Pretreatment method for detecting various vitamins in food

Abstract

The invention provides a pretreatment method for detecting various vitamins in food, and relates to the technical field of vitamin detection. Comprising the following steps: a) mixing a food sample with a sodium acetate buffer solution according to a volume ratio of 1: (1-5), adding a combined enzyme for enzymolysis to obtain enzymatic hydrolysate, and collecting filtrate after filtering a membrane; the combined enzyme comprises neutral protease and lipase; the mass volume ratio of the combined enzyme to the sodium acetate buffer solution is 24-32%; b) adding a mixed solvent of normal hexane and acetonitrile into the filtrate, carrying out vortex mixing, centrifuging, and separating to obtain an upper-layer normal hexane phase or a lower-layer acetonitrile phase; and c) transferring the upper-layer normal hexane phase or the lower-layer acetonitrile phase, adding silanized magnetic nanoparticles, oscillating and adsorbing for 15-20 minutes, and removing the silanized magnetic nanoparticles to obtain a sample to be detected. The pretreatment method disclosed by the invention can meet the requirements of detecting various vitamins in different foods, and is high in detection accuracy and precision.

Description

Technical Field

[0001] The invention belongs to the technical field of vitamin detection, and in particular relates to a pre-treatment method for detecting multiple vitamins in food. Background Art

[0002] Existing technologies have obvious shortcomings in the pre-treatment of simultaneous detection of multi-component vitamins. Traditional methods such as liquid-liquid extraction and solid-phase extraction are not only cumbersome and time-consuming, but also difficult to effectively remove interfering substances when faced with complex food matrices, resulting in poor repeatability of test results. With the widespread application of high-precision detection technologies such as liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS), higher requirements are placed on the purification efficiency and compatibility of pre-treatment methods. However, the pre-treatment technologies currently on the market can often only be used to treat a certain type of food (such as dairy products, fruit and vegetable products) or a few specific vitamins, and cannot meet the increasingly diverse needs of food testing.

[0003] At a time when food safety supervision is becoming increasingly stringent and food varieties are constantly enriching, food companies and testing agencies are in urgent need of a universal and efficient pretreatment method that can quickly and accurately process multiple vitamins (including fat-soluble and water-soluble vitamins) in food samples of different matrices (such as grains, meats, beverages, etc.). This can not only improve detection efficiency and reduce detection costs, but also ensure the accuracy and reliability of test results, providing strong support for food safety risk assessment and quality control. Therefore, the development of a pretreatment method suitable for the detection of multiple vitamins in a variety of food samples has become a key issue that needs to be urgently addressed in the current field of analytical testing. Summary of the Invention

[0004] In view of this, the main purpose of the present invention is to provide a pretreatment method for detecting multiple vitamins in food. By using this pretreatment method, the requirements for detecting multiple vitamins in different food samples can be met, the influence of the matrix on the test results can be reduced, and the detection accuracy and precision are high.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a pre-treatment method for detecting multiple vitamins in food, comprising the following steps:

[0007] a) mixing a food sample with sodium acetate buffer in a volume ratio of 1:(1-5), adding a combined enzyme for enzymatic hydrolysis to obtain an enzymatic hydrolyzate, and filtering through a membrane to collect the filtrate;

[0008] The enzyme combination includes neutral protease and lipase; the mass volume ratio of the enzyme combination to sodium acetate buffer is 24% to 32%;

[0009] b) adding a mixed solvent of n-hexane and acetonitrile to the filtrate, vortex mixing, and centrifuging to separate and obtain an upper n-hexane phase or a lower acetonitrile phase;

[0010] c) removing the upper n-hexane phase or the lower acetonitrile phase, adding silanized magnetic nanoparticles, shaking and adsorbing for 15 to 20 minutes, and then removing the silanized magnetic nanoparticles to obtain a sample to be tested.

[0011] Preferably, in step c), the method for preparing the silanized magnetic nanoparticles comprises the following steps:

[0012] Fe3O4 nanoparticles and silane mixture were added to anhydrous ethanol, and after ultrasonication, the mixture was centrifuged and the supernatant was removed. The product was vacuum dried, crushed and sieved to obtain silanized magnetic nanoparticles.

