Method for simultaneous determination of food additives and heavy metals in food by reversed-phase HPLC-ICP-MS

Abstract

The present invention discloses a method for synchronously detecting food additives and heavy metal content in food by reversed-phase HPLC-ICP-MS method, wherein the method connects HPLC and ICP-MS equipment together through an interface system (5) dedicated to Agilent, and then the processed food sample is first subjected to food additive content detection in HPLC, and then the food sample flowing through HPLC is input into ICP-MS through the interface system (5) for heavy metal content detection. The method can quickly and accurately synchronously measure the content of food additives and heavy metals in food, not only reducing the sampling amount of the sample to be detected and the pretreatment amount of the sample, but also reducing the cumbersomeness of the operation brought by the separate detection and sampling of different instruments, greatly shortening the detection time and improving work efficiency. Usually, it takes about 6 hours for the two instruments to detect separately, and it takes about 4.8 hours after the combination, saving about 20% of time.

Description

[0001] This application is a divisional application with application date of January 13, 2017, application number 201710026096.2, and invention name “Method for simultaneous detection of food additives and heavy metal content in food by reversed-phase HPLC-ICP-MS”. Technical Field

[0002] The invention belongs to the technical field of food detection, and particularly relates to a method for detecting food additives and heavy metal contents in food. Background Art

[0003] Food additives are an additive added to food to ensure the appearance and taste of food. They are important raw and auxiliary materials that are indispensable in the modern food industry. They can improve the quality, color, taste, extend the shelf life, and increase the nutritional value of food. The main raw materials are chemical synthetic substances and natural substances. Both chemical additives and natural additives have certain toxic components, but the harmfulness of these toxic components is related to the dosage of the additives and the chemical reaction of the human body. They will affect human health only when they reach a certain level or dosage. my country's "Food Additives Hygiene Standard" GB2760-1996 divides them into 22 categories, including preservatives, sweeteners, colorants, coloring agents, flour treatment agents, bleaching agents and antioxidants, and stipulates the scope and amount of use of food additives. At present, problematic foods have occurred repeatedly, exposing various problems with food additives, mainly reflected in the use of preservatives, sweeteners, colorants, coloring agents, flour treatment agents and bleaching agents beyond the scope and limit. How to accurately and quickly detect the content of food additives in food to ensure that people can eat it safely and prevent problematic food from endangering people's physical and mental health has become a hot issue of common concern to consumers and researchers.

[0004] The use of food additives beyond the scope and limit has seriously affected the safety of food. If they can be added legally and reasonably, it can be prevented before it happens. However, another invisible killer of food safety - excessive heavy metal content has caught the majority of producers and consumers by surprise. This is mainly due to the wide range of sources of heavy metals in food. Heavy metals refer to metals with a specific gravity of more than 5, such as copper, lead, zinc, nickel, cobalt, cadmium, chromium, mercury, bismuth, tin, antimony, niobium, molybdenum, etc. Under normal circumstances, the natural background concentration of heavy metals will not reach a harmful level, but with the rapid development of social industrialization, the amount of toxic and harmful heavy metals such as lead, mercury, cadmium, chromium, etc. entering the atmosphere, water and soil continues to increase. Exceeding the normal range will cause heavy metal pollution in the environment. From the perspective of food safety, the most concerning heavy metal pollution is currently mercury, cadmium, lead, chromium, and heavy metals with significant biological toxicity such as metalloid arsenic. According to research, heavy metal pollution is amplified through the food chain and enters the human body through food, mainly causing chronic damage to the body. After daily food (including vegetables, fruits, fish, etc.) is contaminated with heavy metals, the heavy metals in it cannot be removed by simple cooking, soaking or washing. And heavy metals that enter the human body will show toxicity after a long period of accumulation. Therefore, it is often not easy to be detected early, and it is difficult to attract enough attention before the toxicity occurs, which aggravates its harmfulness. Therefore, how to accurately and quickly detect the content and morphological distribution of heavy metals in food is also the focus of attention of consumers and research staff.

