Method for separating, enriching and detecting maltol or ethyl maltol in food

Abstract

The invention discloses a method for separating, enriching and detecting maltol or ethyl maltol in food, which comprises the following steps of: constructing a liquid membrane extraction system containing a polymer porous membrane and an organic solvent, taking a Fe < 3 + > aqueous solution as a receiving phase, and chelating ethyl maltol and Fe < 3 + > to realize efficient separation and enrichment of a target object; meanwhile, the concentration of maltol is measured through a spectrophotometric method or a smartphone colorimetric method by utilizing a specific chromogenic reaction generated after a target object and Fe < 3 + > are chelated. The detection limit of the spectrophotometric method reaches 0.67 nmol / mL, the detection limit of the smartphone colorimetric method reaches 29.4 nmol / mL, and the standard recovery rate of the spectrophotometric method food sample is 81.81%-107.77% by optimizing parameters such as the type of an organic liquid film, the concentration of Fe < 3 + >, the pH values of a sample solution and a receiving phase, the extraction time and the like. The method is suitable for detecting maltol in complex matrix samples such as beverages, dairy products, baked food and meat, has the advantages of high sensitivity, simplicity in operation, short detection period and the like, and provides a new way for rapid detection of maltol food additives.

Description

Technical Field

[0001] The invention belongs to the technical field of food additive detection, and particularly relates to a method for separating, enriching and detecting maltol or ethyl maltol in food. Background Art

[0002] Maltol compounds primarily include maltol and ethyl maltol, whose common molecular structure is 3-hydroxy-4H-pyran-4-one (or γ-pyrone derivatives). Maltol compounds are widely used food flavor enhancers that significantly enhance food flavor and are approved for use in a variety of food categories, including dairy products, baked goods, and meat products. However, excessive intake of these food additives may lead to health risks such as neurotoxicity and liver and kidney damage. Therefore, the Food and Agriculture Organization of the United Nations and the World Health Organization's Expert Committee on Food Additives stipulate that the average daily intake of ethyl maltol per person based on body weight should not exceed 2 mg / kg. -1 , and the recommended dosage in food processing is 100-200 μg·kg -1 As food processing technology becomes more complex, illegal excessive addition and abuse are common, and there is an urgent need to establish efficient and accurate detection methods to ensure food safety.

[0003] At present, the main methods for detecting maltol food additives in food include chromatography, chromatography-mass spectrometry, electrochemical method, Raman spectroscopy, fluorescence analysis, spectrophotometry, etc. Among these methods, chromatography and chromatography-mass spectrometry have good accuracy and high sensitivity, but these two methods have high technical barriers and require expensive large instruments, which are not suitable for rapid on-site detection; electrochemical method, Raman spectroscopy and fluorescence analysis have lower technical barriers, but the detection accuracy of these methods is easily affected by many factors. Spectrophotometry is a method that uses the reaction of maltol and Fe 3+ The determination is based on the principle of color development after chelation. The molecular structure of maltol contains an adjacent hydroxyl group (-OH) and a ketone group (C=O). These two functional groups can act as coordination groups and react with Fe 3+ A stable purple-red chelate complex is formed. Spectrophotometry offers advantages such as ease of operation, low cost, and the absence of large instrumentation. However, coexisting components in food samples, such as pigments, reducing substances, and metal ions, can easily interfere with the color development reaction. Direct determination of the color is insufficient (detection limit > 0.5 mg / mL), making it difficult to meet the requirements for highly sensitive detection.

[0004] Liquid membrane extraction (LME) is an environmentally friendly separation technology that achieves the selective migration and separation of target compounds through a three-phase system: sample phase, liquid membrane phase, and receiving phase. It offers the advantages of low solvent consumption (μL-level), excellent sample cleanup, and simple operation. During the LME process, the target compound is first extracted from the sample phase into the organic LME phase and then back-extracted into the receiving phase. Existing LME technologies face two challenges in food testing. First, the mass transfer driving force is limited. Existing technologies often rely on concentration or pH gradients to drive the migration of target compounds. This results in low transmembrane efficiency (<70%) for weakly acidic compounds such as ethyl maltol, making high-fold enrichment difficult. Second, poor detection compatibility exists. LME is often used as a standalone pretreatment module, requiring integration with subsequent detection equipment (such as chromatographs and mass spectrometers). This inability to directly output a detectable signal hinders further improvements in detection efficiency. Summary of the Invention

[0005] In view of the problems and shortcomings in the prior art, the object of the present invention is to provide a method for separating, enriching and detecting maltol or ethyl maltol in food.

[0006] Based on the above purpose, one of the technical solutions adopted by the present invention is as follows: A method for separating, enriching and detecting maltol or ethyl maltol in food comprises the following steps: (1) Sample pretreatment: Liquid samples were ultrasonically degassed at room temperature, diluted with hydrochloric acid, and filtered through a microporous membrane to obtain the sample solution to be tested; solid samples were crushed and added with deionized water, ultrasonicated at 60°C to 70°C for 10 to 30 min, fixed to volume with hydrochloric acid, and the supernatant was filtered through a microporous membrane to obtain the sample solution to be tested; the concentration of hydrochloric acid used was 0.1 to 5 mol / L, and the pH value of the sample solution to be tested was 1.0 to 5.0; (2) Extraction: Construct a liquid membrane extraction system consisting of a sample phase, a polymer porous membrane, an organic liquid membrane phase, and a receiving phase; use the sample solution pretreated in step (1) as the sample phase, and Fe 3+ The aqueous solution is the receiving phase and the extraction is carried out under continuous shaking conditions; the Fe 3+ Fe in aqueous solution 3+ The concentration of Fe is 5~50 mmol / L, 3+ The pH value of the aqueous solution is 1.5~3.0; (3) Detection: After extraction, based on maltol-Fe 3+ The specific color reaction of the chelate is carried out by measuring the absorbance of the receiving phase in the wavelength range of 450-600nm or taking a photo with a smartphone and reading the G value of the receiving phase photo using RGB analysis software. Quantitative analysis is achieved by comparing with the respective standard curves.

