Method for determining zearalenone based on dispersion extraction technology and application

Abstract

The invention relates to a method for determining zearalenone based on a dispersion extraction technology and application, and the method comprises the following steps: S101, weighing 5.0 g of a to-be-detected sample, and placing the to-be-detected sample in a 50 mL centrifuge tube; s102, adding 25 mL of an extracting solution into the centrifugal tube, placing the centrifugal tube in a vortex oscillator to oscillate for 10 min, then adding 5 g of sodium chloride, uniformly shaking, and centrifuging; s103, putting all acetonitrile layers into a new centrifugal tube, adding 1-2g of a purification adsorption material, putting the new centrifugal tube into a vortex oscillator, oscillating for 5 minutes, and standing to obtain a purified solution; s104, taking 10mL of the purified liquid, transferring the purified liquid into a new centrifuge tube with a plug, and blow-drying; adding 1mL of acetonitrile aqueous solution for redissolving to obtain reaction liquid; and S105, filtering the reaction liquid, and quantitatively analyzing the filtered reaction liquid and a derived standard solution by using a high performance liquid chromatograph to obtain a detection result. According to the technical scheme, impurities are adsorbed and purified by utilizing a dispersion extraction technology, purification through a solid-phase extraction small column is not needed, purification materials are cheap, and time and money are saved.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, and particularly relates to a method and application for determining zearalenone based on dispersive liquid-liquid microextraction technology. Background Art

[0002] Zearalenone is highly toxic to humans and several animals. Its toxic effect is mainly damage to the liver. It is most common in natural foods and has the strongest harmfulness. The polar toxicity of zearalenone is 10 times that of potassium cyanide and 68 times that of arsenic trioxide. Its chronic toxicity can induce cancer, and its carcinogenic ability is 75 times that of dimethylnitrosamine and 900 times higher than that of dimethylazobenzene. Zearalenone threatens physical health.

[0003] Currently, methods for detecting zearalenone include thin layer chromatography (TLC), high performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA), and liquid chromatography-tandem mass spectrometry. The HPLC method is the most authoritative detection method used at home and abroad. This method has accurate determination, high resolution, can simultaneously determine multiple aflatoxin components, and complete qualitative and quantitative determinations. However, the sample extraction and purification procedures in the experimental process are cumbersome, the experimental process takes a long time, the stability is poor, and the recovery rate is not high. Therefore, it is necessary to establish a simple, fast, stable, and high-recovery sample pretreatment extraction method.

[0004] Through the above analysis, the problems and defects existing in the prior art are as follows: The existing methods for determining zearalenone in foods have cumbersome sample extraction and purification procedures, long experimental processes, poor stability, and low sensitivity and recovery rates.

[0005] The difficulty in solving the above problems and defects is as follows: It is necessary to screen out suitable purification and adsorption materials through a large number of experiments to remove interfering substances such as fat, protein, pigment, and carbohydrate; find a suitable derivatization solution to increase the fluorescence response and be able to directly inject the sample for determination without secondary nitrogen blowing after derivatization; screen out the optimal temperature and time for zearalenone derivatization through experiments to ensure the best derivatization effect.

[0006] The significance of solving the above problems and defects is as follows: Simplify the sample extraction and purification procedures in the method for determining zearalenone in foods, eliminate the need for passing through a solid-phase extraction cartridge, have stable derivatization and high fluorescence response, short experimental process, good stability, and high recovery rate. Summary of the Invention

[0007] In view of this, the purpose of the present invention is to provide a method and application for determining zearalenone based on dispersive liquid-liquid microextraction technology to solve the problems of cumbersome sample extraction and purification procedures, long experimental process, poor stability, and low sensitivity and recovery rate in the existing methods for determining zearalenone in foods.

[0008] The present invention is realized through the following technical solutions:

[0009] A method for determining zearalenone based on dispersive extraction technology, comprising the following steps:

[0010] S101, Weigh 5.0 g of the sample to be detected and place it in a 50 mL centrifuge tube;

[0011] S102, Add 25 mL of extraction solution to the centrifuge tube, place it on a vortex oscillator and shake for 10 min, then add 5 g of sodium chloride, shake well and centrifuge;

[0012] S103, Take all the acetonitrile layer into a new centrifuge tube, add 1 - 2 g of purification and adsorption material, place it on a vortex oscillator and shake for 5 min, let it stand to obtain a purified solution;

[0013] S104, Transfer 10 mL of the purified solution to a new stoppered centrifuge tube and dry it; Add 1 mL of acetonitrile aqueous solution to redissolve to obtain a reaction solution;

[0014] S105, Filter the reaction solution, and then quantitatively analyze the filtered reaction solution and the derived standard solution using a high performance liquid chromatograph to obtain the detection result.

