Method for quantifying exogenous flavors and fragrances in sesame oil

Abstract

The invention relates to a method for quantifying exogenous flavors and fragrances in sesame oil. The method comprises the following steps: preparing a gradient standard solution of a reference substance; preprocessing an object to be detected; determining the gradient standard solution through a liquid chromatography-tandem mass spectrometer to obtain a chromatographic peak-reference substance contrast relation diagram of the reference substance, drawing a standard curve and obtaining a linear regression equation; and measuring the to-be-measured object with the same instrument parameters to obtain a liquid chromatogram of the to-be-measured object, and comparing the liquid chromatogram of the to-be-measured object with the liquid chromatogram of the reference substance solution. According to the method, the detection limit of the ethyl maltol and the ethyl vanillin is 0.1 mg / kg, and the quantification limit is 0.25 mg / kg; the detection limit of the difurfuryl sulfide is 0.5 mg / kg, and the quantification limit of the difurfuryl sulfide is 1.0 mg / kg; the detection limit of the difurfuryl disulfide is 2.5 mg / kg, the quantitation limit is 5.0 mg / kg, and the determination of the flavors and fragrances in the sesame oil is completely met.

Description

Technical Field

[0001] The present invention relates to the technical field of food detection, and particularly to a method for quantitatively determining exogenous flavoring substances in sesame oil. Background Art

[0002] Sesame oil, also known as fragrant oil and sesame seed oil, is an oil with a special fragrance extracted from sesame seeds. It is a seasoning product in daily life and is commonly used in hot pot dipping sauces, cold dishes, etc. Sesame oil contains relatively high levels of active substances such as sesamol and lignans, which have antioxidant and lipid metabolism-regulating effects, can prevent cardiovascular and cerebrovascular diseases, and have a good promoting effect on human health. At the same time, the good antioxidant properties of sesame oil also endow it with better storage characteristics and a longer shelf life. Due to the advantages of sesame oil in flavor and physiological effects, it has become a high-value-added edible vegetable oil variety, and its price is much higher than that of other edible vegetable oils. Therefore, many illegal merchants use cheap oils to blend and add flavor enhancers to counterfeit sesame oil in order to obtain huge profits. With the increasing crackdown on the production and sale of counterfeit sesame oil, the illegal adulteration methods of illegal merchants have become more and more concealed. Currently, there are many sales channels for compound vegetable oil flavors in the market. This shows that the flavor adulteration of sesame oil is no longer limited to maltol compounds, and its adulteration behavior may develop in the direction of diversification and complexity.

[0003] At present, the research on the differences in the volatile substance composition between pure sesame oil and sesame oil added with flavorings is relatively limited. The unique flavor of sesame oil mainly stems from its specific volatile components, while sesame flavor is formulated by simulating these components. Therefore, the key to distinguishing sesame oil from sesame flavor lies in identifying the marker compounds in the flavor that are different from natural sesame oil. With the increasing prominence of the sesame oil adulteration problem, how to accurately identify natural flavor components and synthetic flavors and effectively curb illegal addition behaviors through rapid qualitative and trace detection technologies has become the focus of current research and an urgent problem to be solved. Summary of the Invention

[0004] In view of the above problems, the present invention provides a method for quantitatively determining exogenous flavoring substances in sesame oil with simple pretreatment operation, high resolution, and high sensitivity.

[0005] The specific technical solutions are as follows:

[0006] A method for quantitatively determining exogenous flavoring substances in sesame oil, comprising:

[0007] Preparing a gradient standard solution of the reference substance;

[0008] Performing pretreatment on the analyte;

[0009] The gradient standard solution was determined by liquid chromatography-tandem mass spectrometry to obtain the chromatographic peak-reference substance correlation diagram of the reference substance, and the standard curve was plotted and the linear regression equation was obtained.

[0010] The analyte was determined with the same instrument parameters to obtain the liquid chromatogram of the analyte, and the liquid chromatogram of the analyte was compared with the liquid chromatogram of the reference substance solution.

[0011] Among them, in liquid chromatography, mobile phase A is 0.1% formic acid aqueous solution, and mobile phase B is methanol; the gradient elution conditions are: 0 min, 90% A, 10% B; 0 - 4 min, 90 - 5% A, 10 - 95% B; 4.1 - 7 min, 5% A, 95% B; 7.1 - 8 min, 5 - 90% A, 95 - 10% B; 8.1 - 10 min, 90% A, 10% A.

