CN113391001B - Detection method of glucosinolate compounds - Google Patents
Detection method of glucosinolate compounds Download PDFInfo
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- CN113391001B CN113391001B CN202110782601.2A CN202110782601A CN113391001B CN 113391001 B CN113391001 B CN 113391001B CN 202110782601 A CN202110782601 A CN 202110782601A CN 113391001 B CN113391001 B CN 113391001B
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Abstract
The invention belongs to the technical field of analytical chemistry, and provides a detection method of a glucosinolate compound. In the invention, the purpose of high-efficiency qualitative determination is achieved by adopting the common secondary mass spectrum fragments of the glucosinolate compounds; in the secondary mass spectrum fragments, a set of parent ion (precursor ion) mode scanning qualitative method is constructed based on the fragments with the m/z of 96.0, so that the detection sensitivity can be ensured, the qualitative accuracy can be improved, and the false positive detection results can be reduced; based on fragments with m/z of 96.0 as quantitative ions, a set of multi-reaction monitoring (MRM) mode scanning quantitative method is constructed by using glucosinolate compound standard substances, so that the quantitative analysis of all glucosinolate compounds in a sample is completed, the problem that the quantitative analysis cannot be performed due to the difficulty in obtaining certain standard substances is solved, and the independent quantification and the total amount determination of the compounds in the sample are efficiently completed.
Description
Technical Field
The invention relates to the technical field of analytical chemistry, in particular to a detection method of a glucosinolate compound.
Background
Glucosinolates (GLS), called glucosinolates for short, are important secondary metabolites in cruciferous vegetables, and more than 100 kinds of glucosinolates are found at present, and the types and the contents of the glucosinolates in different varieties of vegetables are obviously different. According to the difference of side chain R groups in the glucosinolate, the glucosinolate can be divided into 3 types, namely aliphatic type, aromatic type and indole type glucosinolates. When the glucosinolate-containing vegetables are eaten or mechanically disrupted, endogenous myrosinase (myrosinase) is released, which cleaves the beta-glucosinolate bonds in the glucosinolates to produce isothiocyanates (isothiocyanates). Research shows that the isothiocyanate has the functions of resisting cancer, resisting oxidation, resisting diabetes, etc. Among them, sulforaphane (sulforaphane), an enzymatic hydrolysis product of 4-methylsulfonylbutylsulfenthioside (glucoraphane), is the strongest inducer for Phase II enzyme so far discovered, and can inactivate oncogenes.
The method for measuring the glucosinolates in the cauliflower reported in the literature at present mainly comprises the following steps: (1) near Infrared Reflectance Spectroscopy (NIRS) is mainly characterized in that an infrared analysis model is established through known standard products, sample data are compared, and the content of glucosinolate is rapidly determined; the near infrared reflectance spectroscopy can be used for nondestructive detection, the sampling amount is small, but a large amount of standard articles are needed to establish a credible model. (2) The fluorescence detection method mainly adopts the principle that glucosinolate is degraded under the action of myrosinase to obtain glucose, the glucose is subjected to the action of glucose oxidase to obtain gluconic acid, and the gluconic acid reacts with a reagent to generate a fluorescence active substance; the fluorescence detection method has many detection steps, which can increase the error of the detection result. (3) The micellar electrokinetic capillary electrophoresis (HPCED) method mainly utilizes the difference of charges carried by glucosinolate molecules, adds a surfactant and separates the glucosinolate in a borate buffer system; the micelle electrokinetic capillary electrophoresis method has few interference factors and low cost, but has long separation time. (4) Gas Chromatography (GC), i.e., carrying out enzymolysis on thioglycoside to obtain isothiocyanate, and then measuring by using gas chromatography; thioglycoside, which is easily decomposed by heat, cannot be detected by gas chromatography. (5) Determining thioglycoside by using High Performance Liquid Chromatography (HPLC) in combination with an ultraviolet detector (UV) or a Diode Array (DAD), performing derivatization modification on isothiocyanate by using pre-column derivatization, and determining by using High Performance Liquid Chromatography (HPLC); (6) measuring glucosinolate and isothiocyanate by High Performance Liquid Chromatography (HPLC) and Mass Spectrometry (MS); the high performance liquid chromatography can simultaneously measure the contents of various glucosinolates, has good reproducibility and precision, and is relatively common in practical application, but the detection limit and the sensitivity of the method are poor, and the method application limitation exists on cauliflower varieties with low glucosinolate contents.
