Aroma rice identification method

Abstract

The application discloses a kind of essence rice identification method, its steps are: taking the intact rice without being pulverized, adding acetonitrile rapid vortex oscillation extraction, take supernatant and filter, carry out HPLC-MS / MS analysis, whether the sample contains vanillin, methyl vanillin, ethyl vanillin, ethyl maltol, 2-acetylpyridine, 2-propionylpyridine, 2-acetyl-1-pyrroline and 5,6,7,8-tetrahydroquinoxaline at least one, if contains, determine sample as essence rice.The application solves the technical problem that endogenous nature flavor component and artificial essence are effectively distinguished in essence rice identification process, can be used for the rapid qualitative identification of essence rice, method is sensitive, high accuracy.

Description

Technical Field

[0001] The invention belongs to the technical field of detection of adulteration in food, and particularly relates to a method for identifying flavored rice. Background Art

[0002] As living standards improve, specialty high-quality rice products like "Thai Fragrant Rice" and "Wuchang Rice" are becoming increasingly popular. Driven by profit, some businesses, to reduce costs, treat ordinary rice with flavorings, turning it into a pricey specialty rice. Some unscrupulous vendors even add flavorings and spices to moldy reserve grain and aged grain to remove the musty smell. This type of flavored rice is called flavored rice.

[0003] The illegal addition of flavorings and spices during rice production and processing poses a risk to food safety. This not only damages the reputation of branded rice and the legitimate rights and interests of legitimate rice processors, but also disrupts market order. Long-term consumption of flavored rice can pose a potential health threat to humans.

[0004] It is important to note that vanillin and 2-acetyl-1-pyrroline are natural flavor compounds in rice. The presence of endogenous vanillin and 2-acetyl-1-pyrroline can have a certain impact on the identification of flavored rice. If they are not distinguished, it may lead to misidentification. Therefore, when testing adulterated flavored rice, which ingredients to use as markers for flavored rice, and how to effectively extract and ensure recovery while avoiding interference caused by endogenous substances are the difficulties in establishing the entire identification method. Summary of the Invention

[0005] Based on the problems existing in the above-mentioned prior art, the object of the present invention is to provide a method for identifying flavored rice to solve the technical difficulties of identifying rice mixed with flavors and avoiding endogenous interference, and to achieve rapid qualitative identification of flavored rice.

[0006] In order to solve the technical problem, the present invention adopts the following technical solution:

[0007] The invention discloses a method for identifying flavored rice. The method is characterized by: weighing whole rice that has not been crushed, adding acetonitrile for rapid vortex oscillation extraction, collecting and filtering the supernatant, and then performing HPLC-MS / MS analysis to detect whether the sample contains at least one of vanillin, methyl vanillin, ethyl vanillin, ethyl maltol, 2-acetylpyridine, 2-propionylpyridine, 2-acetyl-1-pyrroline and 5,6,7,8-tetrahydroquinoxaline. If the sample contains at least one of vanillin, methyl vanillin, ethyl vanillin, ethyl maltol, 2-acetylpyridine, 2-propionylpyridine, 2-acetyl-1-pyrroline and 5,6,7,8-tetrahydroquinoxaline, and if so, determining that the sample is flavored rice.

[0008] Furthermore, before extraction, the rice was sieved through a 12-14 mesh screen to remove broken rice and debris, and only full, intact rice was collected as the sample. The rice should not be crushed to avoid the release of endogenous vanillin.

[0009] Furthermore, the extraction conditions are as follows: 5-10 g of sample is weighed, placed in a stoppered centrifuge tube, 10-20 mL of acetonitrile is added, and vortexed for 30-60 seconds. Using acetonitrile as the extraction reagent for vortexing can avoid endogenous interference caused by the extraction of endogenous vanillin.

[0010] Furthermore, the filtration is through a 0.22 μm organic phase filter membrane.

