CN115452997A - Method for simultaneously detecting forbidden pesticides, mycotoxins and effective components in traditional Chinese medicinal materials - Google Patents
Method for simultaneously detecting forbidden pesticides, mycotoxins and effective components in traditional Chinese medicinal materials Download PDFInfo
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Abstract
The invention belongs to the technical field of analysis and detection of traditional Chinese medicinal materials, and relates to a method for simultaneously detecting forbidden pesticides, mycotoxins and effective components in traditional Chinese medicinal materials. According to the invention, the sample preparation is carried out through the HLB solid-phase extraction column, and the LC-Q-TOF/MS and the GC-Q-TOF/MS are combined to use, so that the effective separation of the pesticide residue and the exogenous pollutants such as mycotoxin is realized by one-time sample introduction, the use efficiency of the instrument is improved, and the data acquisition and analysis time is shortened; meanwhile, by comparing the purification technology, the pretreatment conditions such as ice bath and oscillation sequence, extraction liquid acidity, constant volume liquid and the like are optimized, the compatibility of sample pretreatment is improved, 33 forbidden pesticides (54 monomers) in the traditional Chinese medicinal materials, 11 mycotoxins and functional components are detected simultaneously, the simultaneous detection of cross-category exogenous pollutants and functional components is possible, the experiment cost is effectively reduced, the detection time is shortened, and the method has a high practical application value.
Description
Technical Field
The invention belongs to the technical field of quality safety detection of traditional Chinese medicinal materials, and particularly relates to a method for simultaneously detecting forbidden pesticides, mycotoxins and effective components in a traditional Chinese medicinal material.
Background
The traditional Chinese medicine is the material basis for inheritance and development of the traditional Chinese medicine cause and is a strategic resource related to the national people. The unique medication principle and the overall therapeutic characteristics of traditional Chinese medicine continuously expand the influence of the traditional Chinese medicine on the world. In order to better complete the international connection between traditional Chinese medicine and the world, the quality safety of the traditional Chinese medicine is necessary and forcefully guaranteed. Except for controlling the quality of the traditional Chinese medicine by establishing scientific quality standards, exogenous pollutants of the traditional Chinese medicine seriously affect the quality safety of the traditional Chinese medicine. Exogenous harmful pollutants of traditional Chinese medicines are residues of animals and plants adsorbed or accumulated from the contacted environments such as soil, water, atmosphere and feed in the growth process and after diseases and insect pests are prevented and controlled in the culture and cultivation processes, and may also be caused by pollution caused by improper processing, processing and storage of decoction pieces, and mainly comprise residues of pesticides and veterinary medicines, residues of heavy metals and harmful elements, residues of mycotoxins, residues of organic pollutants, pollution of pathogenic microorganisms and the like. In addition, the content of the effective components is a key index for evaluating the medicinal quality of the medicinal composition, but a broad-spectrum detection technology and an evaluation grading standard are still lacking at present.
Traditional Chinese medicinal materials are improperly treated in the field growth, storage or processing processes, and all the traditional Chinese medicinal materials can pollute various fungi and produce mycotoxins. The contaminated fungi include not only Aspergillus and Penicillium, but also Fusarium and the like. The produced toxins comprise various mycotoxins closely related to human health safety, such as aflatoxin, ochratoxin, fumonisin, zearalenone and the like. In order to ensure the safety of the medicine for people, the Chinese pharmacopoeia (2020 edition, one) stipulates that the aflatoxin B1 (AFB 1) in 24 traditional Chinese medicinal materials such as coix seed and the like is not higher than 5 mug/kg, and the total amount of the aflatoxin (AFB 1+ AFB2+ AFG1+ AFG 2) is not higher than 10 mug/kg; the content of zearalenone in Coicis semen should not exceed 500 μ g per 1000 g.
According to Chinese pharmacopoeia (2020), pesticide residues, mycotoxins and effective components in traditional Chinese medicinal materials need to be divided into 3 independent experiments from pretreatment to instrument analysis. The pretreatment of pesticide residues adopts a QuEChERS method, the determination of mycotoxin adopts an HLB solid phase extraction method, the effective components mostly adopt a heating reflux method, an ultrasonic extraction method and the like to extract the substance to be detected, and different sample preparation processes and different analytical instruments are needed, so that the experimental consumables and the labor and the time are wasted; moreover, the traditional Chinese medicinal materials are complex in components and different in medicinal parts, and if a rapid and effective mode is not adopted for pretreatment, target loss can be caused, and an experimental result is influenced.
The invention content is as follows:
aiming at overcoming the defects in the prior art, the invention provides a method for simultaneously detecting forbidden pesticides, mycotoxins and effective components (shown in table 1) in traditional Chinese medicinal materials, gives consideration to the chemical properties of various pesticide residues, mycotoxins and different compounds of the effective components, and designs an integrated scheme capable of being compatible with different technologies of different classes of compounds by optimizing a purification technology, extraction liquid acidity, constant volume liquid and the like.
