UHPLC-Q-Orbitrap HRMS (Ultra High Performance Liquid Chromatography-Quantitative Orbitrap High Resolution Mass Spectrometry) synchronous detection method for sulforaphene and sulforaphane in brassicaceous vegetables

Abstract

The invention discloses a UHPLC-Q-Orbitrap HRMS (Ultra High Performance Liquid Chromatography-Quantitative Orbitrap High Resolution Mass Spectrometry) synchronous detection method for sulforaphane and sulforaphane in brassicaceous vegetables, which comprises the following steps: (1) selecting a clean brassicaceous vegetable sample, and preparing homogenate; (2) weighing a quantitative homogenate sample, adding the prepared phosphate buffer, uniformly mixing, performing enzymolysis in a constant-temperature oscillator at 25 DEG C, adding an organic solvent for extraction, performing high-speed centrifugation, taking an organic phase, putting the organic phase into a 150mL flat-bottomed flask, concentrating until the organic phase is dry, fixing the volume by using a methanol solution, passing through an organic phase filter membrane, and performing low-temperature preservation for analysis and determination on a machine; (3) preparing a standard curve: preparing mixed standard solutions of sulforaphane and sulforaphane with different concentration gradients; (4) sample detection: carrying out qualitative and quantitative analysis on sulforaphane and sulforaphane compounds by adopting ultra-high performance liquid chromatography-electrostatic field orbitrap mass spectrometry; the invention establishes an efficient, accurate and short-time-consumption synchronous detection method for the two compounds, and correlation coefficients R2 of a standard working curve are both greater than 0.999.

Description

Technical Field

[0001] The present invention relates to the technical field of food detection, and specifically to a UHPLC-Q-Orbitrap HRMS synchronous detection method for sulforaphane and glucoraphanin in cruciferous vegetables. Background Art

[0002] Vegetables are essential important non-staple foods in people's daily lives, and can provide rich and inexpensive dietary nutrients such as various vitamins, minerals, and cellulose for the human body. With the improvement of living standards, the public's demand for vegetables has gradually shifted from quantity to quality. The simple safety index evaluation system cannot fully meet the requirements of the public, and green and high-quality agricultural products have gradually gained popularity. Non-safety indicators such as nutritional quality, taste, color, aroma, etc. have become important factors determining their market competitiveness.

[0003] Regular consumption of cruciferous herbs such as radish, Chinese cabbage, cauliflower, and broccoli can reduce the risk of cancer. This protective effect stems from the isothiocyanate components they contain. Isothiocyanate is one of the hydrolysis products of glucosinolate. Glucosinolate does not have biological activity. When its β-thioglucoside bond is destroyed by glucosinolate hydrolase (myrosinase), hydrolysis products such as glucose, sulfate, and isothiocyanate are produced, providing a unique smell and taste for cruciferous vegetables such as radish. It is the natural isothiocyanate compound with the highest anti-cancer activity and has effects such as antioxidant, anti-inflammatory and antibacterial, and immune enhancement.

[0004] In recent years, the functional active components of vegetables have received extensive attention, and sulforaphane and glucoraphanin have become important indicators for evaluating the nutritional quality of cruciferous vegetables. However, at present, there is a lack of separation and detection methods for glucoraphanin and sulforaphane, mainly including chemical titration method, spectrophotometry, gas chromatography, high performance liquid chromatography, etc. The chemical titration method and spectrophotometry have cumbersome steps and low detection sensitivity; in the gas chromatography method, high temperature will destroy the structures of sulforaphane and glucoraphanin during the detection process, and the quantitative accuracy is not high; the high performance liquid chromatography method qualitatively determines by the peak emergence time, and it is difficult to achieve baseline separation of these two substances with similar molecular structures, and the elution time is long, and the detection is time-consuming. For example, CN111983099 A discloses a method for determining glucoraphanin and sulforaphane in cruciferous vegetables by HPLC. Sulforaphane and glucoraphanin are detected at two different wavelengths, and the detection time for one sample reaches 1 h, and the detection time is long. In view of the increasing demand for the identification of the characteristic nutritional quality of cruciferous vegetables in the current market, it is urgent to develop a convenient and fast synchronous detection technology. Summary of the Invention

[0005] The object of the present invention is to provide a method for simultaneously detecting sulforaphane and glucoraphanin in cruciferous vegetables by UHPLC-Q-Orbitrap HRMS. Based on ultra-high performance liquid chromatography - electrostatic field orbitrap high-resolution mass spectrometry technology, the present invention establishes a method for simultaneously detecting two types of compounds with high efficiency, accuracy and short time consumption. The correlation coefficient R of the standard working curve 2 is greater than 0.999.