[0005] High performance liquid chromatography (HPLC) is a detection method based on liquid chromatography and gas chromatography. It mainly uses the method of high-pressure infusion to perform chromatographic separation on different mobile phases, and finally analyzes the separated samples using a detector. This detection method is widely used in the field of food trace analysis due to its high recovery rate, good separation effect, high sensitivity and accuracy. Reversed phase high performance liquid chromatography (HPLC) is a reversed phase bonded phase chromatography, which is a type of chemical bonded phase chromatography. Its main feature is that the polarity of the bonded stationary phase is less than that of the mobile phase, and it is suitable for separating non-polar, polar or ionic compounds. It generates a chemically bonded stationary phase by covalently bonding different organic functional groups to the free hydroxyl groups on the surface of the silica gel carrier through chemical reactions. The chemically bonded stationary phase has good chemical stability and thermal stability to various polar solvents. The chromatographic column prepared by it has high column efficiency, long service life, good reproducibility, and good selectivity for almost all types of organic compounds. It can be operated with gradient elution, eliminating the shortcomings of distribution chromatography, which lays the foundation for the simultaneous determination of multiple compounds.

[0006] Traditional heavy metal quantitative detection methods mainly include atomic absorption spectroscopy, ultraviolet visible spectrophotometry, atomic fluorescence spectroscopy, electrochemical method, X-ray fluorescence spectroscopy and inductively coupled plasma mass spectrometry (ICP-MS). ICP-MS has the advantages of simultaneous determination of multiple elements, fast analysis speed, wide linear range and high sensitivity, and has attracted widespread attention from scholars at home and abroad. Its detection limit for metal ions can reach ppm level.

[0007] As can be seen from the above, HPLC and ICP-MS are both advanced large-scale precision instruments in the field of food safety inspection. With the development of detection technology and the actual needs of food detection, single-machine detection often cannot meet the requirements of complex samples, and online detection technology is the general trend of development. Regarding the application of HPLC and ICP-MS, Zhou Ying et al. used HPLC-ICP-MS to test the form analysis and bioavailability of selenium and arsenic in food; Wang Bingtao et al. used HPLC-ICP-MS to detect the distribution of five forms of selenium in food. Although there have been relevant reports and studies on HPLC-ICP-MS detection technology, there have been no reports on the simultaneous detection of multiple food additives and heavy metal contents in food by reversed-phase HPLC-ICP-MS. Summary of the invention

[0008] In view of the above problems in the prior art, the present invention provides a method for simultaneously detecting the contents of multiple food additives and multiple heavy metals in food by using reversed-phase HPLC-ICP-MS.

[0009] The specific scheme of the present invention is as follows:

[0010] A method for synchronously detecting food additives and heavy metal content in food by reverse phase HPLC-ICP-MS method, comprising the following steps: connecting HPLC and ICP-MS equipment together through an interface system (5) dedicated to Agilent, then firstly detecting the content of food additives in the processed food sample on HPLC, and then inputting the food sample flowing through HPLC into ICP-MS through the interface system (5) for heavy metal content detection; the food additives include one or more of preservatives, sweeteners and colorants; the preservatives include one or more of benzoic acid, sodium benzoate, sorbic acid and potassium sorbate; the sweeteners include one or more of acesulfame potassium, saccharin sodium and sodium cyclamate; the colorants include one or more of amaranth, carmine, tartrazine, sunset yellow and brilliant blue; the heavy metals include one or more of arsenic, lead and cadmium;

[0011] The food additive content detection steps are as follows:

[0012] (1) Use food additive standards to debug and determine HLPC chromatographic conditions;

[0013] (2) Draw the standard curve of food additives and determine the minimum detection amount;

[0014] (3) Detect the content of food additives in the processed food samples by HPLC and draw a curve for the samples to be tested;

[0015] (4) Calculate the content of food additives by calculating the peak area of ​​the curve of the sample to be tested;

[0016] Determining the HLPC chromatographic conditions includes selecting and determining the reverse chromatographic column, selecting and determining the mobile phase, and selecting and determining the detection wave;

[0017] The reverse chromatographic column is selected as C18; the mobile phase is selected as a mixed solution of methanol and ammonium acetate in a volume ratio of 10-30:90-70, the mobile phase flow rate range is 2%-3%, and the ammonium acetate concentration is 0.02-0.05 mol / L; the detection wavelength is selected as 210-320 nm;