Disclosure of Invention
In view of this, the present invention aims to provide a method for detecting glucosinolates. The detection method provided by the invention has low detection limit and high sensitivity.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a detection method of a glucosinolate compound, which comprises the following steps:
mixing a sample to be detected with an extracting agent, and sequentially carrying out extraction and solid-liquid separation to obtain supernatant serving as a solution to be detected;
performing qualitative analysis on the solution to be detected by utilizing a first ultra-high liquid chromatography and mass spectrometry;
carrying out quantitative analysis on the solution to be detected by utilizing a second ultra-high liquid chromatography and mass spectrometry to obtain the chromatographic peak area of the glucosinolate compound in the sample to be detected;
substituting the chromatographic peak area of the glucosinolate compound in the sample to be detected into a concentration-chromatographic peak area standard curve of a glucosinolate compound standard substance to obtain the content of the glucosinolate compound in the sample to be detected;
the parameters of the first ultra-high liquid chromatography and mass spectrometry comprise a first ultra-high liquid chromatography parameter and a first mass spectrometry parameter:
the first ultra-high liquid chromatography parameter comprises:
the chromatographic column is Merck ZIC-HILIC column, 100mm × 2.1mm, 3.5 μm
The temperature of the chromatographic column is 30-50 ℃;
the mobile phase A is acetonitrile, and the mobile phase B is 5-10 mmol/L ammonium acetate water solution; the flow rate is 0.2-0.5 mL/min; the sample injection volume is 3-6 mu L;
the elution mode is gradient elution;
the gradient elution is:
0～1min，90％A；
1～9min，90％～65％A；
the first mass spectral parameter comprises:
the scanning mode is a parent ion scanning mode and a negative ion mode;
the electrospray voltage is-4.5 kV, the ion source temperature is 450 ℃, the daughter ions are 96.0Da, the scanning range of the mother ions is 300.0-800.0 Da, the scanning speed is 200Da/s, the gas curtain gas is 40.00psi, the spraying gas is 45psi, and the auxiliary heating gas is 45 psi;
the parameters of the second ultra-high liquid chromatography and mass spectrometry comprise a second ultra-high liquid chromatography parameter and a second mass spectrometry parameter:
the second ultra-high liquid chromatography parameter comprises:
the chromatographic column is Merck ZIC-HILIC column, 100mm × 2.1mm, 3.5 μm
The temperature of the chromatographic column is 30-50 ℃;
the mobile phase A is acetonitrile, and the mobile phase B is 5-10 mmol/L ammonium acetate water solution; the flow rate is 0.2-0.5 mL/min; the sample injection volume is 3-6 mu L;
the elution mode is gradient elution;
the gradient elution comprises:
0～1min，90％A；
1～9min，90％～65％A；
the second mass spectral parameters include:
scanning mode: MRM;
electrospray voltage was-4.5 kV, ion source temperature was 450 ℃, scan rate was 200Da/s, curtain gas was 40.00psi, spray gas was 45psi, and auxiliary heating gas was 45 psi.
Preferably, the particle size of the sample to be detected is 250-830 mu m.
Preferably, the sample to be tested is prepared by the following steps:
and sequentially carrying out freeze drying and crushing on the raw materials to be detected to obtain the sample to be detected.
Preferably, the raw material to be detected comprises cauliflower or green cauliflower leaves.
Preferably, the temperature of the freeze drying is-40 to-60 ℃, and the time is 12 to 48 hours.
Preferably, the extracting agent is a methanol aqueous solution with the volume concentration of 60-90%.
Preferably, the dosage ratio of the sample to be detected and the extracting agent is (0.1-0.5) g: (5-50) mL.
Preferably, the extraction comprises leaching and ultrasound-assisted extraction in sequence; the leaching temperature is 60-90 ℃, and the leaching time is 20-60 min.
Preferably, the temperature of the ultrasonic-assisted extraction is 30-60 ℃, the frequency is 30-50 kHz, and the time is 10 min.
