Method for simultaneously determining seven spices and essences in rice

Abstract

The invention discloses a method for simultaneously determining seven spices and essences in rice, which adopts a liquid chromatography-tandem mass spectrometry method, and comprises the following steps: 1) mixing a rice sample with an extracting agent, carrying out vortex, ultrasonic extraction and low-temperature high-speed centrifugation, passing the supernatant through a 0.2 mu m wwPTFE (polytetrafluoroethylene) microfiltration membrane, and loading the supernatant into a machine for determination, the extracting agent is a mixture of water and 0.1% formic acid methanol in a volume ratio of 3: 7; (2) separating by using a Bipheyl chromatographic column under the chromatographic condition, and carrying out gradient elution by using 0.1% formic acid water and 0.1% formic acid methanol as mobile phases; the mass spectrometry is detected in a positive ion scanning multi-reaction monitoring mode, and is quantified by an external standard method. According to the method, seven spices such as 2-acetylpyrazine, 2-acetylpyridine, ethyl maltol, vanillin, methyl vanillin, ethyl vanillin and coumarin which may be maliciously added in the rice can be simultaneously determined, and the seven spices are simultaneously determined by adopting the same detection means, so that the cost is saved, the efficiency is improved, and the method is suitable for large-scale popularization and application. The method has high separation degree and good accuracy and precision.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for simultaneously determining 7 kinds of rice spices, and belongs to the technical field of testing methods for rice spices. BACKGROUND

[0002] Rice is divided into ordinary rice and fragrant rice, and 2-acetyl-1-pyrroline is a characteristic flavor component of fragrant rice. Fragrant rice is deeply loved by consumers because of its popcorn-like aroma, but its price is much higher than that of ordinary rice.

[0003] Fragrant rice essence is a kind of compound spice with similar aroma to natural fragrant rice. The main components of fragrant rice essence include 2-acetylpyridine, 2-acetylpyrazine, vanillin, ethyl vanillin and ethyl maltol, etc. According to the GB 2760-2014 standard for the use of food additives, “food spices and essences shall not be added to rice”. In order to strengthen the supervision of rice production and processing, a simultaneous testing method for commonly used spices and essences in rice needs to be established.

[0004] At present, the detection methods for ethyl maltol, vanillin, methyl vanillin, ethyl vanillin and coumarin in food include liquid chromatography-mass spectrometry / mass spectrometry, liquid chromatography, gas chromatography-mass spectrometry, and gas chromatography. The detection methods for 2-acetylpyrazine and 2-acetylpyridine include only liquid chromatography, gas chromatography and ultraviolet spectrophotometry, and there is no report on the determination of 2-acetylpyrazine and 2-acetylpyridine by liquid chromatography-tandem mass spectrometry. According to the main components of fragrant rice essence reported, the present application first establishes a liquid chromatography-tandem mass spectrometry method for simultaneously determining 2-acetylpyrazine, 2-acetylpyridine, ethyl maltol, vanillin, methyl vanillin, ethyl vanillin and coumarin in rice by optimizing the pretreatment and chromatography-mass spectrometry conditions. SUMMARY

[0005] The present application provides a method for simultaneously determining 7 kinds of spices in rice, which can simultaneously determine 2-acetylpyrazine, 2-acetylpyridine, ethyl maltol, vanillin, methyl vanillin, ethyl vanillin and coumarin in rice, and has high resolution, good accuracy and precision.

[0006] To solve the above technical problems, the technical solution adopted by the present application is as follows:

[0007] A method for simultaneously determining 7 kinds of spices in rice, which adopts liquid chromatography-tandem mass spectrometry, comprising the following steps:

[0008] 1) Mix the rice sample with the extractant, and after vortexing, ultrasonic extraction and low-temperature high-speed centrifugation, the supernatant is filtered through a 0.2μm PTFE microporous membrane and then tested. All percentages mentioned above are volume percentages. The extractant is a mixture of water and 0.1% formic acid methanol in a volume ratio of 3:7.

[0009] 2) Chromatographic conditions used Separation was performed using a Biphenyl column with gradient elution using 0.1% formic acid in water and 0.1% formic acid in methanol as the mobile phase. All percentages mentioned above are volume percentages. Mass spectrometry analysis was performed using positive ion scanning multiple reaction monitoring (+MRM) mode, and quantification was performed using the external standard method.