[0006] To achieve the above object, the present invention provides the following technical solution: A method for simultaneously detecting sulforaphane and glucoraphanin in cruciferous vegetables by UHPLC-Q-Orbitrap HRMS, characterized in that it is carried out according to the following steps:

[0007] (1) Vegetable pretreatment: Select clean cruciferous vegetable samples and prepare homogenates;

[0008] (2) Extraction and separation: Weigh a quantitative homogenate sample, add the prepared phosphate buffer solution, mix evenly, place it in a constant temperature shaker at 25°C for enzymatic hydrolysis, add an organic solvent for extraction, centrifuge at high speed, take the organic phase, place it in a 150 mL flat-bottom flask, repeat the extraction once, combine the extraction solutions, concentrate to dryness, dissolve in a methanol solution, filter through an organic phase filter membrane, and store at low temperature for on-machine analysis and determination;

[0009] (3) Preparation of standard curve: Prepare mixed standard solutions of sulforaphane and glucoraphanin with different concentration gradients;

[0010] (4) Sample detection: Qualitative and quantitative analysis of sulforaphane and glucoraphanin compounds is carried out by ultra-high performance liquid chromatography - electrostatic field orbitrap mass spectrometry;

[0011] Among them, the liquid chromatography column selects an ACQUITY UPLC BEH C18 chromatographic column with a column temperature of 40°C; the mobile phase: water is phase A and methanol is phase B; the flow rate is 0.3 mL / min, and the injection volume is 1 μL; the initial mobile phase is 95% phase A and 5% phase B, maintained for 1 min, the phase B is gradually adjusted to 20% within 1 - 2 min, the phase B is adjusted to 40% within 2 - 3 min, maintained for 3 min, the phase B is reduced to 5% within 1 min, and maintained for 3 min;

[0012] Mass spectrometry conditions: Electrospray ionization source ESI + Combined with the Full mass mode, the spray voltage is 3.8 kV; the capillary temperature is 275°C; the auxiliary gas heater temperature is 300°C; the sheath gas flow rate is 35 arb; the auxiliary gas flow rate is 6 arb; the S-lens RF level is 50 V; the detector is Orbitrap; the mass spectrometry resolution is 70000 FWHM; the scanning range is 50 - 550 m / z; the maximum injection time is 100 ms; the mass deviation is 5 ppm; the MS automatic gain control target is 2.0×105 ; The monitored ion is [M+H] + .

[0013] In the above solution: the cruciferous vegetable is one of radish, broccoli, cauliflower, and Chinese cabbage. The method of the present invention is applicable to cruciferous vegetables and is not limited to the several kinds listed above.

[0014] In the above solution: the operation in step (1) is as follows: select multiple fresh cruciferous vegetables growing normally, wash them clean with tap water and deionized water respectively, dry them, take samples by the quartering method and mix them evenly, and put them into a high-speed tissue homogenizer to break them evenly.

[0015] In the above solution: in step (2), the pH of the phosphate buffer solution is 5.6, the oscillation parameter of the constant temperature shaker is 150 r / min, the enzymatic hydrolysis time is 1 h, and 3 mL of phosphate buffer solution is added to every 0.2 g of the homogenized product.

[0016] In the above solution: the organic solvent used for extraction is ethyl acetate, and the extraction method is ultrasonic extraction for 30 min; the centrifugation rate is 6000 r / min and the time is 5 min.

[0017] In the above solution: in step (2), filtration is carried out using a Nylon66 0.22 μm organic system needle-type filter membrane; the storage temperature is -20 °C.

[0018] In the above solution: in step (3), the concentration gradients of the compounds in the standard curve solution are 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, 1000 ng / mL, 5000 ng / mL, and 10000 ng / mL.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) Based on the ultrasonic extraction method, the present invention extracts, purifies, and separates two compounds, sulforaphane and glucoraphanin, from the homogenized samples of cruciferous vegetables. The pretreatment process takes a short time, is simple and easy to operate; and the amount of organic solvent used is small, and the environmental pollution is relatively light.