[0013] The silane mixture is 3-aminopropyltriethoxysilane and octadecyltrichlorosilane.

[0014] Preferably, the particle size of the Fe3O4 nanoparticles is 10 to 20 nm; the molar ratio of the Fe3O4 nanoparticles to the silane mixture is 1:2.5 to 3.5; the mass ratio of 3-aminopropyltriethoxysilane to octadecyltrichlorosilane in the silane mixture is 1:1; the mass volume ratio of the Fe3O4 nanoparticles to anhydrous ethanol is 1 g: (45 to 55) mL.

[0015] Preferably, the ultrasonication time is 3 to 5 hours, the centrifugal speed is 4000 to 5000 rpm, the product is vacuum dried at a temperature of 75 to 85° C. for 3 to 5 hours, and an 80 to 120 mesh sieve is used for the crushing and screening.

[0016] Preferably, in step a), the sodium acetate buffer is 0.1-0.3 M and has a pH of 5.8-6.0.

[0017] Preferably, in step a), the enzyme combination comprises a neutral protease and a lipase; the mass ratio of the neutral protease to the lipase is (1-2): (1-2), and the enzymatic activities of the neutral protease and the lipase are both 8000-12000 U / mg.

[0018] Preferably, in step a), the enzymatic hydrolysis temperature is 40-50° C., the enzymatic hydrolysis time is 20-40 min; the enzymatic hydrolysis is assisted by ultrasound, and the ultrasound frequency is 30-50 kHz; the pore size of the filter membrane is 0.22 μm or 0.45 μm.

[0019] Preferably, in step b), the volume ratio of n-hexane to acetonitrile in the mixed solvent of n-hexane and acetonitrile is (2-3):(2-3); and the rotation speed during the centrifugation is 4000-5000 rpm.

[0020] Preferably, in step c), 3 to 5 mL of the upper n-hexane phase or the lower acetonitrile phase is transferred, and the amount of the silanized magnetic nanoparticles added is 300 to 500 mg.

[0021] Preferably, the food comprises milk powder and a compound fruit and vegetable juice beverage; when the food is milk powder, step c) removes the upper n-hexane phase; when the food is a compound fruit and vegetable juice beverage, step c) removes the lower acetonitrile phase.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention provides a pretreatment method for detecting multiple vitamins in food. Sodium acetate buffer is used to maintain pH stability during the subsequent enzymatic hydrolysis process, ensuring the optimal activity of the combined enzyme, while protecting the vitamins from degradation and providing a suitable ionic environment for the reaction. The combined enzyme can decompose protein and fat, release bound vitamins, reduce side reactions of substances such as pectin and cellulose, and eliminate matrix interference. Silanized magnetic nanoparticles are used to adsorb and remove impurities and matrices in different food samples, which can meet the detection requirements of multiple vitamins in different food samples. The above pretreatment processes work synergistically to reduce the impact of different impurities and matrices in different samples on the detection results, and the detection accuracy and precision are high. DETAILED DESCRIPTION

[0024] The present invention provides a pre-treatment method for detecting multiple vitamins in food, comprising the following steps:

[0025] a) mixing a food sample with sodium acetate buffer in a volume ratio of 1:(1-5), adding a combined enzyme for enzymatic hydrolysis to obtain an enzymatic hydrolyzate, and filtering through a membrane to collect the filtrate;

[0026] The enzyme combination includes neutral protease and lipase; the mass volume ratio of the enzyme combination to sodium acetate buffer is 24% to 32%;

[0027] b) adding a mixed solvent of n-hexane and acetonitrile to the filtrate, vortex mixing, and centrifuging to separate and obtain an upper n-hexane phase or a lower acetonitrile phase;

[0028] c) removing the upper n-hexane phase or the lower acetonitrile phase, adding silanized magnetic nanoparticles, shaking and adsorbing for 15 to 20 minutes, and then removing the silanized magnetic nanoparticles to obtain a sample to be tested.