Preferably, the glucosinolate-based compound standard comprises a 3-butenyl thioglycoside standard, a pentylthioglycoside standard, a 1-methoxy-3-indolyl methylthioglycoside standard, a 3- (methylsulfinyl) propylthioglycoside standard, a 4-methoxy-3-indolyl methylthioglycoside standard, a 4-hydroxybenzyl thioglycoside standard, a 4- (methylsulfinyl) butylthioglycoside standard, a sinigrin thioglycoside standard, a 1, 4-dimethoxy-3-indolyl methylthioglycoside standard, a hexyl thioglycoside standard, a 3-methylthio-propylthioglycoside standard, a 5- (methylsulfinyl) pentylthioglycoside standard, a 3-indolyl methylthioglycoside standard, a 4- (methylthio) butylthioglycoside standard, a methyl thioglycoside standard, a methyl glucoside standard, a methyl derivative, a compound standard, a compound, a compound, a compound, a compound, a, A standard substance of the watercresin, a standard substance of the 4-hydroxy-3-indolylmethylthioglycoside or a standard substance of the 2-hydroxy-3-butenyl thioglycoside.
The invention provides a detection method of a glucosinolate compound, which comprises the following steps: mixing a sample to be detected with an extracting agent, and sequentially carrying out extraction and solid-liquid separation to obtain supernatant serving as a solution to be detected; performing qualitative analysis on the solution to be detected by utilizing a first ultra-high liquid chromatography and mass spectrometry; carrying out quantitative analysis on the solution to be detected by utilizing a second ultra-high liquid chromatography and mass spectrometry to obtain the chromatographic peak area of the glucosinolate compound in the sample to be detected; substituting the chromatographic peak area of the glucosinolate compound in the sample to be detected into a concentration-chromatographic peak area standard curve of a glucosinolate compound standard substance to obtain the content of the glucosinolate compound in the sample to be detected; the parameters of the first ultra-high liquid chromatography and mass spectrometry comprise a first ultra-high liquid chromatography parameter and a first mass spectrometry parameter: the first ultra-high liquid chromatography parameter comprises: the chromatographic column is Merck ZIC-HILIC column, 100mm × 2.1mm, 3.5 μm
The temperature of the chromatographic column is 30-50 ℃; the mobile phase A is acetonitrile, and the mobile phase B is 5-10 mmol/L ammonium acetate water solution; flow rate of 0.20.5 mL/min; the sample injection volume is 3-6 mu L; the elution mode is gradient elution; the gradient elution is: 0-1 min, 90% A; 1-9 min, 90-65% A; the first mass spectral parameter comprises: the scanning mode is a parent ion scanning mode and a negative ion mode; the electrospray voltage is-4.5 kV, the ion source temperature is 450 ℃, the daughter ions are 96.0Da, the scanning range of the mother ions is 300.0-800.0 Da, the scanning speed is 200Da/s, the gas curtain gas is 40.00psi, the spraying gas is 45psi, and the auxiliary heating gas is 45 psi; the parameters of the second ultra-high liquid chromatography and mass spectrometry comprise a second ultra-high liquid chromatography parameter and a second mass spectrometry parameter: the second ultra-high liquid chromatography parameter comprises: the chromatographic column is Merck ZIC-HILIC column, 100mm × 2.1mm, 3.5 μm
The temperature of the chromatographic column is 30-50 ℃; the mobile phase A is acetonitrile, and the mobile phase B is 5-10 mmol/L ammonium acetate water solution; the flow rate is 0.2-0.5 mL/min; the sample injection volume is 3-6 mu L; the elution mode is gradient elution; the gradient elution comprises: 0-1 min, 90% A; 1-9 min, 90-65% A; the second mass spectral parameters include: scanning mode: MRM; electrospray voltage was-4.5 kV, ion source temperature was 450 ℃, scan rate was 200Da/s, curtain gas was 40.00psi, spray gas was 45psi, and auxiliary heating gas was 45 psi.
In the invention, the purpose of high-efficiency qualitative determination is achieved by adopting a secondary mass spectrum fragment shared by glucosinolates; in the secondary mass spectrum fragments, a set of parent ion (precursor ion) mode scanning qualitative method is constructed based on the fragments with the m/z of 96.0, so that the detection sensitivity can be ensured, the qualitative accuracy can be improved, and the false positive detection results can be reduced; based on fragments with m/z of 96.0 as quantitative ions, a set of multi-reaction monitoring (MRM) mode scanning quantitative method is constructed by using glucosinolate compound standard substances, so that the quantitative analysis of all glucosinolate compounds in a sample is completed, the problem that the quantitative analysis cannot be performed due to the difficulty in obtaining certain standard substances is solved, and the independent quantification and the total amount determination of the compounds in the sample are efficiently completed. The data of the examples show that: the detection limit of the detection method provided by the invention to different glucosinolates is 0.1 nM; the limit of quantitation is 0.5nM, with higher sensitivity.
