Method for determining glucosinolate

Abstract

The invention relates to the field of analytical chemistry, and discloses a glucosinolate determination method, which comprises: (1) mixing a sample to be determined with an extraction agent, and carrying out first extraction and first solid-liquid separation to obtain a supernatant; (2) mixing the supernate with a first solvent, and carrying out second extraction to obtain a lower-layer extracting solution; (3) measuring the content of glucosinolate in the lower-layer extracting solution; wherein the extracting agent comprises sodium chloride, sodium sulfate, HCl, methanol and water. The method can inhibit degradation of glucosinolate and further increase the content of glucosinolate in the extracting solution, the accuracy of the detection result is high, and the operation is simple, convenient and efficient.

Description

Technical Field

[0001] The present invention relates to the technical field of analytical chemistry, and in particular to a method for determining glucosinolates. Background Art

[0002] Glucosinolates (GLs), abbreviated as glucosinolates, are one of the main active ingredients of cruciferous plants and a class of sulfur-containing secondary metabolites. GLs can be divided into three categories based on the side chain R group: aliphatic GLs, aromatic GLs, and indole GLs, corresponding to aliphatic amino acids such as methionine, aromatic amino acids such as phenylalanine, and heterocyclic amino acids such as tryptophan, respectively. Currently, over 100 glucosinolate species have been discovered. Glucosinolates can be decomposed by myrosinase or microorganisms in the human intestine, initially generating unstable intermediates (β-glycosides), which then undergo Losen rearrangement to form products such as thiocyanates (TCs), isothiocyanates (ITCs), oxazolidinethiones (OZTs), and nitriles (RCNs). These metabolites have strong antioxidant, anticancer, and antibacterial properties.

[0003] There are several main methods for determining glucosinolates in rapeseed: (1) Glucose and sulfate method. The main principle is that glucosinolates (GLs) are hydrolyzed by myrosinase (MYR) to produce sulfate ions and glucose. The former can be quantitatively analyzed by barium chloride titration and the barium sulfate crystals produced by X-ray emission spectroscopy. The latter undergoes dehydration and ring closure under the action of strong acid to generate furfural derivatives, which then condense with thymol to form a red product for quantitative analysis. However, when quantifying GLs, care must be taken to exclude the influence of endogenous glucose in the sample. The sample must also be pre-purified during the determination, and the sample preparation and analysis steps are relatively cumbersome. (2) Near-infrared reflectance spectroscopy (NIRS) is a fast, accurate, and sample-nondestructive analysis method. It establishes a model by analyzing a large number of samples and then performs relevant analysis. Therefore, it has high requirements for the accuracy of the sample standard chemical value and the representativeness of the sample. High-quality samples are needed to improve accuracy. (3) High-performance capillary electrophoresis technology (HPCE method) has the main principle of directly determining the complete glucosinolate content based on the difference in charge and polarity of different glucosinolate components using high-performance capillary electrophoresis. It is an efficient and low-cost analysis method. This method can save the process of desulfurization treatment, has the advantages of low reagent and instrument prices, few experimental interference factors, and easy control of separation conditions. However, as it is a newly developed separation and analysis technology, its application in the quantification of GLs is immature and has not yet been widely used. (4) Gas chromatography method: first trimethylsilane derivatization or sulfatase desulfurization treatment is performed, and then the sample is quantitatively analyzed. However, indole GLs will decompose under heat, and the instrument analysis parameters need to be carefully optimized, which is not conducive to the detection and analysis of GLs. Therefore, the application range of gas chromatography is relatively narrow. (5) High performance liquid chromatography (HPLC method) is currently the most widely used method for the quantitative determination of glucosinolates in the world. It can avoid the conversion of GLs into volatile derivatives. When combined with mass spectrometry, it can also avoid the desulfurization step. It is an efficient and accurate method. However, most of the pre-treatment methods for the machine use freezing treatment conditions, which is relatively complicated. Therefore, it is very necessary to establish an identification method that can detect multiple glucosinolate substances and is efficient, rapid, with a low detection limit and high sensitivity. Summary of the Invention

[0004] The present invention aims to overcome the problems in the prior art of glucosinolate detection such as complex pretreatment and severe degradation, which lead to low content of extracted glucosinolates, large errors in detection results, and low accuracy. The present invention provides a method for determining glucosinolates, which can inhibit the degradation of glucosinolates, has high accuracy in detection results, is simple to operate, and is highly efficient.