In order to achieve the purpose, the method comprises the following specific steps:
TABLE 1 Simultaneous determination of pesticide, mycotoxin and active ingredient species for one sample preparation
The method comprises the following steps: sample preparation:
(1) Weighing traditional Chinese medicine powder, adding formic acid water solution, mixing, performing vortex to fully soak the traditional Chinese medicine powder, placing for a period of time, adding acetonitrile, performing vortex mixing, performing oscillation treatment, adding anhydrous magnesium sulfate and anhydrous sodium acetate mixed powder, shaking, mixing uniformly, cooling for a period of time in an ice water bath, performing secondary oscillation treatment after cooling, performing centrifugation after treatment to obtain supernatant, marking the supernatant as sample liquid A, and concentrating the VML sample liquid A to V/5 by using a parallel concentrator or a rotary evaporator to obtain concentrated solution;
the dosage relation of the mixed powder of the medicinal material powder, the aqueous solution of formic acid, acetonitrile, anhydrous magnesium sulfate and anhydrous sodium acetate is 3g:15mL of: 30mL of: 7.5g; the volume concentration of the formic acid aqueous solution is 5-10%; the mass ratio of the anhydrous magnesium sulfate to the anhydrous sodium acetate in the anhydrous magnesium sulfate and anhydrous sodium acetate mixed powder is 4;
(2) The PRIME HLB column does not need to be activated before use, the concentrated solution prepared in the step (1) is transferred into the column, a centrifugal tube is connected below the column until air passes through the column, methanol is used for eluting for a plurality of times after all the liquid is collected, the eluents for a plurality of times are combined, and the centrifugal tube is taken down and dried on a nitrogen blowing instrument; adding methanol for redissolution, taking out 1/2 of the liquid, and respectively filling the liquid into another small test tube, wherein the two small test tubes are dried by nitrogen and respectively marked as a test tube I and a test tube II; adding a constant volume solution I into the test tube I, wherein the constant volume solution I contains 200ng/mL atrazine D5, shaking after ultrasonic redissolution, filtering through an organic membrane to obtain a solution marked as a sample solution B, and placing the sample solution B into a sample injection vial for measurement by LC-Q-TOF/MS;
adding a volume fixing solution II into a test tube II, wherein the volume fixing solution II contains 200ng/mL ethyl acetate of epoxy heptachlor; shaking after ultrasonic redissolution, filtering by an organic membrane to obtain a solution marked as sample solution C, and placing the sample solution C into a sample injection vial for GC-Q-TOF/MS determination;
preferably, the concentration of the aqueous formic acid solution in step (1) in volume is 5%.
Preferably, the placing time in the step (1) in the first step is 20-30min; the conditions of the oscillation treatment are as follows: 300r/min,10-15min; the cooling time is 10-15min; the conditions of the second oscillation treatment are as follows: 300r/min,5-8min; the centrifugation conditions were: 4200rpm, 5-8 min.
Preferably, the Chinese medicinal material in the step (1) in the first step is angelica.
Preferably, in the step (2) of the first step, the volume ratio of the concentrated solution, the methanol for elution, the methanol for redissolution, the constant volume solution i and the constant volume solution ii is 2: 0.5:0.5; the constant volume liquid I and the constant volume liquid II are both formed by mixing methanol and water according to a volume ratio of 3;
preferably, the size of the organic film in step one (2) is 0.22 μm.
Step two, detecting conditions of pesticide and mycotoxin:
taking the sample liquid B prepared in the step one (2) to detect through LC-Q-TOF/MS; detecting the sample liquid C by GC-Q-TOF/MS;
LC-Q-TOF/MS detection conditions:
and (3) chromatographic column: SB-C18:100 mm. Times.2.1 mm. Times.3.5. Mu.m, column temperature: at 40 ℃; mobile phase: the phase A is 0.1% (v/v) formic acid aqueous solution (the solution contains 5mmol/L ammonium acetate), and the phase B is acetonitrile; the flow rate is 0.4mL/min;
gradient elution procedure: 0-3min, 1-30 percent of C; 3-6 min, 30-40 percent of B;6 to 9min,40% by weight; 9 to 15min,40 to 60% by weight of B;15 to 19min,60 to 90 percent of B; 19-23min, 90%; 23 to 23.01min,90 to 1 percent of B;23.01 to 27.01min,1% by weight B; sample introduction volume: 5 mu L of the solution;
dual AJS ESI source; the scanning mode comprises the following steps: positive ion full scanning; full scan range: m/z is 50-1000; capillary voltage: 4000V; atomizing gas: nitrogen gas; atomizing gas pressure: 0.14Mpa; temperature of sheath gas: 375 ℃; flow rate of sheath gas: 11.0L/min; flow rate of drying gas: 12.0L/min; temperature of the drying gas: 325 ℃; fragmentation voltage: 145V;
all ion MS/MS mode conditions: at 0min, the collision energy is 0V; at 0.5min, the collision energies were 0, 15 and 35V, respectively.