Furthermore, the invention adopts direct centrifugal filtration and loading after leaching for 30min at 60-90 ℃, the efficiency is far higher than that of the existing detection method, the detection time is greatly shortened, and the detection flux is improved.
The detection method provided by the invention has the advantages of simple operation, high sample flux and high detection accuracy, can efficiently and accurately complete qualitative and quantitative analysis of the glucosinolates in the sample, and is suitable for qualitative and quantitative analysis of the glucosinolates in plants such as cruciferous vegetables and the like and application of the glucosinolates in the fields of breeding, nutritional quality evaluation and the like.
Drawings
FIG. 1 is a diagram showing the mass spectrum cracking law and the structure of main fragments of glucosinolates;
FIG. 2 is a detection chromatogram for qualitatively analyzing cauliflower samples for corresponding ions using a first ultra-high liquid chromatography and mass spectrometry;
FIG. 3 is a chromatogram obtained from quantitative analysis of a cauliflower sample using a second ultra-high liquid chromatography and mass spectrometry;
FIG. 4 is a graph of the standard graph of sinigrin in 70% strength by volume aqueous methanol;
FIG. 5 is a graph of the standard graph of sinigrin in the matrix of a green cauliflower leaf sample.
Detailed Description
The invention provides a detection method of a glucosinolate compound, which comprises the following steps:
mixing a sample to be detected with an extracting agent, and sequentially carrying out extraction and solid-liquid separation to obtain supernatant serving as a solution to be detected;
performing qualitative analysis on the solution to be detected by utilizing a first ultra-high liquid chromatography and mass spectrometry;
carrying out quantitative analysis on the solution to be detected by utilizing a second ultra-high liquid chromatography and mass spectrometry to obtain the chromatographic peak area of the glucosinolates in the sample to be detected;
substituting the chromatographic peak area of the glucosinolate compound in the sample to be detected into a concentration-chromatographic peak area standard curve of a glucosinolate compound standard substance to obtain the content of the glucosinolate compound in the sample to be detected.
In the present invention, the raw materials are preferably those commercially available, unless otherwise specified.
According to the invention, a sample to be detected and an extracting agent are mixed, and extraction and solid-liquid separation are sequentially carried out to obtain a supernatant serving as a solution to be detected.
In the invention, the particle size of the sample to be detected is preferably 250-830 mu m. In the present invention, the sample to be tested is preferably prepared by the following steps:
and sequentially carrying out freeze drying and crushing on the raw materials to be detected to obtain the sample to be detected.
In the invention, the raw material to be detected is preferably cauliflower or broccoli leaves. In the invention, the temperature of the freeze drying is preferably-40 to-60 ℃, and the time is preferably 12 to 48 hours. The crushing operation is not particularly limited, and the particle size of the sample to be detected is ensured to be 250-830 mu m.
In the present invention, the extractant is preferably a methanol aqueous solution with a volume concentration of 60 to 90%, more preferably a methanol aqueous solution with a volume concentration of 70 to 80%, and in a specific embodiment of the present invention, the extractant is preferably a methanol aqueous solution with a volume concentration of 70%.
In the invention, the dosage ratio of the sample to be detected and the extracting agent is preferably (0.1-0.5) g: (5-50) mL, more preferably 0.2 g: (10-30) mL, more preferably 0.2 g: 20 mL.
In the present invention, the extraction preferably comprises leaching and ultrasound-assisted extraction in sequence.
In the invention, the leaching temperature is preferably 60-90 ℃, and more preferably 70-80 ℃; the leaching time is preferably 20-60 min, more preferably 30-50 min, and even more preferably 40 min. In the present invention, the leaching is preferably performed under the condition of a water bath. In the present invention, the leaching process is preferably: shaking every 5min until leaching is finished.
In the invention, the temperature of ultrasonic-assisted extraction is preferably 30-60 ℃, and further preferably 40-50 ℃; the frequency is preferably 30-50 kHz, more preferably 35-45 kHz, and even more preferably 40 kHz; the time is preferably 10 min.
In the present invention, the sample to be tested and the extractant are preferably heated to the leaching temperature separately before being mixed.
In the invention, the solid-liquid separation mode is preferably centrifugation, and the rotation speed of the centrifugation is preferably more than or equal to 4000rpm, and more preferably 4000-6000 rpm; the time for centrifugation is preferably 5-10 min.