[0018] The heavy metal content detection steps are:

[0019] (1) Use heavy metal standards to debug and determine ICP-MS conditions;

[0020] (2) Draw a heavy metal standard curve;

[0021] (3) The food sample flowing through the HPLC is input into the ICP-MS through the interface system (5) to detect the heavy metal content, and a curve of the sample to be tested is drawn;

[0022] (4) Calculate the content of heavy metals by calculating the peak area of ​​the curve of the sample to be tested;

[0023] The heavy metal content detection steps are:

[0024] (1) Use heavy metal standards to debug and determine ICP-MS conditions;

[0025] (2) Draw a heavy metal standard curve;

[0026] (3) The food sample flowing through the HPLC is input into the ICP-MS through the interface system (5) to detect the heavy metal content, and a curve of the sample to be tested is drawn;

[0027] (4) Calculate the content of heavy metals by calculating the peak area of ​​the curve of the sample to be tested;

[0028] The mobile phase of the ICP is a mixed solution of methanol and ammonium acetate in a volume ratio of V methanol: V ammonium acetate = 1:9 to 3:7; the ICP measurement parameters are determined as follows: high frequency power 1.45 kW; plasma gas flow rate 14.0 L.min -1 ; Carrier gas flow rate 0.8L.min -1 , He flow rate 4.0mL.min -1 ; Sample lifting volume 0.1mL.min -1 ; Sampling depth 10.0mm; Sampling nickel cone: orifice diameter 1.0mm; Intercepting nickel cone: orifice diameter 0.4mm; Integration time 3s; Repeat sampling 3 times;

[0029] The food additive test results: relative standard deviation RSD <4%; sample recovery rate is between 93%-109%, and the detection limit is 2-28ng / L;

[0030] The heavy metal detection results: relative standard deviation RSD<3.5%; sample recovery rate is between 93%-104%, and the detection limit is 7-12ng / L.

[0031] The present invention has the following beneficial effects:

[0032] 1. The present invention combines reversed-phase high performance liquid chromatography with inductively coupled plasma mass spectrometry (reverse-phase HPLC-ICP-MS), which can quickly and accurately measure the content of food additives and heavy metals in food simultaneously, not only reducing the sampling amount of the sample to be tested and the amount of sample pretreatment, but also reducing the cumbersome operation caused by different instruments for separate testing and separate sampling, greatly shortening the detection time and improving work efficiency. Usually, it takes about 6 hours to detect the two instruments separately, and it takes about 4.8 hours after the combination, saving about 20% of time.

[0033] 2. The present invention can detect multiple food additives and heavy metal contents simultaneously. Traditional methods cannot detect multiple food additives and heavy metal contents simultaneously, and usually can only detect each food additive or heavy metal individually.

[0034] 3. In the present invention, HPLC is combined with ICP-MS. After the food sample is subjected to HPLC, the form of heavy metal ions does not change, and the accuracy of subsequent ICP-MS detection is not affected. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Attached Figure 1 It is a schematic diagram of the coupling of high performance liquid chromatography and electroreceptor coupled plasma mass spectrometry of the present invention;

[0036] Attached Figure 2This is a flow chart for the simultaneous determination of food additives and heavy metals in standard samples by HPLC-ICP-MS;

[0037] In the figure: A is a high performance liquid chromatograph (HPLC); B is an inductively coupled plasma mass spectrometer (ICP-MS);

[0038] 1. Mobile phase; 2. High pressure chromatographic column; 3. Inlet; 4. Reversed phase chromatographic column; 5. Agilent's dedicated interface system; 6. Argon gas bottle; 7. Nebulizer / spray chamber; 8. Fire source; 9. Rotating chromatographic column; 10. Turbine chromatographic column. DETAILED DESCRIPTION

[0039] In order to better understand the present invention, the present invention is further described below in conjunction with implementation examples. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art make various changes or modifications to the present invention, and these equivalent forms also fall within the scope limited by the appended claims of the application.

