Method for determining flavourings in an oil and use thereof

Abstract

The application provides a method for determining essence and flavor in oil, which comprises three main steps of sample pretreatment, purification and analysis and test, the influence of the presence of lipids and proteins in the oil on the extraction efficiency of the target essence and flavor is avoided by removing grease and impurities in the sample pretreatment stage; the determination method adopts a solid phase extraction-gas chromatography analysis method, the extract is purified through a solid phase extraction column and separated through a polar chromatographic column, has the advantages of small matrix interference and fast analysis speed, and through reasonable purification and purification path and analysis and test means, the detection sensitivity is improved, and the detection limit and the quantitative limit are reduced. The application can simultaneously determine vanillin, methyl vanillin, ethyl vanillin, ethyl maltol and 2,3,5-trimethylpyrazine, five kinds of essence and flavor in oil, realizes simple pretreatment operation, good purification effect and higher sensitivity quantitative detection, and provides technical support for oil quality evaluation and edible oil safety supervision.

Description

Technical Field

[0001] This invention belongs to the field of fragrance and flavor detection technology, specifically relating to a method for simultaneously determining vanillin, methyl vanillin, ethyl vanillin, ethyl maltol and 2,3,5-trimethylpyrazine in oilseeds, and also relating to the application of this method in determining the fragrance and flavor content in oilseeds such as rapeseed, sesame and peanut. Background Technology

[0002] Oilseeds are crops containing a large amount of fat, which can be used to extract oil. Peanuts, sesame seeds, and rapeseed are the main oilseed crops for edible oil in my country. Their flavor changes during the seed growth and maturation, later oil storage, processing, and oil production stages.

[0003] Vanillin, methyl vanillin, ethyl vanillin, ethyl maltol, and 2,3,5-trimethylpyrazine are commonly used flavor enhancers in food, widely used in the food industry to improve flavor, modify bitterness, and enhance texture. They are characterized by their significant flavor-enhancing effects even in very small quantities. However, studies have shown that high doses of flavorings can cause adverse symptoms such as dizziness, nausea, and vomiting, and in severe cases, even damage to organs such as the liver and kidneys. Currently, GB2760-2014, the National Food Safety Standard for the Use of Food Additives, strictly regulates the usage limits of vanillin and other flavorings, and stipulates that they cannot be added to foods containing vegetable oils or animal fats. However, unscrupulous vendors illegally add these substances to food to pass off inferior or counterfeit products.

[0004] Meanwhile, research has also shown that edible vegetable oils contain endogenous flavorings. For example, sesame oil and rapeseed oil naturally contain vanillin, which may originate from oil crops or the oil processing process. Therefore, in order to identify the true source of flavorings in edible oils and based on the needs of routine supervision, it is urgent to establish detection methods for flavorings in oil crops.

[0005] Currently, there is limited information on the content and detection of flavorings and fragrances in oilseeds, with the focus mainly on oils and their downstream product, milk powder. Pretreatment extraction techniques for flavorings and fragrances primarily include solid-phase extraction, distillation extraction, stir bar adsorption extraction, solvent-assisted flavor evaporation, and static headspace analysis. Analytical techniques for flavorings mainly include high-performance liquid chromatography (HPLC), gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), electrochemical methods, ion chromatography, and electronic nose and electronic tongue methods. However, few of these methods can simultaneously measure five flavorings and fragrances, resulting in low extraction efficiency. GB5009.284-2021, "National Food Safety Standard - Determination of Vanillin, Methyl Vanillin, Ethyl Vanillin and Coumarin in Food", and BJS 201705, "Determination of Vanillin, Methyl Vanillin and Ethyl Vanillin in Food", can detect three substances simultaneously. However, their detection matrices are mainly dairy products, pastries, candies, beverages, and oils, and do not include oil crops rich in oil. In addition, the instruments used in these standards are relatively expensive, resulting in high costs, which are not suitable for grassroots supervision and enterprises to carry out routine monitoring.