[0012] Furthermore, the reference substance is one or several of dithiodifurfuryl, furfuryl sulfide, ethyl maltol, and ethyl vanillin.

[0013] Furthermore, when the reference substance is furfuryl sulfide, the concentrations of the gradient standard solution of the reference substance are 0.1 μg / mL, 0.2 μg / mL, 0.5 μg / mL, 1 μg / mL, and 2 μg / mL respectively. 。

[0014] Furthermore, when the reference substance is dithiodifurfuryl, the concentrations of the gradient standard solution of the reference substance are 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, and 10 μg / mL respectively. 。

[0015] Furthermore, when the reference substance is ethyl maltol, the concentrations of the gradient standard solution of the reference substance are 0.02 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.2 μg / mL, and 0.5 μg / mL respectively.

[0016] Furthermore, when the reference substance is ethyl vanillin, the concentrations of the gradient standard solution of the reference substance are 0.02 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.2 μg / mL, and 0.5 μg / mL respectively.

[0017] Furthermore, the pretreatment method of the analyte is: weigh the analyte into a centrifuge tube, add methanol, vortex to mix evenly, centrifuge, take the supernatant and transfer it to a brown volumetric flask, make up the volume to the scale with methanol, take a part of the supernatant, add methanol-saturated n-hexane for extraction, centrifuge, take the lower layer supernatant, filter and then measure it on the machine.

[0018] Further, the chromatographic conditions are as follows: ACQUITY UPLC HSS T3 chromatographic column; column temperature 30 - 45°C; injection volume 8 - 12 μL; flow rate 0.2 - 0.4 mL / min 。

[0019] Further, the mass spectrometry conditions are as follows: electrospray ionization source; positive ion monitoring mode; multiple reaction monitoring; ion spray voltage 4500 - 5500 V; ion source temperature 500 - 650°C 。

[0020] Further, the method for drawing the standard curve is as follows: taking the peak area of the quality chromatogram of the reference substance solution as the ordinate and the concentration of the reference substance solution as the abscissa, drawing the standard curve to obtain the linear regression equation.

[0021] In the present invention, more than 10 kinds of commercially available flavors and 40 - 50 kinds of self - made sesame oils from different origins and processes are used for difference analysis. It is found that most of the commercially available flavors contain one or several of bis(2 - furyl) sulfide, bis(2 - furyl) disulfide, ethyl maltol, and ethyl vanillin. Therefore, the above substances can be selected as the reference substance targets. Based on the above principle, a high - performance liquid chromatography - tandem mass spectrometry analysis method for determining four flavoring agents in sesame oil and sesame blended oil is established in the present invention, providing reliable technical support for the quality control and adulteration identification of sesame oil.

[0022] In the present invention, a small amount of methanol is directly used for extraction twice, followed by centrifugation and filtration. The supernatant is then directly injected into the instrument. The method is simple to operate and has low cost. The detection limits of ethyl maltol and ethyl vanillin obtained in the present invention are 0.1 mg / kg, and the quantification limits are 0.25 mg / kg; the detection limit of bis(2 - furyl) sulfide is 0.5 mg / kg, and the quantification limit is 1.0 mg / kg; the detection limit of bis(2 - furyl) disulfide is 2.5 mg / kg, and the quantification limit is 5.0 mg / kg, which fully meets the determination of flavoring agents in sesame oil. Description of the Drawings

[0023] Figure 1 It is the total ion current chromatogram of 4 kinds of exogenous flavoring agents provided in the present invention. Detailed Embodiments

[0024] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the protection scope of the present invention.

[0025] It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other.

[0026] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but it is not intended to limit the present invention.

[0027] In the present invention, sample analysis was carried out on a QTRAP 4500 liquid chromatography-tandem mass spectrometer (equipped with an electrospray ionization source). Analyst software was used for data acquisition and processing. An ACQUITY UPLC HSS T3 chromatographic column (2.1 mm × 100 mm, 1.8 μm) was used to separate the analytes. The chromatographic conditions were as follows: ACQUITY UPLC HSS T3 chromatographic column (2.1 mm × 100 mm, 1.8 μm); column temperature: 40 °C; injection volume: 10 μL; flow rate: 0.3 mL / min; mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was methanol. The gradient elution conditions were: 0 min, 90% A, 10% B; 0 - 4 min, 90 - 5% A, 10 - 95% B; 4.1 - 7 min, 5% A, 95% B; 7.1 - 8 min, 5 - 90% A, 95 - 10% B; 8.1 - 10 min, 90% A, 10% A; the mass spectrometry conditions were: ion source: electrospray ionization source; monitoring mode: positive ion monitoring mode; monitoring method: multiple reaction monitoring (MRM); ion spray voltage: 5000 V; ion source temperature: 600 °C.