[0010] The seven flavorings and fragrances mentioned above are 2-acetylpyrazine, 2-acetylpyridine, ethyl maltol, vanillin, methyl vanillin, ethyl vanillin, and coumarin.

[0011] The 0.1% formic acid methanol of this application refers to methanol with 0.1% formic acid added, where % is a volume percentage.

[0012] In step 1), a mixture of water and 0.1% formic acid in a volume ratio of 30:70 is used as the extractant.

[0013] The 0.1% formic acid water in this application refers to a mixture of 0.1% formic acid and 99.9% water, where % is a volume percentage.

[0014] The 0.1% formic acid methanol of this application refers to a mixture of 0.1% formic acid and 99.9% methanol, where % is a volume percentage.

[0015] The gradient elution program in step 2) above is as follows: 0–0.5 min, 25% B, 75% A; 0.5–2.0 min, 25%–60% B, 75%–40% A; 2.0–5.0 min, 60%–90% B, 40%–10% A; 5.0–7.0 min, 90% B, 10% A; 7.0–7.1 min, 25% B, 75% A; 7.1–10.0 min, 25% B, 75% A; the percentages mentioned above are all volume percentages, B is 0.1% formic acid in methanol, and A is 0.1% formic acid in water.

[0016] The mass spectrometry conditions described above are: electrospray ionization (ESI) source, positive ion scanning multiple reaction monitoring (+MRM) mode; electrospray voltage: 5500 V; ion source temperature: 550 °C; spray gas pressure: 4.137 × 10⁻⁶. 5 Pa, auxiliary gas pressure: 4.137 × 10 5 Pa, air curtain pressure: 2.068 × 10 5 Pa; Collision gas: 7 mL / min.

[0017] The mass spectrometry parameters of the above target analytes are as follows:

[0018]

[0019]

[0020] For the above-mentioned 2-acetylpyrazine in the range of 10.0–1000.0 ng / mL, the linear regression equation is y = 1.821 × 10⁻⁶. 3 x-4.330×10 3 The correlation coefficient is 0.9995, where y is the chromatographic peak area and x is the concentration of 2-acetylpyrazine.

[0021] For the above-mentioned 2-acetylpyridine in the range of 1.5–300.0 ng / mL, the linear regression equation is y = 6.415 × 10⁻⁶. 3 x-6.107×10 2 The correlation coefficient is 0.9999, where y is the chromatographic peak area and x is the concentration of 2-acetylpyridine.

[0022] For the above-mentioned ethyl maltol in the range of 0.2–100.0 ng / mL, the linear regression equation is y = 2.317 × 10⁻⁶. 4 x + 9.701 × 10 2 The correlation coefficient is 0.9996, where y is the chromatographic peak area and x is the concentration of ethyl maltol.

[0023] For the vanillin in the range of 1.0–100.0 ng / mL, the linear regression equation is y = 1.076 × 10⁻⁶. 4 x + 5.927 × 10 3 The correlation coefficient is 0.9995, where y is the chromatographic peak area and x is the vanillin concentration.

[0024] For the above-mentioned methyl vanillin in the range of 0.2–100.0 ng / mL, the linear regression equation is y = 3.656 × 10⁻⁶. 4 x + 8.823 × 10 3 The correlation coefficient is 0.9987, where y is the chromatographic peak area and x is the concentration of methyl vanillin.

[0025] For the above-mentioned ethyl vanillin in the range of 0.2–100.0 ng / mL, the linear regression equation is y = 1.461 × 10⁻⁶. 4 x + 1.237 × 10 3 The correlation coefficient is 0.9997, where y is the chromatographic peak area and x is the concentration of ethyl vanillin.

[0026] For the coumarin in the range of 0.2–100.0 ng / mL, the linear regression equation is y = 3.380 × 10⁻⁶. 4x + 1.202 × 10 4 The correlation coefficient is 0.9990, where y is the chromatographic peak area and x is the coumarin concentration.

