Method for qualitatively or quantitatively detecting urea in grains or distiller's yeast
By using activated carbon, zinc acetate, and potassium ferrocyanide precipitation combined with 9-hydroxyton derivatization reagent, the problems of low extraction efficiency and matrix interference in urea detection in brewing raw materials were solved, achieving high sensitivity and high accuracy in qualitative or quantitative detection.
Patent Information
- Application Number
- CN202511034188.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-25
- Publication Date
- 2025-10-17
AI Technical Summary
Existing urea detection methods have problems with low extraction efficiency, insufficient detection sensitivity and severe matrix interference in brewing raw materials, resulting in inaccurate test results.
Interfering substances in the sample were removed by activated carbon adsorption, zinc acetate and potassium ferrocyanide precipitation. 9-hydroxyton was used as a derivatizing reagent to react with urea to generate a fluorescent derivative, which was then qualitatively or quantitatively detected by high performance liquid chromatography.
It significantly improves the sensitivity and accuracy of detection, simplifies the operation process, reduces costs, and enables accurate and rapid qualitative or quantitative detection of urea in grains or yeast.
Smart Images
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Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of food detection, and particularly relates to a method for qualitatively or quantitatively detecting urea in grain or wine starter. BACKGROUND
[0002] Brewing raw materials (such as sorghum, wheat, etc.) have complex components, containing a large amount of organic substances such as protein, starch, cellulose, and fat. These components are prone to matrix effect during the extraction process, which interferes with the accurate detection of urea. Matrix effect can cause deviation in the detection results, reducing the sensitivity and accuracy of the detection. In addition, most existing urea detection methods are for liquid samples or standard samples, lacking effective extraction and detection technology for solid samples (such as grain or wine starter). The extraction process of solid samples is complex, the content of urea in grain or wine starter is low, and it is easily interfered by other components, resulting in inaccurate detection results. Therefore, the existing urea detection method has problems such as low extraction efficiency, insufficient detection sensitivity, and serious matrix interference when applied to brewing raw materials. Therefore, it is of great significance to develop a rapid quantitative detection method for urea in grain or wine starter to perfect the food safety supervision system and prevent the risk of urea abuse. SUMMARY
[0003] Therefore, one of the purposes of the present application is to provide a pretreatment method for detecting urea in grain or wine starter, which can effectively remove interfering substances, has high extraction efficiency, and can significantly improve the accuracy and sensitivity of subsequent detection.
[0004] The second purpose of the present application is to provide a method for qualitatively or quantitatively detecting urea in grain or wine starter, which is simple to operate, low in cost, can effectively avoid the interference of complex components in grain or wine starter, and can realize accurate and rapid qualitative or quantitative detection of urea in grain or wine starter, filling the technical gap in urea detection in grain or wine starter.
[0005] In order to achieve the above-mentioned purposes of the application, the present application provides the following technical solutions:
[0006] The present application provides a pretreatment method for detecting urea in grain or wine starter, comprising urea extraction and derivatization steps: the urea extraction step is to mix the sample, activated carbon, zinc acetate solution and potassium ferrocyanide solution, ultrasonic, filter with medium-speed filter paper, discard 1-2 mL of the initial filtrate, and collect the subsequent filtrate as the sample solution; the derivatization step is to mix the sample solution, 9-hydroxythrene solution and hydrochloric acid solution, avoid light, place, filter to obtain the test solution; the dosage ratio of the sample, activated carbon, zinc acetate and potassium ferrocyanide is 5g:0.2g:0.0025mol:0.000625mol; the sample is grain or wine starter.
[0007] Preferably, the ultrasonic time is 20-30 min, the ultrasonic temperature is 25-30 DEG C, and the ultrasonic frequency is 40 KHz.
[0008] Preferably, the pore size of the medium-speed filter paper is 10 mu m.
[0009] Preferably, the sample solution, 9-hydroxythioxene and hydrochloric acid are used in a ratio of 400 mu L:0.000012 mol:0.00015 mol.
[0010] Preferably, the light-shielded storage time is 30-40 min.
[0011] The application further provides a method for qualitatively detecting urea in grain or wine starter, which is based on the pretreatment method and further comprises the following steps: detecting the to-be-detected liquid by using high performance liquid chromatography; treating urea standard by using the derivatization step in the pretreatment method, and then detecting the urea standard by using high performance liquid chromatography to obtain a urea standard spectrum and retention time; comparing the detection result of the to-be-detected liquid with the urea standard spectrum and retention time; if the spectrum and retention time are consistent, it is indicated that the grain or wine starter contains urea; if at least one of the spectrum and retention time is inconsistent, it is indicated that the grain or wine starter does not contain urea.