[0040] 1. Determination of chromatographic conditions and drawing of standard curve of mixed solution

[0041] (I) Preparation of mixed standard solution of food additives and heavy metals

[0042] 1. Preparation of standard solutions of food additives: accurately weigh 0.1 g each of tartrazine, sunset yellow, carmine, saccharin sodium, acesulfame potassium, benzoic acid, sodium benzoate, sorbic acid and potassium sorbate; dissolve benzoic acid and sorbic acid in a small amount of ethanol; add water with pH = 6 to the mark of a 100 mL volumetric flask to tartrazine, sunset yellow and carmine; dissolve other additives in distilled water and then make up to the volume in a 100 mL volumetric flask with distilled water; then prepare a total of 9 standard solutions with a concentration of 1000 mg / L.

[0043] 2. Preparation of heavy metal standard solutions of Pb, As and Cd: Accurately transfer 5 mL of hydrochloric acid to a 100 mL volumetric flask, and add Pb, As and Cd standard substance solutions (1000 ug.mL -1 , National Center for Standard Materials Research) pipetted 0.2mL, 0.4mL, 0.6mL, 0.8mL, and 1.0mL into a volumetric flask filled with 5mL of hydrochloric acid, and added distilled water to make a total of 15 standard solutions of different concentrations of the three heavy metals Pb, As, and Cd.

[0044] 3. Preparation of mixed standard solution:

[0045] (1) Add 5 mL of hydrochloric acid to a 100 mL volumetric flask, and take 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of the standard solutions of heavy metals Pb, As, and Cd prepared in step 2, respectively, to obtain 75 in total; for the 9 food additive standard solutions prepared in step 1, take 0.1 mL, 0.5 mL, 1 mL, 2 mL, 5 mL, and 10 mL of each, respectively, to obtain 6 types of mixed food additive standard solutions; then, add the 6 types of mixed food additive standard solutions to the 75 heavy metal standard solution samples in sequence, and add distilled water to the scale line to obtain 75 mixed standard solutions of different concentrations.

[0046] (2) Pipette 0.1mL, 0.5mL, 1mL, 2mL, 5mL, and 10mL of the food additive standard solution prepared in step 1 into a 100mL volumetric flask, and then add 5mL of hydrochloric acid to obtain 54 in total; for the standard solutions of heavy metals Pb, As, and Cd prepared in step 2, pipette 0.2mL, 0.4mL, 0.6mL, 0.8mL, and 1.0mL of each to obtain 5 types of heavy metal standard solutions of Pb, As, and Cd; then, add the 5 types of heavy metal standard solutions of Pb, As, and Cd to the 54 food additive standard solution samples in sequence, add distilled water to the scale, and obtain 54 mixed standard solutions of different concentrations.

[0047] (II) Preparation of standard solutions for other experiments

[0048] (1) Methanol: chromatographically pure, filtered through a microporous filter membrane (0.45 μm);

[0049] (2) Acetonitrile: chromatographically pure, filtered through a microporous filter membrane (0.45 μm);

[0050] (3) Dilute ammonia (1+1) Add equal volumes of ammonia and water and mix well;

[0051] (4) 0.02-0.05 mol / L ammonium acetate (dipotassium hydrogen phosphate) solution Weigh 1.54-3.85 g of ammonium acetate, dissolve it in water and make up to 1000 mL. Filter the solution through a filter membrane (0.45 μm);

[0052] (5) For neutral alumina chromatography, 100-200 mesh;

[0053] (6) Polyamide powder, 100-200 mesh;

[0054] (7) Potassium ferrocyanide solution Weigh 10 g of potassium ferrocyanide, dissolve it in water, and dilute to 100 mL;

[0055] (8) Zinc sulfate solution: Weigh 15 g of zinc sulfate, dissolve it in water, and dilute to 100 mL;

[0056] (9) n-Hexane, analytical grade;

[0057] (10) Hydrochloric acid, analytical grade;

[0058] (11) Ammonia water: take 2 mL of ammonia water, add water to 100 mL, and mix well;

[0059] (12) Methanol + formic acid (6+4) solution: measure 60 mL of methanol and 40 mL of formic acid and mix them well;