[0015] Further defined, the purification and adsorption material is a mixture of PSA and C18, and the mass ratio of PSA to C18 in the purification and adsorption material is 1:1 - 2:1.

[0016] Further defined, in S102, the preparation method of the extraction solution is: Measure 200 mL of acetonitrile, add 50 mL of water, and then add 5 mL of 1 - butyl - 3 - methylimidazolium bromide, and mix well.

[0017] Further defined, in S102, the centrifugation speed and time are respectively: 8000 r / min and 5 min.

[0018] Further defined, in S105, the reaction solution is filtered using a 0.22 μm microporous filter membrane.

[0019] Further defined, the quantitative analysis parameters of the high performance liquid chromatograph include:

[0020] C18 chromatographic column: 250 mm * 4.6 mm, particle size 5 μm;

[0021] Mobile phase: acetonitrile + methanol + water = 20 + 10 + 70;

[0022] Flow rate 1 mL / min;

[0023] ​

[0024] Column temperature: 30 °C; injection volume: 20 μL.

[0025] The above method for determining zearalenone based on dispersive extraction technology, and its application for determining zearalenone in food using a purification and adsorption material composed of PSA and C18.

[0026] The beneficial effects of the present invention are as follows:

[0027] The advantages and positive effects of the method and application for determining zearalenone based on dispersive extraction technology are as follows:

[0028] Using dispersive extraction technology to adsorb and purify impurities, there is no need to pass through a solid-phase extraction cartridge for purification, and the purification material is also relatively cheap, saving both time and money.

[0029] The zearalenone extract is stable, and the extraction efficiency is relatively high. Adding the ionic liquid 1-butyl-3-methylimidazolium bromide increases the solubility of zearalenone and also increases the fluorescence response.

[0030] The present invention provides a rapid detection method for zearalenone in food with short sample treatment time, no need to pass through a solid-phase extraction cartridge for purification, stable and efficient derivatization.

[0031] The present invention adsorbs and purifies the sample through dispersive extraction technology, effectively removing impurities, without passing through a solid-phase extraction cartridge, and realizes a rapid detection method for zearalenone in food with short sample treatment time, stable and efficient derivatization.

[0032] Other advantages, objectives and features of the present invention will be elaborated to some extent in the subsequent specification, and to some extent, will be obvious to those skilled in the art based on the study of the following text, or can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be realized and obtained through the following specification. Description of the Drawings

[0033] [[ID=3s1]] Figure 1 is the flowchart of the method for rapidly determining zearalenone in food provided by the embodiment of the present invention;

[0034] Figure 2 is the detection chromatogram provided by the embodiment of the present invention; Detailed Embodiments

[0035] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. The components of the embodiments of the present invention usually described and illustrated in the drawings here can be arranged and designed in various different configurations.

[0036] Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0037] It should be noted that similar reference numerals and letters denote similar items in the following drawings. Therefore, once an item is defined in one drawing, it does not need to be further defined and explained in subsequent drawings.

[0038] In the above description of the present invention, it should be noted that the orientation or positional relationship indicated by terms such as "one side" and "the other side" is based on the orientation or positional relationship shown in the drawings, or the orientation or positional relationship in which the product of the invention is usually placed during use. It is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be construed as a limitation of the present invention. In addition, terms such as "first" and "second" are only used for distinguishing descriptions and cannot be understood as indicating or implying relative importance.

[0039] In addition, terms such as "the same" do not mean that the components are required to be absolutely the same, but there may be slight differences. The term "vertical" only means that the positional relationship between components is more vertical relative to "parallel", and does not mean that the structure must be completely vertical, but can be slightly inclined.

[0040] Please refer to Figure 1-2 , the present invention provides a technical solution: a method for determining zearalenone based on dispersive extraction technology, including the following steps:

[0041] S101, prepare a zearalenone derivative solution; weigh 5.0 g of the sample to be detected and place it in a 50 mL centrifuge tube;

[0042] S102, add 25 mL of extraction solution to the centrifuge tube, place it on a vortex oscillator and shake for 10 min, then add 5 g of sodium chloride, shake well and centrifuge at 8000 r / min for 5 min;

[0043] S103. Take all the acetonitrile layer into a new centrifuge tube, add 1 - 2 g of purification and adsorption material, place it in a vortex oscillator and shake for 5 min, then let it stand to obtain a purified solution; take 10 mL of the purified solution and transfer it to a new stoppered centrifuge tube, and blow it dry with nitrogen in a water bath at 50 °C; add 1 mL of an acetonitrile aqueous solution composed of 90% acetonitrile and 10% water for reconstitution;

[0044] S104. Filter the reaction solution with a 0.22 μm microporous filter membrane;

[0045] S105. Quantitatively analyze the filtered reaction solution and the derived standard solution using a high performance liquid chromatograph to obtain the detection result.