[0005] In the present invention, since there is no rapeseed raw material without glucosinolates and it is impossible to remove glucosinolates without destroying myrosinase, it is impossible to carry out spiked recovery experiments of glucosinolates in rapeseed matrix and measure the accuracy of the method by the recovery rate. Therefore, the extraction effect and accuracy of the present invention are measured by measuring the content of glucosinolates in the extracts prepared by different extraction methods.

[0006] In order to achieve the above object, the present invention provides a method for determining glucosinolates, which comprises the following steps:

[0007] (1) mixing a sample to be tested with an extractant, performing a first extraction and a first solid-liquid separation to obtain a supernatant;

[0008] (4) mixing the supernatant with a second solvent and performing a second extraction to obtain a lower layer extract;

[0009] (5) determining the content of glucosinolates in the lower layer extract;

[0010] Wherein, the extractant includes sodium chloride, sodium sulfate, hydrochloric acid, methanol and water.

[0011] Through the above technical solution, at least the following beneficial effects can be achieved:

[0012] (1) The method of the present invention can reduce sample moisture absorption, reduce the degradation of glucosinolates after oilseed crushing, and further increase the content of glucosinolates in the extract. In a preferred embodiment, the content of 14 glucosinolates in the extract is as high as 22124.5 μg / g. In addition, the method of the present invention can completely separate the 14 glucosinolates during detection, with good separation effect, further improving the sensitivity and accuracy of the detection results.

[0013] (2) The reagents used in the method of the present invention are environmentally friendly, and the entire experiment can be completed at room temperature without heating, which is more energy-efficient, safer, and takes less time to detect;

[0014] (3) The method of the present invention can further reduce the interference of oil and protein in the sample liquid on the target substance (glucosinolate), reduce the contamination of the chromatographic column and instrument, and extend the service life of the chromatographic column and instrument. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1(a) to Figure 1(n) These are the ion chromatograms of 14 glucosinolates in rapeseed seeds; Figure 1(a)-Figure 1(n) The glucosinolates in the formula correspond to the 14 glucosinolates numbered 1-14 in Table 2.

[0016] Figure 2(a), Figure 2(b) and2(c) The mass spectrometry TIC images of glucosinolates in rapeseed seeds separated by the methods in Example 1, Example 3, and Example 4 are respectively shown; wherein, Figure 2(a) is the UPLC T3 column used in Example 1, Figure 2(b) is the Waters BEH C18 column used in Example 3, and Figure 2(c) is the ACQUITY UPLC BEHHillic column used in Example 4. DETAILED DESCRIPTION

[0017] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0018] The present invention provides a method for determining glucosinolates, which comprises the following steps:

[0019] (1) mixing a sample to be tested with an extractant, performing a first extraction and a first solid-liquid separation to obtain a supernatant;

[0020] (2) mixing the supernatant with a second solvent and performing a second extraction to obtain a lower layer extract;

[0021] (3) determining the content of glucosinolates in the lower layer extract;

[0022] Wherein, the extractant contains sodium chloride, sodium sulfate, HCl, methanol and water.

[0023] According to some embodiments of the present invention, based on 1 g of the sample to be tested, the amount of the extractant used is 5-20 mL, preferably 7-10 mL.

[0024] Preferably, based on 1 g of the sample to be tested, the amount of water in the extractant is 2-7 mL, preferably 3-6 mL.

[0025] Preferably, based on 1 g of the sample to be tested, the amount of methanol in the extractant is 5-14 mL, preferably 6-10 mL.

