A method for detecting additives in food by using pressurized capillary electrochromatography

Abstract

The application relates to a method for detecting additives in food by using pressurized capillary electrochromatography, which comprises the following steps: (1) preparation of mixed series standard solution: standard substances of benzoic acid, sorbic acid and sodium saccharin are weighed respectively, mixed standard solution is prepared by dissolving and constant volume, the mixed series standard solution is obtained by diluting in different proportions; (2) sample treatment and preparation of the liquid to be measured: the weighed food sample is diluted, constant volume is performed after ultrasonic treatment, and the sample liquid to be measured is obtained by filtration; (3) determination: the mixed series standard solution and the liquid to be measured are analyzed by using pressurized capillary electrochromatography, the standard curve is drawn according to the chromatographic peak height and the concentration of the mixed series standard solution, the chromatographic peak height of the liquid to be measured is determined, and the concentration of benzoic acid, sorbic acid and sodium saccharin in the liquid to be measured is obtained according to the standard curve. Compared with the prior art, the pressurized capillary electrochromatography is applied to the simultaneous detection of benzoic acid, sorbic acid and sodium saccharin for the first time, sample treatment is simple, the consumption of the mobile phase and the sample is small, and the detection time is short.

Description

Technical Field

[0001] The present invention relates to the technical field of food detection methods, in particular to a method for detecting additives in food by using pressurized capillary electrochromatography. Background Art

[0002] Benzoic acid and sorbic acid are two commonly used acidic preservatives that prevent food spoilage and extend its shelf life by inhibiting microbial metabolism. Saccharin sodium is the longest-used artificial sweetener, enhancing food flavor and improving taste. However, excessive intake can lead to stunted growth, organ damage, and chronic poisoning. According to the "Standard for the Use of Food Additives of the People's Republic of China" (GB 2760-2014), saccharin sodium is not permitted in beverages, the maximum amount of benzoic acid used is 0.2g / kg, and the maximum amount of sorbic acid used is 0.5g / kg.

[0003] The current national food safety standard GB5009.29-2016 uses high-performance liquid chromatography to detect benzoic acid, sorbic acid, and saccharin sodium. However, the national standard method takes a long time to detect and consumes a large amount of mobile phase and sample. Based on this, it is necessary to provide a greener and faster detection method. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for detecting additives in food by using pressurized capillary electrochromatography.

[0005] The object of the present invention can be achieved by the following technical solution: A method for detecting additives in food using pressurized capillary electrochromatography, comprising the following steps:

[0006] (1) Preparation of mixed series standard solution: Weigh benzoic acid, sorbic acid, and saccharin sodium standard substances separately, dissolve to a fixed volume to obtain a mixed standard solution, and dilute according to different proportions to obtain a mixed series standard solution;

[0007] (2) Sample processing and preparation of the test solution: dilute the weighed food sample, fix the volume after ultrasonication, and filter to obtain the sample test solution;

[0008] (3) Determination: The mixed series standard solution and the test solution were analyzed by pressurized capillary electrochromatography. A standard curve was drawn based on the chromatographic peak height and concentration of the mixed series standard solution. The chromatographic peak height of the test solution was measured. The concentrations of the three food additives, benzoic acid, sorbic acid, and saccharin sodium, in the test solution were obtained based on the standard curve.

[0009] Preferably, the method for detecting additives in food using pressurized capillary electrochromatography comprises the following steps:

[0010] (1) Preparation of mixed series standard solution: Weigh the standard substances of benzoic acid, sorbic acid and saccharin sodium respectively, dissolve them in ultrapure water and then dilute to volume in a volumetric flask to obtain a mixed standard solution. Dilute to volume at different ratios to obtain a mixed series standard solution.

[0011] (2) Sample processing and preparation of test solution: The weighed sample was diluted with ultrapure water, transferred to a volumetric flask after ultrasonication to a fixed volume, and filtered to obtain the sample test solution.

[0012] (3) Determination: The mixed series standard solution and the test solution were analyzed using a pressurized capillary electrochromatograph with a UV-visible detector. A standard curve was drawn using the chromatographic peak height and concentration of the mixed series standard solution. The chromatographic peak height of the test solution was measured, and the concentrations of the three food additives in the test solution were obtained based on the standard curve.