[0006] Furthermore, oilseed crops are rich in both oils and fiber, resulting in a complex matrix. Directly introducing untreated samples into the instrument can cause severe matrix inhibition and even contaminate the entire instrument system, making sample preparation challenging. To avoid matrix interference and ensure accurate quantification, the detection of various fragrances and flavorings typically employs expensive mass spectrometry, a method with high operating costs, making it unsuitable for grassroots and factory-level testing. Therefore, achieving accurate quantification using low-cost instruments remains a challenge.

[0007] Based on this, a method is provided for the simultaneous detection of five flavorings in oil crops: vanillin, methyl vanillin, ethyl vanillin, ethyl maltol, and 2,3,5-trimethylpyrazine. This method provides a more convenient, faster, more precise, and more accurate detection of endogenous and exogenous flavorings in oil crops, which is of great significance for oil quality assessment and edible oil safety supervision, and is also a technical problem that urgently needs to be solved. Summary of the Invention

[0008] One of the objectives of this invention is to provide a method for simultaneously determining vanillin, methyl vanillin, ethyl vanillin, ethyl maltol and 2,3,5-trimethylpyrazine in oils.

[0009] The second objective of this invention is to provide a method for determining the content of flavorings and fragrances in oilseeds, and its application in determining the content of flavorings and fragrances in oilseeds such as rapeseed, sesame, and peanut.

[0010] One of the technical solutions adopted to achieve the objective of this invention is: to provide a method for determining the flavorings and fragrances in oils, comprising the following steps:

[0011] S1. Sample pretreatment: Mix the crushed oil with water, then add acetonitrile and sonicate to obtain the first mixture; add sodium chloride and n-hexane to the first mixture to obtain the second mixture; separate the solid and liquid of the second mixture and take the intermediate liquid; treat the intermediate liquid with nitrogen to obtain the first residue, dissolve the first residue with methanol, add water to make up to volume, and obtain the sample solution.

[0012] S2. Purification and Refinement: The sample solution is passed through a solid-phase extraction column, then rinsed and eluted with water, and the eluent is collected; the eluent is treated with nitrogen to obtain a second residue; the second residue is dissolved in methanol and filtered through a microporous membrane to obtain a filtrate;

[0013] S3. Analysis and testing: The filtrate is injected into a gas chromatograph for detection. Based on the detection results, the vanillin, methyl vanillin, ethyl vanillin, ethyl maltol and 2,3,5-trimethylpyrazine contained therein are qualitatively and quantitatively analyzed.

[0014] The general idea of ​​the method for determining flavorings and fragrances in oils provided by this invention is as follows:

[0015] This invention addresses the problems of existing methods for detecting fragrances and flavorings in vegetable oils, particularly those targeting endogenous fragrances in oilseed crops, such as complex matrices, susceptibility to contamination, difficult sample preparation, high operating costs, and numerous interfering factors. It provides a method for the accurate quantitative detection of fragrances and flavorings contained in oilseeds. This method comprises three main steps: sample pretreatment, purification, and analysis. Sample pretreatment effectively removes impurities, avoiding the impact of lipids and proteins in the oilseeds on the extraction efficiency of the target fragrances and flavorings. The pretreatment process also achieves better purification of the sample solution, providing necessary prerequisites for improving detection sensitivity and lowering the limits of detection and quantification. Furthermore, this invention establishes a solid-phase extraction-gas chromatography (SPE-GC) analytical method. Through a rational purification pathway combined with analytical testing, it meets the requirement for the simultaneous accurate quantitative detection of five fragrance and flavoring agents (vanillin, methyl vanillin, ethyl vanillin, ethyl maltol, and 2,3,5-trimethylpyrazine) from oilseed crops.

[0016] In step S1 of this invention, considering that oilseeds are rich in fat and contain a large amount of fat and protein, the extracted solution will contain a certain amount of lipids, which will greatly affect the extraction efficiency of the target flavorings and concentrates, and will also have an adverse effect on the performance of the quantitative detection instrument. In step S1 of this invention, the pulverized sample is first extracted with water and acetonitrile respectively, then treated with sodium chloride and n-hexane, and finally separated into solid and liquid to remove fats and impurities. The liquid in the intermediate layer obtained from the solid-liquid separation (intermediate liquid) is treated with nitrogen blowing and then dissolved in methanol to prepare a high-purity sample solution. This not only helps to improve the extraction efficiency of subsequent solid phase extraction operations, but also avoids impurities from causing pollution and damage to the solid phase extraction column and instrument.