[0026] Preferably, based on 1 g of the sample to be tested, the molar amount of sodium chloride in the extractant is 3-15 mmol, more preferably 6-12 mmol.

[0027] Preferably, the molar ratio of sodium chloride, sodium sulfate and HCl in the extractant is 1:(0.2-0.4):(0.2-0.5).

[0028] In the present invention, sodium chloride, sodium sulfate and HCl in the extractant can be added in the form of chemical reagents or in the form of solutions.

[0029] In the present invention, the amount of water in the extractant refers to the total amount of water used in preparing the extractant. For example, when preparing the extractant, sodium chloride, sodium sulfate and HCl are added in the form of a solution, then the amount of water in the extractant is understood to be the total volume of the sodium chloride solution, sodium sulfate solution and hydrochloric acid; when preparing the extractant, sodium chloride, sodium sulfate and HCl are added in the form of chemical reagents, then the amount of water in the extractant is understood to be the additional amount of water required.

[0030] In the present invention, there is no particular limitation on the preparation method of the extractant. However, considering that the solubility of each substance in the extractant in water or methanol is different, resulting in a large deviation between the actual molar concentration of each substance in the extractant and the theoretical molar concentration, thereby affecting the extraction efficiency of glucosinolates, preferably, the preparation method of the extractant includes separately preparing sodium chloride, sodium sulfate and HCl into solutions.

[0031] In the present invention, the methanol in the extractant can be added in the form of a methanol reagent or a methanol aqueous solution, preferably a methanol aqueous solution.

[0032] Preferably, the volume concentration of methanol in the methanol aqueous solution is 90-95 vol%.

[0033] The inventors of the present invention have discovered that adding the extractant of the present invention before extracting the test sample (oil seeds) can quickly inactivate the myrosinase in the test sample, thereby inhibiting the degradation of glucosinolates after the test sample (oil seeds) is crushed, further improving the sensitivity and accuracy of the test results.

[0034] According to some embodiments of the present invention, the first solvent is selected from at least one of n-hexane, chloroform and carbon tetrachloride.

[0035] According to some embodiments of the present invention, based on 2 mL of the supernatant, the amount of the second solvent used is 2-4 mL.

[0036] In the present invention, the first solvent described in the present invention is used for extraction, which can effectively reduce the interference of oil, protein, etc. on the target (glucosinolate), and at the same time reduce the entry of these substances into the detection system, thereby protecting the chromatographic column and instrument and extending the service life of the instrument.

[0037] According to some embodiments of the present invention, the first extraction method includes sequentially performing leaching extraction and vortex extraction.

[0038] Preferably, the extraction method includes homogenizing and crushing in a high-speed homogenizer.

[0039] More preferably, the homogenizing and crushing conditions include: a rotation speed of 5000-10000 rpm, preferably 6000-8000 rpm; and a time of 1-5 min, preferably 1-3 min.

[0040] Preferably, the conditions for the vortex extraction include: a rotation speed of 1000-3000 rpm, preferably 2000-3000 rpm, and a time of 10-240 min, preferably 10-30 min.

[0041] According to some embodiments of the present invention, the second extraction method is vortex extraction, and the conditions of the vortex extraction include: a rotation speed of 1000-3000 rpm; and a time of 1-3 min.

[0042] According to some embodiments of the present invention, the first solid-liquid separation method is centrifugation.

[0043] Preferably, the centrifugal conditions include: a rotation speed ≥ 4000 rpm, preferably 5000-6000 rpm; and a time of 5-20 min, preferably 5-10 min.

[0044] In the present invention, preferably, the method further comprises mixing the supernatant with the first solvent for the second extraction and centrifuging the mixed solution to obtain a lower layer extract.

[0045] Preferably, the centrifugal conditions include: a rotation speed ≥ 4000 rpm, preferably 5000-6000 rpm; and a time of 5-20 min, preferably 5-10 min.

[0046] According to some embodiments of the present invention, the sample to be tested is selected from oil seeds and / or oil cakes after oil is pressed from oil seeds.