[0012] The application further provides a method for quantitatively detecting urea in grain or wine starter, which is based on the pretreatment method and further comprises the following steps: detecting the to-be-detected liquid by using high performance liquid chromatography; preparing a series of urea standard working solutions with a mass concentration of 0.5-10 mg / L, and then treating each series of urea standard working solutions by using the derivatization step in the pretreatment method, and then detecting the urea standard working solutions by using high performance liquid chromatography, so as to draw a standard curve of urea with the peak area of urea as the ordinate and the mass concentration as the abscissa; and substituting the detection result of the to-be-detected liquid into the standard curve to calculate the content of urea in the grain or wine starter.
[0013] Preferably, the stationary phase of the liquid chromatography column of the high performance liquid chromatography is C18-A; the mobile phase A is acetonitrile, the mobile phase B is a 0.02 mol / L sodium acetate solution with a pH of 7.2, and the gradient elution conditions are shown in Table 1.
[0014] Table 1 Gradient elution conditions
[0015]
[0016]
[0017] Preferably, the standard curve is Y=2x10 -5 -0.6010, wherein Y is the peak area of urea, and x is the mass concentration of urea.
[0018] Preferably, the grain includes brewing grain, the brewing grain includes sorghum and / or wheat, and the starter includes Daqu.
[0019] Advantages of the present application:
[0020] The pre-treatment method provided by the present application removes interfering substances in the sample through the steps of activated carbon adsorption, zinc acetate and potassium ferrocyanide precipitation, reduces the matrix effect, and further uses 9-hydroxythymol as a derivatization reagent to react with urea to generate a fluorescent derivative, which has high extraction efficiency and can improve the detection sensitivity and accuracy. Under the condition of keeping other pre-treatment conditions consistent, the urea content of a certain grain sample measured by the extraction method of the present application is 2.9 mg / L, which is 20.8% higher than the content of 2.4 mg / L measured by the aqueous solution extraction method reported in the literature. The pre-treatment method provided by the present application significantly improves the recovery rate of the target analyte by optimizing the extraction parameters, which confirms that the pre-treatment method of the present application has significant technical advantages in urea detection.
[0021] The method for qualitatively or quantitatively detecting urea in grain or starter provided by the present application has high sensitivity and accuracy, is simple and low in cost, can effectively avoid the interference of complex components in grain or starter, realizes accurate and rapid qualitative or quantitative detection of urea in grain or starter, fills the technical gap in urea detection in grain or starter, and provides a scientific basis for accurate and rapid quantitative detection of urea in brewing raw materials. BRIEF DESCRIPTION OF DRAWINGS
[0022] Figure 1 The chromatogram of the urea standard working solution is shown in the following figure;
[0023] Figure 2 The influence of different activated carbon addition amounts on the urea content is shown in the following figure, wherein A represents Comparative Example 3, B represents Example 5, and C represents Comparative Example 4;
[0024] Figure 3 The urea detection chromatogram of highland 1 in the sample of Example 7 is shown in the following figure. DETAILED DESCRIPTION
[0025] The present application provides a pre-treatment method for detecting urea in grain or starter, which comprises urea extraction and derivatization steps: the urea extraction step is to mix the sample, activated carbon, zinc acetate solution and potassium ferrocyanide solution, ultrasonic, filter with medium-speed filter paper, discard 1-2 mL of the initial filtrate, and collect the subsequent filtrate as the sample solution; the derivatization step is to mix the sample solution, 9-hydroxythymol solution and hydrochloric acid solution, place in the dark, and filter to obtain the test solution; the dosage ratio of the sample, activated carbon, zinc acetate and potassium ferrocyanide is 5g:0.2g:0.0025mol:0.000625mol; and the sample is grain or starter.
[0026] In the present application, the grain sample preferably comprises brewing grain, the brewing grain preferably comprises sorghum and / or wheat, and the starter preferably comprises Daqu. In the present application, the grain or starter sample is preferably pulverized before being mixed with the activated carbon zinc acetate solution and the potassium ferrocyanide solution. The present application is not particularly limited to the specific manner of pulverization, and a conventional pulverization manner in the art can be used. In the present application, the pulverized sample is preferably passed through a 60-mesh sieve. In some embodiments of the present application, the concentration of the zinc acetate solution is 1 mol / L, and the concentration of the potassium ferrocyanide solution is 0.25 mol / L. The present application can remove interfering substances in the sample, reduce matrix effects, and improve the accuracy of detection by the steps of activated carbon adsorption, zinc acetate and potassium ferrocyanide precipitation.