[0007] Furthermore, the specific process of step (2) is as follows: (a) affixing a polymer porous membrane to one end of a hollow tube with two open ends or affixing the polymer porous membrane to the lower open end of a sample well of a microplate, and then evenly applying an organic liquid membrane phase solvent to at least one side of the polymer porous membrane to form an organic liquid membrane phase; the space formed by the polymer porous membrane and the hollow tube or sample well serves as a receiving phase container, and injecting a receiving phase into the inner cavity of the receiving phase container; (b) adding a sample solution to be tested into the sample phase container, and then inserting the receiving phase container into the sample phase container, so that the organic liquid membrane phase just contacts the liquid surface of the sample solution to be tested, thereby forming an extraction device; (c) The extraction device is fixed on a shaker and the maltol or ethyl maltol in the sample phase is extracted under continuous shaking conditions.

[0008] Furthermore, in step (2), the polymer porous membrane is a polypropylene membrane, a polytetrafluoroethylene membrane or a polyvinylidene fluoride membrane. Preferably, the polymer porous membrane is a polypropylene membrane.

[0009] Furthermore, in step (2), the organic liquid membrane phase solvent is selected from at least one of 2-octanone, n-octanol, n-decanol, n-nonanol, nonanone, undecanol, n-heptanol, tributyl phosphate, 2-nitrophenyl octyl ether, and diethyl ether. Preferably, the organic liquid membrane phase solvent is 2-octanone.

[0010] Furthermore, in step (2), the amount of the organic liquid film solvent applied is 0.1~0.3 μL / mm 2 Preferably, the amount of organic liquid film solvent applied is 0.2~0.25 μL / mm 2 .

[0011] Furthermore, in step (2), the volume ratio of the sample phase to the receiving phase is 10:(1-5), preferably, the volume ratio of the sample phase to the receiving phase is 10:(1-3).

[0012] Preferably, in step (1), the pH value of the sample solution is 1.0-5.0, and in step (2), the pH value of the receiving phase is adjusted to 2.3.

[0013] Furthermore, in step (2), the extraction time is 10-150 minutes, preferably, the extraction time is 30-60 minutes; Furthermore, in step (2), the Fe 3+ The concentration of the aqueous solution is 5 to 50 mmol / L. Preferably, the Fe 3+ The concentration of the aqueous solution is 30 mmol / L; Furthermore, in step (2), the oscillation speed is 10-300 rpm, preferably, the oscillation speed is 240-300 rpm.

[0014] Furthermore, in step (1), the liquid sample is a beverage or dairy product; and the solid sample is baked food or meat.

[0015] Furthermore, in step (1), the liquid sample is ultrasonically degassing and then diluted 2 to 10 times with 0.1 to 0.2 mol / L hydrochloric acid; the solid sample is fixed to a concentration of 0.1 to 0.2 g / mL with 1 to 5 mol / L hydrochloric acid.

[0016] Furthermore, in step (2), FeCl3 is used as the iron source to prepare Fe 3+ After the aqueous solution is prepared, adjust the pH to 1.5-3.0 with 0.5-2 mol / L hydrochloric acid or sodium hydroxide solution.

[0017] Furthermore, in step (3), the wavelength of the received phase absorbance value is measured to be 525-530 nm.

[0018] Wherein, when measuring the absorbance value of the receiving phase, the drawing process of the standard curve is as follows: (1) Prepare ethyl maltol or maltol solution with a gradient concentration of 0-8 μmol / mL using 0.1 M HCl as the sample phase and add it to the sample phase container; (2) A polypropylene membrane was fixed on one end of a hollow tube with two ends open, and 2-octanone was applied to the surface of the polypropylene membrane. After application, the membrane was allowed to stand for 1 to 5 minutes to form an organic liquid membrane phase. The space formed by the polypropylene membrane and the hollow tube was used as a receiving phase container. 0.1 to 0.5 mL of a 30 μmol / mL FeCl3 solution (pH = 2.3) was taken as the receiving phase and added to the extraction device for extraction. The oscillation speed was 240 rpm, the extraction temperature was 25 ± 1 °C, and the extraction time was 60 min. The volume ratio of the sample phase to the receiving phase was 10: (1 to 3). The amount of organic liquid membrane solvent applied was 0.2 to 0.25 μL / mm 2 ; (3) Take 80 ~ 300 μL of the receiving phase and add it to a 96-well plate. Use an enzyme-labeled instrument to measure the absorbance of the receiving phase at 525 ~ 530 nm. Draw a standard curve with the concentration of ethyl maltol or maltol as the horizontal axis and the absorbance as the vertical axis.

[0019] When taking photos for colorimetry with a smartphone, the process of drawing the standard curve is as follows: (1) Prepare ethyl maltol or maltol solution with a gradient concentration of 0-0.5 μmol / mL using 0.1 M HCl as the sample phase and add it to the sample phase container; (2) Fix the polypropylene membrane to the lower open end of the sample hole of the microplate, take 2-octanone and apply it to the surface of the polypropylene membrane. After application, let it stand for 1~5 minutes to form an organic liquid membrane phase. The space formed by the polypropylene membrane and the sample hole is used as a receiving phase container. Take 30~70 μL of FeCl3 solution with a concentration of 30 μmol / mL (pH=2.3) as the receiving phase and add it to the extraction device for extraction. The oscillation speed is 300 rpm, the extraction temperature is 25±1℃, the extraction time is 30 min, and the volume ratio of the sample phase to the receiving phase is 10: (1~3) The amount of organic liquid membrane solvent applied is 0.2~0.25μL / mm 2 .