[0017] In step S1, the addition of sodium chloride can promote the stratification of hexane, acetonitrile and water in subsequent processing, increase the oil carrying capacity of hexane, and thus reduce the oil content of the intermediate layer obtained after solid-liquid separation. This not only facilitates subsequent extraction and purification, but also avoids the contamination of the instrument caused by oil residue.

[0018] Further, in step S1, the oil is pulverized using a pulverizer and passed through a 30-mesh sieve to obtain pulverized oil with a particle size of 100-600 μm.

[0019] Preferably, the mass-to-volume ratio of the pulverized oil to water is 1:(1-5) g / mL; the mass-to-volume ratio of the pulverized oil to acetonitrile is 1:(5-10) g / mL. Preferably, the mass-to-volume ratio of the pulverized oil to water is 2:5 g / mL; the mass-to-volume ratio of the pulverized oil to acetonitrile is 1:10 g / mL; and the ultrasonic treatment time after adding acetonitrile is 10-40 min.

[0020] Further, in step S1, the mass ratio of the pulverized oil to sodium chloride is 1:(1-3); the mass-to-volume ratio of the pulverized oil to n-hexane is 1:(1-5) g / mL. Preferably, the mass ratio of the pulverized oil to sodium chloride is 2:3; the mass-to-volume ratio of the pulverized oil to n-hexane is 2:3 g / mL.

[0021] Further, in step S1, the solid-liquid separation is performed by centrifugation at a speed of 2000–9000 r / min for a time of 5–20 min. Preferably, the centrifugation speed is 3000–5000 r / min for a time of 8–12 min.

[0022] Preferably, in step S1, after adding water, acetonitrile, sodium chloride, and n-hexane, they are mixed by vortex oscillation for 0.5 to 2 minutes.

[0023] Further, in step S1, the intermediate liquid is treated by nitrogen blowing (nitrogen gas is blown into the surface of the heated sample to concentrate and dry the sample), and the temperature of the nitrogen blowing is 30-50°C.

[0024] In step S2 of this invention, the sample solution is purified and refined. The sample solution, after being degreased and impurity-removed, is purified by a solid-phase extraction column and separated by a polar chromatography column, which has the advantages of low matrix interference and fast analysis speed.

[0025] Further, in step S2, the solid-phase extraction column is a C18 solid-phase extraction column LC-C18 (1g / 6mL); the pore size of the microporous filter membrane is 0.22μm.

[0026] Further, in step S2, the flow rate of the sample solution in the solid-phase extraction column is 0.1–2 mL / min; the eluent is a mixture of methanol and water in a volume ratio of (2–5):1; and the nitrogen blowing temperature is 30–50 °C. Preferably, the eluent is a mixture of methanol and water in a volume ratio of 4:1.

[0027] Furthermore, in step S3, before injecting the filtrate into a gas chromatograph for detection, the method further includes: preparing a mixed standard working solution of vanillin, methyl vanillin, ethyl vanillin, ethyl maltol and 2,3,5-trimethylpyrazine, and using gas chromatography to determine the mixed standard working solution to obtain standard curves for the five fragrances.

[0028] Furthermore, the test solution (filtrate) is tested using the same instrument parameters as the mixed standard working solution to obtain the chromatogram of the fragrance and flavoring in the sample, and then qualitative and quantitative analysis is performed.

[0029] Furthermore, in step S3, the gas chromatography uses a CD-WAX capillary column with dimensions of 30m × 0.25mm × 0.25um. The CD-WAX capillary column is used to determine five fragrances in oils and has the advantages of high sensitivity and good reproducibility of the target analytes.

[0030] The gas chromatography conditions include: an injection port temperature of 260–280°C, an initial column temperature of 70–90°C, and a stepwise increase to 230–250°C. Preferably, the specific gas chromatography conditions are as follows: a programmed temperature ramp is used, with an initial column temperature of 70–90°C, held for 1–10 min, increased to 200–230°C at a rate of 5–10°C / min, held for 1–10 min, and then increased to 230–250°C at a rate of 10–30°C / min, held for 1–10 min; and an FID detector temperature of 270–300°C.