[0047] Preferably, the oilseeds are selected from at least one of rapeseed, linseed, peanut, sunflower seed, corn and soybean.

[0048] According to some embodiments of the present invention, in step (3), the content of glucosinolates in the lower layer extract is determined by high performance liquid chromatography-tandem mass spectrometry.

[0049] In the present invention, the method of determining the content of glucosinolates in the lower layer extract by high performance liquid chromatography-tandem mass spectrometry includes the following steps: 1) using high performance liquid chromatography-tandem mass spectrometry to perform qualitative and quantitative analysis on 14 glucosinolates in the test solution to obtain the chromatographic peak area of ​​each glucosinolate compound in the test sample; 2) establishing a concentration-chromatographic peak area standard curve of a glucosinolate compound standard, substituting the obtained chromatographic peak area of ​​the test sample into the curve and calculating to obtain the content of the glucosinolate compound in the test sample.

[0050] In the present invention, the 14 glucosinolate compounds include: trans-2-hydroxy-3-butenyl glucosinolate; propenyl glucosinolate; 3-butenyl glucosinolate; 4-hydroxy-3-indolemethyl glucosinolate; 4-pentenyl glucosinolate; benzyl (benzyl) glucosinolate; 4-methylsulfoxide butyl glucosinolate; 5-methylsulfoxide pentyl glucosinolate; 3-indolemethyl glucosinolate; phenethyl glucosinolate; 4-methoxy-3-indolemethyl glucosinolate; 1-methoxy-3-indolemethyl glucosinolate; 4-methylsulfoxide butyl glucosinolate; and 2-hydroxy-3-butenyl glucosinolate.

[0051] In the present invention, the parameters of the high performance liquid chromatography include: a UPLC T3 (2.1×100 mm, 1.8 μm) column or an equivalent column; a column temperature of 30-50° C.; mobile phase A of 0.05%-0.2% formic acid aqueous solution, and mobile phase B of methanol; a flow rate of 0.2-0.5 mL / min; and an injection volume of 1-2 μL.

[0052] In the present invention, the elution mode of high performance liquid chromatography is gradient elution, specifically:

[0053] 0-2.99 min, 95% A;

[0054] 3.00-4.99 min, 50% A;

[0055] 5.00min-8.00min, 5%A;

[0056] 8.01min-11min, 95%A.

[0057] In the present invention, the parameters of mass spectrometry detection are specifically as follows: ion source: electrospray ion source (ESI), ion source temperature 300-350°C; electrospray voltage is -4 to -2 kV; scanning mode is parent ion scanning, negative ion mode; detection method is multiple reaction monitoring mode (MRM); capillary voltage: 1.5 kV; cone voltage: 20 V.

[0058] According to some implementation methods of the present invention, the method further comprises filtering and diluting the lower supernatant in sequence.

[0059] In the present invention, the concentration of the glucosinolate compound in the test solution is preferably within the concentration range of the glucosinolate compound in the standard solution when establishing the standard curve; if the concentration of the glucosinolate compound in the test solution exceeds the concentration range of the glucosinolate compound in the standard solution, the test solution needs to be diluted.

[0060] Preferably, the dilution method comprises mixing the filtered clear liquid with a diluent.

[0061] Preferably, the amount of the diluent is such that the volume of the filtered clear liquid increases by 9-99 times.

[0062] According to some embodiments of the present invention, the diluent comprises methanol, formic acid and water.

[0063] Preferably, in the diluent, the volume ratio of the total volume of formic acid and water to methanol is (90-95):1.

[0064] Preferably, the volume concentration of formic acid in the diluent is 0.1-0.5 vol%.

[0065] In the present invention, there is no particular limitation on the preparation method of the diluent. The preparation method can be: accurately pipette 5-10 mL of methanol into a 100 mL volumetric flask, dilute to volume with a 0.1 vol% formic acid aqueous solution, mix well, and store in a refrigerator at 0-4°C away from light.

[0066] Preferably, the filtering conditions include: the pore size of the filter membrane is less than 2 μm.