[0046] The preparation of the zearalenone extract provided by the embodiment of the present invention includes:

[0047] Measure 200 mL of acetonitrile, add it to 50 mL of water, and then add 5 mL of 1 - butyl - 3 - methylimidazolium bromide and mix well.

[0048] The standard solution provided by the embodiment of the present invention includes:

[0049] Dilute the standard stock solution with acetonitrile to prepare a standard series with concentrations of 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, and 20 ng / mL respectively. Accurately measure different concentrations of the standard solution into different stoppered centrifuge tubes, blow it dry with nitrogen in a water bath at 50 °C, add acetonitrile aqueous solution for reconstitution, and add zearalenone derivative solution, stopper and mix well. After subjecting it to a constant temperature water bath for derivatization reaction at 40 °C for 30 min, filter it with a 0.22 μm microporous filter membrane.

[0050] The quantitative analysis of the high performance liquid chromatograph provided by the embodiment of the present invention includes:

[0051] C18 chromatographic column: 250 mm * 4.6 mm, particle size 5 μm;

[0052] Mobile phase: acetonitrile + methanol + water = 20 + 10 + 70;

[0053] Flow rate 1 mL / min;

[0054] Excitation wavelength 274 nm, emission wavelength 440 nm;

[0055] Column temperature 30 °C; injection volume 20 μL.

[0056] The technical solution of the present invention will be further described below in conjunction with specific embodiments.

[0057] Example 1:

[0058] I. Reagents selected:

[0059] 1. Zearalenone standard solution

[0060] 2. Acetonitrile, methanol

[0061] 3. PSA, C18 (both 40 - 60μm)

[0062] 4. Sodium chloride

[0063] 5. 1 - Butyl - 3 - methylimidazolium bromide

[0064] II. Instruments Used

[0065] 1. Centrifuge tube, 2. Vortex shaker, 3. Centrifuge, 4. Constant temperature water bath, 5. Pipette, 6. C18 chromatographic column, 7. High - performance liquid chromatograph, 8. One - percent balance, 9. 0.22μm organic filter membrane.

[0066] III. Experiments

[0067] (1) Preparation of standard solution: Take 0.1 mL of zearalenone standard stock solution (10 μg / mL), dilute it to a volume of 10 mL with acetonitrile to obtain a concentration of about 100 ng / mL, and then dilute it with acetonitrile to prepare standard series solutions with concentrations of 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, and 20 ng / mL for standby use.

[0068] (2) Pretreatment: Weigh 5.00 g of the sample (both solid and liquid samples are acceptable) and place it in a 50 - mL centrifuge tube. Add 25 mL of the extraction solution, shake it on a vortex oscillator for 10 min, then add 5 g of sodium chloride, shake well for 5 min, and centrifuge at 8000 r / min for 5 min; Take all the acetonitrile layer and transfer it to a 50 - mL centrifuge tube, add 1 - 2 g of purification and adsorption material, shake it on a vortex oscillator for 5 min, and let it stand; Accurately measure 10 mL of the purified solution and transfer it to a 15 - mL stoppered centrifuge tube, dry it under nitrogen in a 50℃ water bath, add 1 mL of acetonitrile - aqueous solution (90 + 10) for re - dissolution, and filter it through a 0.22μm microporous filter membrane for further analysis.

[0069] (3) Quantitatively analyze the pretreated sample using a high - performance liquid chromatograph. The HPLC conditions are as follows: C18 chromatographic column, 250 mm * 4.6 mm, particle size 5μm; Mobile phase: acetonitrile + methanol + water = 30 + 10 + 60; Flow rate: 1 mL / min; Excitation wavelength: 274 nm, emission wavelength: 440 nm; Column temperature: 30℃; Injection volume: 20 μL, and obtain the liquid chromatogram of zearalenone (see Figure 2 ).

[0070] (4) Analyze and detect standard solution of different concentrations using a high performance liquid chromatograph under the same detection conditions as the sample solution to be measured, obtain the liquid chromatogram of zearalenone in the standard series solution, plot a graph with peak area as the ordinate and concentration as the abscissa, and obtain the linear range, regression equation, and correlation coefficient of the standard curve.

[0071] Component Name Linear Range Regression Equation Correlation Coefficient Zearalenone 0.1 - 20 ng / mL Y = 152677x + 11275.2 0.9999

[0072] (6) Based on the chromatographic peak of zearalenone in the sample solution to be measured and combined with the standard curve, obtain the concentration C of zearalenone in the sample solution to be measured, and calculate the content X of zearalenone in the food according to the following formula. The content calculation formula is:

[0073]

[0074] Where X is the content of zearalenone in the sample solution to be measured, with the unit of μg / kg; C is the concentration of zearalenone in the sample solution to be measured, with the unit of ng / mL; V1 is the volume of the extraction solution, with the unit of mL; V2 is the volume taken for concentration, with the unit of mL; V3 is the final volume of the purified solution after volume fixation, with the unit of mL; m is the mass of the sample to be measured, with the unit of g.