[0060] (13) Citric acid solution: weigh 20 g of citric acid, add water to 100 mL, and dissolve and mix;

[0061] (14) Anhydrous ethanol + ammonia water + water (7+2+1) Take 70 mL of anhydrous ethanol, 20 mL of ammonia water, and 10 mL of distilled water and mix well;

[0062] (15) Tri-n-octylamine n-butanol solution (5%) Take 5 mL of tri-n-octylamine, add n-butanol to 100 mL, and mix well;

[0063] (16) pH = 6 water, distilled water plus citric acid solution to adjust the pH to 6;

[0064] (17) Saturated sodium sulfate solution, analytical grade;

[0065] (18) Acetic acid, analytical grade;

[0066] (19) Chromatography column: Use a 10 mL syringe instead and fill it with 3 cm high neutral alumina.

[0067] Mobile phase: Methanol: chromatographic grade, filtered through a microporous membrane (0.45 μm);

[0068] Acetonitrile: chromatographically pure, filtered through a microporous filter membrane (0.45 μm);

[0069] 0.02-0.05 mol / L ammonium acetate (dipotassium hydrogen phosphate) solution: Weigh 1.54-3.85 g of ammonium acetate, dissolve it in water and make up to 1000 mL. Filter the solution through a filter membrane (0.45 μm).

[0070] (III) Determination of chromatographic conditions and drawing of standard curve of mixed solution

[0071] The 129 prepared mixed standard solution samples were sequentially passed through the membrane and entered the HLP-ICP-MS coupling instrument. The chromatographic columns were screened with Shim-pack CLC-ODS6.0mmi.d.*150mm, ZORBAX Eclipse XDB-C18 Analytical 4.6×250nm 5-Micron and ODS-SP250mm×4.6mm×5um, GL Science. The mobile phases were screened with organic phase (methanol or acetonitrile, respectively) and buffer salt system (potassium dihydrogen phosphate and ammonium acetate, respectively). The volume ratio of the mobile phase was screened from 1-100:100:1. At the same time, the detection wavelength and column temperature were screened, and the chromatographic mass spectrometry conditions were obtained as follows: the reverse chromatographic column was selected as ZORBAX Eclipse XDB-C18 Analytical 4.6×250nm 5-Micron; the mobile phase selection includes the mobile phase selection of a mixed solution of methanol and ammonium acetate in a volume ratio of 10-30:90-70, the mobile phase flow rate range is 2%-3%, and the ammonium acetate concentration is 0.02-0.05 mol / L; the detection wavelength selection is determined to be 210-320 nm. Then, 129 samples were flowed through the HLP-ICP-MS coupling instrument under the determined chromatographic column conditions, and the origin software was used to fit the standard curve. The fitted standard curve equation and correlation coefficient are listed in Table 1.

[0072] Table 1 Standard curves and correlation coefficients of food additives and heavy metals

[0073]

[0074] 2. Composition and test results of mixed standard spiked sample example

[0075] In order to verify the accuracy and precision of the detection method, samples were randomly selected from the above 129 mixed standard solutions, and mixed standards were prepared manually for mixed standard spike detection. The specific composition and detection conditions of each embodiment are listed in Table 2.

[0076] Table 2 Sample solution composition and chromatographic conditions for mixed standard spiked detection example

[0077]

[0078]

[0079] The chromatographic column used in this experiment is: ZORBAX Eclipse XDB-C18 Analytical 4.6×250nm 5-Micron, and a UV detector is used. The mixed standard samples are formulated according to the formulas of each example in Table 1 and mixed evenly. After being fully mixed, they are sampled and tested after passing through a microporous filter membrane (0.45μm). The first step is to select the mobile phase conditions to determine the types and ratios of the organic phase and inorganic salt phase in the best mobile phase, and then determine the ICP measurement parameters. The experimental results show that methanol and ammonium acetate are more suitable mobile phases, and their volume ratio is Vmethanol:Vammonium acetate=1:9~3:7; ICP measurement parameters are determined as: high frequency power 1.45kW; plasma gas flow rate 14.0L.min -1 ; Carrier gas flow rate 0.8L.min -1 ,He flow rate 4.0mL.min -1 ; Sample lifting volume 0.1mL.min -1 ; Sampling depth 10.0mm; Sampling nickel cone: orifice diameter 1.0mm; Intercepting nickel cone: orifice diameter 0.4mm; Integration time 3s; Repeat sampling 3 times. Each sample was measured 8 times in parallel, and a spike recovery experiment was performed to examine the precision and accuracy of the method, and the recovery rate and relative standard deviation (RSD) were calculated. The results are shown in Table 3. The test results are listed in Table 4.

