Method for detecting urea in bean sprouts and application thereof

Abstract

The invention relates to the technical field of food safety detection, in particular to a method for detecting urea in bean sprouts and application thereof.The method comprises the following steps that smashed bean sprouts are subjected to two times of ultrasonic extraction and centrifugation, supernate is combined, and an extracting solution is obtained; fixing the volume and filtering to obtain a to-be-detected solution; detecting the liquid to be detected by using liquid chromatography-tandem mass spectrometry; an extraction solvent for ultrasonic extraction is a 5% formic acid-acetonitrile composite solution; the conditions of the liquid chromatography-tandem mass spectrometry are as follows: the chromatographic conditions are as follows: a chromatographic column is an HILIC column; the sample size is 10 [mu] L; a mobile phase is a mixed solution of a 0.02 mmol / L ammonium acetate buffer salt solution and acetonitrile, and isocratic elution is carried out; the flow rate is 0.3 mL / min; the column temperature is 40 DEG C; mass spectrum conditions are as follows: an electrospray ionization (ESI) positive ion mode, electrospray voltage of 4000 V, sheath gas pressure of 30 psi, auxiliary gas pressure of 5 psi, capillary heating temperature of 300 DEG C and scanning mode of MRM. The method disclosed by the invention is free from substance interference, high in sensitivity, accurate in result, low in cost and high in efficiency, and can be used as a standard detection method.

Description

Technical Field

[0001] This invention relates to the field of food safety testing technology, and more specifically, to a method for detecting urea in bean sprouts and its application. Background Technology

[0002] Bean sprouts are a common vegetable. To promote rapid and vigorous growth, some unscrupulous vendors illegally soak or spray bean sprouts with urea during production, resulting in excessively high urea content and causing "toxic bean sprout" food safety incidents. The lack of a method for detecting urea content in bean sprouts is a significant reason why "toxic bean sprouts" reach the market and cause these food safety incidents. Therefore, establishing a method for detecting urea residues in bean sprouts is crucial.

[0003] Currently, the main methods for determining urea in food include spectrophotometry, high-performance liquid chromatography (HPLC), and enzyme-linked immunosorbent assay (ELISA). Industry standard SN / T1004-2013, "Determination of Urea Residue in Exported Canned Foods," provides methods for the determination of urea residues in canned foods using thionyl alcohol derivatization-HPLC fluorescence detection and diacetyl oxime derivatization colorimetric method. Other studies have also employed thionyl alcohol derivatization HPLC-MS and ELISA methods. The derivatization principle in these methods is the condensation reaction between the derivatizing reagent and the amide bond. These methods determine compounds containing amide bonds in the sample but cannot distinguish whether the amide bond originates from urea or other nitrogen-containing organic compounds. Furthermore, the derivatization reaction conditions and time affect the stability of the derivatized product, and the use of derivatization reactions also complicates the pretreatment process. Summary of the Invention

[0004] To address the deficiencies in the aforementioned background technology, this invention provides a method for detecting urea in bean sprouts and its application.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0006] In a first aspect, the present invention provides a method for detecting urea in bean sprouts, comprising the following steps:

[0007] S1. Pretreatment: Add urea to the crushed bean sprouts. 13 The internal standard and the extraction solution were used to perform two ultrasonic extractions on the crushed bean sprouts. After centrifugation, the supernatants were combined to obtain the extraction solution. The extraction solution was diluted to a certain volume, filtered, and the solution to be tested was obtained. The extraction solvent used for ultrasonic extraction was a 5% formic acid-acetonitrile composite solution.

[0008] S2. Preparation of standard solution: using ultrapure water as solvent, and urea- 13 C is the internal standard. A series of urea mixed standard solutions of different concentrations are prepared, and a standard curve is plotted based on the mixed standard solutions to determine the linear range for the quantitative determination of urea.

[0009] S3, LC-MS / MS detection: LC-MS / MS is used to detect the sample to be tested. Qualitative analysis is used to determine whether there is urea residue in the sample to be tested, and quantitative analysis is used to determine the amount of urea residue in the sample to be tested.

[0010] The conditions for liquid chromatography-tandem mass spectrometry are as follows:

[0011] Chromatographic conditions: Column: ACQUITYBEH HILIC column (2.1 mm × 100 mm, particle size 1.7 μm); Injection volume: 10 μL; Mobile phase: a mixture of 0.02 mmol / L ammonium acetate buffer solution and acetonitrile, isocratic elution; Flow rate: 0.3 mL / min; Column temperature: 40℃;

[0012] Mass spectrometry conditions: Electrospray ionization (ESI) positive ion mode, electrospray voltage: 4000V, sheath gas pressure: 30psi, auxiliary gas pressure: 5psi, heating capillary temperature: 300℃, scan mode: MRM.