[0020] (3) After the extraction is completed, take a photo of the receiving phase directly with a smartphone, and then use the image RGB analysis app or WeChat applet (Colorimetric analysis) to read the G value of the receiving phase photo. Use the concentration of ethyl maltol or maltol as the horizontal axis and the G value as the vertical axis to draw a standard curve.

[0021] Compared with the prior art, the present invention has the following beneficial effects: (1) Constructing Fe 3+ The aqueous solution is used as the receiving phase in the liquid membrane extraction system, using malt phenol-Fe 3+ The chelation reaction simultaneously achieves selective transmembrane migration, enrichment, and colorimetric signal generation of the target, breaking through the efficiency bottleneck of traditional step-by-step processing; (2) By optimizing the type of organic liquid membrane, pH value of sample phase and receiving phase, Fe 3+ concentration, significantly improving the mass transfer efficiency and enrichment multiple of maltol, and reducing the detection limit to 0.67 nmol / mL; (3) Develop a rapid pretreatment method suitable for complex matrices such as dairy products and meat products, which has good sample purification effect and strong resistance to matrix interference. Combined with smartphone photo analysis, it can realize a simple "sample in - result out" operation process, providing a feasible solution for on-site testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a schematic diagram of the liquid membrane extraction device and its structure used in the present invention; Figure 2 Optimize experimental results for the selection of organic liquid membrane phase solvents; Figure 3 Fe is the receiving phase 3+ Concentration optimization experiment results; Figure 4 Optimize the experimental results for the receiving phase pH; Figure 5 Optimize the pH of the sample phase. Figure 6 Optimize the experimental results for the oscillation speed; Figure 7 Optimize results for extraction time; Figure 8 The standard curves and detection limits of different volumes of receiving phase are shown in Figure 2. A, B, and C correspond to 0.5, 0.3, and 0.1 mL of receiving phase, respectively; Figure 9 Schematic diagram of the structure of a liquid membrane extraction device based on a 96-well plate; Figure 10 This is the standard curve for ethyl maltol detection based on 96-well plate liquid membrane extraction and smartphone colorimetry. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below through embodiments in conjunction with the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0024] The polymer porous membrane used in the following examples is a polypropylene membrane (thickness 200 μm, pore size about 0.2 μm).

[0025] (I) Optimization of separation, enrichment and detection conditions of ethyl maltol Example 1: Selection and optimization experiment of organic liquid membrane phase solvent (1) Experimental purpose: To screen the organic liquid membrane phase with the best separation and enrichment effect of ethyl maltol, and to investigate the effects of different organic liquid membranes on the transmembrane migration efficiency of the target substance.

[0026] (2) Experimental design: First, 10 typical organic solvents including 2-octanone (2-Octanone), 1-octanol (1-Octanol), 1-decanol (1-Decanol), 1-nonanol (1-Nonanol), 2-nonanone (2-Nonanone), 1-undecanol (1-Undecanol), 1-heptanol (1-Heptanol), tributyl phosphate (TBP), 2-nitrophenyl octyl ether (NPOE), and diethyl ether (DHE) were selected as organic liquid membrane phase solvents. After extraction for the same time, the concentration of ethyl maltol in the receiving phase was determined, and the extraction rate was calculated. The one with the highest extraction rate was selected as the best organic liquid membrane phase solvent.

[0027] ①Configure the experimental system: Sample phase: Prepare a 1.0 μmol / mL ethyl maltol aqueous solution using 0.1 M hydrochloric acid (the final solution pH is approximately 1.0); Liquid membrane phase: 10 μL of organic liquid membrane phase solvent was applied to the surface of a polymer porous membrane (7.5 mm in diameter) and allowed to stand for 3 min. Receiving phase: 0.5 mL of 10 μmol / mL FeCl3 solution (pH = 2, adjusted with 1 mol / L hydrochloric acid or 1 mol / L sodium hydroxide solution).

[0028] ② Experimental process: Figure 1 As shown, (a) A porous polymer membrane was fixed to one end of a hollow tube (7.5 mm diameter, approximately 1 mL volume) with openings at both ends. 10 μL of organic liquid membrane phase solvent was then evenly applied to one side of the porous polymer membrane to form an organic liquid membrane phase. The space formed by the porous polymer membrane and the hollow tube served as a receiving phase container. 0.5 mL of receiving phase was injected into the inner cavity of the receiving phase container, and the other opening of the receiving phase container was sealed with a sealing film. (b) Using a 2 mL Eppendorf tube as the sample phase container, add 1 mL of the sample solution to be tested into the sample phase container, then insert the receiving phase container into the sample phase container so that the organic liquid membrane phase just contacts the liquid surface of the sample solution to be tested, thereby forming an extraction device; (c) fixing the extraction device on a shaker and extracting ethyl maltol from the sample phase under continuous shaking; Experimental condition control: The shaking speed was fixed at 200 rpm, the extraction temperature was 25±1℃, and the extraction time was 30 min. Parallel experiments (n=3) were performed.

[0029] (3) Evaluation method: ① Take 80 μL of the receiving phase and add it to a 96-well plate. Use a microplate reader to measure the absorbance of the receiving phase at 525-530 nm. Use the standard curve method to determine the concentration of ethyl maltol in the receiving phase.