[0027] The seven fragrances (2-acetylpyrazine, 2-acetylpyridine, ethyl maltol, vanillin, methyl vanillin, ethyl vanillin, and coumarin) exhibited good linearity within a certain concentration range, with correlation coefficients (R) greater than 0.998. The limits of detection (LODs) ranged from 2.0 to 100.0 μg / kg, and the limits of quantitation (LOQs) ranged from 5.0 to 300.0 μg / kg. The recoveries ranged from 79.4% to 113.0%, and the relative standard deviations (n=6) ranged from 2.3% to 10.0%. This method is simple, rapid, accurate, sensitive, and reproducible.

[0028] Any techniques not mentioned in this invention are based on existing technologies.

[0029] This invention provides a method for simultaneously determining seven flavorings in rice. It can simultaneously determine seven flavorings that may be maliciously added to rice, including 2-acetylpyrazine, 2-acetylpyridine, ethyl maltol, vanillin, methyl vanillin, ethyl vanillin, and coumarin. These seven flavorings are simultaneously determined using the same detection method, which saves costs, improves efficiency, and has high separation, accuracy, and precision. Furthermore, it achieves the first determination of 2-acetylpyrazine and 2-acetylpyridine using liquid chromatography-mass spectrometry / mass spectrometry. Attached Figure Description

[0030] Figure 1 Chromatograms of a mixed standard working solution of seven flavorings (+MRM).

[0031] Figure 2 The effect of different flow rates on the response values ​​of seven spices; Detailed Implementation

[0032] To better understand the present invention, the following embodiments further illustrate the content of the present invention, but the content of the present invention is not limited to the following embodiments.

[0033] Example 1

[0034] Materials and reagents

[0035] The purity of 2-acetylpyrazine, 2-acetylpyridine, vanillin, methyl vanillin, ethyl vanillin, and coumarin standards were all greater than 99.0%. Ethyl maltol standard solution (mass concentration of 1000 μg / mL) was obtained from Beijing Tanmo Quality Inspection Standard Material Center. Methanol and acetonitrile (chromatographic grade) were obtained from Fisher Scientific, USA. Formic acid and ammonium formate (chromatographic grade) were obtained from MREDA, USA. 0.2 μm 13 mm wtPTFE microporous filter membrane was obtained from PALL, USA. The test water was ultrapure water. All rice samples used were randomly selected.

[0036] Instruments and equipment

[0037] AB API4000+ HPLC-MS / MS (with ESI ion source), SCIEX Corporation, USA; Talboys heavy-duty digital display vortex mixer, Talboys Corporation, USA; KH20R-Ⅱ high-speed refrigerated centrifuge, Hunan Kaida Scientific Instruments Co., Ltd.; KQ-500DE ultrasonic cleaner, Kunshan Ultrasonic Instruments Co., Ltd.; AL204 0.01% electronic balance, Mettler Toledo Instruments Co., Ltd., Shanghai; WP-UP-YJ-40 ultrapure water system, Sichuan Wotel Water Treatment Equipment Co., Ltd.

[0038] Test methods

[0039] Preparation of standard solutions

[0040] Single-standard stock solution: 1000 μg / mL. Accurately weigh 0.0100 g each of 2-acetylpyrazine, 2-acetylpyridine, vanillin, methyl vanillin, ethyl vanillin, and coumarin standards, dissolve in methanol, and dilute to 10 mL in a volumetric flask. Store frozen in a refrigerator.

[0041] Ethyl maltol single standard solution in methanol: mass concentration of 1000 μg / mL, stored frozen in a refrigerator.

[0042] Mixed standard solution: Accurately pipette appropriate amounts of the above 7 single standard stock solutions, dilute with 70% (volume fraction) methanol solution and make up to 10 mL in a volumetric flask to prepare a mixed standard solution with a mass concentration of 10.0 μg / mL for 2-acetylpyridine, a mass concentration of 3.0 μg / mL for 2-acetylpyridine, and a mass concentration of 1.0 μg / mL for ethyl maltol, vanillin, methyl vanillin, ethyl vanillin and coumarin.