[0075] (7) Spike negative miscellaneous grain samples: Spike and measure at 0.1 μg / kg, 5 μg / kg, and 10 μg / kg respectively. The detection results of zearalenone in the samples are as follows:

[0076]

[0077]

[0078] (8) Spike negative oil samples: Spike and measure at 0.1 μg / kg, 5 μg / kg, and 10 μg / kg respectively. The detection results of zearalenone in the samples are as follows:

[0079]

[0080]

[0081] (9) Spike negative infant formula samples: Spike and measure at 0.1 μg / kg, 5 μg / kg, and 10 μg / kg respectively. The detection results of zearalenone in the samples are as follows:

[0082]

[0083]

[0084] The following is a comparison between the rapid determination method of the present invention and the determination method of GB 5009.209-2016 to highlight the superiority of the present invention in the detection of zearalenone.

[0085] 1 Comparison of experiment time and consumables:

[0086] For the purification process of the sample pretreatment of zearalenone by the method specified in the national standard GB 5009.209-2016, it is necessary to pass through a solid-phase extraction cartridge, and the special solid-phase extraction cartridge used for purifying the sample in the standard is relatively expensive. However, for the sample pretreatment by the rapid purification of the present invention, it takes less time and does not require passing through a solid-phase extraction cartridge, and the purification material is also cheaper.

[0087] 2 Comparison of recovery rates:

[0088] According to the method specified in the national standard GB 5009.209-2016, due to passing through a solid-phase extraction cartridge, the final recovery rate is between 75% and 86%. It can be seen that when using the existing method to determine zearalenone in food, the recovery rate of zearalenone is not stable enough, which is also the drawback of the solid-phase extraction column. The recovery rate of the present invention is between 87.6% and 100.6%. It can be seen that the recovery rate of the rapid determination method of the present invention is high. Therefore, this rapid determination method fully utilizes the efficient extraction technology, which is more stable and faster.

[0089] The above method for determining zearalenone based on the dispersive extraction technology is the application of using the purification and adsorption material composed of PSA and C18 to determine zearalenone in food.

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the purpose and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. A method for determining zearalenone based on dispersive liquid-liquid microextraction technology, characterized in that: It includes the following steps: S101, Weigh 5.0 g of the sample to be detected and place it in a 50 mL centrifuge tube; S102, Add 25 mL of extraction solution to the centrifuge tube, place it on a vortex oscillator and shake for 10 min, then add 5 g of sodium chloride, shake well and centrifuge; S103, Take all the acetonitrile layer into a new centrifuge tube, add 1 - 2 g of purification and adsorption material, place it on a vortex oscillator and shake for 5 min, let it stand to obtain a purified solution; S104, Transfer 10 mL of the purified solution to a new stoppered centrifuge tube and dry it; Add 1 mL of acetonitrile aqueous solution to redissolve to obtain a reaction solution; S105, Filter the reaction solution, and then quantitatively analyze the filtered reaction solution and the derived standard solution using a high performance liquid chromatograph to obtain the detection result.

2. The method for determining zearalenone based on the dispersive extraction technique according to claim 1, wherein: The purification and adsorption material is a mixture of PSA and C18, and the mass ratio of PSA to C18 in the purification and adsorption material is 1:1 - 2:

1.

3. The method for determining zearalenone based on the dispersive extraction technique according to claim 1, wherein: The preparation method of the extraction solution in S102 is: Measure 200 mL of acetonitrile, add 50 mL of water, and then add 5 mL of 1 - butyl - 3 - methylimidazolium bromide and mix well.

4. The method for determining zearalenone based on the dispersive extraction technique according to claim 1, wherein: In S102, the centrifugation speed and time are respectively: 8000 r / min and 5 min.

5. The method for determining zearalenone based on the dispersive extraction technique according to claim 1, characterized in that: In S105, the reaction solution is filtered using a 0.22 μm microporous membrane.

6. The method for determining zearalenone based on the dispersive extraction technique according to claim 1, wherein: The quantitative analysis parameters of the high performance liquid chromatograph include: C18 chromatographic column: 250 mm * 4.6 mm, particle size 5 μm; Mobile phase: acetonitrile + methanol + water = 20 + 10 + 70; Flow rate 1 mL / min; Excitation wavelength 360 nm, emission wavelength 440 nm; Column temperature 30 °C; injection volume 20 μL.

7. The method for determining zearalenone based on the dispersive extraction technique according to any one of claims 1 to 6, characterized in that: The application of using a purification and adsorption material composed of PSA and C18 to determine zearalenone in food.

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