GC-Q-TOF/M detection conditions:
a chromatographic column: HP-5MS UI:30m × 0.25mm × 0.25 μm, column temperature: maintaining at 40 deg.C for 1min, heating to 130 deg.C at 30 deg.C/min, heating to 250 deg.C at 5 deg.C/min, heating to 300 deg.C at 10 deg.C/min, and maintaining for 7min; carrier gas: helium purity is more than or equal to 99.999%, flow rate: 1.2mL/min; sample inlet temperature: 270 ℃; sample injection amount: 1.0 μ L; and (3) sample introduction mode: sampling without shunting, and opening a purge valve for 1 min;
ionization mode: electron Impact (EI); ion source voltage: 70eV; ion source temperature: 280 ℃; quadrupole rod temperature: 180 ℃; solvent retardation: 4min; ion monitoring mode: full scan (TOF MS); scanning range: m/z is 45-550; scanning speed: 5Hz.
Step three, the detection condition of the effective components:
taking the sample liquid A prepared in the step one (1) to perform machine detection; and the instrument conditions of the functional components are the same as those of the LC-Q-TOF/MS in the second step, and the specific steps are as follows:
and (3) chromatographic column: SB-C18:100 mm. Times.2.1 mm. Times.3.5. Mu.m, column temperature: 40 ℃; mobile phase: the phase A is 0.1% (v/v) formic acid aqueous solution (the solution contains 5mmol/L ammonium acetate), and the phase B is acetonitrile; the flow rate is 0.4mL/min;
gradient elution procedure: 0-3min, 1-30 percent of C; 3-6 min, 30-40 percent of B;6 to 9min,40% by weight; 9 to 15min,40 to 60% by weight; 15 to 19min,60 to 90 percent of B;19 to 23min,90% by weight B;23 to 23.01min,90 to 1 percent of B;23.01 to 27.01min,1% by weight B; sample introduction volume: 5 mu L of the solution;
dual AJS ESI source; the scanning mode comprises the following steps: positive ion full scanning; full scan range: m/z is 50-1000; capillary voltage: 4000V; atomizing gas: nitrogen gas; atomizing gas pressure: 0.14Mpa; temperature of sheath gas: 375 ℃; flow rate of sheath gas: 11.0L/min; flow rate of drying gas: 12.0L/min; temperature of the drying gas: 325 ℃; fragmentation voltage: 145V;
all ion MS/MS mode conditions: at 0min, the collision energy is 0V; at 0.5min, the collision energy is 0, 15 and 35V respectively;
step four, calculating a result:
(1) Pesticide and mycotoxin detection:
(a) Standard working curve configuration: selecting blank Chinese medicinal materials, carrying out sample pretreatment according to the first step, adding mixed standard working solutions with different concentrations into a sample solution B and a sample solution C after pretreatment based on the difference of compound quantification limits, and establishing a series of concentration matrix matching standard curves to obtain a linear regression equation;
the preparation of the mixed standard working solution comprises the following steps: preparing a mixed standard solution by using methanol and water in a volume ratio of 3;
(b) Quantitative calculation
Quantification by an internal standard method: according to the quantitative ion pair peak areas of the corresponding compounds in the sample, adopting a linear regression equation in the step (a), and calculating by using MassHunter quantitative analysis software to obtain the concentrations of the corresponding compounds;
(2) And (3) detection of functional components:
(a) Standard working curve configuration: absorbing the sample solution A in the step I, adding an effective component standard solution, diluting the sample solution A with acetonitrile to a series of standard solutions with gradient, and entering a mass spectrum detector; drawing a standard curve by taking the concentration (X) of the standard substance as a horizontal coordinate and the peak area (Y) as a vertical coordinate to obtain a linear regression equation;
(b) And (3) quantitative calculation: calculating the concentration of the effective component of the corresponding compound by using the corresponding peak area in the sample and adopting the linear regression equation in the step (2) (a).
Based on the steps, the simultaneous detection of the forbidden pesticides, the mycotoxins and the effective components in the Chinese medicinal herb angelica can be realized.
The invention has the following beneficial effects:
(1) The invention prepares a sample through an HLB solid-phase extraction column, combines two high-resolution mass spectra of LC-Q-TOF and GC-Q-TOF, optimizes conditions such as comparison, ice bath and oscillation sequence, extraction liquid acidity, constant volume liquid and the like through a purification method, improves the compatibility of sample pretreatment, can realize the simultaneous detection of 33 forbidden pesticides (54 monomers), 11 mycotoxins and effective components in the traditional Chinese medicinal materials, effectively reduces the experiment cost, shortens the experiment time and has stronger practical application value.