[0043] Series of mixed standard working solutions: The mixed standard solutions were gradually diluted with 70% (volume fraction) methanol solution to prepare 2-acetylpyrazine with mass concentrations of 10.0, 20.0, 50.0, 100.0, 200.0, 500.0, 800.0, and 1000.0 ng / mL, and 2-acetylpyridine with mass concentrations of 1.5, 3.0, 6.0, 15.0, 30.0, 60.0, 150.0, and 240. A series of mixed standard working solutions with concentrations of 0, 300.0 ng / mL, vanillin concentrations of 1.0, 2.0, 5.0, 10.0, 20.0, 50.0, 80.0, and 100.0 ng / mL, and ethyl maltol, methyl vanillin, ethyl vanillin, and coumarin concentrations of 0.2, 0.5, 1.0, 2.0, 5.0, 10.0, 20.0, 50.0, 80.0, and 100.0 ng / mL.

[0044] Sample pretreatment

[0045] Weigh 1.00 g of rice sample into a 50 mL centrifuge tube, add 3 mL of water, vortex for 1 min, add 7 mL of 0.1% (volume fraction) formic acid methanol, vortex for 2 min, extract with ultrasound for 20 min, centrifuge at 10000 r / min at low temperature (4℃) for 5 min, filter the supernatant through a 0.2 μm wwPTFE microporous membrane, and then determine it by liquid chromatography-tandem mass spectrometry.

[0046] Instrument conditions

[0047] Chromatographic conditions: Biphenyl The chromatographic column was 100 mm × 3.0 mm, 2.6 μm. Mobile phase A was 0.1% (v / v) formic acid aqueous solution, and mobile phase B was 0.1% (v / v) formic acid methanol. The gradient elution program was: 0–0.5 min, 25% B; 0.5–2.0 min, 25%–60% B; 2.0–5.0 min, 60%–90% B; 5.0–7.0 min, 90% B; 7.0–7.1 min, 25% B; 7.1–10.0 min, 25% B (all v / v). Column temperature was 40 °C; flow rate was 0.4 mL / min; injection volume was 5 μL.

[0048] Mass spectrometry conditions: Electrospray ionization (ESI) source, positive ion scanning multiple reaction monitoring (+MRM) mode; Electrospray voltage: 5500 V; Ion source temperature: 550 °C; Spray gas pressure: 4.137 × 10⁻⁶ 5 Pa, auxiliary gas pressure: 4.137 × 10 5 Pa, air curtain pressure: 2.068 × 10 5Pa; Collision gas: 7 mL / min. Other mass spectrometry parameters are shown in Table 1, where “*” represents quantitative ions.

[0049] Table 1 Mass Spectrometry Parameters

[0050]

[0051] Optimization of mass spectrometry conditions

[0052] Based on the compound properties of the seven fragrances, a positive ion scanning mode was used, and all seven fragrances were ionized as [M+H]. + The adducted ion was used as the parent ion. The declustering voltage (DP) was adjusted to ensure a high response value for each compound's parent ion. The collision energy (CE) was then adjusted to break up the parent ions. Two to three daughter ions with high response values ​​were selected to form +MRM monitoring ion pairs. The CE and DP values ​​were further optimized to achieve the best response signal for the formed ion pairs. Daughter ions with high response values ​​were selected as quantitative ions, and the rest as qualitative ions. The optimized mass spectrometry parameters for the seven fragrances are shown in Table 1. The multiple reaction monitoring (+MRM) chromatograms of the mixed standard working solutions of the seven fragrances are shown in [Table 1]. Figure 1 .

[0053] Selection of mobile phase

[0054] Based on the characteristics of positive ion scanning, adding formic acid to the mobile phase can improve ionization efficiency, thereby increasing the response value. First, different volume fractions of formic acid were added to the water-acetonitrile mobile phase system, and the effects of different formic acid contents on the chromatographic peaks were compared. The results showed that under a 0.2% (volume fraction) formic acid-water-0.2% (volume fraction) formic acid-acetonitrile mobile phase, all seven fragrances were completely separated, but the 2-acetylpyridine peak was a leading peak, and the ethyl maltol peak was a tailing peak; the peak shapes of the other fragrances were relatively good. Under a 0.1% (volume fraction) formic acid-water-0.1% (volume fraction) formic acid-acetonitrile mobile phase, the leading peak of 2-acetylpyridine and the tailing of the ethyl maltol peak were more obvious, and the peaks showed bifurcation. Under a 0.5% (volume fraction) formic acid-water-acetonitrile mobile phase, the peaks of 2-acetylpyrazine and 2-acetylpyridine overlapped, while the peak shapes of 2-acetylpyridine and ethyl maltol showed no improvement and bifurcation. To improve peak shape, 5 mmol / L ammonium formate was added to a mobile phase of 0.2% (v / v) formic acid-water-0.2% (v / v) formic acid-acetonitrile. The results showed that while the peak shapes of 2-acetylpyridine and ethyl maltol were improved, the response values ​​of all seven fragrances decreased significantly, and the 2-acetylpyrazine peak showed no response. In contrast, a mobile phase of 0.2% (v / v) formic acid-water-0.2% (v / v) formic acid-acetonitrile was chosen.

