synthetic colorants are allowed to be added to tea products. However, the illegal use of synthetic colorants in tea for coloring has been repeatedly banned. In February 2022, the China Tea Circulation Association released the latest China Tea Quality and Safety Development Report, which once again emphasized the problem of illegal use of food additives in tea, posing a safety hazard to tea products.

[0003] At present, most of the research on the determination of synthetic colorants in food focuses on foods with relatively simple matrices such as beverages. There are few studies on tea, which has high natural pigments and high small molecule matrix interference such as flavonoids, theaflavins, anthocyanins, etc. The existing determination methods are mainly high performance liquid chromatography, liquid chromatography-mass spectrometry, etc. The common pretreatment method used is mostly solid phase extraction. For example, in 2016, Chen Wei, Qiu Yajing, Qiao Yongsheng et al. used HLB solid phase extraction-reverse phase high performance liquid chromatography to determine the colorants in tea; in 2016, Lin Changhong, Yang Jun, Chen Jiefeng, Dong Chaoxian, Du Yegang et al. washed the tea with ethyl acetate and n-hexane and then extracted it with deionized water, and used high performance liquid chromatography to determine the synthetic colorants in tea; in 2019, Deng Liuai, Zhang Nianying, Guo Bing, Zuo Huanhuan, Yang Xiaomin et al. used Proelut PWA-2 solid phase extraction-liquid chromatography to determine the synthetic colorants in tea; in 2020, Guo Xiaolei, Liu Tao, Lin Zhaosheng, Chen Zhenbiao, Yang Jingjing, Jia Yanyan, Zhang Xiuqin et al. used PWAX solid phase extraction-high performance liquid chromatography to determine the artificial synthetic pigments in commercially available tea. Relatively speaking, pretreatment requires a lot of manpower, materials and time. For example, the routine operations of solid-phase extraction include pretreatment of the extraction column, sample loading, washing of the extraction column (and drying of the extraction column), elution of the extract, etc. There are many steps and the operation is time-consuming, which limits its application in daily detection.

[0004] The present invention is an effective improvement on the applicant's original authorized patent CN201410749917.1, and establishes a QuEChERS determination method for 5 commonly used food synthetic colorants in tea. While maintaining the original technology's simple, fast and easy-to-operate characteristics, it significantly reduces matrix interference and optimizes the method's accuracy and sensitivity. Summary of the invention

[0005] In view of the problems existing in the prior art, the purpose of the present invention is to design and provide a technical solution for the QuEChERS determination of five commonly used food synthetic colorants in tea.

[0006] To achieve the above object, the present invention specifically adopts the following technical solutions:

[0007] QuEChERS determination method for 5 common synthetic food colorants in tea leaves, wherein the 5 common synthetic food colorants are tartrazine, amaranth, ponceau 4R, sunset yellow and brilliant blue, and it includes the following steps:

[0008] 1) Preparation of standard solution: Prepare a single synthetic colorant standard solution of 5 synthetic colorants at 1000 mg / L for standby; use the single synthetic colorant standard solution to synthesize a synthetic colorant mixed standard solution for standby;

[0009] 2) Preparation of test sample: Take not less than 100 g of tea leaf sample, put it into a pulverizer for pulverization to make a sample to be measured, and put it into a sub-packaging container for standby;

[0010] 3) Extraction and purification: Accurately weigh tea leaves and polyvinylpyrrolidone, mix them, add them to a methanol / water / ammonia water mixed solution, quickly shake on a shaker, then perform ultrasonic-assisted extraction, take the supernatant, add acetic acid, and after passing through a 0.45 μm PTFE filter membrane, perform instrumental determination;

[0011] 4) Chromatographic analysis: The standard mixed solution or sample is aspirated by an automatic sampler and injected into the chromatograph. Qualitative analysis is carried out based on the retention time, and quantitative analysis is carried out by comparing the peak area of the sample solution with that of the standard solution;

[0012] 5) Qualitative and quantitative analysis.

[0013] Further, in step 3), the pH value of the methanol / water / ammonia water mixed solution is 8 - 9.

[0014] Further, in step 3), polyvinylpyrrolidone is used as a purification adsorbent, and the dosage of the adsorbent is 0.1 - 1 g / g based on the mass of the tea leaf sample.