[0028] Accurately weigh 10 mg of dithiodifurfuryl into a 10 mL volumetric flask, dissolve it with methanol and make up to the mark to obtain a standard stock solution of dithiodifurfuryl with a mass concentration of about 1 mg / mL; take 1.0 mL of the dithiodifurfuryl standard stock solution into a 10 mL volumetric flask, and make up to the mark with methanol to obtain a standard intermediate solution of dithiodifurfuryl with a mass concentration of about 100 μg / mL; take 1.0 mL of the dithiodifurfuryl standard solution into a 10 mL volumetric flask, and make up to the mark with methanol to obtain a standard intermediate solution of dithiodifurfuryl with a mass concentration of about 10 μg / mL; respectively take 1.0 mL of the ethyl maltol and ethyl vanillin standard solutions into a 10 mL volumetric flask, and make up to the mark with methanol to obtain a mixed standard intermediate solution with a mass concentration of 10 μg / mL.

[0029] Appropriately take appropriate amounts of the standard intermediate solutions of the four substances into 10 mL volumetric flasks, and dilute to the mark with the blank matrix solution to obtain standard working solutions with mass concentrations of ethyl maltol and ethyl vanillin being 0.02 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 0.5 μg / mL, and the mass concentration of dithiolane being 0.1 μg / mL, 0.2 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, and the mass concentration of dithiodiethylacetal being 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL (the responses of the four substances on the instrument are different, and the method provided in the present invention can simultaneously determine multiple substances).

[0030] Weigh 2 g (accurate to 0.0001 g) of the sample into a 10 mL centrifuge tube, accurately add 5 mL of methanol, vortex for 10 min, mix evenly, centrifuge at 3900 r / min for 5 min, and take the supernatant into a 10 mL brown volumetric flask. Then add 5 mL of methanol to the lower oil sample and repeat the extraction once. Combine the supernatants into the above 10 mL brown volumetric flask and dilute to the mark with methanol. Take 2 mL of the supernatant, add 2 mL of methanol-saturated n-hexane for extraction (due to its strong hydrophilicity, methanol can effectively form hydrogen bonds with n-hexane, thereby promoting the conversion of n-hexane into a more stable saturated state, significantly improving the stability and efficiency of the extract). After centrifugation, take the lower-layer supernatant, filter it through a 0.22 μm microporous filter membrane, and directly inject it into the instrument for determination.

[0031] The experiment optimized several factors such as mass spectrometry conditions, mobile phase conditions, extraction solvent, extraction time, matrix standard curve, etc. After obtaining the optimal conditions, external standard method was used for quantitative method validation, and the method validation was carried out in accordance with GB 5009.295-2023 "General Rules for Method Validation of National Food Safety Standards Chemical Analysis Methods". The linear regression equation (draw a standard curve with the mass concentration (X, μg / mL) of the compound as the abscissa and the peak area (Y, counts) as the ordinate, and fit it into a linear equation), linear range, LOD, and LOQ are shown in Table 1.

[0032] Table 1

[0033]

[0034]

[0035] As can be seen from Table 1, the four compounds have a good linear relationship within their linear ranges, and the correlation coefficients (r) are 0.998 - 0.999. Taking 3 times the signal-to-noise ratio (S / N) as the detection limit and 10 times as the quantification limit, the detection limits of ethyl maltol and ethyl vanillin are 0.1 mg / kg, and the quantification limits are 0.25 mg / kg; the detection limit of dithiophene is 0.5 mg / kg, and the quantification limit is 1.0 mg / kg; the detection limit of bis(2-furyl) disulfide is 2.5 mg / kg, and the quantification limit is 5.0 mg / kg.

[0036] In the present invention, according to the optimized chromatographic conditions, a mixed standard solution with a mass concentration of 0.5 μg / mL was continuously injected 6 times, and the RSD of the peak areas of the four flavor substances was calculated. The results are shown in Table 2.

[0037] Table 2

[0038]

[0039] As can be seen from Table 2, the relative standard deviations of the four flavor substances are between 1.56% - 7.27%, indicating that the precision of this method is good.