[0021] (2) The present invention successfully established a synchronous qualitative and quantitative analysis method for sulforaphene and sulforaphane by using ultra-high performance liquid chromatography coupled with electrostatic field orbitrap high-resolution mass spectrometry. The method was used to detect the extracted samples of cruciferous vegetables, with a relatively low detection limit, high sensitivity and reproducibility. The spiked recoveries of sulforaphene and sulforaphane were 100.25% ± 8.25% and 98.08% ± 8.18% respectively. The detection limits of sulforaphene and sulforaphane were 0.172 ng / mL and 0.076 ng / mL respectively, and the quantitative limits were 0.344 ng / mL and 0.152 ng / mL respectively. The detection time was short.

[0022] (3) The detection results are more accurate, and it is applicable to the rapid detection and analysis of two compounds, sulforaphene and sulforaphane, in cruciferous vegetables such as radish, Chinese cabbage, and broccoli, providing a strong guarantee for meeting the market demand for the identification of the characteristic nutritional quality of cruciferous vegetables. Description of the Drawings

[0023] Figure 1 It is the response intensity of the UHPLC-ESI-Q-Orbitrap HRMS chromatographic peaks of sulforaphene and sulforaphane under different mobile phase solvents in the embodiment of the present invention.

[0024] Figure 2 It is the extracted ion chromatogram of sulforaphene and sulforaphane by UHPLC-ESI-Q-Orbitrap HRMS in the embodiment of the present invention.

[0025] Figure 3 It is the comparison of the extraction contents of sulforaphene and sulforaphane in different radish varieties with different extraction solvents in the embodiment of the present invention.

[0026] Figure 4 It is the comparison of the contents of sulforaphene and sulforaphane in white radishes of different varieties from different regions in the embodiment of the present invention. Detailed Embodiments

[0027] The present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments.

[0028] Example 1

[0029] I. Detection Method

[0030] 1. Main Instruments and Reagent Materials

[0031] 1.1 Instruments

[0032] Ultra-high performance liquid chromatography-electrostatic field orbitrap high-resolution mass spectrometer (UHPLC-Q-Orbitrap HRMS, Thermo Fisher Scientific, USA); ACQUITY UPLC BEH C18 chromatographic column, specification: 100 mm × 2.1 mm, 1.7 μm; high-speed refrigerated centrifuge; rotary evaporator ( BUCHI Labortechnik AG, Switzerland); vortex mixer ( VORTEX 3, Germany); RDK JRES-02B laboratory ultrapure water machine of Chongqing Runde Kangjian Water Treatment Equipment Co., Ltd., homogenizer; analytical balance, Hamilton glass microsyringe (USA).

[0033] 1.2 Reagents and consumables

[0034] Sulforaphene (CAS: 592-95-0), purity HPLC ≥ 98%, purchased from Zeye Biotechnology Co., Ltd.; D,L-Sulforaphane (CAS: 4478-93-7), purity 90-95%, purchased from Sigma-Aldrich (USA); methanol, HPLC grade, purchased from Fisher Chemical (USA); ethyl acetate and dichloromethane, both chromatographic grade, purchased from Anaqua Chemicals Supply (USA); Nylon66 organic syringe filter membrane (13 mm, 0.22 μm) purchased from Gaopan Technology, sodium dihydrogen phosphate (CAS: 7758-80-7), purity ≥ 99.9%, disodium hydrogen phosphate (CAS: 10028-24-7), purity chromatographic grade ≥ 99%, both purchased from Macklin.

[0035] 2. Determination of standard working curve

[0036] Prepare the standard working curve: Take the standard reagents of sulforaphene and sulforaphane to prepare a mixed standard solution, and add methanol solvent for stepwise dilution to obtain 9 standard working curves at different concentration levels. The concentrations of sulforaphene and sulforaphane in the standard solution are in turn: 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, 1000 ng / mL, 5000 ng / mL and 10000 ng / mL. Qualitative analysis is carried out by monitoring ions and retention time, and quantitative analysis is carried out by the external standard method. Taking the concentration of the target compound standard solution as the abscissa and the corresponding chromatographic peak area as the ordinate, draw the standard curve, and obtain the concentrations of sulforaphene and sulforaphane in the subsequent test samples according to the standard curve.