[0029] In the present invention, the food sample is obtained after preliminary processing of the food to be tested. Preferably, the food includes milk powder and compound fruit and vegetable juice beverages. When the food is milk powder, the food sample is prepared by mixing milk powder and ultrapure water at a mass volume ratio of 1:5 (w / v = g / mL). The water temperature of the ultrapure water is 40-60°C, preferably 50°C, and vortexing is performed for 5 minutes during the preparation. When the food is a compound fruit and vegetable juice beverage, it is directly used as the food sample.

[0030] In step a) of the present invention, the sodium acetate buffer is preferably 0.1-0.3M, more preferably 0.1, 0.2, 0.3M, and most preferably 0.2M; the pH value is preferably 5.8-6.0, more preferably 5.8, 5.9, 6.0, and most preferably 5.8. As an optional embodiment, when the food is milk powder, the volume ratio of the food sample to the sodium acetate buffer is preferably 1:5 (v / v). As an optional embodiment, when the food is a compound fruit and vegetable juice beverage, the volume ratio of the food sample to the sodium acetate buffer is preferably 1:1 (v / v). Ultrasound is used to assist in the enzymatic hydrolysis, and the ultrasonic frequency is preferably 30-50 kHz, more preferably 40 kHz. The combined enzyme is preferably a neutral protease and a lipase. The mass ratio of the neutral protease and the lipase is preferably (1-2): (1-2), more preferably 1:1 or 1:2 or 2:1 or 2:2. The enzymatic activities of the neutral protease and lipase are preferably 8000-12000, more preferably 9000-11000, and most preferably 10000 U / mg. As an optional embodiment, when the food is milk powder, the mass ratio of the neutral protease and lipase is preferably 1:2. When the food is a compound fruit and vegetable juice beverage, the mass ratio of the neutral protease and lipase is preferably 2:1. As an optional embodiment, when the food is milk powder, the combined enzyme is added at a sodium acetate buffer mass volume ratio of 24% (w / v=mg / mL). As an optional embodiment, when the food is a compound fruit and vegetable juice beverage, the combined enzyme is added at a sodium acetate buffer mass volume ratio of 32% (w / v=mg / mL). The enzymatic hydrolysis temperature is preferably 40-50°C, more preferably 45°C. The enzymatic hydrolysis time is preferably 20-40 min. As an optional embodiment, when the food is milk powder, the enzymatic hydrolysis time is more preferably 30 min. As an optional embodiment, when the food is a composite fruit and vegetable juice beverage, the enzymatic hydrolysis time is further preferably 40 minutes. When filtering the enzymatic hydrolyzate, the pore size of the filter membrane is preferably 0.22 μm or 0.45 μm, more preferably 0.45 μm, and most preferably 0.45 μm PTFE (polytetrafluoroethylene) filter membrane.

[0031] In step b) of the present invention, the rotation speed during centrifugation is preferably 4000-5000 rpm. The vortex oscillation time is preferably 5-15 min, and more preferably 10 min. In the mixed solvent of n-hexane and acetonitrile, the volume ratio of n-hexane and acetonitrile is preferably (2-3): (2-3). As an optional embodiment, when the food is milk powder, the volume ratio of n-hexane and acetonitrile is more preferably 3:2 (v / v). As an optional embodiment, when the food is a compound fruit and vegetable juice beverage, the volume ratio of n-hexane and acetonitrile is more preferably 2:3 (v / v). The volume ratio of the filtrate and the n-hexane-acetonitrile mixed solvent is preferably 3:2.

[0032] In step c) of the present invention, 3 to 5 mL, more preferably 4 mL, of the upper n-hexane phase or the lower acetonitrile phase is preferably transferred; the amount of the silanized magnetic nanoparticles added is preferably 300 to 500 mg, more preferably 400 mg. As an alternative embodiment, when the food is milk powder, step c) transfers the upper n-hexane phase; as an alternative embodiment, when the food is a composite fruit and vegetable juice beverage, step c) transfers the lower acetonitrile phase.

