Method for determining sesame content based on labeled peptide combined with liquid chromatography-mass spectrometry technology

Abstract

The invention relates to a method for measuring sesame content based on labeled peptide combined with a liquid chromatography-mass spectrometry technology. The method comprises the following steps: selecting wheat flour, cocoa powder and mixed nut powder as food matrixes, respectively adding sesame powder with different masses, mixing to obtain a sample, extracting protein by adopting a buffer solution with specific conditions, performing reduction and alkylation reaction on the protein, and performing trypsin digestion to obtain a to-be-detected solution; based on targeted proteomics, the enzymolysis peptide fragment is analyzed through high performance liquid chromatography-triple quadrupole tandem mass spectrometry, the linear relation between the ratio of the peak area of the sesame quantitative peptide fragment to the peak area of the internal standard peptide fragment and the sesame content is explored, and sesame quantitative curves under three matrixes are established. The content of sesame in food can be detected according to the quantitative curve, and the quantitative limits of sesame under three matrixes of wheat, cocoa and nut powder are respectively 1mg / kg, 1.5 mg / kg and 2mg / kg. According to the method, the labeled peptide fragment is introduced, the loss caused by instability in the analysis process of a mass spectrometer is corrected by using the peak area ratio of the sample enzymolysis peptide fragment to the labeled peptide fragment, the sesame content in the food can be sensitively and accurately detected, the precision is good, and a certain technical support is provided for food allergen label identification.

Description

Technical Field

[0001] The present invention relates to a mass spectrometry method for determining sesame content in food. Specifically, it relates to a method for accurately determining sesame content in three common food matrices by establishing an external standard curve using isotope-labeled peptides as internal standards based on liquid chromatography-mass spectrometry. Background Art

[0002] Sesame (Sesamumindicum L.), a member of the genus Sesame in the family Pedulaceae, is widely cultivated in Henan, Hubei, Anhui, Jiangxi, and other regions of my country. According to the National Bureau of Statistics, my country's sesame planting area reached nearly 300,000 hectares in 2023, with an annual output of approximately 500,000 tons. Sesame contains a rich composition of substances, including 44%-57% lipids, 18%-25% protein, 13-14% carbohydrates, 2.46% minerals, and 0.088% polyphenols. When heated, sesame emits a strong aroma and is widely used in the food industry. For example, sesame is often added to the production of baked goods such as bread and biscuits to enhance flavor. Seasoning sauces or sesame oil are often made with variegated sesame seeds such as yellow sesame seeds. Foods such as sesame paste and sesame powder are generally made by grinding black sesame seeds. Studies have shown that sesame has anti-aging, antioxidant, and immune-enhancing properties, and therefore is also used as a functional ingredient in the development of health foods or new food products.

[0003] At the 2021 Food and Agriculture Organization of the United Nations and World Health Organization (FAO / WHO) Expert Consultation on Food Allergen Risk Assessment, sesame is proposed to be listed as one of the eight major categories of food allergens ("Big 8"). The prevalence of sesame allergies has been reported in many countries, including the United Kingdom, Israel, and the United States. In Israel, sesame allergy is the second most common food allergy in children, accounting for 43% of childhood allergy cases. In Lebanon, the prevalence of sesame allergies in infants, children, and adults is 3.79%, 2.65%, and 1.91%, respectively. In recent years, the prevalence of sesame allergies has continued to rise. Many countries, including the European Union, Canada, and the United States, have successively introduced allergen regulations on sesame labeling, requiring sesame allergen information to be indicated on food labels. However, during food production and transportation, sesame may be unintentionally mixed into unlabeled foods, threatening the health of allergic people.

[0004] Sesame can be detected at both the nucleic acid and protein levels. Detection methods at the nucleic acid level target sesame DNA, and mainly include molecular biology methods such as polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP); immunological detection methods at the protein level rely on the specific binding of antigens and antibodies to identify target proteins, and mainly include enzyme-linked immunosorbent assay (ELISA); protein-level detection methods also include liquid chromatography-mass spectrometry (LC-MS / MS). Due to the complexity of food matrices and the fact that processing may affect protein structure, the application of molecular biology and immunology methods in sesame detection in food is limited. By identifying the characteristic peptides of the target species, LC-MS / MS can sensitively and accurately determine the sesame content in food.