[0037] 3. Sample preparation

[0038] Select multiple fresh cruciferous vegetables that are growing normally. After rinsing them clean with tap water and deionized water and drying them, take samples by the quartering method, mix them evenly, and put them into a high-speed tissue homogenizer to break them evenly.

[0039] 4. Extraction

[0040] Weigh 0.2 g of the vegetable homogenate sample into a 50 mL centrifuge tube, add 3 mL of phosphate buffer solution with pH = 5.6 prepared from sodium dihydrogen phosphate and disodium hydrogen phosphate, place it in a constant temperature shaker at 25 °C with a speed of 150 r / min, and the enzymatic hydrolysis time is 1 h. Add 40 mL of ethyl acetate and perform ultrasonic extraction for 30 min, centrifuge at a high speed of 6000 r / min for 5 min, take the upper organic phase, place it in a 150 mL flat-bottom flask, repeat the extraction once, combine the extraction solutions, concentrate them to nearly dry using a rotary evaporator, make up the volume to 5 mL with methanol, filter through a 0.22 μm organic syringe filter, and store at -20 °C for later analysis and determination by machine.

[0041] 5. Analytical detection

[0042] Chromatographic conditions: Select an ACQUITY UPLC BEH C18 chromatographic column for the liquid chromatograph, specifications: 100 mm × 2.1 mm, 1.7 μm; column temperature 40 °C; mobile phase: water is phase A, methanol is phase B; flow rate is 0.3 mL / min, injection volume is 1 μL; the initial mobile phase is 95% phase A and 5% phase B, hold for 1 min, gradually adjust phase B to 20% within 1 - 2 min, adjust phase B to 40% within 2 - 3 min, hold for 3 min, reduce phase B to 5% within 1 min, hold for 3 min, as shown in Table 1.

[0043] Table 1 Mobile phase gradient elution program for the detection of sulforaphane and glucoraphanin

[0044]

[0045] Mass spectrometry conditions: Electrospray ionization source ESI + Combined with the Full mass mode, spray voltage 3.8 kV; capillary temperature 275 °C; auxiliary gas heater temperature 300 °C; sheath gas flow rate 35 arb; auxiliary gas flow rate 6 arb; S-lens RF level 50 V; detector is Orbitrap; mass spectrometry resolution is 70000 FWHM; scanning range is 50 - 550 m / z; maximum injection time is 100 ms; mass deviation is 5 ppm; MS automatic gain control target is 2.0×10 5 ; Monitoring ion is [M + H] + .

[0046] Table 2 [M + H] of sulforaphane and glucoraphanin +Molecular weights and molecular formulas of quantitative and qualitative ions

[0047]

[0048] 6. Result calculation

[0049] The contents of sulforaphane and sulforaphane in fresh vegetable samples are calculated according to the following formula:

[0050]

[0051] Where: ω——Content of sulforaphane or sulforaphane in fresh vegetable samples (ug / g);

[0052] ρ——Concentration of sulforaphane or sulforaphane in the test solution (ng / mL);

[0053] V——Volume of the test solution (mL);

[0054] m——Mass of the sample weighed (g).

[0055] 6. Results and analysis

[0056] 6.1 Determination of mobile phase conditions

[0057] Matrix effect will seriously affect the peak shape and sensitivity of sulforaphane and sulforaphane. The matrix and target compounds need to be effectively separated by gradient elution through the chromatographic column. This invention focuses on studying the effects of different mobile phase combinations on the retention time, peak width and response of sulforaphane and sulforaphane. The specific mobile phase settings are as follows: (a) Phase A is 0.1% formic acid - aqueous solution, Phase B is acetonitrile; (b) Phase A is 0.1% formic acid - aqueous solution, Phase B is methanol; (c) Phase A is pure water, Phase B is methanol. As Figure 1 shown, the results show that when using test (a) as the mobile phase combination, there is serious tailing. When using test (b) as the mobile phase combination, the tailing phenomenon is improved, and the peak area is higher. Compared with methanol, acetonitrile has an inhibitory effect on the response of sulforaphane and sulforaphane. When using test (c) as the mobile phase combination, the chromatographic peak is symmetric, there is no tailing phenomenon, and the peak area is the highest, about twice that of test (b).