[0033] In step c) of the present invention, preferably, the method for preparing the silanized magnetic nanoparticles comprises the following steps: adding Fe3O4 nanoparticles and a silane mixture to anhydrous ethanol, ultrasonicating, centrifuging, removing the supernatant, vacuum drying the product, crushing and sieving to obtain silanized magnetic nanoparticles; the silane mixture is 3-aminopropyltriethoxysilane and octadecyltrichlorosilane. The particle size of the Fe3O4 nanoparticles is preferably 10-20 nm; the molar ratio of the Fe3O4 nanoparticles to the silane mixture is 1:2.5-3.5, more preferably 1:3; the mass ratio of 3-aminopropyltriethoxysilane to octadecyltrichlorosilane in the silane mixture is preferably 1:1; the mass volume ratio of the Fe3O4 nanoparticles to anhydrous ethanol is preferably 1 g:(45-55) mL, more preferably any one of 1 g:46 mL, 1 g:47 mL, 1 g:48 mL, 1 g:49 mL, 1 g:50 mL, 1 g:51 mL, 1 g:52 mL, 1 g:53 mL, and 1 g:54 mL, and most preferably 1 g:50 mL. The ultrasonication time is preferably 3-5 hours, more preferably 4 hours. The centrifugal speed is preferably 4000-5000 rpm. The temperature for vacuum drying the product is preferably 75-85°C, more preferably 80°C; the time for vacuum drying the product is preferably 3-5h, more preferably 4h; and the 80-120 mesh sieve is preferably used for the crushing and screening, more preferably 100 mesh sieve.

[0034] In the present invention, under the action of ultrasound, a mixture of 3-aminopropyltriethoxysilane and octadecyltrichlorosilane in a molar ratio of 1:3 in an anhydrous ethanol medium is assembled with Fe3O4 nanoparticles to form silanized magnetic nanoparticles with surface modifications of C18 and amino groups.

[0035] In the present invention, the main function of the sodium acetate buffer is to maintain pH stability during the subsequent enzymatic hydrolysis process, ensure the optimal activity of neutral protease and lipase, protect vitamins from degradation, and provide a suitable ionic environment for the reaction. The combined enzyme can decompose protein and fat, release bound vitamins, reduce side reactions of substances such as pectin and cellulose, and eliminate matrix interference. The present invention uses the silanized magnetic nanoparticles to adsorb and remove impurities and matrices in different food samples, which can meet the detection requirements of multiple vitamins in different food samples. The above pretreatment processes work synergistically to reduce the influence of different impurities and matrices in different samples on the test results. The detection accuracy and precision are high, and it has strong application and promotion value.

[0036] The pretreatment method of the present invention can detect five vitamins, including vitamin D3, vitamin E, vitamin B1, vitamin B2, and vitamin C, in samples to be tested. The recovery rates of the five vitamins in milk powder samples ranged from 90% to 110%, and the relative standard deviations of six replicate tests for the five vitamins were less than 5%, demonstrating satisfactory results. The recovery rates of the five vitamins in composite fruit and vegetable juice samples ranged from 85% to 120%, and the relative standard deviations of six replicate tests for the five vitamins were less than 5%, demonstrating satisfactory results.

[0037] The following will be a clear and complete description of the technical solutions of the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0038] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0039] Example 1

[0040] A pretreatment method for detecting multiple vitamins in food:

[0041] 1. Preparation of silanized magnetic nanoparticles

[0042] 1 g of Fe3O4 nanoparticles with a particle size of 10 to 20 nm, 1.81 g of 3-aminopropyltriethoxysilane (CAS No. 3663-44-3, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number A151452) and 1.81 g of octadecyltrichlorosilane (CAS No. 112-04-9, Shanghai Aladdin Biochemical Technology Co., Ltd., product number T463158) were ultrasonically mixed in 50 mL of anhydrous ethanol for 4 h, and then centrifuged at 4000 to 5000 rpm to remove the supernatant. The product was placed in a vacuum at 80°C, dried for 4 h, crushed and passed through a 100-mesh sieve to obtain silanized magnetic nanoparticles.

[0043] Under the action of ultrasound, a mixture of 3-aminopropyltriethoxysilane and octadecyltrichlorosilane in a molar ratio of 1:3 in anhydrous ethanol medium was assembled with Fe3O4 nanoparticles to form silanized magnetic nanoparticles with surface modified with C18 and amino groups.