[0015] Further, in step 3), polyvinylpyrrolidone is used as a purification adsorbent, and the dosage of the adsorbent is 0.4 g / g based on the mass of the tea leaf sample.

[0016] Further, in step 3), acetic acid is added to adjust the pH value of the sample solution to 6 - 7.

[0017] Further, in step 4), the target substances are detected according to the spectral characteristic wavelengths and chromatographic retention times of each synthetic colorant in time segments.

[0018] Further, in step 5), the qualitative analysis is specifically: the retention time RT of the unknown component in the sample is compared with the retention time RT of the standard sample on the same chromatographic column. If the difference is within ±0.1 min and the response values at different wavelengths are synchronously correlated, it can be determined as the target substance.

[0019] Further, the quantitative analysis in step 5) is specifically as follows: the mass fraction ω of the synthetic colorant to be measured in the sample, with the value expressed in milligrams per kilogram, is calculated according to formula (1).

[0020] ω = V 1 × A × V 3 × Ψ / (V 2 × A S × m) (1)

[0021] In the formula:

[0022] Ψ - the content of the pigment in the standard solution, with the unit of milligrams per liter;

[0023] A - the peak area of the pigment to be measured in the sample;

[0024] A s - the peak area of the pigment to be measured in the pigment standard solution;

[0025] V l - the total volume of the extraction solvent;

[0026] V 2 - the volume of the extraction solution taken out for detection;

[0027] V 3 - the volume for sample constant volume;

[0028] m - the mass of the sample;

[0029] The calculation result is retained to three significant figures.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) The present invention first proposes a QuEChERS determination method for 5 common synthetic food colorants in tea. Using matrix solid-phase dispersion extraction as the purification means, the interference of the tea matrix is further reduced. For the comparison of the specific chromatograms before and after purification, see Figure 1 .

[0032] (2) The present invention proposes a QuEChERS determination method for 5 common synthetic food colorants in tea. Without increasing personnel and time, using a commercially available material - polyvinylpyrrolidone for purification, the method detection limits of the 5 synthetic colorants in tea are reduced to 0.094 - 0.281 mg / kg, which is 3.1 - 16.5 times lower than the indexes of the original authorized patent CN201410749917.1, and still maintains the characteristics of being fast, simple and easy to operate of the original authorized patent.

[0033] (3) The present invention does not require high-end instrument equipment and reagent materials, and is simple, fast and easy to operate. Matrix solid-phase dispersion extraction and purification are carried out with polyvinylpyrrolidone, combined with pH value adjustment and selective detection at characteristic wavelengths, effectively reducing the interference of complex matrices such as tea leaves. The average spiked recovery rate is 84.2-97.9%, and the relative standard deviation RSD is 1.2-5.2%. The pretreatment of 20-30 tea samples can be completed within 60 minutes, meeting the requirements of daily detection. Description of the Drawings

[0034] Figure 1 Comparison of chromatograms of tea sample solutions with pH 6-7 purified by QuEChERS, tea sample solutions with pH 6-7 not purified, tea sample solutions with pH 8-9 not purified, and standard sample chromatograms;

[0035] Figure 2 Chromatogram of the standard sample in Example 1;

[0036] Figure 3 Chromatogram of the tea sample in Example 1. Detailed Description of the Invention

[0037] The present invention will be further described below in conjunction with examples.

[0038] Example 1:

[0039] 1. Scope

[0040] Specifies the QuEChERS determination method for 5 commonly used synthetic food colorants such as tartrazine, amaranth, ponceau 4R, sunset yellow, and brilliant blue in tea leaves.

[0041] 2. Principle

[0042] Weigh the tea sample and PVPP, and ultrasonically assist in extracting 5 commonly used synthetic food colorants such as tartrazine, sunset yellow, amaranth, ponceau 4R, and brilliant blue with a methanol / water / ammonia mixture with a pH of 8-9 and a corresponding volume ratio of 70 / 27 / 3 (V / V / V). After adjusting the pH with acetic acid and filtering, it is introduced into the liquid chromatograph, and the response values at wavelengths of 426 nm, 510 nm, and 610 nm are monitored in time segments according to the retention times and characteristic wavelengths of each synthetic colorant. Qualitative and quantitative analysis are carried out by the external standard method.