(2) According to the invention, by optimizing an instrument analysis method, combining LC-Q-TOF and GC-Q-TOF and injecting sample once, the pesticide residue and exogenous pollutants such as mycotoxin can be effectively separated, the use efficiency of the instrument is improved, the data acquisition and data analysis time is shortened, and the experiment efficiency is improved to a great extent.
(3) The invention uses high-resolution mass spectrometry to facilitate retrospective detection, and can continuously update the screening capability and the screening range of exogenous pollutants. An unlimited number of analytes can be extracted from the full scan data without affecting the sensitivity of the results. The amount of compound that can be monitored in one run by conventional low resolution mass spectrometry severely limits its screening ability.
(4) In the traditional method, the detection of the triolein in the coix seeds needs to be equipped with the liquid chromatogram of an evaporative light scattering detector, and when the ferulic acid in the angelica is measured, a heating reflux device is needed for sample preparation, and the detection is carried out by using the liquid chromatogram. The experiment realizes the normalized high-flux detection of cross-category exogenous pollutants and functional components by optimizing extraction and purification conditions and instrument analysis parameters.
Drawings
FIG. 1 is a chromatogram of 4 kinds of mycotoxins in different constant volume solutions, wherein, a graph A is a deoxynivalenol (Don) chromatogram, a graph B is a Patulin chromatogram (Patulin), a graph C is a fumonisin B1 chromatogram (FB 1), and a graph D is a fumonisin B2 chromatogram (FB 2); wherein (3.
FIG. 2 is a comparison of the chromatograms of deoxynivalenol and patulin against the respective volumetric solutions of aqueous methanol (methanol: water = 3) and aqueous acetonitrile (acetonitrile: water = 3);
wherein, the A and B are deoxynivalenol (Don) chromatograms, the A is acetonitrile: water (3; b is methanol: water (3; panels C and D are patulin chromatograms, panel C is acetonitrile: water (3; b is methanol: and (3) water (2) is used as a constant volume liquid.
The specific implementation mode is as follows:
the present invention will be further described with reference to the following specific examples;
sample preparation:
weighing 3g of traditional Chinese medicine powder, adding 15mL of formic acid aqueous solution with volume concentration of 5% into a 80mL polystyrene centrifuge tube with a plug, swirling to fully infiltrate the medicinal powder, and standing for 30min. And then adding 30mL of acetonitrile, uniformly mixing by vortex, placing on an oscillator (300 r/min), oscillating for 10min, adding 7.5g of anhydrous magnesium sulfate and anhydrous sodium acetate mixed powder (mass ratio is 4.
The PRIME HLB column needs no activation before use, the concentrated solution is transferred to the column, a centrifugal tube is connected below the column until air passes through the column, after all liquid is collected, 3mL methanol is used for eluting for 2 times, the eluents for 2 times are combined, and the centrifugal tube is taken down and dried on a nitrogen blowing instrument. Adding 1mL of methanol for redissolution, taking out 0.5mL of the redissolved solution, putting the redissolved solution into another small test tube, drying the two test tubes by using nitrogen, adding 0.5mL of a constant volume solution (methanol: water = 3. And adding 0.5mL of ethyl acetate constant volume solution containing 200ng/mL of epoxy heptachlor into the other test tube, shaking after ultrasonic redissolution, passing through a 0.22-micron organic membrane to obtain a solution, marking as sample solution C, and placing the solution into a sample injection vial for GC-Q-TOF/MS determination.
1 optimization of pretreatment method (Chinese medicinal materials selected from Chinese angelica and coix seed)
1.1 purification method optimization
Chinese angelica and coix seed two Chinese medicinal material matrixes, wherein each matrix is Carb/NH 2 HLB (200mg, 6 mL) column purification mode and 2 QuEChERS purification modes, the addition level is 10 mug/kg, and 3 parallels, namely 3 matrix front labeling and 1 rear labeling are examined; and inspecting the quantity of pesticides which meet the requirements of 70-120% recovery rate and 20% RSD by different pretreatment methods.
The result shows that the recovery rate of HLB on most compounds, particularly sulfometuron pesticides and aflatoxins B1 and B2 is better, and HLB is finally selected as a pretreatment purification method.
1.2 optimization of ice bath oscillation sequence by HLB method
The angelica adopts two pre-treatments of firstly oscillating and then ice-bathing and firstly ice-bathing and then oscillating, and the difference between the detected compound quantity and the qualified compound quantity is not large. The coix seeds adopt two modes, the difference of the number of compounds detected by LC is not large, but the number of the compounds qualified by GC verification is large, 26 compounds are firstly subjected to ice bath verification and 16 compounds are firstly subjected to oscillation verification, and the oscillation is performed after the ice bath, so that the oscillation and heat release can be relieved in a low-temperature environment, and the extraction of the thermally unstable compounds is facilitated.