[0055] Secondly, different volume fractions of formic acid were added to the water-methanol mobile phase system, and the effects of different formic acid contents in the methanol system on the chromatographic peaks were compared. The results showed that with a mobile phase of 0.1% (v / v) formic acid in water and 0.1% (v / v) formic acid in methanol, the peaks of 2-acetylpyridine and vanillin overlapped. Except for 2-acetylpyridine showing a slightly leading peak and ethyl maltol showing a slightly tailing peak, the peak shapes of the other fragrances were good. Changing the mobile phase to 0.2% (v / v) formic acid in water and 0.2% (v / v) formic acid in methanol did not significantly improve the peak shape or resolution. Considering the column lifespan, the concentration of formic acid in the mobile phase should be minimized to achieve the same separation effect; therefore, a mobile phase of 0.1% (v / v) formic acid in water and 0.1% (v / v) formic acid in methanol was selected.

[0056] Seven fragrance compounds were analyzed by injection of mixed standard working solutions. The effects of mobile phases of 0.1% (v / v) formic acid water-0.1% (v / v) formic acid methanol and 0.2% (v / v) formic acid water-0.2% (v / v) formic acid acetonitrile were compared on the response values ​​of the seven fragrance compounds. The results showed that when using 0.1% (v / v) formic acid water-0.1% (v / v) formic acid methanol as the mobile phase, the response values ​​of the seven fragrance compounds were 1.3 to 6.7 times higher than those using 0.2% (v / v) formic acid water-0.2% (v / v) formic acid acetonitrile as the mobile phase (see [link to article]). Figure 2 Although the peaks of 2-acetylpyridine and vanillin overlapped when using a mobile phase of 0.1% (v / v) formic acid water-0.1% (v / v) formic acid methanol, this did not affect the qualitative and quantitative analysis for mass spectrometry. Based on the above analysis, 0.1% (v / v) formic acid water-0.1% (v / v) formic acid methanol was ultimately chosen as the mobile phase.

[0057] Extraction solvent selection

[0058] Rice samples generally have low moisture content (≤15%), and direct addition of extraction solvent results in poor extraction efficiency. Therefore, 3 mL of water (1.00 g of rice sample) is added first, and the sample is vortexed to moisten and disperse it before adding the extraction solvent to improve extraction efficiency. The experiment involved extracting and determining the recovery rate of spiked blank rice samples using 7 mL of methanol and 7 mL of acetonitrile, respectively, to investigate the effects of two extraction solvents: 70% (volume fraction) methanol-water and 70% (volume fraction) acetonitrile-water. The results (see Table 2) show that with 70% methanol-water as the extractant, the recoveries of the seven spices ranged from 91.8% to 109.4%, while with 70% acetonitrile-water as the extractant, the recoveries ranged from 69.2% to 91.0%. The experiment also found that the 2-acetylpyrazine peak in the spiked sample extracted with 70% acetonitrile-water exhibited solvent effects, resulting in peak broadening and bifurcation. Therefore, 70% methanol-water was chosen as the extraction solvent.

[0059] Table 2 Effect of different extraction solutions on recovery rate

[0060]

[0061] Matrix effect evaluation

[0062] A series of mixed standard solutions of seven spices were prepared using matrix sample solution extracted from blank rice. Rice matrix standard solutions at six different concentration points (corresponding to the first six concentration points in the mixed standard working solution series) were injected and analyzed. The measured values ​​were calculated using the solvent standard curve, and the matrix effect (ME) was evaluated as the percentage of the measured value to the theoretical value. When the ME was within 85%–115%, the matrix effect was considered insignificant and its influence could be ignored. The results (see Table 3) showed that the average matrix effect of the seven spices ranged from 92.4% to 99.9%, indicating that accurate quantification could be achieved using the solvent standard curve without the need for correction using the matrix standard curve.