[0067] According to a particularly preferred embodiment of the present invention, a method for determining glucosinolates is provided, the method comprising the following steps:

[0068] (1) mixing the oilseeds to be tested with an extractant, and sequentially performing a first extraction and a first solid-liquid separation to obtain a supernatant;

[0069] The first extraction method includes leaching and vortex extraction; the leaching method includes homogenizing and crushing in a high-speed homogenizer, and the homogenizing and crushing conditions include: a speed of 6000-8000 rpm and a time of 1-3 minutes; the vortex extraction conditions include: a speed of 2000-3000 rpm and a time of 15-30 minutes;

[0070] Wherein, the first solid-liquid separation method is centrifugation; the centrifugation conditions include: a rotation speed of 5000-6000 rpm; a time of 5-10 minutes;

[0071] wherein the extractant contains sodium chloride, sodium sulfate, HCl, methanol and water;

[0072] Wherein, based on 1g of the sample to be tested, the amount of the extractant used is 9-11mL;

[0073] Wherein, based on 1g of the sample to be tested, the amount of water in the extractant is 3-4mL, the amount of methanol is 6-7mL, and the molar amount of sodium chloride is 3-6mmol;

[0074] wherein the molar ratio of sodium chloride, sodium sulfate, and HCl in the extractant is 1:(0.3-0.4):(0.4-0.5);

[0075] (2) mixing the supernatant with the first solvent, performing a second extraction and centrifugation in sequence to obtain a lower layer extract; filtering the lower layer extract in sequence (the pore size of the filter membrane is less than 2 μm) and diluting the lower layer extract to prepare a sample solution for later use;

[0076] Wherein, the first solvent is n-hexane; based on 2 mL of the supernatant, the amount of n-hexane used is 2-4 mL;

[0077] Wherein, the second extraction method is vortex extraction, and the conditions of the vortex extraction include: a rotation speed of 2000-3000 rpm and a time of 1-3 min;

[0078] The centrifugal conditions include: a rotation speed of 5000-6000 rpm; a time of 5-10 min;

[0079] The dilution method includes mixing the filtered clear liquid with a diluent, wherein the amount of the diluent is such that the volume of the filtered clear liquid increases by 9-19 times;

[0080] (3) Liquid chromatography-tandem mass spectrometry (HPLC-MS) was used to qualitatively and quantitatively analyze the contents of 14 glucosinolates in the test sample.

[0081] The present invention will be described in detail below through examples.

[0082] In the following examples and comparative examples, the qualitative and quantitative analysis of 14 glucosinolates in oilseeds was performed using liquid chromatography-tandem mass spectrometry (HPLC-MS). The mass spectrometry conditions for the 14 glucosinolates are shown in Table 2, and the ion chromatograms are shown in Figure 1.

[0083] The liquid chromatography-tandem mass spectrometry instrument used in the following examples and comparative examples was purchased from AB SCIEX, and the instrument model is AB 5500.

[0084] Unless otherwise specified, all reagents were of chromatographic grade, the purity of glucosinolate standards was ≥98%, and the water was Grade 1 water as specified in GB / T6682-2008 Specifications and Test Methods for Water Used in Analytical Laboratories.

[0085] Example 1

[0086] A method for extracting and detecting rapeseed glucosinolates, comprising the following steps:

[0087] S1: Sample pretreatment and glucosinolate extraction: Weigh 1.0 g (accurate to 0.01 g) of rapeseed in a 50 mL plastic centrifuge tube, add 1 mL of sodium chloride solution (molar concentration of 6 mol / L), 1 mL of sodium sulfate solution (molar concentration of 2.4 mol / L), and 1 mL of hydrochloric acid (molar concentration of 3 mol / L), then add 7 mL of methanol, homogenize and break at 8000 rpm for 1 min, vortex extract for 30 min (vortex extraction speed is 2000 rpm), and then centrifuge at 5000 rpm for 5 min; then take 2 mL of methanol-water extract, add 2 mL of n-hexane and vortex extract for 1 min (vortex extraction speed is 2000 rpm), centrifuge at 5000 rpm for 5 min, take the lower layer of methanol-water extract, filter through a 0.22 μm organic phase filter membrane, and dilute 20 times with diluent as the sample solution to be tested;