[0005] Based on LC-MS / MS, the external standard method can be used to determine the sesame content in food. Previous studies have reported establishing a quantitative method for five food allergens, including sesame, in bread and biscuit matrices by determining an external standard curve. However, during the establishment of the external standard curve, interference from the food matrix and instrument instability can affect the accuracy of the quantitative results. Therefore, it is necessary to establish an external standard curve based on different food matrices. To minimize interference caused by instrument instability, isotope-labeled peptides are added as internal standards. External standard curves are established based on the peak area ratio of the sample protein hydrolyzed peptides to the internal standard and the sesame content, thereby improving detection accuracy. Summary of the Invention

[0006] The purpose of the present invention is to use isotope-labeled peptides as internal standards to correct for unstable losses in mass spectrometry analysis and establish quantitative external calibration curves for sesame suitable for three common food matrices, thereby providing a method for determining the sesame content in food, realizing trace detection of sesame in food, and ensuring the accuracy and stability of the test results.

[0007] To achieve the above object, the present invention is implemented through the following technical solutions:

[0008] A method for determining sesame content based on labeled peptide combined with liquid chromatography-mass spectrometry technology, comprising the following steps:

[0009] (1) Sample preparation: Sesame powder, wheat flour, cocoa powder, and mixed nut powder were defatted and dried, and wheat flour, cocoa powder, and nut powder were added with different amounts of sesame powder and mixed evenly;

[0010] (2) Protein extraction: Weigh the sample and add protein extraction buffer solution at a ratio of 1 g:10 mL. Oscillate for 20 min and then ultrasonicate in a water bath for 20 min to assist protein extraction to obtain a crude protein extract.

[0011] (3) Enzymatic digestion: Trypsin was added at a mass ratio of 1:50 and digested in a 37°C water bath overnight to obtain hydrolyzed peptides;

[0012] (4) Mass spectrometry detection: Determine the liquid chromatography and mass spectrometry conditions, establish a scheduled MRM method based on the quantitative peptide ion pair information, and analyze the hydrolyzed peptides;

[0013] (5) Establishment of quantitative curve: A certain concentration of isotope-labeled peptide was added to the hydrolyzed peptide of the sample as an internal standard. The ratio of the peak area of ​​the quantitative peptide to that of the internal standard was calculated. The linear relationship between the peak area ratio and the sesame content was analyzed to establish a standard curve.

[0014] (6) Methodological evaluation: The linear regression coefficient R 2 The linearity was evaluated; the mass concentrations at which the signal-to-noise ratio (S / N) of the quantitative peptide ion current extraction peak was 3 and 10 were used as the detection limit (LOD) and quantification limit (LOQ); the recovery rates of samples with mass concentrations of 10LOQ, 50LOQ, and 100LOQ were used to evaluate the accuracy; the reproducibility was evaluated by calculating the intra-day and inter-day precision by repeating the test six times within a day and five times between days.

[0015] The detection method of the present invention is further preferably:

[0016] (1) Sample preparation: Grind the freeze-dried sample in liquid nitrogen. Add hexane at a ratio of 1 g:5 mL, stir at 25°C for 20 min, centrifuge (4000 rpm, 10 min), discard the supernatant, add hexane, repeat this process three times, and place the precipitate in a fume hood to dry overnight.

[0017] (2) Protein Extraction: Weigh 1 g of defatted sample powder and add 10 mL of protein extraction buffer (50 mM Tris-HCl + 7 M urea + 2 M thiourea + 4% CHAPS, pH 8.5). Extract at room temperature with shaking for 20 min, followed by water bath sonication for 20 min. Centrifuge (12,000 rpm, 20 min), and filter the supernatant through a 0.22 μm filter to obtain a standard stock solution. The sesame standard stock solution was diluted with three blank matrix solutions to a gradient concentration range of 1 to 1000 mg / kg.

[0018] (3) Enzyme digestion: Pipette about 200 μL of protein solution into a centrifuge tube. Add 10 μL of 120 mM dithiothreitol (DTT) solution and react in a 37°C water bath for 1 hour. Add 10 μL of 600 mM iodoacetamide (IAA) solution and react in the dark for 15 minutes. After the reaction is completed, transfer the liquid to an ultrafiltration membrane (10 kDa), centrifuge (12000 rpm, 20 minutes), add 100 μL of 50 mM ammonium bicarbonate (ABC) solution three times and centrifuge (12000 rpm, 15 minutes). Discard the waste liquid at the bottom of the tube and wash with mass spectrometry water. Add 4 μL of trypsin to the crude protein extract, vortex and incubate in a 37°C water bath overnight. Centrifuge the ultrafiltration tube (12000 rpm, 20 minutes), add 100 μL of 25 mM ABC solution three times and centrifuge (12000 rpm, 15 minutes). The enzymatic peptide solution is vacuum dried. Add 100 μL of mobile phase A, vortex centrifuge (10,000 rpm, 10 min), and store the liquid at 4°C for a short term.