[0040] In the present invention, standard solutions of the four flavor substances were added to blank sesame oil samples to prepare spiked samples at three concentration levels: low, medium, and high. Six parallel experiments were carried out according to the pretreatment method to investigate the recovery rate of the method. The results are shown in Table 3.

[0041]

[0042]

[0043] As can be seen from Table 3, the recovery rates of the four compounds are 84.2% - 101%, indicating that this method has a good recovery rate and can meet the test requirements. Therefore, the verified method fully meets the precise quantification requirements of flavor substances in sesame oil.

[0044] The above are only the preferred embodiments of the present invention, and do not limit the implementation manners and protection scope of the present invention accordingly. For those skilled in the art, it should be able to realize that all equivalent replacements and obvious changes made by using the description and illustration content of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for quantifying exogenous flavors and fragrances in sesame oil, characterized in that: include: Prepare a gradient standard solution of a reference substance; Pre-treat the test object; The gradient standard solution is measured by liquid chromatography-tandem mass spectrometry to obtain a chromatographic peak-reference substance comparison relationship diagram of the reference substance, a standard curve is drawn and a linear regression equation is obtained; The analyte is measured with the same instrument parameters to obtain a liquid chromatogram of the analyte, and the liquid chromatogram of the analyte is compared with the liquid chromatogram of the reference solution; In the liquid chromatography method, mobile phase A is 0.1% formic acid aqueous solution, and mobile phase B is methanol; the gradient elution conditions are: 0 min, 90% A, 10% B; 0-4 min, 90-5% A, 10-95% B; 4.1-7 min, 5% A, 95% B; 7.1-8 min, 5-90% A, 95-10% B; 8.1-10 min, 90% A, 10% A.

2. The quantitative method for exogenous flavors and fragrances in sesame oil according to claim 1, characterized in that: The reference substance is one or more of difurfuryl disulfide, difurfuryl sulfide, ethyl maltol and ethyl vanillin.

3. The quantitative method for exogenous flavors and fragrances in sesame oil according to claim 2, characterized in that: When the reference substance is diffuryl sulfide, the concentrations of the gradient standard solutions of the reference substance are 0.1 μg / mL, 0.2 μg / mL, 0.5 μg / mL, 1 μg / mL, and 2 μg / mL, respectively. 。 4. The quantitative method for exogenous flavors and fragrances in sesame oil according to claim 2, characterized in that: When the reference substance is diffuryl disulfide, the concentrations of the gradient standard solutions of the reference substance are 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, and 10 μg / mL, respectively. 。 5. The quantitative method for exogenous flavors and fragrances in sesame oil according to claim 2, characterized in that: When the reference substance is ethyl maltol, the concentrations of the gradient standard solutions of the reference substance are 0.02 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.2 μg / mL, and 0.5 μg / mL, respectively.

6. The quantitative method for exogenous flavors and fragrances in sesame oil according to claim 2, characterized in that: When the reference substance is ethyl vanillin, the concentrations of the gradient standard solutions of the reference substance are 0.02 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.2 μg / mL, and 0.5 μg / mL, respectively.

7. The quantitative method for exogenous flavors and fragrances in sesame oil according to claim 1, characterized in that: The pretreatment method of the analyte is as follows: weigh the analyte into a centrifuge tube, add methanol, vortex, mix evenly, centrifuge, take the upper clear liquid into a brown volumetric flask, dilute to the mark with methanol, take part of the supernatant, add methanol-saturated n-hexane for extraction, centrifuge, take the lower clear liquid, filter it, and then measure it on the machine.

8. The method for quantifying exogenous flavors and fragrances in sesame oil according to claim 1, characterized in that: Chromatographic conditions: ACQUITY UPLC HSS T3 column; column temperature 30-45°C; injection volume 8-12 μL; flow rate 0.2-0.4 mL / min 。 9. The method for quantifying exogenous flavors and fragrances in sesame oil according to claim 1, characterized in that: The mass spectrometry conditions were: electrospray ion source; positive ion monitoring mode; multiple reaction monitoring; ion spray voltage 4500-5500 V; ion source temperature 500-650 °C 。 10. The method for quantifying exogenous flavors and fragrances in sesame oil according to any one of claims 1 to 6, characterized in that: The method for drawing the standard curve is: using the peak area of ​​the mass chromatogram of the reference solution as the ordinate and the concentration of the reference solution as the abscissa to draw the standard curve and obtain a linear regression equation.

Images