A sample processing method for detecting neotame content in food

Abstract

The invention discloses a sample processing method for detecting neotame content in food, which is characterized by comprising the following steps: 1) preparing a sample to be tested; 2) extracting the sample: 2.1) adding the sample into a container, adding an extracting solution, and adjusting the pH value to 4-5; the addition amounts of the sample and the extracting solution satisfy the following ratio: 1 g: 5-10 mL; 2.2) heating the sample in a water bath at 50-70° C., adding n-hexane, centrifuging, and discarding the n-hexane layer; the addition amounts of the sample and n-hexane satisfy the following ratio: 1 g: 0.8-1.2 mL; 2.3) adding an 80-100 g / L potassium ferrocyanide aqueous solution and a 150-200 g / L zinc acetate aqueous solution, centrifuging, and transferring the supernatant; the addition amounts of the sample, potassium ferrocyanide aqueous solution, and zinc acetate aqueous solution satisfy the following ratio: 1 g: 0.1 mL-0.4 mL: 0.1 mL-0.4 mL. This sample pretreatment method is suitable for testing a variety of food matrices, covering the 14 major food categories specified in GB 2760-2014. It is highly versatile and improves detection accuracy and efficiency. The recovery rate of Niutian is 80% to 110%.

Description

Technical Field

[0001] The present invention belongs to the field of food testing, and in particular relates to a sample processing method for detecting neotame content in food. Background Art

[0002] Neotame, chemically known as N-[N-(3,3-dimethylbutyl)-L-α-aspartyl]-L-phenylalanine-1-methyl ester, is a white crystalline powder containing approximately 4.5% water of crystalline water. It is a functional sweetener added to foods to improve their sweetness. my country has strict restrictions on the use of the sweetener neotame in foods. GB 2760-2014, "National Food Safety Standard for the Use of Food Additives," lists the use of neotame in 56 major food categories and clearly defines the types of foods in which it can be used and the limits on its use. Neotame can be used in a wide range of dosages, from 0.01g / kg to 1.0g / kg.

[0003] Currently, the main domestic testing method for neotame in food is based on GB 5009.247-2016, "National Food Safety Standard - Determination of Neotame in Foods," which uses high-performance liquid chromatography to determine neotame in beverages, preserved fruits, pastries, roasted seeds and nuts, pickled vegetables, jams, jellies, and compound seasonings. SN / T 3538-2013, "Determination of Six Synthetic Sweeteners in Exported Foods - Liquid Chromatography-Mass Spectrometry / Mass Spectrometry," uses liquid chromatography-tandem mass spectrometry / mass spectrometry to determine six synthetic sweeteners, including neotame, in milk powder, liquid milk, yogurt, cream, cheese, ice cream, red wine, juice, pastries, preserved fruits, and sweet and sour radish. The GB5009.247-2016 method pre-treats samples using a formic acid-triethylamine mixed extraction solution followed by centrifugation. Samples are then extracted using a C18 solid-phase extraction cartridge and concentrated with nitrogen purging prior to analysis. This method uses water as the extraction fluid. Extraction of matrices with high protein and fat content after matrix expansion cannot be clarified by centrifugation, resulting in poor separation of impurities during instrumental testing and biased qualitative and quantitative results. Furthermore, the formic acid-triethylamine elution used during the C18 solid-phase extraction (SPE) column fails to remove most impurities. The strong elution capacity of pure methanol, used for elution, results in a high concentration of impurities in the eluate, which can easily cause interference. The SN / T 3538-2013 method only adds a precipitant to dairy products during pretreatment, which provides some purification of impurities. However, the precipitant is ineffective and fails to effectively remove impurity interference during SPE cleanup, making it unsuitable for liquid chromatography.

[0004] The detection methods in both GB 5009.247-2016 and SN / T 3538-2013 are only targeted at specific food matrices and cannot cover the 14 major food categories specified in GB 2760-2014, as well as the 72 typical matrices of allowable additives and common illegal additives. They have poor versatility and low detection efficiency. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a sample processing method suitable for detecting the neotame content in foods with various food matrices.

[0006] The technical solution adopted by the present invention to solve the above technical problems is: a sample processing method for detecting the content of neotame in food, characterized by comprising the following steps:

[0007] 1) Preparation of samples to be tested;

[0008] 2) Sample extraction:

[0009] 2.1) Add the sample to the container, add the extract, and adjust the pH to 4-5; the sample and extract are added in an amount of 1g: 5-10mL;

[0010] 2.2) Heat the sample in a water bath at 50-70°C, add n-hexane, centrifuge, and discard the n-hexane layer. The amount of sample and n-hexane added should be 0.8-1.2 mL for 1 g.