[0030] ② Calculate the extraction rate of ethyl maltol (E): Among them, V a and V d are the volumes of the receiving phase and sample phase, respectively, C a and C d are the concentrations of ethyl maltol in the receiving phase and the sample phase, respectively.

[0031] (4) Experimental results and conclusions: The results are as follows Figure 2As shown, the extraction yield of the 2-octanone group reached 45.86% ± 2.62%, significantly superior to the other solvents. Analysis revealed that 2-octanone's moderate polarity (log P = 2.585) ensured the solubility of p-ethyl maltol while effectively blocking interfering substances in the aqueous phase. Its viscosity (1.02 mPa·s) facilitated the formation of a stable liquid film, while its low volatility (boiling point 173°C) helped reduce experimental losses. Therefore, 2-octanone was the optimal organic liquid film phase.

[0032] Example 2: Receiving phase Fe 3+ Concentration optimization experiment (1) Experimental purpose: To investigate the effect of FeCl3 concentration in the receiving phase on the transmembrane migration efficiency of the target substance and to determine the optimal concentration of FeCl3.

[0033] (2) Experimental design: First, FeCl3 solutions of different concentrations were prepared (5, 10, 20, 30, 40, 50 μmol / mL, adjusted to pH = 2 with 1 mol / L hydrochloric acid or 1 mol / L sodium hydroxide solution). After extraction for the same time, the concentration of ethyl maltol in the receiving phase was measured, and the extraction rate was calculated. The FeCl3 concentration with the highest extraction rate was the optimal concentration.

[0034] ①Configure the experimental system: Sample phase: Prepare a 1.0 μmol / mL ethyl maltol aqueous solution using 0.1 M hydrochloric acid (the final solution pH is approximately 1.0); Liquid film phase: 10 μL of 2-octanone was applied to the surface of the polymer porous membrane and allowed to stand for 3 min; Receiving phase: 0.5 mL of FeCl3 solution with different concentrations (pH = 2).

[0035] ②The experimental process is the same as in Example 1.

[0036] Experimental condition control: The shaking speed was fixed at 200 rpm, the extraction temperature was 25±1℃, and the extraction time was 30 min. Parallel experiments (n=3) were performed.

[0037] (3) Evaluation method: ① Take 80 μL of the receiving phase and add it to a 96-well plate. Use a microplate reader to measure the absorbance of the receiving phase at 525-530 nm. Use the standard curve method to determine the concentration of ethyl maltol in the receiving phase.

[0038] ② Calculate the extraction rate of ethyl maltol (E): Among them, V a and V dare the volumes of the receiving phase and sample phase, respectively, C a and C d are the concentrations of ethyl maltol in the receiving phase and the sample phase, respectively.

[0039] (4) Experimental results and conclusions: The results, shown in Figure 3, show that the extraction rate of ethyl maltol increased with increasing FeCl₃ concentration within the range of 5 to 30 μmol / mL. When the FeCl₃ concentration exceeded 30 μmol / mL, the extraction rate stabilized, but the color of the FeCl₃ solution darkened significantly, leading to a significant increase in background absorbance. To minimize background interference in absorbance detection, a 30 μmol / mL FeCl₃ solution was used as the receiving phase.

[0040] Example 3: Receiving phase pH optimization experiment (1) Experimental purpose: To investigate the effect of the pH of the receiving phase on the transmembrane migration efficiency of the target substance and to determine the optimal pH of the receiving phase.

[0041] (2) Experimental design: First, prepare a 30 μmol / mL FeCl3 solution, and use 1 mol / L hydrochloric acid or 1 mol / L sodium hydroxide solution to adjust the solution pH to 1.5, 1.8, 2.0, 2.3, or 3.0. After extraction for the same time, measure the concentration of ethyl maltol in the receiving phase and calculate the extraction rate. The pH with the highest extraction rate is the optimal pH.

[0042] ①Configure the experimental system: Sample phase: Prepare a 1.0 μmol / mL ethyl maltol aqueous solution using 0.1 M hydrochloric acid (the final solution pH is approximately 1.0); Liquid film phase: 10 μL of 2-octanone was applied to the surface of the polymer porous membrane and allowed to stand for 3 min; Receiving phase: 0.5 mL of a 30 μmol / mL solution, adjusting the solution pH to 1.5, 1.8, 2.0, 2.3, or 3.0.

[0043] ②The experimental process is the same as in Example 1.

[0044] ③ Experimental condition control: The shaking speed was fixed at 200 rpm, the extraction temperature was 25±1℃, and the extraction time was 30 min. Parallel experiments (n=3) were performed.

[0045] (3) Evaluation method: ① Take 80 μL of the receiving phase and add it to a 96-well plate. Use a microplate reader to measure the absorbance of the receiving phase at 525-530 nm. Use the standard curve method to determine the concentration of ethyl maltol in the receiving phase.

[0046] ② Calculate the extraction rate of ethyl maltol (E): Among them, V a and V d are the volumes of the receiving phase and sample phase, respectively, C a and C d are the concentrations of ethyl maltol in the receiving phase and the sample phase, respectively.

[0047] (4) Experimental results and conclusions: The results are as follows Figure 4 As shown in the figure, within the pH range of 1.5 to 2.0, the extraction rate of ethyl maltol did not change significantly with the increase of pH, fluctuating between 47% and 49%. When the pH increased to 2.3, the extraction rate increased to 57%. When the pH value continued to increase, the extraction rate showed a downward trend, which may be due to the presence of Fe 3+ At high pH values, hydrolysis occurs, resulting in Fe 3+ It is unable to chelate with ethyl maltol. Based on the above research results, FeCl3 solution with pH=2.3 was used as the receiving phase.