[0031] Furthermore, the injection process employs splitless injection with an injection flow rate of 1.0–3.0 mL / min and an injection volume of 1–2 μL. Nitrogen is used as the carrier gas with a flow rate of 10–40 mL / min; in the combustion gas supply for the FID detector, the hydrogen flow rate is 350–400 mL / min; and the air flow rate is 35–40 mL / min.

[0032] In step S3 of this invention, a unique heating program combined with an appropriate carrier gas flow rate effectively separates five similar fragrances and flavorings while avoiding interference from other substances in the matrix. This results in shorter peak times and symmetrical, sharp peak shapes. Preferably, the carrier gas nitrogen flow rate is 25 mL / min; the detector's combustion gas contains 400 mL / min hydrogen and 40 mL / min air.

[0033] Furthermore, this invention employs the external standard method for quantification.

[0034] The second objective of this invention is to provide an application of the determination method described in one objective of this invention in determining the content of fragrances and flavorings in oils.

[0035] Furthermore, the oilseeds include one or more of rapeseed, sesame, peanut, sunflower seeds, and soybeans.

[0036] Furthermore, the flavorings include vanillin, methyl vanillin, ethyl vanillin, ethyl maltol, and 2,3,5-trimethylpyrazine.

[0037] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0038] (1) This invention provides a method for determining fragrances and flavorings in oilseeds, comprising three main steps: sample pretreatment, purification and refinement, and analytical testing. In the sample pretreatment stage, water and acetonitrile are used for extraction, supplemented with sodium chloride and n-hexane. Solid-liquid separation is combined to remove lipids and impurities, avoiding the influence of lipids and proteins in the oilseeds on the extraction efficiency of the target fragrances and flavorings. This results in better purification of the sample solution, providing necessary prerequisites for improving detection sensitivity and lowering the limits of detection and quantitation. Furthermore, this invention also establishes a solid-phase extraction-gas chromatography analytical method. The extract is purified using a C18 solid-phase extraction column and separated using a polar chromatographic column, offering advantages such as low matrix interference and fast analysis speed. Through a reasonable purification and refinement pathway combined with analytical testing methods, the method meets the requirement for accurate simultaneous quantitative detection of five fragrances and flavorings from oilseed crops.

[0039] (2) The present invention provides a method for determining flavorings and fragrances in oilseeds. In view of the serious lack of research on the detection methods of flavorings and fragrances in oilseeds at home and abroad, this invention provides a method that can simultaneously determine vanillin, methyl vanillin, ethyl vanillin, ethyl maltol and 2,3,5-trimethylpyrazine in oilseeds. This method achieves simple pretreatment operation, good purification effect and higher sensitivity quantitative detection, providing technical support for oilseed quality evaluation and edible oil safety supervision. Attached Figure Description

[0040] Figure 1 A schematic flowchart of a method for determining flavorings and fragrances in oils provided by the present invention;

[0041] Figure 2 This is a schematic diagram of the operation process of the method for determining the fragrance and flavoring in oils used in Examples 1-3 of the present invention;

[0042] Figure 3 Gas chromatogram of a mixed standard working solution of five flavorings and fragrances prepared for an embodiment of the present invention. Detailed Implementation

[0043] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0044] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0045] The present invention will be further described below with reference to specific embodiments, but these are not intended to limit the scope of the invention.

[0046] In all embodiments of the present invention, sample analysis was performed on a gas chromatograph (Agilent 8890, USA). Data acquisition and processing were performed using Agilent OpenLab CDS software. Analytes were separated using an Anpel CD-WAX capillary column (30m × 0.25mm × 0.25um).

[0047] The preparation methods for the mixed standard working solutions of the five fragrances and flavorings used in each embodiment are as follows:

[0048] (1) Preparation of standard stock solutions

[0049] Accurately weigh 20 mg each of vanillin, methyl vanillin, ethyl vanillin, ethyl maltol, and 2,3,5-trimethylpyrazine (accurate to 0.1 mg), and place them separately in 10 mL volumetric flasks. Dissolve each flask in methanol and dilute to the mark. Mix well to prepare a 2000 mg / L standard stock solution. Transfer the solution to a brown glass container and store at -18°C protected from light.