In the invention, the concentration of the glucosinolate compound in the solution to be detected is preferably within the concentration range of the glucosinolate compound in the standard solution when a standard curve is established; preferably diluting if the biological concentration of the glucosinolates in the solution to be measured is not within the concentration range of the glucosinolates in the standard solution when the standard curve is established; the diluted reagent is preferably in accordance with the extractant.
After the solution to be detected is obtained, the qualitative analysis is carried out on the solution to be detected by utilizing a first ultra-high liquid chromatography and mass spectrometry.
In the present invention, the parameters of the first hplc and ms analysis include a first hplc parameter and a first ms parameter:
the first ultra-high liquid chromatography parameter comprises:
the chromatographic column is Merck ZIC-HILIC column, 100mm × 2.1mm, 3.5 μm
The temperature of the chromatographic column is 30-50 ℃, and preferably 40 ℃;
the mobile phase A is acetonitrile; the mobile phase B is 5-10 mmol/L ammonium acetate aqueous solution, preferably 6-9 mmol/L ammonium acetate aqueous solution, and further preferably 7-8 mmol/L ammonium acetate aqueous solution, and in a specific embodiment of the present invention, the mobile phase B is preferably 5mmol/L ammonium acetate aqueous solution;
the flow rate is 0.2-0.5 mL/min, preferably 0.3 mL/min; the sample injection volume is 3-6 muL, preferably 4-5 muL, and in the specific embodiment of the invention, the sample injection volume is preferably 3 muL;
the elution mode is gradient elution;
the gradient elution is:
0～1min，90％A；
1～9min，90％～65％A；
the first mass spectral parameter comprises:
the scanning mode is a parent ion scanning mode and a negative ion mode;
the electrospray voltage is-4.5 kV, the ion source temperature is 450 ℃, the daughter ions are 96.0Da, the scanning range of the mother ions is 300.0-800.0 Da, the scanning speed is 200Da/s, the gas curtain gas is 40.00psi, the spraying gas is 45psi, and the auxiliary heating gas is 45 psi.
In the present invention, the mass spectrometer for the first hplc and mass spectrometry is preferably an AB Science 5500 triple quadrupole mass spectrometer.
After the solution to be detected is obtained, the quantitative analysis is carried out on the solution to be detected by utilizing a second ultra-high liquid chromatography and mass spectrometry, so as to obtain the chromatographic peak area of the glucosinolates in the sample to be detected.
In the present invention, the second parameters of the ultra high liquid chromatography and mass spectrometry include a second ultra high liquid chromatography parameter and a second mass spectrometry parameter:
the second ultra-high liquid chromatography parameter comprises:
the chromatographic column is Merck ZIC-HILIC column, 100mm × 2.1mm, 3.5 μm
The temperature of the chromatographic column is 30-50 ℃, and preferably 40 ℃;
the mobile phase A is acetonitrile; the mobile phase B is 5-10 mmol/L ammonium acetate aqueous solution, preferably 6-9 mmol/L ammonium acetate aqueous solution, and further preferably 7-8 mmol/L ammonium acetate aqueous solution, and in a specific embodiment of the present invention, the mobile phase B is preferably 5mmol/L ammonium acetate aqueous solution;
the flow rate is 0.2-0.5 mL/min, preferably 0.3 mL/min; the sample injection volume is 3-6 muL, preferably 4-5 muL, and in the specific embodiment of the invention, the sample injection volume is preferably 3 muL;
the elution mode is gradient elution;
the gradient elution includes:
0～1min，90％A；
1～9min，90％～65％A；
the second mass spectral parameters include:
scanning mode: MRM;
electrospray voltage was-4.5 kV, ion source temperature was 450 ℃, scan rate was 200Da/s, curtain gas was 40.00psi, spray gas was 45psi, and auxiliary heating gas was 45 psi.
In the present invention, the mass spectrometer for the second hplc and mass spectrometry is preferably an AB Science 5500 triple quadrupole mass spectrometer.
After the chromatographic peak area of the glucosinolates in the sample to be detected is obtained, substituting the chromatographic peak area of the glucosinolates in the sample to be detected into a standard curve of the concentration of the glucosinolates standard substance-the chromatographic peak area to obtain the content of the glucosinolates in the sample to be detected.