[0027] In the present application, the ultrasonic time is preferably 20-30 min, more preferably 22-28 min, and further preferably 24-26 min; the ultrasonic temperature is preferably 25-30℃, more preferably 26-29℃, and further preferably 27-28℃; and the ultrasonic frequency is preferably 40 KHz. In the present application, the pore size of the medium-speed filter paper is preferably 10 μm. In some embodiments of the present application, the medium-speed filter paper is filtered at normal pressure, and the filtration speed of the medium-speed filter paper is 6-9 mL / min.
[0028] In some embodiments of the present application, the concentration of the 9-hydroxythion solution is 0.02 mol / L, and the concentration of the hydrochloric acid solution is 1.5 mol / L. In some embodiments of the present application, the volume ratio of the sample solution, the 9-hydroxythion solution and the hydrochloric acid solution is 400 μL:600 μL:100 μL. In the present application, the usage ratio of the sample solution, the 9-hydroxythion and the hydrochloric acid is preferably 400 μL:0.000012 mol:0.00015 mol. In the present application, the light-avoiding placement time is preferably 30-40 min, more preferably 32-38 min, and further preferably 34-36 min. The present application uses 9-hydroxythion solution as a derivatization reagent to react with urea to generate a fluorescent derivative, which can improve the detection sensitivity. After light-avoiding placement, the sample solution is filtered to obtain a test solution. In some embodiments of the present application, the filtration is performed by filter membrane filtration, and the pore size of the filter membrane is 0.22 μm.
[0029] The application further provides a method for qualitatively detecting urea in grains or wine leaven, based on the pretreatment method, further comprising the following steps: detecting the to-be-detected liquid by using high performance liquid chromatography; treating the urea standard by using the derivatization step in the pretreatment method, and then detecting the urea standard by using high performance liquid chromatography to obtain a urea standard spectrum and retention time; comparing the detection result of the to-be-detected liquid with the urea standard spectrum and retention time, if the spectrum and retention time are consistent, it indicates that the grains or wine leaven contains urea, if at least one of the spectrum and retention time is inconsistent, it indicates that the grains or wine leaven does not contain urea.
[0030] In the application, the stationary phase of the liquid chromatography column of the high performance liquid chromatography is preferably C18-A, the column size of the liquid chromatography column is preferably long x inner diameter = 250 mm x 4.6 mm, and the packing particle size of the liquid chromatography column is preferably 5 μm. In the application, the mobile phase A of the high performance liquid chromatography is preferably acetonitrile, the mobile phase B is preferably a 0.02 mol / L sodium acetate solution with a pH of 7.2, and the gradient elution condition is preferably as shown in Table 1; the column temperature of the high performance liquid chromatography is preferably 35°C, the excitation wavelength is preferably 213 nm, the emission wavelength is preferably 308 nm, and the injection amount is preferably 40 μL.
[0031] The application further provides a method for quantitatively detecting urea in grains or wine leaven, based on the pretreatment method, further comprising the following steps: detecting the to-be-detected liquid by using high performance liquid chromatography; preparing a series of urea standard working solutions with a mass concentration of 0.5-10 mg / L, and then treating each series of urea standard working solutions by using the derivatization step in the pretreatment method, and then detecting by using high performance liquid chromatography, to draw a standard curve of urea by taking the peak area of urea as the ordinate and the mass concentration as the abscissa; substituting the detection result of the to-be-detected liquid (the detection result refers to the peak area of the to-be-detected liquid) into the standard curve to calculate the content of urea in the grains or wine leaven.
[0032] In the method for quantitatively detecting urea in grains or wine leaven, the specific conditions of the high performance liquid chromatography are as shown above, which will not be repeated here. In the application, the standard curve is preferably Y = 2 x 10 -5 x-0.6010, wherein Y is the peak area of urea, and x is the mass concentration of urea.
[0033] The technical solutions provided by the application will be described in detail below in combination with the embodiments, but they should not be understood as limitations to the protection scope of the application.
[0034] In the following examples, the conventional methods are used unless otherwise specified.
[0035] The materials, reagents and the like used in the following examples, unless otherwise specified, can be obtained commercially.