[0050] (2) Preparation of standard intermediate solution

[0051] Accurately pipette 5 mL each of vanillin, methyl vanillin, ethyl vanillin, ethyl maltol, and 2,3,5-trimethylpyrazine standard stock solutions into 10 mL volumetric flasks, dissolve them in methanol and dilute to the mark, mix well, and prepare a 1000 mg / L standard intermediate solution.

[0052] (3) Preparation of mixed standard working solution

[0053] Pipette 0.005 mL, 0.010 mL, 0.050 mL, 0.100 mL, 0.500 mL, 1.000 mL, and 2.000 mL of the standard intermediate solution into 10 mL volumetric flasks, respectively, and dilute to the mark with methanol solution. Mix well to obtain mixed standard working solutions with concentrations of 0.5 mg / L, 1.0 mg / L, 5.0 mg / L, 10.0 mg / L, 50.0 mg / L, 100.0 mg / L, and 200.0 mg / L, respectively.

[0054] Example 1

[0055] This embodiment provides a method for the simultaneous determination of five flavorings and fragrances contained in peanuts, an oilseed crop, including vanillin, methyl vanillin, ethyl vanillin, ethyl maltol, and 2,3,5-trimethylpyrazine. The method comprises the following steps:

[0056] Step 1, Sample pretreatment: Weigh 2.00g of evenly crushed peanuts, add 5mL of water, vortex for 1min, add 20mL of acetonitrile, vortex for 2min, ultrasonically extract for 30min, add 3g of sodium chloride, vortex for 30s, add 3mL of n-hexane, vortex for 1min, centrifuge at 4000r / min for 10min at 5℃, take 10mL of intermediate liquid into a test tube, blow the clear liquid with nitrogen at 40℃ until nearly dry, add 1mL of methanol to dissolve the residue, and dilute with water to 10mL;

[0057] Step 2, purification and refinement:

[0058] The sample solution was passed through a C18 solid-phase extraction column at a flow rate of less than 1 mL / min. After all the sample solution had eluted, it was rinsed with 5 mL of water, dried under vacuum, eluted with 10 mL of methanol-water solution (methanol to water volume ratio of 4:1), dried under vacuum, and the eluent was collected. The eluent was then purged with nitrogen at 40 °C until nearly dry. 1 mL of methanol was accurately added to dissolve the residue, and the solution was filtered through a 0.22 μm microporous membrane. 1 μL of the filtrate was then injected into the GC system.

[0059] Step 3, Analysis and Testing:

[0060] A mixed standard working solution of vanillin, methyl vanillin, ethyl vanillin, ethyl maltol, and 2,3,5-trimethylpyrazine was prepared and analyzed by gas chromatography to obtain standard curves for the five fragrances. The filtrate obtained in step 2 was analyzed with the mixed standard working solution using the same instrument parameters to obtain chromatograms of the fragrances in the sample, followed by qualitative and quantitative analysis. The specific method is as follows:

[0061] The filtrate was injected into a gas chromatograph for detection. The gas chromatographic conditions were as follows: CD-WAX capillary column (30m×0.25mm×0.25um); injection port temperature 270℃, temperature programmed, initial column temperature 90℃, held for 3 min, increased to 220℃ at a rate of 10℃ / min, held for 3 min, then increased to 240℃ at a rate of 20℃ / min, held for 3 min; FID detector temperature 280℃; splitless injection, flow rate: 2.0mL / min, injection volume 1μL; carrier gas: nitrogen, hydrogen flow rate: 400mL / min, air flow rate: 40mL / min, nitrogen flow rate: 25mL / min.

[0062] The external standard method was used for quantitative analysis. Based on the test results, the vanillin, methyl vanillin, ethyl vanillin, ethyl maltol and 2,3,5-trimethylpyrazine contained in peanuts were qualitatively and quantitatively analyzed.