In the present invention, the glucosinolate-based compound standard preferably includes a 3-butenylthioglycoside standard, a pentylthioglycoside standard, a 1-methoxy-3-indolylmethylthioglycoside standard, a 3- (methylsulfinyl) propylthioglycoside standard, a 4-methoxy-3-indolylmethylthioglycoside standard, a 4-hydroxybenzylthio glycoside standard, a 4- (methylsulfinyl) butylthioglycoside standard, a sinigrin standard, a 1, 4-dimethoxy-3-indolylmethylthioglycoside standard, a hexylthioglycoside standard, a 3-methylthio-propylthioglycoside standard, a 5- (methylsulfinyl) pentylthioglycoside standard, a 3-indolylmethylthioglycoside standard, a 4- (methylthio) butylthioglycoside standard, a thioglucoside standard, A standard of phaseolin, a standard of 4-hydroxy-3-indolylmethylthioglycoside or a standard of 2-hydroxy-3-butenylthioglucoside, and more preferably a standard of sinigrin.
The establishing mode of the standard curve of the concentration-chromatographic peak area of the thioglucoside compound standard substance is not particularly limited, and the standard curve is established by adopting a mode well known by the technical personnel in the field.
In the invention, the concentration-chromatographic peak area standard curve of the thioglucoside compound standard preferably takes the chromatographic peak area of the thioglucoside compound standard as an axis Y and takes the molar concentration of the thioglucoside compound standard as an axis X.
FIG. 1 is a diagram showing the mass spectrum cracking law and the structure of main fragments of glucosinolates.
The detection method of the glucosinolates provided by the present invention is described in detail below with reference to examples, but these should not be construed as limiting the scope of the present invention.
EXAMPLE 1 sample treatment
Weighing 0.20g of a sample of freeze-dried (freeze-dried at-50 ℃ for 48h) crushed cauliflower powder (250-830 microns) and preheating the sample at 75 ℃ for 10min, adding 20mL of 70 vol% methanol aqueous solution preheated to 75 ℃, leaching for 20min under the condition of 75 ℃ water bath, shaking up the sample at intervals of 5min in the leaching process, and performing ultrasonic-assisted extraction at room temperature for 10min after leaching with the frequency of 40 kHz; centrifuging at 4000rpm for 5 min. Taking 100 mu L of supernatant fluid, putting the supernatant fluid into a 1.5mL centrifuge tube, adding 900 mu L of methanol solution with volume concentration of 70%, shaking up, filtering by a filter membrane (0.22 mu m), and storing at 4 ℃ for standby.
Example 2 Instrument methods and standards preparation and preservation
1. Instrumentation and equipment
Waters Acquity UPLC liquid chromatograph (Waters corporation, usa), AB SCIEX 5500 triple quadrupole mass spectrometer (AB SCIEX corporation, usa), vortex mixer, high speed centrifuge, nitrogen blow dryer, electronic analytical balance, pipette gun, etc.
2. Materials and reagents
Standard substances such as 3-butenylthiooside (Gluconapin), 4-hydroxybenzylthiooside (Sinalbin), Sinigrin (Sinigrin), etc. are purchased from Shanghai-derived leaf Biotech, Inc.; chromatographically pure acetonitrile, methanol, and the like were purchased from Shanghai Tantake Technology, Inc.
3. Preparing and storing standard solution
Accurately weighing 5mg of Gluconapin, Sinalbin and Sinigrin standard products into volumetric flasks respectively, fixing the volume to 10mL by using ultrapure water, and storing the volume as a standard stock solution at-20 ℃ in a dark place. A suitable amount of the standard stock solution was prepared into a 10. mu.M mixed working solution using a 70 vol% aqueous solution of methanol, and gradient serial dilution was performed.
Example 3 qualitative analysis of glucosinolates
Qualitative analysis of the above-machine sample obtained in example 1 was performed using a first ultra-high liquid chromatography and mass spectrometry.
The parameters of the first ultra-high liquid chromatography and mass spectrometry comprise a first ultra-high liquid chromatography parameter and a first mass spectrometry parameter;
the first ultra-high liquid chromatography parameter comprises: the chromatographic column is Merck ZIC-HILIC column, 100mm × 2.1mm (i.d.), 3.5 μm
The temperature of the chromatographic column is preferably 40 ℃; the mobile phase A is acetonitrile, and the mobile phase B is 5mmol/L ammonium acetate aqueous solution; the flow rate is 0.3 mL/min; the sample injection volume is 3 mu L; gradient elution conditions: 0-1 min, 90% A; 1-9 min, 90-65% A;
the first mass spectral parameters include: scanning mode: a parent ion scan (precursor ion) mode scan, preferably a negative ion mode; the electrospray voltage was-4.5 kV; the ion source temperature is 450 ℃; the scanning range of the parent ions is 300.0-800.0 Da, the scanning speed is 200Da/s, the air curtain gas (CUR) is 40.00psi, the spraying gas is 45psi, and the auxiliary heating gas is 45 psi.