[0011] 2.3) Add 80-100 g / L potassium ferrocyanide aqueous solution and 150-200 g / L zinc acetate aqueous solution, centrifuge, and transfer the supernatant, wherein the amount of sample, potassium ferrocyanide aqueous solution, and zinc acetate aqueous solution added is 1 g: 0.1 mL-0.4 mL: 0.1 mL-0.4 mL;

[0012] 2.4) Add the extract to the residue in the container, centrifuge, combine with the supernatant, and adjust the volume to 50-100 mL with the extract. Filter with filter paper to obtain a filtrate, wherein the amount of extract added to the sample and residue is 4-9 mL for 1 g of sample.

[0013] 3) Purification:

[0014] Before use, the solid phase extraction column was activated with methanol and water in sequence. 5-10 mL of the filtrate was passed through the activated solid phase extraction column, and then rinsed with 2.5-5 mL of buffer. All the effluent was discarded, and the column was eluted with 7-9 mL of eluent. The eluate was collected and concentrated to 0.5-1.5 mL in a 55-65°C water bath with nitrogen purge. The volume was then made up to 1-2 mL with buffer, and the column was filtered through a membrane before injection.

[0015] The buffer solution is a formic acid-triethylamine aqueous solution, and the volume addition amounts of formic acid, triethylamine and water meet the following ratio: 0.6-1.0:2.0-3.0:1000;

[0016] The extract is a mixture of methanol and buffer, with a pH value of 4 to 5, and the volume proportion of methanol in the extract is 30% to 50%;

[0017] The eluent is a mixture of methanol and buffer solution, with a pH value of 4-5, and the volume proportion of methanol in the eluent is 70%-90%.

[0018] The sample extraction method of the present invention is applicable to a variety of food matrices, such as those containing proteins, oils, and gum bases. It is suitable for the extraction of a single food matrix, and is more suitable for the extraction of a variety of mixed food matrices. After the sample is added to the extracting solution, the pH value is adjusted to 4-5 to ensure that neotame will not degrade during the extraction process. For oil samples, n-hexane is added to the sample after the sample is water-bathed to ensure that the sample is defatted while neotame is retained in the sample without loss. Gum base samples can be dissolved by heating. Adding a precipitation reagent, namely a mixed aqueous solution of potassium ferrocyanide and zinc acetate, to the methanol-containing extracting solution can ensure that impurities such as proteins are further precipitated and protein interference is reduced. By performing a secondary extraction on the residue, sufficient neotame extraction is ensured.

[0019] Adding a specific proportion of methanol to the extract changes the polarity of the extract, which can precipitate proteins during sample extraction and ensure that neotame is retained on the solid phase extraction column, while impurities with weaker hydrophobicity are not retained on the solid phase extraction column, thereby improving the detection accuracy of neotame.

[0020] Adding a specific proportion of methanol to the eluent changes the polarity of the eluent, allowing neotame to be eluted from the solid phase extraction column while impurities with greater hydrophobicity remain on the solid phase extraction column, thereby improving the detection accuracy of neotame.

[0021] Preferably, in step 2) of 2.1), the pH value is adjusted to 4-5 using a 10-30% v / v formic acid aqueous solution or a 10-30% v / v ammonia aqueous solution.

[0022] Preferably, in step 2), the centrifugal treatment is performed at a speed of 3000 to 5000 r / min and for a time of 3 to 10 min.

[0023] Preferably, the recovery rate of neotame is 80% to 110%.

[0024] Compared with the existing technology, the advantages of the present invention are: the sample pretreatment method is applicable to the detection of various food matrices, covering the 14 major categories of food specified in GB 2760-2014, has strong versatility, improves detection accuracy and efficiency, and the recovery rate of neotame is 80% to 110%. DETAILED DESCRIPTION

[0025] The present invention is described in further detail below with reference to the examples.

[0026] The present invention provides 10 examples, in which a certain concentration of neotame is added to a blank sample. The specific samples and added concentrations are shown in Table 3.

[0027] The embodiment includes the following steps:

[0028] 1) Sample preparation: Liquid samples should be shaken well before extraction. Gas-containing samples such as carbonated beverages and red wine should be slightly heated first, stirred to remove carbon dioxide from the sample, or ultrasonically degassed before extraction. Solid or semi-solid samples should be crushed evenly before extraction. The prepared samples should be stored at 0°C to 5°C and measured as soon as possible.

[0029] 2) Sample extraction:

[0030] 2.1) Add 5 g of sample to a container, add the extract, and adjust the pH to 4.5;

[0031] 2.2) Heat the sample in a 60°C water bath, add n-hexane, and centrifuge at 4000 rpm for 5 min. Discard the n-hexane layer.