[0013] In order to achieve the simultaneous detection of benzoic acid, sorbic acid, and saccharin sodium, the chromatographic conditions need to be optimized to achieve the following results: (1) The three target peaks are baseline-separated, with symmetrical peak shapes and no tailing; (2) The solvent peak and other components in the sample have no interference with the target peaks, allowing accurate quantification; (3) The detection time is shorter than that of the national standard method, and the detection efficiency is high.

[0014] Preferably, in step (3), the pressurized capillary electrochromatograph uses TriSep TM -3000, and the chromatographic column used was C18 column.

[0015] Further preferably, the specification of the chromatographic column is 100 μm×20 cm (ie, the inner diameter of the chromatographic column is 100 μm and the effective length is 20 cm), and the particle size of the chromatographic column filler is 3 μm.

[0016] Further preferably, the model of the chromatographic column is EP-100-20 / 45-3-C18-BP (3 μm, 100 μm×20 cm).

[0017] Preferably, in step (3), the pressurized capillary electrochromatograph is equipped with a pre-column quantitative loop with a volume of 20 nL, a split ratio of 1:300, and microinjection.

[0018] Further preferably, in step (3), the single injection volume is 10 μL.

[0019] Preferably, in step (3), the mobile phase is a mixed solution of 0.02 mol / L ammonium acetate solution and methanol in a volume ratio of 50:50, isocratic elution, and the flow rate of the mobile phase is 0.05 mL / min. Under these conditions, the solvent peak interference is small and the peak elution time is short.

[0020] Preferably, in step (3), the detector used is a UV-visible detector with a detection wavelength of 230 nm, high sensitivity, low noise, and strong target peak signal.

[0021] Preferably, in step (3), the separation voltage is -20.0 kV, and electrophoresis combined with pressure flow is used as the driving force to achieve baseline separation of the three target peaks in a short time.

[0022] Preferably, the mixed series standard solution and the test solution are filtered into the sample tube using a 13mm*0.22μm nylon syringe filter after constant volume. The filter can effectively remove residues in the solution, exclude impurities, reduce detection interference, and avoid clogging of the capillary.

[0023] Preferably, the mixed series standard solutions are mixed series standard solutions with concentrations of 4, 8, 12, 16, and 20 mg / L.

[0024] Preferably, the food comprises a beverage.

[0025] The pressurized capillary electrochromatography used in the present invention is a new microcolumn separation technology developed on the basis of high-performance liquid chromatography and capillary electrophoresis. It combines the advantages of both and has high selectivity and high sensitivity. Currently, there are no reports on the simultaneous detection of benzoic acid, sorbic acid and saccharin sodium by pressurized capillary electrochromatography.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. The present invention applies pressurized capillary electrochromatography to the simultaneous detection of benzoic acid, sorbic acid and saccharin sodium for the first time.

[0028] 2. The present invention eliminates the interference of other components by optimizing the mobile phase ratio and detection parameters, achieves good separation of the chromatographic peaks of the three food additives, and performs accurate quantitative analysis using a standard curve.

[0029] 3. The present invention provides a method for rapid detection of food additives using pressurized capillary electrochromatography. By optimizing the mobile phase ratio and detection parameters, the separation and quantification of three food additives are achieved, which greatly shortens the detection cycle, reduces the amount of various solvents used, and improves detection efficiency.

[0030] 4. The present invention determines the optimal mobile phase ratio, separation voltage and detection wavelength through condition optimization, and can achieve effective separation of three food additives within 3.4 minutes, with good separation effect, accurate quantification, good repeatability and short detection time.

[0031] 5. The present invention only requires ultrasound and filtration to pre-treat the sample, and the operation is simple and easy.

[0032] 6. The detection method of the present invention has simple sample processing, low consumption of mobile phase and sample, short detection time, and by optimizing the mobile phase ratio and detection parameters, the target peaks do not interfere with each other, which can provide new technical support for the rapid separation of additives in food.

[0033] 7. The present invention adopts pressurized capillary electrochromatography, a new micro-separation technology, as the detection method. This technology meets the requirements of green and environmentally friendly detection and consumes less mobile phase and sample than other liquid chromatography methods.