Based on the comprehensive analysis of the chemical structure of the glucosinolate compounds, daughter ions with m/z values of 74.99, 95.95, 96.96, 195.00, 259.00, 275.00 and the like can be generated, and the result of using the daughter ions to perform precorsor ion mode analysis is shown in FIG. 2. As can be seen from fig. 2: m/z 95.95 has higher sensitivity and qualitative accuracy.
Example 4 quantitative analysis conditions for glucosinolates
The upper computer sample obtained in example 1 was subjected to quantitative analysis using a second ultra high liquid chromatography and mass spectrometry.
The parameters of the second ultra-high liquid chromatography and mass spectrometry comprise second ultra-high liquid chromatography parameters and second mass spectrometry parameters
The second ultra-high liquid chromatography parameter is the same as the first ultra-high liquid chromatography parameter;
the second mass spectral parameters include: scanning mode: MRM, the electric spraying voltage is-4.5 kV; the ion source temperature is 450 ℃; the scan rate was 200Da/s, the air curtain air (CUR)40.00psi, the spray air 45psi, and the supplemental heating air 45 psi.
The thioglucoside mass spectral parameters are shown in table 1.
TABLE 1 Mass Spectrometry parameters for MRM analysis of thioglucoside Compounds
The obtained spectrum is shown in fig. 3, and it can be seen from fig. 3 that: the detection method provided by the invention can ensure that the peak patterns of all compounds in the sample are symmetrical and the separation degree is better.
Example 5 matrix Effect analysis
First, a myrosinase standard substance, 358/96, was selected as a quantitative ion pair, a matrix standard curve was established using a green cauliflower leaf extract, and a solvent standard curve was established using a 70% methanol solution, and the results are shown in fig. 4 and 5, respectively. As can be seen in fig. 4 and 5: the matrix effect is k (matrix)/k (solvent) -1-0.098, which is a weak matrix effect, indicating that the matrix effect of the extraction method is not significant.
Example 6 quantitative accuracy verification
The other two standards, 4-hydroxybenzylthio glycoside and 3- (methylsulfinyl) propylthio glycoside, were formulated to the same concentration as myrosinase glycoside, and the measured concentration was calculated using the standard curve of myrosinase glycoside, whereby the reliability of the method using the equivalent quantitative ion method was examined, and the results are shown in Table 2.
TABLE 2 quantitative accuracy verification
As can be seen from table 2: the deviation between the detected concentration and the actual concentration of the two standards is within 15%, which indicates that the method is reliable and can be used for quantitative analysis of other glucosinolates.
Example 7 validation of tagged recovery
Based on the above results, a labeling recovery experiment was conducted for 3 kinds of standards. The results are shown in Table 3, where 3 standards (4-hydroxybenzylthio glycoside, 3-butenyl thioglycoside and myrosinoside) were added to the green cauliflower leaf samples at 2nmol to give 100nmol/L concentrations of each standard in the extract, and the measured concentrations of the 3 standards were also calculated using the myrosinoside standard curve.
TABLE 3 Bidding recovery validation
As can be seen from table 3: the deviation between the detection concentration and the actual concentration of the 3 standard substances is within 15 percent, the recovery rate of the method is reliable, and the method can be applied to carry out quantitative analysis on the glucosides in the cauliflowers.
Example 8 method sensitivity
The limit of detection (LOD) is the concentration at which the target quantitation ion is 3-fold signal-to-noise (S/N10) in the corresponding solution of the sample matrix. Limit of quantitation (LOQ) refers to the concentration at which the target quantitation ion is 10 times the signal-to-noise ratio (S/N10) in the corresponding solution of the sample matrix. The detection limit of the method on different glucosinolates is measured to be 0.1nM through experiments; the limit of quantitation was 0.5 nM.