[0063] Example 2

[0064] This embodiment provides a method for the simultaneous determination of five flavorings and fragrances contained in sesame, an oilseed crop: vanillin, methyl vanillin, ethyl vanillin, ethyl maltol, and 2,3,5-trimethylpyrazine. The method includes the following steps:

[0065] Step 1, Sample pretreatment: Weigh 1.00g of evenly ground sesame seeds, add 3mL of water, vortex for 2min, add 20mL of acetonitrile, vortex for 3min, ultrasonically extract for 30min, add 3g of sodium chloride, vortex for 30s, add 3mL of n-hexane, vortex for 1min, centrifuge at 4000r / min for 10min at 5℃, take 10mL of intermediate liquid into a test tube, blow the clear liquid with nitrogen at 45℃ until nearly dry, add 1mL of methanol to dissolve the residue, and dilute with water to 10mL;

[0066] Step 2, purification and refinement:

[0067] The sample solution was passed through a C18 solid-phase extraction column at a flow rate of less than 1 mL / min. After all the sample solution had eluted, it was rinsed with 5 mL of water, dried under vacuum, eluted with 10 mL of methanol-water solution (methanol to water volume ratio of 4:1), dried under vacuum, and the eluent was collected. The eluent was then purged with nitrogen at 40 °C until nearly dry. 1 mL of methanol was accurately added to dissolve the residue, and the solution was filtered through a 0.22 μm microporous membrane. 1 μL of the filtrate was then injected into the GC system.

[0068] Step 3, Analysis and Testing:

[0069] A mixed standard working solution of vanillin, methyl vanillin, ethyl vanillin, ethyl maltol, and 2,3,5-trimethylpyrazine was prepared and analyzed by gas chromatography to obtain standard curves for the five fragrances. The filtrate obtained in step 2 was analyzed with the mixed standard working solution using the same instrument parameters to obtain chromatograms of the fragrances in the sample, followed by qualitative and quantitative analysis. The specific method is as follows:

[0070] The filtrate was injected into a gas chromatograph for detection. The gas chromatographic conditions were as follows: CD-WAX capillary column (30m×0.25mm×0.25um); injection port temperature 280℃, temperature programmed, initial column temperature 90℃, held for 3 min, increased to 220℃ at a rate of 10℃ / min, held for 3 min, then increased to 240℃ at a rate of 20℃ / min, held for 3 min; FID detector temperature 290℃; splitless injection, flow rate: 2.0mL / min, injection volume 1μL; carrier gas: nitrogen, hydrogen flow rate: 400mL / min, air flow rate: 40mL / min, nitrogen flow rate: 25mL / min.

[0071] The external standard method was used for quantitative analysis. Based on the test results, the vanillin, methyl vanillin, ethyl vanillin, ethyl maltol and 2,3,5-trimethylpyrazine contained in sesame were qualitatively and quantitatively analyzed.

[0072] Example 3

[0073] This embodiment provides a method for simultaneously determining five flavorings and fragrances contained in rapeseed, an oilseed crop: vanillin, methyl vanillin, ethyl vanillin, ethyl maltol, and 2,3,5-trimethylpyrazine. The method includes the following steps:

[0074] Step 1, Sample pretreatment: Weigh 1.00g of evenly crushed rapeseed, add 4mL of water, vortex for 1min, add 20mL of acetonitrile, vortex for 2min, ultrasonically extract for 25min, add 3g of sodium chloride, vortex for 30s, add 4mL of n-hexane, vortex for 1min, centrifuge at 4000r / min for 15min at 5℃, take 10mL of intermediate liquid into a test tube, blow the obtained clear liquid with nitrogen at 45℃ until nearly dry, add 1mL of methanol to dissolve the residue, and dilute with water to 10mL;

[0075] Step 2, purification and refinement:

[0076] The sample solution was passed through a C18 solid-phase extraction column at a flow rate of less than 1 mL / min. After all the sample solution had eluted, it was rinsed with 5 mL of water, dried under vacuum, eluted with 10 mL of methanol-water solution (methanol to water volume ratio of 4:1), dried under vacuum, and the eluent was collected. The eluent was then purged with nitrogen at 40 °C until nearly dry. 1 mL of methanol was accurately added to dissolve the residue, and the solution was filtered through a 0.22 μm microporous membrane. 1 μL of the filtrate was then injected into the GC system.