[0043] 3. Reagents and Materials

[0044] 3.1 Reagents

[0045] The reagents for preparing the standard sample are chromatographically pure, and the rest are analytically pure.

[0046] 3.2 Preparation of Standard Solutions

[0047] Single synthetic colorant standard solution: Weigh accurately a certain amount of pigment standard, dilute it with 50% methanol, and prepare single standard stock solutions of 5 synthetic colorants at 1000 mg / L one by one, and store them in a refrigerator at -18°C.

[0048] Mixed standard solution of synthetic colorants: According to the response values of the 5 synthetic colorants on the instrument, pipette a certain volume of single synthetic colorant standard solution into the same volumetric flask one by one, dilute it to the mark with 50% methanol, prepare a mixed standard stock solution, and store it in a refrigerator at about 4°C for later use. Before use, dilute it with mobile phase A to the standard working solution of the required concentration.

[0049] 4. Analysis steps

[0050] 4.1 Sample preparation

[0051] Take no less than 100 g of tea samples, put them into a pulverizer to pulverize, make them into samples to be measured, and put them into a dispensing container for standby. Among them, the tea samples are blank tea samples added with 5 synthetic colorants.

[0052] 4.2 Extraction

[0053] Accurately weigh 1.00 g of tea and 0.4 g of polyvinylpyrrolidone and put them into a 15 mL stoppered tube, add 10 mL of methanol / water / ammonia water (70 / 27 / 3, V / V / V, pH value is 8 - 9), shake rapidly on an oscillator for 30 s, then extract with ultrasonic assistance for 20 min. Take 1 mL of the supernatant, add 20 μL of acetic acid, adjust the pH value to 6 - 7, filter through a 0.45 μm PTFE filter membrane, and then conduct instrumental determination.

[0054] 4.3 Determination

[0055] 4.3.1 Chromatographic reference conditions

[0056] 4.3.1.1 The chromatographic column is a C18 liquid chromatographic column, 5 μm × 4.6 μm × 250 mm; the column temperature is 40°C; the flow rate is 1 mL / min; the detection wavelengths are 426 nm, 510 nm, and 610 nm.

[0057] 4.3.1.2 Mobile phase A (20 mmol ammonium acetate aqueous solution), mobile phase B (acetonitrile); initially 0% A; 70% A at 15 min; 0% A at 15.01 min.

[0058] 4.3.2 Chromatographic analysis

[0059] Pipette 10 μL of the standard mixed solution (or sample) into the chromatograph by an autosampler, identify by retention time, and quantify by comparing the peak area of the sample solution with that of the standard solution.

[0060] 5. Result expression

[0061] 5.1 Qualitative analysis

[0062] The retention time (RT) of the unknown components in the sample is compared with the retention time (RT) of the standard sample on the same chromatographic column. If the difference is within ±0.1 min and the response values at different wavelengths are synchronously correlated, it can be identified as the target substance.

[0063] 5.2 Calculation

[0064] The mass fraction ω of the synthetic coloring agent to be measured in the sample, expressed in milligrams per kilogram (mg / kg), is calculated according to formula (1).

[0065] ω = V 1 × A × V 3 × Ψ / (V 2 × A S × m) (1)

[0066] In the formula:

[0067] Ψ - The content of the pigment in the standard solution, in milligrams per liter (mg / L);

[0068] A - The peak area of the pigment to be measured in the sample;

[0069] A s - The peak area of the pigment to be measured in the pigment standard solution;

[0070] V 1 - The total volume of the extraction solvent;

[0071] V 2 - The volume of the extraction solution taken for detection;

[0072] V 3 - The volume for sample constant volume;

[0073] m - The mass of the sample;

[0074] The calculation result is retained to three significant figures.

[0075] 6. Conclusion

[0076] The chromatograms of the standard sample and the spiked sample (added concentration is 10 mg / kg) are shown in Figure 2 and Figure 3 , and the detection results are shown in Table 1. It can be seen from Table 1 that the recovery rates of applying this method to detect the blank tea samples added with 5 common synthetic food colorants are in the range of 84.2 - 97.9%, the relative standard deviation RSD is 1.24 - 5.23%, and the method detection limit is 0.094 - 0.281 mg / kg.