[0013] In the preferred embodiment, the solvent used for volume adjustment is a 5% formic acid-acetonitrile composite solution.

[0014] In the preferred embodiment, the mass-to-volume ratio of the pulverized bean sprouts to the extraction solvent used for ultrasonic extraction is 1:1, and the ultrasonic extraction time is 5 minutes; the centrifugation time is 5 minutes, and the rotation speed is 10,000 rpm.

[0015] In the preferred embodiment, the volume ratio of the extract to the volume-fixing solvent is 95:5.

[0016] In the preferred embodiment, the mobile phase has a pH of 4.0, and the volume ratio of 0.02 mmol / L ammonium acetate buffer solution to acetonitrile is 15:85 (V / V).

[0017] A second aspect of the present invention provides an application of the above-mentioned method for detecting urea in bean sprouts as a standard detection method.

[0018] The beneficial effects of this invention are as follows:

[0019] The method for detecting urea in bean sprouts provided by this invention is free from material interference, has high sensitivity, is simple, easy to operate, produces accurate results, is low in cost, and is highly efficient.

[0020] The method for detecting urea in bean sprouts provided by this invention can be used as a standard detection method. It meets the requirements of my country's vegetable product quality and safety standards, and its main technical parameters meet the requirements of GB / T 27414-2017 "Guideline for Conformity Assessment and Validation of Chemical Analysis Methods", ensuring the uniformity, scientific nature, systematicness and practicality of the standard. Attached Figure Description

[0021] Figure 1 This is the total ion chromatogram (TIC) of characteristic ions in a mixed standard solution of urea and internal standard (5 ng / mL).

[0022] Figure 2 This is a secondary mass spectrum of urea standard;

[0023] Figure 3 This is a chromatogram of a urea standard solution under a C18 column;

[0024] Figure 4 This is a chromatogram of a urea standard solution under a HILIC column;

[0025] Figure 5 These are chromatograms of urea standard solutions in the range of 0.1–1 mg / L using 0.1% formic acid and acetonitrile as the mobile phase; A: 1 mg / L urea standard solution; B: 0.5 mg / mL urea standard solution; C: 0.1 mg / mL urea standard solution;

[0026] Figure 6 Chromatography was performed using 0.2 mmol / L NH4OAc (pH 4.0) and acetonitrile as the mobile phase with a 5 ng / mL urea standard solution.

[0027] Figure 7 This is a quantitative ion chromatogram of a 5 ng / mL mixed standard solution. Detailed Implementation

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0029] In this invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise specified, the experimental methods in the embodiments are conventional methods. Where specific conditions are not specified in the embodiments, they should be performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0030] A method for detecting urea in bean sprouts includes the following steps:

[0031] After pulverizing, bean sprout samples were extracted with acetonitrile aqueous solution by ultrasonication. The supernatant was collected after centrifugation, and the residue was extracted again, with the supernatants combined. Urea was separated using affinity chromatography, and determined by triple quadrupole mass spectrometry in electrospray ionization (ESI) mode, positive ion mode, and MRM (multiple reaction monitoring) mode.13 C is an internal standard, and quantification is performed using the internal standard method.

[0032] Pretreatment conditions were as follows: 20 g of sample was weighed into a 50 mL centrifuge tube, and 100 μL of 100 ng / mL urea solution was added. 13 Add 20 mL of 5% formic acid-acetonitrile composite solution to the C internal standard, sonicate for 5 min, centrifuge at 10000 rpm at 4℃ for 5 min, transfer the supernatant to a 50 mL volumetric flask, add another 20 mL of 5% formic acid-acetonitrile composite solution to the precipitate and repeat the extraction once more, combine the supernatants, and finally dilute to volume with 5% formic acid-acetonitrile composite solution, mix well, filter through a 0.22 μm filter membrane, and perform LC-MS / MS analysis.

[0033] The instrument conditions are as follows:

[0034] Chromatographic conditions:

[0035] Chromatographic column: ACQUITYBEH HILIC column (2.1 mm × 100 mm, particle size 1.7 μm); injection volume: 10 μL; mobile phase: 0.02 mmol / L ammonium acetate (pH 4.0): acetonitrile (15:85, V / V), isocratic elution; flow rate: 0.3 mL / min; column temperature: 40 °C.