1.3 optimization of extracts of different acidity
The different acidity of the sample liquid A has great influence on the extraction efficiency of the fumonisins B1 and B2, so that the acidity of the sample liquid A needs to be adjusted to improve the recovery rate of the fumonisins B1 and B2 on the premise of ensuring that the recovery rate of most compounds is not influenced. Comparing 1% acetic acid aqueous solution, 2% acetic acid aqueous solution, 1% formic acid aqueous solution, 2% formic acid aqueous solution, 3% formic acid aqueous solution, 5% formic acid aqueous solution, 10% formic acid aqueous solution as extraction solvent, and examining the influence of different acidity sample solution A on extraction efficiency.
Experiments show that the recovery rates of fumonisins B1 and B2 are obviously improved along with the increase of the concentration of formic acid in the sample liquid A. The acidity of the sample liquid A is too small, the recovery rates of fumonisins B1 and B2 are poor, the acidity is too large, and the rotary evaporation and parallel concentration time can be prolonged, so that the stability of the heat-sensitive compound is influenced. When the concentration of formic acid in the angelica is more than 5 percent, the recovery rates of fumonisins B1 and B2 are qualified; when the concentration of formic acid in the coix seeds is more than 3 percent, the recovery rates of fumonisins B1 and B2 are qualified.
And (3) comprehensively considering the influence of different experimental factors and different acidity on other substances to be detected, and finally selecting a 5% formic acid solution as an extraction solution.
1.4 optimization of different constant volume liquids (the related solution proportions are volume ratio, v/v)
(1) Methanol, acetonitrile, methanol: water (3
With methanol, acetonitrile and methanol, respectively: water (3. It was found that for most compounds the peak areas of the different volumetric solutions differed little.
However, the response value of partial mycotoxins such as fumonisin B1, fumonisin B2, ochratoxin A, deoxynivalenol and pavotoxin in ACN is obviously lower than that of methanol: water (3. Comparing the peak appearance time, peak area and peak shape of the chromatographic peaks of fumonisins B1, fumonisins B2, deoxynivalenol and patulin in different constant volume liquids, as can be seen from figure 1, the deoxynivalenol and the patulin have double peaks in acetonitrile constant volume liquid, and the ratio of methanol to methanol is as follows: the water (3. Fumonisins B1 and B2 are not greatly different from each other in peak shape, and the fumonisins B1 are shown in the following formula: peak areas in water (3; fumonisins B2 in methanol: peak areas in water (3. Therefore, for fumonisins B1, fumonisins B2, deoxynivalenol, pavlosin, methanol is selected: the water (3.
(2) Methanol: water (3: water (3
The experiment compares acetonitrile: water (3: effect of water (3. Aiming at four mycotoxins of fumonisin B1, fumonisin B2, vomitoxin and patulin, under the concentration of 1ppm, acetonitrile: water (3: water (3: the chromatographic peak shape of water (3: water (3: water (3.
1.5 optimization of effective component experiment conditions
The representative functional component of coix seed, namely glycerol trioleate, is determined by using high performance liquid chromatography (general rule 0512) according to the specification of Chinese pharmacopoeia (2020 edition), samples are ultrasonically extracted by using acetonitrile-dichloromethane (65) and octadecylsilane chemically bonded silica is used as a chromatographic column; acetonitrile-dichloromethane (65; detection by an evaporative light scattering detector.
Compared with acetonitrile, dichloromethane, isopropanol and water as extraction solvents, the extraction efficiency of the acetonitrile, the dichloromethane and the isopropanol on the triolein is not very different, but the effective extraction of the triolein is influenced by the water as the extraction solvent or the water contained in the extraction solvent. Therefore, the invention adopts acetonitrile as an extraction solvent and combines a liquid chromatography-mass spectrometer for detection.
The representative effective component ferulic acid of radix Angelicae sinensis in Chinese pharmacopoeia (2020 edition) is determined by high performance liquid chromatography (general rule 0512), and octadecylsilane chemically bonded silica is used as filler; acetonitrile-0.085% phosphoric acid solution (17; the detection wavelength is 316nm; the column temperature was 35 ℃ and the sample was heated to reflux for 30min.
The method only needs to be operated according to the extraction step in the sample pretreatment process, and the liquid chromatography-mass spectrometer is directly used for detection, so that the detection sensitivity of the traditional method is improved, meanwhile, the integrated pretreatment operation can be carried out on pesticide residues and mycotoxins, the reasonable configuration of the instrument is realized, and the labor cost, the time cost and the economic cost are saved.