[0058] In summary, according to the test results and considering the influence of solvent effect, this invention finally selects methanol - ultrapure water as the mobile phase, establishes a gradient elution process based on methanol and pure water, and the elution program is shown in Table 1. The chromatograms of sulforaphane and sulforaphane standard solutions are as Figure 2 shown.

[0059] 6.2 Influence of solvents on the extraction effects of sulforaphane and sulforaphane in cruciferous vegetables

[0060] The present invention focuses on comparing the extraction effects of two extraction solvents, dichloromethane and ethyl acetate. As Figure 3 shown, for two different varieties of radish samples, S-1 (Chunbulao) and S-2 (Caodoujiushi), ultrasonic extraction was carried out using two different solvents, dichloromethane and ethyl acetate. It can be clearly seen that ethyl acetate has a better extraction effect on the two compounds and a higher detected content. In addition, it can be clearly seen from the figure that the content of sulforaphene in variety S-2 (Caodoujiushi) is significantly higher than that in S-1 (Chunbulao), while the content of glucoraphanin is the opposite. Therefore, ethyl acetate was finally determined as the extraction organic solvent.

[0061] II. Methodological verification

[0062] Based on the above mixed standard working solutions of sulforaphene and glucoraphanin with concentrations of 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, 1000 ng / mL, 5000 ng / mL, and 10000 ng / mL, qualitative analysis was carried out by monitoring ions and retention times, and quantitative analysis was carried out by the external standard method. With the concentration of the target compound standard solution as the abscissa and the corresponding chromatographic peak area as the ordinate, a standard curve was plotted. The detection limit LOD of the instrument was calculated as 3 times the standard deviation of the response values of the lowest concentration point injected continuously 7 times, and 2 times the LOD value was used as the quantitation limit LOQ. The information of the standard working curve is shown in Table 3. The two compounds, sulforaphene and glucoraphanin, have a good linear relationship in the range of 1 - 10000 ng / mL, and R 2 is greater than 0.999. The detection limits are 0.172 ng / mL and 0.076 ng / mL respectively, and the quantitation limits are 0.344 ng / mL and 0.152 ng / mL respectively. The accuracy and precision of the method were verified by the recovery experiment. The spiked recovery rates of sulforaphene and glucoraphanin were 100.25% ± 8.25% and 98.08% ± 8.18% respectively, indicating that the method of the present invention has a good detection effect on the two target compounds, sulforaphene and glucoraphanin, and can meet the detection requirements.

[0063] Table 3 Standard curve

[0064]

[0065] III. Detection of actual samples

[0066] Fifteen white radish samples of three varieties (Baifen Tuan, Chun Bu Lao, and Jiu Guan) from three production areas were collected separately, homogenized, and 0.2 g of the radish homogenate sample was weighed into a 50 mL centrifuge tube. 3 mL of phosphate buffer with pH = 5.6 was added, mixed well, and enzymatically digested in a constant temperature shaker at 25 °C for 1 h. 40 mL of ethyl acetate was added, and ultrasonic extraction was carried out for 30 min. Centrifugation was performed at 6000 r / min for 5 min, and the upper organic phase was taken and placed in a 150 mL flat-bottom flask. The extraction was repeated 2 times, and the extraction solutions were combined, concentrated and evaporated to dryness using a rotary evaporator, fixed volume was made with 5 mL of methanol solution, and it was filtered through a 0.22 μm organic filter membrane. The contents of raphanin and sulforaphane in radish were qualitatively and quantitatively detected based on a UPLC-ESI-Q-Orbitrap HRMS combined instrument.

[0067] The detection results of 15 radish samples are shown in Figure 4 , the content of raphanin in Baifen Tuan was significantly lower than that in Chun Bu Lao and Jiu Guan, and the content of raphanin in Chun Bu Lao and Jiu Guan was close. For sulforaphane, the content of sulforaphane in Baifen Tuan radishes from three different production areas fluctuated greatly and there were significant differences, indicating that the production area has an important impact on the content of characteristic nutritional components in radish, and the contents of raphanin and sulforaphane in the three kinds of radishes in Production Area 1 were both high. In order to ensure the accurate identification and quantitative analysis of raphanin and sulforaphane, a procedural blank analysis was carried out simultaneously during the analysis of actual samples, and neither raphanin nor sulforaphane was detected in the blank samples.