[0011] Furthermore, the conditions for HPLC-MS / MS analysis were set as follows:

[0012] (1) Liquid chromatography conditions: C 18 Chromatographic column; mobile phase: phase A is 0.1% formic acid in water, phase B is methanol; flow rate: 0.2-0.5 mL / min; column temperature: 30-40°C; injection volume: 1-20 μL; gradient elution;

[0013] (2) Mass spectrometry conditions: electrospray ionization source, positive ion scan mode, multiple reaction monitoring mode.

[0014] Compared with the prior art, the beneficial effects of the present invention are embodied in:

[0015] The present invention solves the technical problems of which ingredients to use as markers for flavored rice when detecting adulterated flavored rice, as well as how to avoid interference from endogenous substances while effectively extracting and ensuring recovery. The method of the present invention consumes less reagents and has a fast analysis speed, effectively saving sample pretreatment time and costs. The detection limit for vanillin, methyl vanillin, ethyl vanillin, ethyl maltol, and 2-acetyl-1-pyrroline can reach 3μg / kg, and the quantification limit can reach 10μg / kg. The detection limit for 2-acetylpyridine, 2-propionylpyridine, and 5,6,7,8-tetrahydroquinoxaline can reach 15μg / kg, and the quantification limit can reach 50μg / kg. The method of the present invention can be used for the rapid qualitative identification of flavored rice, and is characterized by being rapid, simple, sensitive, with little interference from endogenous substances, and high accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The figure is an extracted ion chromatogram of a standard solution of vanillin, methyl vanillin, ethyl vanillin, ethyl maltol, 2-acetylpyridine, 2-propionylpyridine, 2-acetyl-1-pyrroline, and 5,6,7,8-tetrahydroquinoxaline obtained by liquid chromatography triple quadrupole tandem mass spectrometry;

[0017] Figure 2 The figure is a liquid chromatography triple quadrupole tandem mass spectrometry extracted ion chromatogram obtained by detecting a blank rice sample using the method of the present invention;

[0018] Figure 3 The figure is a liquid chromatography triple quadrupole tandem mass spectrometry extracted ion chromatogram obtained by testing a flavor rice sample using the method of the present invention;

[0019] Figure 4 This is the extracted ion chromatogram of the blank rice sample obtained by extraction and detection using the BJS 201705 method, using liquid chromatography triple quadrupole tandem mass spectrometry. DETAILED DESCRIPTION

[0020] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process. However, the protection scope of the present invention is not limited to the following embodiment.

[0021] Example 1

[0022] The method for identifying flavored rice provided in this embodiment comprises the following specific steps:

[0023] Step 1: Sieve

[0024] Pass the uncrushed rice through a 12-mesh sieve, discard the broken rice and debris, and collect the full and complete rice as the sample.

[0025] Step 2: Extraction and determination

[0026] Weigh 5 g of sample and place it in a stoppered centrifuge tube. Add 10 mL of acetonitrile and extract by vortexing for 30 seconds. Pass the supernatant through a 0.22 μm organic phase filter membrane for HPLC-MS / MS determination of whether the sample contains at least one of vanillin, methyl vanillin, ethyl vanillin, ethyl maltol, 2-acetylpyridine, 2-propionylpyridine, 2-acetyl-1-pyrroline, and 5,6,7,8-tetrahydroquinoxaline. If so, the sample is determined to be flavored rice.

[0027] The specific measurement conditions of HPLC-MS / MS are as follows:

[0028] (1) Liquid chromatography conditions: Agilent ZORBAX Eclipse Plus C 18 Chromatographic column (2.1 mm × 50 mm, 1.8 μm); mobile phase: phase A is 0.1% formic acid aqueous solution, phase B is methanol; flow rate: 0.3 mL / min; column temperature: 35°C; injection volume: 10 μL.

[0029] (2) Gradient elution program, according to volume fraction: 0 min, 25% B; 0.25 min, 25% B; 1.0 min, 75% B; 1.5 min, 75% B; 2.5 min, 95% B; 3.0 min, 95% B; 3.01 min, 25% B; 7 min, 25% B.