[0019] (4) Mass spectrometry: The enzymatic peptide solution was analyzed using high performance liquid chromatography-triple quadrupole tandem mass spectrometry. The required liquid chromatography and mass spectrometry conditions were as follows:

[0020] ①Chromatographic conditions

[0021] Column: Waters Xbridge Peptide BEH C 18 , 4.6mm×150mm, 3.5μm,

[0022] Mobile phase: A: 98% water-2% acetonitrile-0.1% formic acid; B: 98% acetonitrile-2% water-0.1% formic acid;

[0023] Flow rate: 0.4 mL / min; column temperature: 40°C; injection volume: 10 μL;

[0024] The gradient elution program is run according to the following table:

[0025] Table 1 Liquid chromatography gradient elution program

[0026]

[0027]

[0028] ②Mass spectrometry conditions

[0029] Mode: ESI+positive ion, scheduled MRM; detection window: 120s, scan time: 3s;

[0030] Curtain gas: 35psi; Nebulizer gas: 65psi; Collision gas: Medium; Auxiliary gas: 50psi;

[0031] Ionization voltage: 4500 V; ion source temperature: 500°C.

[0032] (5) Establishment of Scheduled MRM Method: Based on the peptide quantification, we commissioned a company to synthesize isotopic internal standard peptides. These two peptides were input into Skyline software to obtain ion pair information. A Scheduled MRM method was established based on HPLC-triple quadrupole tandem mass spectrometry. The quantitative peptide information is as follows:

[0033] Table 2 Sesame quantitative peptide information

[0034]

[0035] (6) Establishment of quantitative curve: Add 30 fmo / μL of isotope-labeled peptide solution to the reconstituted enzymatic solution at a volume ratio of 1:1 and vortex to mix. Transfer a certain volume of the mixed liquid to a liquid phase vial for mass spectrometry analysis. Calculate the peak area ratio of the hydrolyzed quantitative peptides and the isotope-labeled peptides of sesame samples with different contents, analyze the linear relationship between the peak area ratio and the sesame content, and establish a standard curve.

[0036] (7) Methodological evaluation: The linear regression coefficient R 2 The linearity was evaluated; the mass concentrations at which the signal-to-noise ratio (S / N) of the quantitative peptide ion current extraction peak was 3 and 10 were used as the detection limit (LOD) and quantification limit (LOQ); the recovery rates of samples with mass concentrations of 10LOQ, 50LOQ, and 100LOQ were used to evaluate the accuracy; the reproducibility was evaluated by calculating the intra-day and inter-day precision by repeating the test six times within a day and five times between days.

[0037] (8) The present invention further relates to the use of isotope-labeled peptides as internal standards to establish external standard curves of sesame hydrolyzed peptides and internal standard peak area ratios and sesame content, so as to determine the sesame content in three common food matrices.

[0038] Beneficial effects of the present invention:

[0039] (1) The present invention selected three common food matrices, namely wheat, cocoa and nuts, and mixed them with sesame to prepare samples. External standard curves of sesame under the three food matrices were established to reduce the interference of matrix effects and make the sesame content determination results more accurate.

[0040] (2) The present invention optimizes the external standard method, using a chemically synthesized isotope-labeled peptide as an internal standard. This is mixed with the sample hydrolyzed peptide solution for mass spectrometry analysis, and a standard curve is established based on the peak area ratio of the two peptides and the sesame content. The presence of the internal standard reduces the impact of instability in the mass spectrometer during the analysis process, facilitates highly sensitive detection of sesame in food, and ensures the accuracy and stability of the test results. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is the standard curve for sesame content determination in wheat matrix;

[0042] Figure 2 This is the standard curve for sesame content determination in cocoa matrix;

[0043] Figure 3 This is the standard curve for sesame content determination in nut matrix. DETAILED DESCRIPTION

[0044] The present invention will be further described by way of examples, but the present invention is not limited to the following examples.