[0048] Example 4: Sample Phase pH Optimization Experiment (1) Experimental purpose: To investigate the effect of sample phase pH on the transmembrane migration efficiency of the target substance and to determine the optimal pH of the sample phase.

[0049] (2) Experimental design: First, prepare a 1.0 μmol / mL ethyl maltol aqueous solution as the sample phase. Use 1 mol / L hydrochloric acid or 1 mol / L sodium hydroxide solution to adjust the pH to 1, 2, 3, 4, 5, 6, or 7. After extraction for the same time, measure the concentration of ethyl maltol in the receiving phase and calculate the extraction rate. The pH value with the highest extraction rate is the optimal sample phase pH value.

[0050] ①Configure the experimental system: Sample phase: 1 mL of 1.0 μmol / mL ethyl maltol standard, adjusted to pH 1, 2, 3, 4, 5, 6, or 7; Liquid film phase: 10 μL of 2-octanone was applied to the surface of the polymer porous membrane and allowed to stand for 3 min; Receiving phase: 0.5 mL of 30 μmol / mL FeCl3 solution, adjusted to pH 2.3 with 1 M sodium hydroxide solution.

[0051] ②The experimental process is the same as in Example 1.

[0052] ③ Experimental condition control: The shaking speed was fixed at 200 rpm, the extraction temperature was 25±1℃, and the extraction time was 30 min. Parallel experiments (n=3) were performed.

[0053] (3) Evaluation method: ① Take 80 μL of the receiving phase and add it to a 96-well plate. Use a microplate reader to measure the absorbance of the receiving phase at 525-530 nm. Use the standard curve method to determine the concentration of ethyl maltol in the receiving phase.

[0054] ② Calculate the extraction rate of ethyl maltol (E): Among them, V a and V d are the volumes of the receiving phase and sample phase, respectively, C a and C d are the concentrations of ethyl maltol in the receiving phase and the sample phase, respectively.

[0055] (4) Experimental results and conclusions: The results are as follows Figure 5 As shown, within the range of pH=1~5, as the pH increases, the extraction rate of ethyl maltol does not change significantly, fluctuating between 56% and 57%. When the pH increases to 6, the extraction rate shows a downward trend. The reason may be that ethyl maltol in the sample phase is ionized at high pH values, which reduces the mass transfer efficiency from the sample phase to the organic phase. Although within the range of pH=1~5, as the pH increases, the extraction rate of ethyl maltol does not change significantly, considering that the food matrix may contain iron ions, iron ions can chelate with ethyl maltol, hindering its migration to the receiving phase. And at low pH values, the stability of the chelation of iron ions and ethyl maltol is reduced, so the present invention takes pH=1 as the optimal sample phase pH value.

[0056] Example 5: Oscillation speed optimization experiment (1) Experimental purpose: To investigate the effect of oscillation speed on the transmembrane migration efficiency of the target substance and determine the optimal oscillation conditions.

[0057] (2) Experimental design: Different oscillation speeds were used during extraction. After the same extraction time, the concentration of ethyl maltol in the receiving phase was measured and the extraction rate was calculated. The one with the highest extraction rate was considered the optimal oscillation speed.

[0058] ①Configure the experimental system: Sample phase: Prepare a 1.0 μmol / mL ethyl maltol aqueous solution using 0.1 M hydrochloric acid (the final solution pH is approximately 1.0); Liquid film phase: 10 μL of 2-octanone was applied to the surface of the polymer porous membrane and allowed to stand for 3 min; Receiving phase: 0.5 mL of 30 μmol / mL FeCl3 solution, adjusted to pH 2.3 with 1 M sodium hydroxide solution.

[0059] ②The experimental process is the same as in Example 1.

[0060] ③ Experimental condition control: Parallel experiments (n=3) were performed with varying shaking speeds of 0, 60, 120, 180, 240, and 300 rpm, extraction temperature of 25±1°C, and extraction time of 30 min.

[0061] (3) Evaluation method: ① Take 80 μL of the receiving phase and add it to a 96-well plate. Use a microplate reader to measure the absorbance of the receiving phase at 525-530 nm. Use the standard curve method to determine the concentration of ethyl maltol in the receiving phase.

[0062] ② Calculate the extraction rate of ethyl maltol (E): Among them, V a and V d are the volumes of the receiving phase and sample phase, respectively, C a and C d are the concentrations of ethyl maltol in the receiving phase and the sample phase, respectively.

[0063] (4) Experimental results and conclusions: The results are as follows Figure 6 As shown in the figure, within the range of 0-240 rpm, as the oscillation speed increases, the extraction rate of ethyl maltol increases accordingly. When the speed exceeds 240 rpm, the extraction rate of ethyl maltol no longer increases. Therefore, the present invention uses 240 rpm as the optimal oscillation speed.

[0064] Example 6: Extraction time optimization (1) Experimental purpose: To investigate the effect of extraction time on the transmembrane migration efficiency of the target substance and determine the optimal extraction time.

[0065] (2) Experimental design: During the extraction process, after different extraction times (10, 20, 30, 60, 90, 120, 150 min), the concentration of ethyl maltol in the receiving phase was measured and the extraction rate was calculated. The optimal extraction time was determined by comprehensively considering the extraction efficiency and time cost.

[0066] ① Configure the experimental system: Sample phase: Prepare a 1.0 μmol / mL ethyl maltol aqueous solution using 0.1 M hydrochloric acid (the final solution pH is approximately 1.0); Liquid film phase: 10 μL of 2-octanone was applied to the surface of the polymer porous membrane and allowed to stand for 3 min; Receiving phase: 0.5 mL of 30 μmol / mL FeCl3 solution, adjusted to pH 2.3 with 1 M sodium hydroxide solution.

[0067] ②The experimental process is the same as in Example 1.