[0030] (3) Mass spectrometry conditions: Electrospray ionization source, positive ion scan mode, sheath gas temperature 320°C, sheath gas flow rate 12 L / min, nebulizer gas pressure 40 psi, drying gas temperature 320°C, drying gas flow rate 6 L / min, capillary voltage positive mode 3000 V, multiple reaction monitoring mode. The qualitative ion pairs, quantitative ion pairs, fragmentor voltage (FP), and collision gas voltage (CE) in the multiple reaction monitoring mode are shown in Table 1, where a represents the quantitative ion pair and b represents the auxiliary qualitative ion pair.

[0031] Table 1 Multiple reaction monitoring parameters of target compounds

[0032]

[0033] To verify the sensitivity and accuracy of the above method, rice without rice flavoring was tested, and no vanillin, methyl vanillin, ethyl vanillin, ethyl maltol, 2-acetylpyridine, 2-propionylpyridine, 2-acetyl-1-pyrroline and 5,6,7,8-tetrahydroquinoxaline were detected. Vanillin, methyl vanillin, ethyl vanillin, ethyl maltol, and 2-acetyl-1-pyrroline at three levels of 0.01 mg / kg, 0.02 mg / kg, and 0.10 mg / kg, and 2-acetylpyridine, 2-propionylpyridine, and 5,6,7,8-tetrahydroquinoxaline at three levels of 0.05 mg / kg, 0.10 mg / kg, and 0.50 mg / kg were added to blank samples for recovery determination. Each level was repeated 6 times. Extraction, purification, and detection were performed using the method of this example. The results are shown in Table 2. The recovery rate of each marker was higher than 60%, and the indoor precision was less than 15%, which met the requirements of the detection methodology.

[0034] Table 2 Average recovery and precision of rice samples (n=6)

[0035]

[0036] Example 2

[0037] The method for identifying flavored rice provided in this embodiment comprises the following specific steps:

[0038] Step 1: Sieve

[0039] Pass the uncrushed rice through a 14-mesh sieve, discard the broken rice and debris, and collect the full and complete rice as the sample.

[0040] Step 2: Extraction and determination

[0041] Weigh 10 g of the sample and place it in a stoppered centrifuge tube. Add 20 mL of acetonitrile and extract by vortexing for 60 seconds. Pass the supernatant through a 0.22 μm organic phase filter membrane for HPLC-MS / MS determination of whether the sample contains at least one of vanillin, methyl vanillin, ethyl vanillin, ethyl maltol, 2-acetylpyridine, 2-propionylpyridine, 2-acetyl-1-pyrroline, and 5,6,7,8-tetrahydroquinoxaline. If so, the sample is determined to be flavored rice.

[0042] The specific measurement conditions of HPLC-MS / MS are as follows:

[0043] (1) Liquid chromatography conditions: Thermo Fisher Scientific Hypersil GOLD C 18 Column (2.1 mm × 100 mm, 1.9 μm); mobile phase: phase A: 0.1% formic acid in water, phase B: methanol; flow rate: 0.4 mL / min; column temperature: 40°C; injection volume: 5 μL;

[0044] (2) Gradient elution program, according to volume fraction: 0 min, 25% B; 0.25 min, 25% B; 1.0 min, 75% B; 1.5 min, 75% B; 2.5 min, 95% B; 3.0 min, 95% B; 3.01 min, 25% B; 7 min, 25% B;

[0045] (3) Mass spectrometry conditions: Electrospray ionization source, positive ion scan mode, dryer gas temperature 300°C, drying gas flow rate 10 L / min, nebulizer gas pressure 40 psi, capillary voltage 3000 V in positive mode, multiple reaction monitoring mode. The qualitative ion pairs, quantitative ion pairs, fragmentor voltage (FP), and collision gas voltage (CE) in the multiple reaction monitoring mode are shown in Table 3, where a represents the quantitative ion pair and b represents the auxiliary qualitative ion pair.