[0088] S2: Liquid chromatography-tandem mass spectrometry (HPLC-MS) was used to qualitatively and quantitatively analyze the contents of 14 glucosinolates in the test sample solution. The test results are shown in Figure 2 and Table 3;

[0089] The specific analysis conditions of HPLC-MS are as follows: chromatographic column: UPLC T3 (2.1×100 mm, 1.8 μm) chromatographic column; mobile phase (see Table 1): A: 0.1% formic acid-water solution, B: methanol; elution mode: gradient elution (see Table 1); flow rate: 0.3 mL / min; column temperature: 30°C; injection volume: 1 μL; mass spectrometry ion source: electrospray ionization source (ESI); ion source temperature: 350°C; electrospray voltage: -4.0 kV; scan mode: parent ion scan, negative ion mode; detection mode: multiple reaction monitoring mode (MRM); capillary voltage: 1.5 kV; cone voltage: 20 V.

[0090] Example 2

[0091] A method for extracting and detecting rapeseed glucosinolates, comprising the following steps:

[0092] S1: Sample pretreatment and glucosinolate extraction: Weigh 1.0 g (accurate to 0.01 g) of rapeseed in a 50 mL plastic centrifuge tube, add 2 mL of sodium chloride solution (molar concentration of 6 mol / L), 2 mL of sodium sulfate solution (molar concentration of 2.4 mol / L), and 2 mL of hydrochloric acid (molar concentration of 3 mol / L), then add 10 mL of methanol, homogenize and break at 5000 rpm for 1 min, vortex for 10 min (vortex extraction speed is 2000 rpm), and then centrifuge at 5000 rpm for 5 min; then take 2 mL of methanol-water extract, add 2 mL of carbon tetrachloride and vortex extract for 1 min (vortex extraction speed is 2000 rpm), centrifuge at 6000 rpm for 5 min, take the lower layer of methanol-water extract, pass through a 0.22 μm organic phase filter membrane, and dilute 20 times with diluent as the sample solution to be tested;

[0093] S2: Liquid chromatography-tandem mass spectrometry (HPLC-MS) was used to qualitatively and quantitatively analyze the contents of 14 glucosinolates in the sample solution. The test results are shown in Table 3;

[0094] The specific analysis conditions of HPLC-MS are as follows: chromatographic column: UPLC T3 (2.1×100 mm, 1.8 μm) chromatographic column, or equivalent chromatographic column; mobile phase (see Table 1): A: 0.1% formic acid-water solution, B: methanol; elution mode: gradient elution (see Table 1); flow rate: 0.3 mL / min; column temperature: 30°C; injection volume: 1 μL; mass spectrometry ion source: electrospray ionization source (ESI); ion source temperature: 350°C; electrospray voltage: -4.0 kV; scan mode: parent ion scan, negative ion mode; detection mode: multiple reaction monitoring mode (MRM); capillary voltage: 1.5 kV; cone voltage: 20 V.

[0095] Example 3

[0096] The method of Example 1 was followed, except that in S2, the UPLC T3 column was replaced with a Waters BEHC18 (2.1×100 mm, 1.7 μm) column. The test results are shown in FIG2 and Table 3.

[0097] Example 4

[0098] The method of Example 1 was followed, except that in S2, the UPLC T3 column was replaced with an ACQUITY UPLC BEH Hillic (2.1×100 mm, 1.7 μm) column. The test results are shown in FIG2 and Table 3.

[0099] Example 5

[0100] The method of Example 1 was followed, except that in S1, a water bath heating method was used for the first extraction. Specifically, 1.0 g (accurate to 0.01 g) of rapeseed was weighed into a 50 mL plastic centrifuge tube. 1 mL of sodium chloride solution, 1 mL of sodium sulfate solution, and 1 mL of hydrochloric acid were added, followed by 7 mL of methanol. The mixture was homogenized at 8000 rpm for 1 min and then incubated in a 75°C water bath for 30 min. The test results are shown in Table 3.