[0045] Example 1:

[0046] (1) Sample preparation: 100 g of sesame, wheat, cocoa, and five common nuts (cashew, almond, walnut, hazelnut, and almond, 1:1 ratio) were weighed, washed, air-dried, and freeze-dried. Four samples of sesame, wheat, cocoa, and mixed nuts were obtained by grinding each sample in a mortar under liquid nitrogen.

[0047] (2) Sample defatting: Weigh 50 g of each of the four samples and place them in a beaker. Add an appropriate amount of n-hexane at a ratio of 1:5 between sample and n-hexane. Stir magnetically at 25°C for 20 min and then centrifuge (4000 rpm, 10 min). Discard the supernatant and add n-hexane. Repeat this process three times. After the final defatting, place the precipitate in a fume hood and air-dry overnight to allow the n-hexane to evaporate completely. Place the four defatted sample powders in a -80°C refrigerator.

[0048] (3) Protein extraction: Weigh 1 g of defatted sample powder from four separate aliquots and add 10 mL of protein extraction buffer (50 mM Tris-HCl + 7 M urea + 2 M thiourea + 4% CHAPS, pH 8.5) to each aliquot. Vortex mix thoroughly, extract by oscillation for 20 min, then sonicate for 20 min. Centrifuge (12,000 rpm, 20 min), aspirate the supernatant, filter through a 0.22 μm filter, and use the four aliquots as standard stock solutions.

[0049] (4) Preparation of standard solutions: 1 mL of sesame standard stock solution was taken and mixed with wheat, cocoa, and nut standard stock solutions at a ratio of 1:1 to obtain three sesame-wheat mixed stock solutions, sesame-cocoa mixed stock solutions, and sesame-nut mixed stock solutions, each with a sesame content of 0.5 g / g. Appropriate amounts of wheat, cocoa, and nut standard stock solutions were taken as blank matrix solutions to dilute the three mixed stock solutions to obtain a series of gradient concentration standard solutions with sesame contents of 1, 2, 5, 10, 20, 50, 100, 200, 500, and 1000 mg / kg.

[0050] (5) Protein reduction: Take 200 μL of the series of gradient concentration standard solutions, add 10 μL of 120 mM dithiothreitol (DTT) solution, vortex mix, and react in a 37°C water bath for 1 h.

[0051] (6) Protein alkylation: Add 10 μL of 600 mM iodoacetamide (IAA) solution to the above solution, vortex mix, and incubate in the dark for 15 minutes to perform protein alkylation reaction.

[0052] (7) Trypsin digestion: All liquids were transferred to a 10kDa ultrafiltration membrane, centrifuged (12000rpm, 20min), 100μL of 50mMABC solution was added to the ultrafiltration tube and centrifuged again (12000rpm, 15min), and the operation was repeated three times. The liquid at the bottom of the ultrafiltration tube was discarded, and an appropriate amount of mass spectrometry water was added for washing and drying. 100μL of 50mMABC solution was added again, and 4μL of trypsin (1μg / μL) dissolved in acetic acid was added, mixed, and digested for 10h in a 37℃ water bath. After the digestion was completed, the ultrafiltration tube was centrifuged (12000rpm, 20min), 100μL of 25mMABC solution was added, and centrifuged (12000rpm, 15min). The above operation was repeated three times so that all the enzyme-cleaved peptide solutions were placed at the bottom of the tube. The ultrafiltration tube was placed in a vacuum rotary evaporator, and after the solution was dried, 100-200μL of mass spectrometry water was added, and the rotary drying was repeated 3 times. Finally, 100 μL of mobile phase A consisting of 98% water, 2% acetonitrile and 0.1% formic acid was added, vortexed and mixed, centrifuged (10,000 rpm, 10 min), and 80 μL of the liquid was transferred to a liquid phase vial and stored at 4°C.

[0053] (8) Open source software analysis: Use the open source software Skyline to obtain peptide ion information. Open Skyline, input the quantitative peptide and isotope-labeled peptide sequences (NVIQPR, NVIQPR*), determine the ion pairs detected in the sample, and export the ion pair information of the two peptides.

[0054] (9) Establishing a Scheduled MRM method: Establish a Scheduled MRM method for mass spectrometry analysis based on the derived ion pair information, place the liquid phase vial to be tested on the sample tray, and set the sample collection method.