[0077] Step 3, Analysis and Testing:

[0078] A mixed standard working solution of vanillin, methyl vanillin, ethyl vanillin, ethyl maltol, and 2,3,5-trimethylpyrazine was prepared and analyzed by gas chromatography to obtain standard curves for the five fragrances. The filtrate obtained in step 2 was analyzed with the mixed standard working solution using the same instrument parameters to obtain chromatograms of the fragrances in the sample, followed by qualitative and quantitative analysis. The specific method is as follows:

[0079] The filtrate was injected into a gas chromatograph for detection. The gas chromatographic conditions were as follows: CD-WAX capillary column (30m×0.25mm×0.25um); injection port temperature 280℃, temperature programmed, initial column temperature 90℃, held for 3 min, increased to 220℃ at a rate of 10℃ / min, held for 3 min, then increased to 240℃ at a rate of 20℃ / min, held for 3 min; FID detector temperature 290℃; splitless injection, flow rate: 2.0mL / min, injection volume 1μL; carrier gas: nitrogen, hydrogen flow rate: 400mL / min, air flow rate: 40mL / min, nitrogen flow rate: 25mL / min.

[0080] The external standard method was used for quantitative analysis. Based on the test results, the vanillin, methyl vanillin, ethyl vanillin, ethyl maltol and 2,3,5-trimethylpyrazine contained in rapeseed were qualitatively and quantitatively analyzed.

[0081] Results Analysis

[0082] (I) Method Validation

[0083] The external standard method was used to quantitatively validate the detection methods of Examples 1-3. The method validation was carried out in accordance with the national standards of the People's Republic of China. The linear regression equation, linear range, limit of detection, and limit of quantitation are shown in Table 1. The accuracy and precision of the detection methods provided in Examples 1-3 of this invention for the detection of five flavorings and fragrances are shown in Table 2.

[0084] Table 1. Linear equations, correlation coefficients (r), limits of detection (LODs), limits of quantitation (LOQs), and retention times for five flavorings and fragrances.

[0085]

[0086] Table 2. Average recovery and precision of five flavorings and fragrances spiked in different oil samples (n=6)

[0087]

[0088] (II) Calibration curve, limit of detection and limit of quantitation

[0089] The external standard method was used to identify and quantify analytes in oilseed (peanut, sesame, and rapeseed) samples spiked at three concentrations: vanillin, methyl vanillin, ethyl vanillin, ethyl maltol, and 2,3,5-trimethylpyrazine at concentrations of 1.5 (LOQ), 15, and 100 mg / kg were all within the linear range of 0.5–200 mg / L, with correlation coefficients (r) exceeding 0.999. The limits of detection (LOD) and quantitation (LOQ) were calculated based on a signal-to-noise ratio (S / N) of 3 and 10, respectively. The LOD and LOQ for vanillin, methyl vanillin, ethyl vanillin, ethyl maltol, and 2,3,5-trimethylpyrazine were 0.5 and 1.5 mg / kg, respectively. To obtain accurate limits of quantitation (LOQs), peanut, sesame, and rapeseed samples were added at the LOQ concentration and tested six times repeatedly. For the three oilseed samples, the average recovery rate of vanillin was 98.1%–98.4%, with an RSD of 3.00–3.35%; the average recovery rate of methyl vanillin was 96.1%–98.9%, with an RSD of 2.95%–4.54%; the recovery rate of ethyl vanillin was 95.9%–97.9%, with an RSD of 2.38%–3.97%; the average recovery rate of ethyl maltol was 96.7%–102.3%, with an RSD of 1.27%–3.25%; and the average recovery rate of 2,3,5-trimethylpyrazine was 96.3%–101.5%, with an RSD of 2.16%–2.57%.

[0090] (III) Accuracy and Precision

[0091] To verify the reproducibility of the method, recoveries were calculated by analyzing spiked oilseed (peanut, sesame, and rapeseed) samples at three concentration levels (1.5 mg / kg, 15 mg / kg, and 100 mg / kg). Six replicate experiments were performed. Among the three oilseed samples, the average recovery rate of vanillin ranged from 95.2% to 98.9%, with an RSD of 1.45% to 4.48%; the average recovery rate of methyl vanillin ranged from 95.5% to 98.9%, with an RSD of 1.76% to 4.54%; the recovery rate of ethyl vanillin ranged from 95.9% to 102.6%, with an RSD of 1.24% to 4.39%; the average recovery rate of ethyl maltol ranged from 96.7% to 104.2%, with an RSD of 1.25% to 4.11%; and the average recovery rate of 2,3,5-trimethylpyrazine ranged from 96.3% to 101.5%, with an RSD of 1.35% to 3.04%. Therefore, the validated method fully meets the monitoring requirements for flavorings and fragrances in oilseeds.