[0101] Example 6

[0102] The method of Example 1 was followed, except that in S1, the molar amount of the sodium chloride solution was 1 mmol, the molar amount of the sodium sulfate solution was 0.5 mmol, and the molar amount of the hydrochloric acid was 1 mmol. The test results are shown in Table 3.

[0103] Example 7

[0104] The method of Example 1 was followed, except that in S1, n-hexane was replaced with isopropanol. The test results are shown in Table 3.

[0105] Comparative Example 1

[0106] The method of Example 1 was followed, except that in S1, the extractant was 3 mL of water and 7 mL of methanol. The test results are shown in Table 3.

[0107] Comparative Example 2

[0108] The method of Example 1 was followed, except that in S1, the extractant was 3 mL of sodium chloride solution (molar concentration of 6 mol / L) and 7 mL of methanol. The test results are shown in Table 3.

[0109] Comparative Example 3

[0110] The method of Example 1 was followed, except that in S1, the extractant was 3 mL of sodium sulfate solution (molar concentration of 2.4 mol / L) and 7 mL of methanol. The test results are shown in Table 3.

[0111] Comparative Example 4

[0112] The method of Example 1 was followed, except that in S1, the extractant was 3 mL of silver nitrate solution (molar concentration of 0.05 mmol / L) and 7 mL of methanol. The test results are shown in Table 3.

[0113] Comparative Example 5

[0114] The method of Example 1 was followed, except that in S1, the extractant was 3 mL of hydrochloric acid (molar concentration of 3 mol / L) and 7 mL of methanol. The test results are shown in Table 3.

[0115] Comparative Example 6

[0116] The method of Example 1 was followed, except that in S1, the sodium chloride in the extractant was replaced by potassium chloride, and the other components remained unchanged. The test results are shown in Table 3.

[0117] Comparative Example 7

[0118] The method of Example 1 was followed, except that in S1, the sodium sulfate in the extractant was replaced with potassium sulfate, and the other components remained unchanged. The test results are shown in Table 3.

[0119] Comparative Example 8

[0120] The method of Example 1 was followed, except that in S1, the hydrochloric acid in the extractant was replaced by sulfuric acid, and the other components remained unchanged. The test results are shown in Table 3.

[0121] Comparative Example 9

[0122] The method of Example 1 was followed, except that in S1, the methanol in the extractant was replaced by ether, and the other components remained unchanged. The test results are shown in Table 3.

[0123] Table 1

[0124]

[0125] Table 2

[0126]

[0127]

[0128] Table 3

[0129]

[0130] Table 3 (continued)

[0131]

[0132]

[0133] Different chromatographic columns were used to separate glucosinolates, and the peak results of the mass spectrum TIC graph are shown in Figure 2. As shown in Figure 2(a), the T3 chromatographic column in Example 1 was used to separate the 14 glucosinolates separated from the oil seeds. The peak shape of the glucosinolates was relatively ideal, the multiple components could be completely separated, and the separation effect was the best. As shown in Figure 2(b), the C18 chromatographic column in Example 3 was used to separate the 14 glucosinolates separated from the oil seeds. The peak time of the glucosinolates was too early, and the retention effect was poor. As shown in Figure 2(c), the Hillic chromatographic column in Example 4 was used to separate the 14 glucosinolates. The peak shape of the glucosinolates was also unsatisfactory, and the multiple components could not be completely separated. In particular, several substances that peaked early could not be separated. In addition, the peak was severely broadened.

[0134] As can be seen from Table 3, by comparing Examples 1-2 with Examples 5-7, it can be seen that the use of the preferred method to extract and detect 14 glucosinolates in oil seeds can further reduce the degradation of glucosinolates after the oil seeds are crushed, thereby improving the sensitivity and accuracy of the glucosinolate detection results.