[0080] Table 3 Spiked recovery and relative standard deviation of food additives and heavy metals

[0081] Serial number food additives Detection limit (μg / L) Spiked recovery rate (%) RSD% 1 Saccharin Sodium 0.006 99.33 2.5 2 Acesulfame K 0.018 100.39 3.8 3 Carmine 0.028 108.21 3.2 4 Sunset Yellow 0.015 103.25 2.8 5 Lemon Yellow 0.025 106.97 2.6 6 Potassium Sorbate 0.008 104.27 2.3 7 benzoic acid 0.012 97.26 2.1 8 Sorbic acid 0.010 98.50 1.8 9 Sodium Benzoate 0.015 93.32 2.7 10 Aspartame 0.008 98.46 2.5 11 Pb 0.007 93.05 1.6 12 As 0.034 97.22 1.7 13 Cd 0.012 103.10 1.8

[0082] Table 4 Test results of mixed standard spiked test example

[0083]

[0084] 3. Simultaneous determination of food additives and heavy metals in food samples

[0085] 1. Sample processing method without colorant:

[0086] (1) Soft drinks and fruit juices: Remove carbon dioxide by warm stirring or ultrasonic degassing. Pipette 2.5 mL of sample into a 25 mL volumetric flask, add mobile phase to the mark, stir well, filter the solution through a microporous filter membrane, and test the filtrate using an HLP-ICP-MS instrument.

[0087] (2) Cola beverages: Warm the sample and stir to remove carbon dioxide or use ultrasonic degassing. Pipette 2.5 mL of the sample from which carbon dioxide has been removed and pass it through a neutral alumina column. When the sample liquid flows to the surface of the column, elute it with mobile phase and collect 25 mL of the eluate. Shake well and degas it by ultrasonication. Filter the solution through a microporous filter membrane (0.45 μm) and detect the filtrate using an HLP-ICP-MS instrument.

[0088] (3) Fruit tea and fruit juice: Take 2.5 mL of sample, add about 20 mL of water, mix, centrifuge for 15 min (4000 r / min), transfer all the supernatant into a neutral alumina column, and elute with mobile phase when the sample liquid flows to the column surface. Collect 25 mL of eluate, mix, degas by ultrasonic, filter the solution through a microporous filter membrane (0.45 μm), and detect the filtrate by HLP-ICP-MS.

[0089] (4) Preparation of alcoholic beverages: Weigh 10.0 g of sample and place it in a small beaker. Heat in a water bath to remove ethanol. Adjust the pH value to about 7 with ammonia water (1+1). Add water to make the volume appropriate. Filter through a filter membrane (0.45 μm). The filtrate is tested by HLP-ICP-MS.

[0090] (5) Other foods: Weigh 2.5 g of sample and add 5 mL of water. Perform ultrasonic extraction for 15 min and centrifuge for 10 min (4000 r / min). Pass the supernatant through a neutral alumina column. When the sample liquid flows to the column surface, elute with mobile phase and collect 25 mL of the eluate. Shake well and degas by ultrasonication. Filter the solution through a microporous filter membrane (0.45 μm) and detect the filtrate using HLP-ICP-MS.

[0091] (6) Other foods (including meat products): weigh 2.5 g of sample, add 1.0 mL of zinc sulfate solution and 1.0 mL of potassium ferrocyanide solution, add water to make up to 25 mL, shake well, extract by ultrasonication for 20 min, centrifuge for 10 min, filter the supernatant through a 0.45 μm microporous membrane, and then analyze it using HLP-ICP-MS.