[0092] The above are merely preferred embodiments of the present invention and are not intended to limit the implementation methods and protection scope of the present invention. Those skilled in the art should recognize that any equivalent substitutions and obvious changes made based on the content of this specification should be included within the protection scope of the present invention.

Claims

1. A method for determining flavorings and fragrances in oilseeds, characterized in that, Includes the following steps: S1. Sample pretreatment: Mix the crushed oil with water, then add acetonitrile and sonicate to obtain the first mixture; add sodium chloride and n-hexane to the first mixture to obtain the second mixture; separate the solid and liquid of the second mixture and take the intermediate liquid; treat the intermediate liquid with nitrogen to obtain the first residue, dissolve the first residue with methanol, add water to make up to volume, and obtain the sample solution. The oilseed is selected from one of peanut, sesame, and rapeseed; the particle size of the crushed oilseed is 100-600 μm; the mass-to-volume ratio of the crushed oilseed to water is 1:1-5 g / mL; the mass-to-volume ratio of the crushed oilseed to acetonitrile is 1:5-10 g / mL; the mass ratio of the crushed oilseed to sodium chloride is 1:1-3; and the mass-to-volume ratio of the crushed oilseed to n-hexane is 1:1-5 g / mL. S2. Purification and Refinement: The sample solution is passed through a solid-phase extraction column, then rinsed with water, eluted with a mixture of methanol and water, and the eluent is collected; the eluent is treated with nitrogen to obtain a second residue; the second residue is dissolved in methanol and filtered through a microporous membrane to obtain a filtrate; the solid-phase extraction column used is a C18 solid-phase extraction column. S3. Analysis and Testing: The filtrate is injected into a gas chromatograph for detection. The gas chromatograph uses a CD-WAX capillary column with an injection port temperature of 260-280℃, an initial column temperature of 70-90℃, and a stepwise increase to 230-250℃. The FID detector temperature is 270-300℃. Based on the detection results, the vanillin, methyl vanillin, ethyl vanillin, ethyl maltol, and 2,3,5-trimethylpyrazine contained in the filtrate are qualitatively and quantitatively analyzed.

2. The determination method according to claim 1, characterized in that, In step S1, the solid-liquid separation is carried out by centrifugation, with a centrifugation speed of 2000-9000 r / min and a centrifugation time of 5-20 min.

3. The determination method according to claim 1, characterized in that, In step S2, the pore size of the microporous filter membrane is 0.22 μm.

4. The determination method according to claim 1, characterized in that, In step S2, the flow rate of the sample solution in the solid-phase extraction column is 0.1-2 mL / min; the volume ratio of methanol to water in the elution mixture is 2-5:

1.

5. The determination method according to claim 1, characterized in that, In step S3, before injecting the filtrate into a gas chromatograph for detection, the method further includes: preparing a mixed standard working solution of vanillin, methyl vanillin, ethyl vanillin, ethyl maltol and 2,3,5-trimethylpyrazine, and using gas chromatography to determine the mixed standard working solution to obtain standard curves for the five fragrances.

6. The determination method according to claim 1, characterized in that, In step S3, the gas chromatography injection process adopts splitless injection, with an injection flow rate of 1.0-3.0 mL / min and an injection volume of 1-2 μL.

7. The determination method according to claim 1, characterized in that, In step S3, nitrogen is used as the carrier gas in the gas chromatograph, with a nitrogen flow rate of 10-40 mL / min; in the combustion gas of the FID detector, the hydrogen flow rate is 350-400 mL / min, and the air flow rate is 35-40 mL / min.

8. The application of the determination method according to any one of claims 1-7 in the determination of the flavor and fragrance content in oils.

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