Implementing a specific scheme based on the optimized conditions:
example 1: the Chinese medicinal material is angelica sinensis, and the detection steps are as follows:
step one, sample preparation:
weighing 3g of angelica powder, adding 15mL of 5% formic acid aqueous solution into an 80mL polystyrene centrifuge tube with a plug, performing vortex to fully infiltrate the medicinal powder, placing for 30min, adding 30mL of acetonitrile, performing vortex mixing, placing on an oscillator (300 r/min), performing oscillation for 10min, adding 7.5g of anhydrous magnesium sulfate and anhydrous sodium acetate mixed powder (mass ratio 4); the obtained supernatant is marked as sample liquid A, 10mL of sample liquid A is added into a large test tube, and the sample liquid A is concentrated to 2mL by a parallel concentrator or a rotary evaporator.
The PRIME HLB column is not required to be activated before use, the concentrated solution is moved into the column, a small test tube is connected downwards until air passes through, after all liquid is collected, 3mL methanol is used for elution, 2 times of eluent is combined, and a centrifuge tube is taken down and dried on a nitrogen blowing instrument. Adding 1mL of methanol for redissolution, taking out 0.5mL of the redissolved solution, placing the redissolved solution into another small test tube, drying the two test tubes by using nitrogen, adding 0.5mL of a constant volume solution containing 200ng/mL of atrazine D5 (methanol: water = 3.
And adding 0.5mL of ethyl acetate constant volume solution containing 200ng/mL of epoxy heptachlor into another test tube, shaking after ultrasonic redissolution, passing through a 0.22-micron organic membrane to obtain a solution, marking as sample solution C, and placing the sample solution C into a sample injection vial for GC-Q-TOF/MS determination.
Step two, detecting conditions of pesticide and mycotoxin samples:
detecting the sample liquid B prepared in the first step through LC-Q-TOF/MS; detecting the sample liquid C by GC-Q-TOF/MS;
(1) Detection conditions of an LC-Q-TOF/MS instrument are as follows:
a chromatographic column: SB-C18:100mm × 2.1mm (i.d.) × 3.5 μm, column temperature: at 40 ℃; mobile phase: the phase A is 0.1% (v/v) formic acid aqueous solution (the solution contains 5mmol/L ammonium acetate), and the phase B is acetonitrile; the flow rate was 0.4mL/min.
Gradient elution procedure: 0 to 3min,1 to 30 percent of B; 3-6 min,30% -40% B;6 to 9min,40 percent B;9 to 15min,40 to 60% by weight of B;15 to 19min,60 to 90 percent of B; 19-23min, 90%; 23 to 23.01min,90 to 1 percent of B;23.01 to 27.01min,1% by weight of B. Sample introduction volume: 5 μ L.
Dual AJS ESI source; the scanning mode is as follows: positive ion full scanning; full scan range: m/z is 50-1000; capillary voltage: 4000V; atomizing gas: nitrogen gas; atomizing gas pressure: 0.14Mpa; temperature of sheath gas: 375 ℃; flow rate of sheath gas: 11.0L/min; flow rate of drying gas: 12.0L/min; temperature of drying gas: 325 ℃; fragmentation voltage: 145V. All ion MS/MS mode conditions: at 0min, the collision energy is 0V; at 0.5min, the collision energies were 0, 15 and 35V, respectively.
(2) And (3) detecting conditions of a GC-Q-TOF/MS instrument:
a chromatographic column: HP-5MS UI:30m × 0.25mm (i.d.) × 0.25 μm, column temperature: maintaining at 40 deg.C for 1min, heating to 130 deg.C at 30 deg.C/min, heating to 250 deg.C at 5 deg.C/min, heating to 300 deg.C at 10 deg.C/min, and maintaining for 7min; carrier gas: helium purity is more than or equal to 99.999%, flow rate: 1.2mL/min; sample inlet temperature: 270 ℃; sample injection amount: 1.0 mu L; and (3) sample introduction mode: sampling without shunting, and opening a purge valve for 1 min;
ionization mode: electron Impact (EI); ion source voltage: 70eV; ion source temperature: 280 ℃; temperature of the quadrupole rods: 180 ℃; solvent retardation: 4min; ion monitoring mode: full scan (TOF MS); scanning range: m/z is 45-550; scanning rate: 5Hz.
Step three, detecting the effective components of the angelica:
taking the sample liquid A obtained in the step one, and detecting on a machine;
detection conditions of an LC-Q-TOF/MS instrument are as follows:
a chromatographic column: SB-C18:100mm × 2.1mm (i.d.) × 3.5 μm, column temperature: 40 ℃; mobile phase: the phase A is 0.1% (v/v) formic acid aqueous solution (the solution contains 5mmol/L ammonium acetate), and the phase B is acetonitrile; the flow rate was 0.4mL/min.
Gradient elution procedure: 0 to 3min,1 to 30 percent of B; 3-6 min,30% -40% B;6 to 9min,40% by weight; 9 to 15min,40 to 60% by weight of B;15 to 19min,60 to 90 percent of B; 19-23min, 90%; 23 to 23.01min,90 to 1 percent of B;23.01 to 27.01min,1% by weight of B. The injection volume is 5. Mu.L.