[0034] 8. Pressurized capillary electrochromatographs are not yet popular. The present invention helps to promote the use of pressurized capillary electrochromatographs and provides new ideas for the application of this technology in the fields of food, life sciences, chemical analysis, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a diagram showing the optimization of mobile phase ratio conditions for pressurized capillary electrochromatography;

[0036] Figure 2 Diagram of optimized voltage conditions for pressurized capillary electrochromatography separation;

[0037] Figure 3 This is the chromatogram after optimization of pressurized capillary electrochromatography;

[0038] Figure 4 are the standard curves of the three additives. DETAILED DESCRIPTION

[0039] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0040] Example 1

[0041] Establishment of chromatographic conditions: Pressurized capillary electrochromatograph using TriSep TM -3000, the detector is a UV-visible detector, the chromatographic column is a C18 column, model EP-100-20 / 45-3-C18-BP, with an inner diameter of 100 μm and an effective length of 20 cm; the flow rate of the mobile phase is 0.05 mL / min; the single injection volume is 10 μL, the quantitative loop volume is 20 nL, the split ratio is 1:300; and the detection wavelength is 230 nm.

[0042] Using methanol-0.02 mol / L ammonium acetate mixed solution as the mobile phase, the retention time and separation of benzoic acid, sorbic acid and saccharin sodium were investigated at different methanol volume fractions (10%, 20%, 30%, 40%, 50%, 60%). Figure 1As shown in the figure, increasing the proportion of methanol in the mobile phase improves the elution capacity of the mobile phase, shortens the retention time, changes the order of peak elution, and maintains little change in the retention time of the solvent peak. At a methanol volume fraction of 60%, the solvent peak overlaps with the saccharin sodium peak, making separation impossible. At a methanol volume fraction of 50%, effective separation of the three components is achieved with a short separation time. Based on these considerations, a mobile phase ratio of methanol: 0.02 mol / L ammonium acetate = 50:50 (v / v) was selected.

[0043] The effects of different voltages (0, -5, -8, -10, -15, -17, -20, -25 kV) on the separation of saccharin sodium, benzoic acid and sorbic acid were investigated. Figure 2 As shown in the figure, the results show that as the voltage increases, the retention time decreases, the resolution decreases, and the plate number decreases. Separation is achieved at -25 kV, but the current is high and the baseline is unstable. In contrast, at -20 kV, the resolution is high and the baseline is more stable. Considering both resolution and column efficiency, -20 kV was selected as the optimal separation voltage, achieving effective separation of the three food additives within 3.4 minutes.

[0044] Establishment of standard curve: Accurately weigh 10 mg of standard substances of benzoic acid, sorbic acid and saccharin sodium respectively in a beaker, add appropriate amount of ultrapure water to dissolve, transfer to a 100 mL volumetric flask and dilute to obtain a 100 mg / L mixed standard solution. Sequentially take 1, 2, 3, 4, and 5 mL of the mixed standard solution into 5 25 mL volumetric flasks, dilute to obtain 4, 8, 12, 16, and 20 mg / L mixed series standard solutions, take the supernatant and filter it with a 0.22 μm nylon syringe filter. Use a pressurized capillary electrochromatograph to detect the mixed series standard solution and obtain the chromatogram of the standard solution (as shown in Figure 2). Figure 3 ), with the peak heights of benzoic acid, sorbic acid and saccharin sodium in the chromatogram as the ordinate and the concentration of the standard solution as the abscissa, a standard curve was prepared (such as Figure 4 ).

[0045] The ammonium acetate, benzoic acid, sorbic acid, and sodium saccharin used were all of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd., and methanol was of chromatographic grade and purchased from Shanghai Titan Technology Co., Ltd.

[0046] The content of benzoic acid, sorbic acid and saccharin sodium is calculated as follows: the average peak height of the measured target component is substituted into the regression equation obtained by fitting the standard curve to calculate the concentration of the component in the test solution, and the content of the component in the beverage is calculated based on the actual sample mass.