[0092] 2. Colorant sample processing method

[0093] (1) Orange juice, fruit-flavored water, sherbet soda, etc.: Weigh 20.0-40.0 g and place in a 100 mL beaker. Heat samples containing carbon dioxide to drive off the carbon dioxide.

[0094] (2) Preparation of alcoholic beverages: Weigh 20.0g-40.0g into a 100mL beaker, add several small porcelain pieces, heat the water to remove ethanol, add 30mL of water, and dissolve in warm water. If the pH value of the sample solution is high, adjust the pH value to about 4 with citric acid solution.

[0095] (3) Hard candies, candied fruits, starch soft candies, etc.: Weigh 5.00g-10.00g, put into a 100mL small beaker, add 30mL of water, and dissolve in warm water. If the pH value of the sample solution is high, adjust the pH value to about 4 with citric acid solution.

[0096] (4) Chocolate beans and colored sugar-coated products: Weigh 5.00 g to 10.00 g of the pigment into a 100 mL beaker and repeatedly wash the pigment with water until the chocolate beans are free of pigment. Combine the pigment rinses to form the sample solution.

[0097] (5) Pigment extraction method

[0098] ① Polyamide adsorption method: add citric acid solution to the sample solution to adjust the pH value to 4, heat to 60℃, add a little water to 1g polyamide powder to make porridge, pour into the sample solution, stir for a while, pour into a chromatography tube with a little sea sand, wash with 60℃ pH=4 water 3-5 times, then wash with water to neutrality, analyze with ethanol-ammonia-water mixed solution 3-5 times, 5mL each time, collect the analysis solution, evaporate to near dryness, add water to dissolve, and adjust the volume to 5mL. Filter through a filter membrane (0.45μm), take 10μL of the filtrate and enter the HLP-ICP-MS coupling instrument for detection.

[0099] ② Liquid-liquid distribution method (applicable to samples containing erythrosine): put the prepared sample solution into a separatory funnel, add 2mL hydrochloric acid, 10-20mL tri-n-octylamine n-butanol solution (5%), shake to extract, separate the organic phase, repeat the extraction until the organic phase is colorless, combine the organic phases, wash twice with saturated sodium sulfate solution, 10mL each time, separate the organic phase, heat in a water bath in an evaporating dish and concentrate to 10mL, transfer to a separatory funnel, add 60mL n-hexane, mix well, extract 2-3 times with ammonia water, 5mL each time, combine the ammonia solution layer (containing water-soluble acidic pigments), wash twice with n-hexane, add acetic acid to the ammonia layer to neutralize, heat in a water bath and evaporate to near dryness, add water to make the volume 5mL. Filter through a filter membrane (0.45μm), and take 10μL of the filtrate into the HLP-ICP-MS coupling instrument for detection.

[0100] 3. The HLP chromatographic conditions and ICP-MS mass spectrometry working parameters are the same as the chromatographic conditions of the mixed standard spiked detection embodiment in Table 2.

[0101] 4. Presentation of analysis results

[0102] The content of additives in food is calculated according to the following formula:

[0103]

[0104] Where X is the content of acesulfame potassium, benzoic acid, sorbic acid and saccharin sodium in the sample, g / kg;

[0105] ρ—the content of benzoic acid (or acesulfame potassium, sorbic acid, saccharin sodium) in the test solution, μg / mL;

[0106] V—total volume of sample dilution, mL;

[0107] m—sample mass, g.

[0108] Calculation method of heavy metal content in food:

[0109]

[0110] Where:

[0111] X——heavy metal content in the sample, in milligrams per kilogram or milligrams per liter (mg / kg or mg / L);

[0112] C1——determine the heavy metal content in the sample solution in micrograms per milliliter (μg / mL);

[0113] C0——Heavy metal content in blank solution, in micrograms per milliliter (μg / mL);

[0114] V——Quantitative total volume of sample digestion solution, in milliliters (mL);

[0115] m——sample mass or volume, in grams or milliliters (g);

[0116] The arithmetic mean of the two independent determination results obtained under the above repeated conditions is expressed with two significant figures retained.

[0117] The test results are listed in Table 5.