Dual AJS ESI source; the scanning mode comprises the following steps: positive ion full scanning; full scan range: m/z is 50-1000; capillary voltage: 4000V; atomizing gas: nitrogen gas; atomizing gas pressure: 0.14Mpa; temperature of sheath gas: 375 ℃; flow rate of sheath gas: 11.0L/min; flow rate of drying gas: 12.0L/min; temperature of the drying gas: 325 ℃; fragmentation voltage: 145V. All ion MS/MS mode conditions: at 0min, the collision energy is 0V; at 0.5min, the collision energies were 0, 15 and 35V, respectively.
Angelica sinensis methodological verification
1. Linear range
Selecting 6 parts of blank angelica, carrying out sample pretreatment according to the step (1), adding mixed standard working solutions with different concentrations into an extracting solution after the pretreatment based on the difference of the compound quantification limit, and establishing matrix matching standard curves of 0.5LOQ, 1LOQ, 2LOQ, 4LOQ, 10LOQ and 20 LOQ; the preparation of the mixed standard working solution comprises the following steps: preparing a mixed standard solution of 100ng/mL by using methanol and water in a volume ratio of 3;
the sample liquid B prepared in the first step was subjected to LC-Q-TOF/MS and the sample liquid C was subjected to GC-Q-TOF/MS to detect 65 compounds in total, and as a result, as shown in Table 1, 38 compounds detected by LC-Q-TOF/MS and 41 compounds detected by GC-Q-TOF/MS showed R2 > 0.9801 in the respective linear ranges.
Table 1 shows the linear ranges, linear equations, correlation coefficients and quantitative limits for 65 compounds
2 limit of quantification
All compounds satisfy the minimum addition level of recovery 70-120% and RSD ≦ 20% at different addition levels as quantitative limits, with reference to the requirements specified in the European Union directive document SANTE/12682/2021.
LC-Q-TOF/MS and GC-Q-TOF/MS jointly detect 65 compounds, wherein the quantitative limit of 1 pesticide (cloxaphos) and 8 fungaltoxins (aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, T-2 toxin, fumonisin B1, fumonisin B2 and patulin) is the same as that in Chinese pharmacopoeia (2020 edition), and the rest 55 compounds can reach the quantitative limit lower than that in Chinese pharmacopoeia (2020 edition).
In LC-Q-TOF/MS and GC-Q-TOF/MS combined detection of 65 compounds, 16 compounds are co-detected compounds, wherein LC-Q-TOF/MS detection of 10 compounds can reach lower limit of quantitation than GC-Q-TOF/MS detection of 1 compound, and LC-Q-TOF/MS detection of 5 compounds can reach the same limit of quantitation as GC-Q-TOF/MS.
3 recovery and relative standard deviation
LC/GC-Q-TOF/MS determined recovery and relative standard deviation of 65 compounds at LOQ, 2LOQ, and 10 LOQ. Each addition level was 6 replicates, while blank experiments were performed to subtract interference from endogenous substances in the matrix. 38 compounds measured by LC-Q-TOF/MS and 41 compounds measured by GC-Q-TOF/MS can meet the recovery rate of 70-120% and the RSD of less than or equal to 20% under three addition levels. The results are shown in Table 2 (including tables 2-1 and 2-2), and Table 2 shows the results of matrix effect, recovery rate and precision for 65 compounds.
TABLE 2-1 LC-Q-TOF/MS measurements at three addition levels of LOQ, 2LOQ, and 10LOQ
TABLE 2-2 GC-Q-TOF/MS results of the tests at three addition levels of LOQ, 2LOQ and 10LOQ
4 verification of methodology of effective components of angelica sinensis
4.1 limit of quantitation
And (3) gradually diluting the sample solution A prepared in the step one by a certain dilution multiple until the signal-to-noise ratio of the ferulic acid is detected to be 10, and taking the signal-to-noise ratio as the lowest quantification limit (S/N is greater than 10) of the ferulic acid, and measuring the result to obtain the quantification limit of the ferulic acid to be 50ng/mL.
4.2 matrix matching Standard Curve
And precisely sucking a proper amount of standard solution, adding the standard solution into the sample solution A to prepare a sample to obtain a matrix matching standard curve solution with ferulic acid concentrations of 0.5, 1, 2, 3, 5 and 10 mu g/mL respectively, and entering a mass spectrum detector. And (3) drawing a standard curve by taking the background concentration (X) of the ferulic acid subtracted from the detected concentration as a horizontal coordinate and the peak area (Y) as a vertical coordinate to obtain a linear regression equation: y =66005X +7362.7 (R) 2 ＝0.9988)，The ferulic acid has good linear relation within 0.5-10 mug/mL.
4.3 precision of the Instrument
And (3) adding a standard sample of 5 mu L of ferulic acid matrix with the standard concentration of 3 mu g/mL, continuously feeding samples for 6 times, recording the standard adding peak area of the matrix, and calculating the RSD value of the peak area to be 0.92 percent, thereby indicating that the precision of the instrument is good.