[0055] (10) High performance liquid chromatography-triple quadrupole tandem mass spectrometry detection: The instrument parameters were set according to the following conditions, and the samples to be tested were analyzed. Each sample was tested three times.

[0056] ①Chromatographic conditions

[0057] Column: Waters Xbridge Peptide BEH C 18 , 4.6mm×150mm, 3.5μm,

[0058] Mobile phase: A: 98% water-2% acetonitrile-0.1% formic acid; B: 98% acetonitrile-2% water-0.1% formic acid;

[0059] Flow rate: 0.4 mL / min; column temperature: 40°C; injection volume: 10 μL;

[0060] The gradient elution program is run according to the following table:

[0061] Table 1 Liquid chromatography gradient elution program

[0062]

[0063] ②Mass spectrometry conditions

[0064] Mode: ESI+positive ion, scheduled MRM; detection window: 120s, scan time: 3s;

[0065] Curtain gas: 35psi; Nebulizer gas: 65psi; Collision gas: Medium; Auxiliary gas: 50psi;

[0066] Ionization voltage: 4500 V; ion source temperature: 500°C.

[0067] (11) Quantitative curve drawing: The mass spectrometry data were imported into Peakview software, and the ion information of the quantitative peptide and the isotope-labeled peptide was input to obtain the peak areas corresponding to the two peptides. Using Graphad software, the sesame content and the peak area ratio of the two peptides were input to analyze the linear relationship and establish the standard curve for sesame content determination under three matrices ( Figure 1-3 ).

[0068] (12) Methodological evaluation: Performance Requirements for Standard Methods for Detection and Quantification of Food Allergens (AOAC 2016.002) stipulates that sensitivity, accuracy and precision should be evaluated.

[0069] ① Sensitivity:

[0070] According to the linear regression coefficient R of the standard curve 2 Linearity was assessed, with a value greater than 0.99 considered good. The ion current peaks of the quantified peptides were extracted, and the signal-to-noise ratio (S / N) was calculated. The sample concentrations at S / N = 3 and 10 were determined as the limits of detection and quantification, respectively. The limits of detection for sesame in wheat, cocoa, and nut matrices were 0.5, 1, and 1.5 mg / kg, respectively, and the limits of quantification were 1, 1.5, and 2 mg / kg, respectively.

[0071] ②Accuracy:

[0072] Based on the quantification limits of sesame in the three matrices, samples at three concentration levels (10LOQ, 50LOQ, and 100LOQ) were prepared. After mass spectrometry analysis, the peak areas were calculated using PeakView software and substituted into the linear regression equation of the standard curve to calculate the sesame content. The recovery rate was calculated by comparing the two values ​​(Table 3). The recovery rate ranged from 75% to 100%, with good accuracy.

[0073] Table 3 Sample recovery results

[0074] 10LOQ 50LOQ 100LOQ Wheat substrate 80.12％ 86.03％ 89.23％ Cocoa base 78.98％ 82.87％ 87.68％ Nut Matrix 78.76％ 82.85％ 84.27％

[0075] ③Precision:

[0076] A sample of a certain concentration was prepared and tested six times a day for five consecutive days. The intra-day and inter-day relative standard deviations were calculated (Table 4). The relative standard deviations were less than 10%, indicating good precision.

[0077] Table 4 Sample precision results

[0078]

[0079]

[0080] The above description is merely one embodiment of the present invention and is not intended to limit the present invention in any other manner. Any person skilled in the art may utilize the above-disclosed technical content to modify or modify the present invention into equivalent embodiments with equivalent variations. However, any simple modifications, equivalent variations, and improvements to the above embodiment that do not depart from the technical content of the present invention and are based on the technical essence of the present invention shall remain within the scope of protection of the present invention.