[0068] ③ Experimental condition control: The shaking speed was set at 240 rpm, the extraction temperature was set at 25±1°C, and the extraction time was set at 10, 20, 30, 60, 90, 120, and 150 min. Parallel experiments were performed (n=3).

[0069] (3) Evaluation method: ① Take 80 μL of the receiving phase and add it to a 96-well plate. Use a microplate reader to measure the absorbance of the receiving phase at 525-530 nm. Use the standard curve method to determine the concentration of ethyl maltol in the receiving phase.

[0070] ② Calculate the extraction rate of ethyl maltol (E): Among them, V a and V d are the volumes of the receiving phase and sample phase, respectively, C a and C d are the concentrations of ethyl maltol in the receiving phase and the sample phase, respectively.

[0071] (4) Experimental results and conclusions: The results are as follows Figure 7 As shown in the figure, within the extraction time range of 0–60 min, the extraction efficiency of ethyl maltol increased rapidly, reaching 78.62% ± 4% in just 60 min. Although the extraction efficiency of ethyl maltol continued to increase after 60 min, the extraction rate slowed significantly. The extraction efficiency reached a maximum of 94% after 120 min. The extraction efficiency increased by only approximately 15% between 60 and 120 min. Considering both extraction efficiency and time cost, 60 min was determined to be the optimal extraction time.

[0072] (II) Establishment of standard curve Example 7: Drawing a standard curve (1) Experimental purpose: To draw the standard curve of the detection method described in the present invention and to clarify the linear range and detection limit (LOD) of the method under different volumes of receiving phase.

[0073] (2) Experimental Design: First, prepare a series of ethyl maltol standard solutions. Use the method described in this invention to extract ethyl maltol. After extraction, measure the absorbance of the receiving phase at 525–530 nm and plot a standard curve of ethyl maltol concentration versus absorbance. The volume of the receiving phase was varied to determine the linear range and limit of detection (LOD) of the method under different receiving phase volumes.

[0074] ①Configure the experimental system: Sample phase: Prepare 0-8 μmol / mL ethyl maltol solution with 0.1 M HCl, and add 1 mL of each concentration to the extraction device for extraction.

[0075] Liquid film phase: 10 μL of 2-octanone was applied to the surface of the polymer porous membrane and allowed to stand for 3 min; Receiving phase: Take 0.1, 0.3 and 0.5 mL of 30 μmol / mL FeCl3 solution (pH = 2.3) as the receiving phase and add it to the extraction device for extraction.

[0076] ②The experimental process is the same as in Example 1.

[0077] ③ Experimental condition control: The shaking speed was set at 240 rpm, the extraction temperature was 25±1°C, and the extraction time was set at 60 min. Parallel experiments were performed for each concentration sample (n=3).

[0078] (3) Evaluation method: ① Take 80 μL of the receiving phase and add it to a 96-well plate. Use a microplate reader to measure the absorbance of the receiving phase at 525-530 nm.

[0079] ② With the absorbance value as the ordinate and the ethyl maltol concentration as the abscissa, draw a standard curve of ethyl maltol concentration-absorbance value, and calculate the limit of detection (LOD) using the following formula: Among them, σ is the detection standard deviation of the blank sample, k is the slope of the standard curve.

[0080] (4) Experimental results and conclusions: The results are as follows Figure 8 As shown in the figure, when the volume of the receiving phase is 0.5 mL, there is a good linear relationship between the concentration of ethyl maltol and the absorbance value at 525-530 nm in the range of 0-4 μmol / mL, and the correlation coefficient R 2 =1, detection limit LOD = 8.48 nmol / mL (1.19 mg / L) ( Figure 8 A). When the volume of the receiving phase was 0.3 mL, there was a good linear relationship between the concentration of ethyl maltol and the absorbance at 525-530 nm in the range of 0-4 μmol / mL, with a correlation coefficient of R 2 =1, the detection limit LOD was reduced to 2.01 nmol / mL (0.28 mg / mL) ( Figure 8B). When the volume of the receiving phase was 0.1 mL, the sample was further enriched. In the range of 0-1.5 μmol / mL, the concentration of ethyl maltol showed a good linear relationship with the absorbance at 525-530 nm, with a correlation coefficient of R 2 =1, the detection limit LOD decreased to 0.67 nmol / mL (0.094 mg / mL) ( Figure 8 C).

[0081] (3) Detection results of actual samples Example 8: Spike recovery experiment of actual samples (1) Experimental purpose: To investigate the effectiveness of the detection method proposed in this invention for detecting ethyl maltol in various food samples.

[0082] (2) Experimental design: First, the sample was spiked and the sample phase was obtained after pretreatment. The method described in the present invention was used to extract and detect ethyl maltol. The concentration of ethyl maltol in the food sample was quantified by combining the standard curve method. The spike recovery rate and the relative standard deviation of the test results were calculated.

[0083] ①Configure the experimental system: Sample Phase: After grinding the solid sample in a mortar, accurately weigh 2 g (accurate to 0.001 g) into a 20 mL graduated stoppered test tube. Ethyl maltol standard was added to concentrations of 0, 0.25, 0.5, and 1.0 μmol / kg, respectively. Subsequently, 5 mL of ultrapure water was added and the mixture was vortexed for 1 min to achieve uniform dispersion. Then, 10 mL of ultrapure water was added and ultrasonic extraction was performed in a 70°C waterbath for 20 min. After ultrasonication, the sample was cooled to room temperature and brought to volume with 4 M HCl. After shaking, the mixture was centrifuged at 6000 rpm for 10 min. The supernatant was filtered through a 0.22 μm filter to obtain the sample phase. Liquid samples were ultrasonically sonicated at room temperature for 10 min to remove CO2. Ethyl maltol standard was then added to concentrations of 0, 0.25, 0.5, and 1.0 μmol / mL, respectively. The sample was then diluted 2-fold with 0.2 M HCl and centrifuged at 6000 rpm for 10 minutes. The supernatant was collected and filtered through a 0.22 μm filter membrane before testing. 1 mL of each sample was added to the extraction device for extraction.