[0046] Table 3 Multiple reaction monitoring parameters of target compounds

[0047]

[0048] To verify the sensitivity and accuracy of the above method, rice without rice flavoring was tested, and no vanillin, methyl vanillin, ethyl vanillin, ethyl maltol, 2-acetylpyridine, 2-propionylpyridine, 2-acetyl-1-pyrroline and 5,6,7,8-tetrahydroquinoxaline were detected. Vanillin, methyl vanillin, ethyl vanillin, ethyl maltol, and 2-acetyl-1-pyrroline at three levels of 0.01 mg / kg, 0.02 mg / kg, and 0.10 mg / kg, and 2-acetylpyridine, 2-propionylpyridine, and 5,6,7,8-tetrahydroquinoxaline at three levels of 0.05 mg / kg, 0.10 mg / kg, and 0.50 mg / kg were added to blank samples for recovery determination. Each level was repeated 6 times. Extraction, purification, and detection were performed using the method of this example. The results are shown in Table 4. The recovery rate of each marker was higher than 60%, and the indoor precision was less than 15%, meeting the requirements of the detection methodology.

[0049] Table 4 Average recovery and precision of rice samples (n=6)

[0050]

[0051]

[0052] Example 3

[0053] The method for identifying flavored rice provided in this embodiment comprises the following specific steps:

[0054] Step 1: Sieve

[0055] Pass the uncrushed rice through a 13-mesh sieve, discard the broken rice and debris, and collect the full and complete rice as the sample.

[0056] Step 2: Extraction

[0057] Weigh 5 g of sample and place it in a stoppered centrifuge tube. Add 20 mL of acetonitrile and extract by vortexing for 45 seconds. Pass the supernatant through a 0.22 μm organic phase filter membrane for HPLC-MS / MS determination of whether the sample contains at least one of vanillin, methyl vanillin, ethyl vanillin, ethyl maltol, 2-acetylpyridine, 2-propionylpyridine, 2-acetyl-1-pyrroline, and 5,6,7,8-tetrahydroquinoxaline. If so, the sample is determined to be flavored rice.

[0058] The specific measurement conditions of HPLC-MS / MS are as follows:

[0059] (1) Liquid chromatography conditions: ZORBAX SB-C 18Column (2.1 mm × 100 mm, 3.5 μm); mobile phase: phase A: 0.1% formic acid in water, phase B: methanol; flow rate: 0.4 mL / min; column temperature: 40°C; injection volume: 5 μL;

[0060] (2) Gradient elution program, according to volume fraction: 0 min, 25% B; 0.5 min, 25% B; 1.0 min, 75% B; 2.0 min, 75% B; 3.0 min, 95% B; 5.0 min, 95% B; 5.01 min, 25% B; 10 min, 25% B;

[0061] (3) Mass spectrometry conditions: Electrospray ionization source, positive ion scan mode, sheath gas temperature 320°C, sheath gas flow rate 12 L / min, nebulizer gas pressure 40 psi, drying gas temperature 320°C, drying gas flow rate 6 L / min, capillary voltage positive mode 3000 V, dynamic multiple reaction monitoring mode. The qualitative and quantitative ion pairs, fragmentor voltage (FP), and collision gas voltage (CE) in dynamic multiple reaction monitoring mode are shown in Table 5.

[0062] Table 5 Multiple reaction monitoring parameters of target compounds

[0063]

[0064]

[0065] Rice without rice flavoring was tested according to the method of this example, and none of vanillin, methyl vanillin, ethyl vanillin, ethyl maltol, 2-acetylpyridine, 2-propionylpyridine, 2-acetyl-1-pyrroline, and 5,6,7,8-tetrahydroquinoxaline were detected. A portion of flavored rice was tested according to the method of this example, and vanillin, ethyl vanillin, and 5,6,7,8-tetrahydroquinoxaline were detected.