[0036] Mass spectrometry conditions:

[0037] Electrospray ionization (ESI) positive ion mode was used. Electrospray voltage: 4000V, sheath gas pressure: 30psi (206.85kPa), auxiliary gas pressure: 5psi (34.48kPa), heating capillary temperature: 300℃, scanning mode: MRM. The retention times and mass spectrometry parameters of urea and internal standard are shown in Table 1.

[0038] Table 1 Retention times and ion-pair mass spectrometry parameters of urea and internal standard.

[0039]

[0040] Example 1: Selection of detection methods

[0041] This embodiment examines the detection results of three methods: thionol derivatization-liquid chromatography (mass spectrometry), diacetyl monooxime colorimetry, and liquid chromatography-mass spectrometry, as detailed below:

[0042] 1. Tonol derivatization-liquid chromatography (mass spectrometry):

[0043] Weigh 5.0 g of the sample into a 50 mL centrifuge tube, add 20 mL of 1% acetic acid solution (3.7), homogenize for 2 min, add 5 mL of chloroform, homogenize for 2 min, centrifuge at 3000 r / m for 3 min, extract the supernatant, and filter. Add another 20 mL of 1% acetic acid aqueous solution to the centrifuge tube containing the residue, homogenize for 2 min, centrifuge again, and filter. Combine the extracts and dilute to 50 mL with water, mix well, and allow for derivatization. Accurately pipette 0.5 mL of the extract and 20 mg / L urea standard solution into separate 2 mL sample vials, add 0.4 mL of methanol, 50 μL of jantonol derivatizing agent, and 50 μL of 1% hydrochloric acid solution sequentially, tighten the caps, mix well, derivatize for 5 min, filter through a 0.22 μm filter membrane, and proceed with HPLC analysis. Separation was performed using a C18 column, with elution using a mobile phase of 20 mM sodium octyl sulfonate aqueous solution and acetonitrile (65:35, v / v) at a flow rate of 1 mL / min.

[0044] Both 9-ureidozenton (m / z = 241) and N,N-diureidozenton (m / z = 421) were detected, but their ratio was not constant, and their responses gradually decreased with increasing derivatization time. The quantitative relationships between 9-ureidozenton and N,N-diureidozenton and urea were also investigated, with correlation coefficients R0 ranging from 1 ng / mL to 500 ng / mL. 2 The values ​​were 0.46 and 0.58. Daughter ion scans using m / z = 241 and m / z = 421 as parent ions did not reveal any corresponding daughter ions.

[0045] 2. Diacetyl monooxime colorimetric method:

[0046] Urea was derivatized with diacetyl oxime under acidic conditions and in the presence of methanol. The product exhibited fluorescence at an excitation wavelength of 213 nm and an emission wavelength of 308 nm. 20.0 g of the sample (accurate to 0.1 g) was weighed and placed in a 100 mL volumetric flask. 5 mL each of zinc acetate solution (11.9) and potassium ferrocyanide solution (11.10) were added, along with 2.5 g of activated carbon. The mixture was shaken well, and 50 mL of water was added. The mixture was shaken for 30 min, and water was added to the mark. The mixture was shaken well, allowed to stand for 1 min, and then filtered through medium-speed filter paper. 5 mL of the filtrate was collected, and the absorbance was measured at a wavelength of 525 nm.

[0047] Accurately pipette 5 mL each of 0 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, and 50 μg / mL urea standard working solutions into separate 20 mL colorimetric tubes. Add 5 mL of acidic diacetyl monooxime reaction solution to each tube, seal the tubes tightly, and incubate in a 95°C water bath for 20 min. After filtration through medium-speed filter paper, use a standard working solution with zero urea concentration as a reference to measure the absorbance at 525 nm and plot a standard curve. The curve shows a low linear correlation coefficient and low correlation. The detection effect of the sample solutions is not stable, and the repeatability is low.

[0048] 3. Liquid chromatography-mass spectrometry (LC-MS):

[0049] Accurately weigh 2 g (accurate to 0.001 g) of sample into a 50 mL stoppered polytetrafluoroethylene centrifuge tube, add 20 mL of acetonitrile-water (85:15, V / V), homogenize, and sonicate for 5 min. Centrifuge at 10000 rpm for 5 min, transfer the supernatant to a 50 mL volumetric flask, and repeat the extraction once more with 20 mL of acetonitrile-water (85:15, V / V). Combine the supernatants, and finally dilute to volume with acetonitrile-water (85:15, V / V), mix well, filter through a 0.22 μm nylon membrane, and perform LC-MS / MS analysis. Elute using an ACQUITY UPLCBEH Amide (100 mm × 2.1 mm, 1.7 μm) column with acetonitrile-0.5% ammonium formate aqueous solution (85:15, V / V) at a flow rate of 0.3 mL / min.