[0036] In the following examples, unless otherwise specified, only analytical pure reagents are used, and the water is the first grade water specified in GB / T 6682. In the following examples, the preparation of some reagents is shown as follows:
[0037] Mobile phase B: sodium acetate solution: accurately weigh 2.72 g of sodium acetate trihydrate, dissolve in deionized water, adjust the pH value to 7.2 with 1% acetic acid, and dilute to 1 L. After ultrasonic filtration, it is ready for use.
[0038] Urea standard stock solution (1000 mg / L): accurately weigh 0.05 g of urea, dissolve in anhydrous ethanol and dilute to 50 mL. Store at -4℃ and the effective period is one month.
[0039] Urea standard working solution (50 mg / L): accurately pipette 0.5 mL of urea standard stock solution and dilute to 10 mL with 10 mL of deionized water.
[0040] 9-hydroxyxanthene solution (0.02 mol / L): accurately weigh 0.198 g of 9-hydroxyxanthene, ultrasonically dissolve in n-propanol, dilute to 50 mL, and store in the dark.
[0041] Hydrochloric acid solution (1.5 mol / L): dilute 6.2 mL of concentrated hydrochloric acid with water to 50 mL.
[0042] Zinc acetate solution: weigh 22 g of zinc acetate dihydrate, dissolve in water, add 3 mL of glacial acetic acid, and dilute to 100 mL.
[0043] Potassium ferrocyanide solution: weigh 10.60 g of potassium ferrocyanide trihydrate, dissolve in water, and dilute to 100 mL. Prepare fresh every time.
[0044] The liquid chromatograph with a fluorescence detector is used in the following examples.
[0045] Example 1
[0046] A pretreatment method for detecting urea in grain, the steps are as follows:
[0047] (1) Urea extraction: place 5 g of ground grain sample in a 25 mL volumetric flask, add 0.2 g of activated carbon, shake well with water, then add 2.5 mL of 1 mol / L zinc acetate solution and 2.5 mL of 0.25 mol / L potassium ferrocyanide solution, shake well, dilute to 25 mL with water, ultrasonicate at 30℃ and 40 KHz for 30 min, filter with a medium-speed filter paper with a pore size of 10 μm, discard the first 2 mL of filtrate, and collect the subsequent filtrate as the sample solution;
[0048] (2) Derivation: 400 μL of sample solution was taken in a centrifuge tube, 600 μL of 0.02 mol / L 9-hydroxythioxanthene solution was added, then 100 μL of 1.5 mol / L hydrochloric acid solution was added, mixed, placed in the dark to avoid light for 30 min, filtered with a 0.22 μm filter membrane, and the resulting solution was the test solution.
[0049] Example 2
[0050] A pretreatment method for detecting urea in grain, the steps are as follows:
[0051] (1) Urea extraction: 5 g of ground grain sample was placed in a 25 mL volumetric flask, 0.2 g of activated carbon was added, shaken with water and placed, then 2.5 mL of 1 mol / L zinc acetate solution and 2.5 mL of 0.25 mol / L potassium ferrocyanide solution were added, shaken with water to 25 mL, 25℃, 40KHz ultrasonic for 20 min, filtered with medium speed filter paper with a pore size of 10 μm, 2 mL of the initial filtrate was discarded, and the subsequent filtrate was collected as the sample solution;
[0052] (2) Derivation: 400 μL of sample solution was taken in a centrifuge tube, 600 μL of 0.02 mol / L 9-hydroxythioxanthene solution was added, then 100 μL of 1.5 mol / L hydrochloric acid solution was added, mixed, placed in the dark to avoid light for 30 min, filtered with a 0.22 μm filter membrane, and the resulting solution was the test solution.
[0053] Example 3
[0054] A pretreatment method for detecting urea in wine starter, the steps are as follows:
[0055] (1) Urea extraction: 5 g of ground wine starter sample was placed in a 25 mL volumetric flask, 0.2 g of activated carbon was added, shaken with water and placed, then 2.5 mL of 1 mol / L zinc acetate solution and 2.5 mL of 0.25 mol / L potassium ferrocyanide solution were added, shaken with water to 25 mL, 28℃, 40KHz ultrasonic for 26 min, filtered with medium speed filter paper with a pore size of 10 μm, 2 mL of the initial filtrate was discarded, and the subsequent filtrate was collected as the sample solution;
[0056] (2) Derivation: 400 μL of sample solution was taken in a centrifuge tube, 600 μL of 0.02 mol / L 9-hydroxythioxanthene solution was added, then 100 μL of 1.5 mol / L hydrochloric acid solution was added, mixed, placed in the dark to avoid light for 30 min, filtered with a 0.22 μm filter membrane, and the resulting solution was the test solution.