[0044] 2. Pre-treatment of multivitamin testing for formula milk powder

[0045] Sample preparation: Accurately weigh 10.00 g of milk powder (Jinlingguan-Zhenhu), add 50 mL of 50°C ultrapure water to reconstitute, and vortex for 5 minutes to obtain the milk powder sample solution;

[0046] Pre-processing steps:

[0047] a) The milk powder treated solution was mixed (evenly) with 250 mL of 0.1 M sodium acetate buffer at pH 5.8 at a ratio of 1:5 (v / v=mL / mL), and 60 mg of a combined enzyme (neutral protease (purchased from Shandong West Asia Chemical Co., Ltd., CAS No.: 9068-59-1) + lipase (purchased from Shandong West Asia Chemical Co., Ltd., CAS No.: 9001-62-1) was added; mass ratio 1:2, and the enzyme activity was 10,000 U / mg); ultrasonic-assisted enzymolysis was performed at 45° C. for 30 minutes at an ultrasonic frequency of 40 kHz to obtain an enzymatic solution, which was filtered through a 0.45 μm PTFE (polytetrafluoroethylene) filter membrane (product name: Meck Millipore Omnipore, PTFE 0.45UM, product model: JHWP04700) to collect the filtrate;

[0048] b) adding a mixed solvent of n-hexane and acetonitrile (3:2, v / v) to the filtrate obtained above, with the volume ratio of the filtrate to the mixed solvent being 3:2; vortexing for 10 minutes and then centrifuging at 4000-5000 rpm to obtain an upper n-hexane phase;

[0049] c) 4 mL of the upper n-hexane phase was removed, and 400 mg of the silanized magnetic nanoparticles prepared above were added. After oscillation and adsorption for 15 minutes, the silanized magnetic nanoparticles carrying impurities were removed by centrifugation to obtain a sample to be tested.

[0050] Example 2

[0051] A pretreatment method for detecting multiple vitamins in food:

[0052] 1. Preparation of silanized magnetic nanoparticles

[0053] 1 g of Fe3O4 nanoparticles with a particle size of 10-20 nm, 1.81 g of 3-aminopropyltriethoxysilane and 1.81 g of octadecyltrichlorosilane were ultrasonically mixed in 50 mL of anhydrous ethanol for 4 h, and then centrifuged at 4000-5000 rpm to remove the supernatant. The product was placed in a vacuum at 80°C, dried for 4 h, crushed and passed through a 100-mesh sieve to obtain silanized magnetic nanoparticles.

[0054] Under the action of ultrasound, a mixture of 3-aminopropyltriethoxysilane and octadecyltrichlorosilane in a molar ratio of 1:3 in anhydrous ethanol medium was assembled with Fe3O4 nanoparticles to form silanized magnetic nanoparticles with surface modified with C18 and amino groups.

[0055] 2. Pretreatment of multivitamin detection in compound fruit and vegetable juice beverages

[0056] Sample preparation: A composite fruit and vegetable juice drink (ONLYTREE - 21 fruits and vegetables a day) purchased from a supermarket was vortexed and allowed to stand for 15 minutes, and 50 mL was taken as the fruit and vegetable juice sample.

[0057] Pre-processing steps:

[0058] a) The above-mentioned fruit and vegetable juice sample liquid was mixed (evenly) with 50 mL of 0.1 M sodium acetate buffer at pH 5.8 at a ratio of 1:1 (v / v = mL / mL), and 80 mg of combined enzyme (neutral protease (purchased from Shandong Xiya Chemical Co., Ltd., CAS No.: 9068-59-1) + lipase (purchased from Shandong Xiya Chemical Co., Ltd., CAS No.: 9001-62-1); mass ratio 2:1, enzyme activity of both 10000 U / mg) was added; ultrasonic-assisted enzymatic hydrolysis was performed at 45°C for 40 minutes at an ultrasonic frequency of 40 kHz to obtain an enzymatic solution, which was filtered through a 0.45 μm PTFE (polytetrafluoroethylene) filter membrane (product name: Meck Millipore Omnipore, PTFE 0.45UM, product model: JHWP04700) to collect the filtrate.