[0050] After urea is ionized in positive ion mode of ESI source, the mass analyzer can measure its parent ion and daughter ion as m / z = 61 and m / z = 44, respectively, which can be identified as the quasi-molecular ion of urea and its fragments. Therefore, mass spectrometry can confirm whether the sample contains urea rather than other amide compounds, with good specificity.

[0051] Based on the evaluation of the stability and specificity of the above methods, the liquid chromatography-mass spectrometry (LC-MS) method has certain advantages. Therefore, this standard adopts this method to determine the urea content.

[0052] Example 2: Selection of LC-MS / MS conditions

[0053] 1. Selection of chromatographic conditions

[0054] 1.1 Selection of chromatographic column

[0055] Separation was performed using a 100 ng / mL urea standard solution on both a C18 and a HILIC column, with isocratic elution at a rate of 0.3 mL / min using a mobile phase of 0.02 mmol / L ammonium acetate (pH 4.0):acetonitrile (15:85, V / V). Results are as follows: Figure 2 As shown, urea was separated using a reverse-bonded phase column C18, and it was found that it was almost completely retained on the C18 column. Considering that urea is a highly polar compound, affinity chromatography columns are more suitable. Figure 3 As shown, using a HILIC affinity column as the stationary phase, experiments showed that urea was well retained in this column, so the HILIC column was chosen as the stationary phase.

[0056] 1.2 Selection of mobile phase

[0057] Urea standard solutions were separated using a HILIC column with isocratic elution at a rate of 0.3 mL / min. Considering that electrospray ionization (ESI+) is a positive ion mode, formic acid was added to the mobile phase to enhance protonation. The separation performance with 0.1% formic acid and acetonitrile as the mobile phase was investigated. The results showed that the response values ​​of standard solutions at different concentration gradients were nonlinear, with poor repeatability, peak broadening, and severe tailing. Within the 0.1–1 mg / L range, the response value of urea decreased significantly from low to high concentrations; the trend of concentration and response value is shown in the figure. Figure 4 .

[0058] This is because the protonation degree of the target compound is significantly affected by the pH of the mobile phase. Therefore, a buffer salt is introduced into the mobile phase to maintain a relatively stable pH. Considering the pH requirements of the HILIC column, the effects of 5 mmol / L ammonium acetate buffer and acetonitrile as mobile phases at pH values ​​of 4.0 and 5.0 were investigated. The results showed that at pH 5.0, although the response value decay of high-concentration compounds was improved, a good linear relationship could not still be obtained. However, under the buffer salt conditions at pH 4.0, the analyte showed a good linear relationship in the range of 0.01–1.0 mg / L, and the peak shape was significantly improved. The optimal pH value was finally selected as 4.0. The addition of the buffer salt can inhibit the ion exchange between the sample and the silanol groups and maintain the relative stability of the acidity of the mobile phase, thereby achieving good separation results. The effects of adding 0.2 mmol / L, 1.0 mmol / L, and 5.0 mmol / L NH4OAc buffer (pH 4.0) to the mobile phase on the mass spectrometry detection signal were investigated. The results showed that the higher the concentration of NH4OAc buffer salt, the more severe the suppression of the mass spectrometry detection signal, while the addition of 0.2 mmol / L NH4OAc resulted in good separation and signal intensity (see...). Figure 5 The linear relationship was good. Therefore, 0.2 mmol / L NH4OAc was chosen to adjust the ionic strength and pH of the mobile phase.

[0059] 2. Selection of mass spectrometry conditions

[0060] like Figure 6 As shown, urea molecules contain amino groups and are inherently highly polar. Therefore, using an ESI source yields better ionization results. Scanning was performed in both positive and negative ion modes. Urea molecules showed a higher response in positive ion mode and a lower response in negative ion mode; therefore, positive ion mode was chosen for determination. Using m / z 61 as the parent ion, a daughter ion scan was performed, yielding only one daughter ion at m / z 44. This fragment should be the remaining portion of urea after the removal of an amino group. Its optimal collision voltage is -21V. The qualitative and quantitative ion pairs for urea are both 61.2 > 44.6. The internal standard urea... 13 The qualitative and quantitative ion pairs of C are 62.2 > 45.6.