[0066] Comparative Example 1

[0067] Referring to the sample preparation and extraction method for grain milling products (rice, etc.) in BJS 201705 "Determination of Vanillin, Methyl Vanillin and Ethyl Vanillin in Foods", the rice was crushed and mixed until it could pass through a 425μm standard mesh sieve. 1g of sample was weighed (accurate to 0.01g) and placed in a 50mL polypropylene centrifuge tube. 3mL of water was added and vortexed for 30s. 7mL of acetonitrile was added and vortexed for 30s. Ultrasonic treatment was performed for 25min. After centrifugation at 10000r / min for 5min, the supernatant was filtered through a 0.22μm filter membrane and analyzed by HPLC-MS / MS. Endogenous vanillin was detected in rice without added flavoring. The main technical issues are as follows:

[0068] 1. Sample preparation: Endogenous vanillin and 2-acetyl-1-pyrroline are released after rice is crushed.

[0069] 2. Extraction: The extraction reagent contains water and ultrasonic extraction causes the endogenous vanillin and 2-acetyl-1-pyrroline in rice to be extracted, causing endogenous interference.

[0070] 3. Detection: The detection limit of vanillin, methyl vanillin and ethyl vanillin in the BJS 201705 method is 30.0 μg / kg, and the quantification limit is 100.0 μg / kg, which are higher than the technology of the present invention. It cannot effectively identify flavored rice with generally low flavor content.

[0071] 4. BJS 201705 cannot detect 2-acetylpyridine, 2-propionylpyridine, 2-acetyl-1-pyrroline, and 5,6,7,8-tetrahydroquinoxaline.

[0072] Comparative Example 2

[0073] Compared with the method in GB 5009.284-2021 "National Food Safety Standard - Determination of Vanillin, Methyl Vanillin, Ethyl Vanillin and Coumarin in Foods", the differences are as follows:

[0074] 1. The scope of application of GB 5009.284-2021 standard does not include grains such as rice.

[0075] 2. Solid samples in GB 5009.284-2021 need to be crushed evenly.

[0076] 3. The sample pretreatment process of GB 5009.284-2021 also requires the addition of water and ultrasonic treatment, which will extract endogenous vanillin and 2-acetyl-1-pyrroline, causing interference.

[0077] 4. GB 5009.284-2021 cannot detect 2-acetylpyridine, 2-propionylpyridine, 2-acetyl-1-pyrroline, and 5,6,7,8-tetrahydroquinoxaline.

[0078] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for identifying flavored rice, characterized in that: Sieve whole, uncrushed rice to discard broken rice and debris, and collect plump, whole rice as a sample; weigh 5-10 g of the sample, place it in a stoppered centrifuge tube, add 10-20 mL of acetonitrile, and vortex extract for 30-60 seconds. Collect the supernatant, filter it, and perform HPLC-MS / MS analysis to detect whether the sample contains at least one of vanillin and 2-acetyl-1-pyrroline, or whether the sample contains at least one of vanillin and 2-acetyl-1-pyrroline and at least one of methyl vanillin, ethyl vanillin, ethyl maltol, 2-acetylpyridine, 2-propionylpyridine, and 5,6,7,8-tetrahydroquinoxaline. If the sample contains these compounds, the sample is determined to be flavored rice; the HPLC-MS / MS analysis conditions are set as follows: (1) Liquid chromatography conditions: C 18 Chromatographic column; mobile phase: phase A is 0.1% formic acid in water, phase B is methanol; flow rate: 0.2-0.5 mL / min; column temperature: 30-40°C; injection volume: 1-20 μL; gradient elution; (2) Mass spectrometry conditions: electrospray ionization source, positive ion scan mode, multiple reaction monitoring mode.

2. a kind of flavor rice identification method according to claim 1, is characterized in that: The sieving is through a 12-14 mesh sieve.

3. A method for identifying flavored rice according to claim 1, characterized in that, The filtration was performed through a 0.22 μm organic phase filter membrane.

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