[0063] Table 3 Matrix effect results

[0064]

[0065]

[0066] Calibration curve, limit of detection and limit of quantitation

[0067] The series of mixed standard working solutions were determined under optimized experimental conditions, and external standard-calibration working curves were plotted. The results showed that the seven flavorings exhibited good linearity within a certain mass concentration range, with correlation coefficients all greater than 0.9985. Low-concentration mixed standard solutions were added to blank rice samples to prepare spiked samples (the mixed standard solutions were added to the blank rice samples so that the spiked concentrations of the six flavorings in the spiked samples were the same as the limits of detection and limits of quantitation in Table 4). The signal-to-noise ratio was determined and calculated using the optimized method. The limit of detection (3S / N) was calculated using a signal-to-noise ratio of 3, and the limit of quantitation (10S / N) was calculated using a signal-to-noise ratio of 10. The results are shown in Table 4.

[0068] Table 4 Linear equation, limit of detection, and limit of quantitation

[0069]

[0070]

[0071] Precision and recovery tests

[0072] Spiked blank rice samples at three concentration levels (low, medium, and high), recovery and precision tests were conducted. Each concentration level was measured six times, and the recovery rate and relative standard deviation (RSD) were calculated. The results are shown in Table 5. The results indicate that the recovery rates of the seven spices in rice ranged from 79.4% to 113.0%, and the RSDs of the measured values ​​ranged from 2.3% to 10.0%, demonstrating that the method is accurate, stable, and reliable.

[0073] Table 5. Precision and recovery test results (n=6)

[0074]

[0075] Sample Analysis

[0076] Ten randomly selected rice samples were tested according to the experimental method. The results showed that vanillin was detected in nine of the rice samples, with contents ranging from 44.3 to 116.9 μg / kg. 2-acetylpyrazine, 2-acetylpyridine, ethyl maltol, methyl vanillin, ethyl vanillin, and coumarin were not detected in any of the ten rice samples. Multiple studies have shown that vanillin can be detected in most types of rice, with a detection rate of 97.5%. Furthermore, research by Chen Renxi et al. found that "rice may contain endogenous vanillin, and the vanillin content is closely related to rice variety, storage time, and storage environment; the vanillin content varies significantly in pre-packaged rice." Due to limitations in current detection technology, it is impossible to distinguish between endogenous and exogenous vanillin. Therefore, the detection of vanillin in rice alone cannot determine whether it is rice with added exogenous vanillin. Since the commonly used fragrant rice flavoring is a compound flavoring, other flavoring components must be detected in the rice at the same time to determine whether it is flavored rice. This highlights the importance of multiple flavoring detection methods.

[0077] The above-described method establishes an analytical technique for the simultaneous determination of seven flavorings in rice using liquid chromatography-tandem mass spectrometry (LC-MS / MS). This method uses water as a dispersant, adds 0.1% (v / v) formic acid in methanol to form a 70% (v / v) methanol-water extraction solvent, and performs vortexing, ultrasonic extraction, and low-temperature high-speed centrifugation. The extract is then filtered through a microporous membrane and analyzed by LC-MS / MS. This method is the first to achieve the simultaneous determination of 2-acetylpyrazine, 2-acetylpyridine, ethyl maltol, vanillin, methyl vanillin, ethyl vanillin, and coumarin in rice, and features simple operation, rapidity, accuracy, sensitivity, and good reproducibility.

Claims

1. A method for simultaneously determining seven flavorings in rice, characterized in that: The determination was performed using liquid chromatography-tandem mass spectrometry, including the following steps: 1) Mix the rice sample with the extractant, which is a mixture of water and 0.1% formic acid methanol in a volume ratio of 3:

7. After vortexing, ultrasonic extraction and low-temperature high-speed centrifugation, the supernatant is filtered through a 0.2μm wwPTFE microporous membrane and then analyzed. All percentages mentioned above are volume percentages. 2) Chromatographic conditions used Separation was performed using a Biphenyl column with gradient elution using 0.1% formic acid in water and 0.1% formic acid in methanol as the mobile phase (all percentages are volume percentages). Mass spectrometry analysis was performed using positive ion scanning multiple reaction monitoring mode, and quantification was performed using the external standard method.