[0084] Liquid film phase: 10 μL of 2-octanone was applied to the surface of the polymer porous membrane and allowed to stand for 3 min; Receiving phase: 0.1 mL of 30 μmol / mL FeCl3 solution, adjusted to pH 2.3 with 1 M sodium hydroxide solution.

[0085] ②The experimental process is the same as in Example 1.

[0086] ③ Experimental condition control: The stirring rate was set at 240 rpm, the extraction temperature was 25±1°C, and the extraction time was set at 60 min. Parallel experiments were performed for each sample (n=3).

[0087] (3) Evaluation method: ① Take 80 μL of the receiving phase and add it to a 96-well plate. Use a microplate reader to measure the absorbance of the receiving phase at 525-530 nm. Use the standard curve method to quantify the concentration of ethyl maltol in the food sample.

[0088] ② Calculate the recovery rate of ethyl maltol (R): in, C i 、 C 0 and C 1 They are respectively the concentration of ethyl maltol in food measured by the method of the present invention after spike addition, the background concentration of ethyl maltol in food measured by the method of the present invention before spike addition, and the spiked concentration of ethyl maltol.

[0089] (4) Experimental results and conclusions: The results are shown in Table 1. As can be seen from Table 1, the method of the present invention is used to detect ethyl maltol in spiked actual samples, and the recovery rate is between 81.81% and 107.77%, and the relative standard deviation is less than 10%, indicating that the method of the present invention has good accuracy in detecting ethyl maltol in actual samples.

[0090] Table 1: Detection results of ethyl maltol in spiked samples by the method of the present invention Example 9: Detection method of ethyl maltol based on 96-well plate liquid membrane extraction and smartphone colorimetry (1) Experimental purpose: To develop a liquid membrane extraction device based on a 96-well microplate, combined with smartphone photography and colorimetry, to achieve high-throughput, on-site detection of ethyl maltol.

[0091] (2) Experimental Design: A 96-well microplate with a single well diameter of 7.0 mm and a volume of 300 μL was used as the sample phase container, and another 96-well microplate with a sample well (6.5 mm diameter) with an open bottom was used as the receiving phase container. The open ends of the sample wells were sealed with a polymer porous membrane. After the extraction was completed using the liquid membrane extraction system described in the present invention, the receiving phase was directly photographed using a smartphone. The G value of the receiving phase photo was then read using an image RGB analysis app or WeChat mini-program (Colorimetric analysis). The content of ethyl maltol in the spiked sample was calculated using a standard curve.

[0092] (3) ①Configure the experimental system: Sample phase: Prepare 0-0.5 μmol / mL ethyl maltol solution in 0.1 M HCl; Liquid film phase: Apply 2-octanone to the surface of the polymer porous membrane at the bottom of the receiving phase container at a coating volume of 0.2 μL / mm 2 , let it stand for 3 minutes after application; Receiving phase: A 30 μmol / mL FeCl3 solution (adjusted to pH 2.3 with 1 M sodium hydroxide solution) was used as the receiving phase.

[0093] For spiked water samples, ultrasonicate the samples at room temperature for 10 minutes and spike them with ethyl maltol standard to concentrations of 0, 0.2, 0.5, and 1.0 μmol / mL. The samples were then diluted 2-fold with 0.2 M HCl and centrifuged at 6000 rpm for 10 minutes. The supernatant was collected and filtered through a 0.22 μm filter before analysis. 250 μL of each sample was added to the extraction apparatus for analysis.

[0094] ② Experimental process: Figure 9 As shown, (a) A polymer porous membrane was fixed to the bottom of a 96-well microplate sample well (the bottom of the microplate sample well was open, and the diameter of each well was 6.5 mm). Then, 2-octanone was applied to one side of the polymer porous membrane surface at the bottom of the receiving phase container at a coating volume of 0.2 μL / mm. 2 After coating, let it stand for 3 minutes to form an organic liquid film phase; the space formed by the polymer porous membrane and the sample hole is used as a receiving phase container, 50 μL of receiving phase is injected into the inner cavity of the receiving phase container, and the opening at the other end of the receiving phase container is sealed with a sealing film; (b) Using a 96-well microplate with a single well diameter of 7.0 mm and a volume of 300 μL as the sample phase container, 250 μL of the sample solution to be tested was added to the sample phase container, and then the receiving phase container was inserted into the sample phase container so that the organic liquid membrane phase just contacted the liquid surface of the sample solution to be tested, thereby forming an extraction device; (c) A 96-well plate liquid membrane extraction device is fixed on a shaker to extract ethyl maltol from the sample phase under continuous shaking conditions; ③ Experimental condition control: The shaking speed was set at 300 rpm, the extraction temperature was set at 25±1°C, and the extraction time was set at 30 min. Parallel experiments were performed for each concentration sample (n=3).

[0095] (3) Evaluation method: ① After the extraction is completed, remove the sealing film of the receiving phase container, take a photo of the receiving phase directly with a smartphone, and then use the image RGB analysis app or WeChat applet (Colorimetric analysis) to read the G value of the receiving phase photo.

[0096] ② With G value as the ordinate and ethyl maltol concentration as the abscissa, draw a standard curve of ethyl maltol concentration-photo G value, and calculate the detection limit (LOD) using the following formula: Among them, σ is the detection standard deviation of the blank sample, k is the slope of the standard curve.