[0032] 2.3) Add 80-100 g / L potassium ferrocyanide aqueous solution and 150-200 g / L zinc acetate aqueous solution, centrifuge at 4000 rpm for 5 min, and transfer the supernatant;

[0033] 2.4) Add the extract to the container residue and centrifuge at 4000 rpm for 5 min. Combine the extract with the supernatant and adjust the volume to 80 mL with the extract. Filter with filter paper to obtain the filtrate.

[0034] 3) Purification:

[0035] Before use, the solid-phase extraction column was activated with 6 mL of methanol and 6 mL of water. 5-10 mL of the filtrate was passed through the activated solid-phase extraction column and rinsed with 2.5-5 mL of buffer. All the effluent was discarded and eluted with 7-9 mL of eluent. The eluate was collected and concentrated to 0.5-1.5 mL in a 55-65°C water bath with nitrogen purge. The volume was then adjusted to 1-2 mL with buffer and filtered through a membrane before injection. The compositions of the buffer, extract, and eluent are shown in Table 1, and the key parameter controls are shown in Table 2.

[0036] The difference between Comparative Example 1 and Example 5 is that the extract is a buffer solution.

[0037] The difference between Comparative Example 2 and Example 2 is that the eluent is pure methanol.

[0038] The difference between Comparative Example 3 and Example 9 is that step 2.2) is omitted.

[0039] The difference between Comparative Example 4 and Example 6 is that step 2.2 is omitted.

[0040] The obtained examples and comparative examples were analyzed using liquid chromatography using a C18 column, 150 mm × 4.6 mm, 5 μm, or equivalent; column temperature: 30°C; mobile phase: methanol (phase A); 20 mmol / L ammonium acetate solution (phase B); flow rate: 1.0 mL / min; detection wavelength: 210 nm; injection volume: 50 μL. The results are shown in Table 3.

[0041] Table 1 Key process parameter control in the embodiment of the present invention

[0042]

[0043] Table 2 Key process parameter control in the embodiment of the present invention

[0044]

[0045] Table 3 Test results of the embodiments of the present invention and comparative examples

[0046]

[0047] Table 3 Test results of the embodiments of the present invention and comparative examples

[0048]

Claims

1. A sample processing method for detecting neotame content in food, characterized in that: The following steps are involved: 1) Preparation of samples to be tested; 2) Sample extraction: 2.1) Add the sample to the container, add the extract, and adjust the pH to 4-5; the sample and extract are added in an amount of 1g: 5-10mL; 2.2) Heat the sample in a water bath at 50-70°C, add n-hexane, centrifuge, and discard the n-hexane layer. The amount of sample and n-hexane added should be 0.8-1.2 mL for 1 g. 2.3) Add 80-100 g / L potassium ferrocyanide aqueous solution and 150-200 g / L zinc acetate aqueous solution, centrifuge, and transfer the supernatant, wherein the amount of sample, potassium ferrocyanide aqueous solution, and zinc acetate aqueous solution added is 1 g: 0.1 mL-0.4 mL: 0.1 mL-0.4 mL; 2.4) Add the extract to the residue in the container, centrifuge, combine with the supernatant, and adjust the volume to 50-100 mL with the extract. Filter with filter paper to obtain a filtrate, wherein the amount of extract added to the sample and residue is 4-9 mL for 1 g of sample. 3) Purification: Before use, the solid phase extraction column was activated with methanol and water in sequence. 5-10 mL of the filtrate was passed through the activated solid phase extraction column, and then rinsed with 2.5-5 mL of buffer. All the effluent was discarded, and the column was eluted with 7-9 mL of eluent. The eluate was collected and concentrated to 0.5-1.5 mL in a 55-65°C water bath with nitrogen purge. The volume was then made up to 1-2 mL with buffer, and the column was filtered through a membrane before injection. The buffer solution is a formic acid-triethylamine aqueous solution, and the volume addition amounts of formic acid, triethylamine and water meet the following ratio: 0.6-1.0:2.0-3.0:1000; The extract is a mixture of methanol and buffer, with a pH value of 4 to 5, and the volume proportion of methanol in the extract is 30% to 50%; The eluent is a mixture of methanol and buffer, with a pH value of 4 to 5, and the volume proportion of methanol in the eluent is 70% to 90%; In step 2) of 2.1), the pH value is adjusted to 4-5 using a 10-30% v / v formic acid aqueous solution or a 10-30% v / v ammonia aqueous solution.

2. The method for sample processing for detecting neotame content in food according to claim 1, wherein: In the step 2), the centrifugal treatment is performed at a speed of 3000 to 5000 r / min and for a time of 3 to 10 minutes.

3. The sample processing method for detecting neotame content in food according to claim 1, characterized in that: The recovery rate of neotame is 80% to 110%.

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