[0118] Table 5 Test results of actual food samples

[0119]

[0120] The test results in Table 5 show that the contents of various food additives and three heavy metals in the Mirinda orange-flavored drinks, cocktails, soy sauce, jelly and other foods selected for the experiment did not exceed the maximum limit requirements of GB2760-2014.

[0121] At the same time, comparative experiments on the same samples were carried out, and the experiments proved that:

[0122] 1. The present invention combines reversed-phase high performance liquid chromatography with inductively coupled plasma mass spectrometry (reverse-phase HPLC-ICP-MS), which can quickly and accurately measure the content of food additives and heavy metals in food simultaneously, not only reducing the sampling amount of the sample to be tested and the amount of sample pretreatment, but also reducing the cumbersome operation caused by different instruments for separate testing and separate sampling, greatly shortening the detection time and improving work efficiency. Usually, it takes about 6 hours to detect the two instruments separately, and it takes about 4.8 hours after the combination, saving about 20% of time.

[0123] 2. The present invention can detect multiple food additives and heavy metal contents simultaneously. Traditional methods cannot detect multiple food additives and heavy metal contents simultaneously, and usually can only detect each food additive or heavy metal individually.

[0124] 3. In the present invention, HPLC is combined with ICP-MS. After the food sample is subjected to HPLC, the form of heavy metal ions does not change, and the accuracy of subsequent ICP-MS detection is not affected.

Claims

1. A method for synchronously detecting food additives and heavy metal contents in food by reversed-phase HPLC-ICP-MS, characterized in that: The HPLC and ICP-MS equipment are connected together through an Agilent company dedicated interface system (5), and then the processed food sample is first tested for food additive content on the HPLC, and then the food sample flowing through the HPLC is input into the ICP-MS through the interface system (5) for heavy metal content detection; the food additives are saccharin sodium, acesulfame potassium, carmine, sunset yellow, tartrazine, potassium sorbate, benzoic acid, sorbic acid, sodium benzoate and aspartame; the heavy metals are arsenic, lead and cadmium; The food additive content detection steps are as follows: (1) Use food additive standards to debug and determine HLPC chromatographic conditions; (2) Draw the standard curve of food additives and determine the minimum detection amount; (3) Detect the content of food additives in the processed food samples by HPLC and draw a curve for the samples to be tested; (4) Calculate the content of food additives by calculating the peak area of ​​the curve of the sample to be tested; Determining the HLPC chromatographic conditions includes selecting and determining the reverse chromatographic column, selecting and determining the mobile phase, and selecting and determining the detection wave; The reverse chromatographic column is selected as ZORBAX Eclipse XDB-C18Analytical4.6×250nm 5-Micron; the mobile phase is selected as a mixed solution of methanol and ammonium acetate in a volume ratio of 10-30:90-70, the mobile phase flow rate range is 2%-3%, and the ammonium acetate concentration is 0.02-0.05 mol / L; the detection wavelength is selected as 210-320nm; The heavy metal content detection steps are: (1) Use heavy metal standards to debug and determine ICP-MS conditions; (2) Draw a heavy metal standard curve; (3) The food sample flowing through the HPLC is input into the ICP-MS through the interface system (5) to detect the heavy metal content, and a curve of the sample to be tested is drawn; (4) Calculate the content of heavy metals by calculating the peak area of ​​the curve of the sample to be tested; The mobile phase of the ICP is a mixed solution of methanol and ammonium acetate in a volume ratio of V methanol: V ammonium acetate = 1:9 to 3:7; the ICP measurement parameters are determined as follows: high frequency power 1.45 kW; plasma gas flow rate 14.0 L.min -1 ; Carrier gas flow rate 0.8L·min -1 , He flow rate 4.0mL·min -1 ; Sample lifting volume 0.1mL·min -1 ; Sampling depth 10.0mm; Sampling nickel cone: orifice diameter 1.0mm; Intercepting nickel cone: orifice diameter 0.4mm; Integration time 3s; Repeat sampling 3 times; The food additive test results: relative standard deviation RSD <4%; sample recovery rate is between 93%-109%, and the detection limit is 2-28ng / L; The heavy metal detection results: relative standard deviation RSD<3.5%; sample recovery rate is between 93%-104%, and the detection limit is 7-12ng / L.

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