[0068] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for synchronous detection of sulforaphane and sulforaphane in cruciferous vegetables by UHPLC-Q-Orbitrap HRMS, characterized in that, Proceed as follows: (1) Vegetable pretreatment: Select clean cruciferous vegetable samples and prepare homogenates; (2) Extraction and separation: Weigh a quantitative homogenate sample, add the prepared phosphate buffer solution, mix evenly, place it in a constant temperature shaker at 25 °C for enzymatic hydrolysis, add an organic solvent for extraction, centrifuge at high speed, take the organic phase, place it in a 150 mL flat-bottom flask, repeat the extraction once, combine the extraction solutions, concentrate to dryness, dissolve to a fixed volume with a methanol solution, filter through an organic phase filter membrane, and store at low temperature for on-machine analysis and determination; (3) Prepare the standard curve: Prepare mixed standard solutions of sulforaphane and sulforaphane at different concentration gradients; (4) Sample detection: Qualitative and quantitative analysis of sulforaphane and sulforaphane compounds is carried out by ultra-high performance liquid chromatography - electrostatic field orbitrap high-resolution mass spectrometry; Among them, the liquid chromatography column is selected as the ACQUITY UPLC BEH C18 chromatographic column, and the column temperature is 40 °C; Mobile phase: water is phase A, and methanol is phase B; The flow rate is 0.3 mL / min, and the injection volume is 1 μL; The initial mobile phase is 95% phase A and 5% phase B, maintained for 1 min, the phase B is gradually adjusted to 20% within 1 - 2 min, the phase B is adjusted to 40% within 2 - 3 min, maintained for 3 min, the phase B is reduced to 5% within 1 min, and maintained for 3 min; Mass spectrometry conditions: Electrospray ionization source ESI + Combined with Full mass mode, spray voltage 3.8 kV; capillary temperature 275 °C; auxiliary gas heater temperature 300 °C; sheath gas flow rate 35 arb; auxiliary gas flow rate 6 arb; S-lens RF level 50 V; detector is Orbitrap; mass spectrometry resolution is 70000 FWHM; scanning range is 50 - 550 m / z; maximum injection time is 100 ms; mass deviation is 5 ppm; MS automatic gain control target is 2.0×10 5 ; monitored ion is [M + H] + .

2. The synchronous detection method of sulforaphane and sulforaphane in cruciferous vegetables by UHPLC-Q-Orbitrap HRMS according to claim 1, characterized in that: The cruciferous vegetables are one of radish, broccoli, cauliflower, and Chinese cabbage.

3. The synchronous detection method of sulforaphane and sulforaphane in cruciferous vegetables by UHPLC-Q-Orbitrap HRMS according to claim 1 or 2, characterized in that: The operation in step (1) is as follows: Select multiple fresh cruciferous vegetables growing normally, wash them clean with tap water and deionized water respectively, dry them, take samples by the quartering method and mix them evenly, and put them into a high-speed tissue homogenizer to break them evenly.

4. The UHPLC-Q-Orbitrap HRMS synchronous detection method for sulforaphane and sulforaphane in cruciferous vegetables according to claim 3, characterized in that: In step (2), the oscillation parameter of the phosphate buffer solution with pH = 5.6 in the constant temperature shaker is 150 r / min, and the enzymatic hydrolysis time is 1 h.

5. The UHPLC-Q-Orbitrap HRMS synchronous detection method for sulforaphane and sulforaphane in cruciferous vegetables according to claim 4, characterized in that: The organic solvent used for extraction is ethyl acetate, and the extraction method is ultrasonic extraction for 30 min; The centrifugation rate is 6000 r / min, and the time is 5 min.

6. The synchronous detection method of sulforaphane and sulforaphane in cruciferous vegetables by UHPLC-Q-Orbitrap HRMS according to claim 5, characterized in that: In step (2), filtration is carried out using a Nylon66 0.22 μm organic system needle filter membrane; The storage temperature is -20 °C.

7. The synchronous detection method of sulforaphane and sulforaphane in cruciferous vegetables by UHPLC-Q-Orbitrap HRMS according to claim 6, characterized in that: In step (3), the concentration gradients of the compounds in the standard curve solution are 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, 1000 ng / mL, 5000 ng / mL, and 10000 ng / mL.

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