[0059] b) adding a mixed solvent of n-hexane and acetonitrile (2:3, v / v) to the filtrate obtained above, with the volume ratio of the filtrate to the mixed solvent being 3:2; vortexing for 10 minutes and then centrifuging at 4000-5000 rpm to obtain the lower acetonitrile phase;

[0060] c) 4 mL of the lower acetonitrile phase was removed, and 400 mg of the silanized magnetic nanoparticles prepared above were added. After oscillation and adsorption for 20 minutes, impurities were removed by magnetic separation to obtain a sample to be tested.

[0061] Application Example 1:

[0062] Multivitamin detection in pre-treated samples

[0063] The samples to be tested prepared in the above Example 1 and Example 2 were respectively subjected to liquid chromatography-mass spectrometry detection.

[0064] For milk powder samples, the test parameters are as follows:

[0065] HPLC detection conditions:

[0066] Column: Waters BEH C 18 (1.7μm,150mm×2.1mm); mobile phase: phase A is acetonitrile aqueous solution, the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 5:95; phase B is acetonitrile aqueous solution containing 0.1% formic acid, the volume ratio of water to acetonitrile in the acetonitrile aqueous solution containing 0.1% formic acid is 5:95; gradient elution program is: 0-1min, 2%-4% B; 1-2min, 4% B-8% B; 2-8min, 8% B; 8-10min, 8% B-18% B; 10-13min, 18% B-40% B; 13-15min, 40% B-70% B; 15-18min, 70% B-90% B.

[0067] Flow rate: 0.4 mL / min; column temperature: 30°C, injection volume: 2 μL, UV detection wavelength: 270 nm.

[0068] The fat-soluble vitamins separated by liquid chromatography were detected by mass spectrometry. The mass spectrometry analysis was performed using a triple quadrupole mass spectrometer (SCIEX 4500MD) for quantitative detection.

[0069] Mass spectrometry conditions: electron impact ion source (EI); electron energy: 70 eV; emission current: 45 μA; ion source temperature: 230°C; transfer line temperature: 280°C; solvent delay: 5 min; primary mass spectrometry mass scan range: m / z 50-600; secondary mass spectrometry mass scan range: m / z 50-600; acquisition rate: 2 spectrum / s; acquisition time: 200 ms / spectrum.

[0070] For composite fruit and vegetable juice samples, the test parameters are as follows:

[0071] The conditions for the high performance liquid chromatography detection include:

[0072] Chromatographic column: Waters Atlantis T3 C18 (3 μm, 100 mm × 2.1 mm); mobile phase: phase A is acetonitrile-water solution, the volume ratio of acetonitrile to water in the acetonitrile-water solution is 10:90; phase B is acetonitrile-water solution containing 0.1% formic acid, the volume ratio of water to acetonitrile in the acetonitrile-water solution containing 0.1% formic acid is 10:90; gradient elution program is: 1-2 min, 2% B to 6% B; 8-9.5 min, 6% B to 18% B; 12-13 min, 18% B to 60% B; 15-17.5 min, 60% B to 85% B; 17.5-18 min, 85% B to 90% B; 20-21 min, 90% B to 2% B.

[0073] Flow rate: 0.3 mL / min; column temperature: 35°C, injection volume: 5 μL, UV detection wavelength: 270 nm.

[0074] The water-soluble vitamins separated by liquid chromatography were detected by mass spectrometry. The mass spectrometry analysis was carried out using a triple quadrupole mass spectrometer (SCIEX4500MD) for quantitative detection.

[0075] Mass spectrometry conditions: electron impact ion source (EI); electron energy: 70 eV; emission current: 45 μA; ion source temperature: 250°C; transfer line temperature: 280°C; solvent delay: 6 min; primary mass spectrometry mass scan range: m / z 50-600; secondary mass spectrometry mass scan range: m / z 50-600; acquisition rate: 3 spectrum / s; acquisition time: 150 ms / spectrum.

[0076] Test results: as shown in Table 1.

[0077] Table 1 Recovery and precision of multivitamins (n=3)

[0078]

[0079] As shown in Table 1, the recoveries of the five vitamins in the milk powder samples ranged from 90% to 110%, and the relative standard deviations of the six replicates for the five vitamins were less than 5%, indicating satisfactory results. The recoveries of the five vitamins in the composite fruit and vegetable juice samples ranged from 85% to 120%, and the relative standard deviations of the six replicates for the five vitamins were less than 5%, indicating satisfactory results.