Example 9 Blind sample testing
Different cruciferous vegetable samples were tested by other experimenters according to the detection method of the present invention. The results show that satisfactory results are achieved for all quantitative results.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (9)
1. A detection method of glucosinolates comprises the following steps:
mixing a sample to be detected with an extracting agent, and sequentially performing extraction and solid-liquid separation to obtain a supernatant serving as a solution to be detected;
performing qualitative analysis on the solution to be detected by utilizing a first ultra-high liquid chromatography and mass spectrometry;
carrying out quantitative analysis on the solution to be detected by utilizing a second ultra-high liquid chromatography and mass spectrometry to obtain the chromatographic peak area of the glucosinolate compound in the sample to be detected;
substituting the chromatographic peak area of the glucosinolate compound in the sample to be detected into a concentration-chromatographic peak area standard curve of a glucosinolate compound standard substance to obtain the content of the glucosinolate compound in the sample to be detected;
the parameters of the first ultra-high liquid chromatography and mass spectrometry comprise a first ultra-high liquid chromatography parameter and a first mass spectrometry parameter:
the first ultra-high liquid chromatography parameter comprises:
the chromatographic column is a Merck ZIC-HILIC column with a diameter of 100mm multiplied by 2.1mm and a diameter of 3.5 mu m 100A;
the temperature of the chromatographic column is 40 ℃;
the mobile phase A is acetonitrile, and the mobile phase B is 5mmol/L ammonium acetate aqueous solution; the flow rate is 0.3 mL/min; the sample injection volume is 3 mu L;
the elution mode is gradient elution;
the gradient elution is:
0~1 min，90%A；
1~9 min，90%~65%A；
the first mass spectral parameter comprises:
the scanning mode is a parent ion scanning mode and a negative ion mode;
the electrospray voltage is-4.5 kV, the ion source temperature is 450 ℃, the daughter ions are 96.0Da, the scanning range of the parent ions is 300.0-800.0 Da, the scanning speed is 200Da/s, the air curtain gas is 40.00psi, the spray gas is 45psi, and the auxiliary heating gas is 45 psi;
the parameters of the second ultra-high liquid chromatography and mass spectrometry comprise a second ultra-high liquid chromatography parameter and a second mass spectrometry parameter:
the second ultra-high liquid chromatography parameter comprises:
the chromatographic column is a Merck ZIC-HILIC column with a diameter of 100mm multiplied by 2.1mm and a diameter of 3.5 mu m 100A;
the temperature of the chromatographic column is 40 ℃;
the mobile phase A is acetonitrile, and the mobile phase B is 5mmol/L ammonium acetate aqueous solution; the flow rate is 0.3 mL/min; the sample injection volume is 3 mu L;
the elution mode is gradient elution;
the gradient elution comprises:
0~1 min，90%A；
1~9 min，90%~65%A；
the second mass spectral parameters include:
scanning mode: MRM;
the electrospray voltage is-4.5 kV, the ion source temperature is 450 ℃, the scanning speed is 200Da/s, the gas curtain gas is 40.00psi, the spraying gas is 45psi, and the auxiliary heating gas is 45 psi;
the glucosinolate compound standard comprises a 3-butenyl glucosinolate standard, a 4-hydroxybenzyl glucosinolate standard or a sinigrin standard;
constructing a set of parent ion mode scanning qualitative method based on fragments with m/z of 96.0;
based on fragments with m/z of 96.0 as quantitative ions, a set of multi-reaction monitoring mode scanning quantitative method is constructed by utilizing glucosinolate compound standard substances.
2. The detection method according to claim 1, wherein the particle size of the sample to be detected is 250 to 830 μm.
3. The detection method according to claim 1 or 2, wherein the sample to be detected is prepared by the following steps:
and sequentially carrying out freeze drying and crushing on the raw materials to be detected to obtain the sample to be detected.
4. The detection method according to claim 3, wherein the raw material to be detected comprises cauliflower or green cauliflower leaves.
5. The detection method according to claim 3, wherein the temperature of the freeze drying is-40 to-60 ℃, and the time is 12 to 48 hours.
6. The detection method according to claim 1, wherein the extractant is a 60 to 90 vol% methanol aqueous solution.
7. The detection method according to claim 6, wherein the ratio of the amount of the sample to be detected to the amount of the extractant is (0.1 to 0.5) g: (5-50) mL.
8. The detection method according to claim 1 or 7, wherein the extraction comprises leaching and ultrasound-assisted extraction in sequence; the leaching temperature is 60-90 ℃, and the leaching time is 20-60 min.
9. The detection method according to claim 8, wherein the temperature of the ultrasonic-assisted extraction is 30-60 ℃, the frequency is 30-50 kHz, and the time is 10 min.
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