[0047] Example 2

[0048] The rapid detection of food additives in beverages by pressurized capillary electrochromatography comprises the following steps:

[0049] (1) Sample treatment and preparation of test solution: Weigh 10.2371 g of beverage sample 1 into a beaker, add 20 mL of ultrapure water to dilute, sonicate for 30 min, transfer to a 100 mL volumetric flask to a fixed volume, and filter the supernatant using a 0.22 μm nylon syringe filter.

[0050] (2) Determination: Analyze the test liquid by pressurized capillary electrochromatography to obtain a sample chromatogram. Substitute the obtained peak height into the regression equation to calculate the content of benzoic acid, sorbic acid and saccharin sodium in the test liquid, and convert it into the content of benzoic acid, sorbic acid and saccharin sodium in the beverage.

[0051] Example 3

[0052] The difference between Example 3 and Example 2 is that: 10.5209 g of beverage sample 2 was weighed, and the contents of benzoic acid, sorbic acid and saccharin sodium were determined by pressurized capillary electrochromatography after pretreatment. Other aspects were the same as in Example 2.

[0053] In this example, a sample spike recovery experiment was conducted on beverage sample 2 to verify the detection method proposed in the present invention. The results are shown in Table 1.

[0054] Table 1

[0055] Components Spiked amount (mg / L) Average recovery rate (%) RSD (%, n=5) benzoic acid 30 112.8 3.1 Sorbic acid 30 104.0 1.3 Saccharin sodium 20 96.3 2.2

[0056] Experimental results: The average recovery rate of each target component was in the range of 96.3% to 112.8%, with RSD ≤ 3.1%, indicating that the method is scientific and effective.

[0057] The test results of the contents of benzoic acid, sorbic acid and saccharin sodium in the beverage samples of Example 2 and Example 3 are shown in Table 2.

[0058] Table 2

[0059]

[0060] The experimental results show that the levels of benzoic acid, sorbic acid and saccharin sodium in the two beverages did not exceed the standard.

[0061] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

Claims

1. A method for detecting additives in food using pressurized capillary electrochromatography, characterized in that: The following steps are involved: (1) Preparation of mixed series standard solution: Weigh benzoic acid, sorbic acid, and saccharin sodium standard substances separately, dissolve them to a fixed volume to obtain a mixed standard solution, and dilute them in different proportions to obtain a mixed series standard solution; (2) Sample processing and preparation of test solution: dilute the weighed food sample, fix the volume after ultrasonication, and filter to obtain the sample test solution; (3) Determination: Analyze the mixed series standard solution and the test solution by pressurized capillary electrochromatography. Draw a standard curve based on the chromatographic peak height and concentration of the mixed series standard solution. Measure the chromatographic peak height of the test solution. According to the standard curve, the concentrations of the three food additives, benzoic acid, sorbic acid, and saccharin sodium, in the test solution are obtained. In step (3), the pressurized capillary electrochromatograph uses TriSep™-3000 and the chromatographic column uses a C18 column; In step (3), the mobile phase is a mixed solution of 0.02 mol / L ammonium acetate solution and methanol in a volume ratio of 50:50, and the elution is isocratic at a flow rate of 0.05 mL / min; In step (3), the detector used is a UV-visible detector with a detection wavelength of 230 nm; In step (3), the separation voltage is -20.0 kV.

2. The method for detecting additives in food using pressurized capillary electrochromatography according to claim 1, wherein: The model of the chromatographic column is EP-100-20 / 45-3-C18-BP.

3. The method for detecting additives in food using pressurized capillary electrochromatography according to claim 1, wherein: In step (3), the pressurized capillary electrochromatograph is equipped with a pre-column quantitative loop with a volume of 20 nL, a split ratio of 1:300, and microinjection.

4. The method for detecting additives in food using pressurized capillary electrochromatography according to claim 3, wherein: In step (3), the single injection volume is 10 μL.

5. The method for detecting additives in food using pressurized capillary electrochromatography according to claim 1, wherein: The mixed series standard solution and the test solution must be filtered into the sample tube using a 13 mm*0.22 μm nylon syringe filter after constant volume.

6. The method for detecting additives in food using pressurized capillary electrochromatography according to claim 1, wherein: The mixed series standard solutions are mixed series standard solutions with concentrations of 4, 8, 12, 16, and 20 mg / L.

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