[0061] Example 3: Optimization of preprocessing methods

[0062] The bean sprout substrate is relatively clean, containing only small amounts of protein and carbohydrates, thus its pretreatment method is relatively simple. The type of extraction solvent, centrifugation time, and number of centrifugation cycles were investigated in the pretreatment method, and the condition with the highest recovery rate was selected as the optimization criterion. The optimization results are shown in Table 2.

[0063] Table 2. Recovery rate of urea under different extraction conditions

[0064]

[0065] Acidified acetonitrile as an extractant has a higher recovery rate than aqueous acetonitrile solution, and acetonitrile containing 5% formic acid has a better recovery rate than acetonitrile containing 1% formic acid. Centrifugation time also affects the recovery rate; shorter centrifugation time results in higher recovery rate. This may be because the analyte is carried away during the sedimentation process of the precipitate, leading to a decrease in recovery rate.

[0066] The optimized method is to weigh 20g of bean sprout sample into a 50mL test tube, and add 100μL of 100ng / mL urea solution to the test tube. 13 Add 20 mL of 5% formic acid-acetonitrile to the C internal standard and sonicate for 5 min. Centrifuge at 10000 rpm for 5 min, transfer the supernatant to a 50 mL volumetric flask, add another 20 mL of 5% formic acid-acetonitrile to the residue, repeat the above extraction process, combine the supernatants, and finally dilute to volume with 5% formic acid-acetonitrile. Take 1 mL of the diluted supernatant, filter through a 0.22 μm filter membrane, and perform LC-MS / MS analysis.

[0067] Example 4: Method suitability testing

[0068] This embodiment systematically examines the reliability parameters of the detection method, such as linear range, precision, and accuracy.

[0069] 1. Linear range

[0070] Table 3 Internal Standard Series Mixture Standard Solutions

[0071]

[0072] As shown in Table 3, a series of mixed standard solutions of urea and internal standard were measured sequentially from low to high concentration to create standard curves. Linear regression equations and correlation coefficients were calculated. The linear standard curve and correlation coefficient of the urea mixed standard solution, as well as the instrument's detection limit and quantitation limit, are shown in Table 4. The standard curve exhibited good linearity in the range of 1–500 ng / mL, with R0... 2 The linearity was 0.998, and the instrument's limit of detection (LOD) and limit of quantitation (LOQ) were 2.0 ng / mL and 5.0 ng / mL, respectively. This indicates that the method exhibits good linearity, reproducibility, high recovery, accuracy, and precision. The entire method demonstrates good stability and can be used for the detection of real-world samples.

[0073] Table 4. Standard curve, detection limit, and quantitation limit of urea.

[0074]

[0075] 2. Sensitivity

[0076] The concentration added to the blank solution is the instrument's limit of quantitation. Figure 7 The TIC plot shows a signal-to-noise ratio (S / N) of 9.85. After calculation, the limit of quantitation for the method is determined to be 2.5 ng / g.

[0077] 3. Accuracy and Precision

[0078] In the spiking recovery experiment, three spiking levels were selected: 2.5 ng / g, 12.5 ng / g, and 25 ng / g. Six parallel experiments were performed at each level. The average recovery rate and relative standard deviation (RSD%) were calculated using the following formulas:

[0079] Precision

[0080]

[0081] In the formula: --The arithmetic mean of n repeated measurements; X i --The i-th measurement in n measurements; n--the number of repeated measurements; S--the standard deviation.

[0082] The results are shown in Table 4. The average recovery rates for all three levels were above 80%, and the RSD% were all less than 5%.

[0083] Table 4. Urea recovery rate and intraday precision

[0084]

[0085]

[0086] Example 5: Application of detection methods

[0087] A method for detecting urea in bean sprouts includes the following steps:

[0088] S1. Pretreatment: Weigh 20g of the crushed bean sprout sample into a 50mL centrifuge tube, and add 100μL of 100ng / mL urea solution to the centrifuge tube. 13 Add 20 mL of 5% formic acid-acetonitrile composite solution to the C internal standard, sonicate for 5 min, centrifuge at 10000 rpm for 5 min at 4℃, transfer the supernatant to a 50 mL volumetric flask, add another 20 mL of 5% formic acid-acetonitrile composite solution to the remaining precipitate, repeat the above steps to extract once more, combine the supernatants, dilute to volume with 5% formic acid-acetonitrile composite solution, filter through a 0.22 μm filter membrane to obtain the sample to be tested.