Claims

1. A method for determining sesame content based on labeled peptide combined with liquid chromatography-mass spectrometry, characterized in that: The following steps are involved: (1) Sample preparation: Sesame powder, wheat flour, cocoa powder, and mixed nut powder were defatted and dried, and different amounts of sesame powder were added to the three matrices and evenly mixed to prepare the samples; (2) Protein extraction: Buffer solution was added to the sample at a ratio of 1 g:10 mL, and the sample was shaken for 20 min and then ultrasonicated in a water bath for 20 min to assist in protein extraction; (3) Enzyme digestion: Determine the concentration of the crude protein extract, add trypsin at a mass ratio of 1:50, and digest in a 37°C water bath overnight; (4) Mass spectrometry: Determine the liquid chromatography and triple quadrupole mass spectrometry conditions, establish a scheduled MRM method, and analyze the hydrolyzed peptides; (5) Establishment of quantitative curve: Isotope-labeled peptides were added to the hydrolyzed peptide solution as internal standards, the peak area ratio of sesame quantitative peptides and internal standard peptides was calculated, and the linear relationship between the peak area ratio and sesame content was analyzed to establish a standard curve; (6) Methodological evaluation: Linearity was determined based on the fitting of the quantitative standard curve and the R 2 The limit of detection (LOD) and limit of quantification (LOQ) were determined based on the peak signal-to-noise ratio (S / N) of 3 and 10 for the quantitative peptide ion current extraction. The accuracy was evaluated based on the recoveries of samples at 10LOQ, 50LOQ, and 100LOQ concentrations. The reproducibility was evaluated by calculating the intra- and inter-day precision using 6 repeated tests within a day and 5 repeated tests between days.

2. The method according to claim 1, wherein the sample powders in step (1) are freeze-dried sesame, wheat, cocoa and five common nuts (cashew, walnut, almond, hazelnut and almond, 1:1), ground and defatted with n-hexane, and placed in a fume hood to dry overnight.

3. The method according to claim 1, wherein the protein extraction buffer solution in step (2) is 50 mM Tris-HCl (pH 8.5) containing 7 M urea, 2 M thiourea and 4% CHAPS.

4. The method according to claim 1, wherein the protein is subjected to reduction and alkylation reactions before step (3). Approximately 200 μg of protein is added to 10 μL of 120 mM dithiothreitol (DTT) solution and reacted in a 37°C water bath for 1 hour. Then, 10 μL of 600 mM iodoacetamide (IAA) solution is added and reacted in the dark for 15 minutes to complete the protein alkylation reaction.

5. The method according to claim 1, wherein the conditions for high performance liquid chromatography-triple quadrupole tandem mass spectrometry in scheduled MRM mode in step (4) are as follows: (1) Chromatographic conditions Column: Waters Xbridge Peptide BEH C 18 , 4.6mm×150mm, 3.5μm, Mobile phase: A: 98% water-2% acetonitrile-0.1% formic acid; B: 98% acetonitrile-2% water-0.1% formic acid; Flow rate: 0.4 mL / min; column temperature: 40°C; injection volume: 10 μL; The gradient elution program is run according to the following table: Table 1 Liquid chromatography gradient elution program (2) Mass spectrometry conditions Mode: ESI+positive ion, scheduled MRM; detection window: 120s, scan time: 3s; Curtain gas: 35psi; Atomizing gas: 65psi; Collision gas: Medium; Auxiliary gas 50psi; Ionization voltage: 4500 V; ion source temperature: 500°C.

6. According to the method of claim 1, the ion pairs, retention times, optimized declustering voltages and collision energies of the peptides required for establishing the MRM method in step (4) are shown in Table 5: Table 2 Sesame quantitative peptide information 7. The method according to claim 1, wherein in step (5), the enzymatic peptide solution is vacuum dried, 50 μL of mobile phase A solution is added for redissolution, 50 μL of isotope-labeled peptide at a concentration of 30 fmol / μL is added as an internal standard, vortexed and mixed, and 80 μL is taken for mass spectrometry detection.

8. The method according to claim 1, wherein in step (5), the quantitative standard curves of sesame in three matrices of wheat, cocoa and mixed nut powder (linear range 1-1000 mg / kg) are as follows: (1) Wheat matrix: y = 0.004607x + 0.06111, linear regression coefficient R 2 =0.9933; (2) Cocoa matrix: y = 0.004150x + 0.05694, linear regression coefficient R 2 =0.9961; (3) Nut matrix: y = 0.003858x + 0.02680, linear regression coefficient R 2 =0.9987.

9. The method according to claim 1, wherein in step (6), the detection limits for sesame in wheat, cocoa, and mixed nut powder matrices are 0.5 mg / kg, 1 mg / kg, and 1 mg / kg, respectively; and the limits of quantification for sesame are 1 mg / kg, 1.5 mg / kg, and 2 mg / kg, respectively. The recovery range is 75%-105%, and the relative standard deviation (RSD) is less than 10%.

10. Use of the method according to any one of claims 1 to 9 in determining the sesame content in food.

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