2. The method for simultaneously determining seven flavorings in rice as described in claim 1, characterized in that: The seven flavorings are 2-acetylpyrazine, 2-acetylpyridine, ethyl maltol, vanillin, methyl vanillin, ethyl vanillin, and coumarin.

3. The method for simultaneously determining seven flavorings in rice as described in claim 1 or 2, characterized in that: In step 2), the gradient elution program is as follows: 0–0.5 min, 25% B, 75% A; 0.5–2.0 min, 25%–60% B, 75%–40% A; 2.0~5.0min, 60%~90%B, 40%-10%A; 5.0~7.0min, 90%B, 10%A; 7.0–7.1 min, 25% B, 75% A; 7.1–10.0 min, 25% B, 75% A; the aforementioned % are all volume percentages, B is 0.1% formic acid methanol, and A is 0.1% formic acid water.

4. The method for simultaneously determining seven flavorings in rice as described in claim 1 or 2, characterized in that: In step 2), the mass spectrometry conditions are as follows: electrospray ionization source, positive ion scanning multiple reaction monitoring mode; electrospray voltage: 5500V; ion source temperature: 550℃; spray gas pressure: 4.137×10⁻⁶. 5 Pa, auxiliary gas pressure: 4.137 × 10 5 Pa, air curtain pressure: 2.068 × 10 5 Pa; Collision gas: 7 mL / min.

5. The method for simultaneously determining seven flavorings in rice as described in claim 1 or 2, characterized in that: The mass spectrometry parameters of the target analyte are as follows: 。 6. The method for simultaneously determining seven flavorings in rice as described in claim 1 or 2, characterized in that: For 2-acetylpyrazine in the range of 10.0–1000.0 ng / mL, the linear regression equation was y = 1.821 × 10⁻⁶. 3 x-4.330×10 3 The correlation coefficient is 0.9995, where y is the chromatographic peak area and x is the concentration of 2-acetylpyrazine.

7. The method for simultaneously determining seven flavorings in rice as described in claim 1 or 2, characterized in that: For 2-acetylpyridine in the range of 1.5–300.0 ng / mL, the linear regression equation was y = 6.415 × 10⁻⁶. 3 x-6.107×10 2 The correlation coefficient is 0.9999, where y is the chromatographic peak area and x is the concentration of 2-acetylpyridine.

8. The method for simultaneously determining seven flavorings in rice as described in claim 1 or 2, characterized in that: For ethyl maltol in the range of 0.2–100.0 ng / mL, the linear regression equation is y = 2.317 × 10⁻⁶. 4 x + 9.701 × 10 2 The correlation coefficient is 0.9996, where y is the chromatographic peak area and x is the concentration of ethyl maltol.

9. The method for simultaneously determining seven flavorings in rice as described in claim 1 or 2, characterized in that: For vanillin in the range of 1.0–100.0 ng / mL, the linear regression equation is y = 1.076 × 10⁻⁶. 4 x + 5.927 × 10 3 The correlation coefficient is 0.9995, where y is the chromatographic peak area and x is the vanillin concentration; For methyl vanillin in the range of 0.2–100.0 ng / mL, the linear regression equation is y = 3.656 × 10⁻⁶. 4 x + 8.823 × 10 3 The correlation coefficient is 0.9987, where y is the chromatographic peak area and x is the concentration of methyl vanillin.

10. The method for simultaneously determining seven flavorings in rice as described in claim 1 or 2, characterized in that: For ethyl vanillin in the range of 0.2–100.0 ng / mL, the linear regression equation is y = 1.461 × 10⁻⁶. 4 x + 1.237 × 10 3 The correlation coefficient was 0.9997, where y is the chromatographic peak area and x is the concentration of ethyl vanillin. For coumarin in the range of 0.2–100.0 ng / mL, the linear regression equation is y = 3.380 × 10⁻⁶. 4 x + 1.202 × 10 4 The correlation coefficient is 0.9990, where y is the chromatographic peak area and x is the coumarin concentration.

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