[0089] S2. Preparation of standard solution: using ultrapure water as solvent, and urea- 13 C is the internal standard (100 μL / mL). Mixed standard solutions with different concentrations of urea (5 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, 250 ng / mL and 500 ng / mL) were prepared, and a standard curve was plotted based on the mixed standard solutions to determine the linear range for the quantitative determination of urea.

[0090] S3, LC-MS / MS detection: Take 1 mL of the sample to be tested and use LC-MS / MS to detect the sample. Qualitative analysis is used to determine whether there is urea residue in the sample, and quantitative analysis is used to determine the amount of urea residue in the sample.

[0091] The LC-MS / MS detection conditions are as follows:

[0092] Chromatographic conditions:

[0093] Chromatographic column: ACQUITYBEH HILIC column (2.1 mm × 100 mm, particle size 1.7 μm); injection volume: 10 μL; mobile phase: 0.02 mmol / L ammonium acetate (pH 4.0): acetonitrile (15:85, V / V), isocratic elution; flow rate: 0.3 mL / min; column temperature: 40 °C.

[0094] Mass spectrometry conditions:

[0095] Electrospray ionization (ESI) positive ion mode, electrospray voltage: 4000V, sheath gas pressure: 30psi (206.85kPa), auxiliary gas pressure: 5psi (34.48kPa), heating capillary temperature: 300℃, scan mode: MRM.

[0096] The optimized method described above was applied to soybean sprouts, mung bean sprouts, spontaneously sprouted soybean sprouts, and spontaneously sprouted mung bean sprouts for additive recovery experiments. The results are shown in Table 5. The recovery rates among the various bean sprouts were not significantly different, with average recovery rates ranging from 79.45% to 86.88% and RSD% ranging from 1.92% to 5.21%.

[0097] Table 54 shows the recovery rate and precision of urea in 54 types of bean sprouts.

[0098]

[0099]

[0100] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for detecting urea in bean sprouts, characterized in that, Includes the following steps: S1. Pretreatment: Add urea to the crushed bean sprouts. 13 The internal standard and the extract were used to perform two ultrasonic extractions on the crushed bean sprouts. After centrifugation, the supernatants were combined to obtain the extract. The extract was diluted to a fixed volume, filtered, and the solution to be tested was obtained; the extraction solvent used for ultrasonic extraction was a 5% formic acid-acetonitrile composite solution. S2. Preparation of standard solution: using ultrapure water as solvent, and urea- 13 C is the internal standard. A series of urea mixed standard solutions of different concentrations are prepared, and a standard curve is plotted based on the mixed standard solutions to determine the linear range for the quantitative determination of urea. S3, LC-MS / MS detection: LC-MS / MS is used to detect the sample to be tested. Qualitative analysis is used to determine whether there is urea residue in the sample to be tested, and quantitative analysis is used to determine the amount of urea residue in the sample to be tested. The conditions for liquid chromatography-tandem mass spectrometry are as follows: Chromatographic conditions: Column: ACQUITYBEH HILIC column (2.1 mm × 100 mm, particle size 1.7 μm); Injection volume: 10 μL; Mobile phase: a mixture of 0.02 mmol / L ammonium acetate buffer solution and acetonitrile, isocratic elution; Flow rate: 0.3 mL / min; Column temperature: 40℃; Mass spectrometry conditions: Electrospray ionization (ESI) positive ion mode, electrospray voltage: 4000V, sheath gas pressure: 30psi, auxiliary gas pressure: 5psi, heating capillary temperature: 300℃, scan mode: MRM.

2. The method for detecting urea in bean sprouts according to claim 1, characterized in that, The solvent used for volume adjustment is a 5% formic acid-acetonitrile composite solution.

3. The method for detecting urea in bean sprouts according to claim 1, characterized in that, The mass-to-volume ratio of the pulverized bean sprouts to the extraction solvent used for ultrasonic extraction was 1:1, and the ultrasonic extraction time was 5 min; the centrifugation time was 5 min, and the rotation speed was 10000 rpm.

4. The method for detecting urea in bean sprouts according to claim 1, characterized in that, The volume ratio of the extract to the solvent used for volume adjustment is 95:

5.

5. The method for detecting urea in bean sprouts according to claim 1, characterized in that, The mobile phase had a pH of 4.0 and a volume ratio of 0.02 mmol / L ammonium acetate buffer solution to acetonitrile of 15:85 (V / V).

6. The application of a method for detecting urea in bean sprouts according to any one of claims 1 to 5 as a standard detection method.

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