[0057] Comparative Example 1
[0058] The difference from Example 1 is that the amount of activated carbon is 0.15 g, and the rest is the same as Example 1.
[0059] Comparative Example 2
[0060] The difference from Example 1 is that the amount of activated carbon is 0.25 g, and the rest is the same as Example 1.
[0061] Example 4
[0062] A method for qualitatively detecting urea in grain, the steps are as follows:
[0063] (1) The grain sample is subjected to the pretreatment method of Example 1 to obtain a test solution;
[0064] (2) The test solution is detected by high performance liquid chromatography, the stationary phase of the liquid chromatography column of the high performance liquid chromatography is C18-A, the column size of the liquid chromatography column is 250 mm x 4.6 mm, the packing particle size of the liquid chromatography column is 5 μm, the mobile phase A of the high performance liquid chromatography is acetonitrile, the mobile phase B is 0.02 mol / L and pH 7.2 sodium acetate solution, and the gradient elution conditions are shown in Table 1:
[0065] Table 1 Gradient elution conditions
[0066] Time (min) Sodium acetate solution (%) Acetonitrile (%) Flow rate (mL / min) 0.00 80 20 0.45 0.06 80 20 0.45 12.06 50 50 0.45 12.80 50 50 0.45 14.60 0 100 0.45 22.60 0 100 0.8 23.80 80 20 0.8 24.50 80 20 0.8 30.00 80 20 0.45 31.00 80 20 0.45
[0067] The column temperature of the high performance liquid chromatography is 35°C, the excitation wavelength is 213 nm, the emission wavelength is 308 nm, and the injection amount is 40 μL.
[0068] (3) The urea standard is treated by the derivatization step in the pretreatment method of Example 1, and then detected by high performance liquid chromatography, the detection method of the high performance liquid chromatography is the same as step (2), and the urea standard spectrum and retention time are obtained;
[0069] (4) The detection results of the test solution of step (1) are compared with the urea standard spectrum and retention time of step (3), if the spectrum and retention time are consistent, it indicates that the grain contains urea, if at least one of the spectrum and retention time is inconsistent, it indicates that the grain does not contain urea.
[0070] Example 5
[0071] A method for quantitatively detecting urea in grain or distiller's yeast, the steps are as follows:
[0072] (1) The grain or distiller's yeast sample is subjected to the pretreatment method of Example 1 to obtain a test solution;
[0073] (2) The test solution is detected by high performance liquid chromatography, the detection method of the high performance liquid chromatography is the same as step (2) of Example 4.
[0074] (3) dilute the urea standard solution with deionized water to prepare urea standard working solutions with mass concentrations of 0.5 mg / L, 1 mg / L, 2 mg / L, 5 mg / L and 10 mg / L, and then treat each urea standard working solution with the derivatization step in the pretreatment method described in Example 1, and then perform high performance liquid chromatography detection, the detection method being the same as step (2) in Example 4, and plot a standard curve of urea with the peak area of urea as the ordinate and the mass concentration as the abscissa;
[0075] (4) substitute the peak area of the detection result of the sample solution in step (1) into the standard curve in step (3) to calculate the content of urea in the grain or distiller's yeast.
[0076] The chromatogram of the urea standard working solution is shown in Figure 1 The linear range, standard curve, detection limit and lower limit of quantification of the method of this example are shown in Table 2.
[0077] Table 2 Linear range, standard curve, detection limit and lower limit of quantification of the quantitative detection method of the present application
[0078]
[0079] Comparative Example 3
[0080] The difference from Example 5 is that the pretreatment method in Example 1 in Example 5 is replaced by the pretreatment method in Comparative Example 1, and the rest are the same as in Example 5.
[0081] Comparative Example 4
[0082] The difference from Example 5 is that the pretreatment method in Example 1 in Example 5 is replaced by the pretreatment method in Comparative Example 2, and the rest are the same as in Example 5.
[0083] Example 6
[0084] The urea content in the same sorghum sample was detected by the methods of Example 5 (denoted as group B), Comparative Example 3 (denoted as group A) and Comparative Example 4 (denoted as group C) respectively, and the results are shown in Figure 2 which shows that the amount of activated carbon added will affect the detection results of urea content, and the highest urea content is detected when the amount is 0.2 g.