[0080] From the above results, it can be seen that the pretreatment method of the present invention can meet the detection requirements of multiple vitamins in different food samples. The pretreatment process can reduce the influence of different matrices in different samples on the detection results. It has high accuracy and precision and has strong application and promotion value.

[0081] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A pretreatment method for detecting multiple vitamins in food, characterized in that: The steps include: a) mixing a food sample with sodium acetate buffer in a volume ratio of 1:(1-5), adding a combined enzyme for enzymatic hydrolysis to obtain an enzymatic hydrolyzate, and filtering through a membrane to collect the filtrate; The enzyme combination includes neutral protease and lipase; the mass volume ratio of the enzyme combination to sodium acetate buffer is 24% to 32%; b) adding a mixed solvent of n-hexane and acetonitrile to the filtrate, vortex mixing, and centrifuging to separate and obtain an upper n-hexane phase or a lower acetonitrile phase; c) removing the upper n-hexane phase or the lower acetonitrile phase, adding silanized magnetic nanoparticles, shaking and adsorbing for 15 to 20 minutes, and then removing the silanized magnetic nanoparticles to obtain a sample to be tested.

2. The pre-treatment method according to claim 1, characterized in that: In step c), the method for preparing the silanized magnetic nanoparticles comprises the following steps: Fe3O4 nanoparticles and silane mixture were added to anhydrous ethanol, and after ultrasonication, the mixture was centrifuged and the supernatant was removed. The product was vacuum dried, crushed and sieved to obtain silanized magnetic nanoparticles. The silane mixture is 3-aminopropyltriethoxysilane and octadecyltrichlorosilane.

3. The pre-treatment method according to claim 2, characterized in that: The particle size of the Fe3O4 nanoparticles is 10 to 20 nm; the molar ratio of the Fe3O4 nanoparticles to the silane mixture is 1:2.5 to 3.5; the mass ratio of 3-aminopropyltriethoxysilane to octadecyltrichlorosilane in the silane mixture is 1:1; the mass volume ratio of the Fe3O4 nanoparticles to anhydrous ethanol is 1g:(45 to 55)mL.

4. The pre-treatment method according to claim 2, characterized in that: The ultrasonic time is 3 to 5 hours, the centrifugal speed is 4000 to 5000 rpm, the temperature of the product vacuum drying is 75 to 85° C., and the time is 3 to 5 hours; and an 80 to 120 mesh sieve is used during the crushing and screening.

5. The pretreatment method according to any one of claims 1 to 4, characterized in that: In step a), the sodium acetate buffer solution is 0.1-0.3 M and has a pH value of 5.8-6.

0.

6. The pretreatment method according to any one of claims 1 to 4, characterized in that: In step a), the enzyme combination includes neutral protease and lipase; the mass ratio of the neutral protease to the lipase is (1-2): (1-2), and the enzyme activities of the neutral protease and the lipase are both 8000-12000 U / mg.

7. The pretreatment method according to any one of claims 1 to 4, characterized in that: In step a), the enzymatic hydrolysis temperature is 40-50° C., and the enzymatic hydrolysis time is 20-40 min. Ultrasound is used to assist the enzymatic hydrolysis, and the ultrasonic frequency is 30-50 kHz. The pore size of the filter membrane is 0.22 μm or 0.45 μm.

8. The pretreatment method according to any one of claims 1 to 4, characterized in that: In step b), the volume ratio of n-hexane to acetonitrile in the mixed solvent of n-hexane and acetonitrile is (2-3):(2-3); and the rotation speed during the centrifugation is 4000-5000 rpm.

9. The pretreatment method according to any one of claims 1 to 4, characterized in that: In step c), 3 to 5 mL of the upper n-hexane phase or the lower acetonitrile phase is transferred, and the amount of the silanized magnetic nanoparticles added is 300 to 500 mg.

10. The pretreatment method according to any one of claims 1 to 9, characterized in that: The food includes milk powder and compound fruit and vegetable juice beverage; when the food is milk powder, step c) removes the upper n-hexane phase; when the food is a compound fruit and vegetable juice beverage, step c) removes the lower acetonitrile phase.

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