[0135] Compared with the embodiment, comparative examples 1-9 respectively changed the composition of the extractant, and the degradation of 14 glucosinolates in oil seeds resulted in inaccurate detection results.

[0136] In summary, the method of the present invention can inhibit the degradation of 14 glucosinolates in oil seeds and further increase the content of glucosinolates in the extract; in addition, the method of the present invention can completely separate the 14 glucosinolates during detection, with good separation effect, high accuracy, simple operation and high efficiency.

[0137] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. A method for determining glucosinolates, characterized in that: The method comprises the following steps: (1) mixing a sample to be tested with an extractant, performing a first extraction and a first solid-liquid separation to obtain a supernatant; (2) mixing the supernatant with the first solvent and performing a second extraction to obtain a lower layer extract; (3) determining the content of glucosinolates in the lower layer extract; Wherein, the extractant contains sodium chloride, sodium sulfate, HCl, methanol and water.

2. The method according to claim 1, wherein Based on 1 g of the sample to be tested, the amount of the extractant used is 5-20 mL, preferably 7-10 mL; Preferably, based on 1 g of the sample to be tested, the amount of water in the extractant is 2-7 mL, preferably 3-6 mL; Preferably, based on 1 g of the sample to be tested, the amount of methanol in the extractant is 5-14 mL, preferably 6-10 mL; Preferably, based on 1 g of the sample to be tested, the molar amount of sodium chloride in the extractant is 3-15 mmol, more preferably 6-12 mmol; Preferably, the molar ratio of sodium chloride, sodium sulfate and HCl in the extractant is 1:(0.2-0.4):(0.2-0.5).

3. The method according to claim 1 or 2, wherein: The first solvent is selected from at least one of n-hexane, chloroform and carbon tetrachloride; And / or, based on 2 mL of the supernatant, the amount of the first solvent used is 2-4 mL.

4. The method according to any one of claims 1 to 3, wherein: The first extraction method includes sequentially performing leaching and vortex extraction; Preferably, the extraction method includes homogenizing and crushing in a high-speed homogenizer; More preferably, the homogenizing and crushing conditions include: a rotation speed of 5000-10000 rpm, preferably 6000-8000 rpm; a time of 1-5 min, preferably 1-3 min; Preferably, the conditions for the vortex extraction include: a rotation speed of 1000-3000 rpm, preferably 2000-3000 rpm; and a time of 10-240 min, preferably 10-30 min.

5. The method according to any one of claims 1 to 4, wherein: The second extraction method is vortex extraction, and the conditions of the vortex extraction include: a rotation speed of 1000-3000 rpm; a time of 1-3 minutes; And / or, the first solid-liquid separation method is centrifugation; Preferably, the centrifugal conditions include: a rotation speed ≥ 4000 rpm, preferably 5000-6000 rpm; and a time of 5-20 min, preferably 5-10 min.

6. The method according to any one of claims 1 to 5, wherein: The sample to be tested is selected from oil seeds and / or oil cakes after oil is pressed from oil seeds; Preferably, the oilseeds are selected from at least one of rapeseed, linseed, peanut, sunflower seed, corn and soybean.

7. The method according to any one of claims 1 to 6, wherein: In step (3), the content of glucosinolates in the lower layer extract is determined by high performance liquid chromatography-tandem mass spectrometry.

8. The method according to any one of claims 1 to 7, wherein: The method further comprises filtering and diluting the lower layer extract in sequence; Preferably, the filtering conditions include: the pore size of the filter membrane is less than 2 μm.

9. The method according to claim 8, wherein The dilution method includes mixing the filtered clear liquid with a diluent; Preferably, the amount of the diluent is such that the volume of the filtered clear liquid increases by 9-99 times.

10. The method according to claim 9, wherein: The diluent includes methanol, formic acid and water; Preferably, in the diluent, the volume ratio of the total volume of formic acid and water to methanol is (90-95):1; Preferably, the volume concentration of formic acid in the diluent is 0.1-0.5 vol%.

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