[0085] Example 7
[0086] The urea content of each grain or distiller's yeast sample in Table 3 was detected by the method of Example 5, and the results are shown in Table 3, and the calculation method of the actual concentration in Table 3 is as follows: In the formula, w: actual concentration of urea in the sample, mg / kg; c: concentration of urea in the sample extract obtained from the standard curve, mg / L; v: sample constant volume, mL; m: sample mass, g. The urea detection chromatogram of sorghum 1 is shown in Figure 3
[0087] Table 3 Detection results of urea content of different grain or koji samples
[0088] Sample name Actual concentration (mg / kg) Sorghum 1 2.27 Sorghum 2 2.30 Sorghum 3 3.62 Sorghum 4 4.12 Sorghum 5 3.39 Sorghum 6 3.395 Daqu 1 82.30 Daqu 2 82.29 Wheat 1 3.96 Wheat 2 6.435
[0089] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.
Claims
1. A pretreatment method for detecting urea in grain or distiller's yeast, characterized in that: The method comprises urea extraction and derivatization steps: the urea extraction step comprises: mixing a sample, activated carbon, a zinc acetate solution and a potassium ferrocyanide solution, ultrasonicating, filtering with medium-speed filter paper, discarding 1 to 2 mL of the initial filtrate, and collecting the subsequent filtrate as the sample solution; The derivatization step is as follows: mixing the sample solution, 9-hydroxyanthene solution and hydrochloric acid solution, placing in the dark, and filtering to obtain the test solution; The usage ratio of the sample, activated carbon, zinc acetate and potassium ferrocyanide is 5g:0.2g:0.0025mol:0.000625mol; the sample is grain or distiller's yeast.
2. The pre-treatment method according to claim 1, characterized in that The ultrasonic time is 20 to 30 minutes, the ultrasonic temperature is 25 to 30° C., and the ultrasonic frequency is 40 KHz.
3. The pre-treatment method according to claim 1, characterized in that The pore size of the medium-speed filter paper is 10 μm.
4. The pre-treatment method according to claim 1, characterized in that: The usage ratio of the sample solution, 9-hydroxyanthene and hydrochloric acid is 400 μL: 0.000012 mol: 0.00015 mol.
5. The pre-treatment method according to claim 1, characterized in that: The time of keeping away from light is 30 to 40 minutes.
6. A method for qualitatively detecting urea in grain or distiller's yeast, characterized in that: Based on the pretreatment method described in any one of claims 1 to 5, the method further includes the following steps: detecting the test liquid by high performance liquid chromatography; treating the urea standard product by the derivatization step in the pretreatment method described in any one of claims 1 to 5, and then detecting the same by high performance liquid chromatography to obtain the spectrum and retention time of the urea standard product; comparing the detection result of the test liquid with the spectrum and retention time of the urea standard product; if the spectrum and the retention time are consistent, it indicates that the grain or distiller's yeast contains urea; if at least one of the spectrum and the retention time is inconsistent, it indicates that the grain or distiller's yeast does not contain urea.
7. A method for quantitatively detecting urea in grain or distiller's yeast, characterized in that: Based on the pretreatment method described in any one of claims 1 to 5, the method further includes the following steps: detecting the test liquid by high performance liquid chromatography; preparing a series of urea standard working solutions with a mass concentration of 0.5 to 10 mg / L, and then treating each series of urea standard working solutions by the derivatization step in the pretreatment method described in any one of claims 1 to 5, and then performing high performance liquid chromatography detection, drawing a standard curve of urea with the peak area of urea as the ordinate and the mass concentration as the abscissa; substituting the test results of the test liquid into the standard curve to calculate the urea content in the grain or distiller's yeast.
8. The method according to claim 6 or 7, characterized in that The stationary phase of the liquid chromatography column of the high performance liquid chromatography method is C18-A; the mobile phase A is acetonitrile, the mobile phase B is a 0.02 mol / L sodium acetate solution with a pH of 7.2, and the gradient elution conditions are:
9. The method according to claim 7, characterized in that The standard curve is Y=2×10 -5 x=0.6010, Y is the peak area of urea, and x is the mass concentration of urea.
10. The pretreatment method according to any one of claims 1 to 5 or the method according to any one of claims 6 to 9, characterized in that: The grains include grains for brewing wine, the grains for brewing wine include sorghum and / or wheat, and the distiller's yeast includes Daqu.