[0077] Table 1 Detection data of spiked tea samples (n = 3)

[0078]

[0079] Example 2

[0080] Referring to the steps and methods of Example 1, 100 commercially available black tea samples were detected for 5 common synthetic food colorants. 7 batches were detected, and the detected substances were tartrazine and sunset yellow. Among them, 7 batches of tartrazine were detected, and the detected value range was 15 mg / kg - 1500 mg / kg; 1 batch of sunset yellow was detected, and the detected value was 220 mg / kg.

Claims

1. QuEChERS Determination Method for 5 Commonly Used Synthetic Food Colorants in Tea, where the 5 commonly used synthetic food colorants are tartrazine, amaranth, ponceau 4R, sunset yellow, and brilliant blue, which is characterized in that it includes the following steps: 1) Preparation of standard solutions: Prepare single synthetic colorant standard solutions of 1000 mg / L for the 5 synthetic colorants for standby; use the single synthetic colorant standard solutions to synthesize synthetic colorant mixed standard solutions for standby; 2) Preparation of test samples: Take no less than 100 g of tea samples, put them into a pulverizer for pulverization, make them into test samples to be measured, and put them into a sub-packaging container for standby; 3) Extraction and purification: Accurately weigh tea and polyvinylpyrrolidone, mix them, add them to a methanol / water / ammonia water mixed solution, the pH value of the methanol / water / ammonia water mixed solution is 8 - 9, quickly shake on a shaker, then use ultrasonic-assisted extraction, take the supernatant and add acetic acid to adjust the pH value of the sample solution to 6 - 7, pass through a 0.45 μm PTFE filter membrane and then carry out instrumental determination; 4) Chromatographic analysis: The automatic sampler aspirates the standard mixed solution or the sample and injects it into the chromatograph, qualitatively analyze by retention time, and quantitatively analyze by comparing the peak area of the sample solution with that of the standard solution; the chromatographic column is a C18 liquid chromatographic column, 5 μm × 4.6 μm × 250 mm; the column temperature is 40 °C; the flow rate is 1 mL / min; the detection wavelengths are 426 nm, 510 nm, 610 nm; mobile phase A: 20 mmol ammonium acetate aqueous solution, mobile phase B: acetonitrile; initially 0% A; 70% A at 15 min; 0% A at 15.01 min; 5) Qualitative and quantitative analysis.

2. The QuEChERS determination method for 5 commonly used synthetic food colorants in tea as described in claim 1, which is characterized in that in step 3), polyvinylpyrrolidone is used as a purification adsorbent, and the dosage of the adsorbent is 0.1 - 1 g / g based on the mass of the tea sample.

3. The QuEChERS determination method for 5 commonly used synthetic food colorants in tea as described in claim 2, which is characterized in that in step 3), polyvinylpyrrolidone is used as a purification adsorbent, and the dosage of the adsorbent is 0.4 g / g based on the mass of the tea sample.

4. The QuEChERS determination method for 5 commonly used synthetic food colorants in tea as described in claim 1, which is characterized in that in step 4), the target substances are detected in time segments according to the spectral characteristic wavelengths and chromatographic retention times of each synthetic colorant.

5. The QuEChERS determination method for 5 commonly used synthetic food colorants in tea as described in claim 4, which is characterized in that in step 5), the qualitative analysis is specifically as follows: The retention time RT of the unknown components in the sample is compared with the retention time RT of the standard sample on the same chromatographic column, and the difference is within ±0.1 min, and the response values at different wavelengths are synchronously correlated, then it can be determined as the target substance.

6. The QuEChERS determination method for 5 commonly used synthetic food colorants in tea as described in claim 1, which is characterized in that in step 5), the quantitative analysis is specifically as follows: The content of the synthetic colorant to be measured in the sample is expressed as a mass fraction ω, and the value is expressed in milligrams per kilogram, and is calculated according to formula (1) ω = V 1 × A × V 3 × Ψ / (V 2 × A S × m) (1) where: Ψ - Content of pigment in the standard solution, unit: mg / L; A - Peak area of the pigment to be measured in the sample; A s - Peak area of the pigment to be measured in the pigment standard solution; V l - Total volume of extraction solvent; V 2 - The volume of the extraction solution for detection is extracted; V 3 - Volume for sample constant volume; m - Mass of the sample; The calculation result is retained to three significant figures.

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