[0097] When measuring spiked samples, substitute the G value of the spiked sample after extraction into the above standard curve to calculate the content of ethyl maltol in the spiked sample. The recovery rate (R) of ethyl maltol is calculated using the following formula: in, C i 、 C 0 and C 1 The concentrations of ethyl maltol in the spiked sample, the background concentration of ethyl maltol in the sample, and the spiked concentration of ethyl maltol measured by the method of the present invention are shown in Table 2.

[0098] (4) Experimental results and conclusions: Standard curve Figure 10 As shown in the figure, in the range of 0~0.5 μmol / mL, the concentration of ethyl maltol and the G value of the receiving phase photo showed a good linear relationship, and the correlation coefficient R 2 =0.998, limit of detection LOD=29.4 nmol / mL.

[0099] Table 2: Detection results of ethyl maltol in water based on 96-well plate liquid membrane extraction and smartphone colorimetry As shown in Table 2, the recovery rates of ethyl maltol in spiked water samples using the 96-well plate liquid membrane extraction and smartphone colorimetry method ranged from 98% to 104.0%, with relative standard deviations less than 5%. This demonstrates that the method described herein can accurately determine the ethyl maltol content in water samples without relying on large laboratory instruments. Furthermore, the method described herein can accurately measure 96 samples within 45 minutes (organic liquid membrane application: <3 minutes, sample and receiver addition: <6 minutes, agitation: 30 minutes, and imaging and G-value reading: <5 minutes). The average processing time per sample is only approximately 0.5 minutes, demonstrating the high-throughput nature of the 96-well plate liquid membrane extraction and smartphone colorimetry method.

Claims

1. A method for separating, enriching and detecting maltol or ethyl maltol in food, characterized in that: The following steps are involved: (1) Sample pretreatment: Liquid samples were ultrasonically degassed at room temperature, diluted with hydrochloric acid, and filtered through a microporous membrane to obtain the sample solution to be tested; solid samples were crushed and added with deionized water, ultrasonicated at 60°C to 70°C for 10 to 30 min, fixed to volume with hydrochloric acid, and the supernatant was filtered through a microporous membrane to obtain the sample solution to be tested; the concentration of hydrochloric acid used was 0.1 to 5 mol / L, and the pH value of the sample solution to be tested was 1.0 to 5.0; (2) Extraction: Construct a liquid membrane extraction system consisting of a sample phase, a polymer porous membrane, an organic liquid membrane phase, and a receiving phase; use the sample solution pretreated in step (1) as the sample phase, and Fe 3+ The aqueous solution is the receiving phase and the extraction is carried out under continuous shaking conditions; the Fe 3+ Fe in aqueous solution 3+ The concentration of Fe is 5~50 mmol / L, 3+ The pH value of the aqueous solution is 1.5~3.0; (3) Detection: After extraction, based on malt phenol-Fe 3+ The specific color reaction of the chelate is carried out by measuring the absorbance of the receiving phase in the wavelength range of 450-600 nm, or taking a photo with a smartphone and using RGB analysis software to read the G value of the receiving phase photo, and quantitative analysis is achieved by comparing with the respective standard curves.

2. The method according to claim 1, characterized in that The specific process of step (2) is as follows: (a) affixing a polymer porous membrane to one end of a hollow tube with two open ends or affixing the polymer porous membrane to the lower open end of a sample well of a microplate, and then evenly applying an organic liquid membrane phase solvent to at least one side of the polymer porous membrane to form an organic liquid membrane phase; the space formed by the polymer porous membrane and the hollow tube or sample well serves as a receiving phase container, and injecting a receiving phase into the inner cavity of the receiving phase container; (b) adding a sample solution to be tested into the sample phase container, and then inserting the receiving phase container into the sample phase container, so that the organic liquid membrane phase just contacts the liquid surface of the sample solution to be tested, thereby forming an extraction device; (c) The extraction device is fixed on a shaker and the maltol or ethyl maltol in the sample phase is extracted under continuous shaking conditions.

3. The method according to claim 1 or 2, characterized in that: The polymer porous membrane is a polypropylene membrane, a polytetrafluoroethylene membrane or a polyvinylidene fluoride membrane.

4. The method according to claim 1 or 2, characterized in that: The organic liquid membrane phase solvent is selected from at least one of 2-octanone, n-octanol, n-decanol, n-nonanol, nonanone, undecanol, n-heptanol, tributyl phosphate, 2-nitrophenyl octyl ether and diethyl ether.

5. The method according to claim 1 or 2, characterized in that: The amount of the organic liquid film solvent applied is 0.1~0.3μL / mm 2 .

6. The method according to claim 1 or 2, characterized in that: The volume ratio of the sample phase to the receiving phase is 10:(1-5).

7. The method according to claim 1 or 2, characterized in that: The extraction time is 10-150 minutes, the extraction temperature is controlled at 5-35°C, and the shaking speed is 10-300 rpm.

8. The method according to claim 1, wherein: The liquid sample is a beverage or dairy product; the solid sample is baked food or meat.

9. The method according to claim 1, wherein: In step (1), the liquid sample is ultrasonically degased and then diluted 2 to 10 times with 0.1 to 0.2 mol / L hydrochloric acid; the solid sample is fixed to volume with 1 to 5 mol / L hydrochloric acid to a sample concentration of 0.1 to 0.2 g / mL.

10. The method according to claim 1, wherein: In step (2), FeCl3 is used as the iron source to prepare Fe 3+ After the aqueous solution is prepared, adjust the pH to 1.5-3.0 with 0.5-2 mol / L hydrochloric acid or sodium hydroxide solution.

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