4.4 stability
And sampling the sample solution A, respectively carrying out sample injection measurement for 0 h, 2h, 4h, 8 h, 12 h and 24h, recording peak areas, and calculating the RSD value to be 1.9%, which indicates that the sample solution A has good stability in 24 h.
4.5 repeatability
Taking 6 parts of angelica sinensis sample powder of the same batch, wherein each part is 0.2g, operating according to a sample preparation method, recording peak areas, and calculating by an external standard method to obtain that the average content of ferulic acid is 0.056% and the RSD value is 3.2% (n = 6), which indicates that the method has good repeatability.
4.6 recovery
Precisely weighing 0.2g of the same batch of angelica powder with known content, precisely adding 100 mu L of 1.229mg/mL of mother liquor and 6 parts of the same addition level respectively, and calculating the recovery rate according to the operation of a sample preparation method. The ferulic acid content is 0.56mg/g, the standard substance addition amount is 0.61mg/g, the average recovery rate is 98.1%, and the RSD value is 3.2%. The results are shown in Table 3, and Table 3 shows the results of the ferulic acid recovery test.
TABLE 3 Ferulic acid recovery Experimental results
Example 2:
the detection method is not limited to angelica sinensis, can also be suitable for other medicinal materials, and can be used for directly detecting pesticides and mycotoxins; the selected traditional Chinese medicinal material is coix seed; the detection steps are as follows:
step one, preparation of pesticide and mycotoxin samples:
weighing 3g of coix seed powder, adding 15mL of 5% formic acid aqueous solution into a 80mL polystyrene centrifuge tube with a plug, swirling to fully infiltrate the powder, and standing for 30min. And then adding 30mL of acetonitrile, uniformly mixing in a vortex manner, placing the mixture on an oscillator (300 r/min) for oscillation for 10min, adding 7.5g of anhydrous magnesium sulfate and anhydrous sodium acetate mixed powder (mass ratio is 4). The obtained supernatant is marked as sample liquid A, 10mL of sample liquid A is added into a large test tube, and the sample liquid A is concentrated to 2mL by a parallel concentrator or a rotary evaporator.
The PRIME HLB column is not required to be activated before use, the concentrated solution is moved into the column, a small test tube is connected downwards until air passes through, after all liquid is collected, 3mL methanol is used for elution, 2 times of eluent is combined, and a centrifuge tube is taken down and dried on a nitrogen blowing instrument. Adding 1mL of methanol for redissolution, taking out 0.5mL of the redissolved solution, placing the redissolved solution into another small test tube, drying the two test tubes by using nitrogen, adding 0.5mL of a constant volume solution containing 200ng/mL of atrazine D5 (methanol: water = 3.
And adding 0.5mL of ethyl acetate constant volume solution containing 200ng/mL of epoxy heptachlor into the other test tube, shaking after ultrasonic redissolution, passing through a 0.22-micron organic membrane to obtain a solution, marking as sample solution C, and placing the solution into a sample injection vial for GC-Q-TOF/MS determination.
Step two, detecting conditions of pesticide and mycotoxin samples:
(1) Detection conditions of an LC-Q-TOF/MS instrument are as follows:
a chromatographic column: SB-C18:100mm × 2.1mm (i.d.) × 3.5 μm, column temperature: 40 ℃; mobile phase: the phase A is 0.1% (v/v) formic acid aqueous solution (the solution contains 5mmol/L ammonium acetate), and the phase B is acetonitrile; the flow rate was 0.4mL/min.
Gradient elution procedure: 0-3min, 1-30 percent of C; 3-6 min,30% -40% B;6 to 9min,40 percent B;9 to 15min,40 to 60% by weight of B;15 to 19min,60 to 90 percent of B; 19-23min, 90%; 23 to 23.01min,90 to 1 percent of B;23.01 to 27.01min,1% by weight of B. The injection volume is 5. Mu.L.
Dual AJS ESI source; the scanning mode is as follows: positive ion full scanning; full scan range: m/z is 50-1000; capillary voltage: 4000V; atomizing gas: nitrogen gas; atomizing gas pressure: 0.14Mpa; temperature of sheath gas: 375 ℃; flow rate of sheath gas: 11.0L/min; flow rate of drying gas: 12.0L/min; temperature of drying gas: 325 ℃; fragmentation voltage: 145V. All ion MS/MS mode conditions: at 0min, the collision energy is 0V; at 0.5min, the collision energies were 0, 15 and 35V, respectively.
(2) Detection conditions of a GC-Q-TOF/MS instrument are as follows:
and (3) chromatographic column: HP-5MS UI 30m.times.0.25 mm (i.d.).times.0.25 μm, column temperature: maintaining at 40 deg.C for 1min, heating to 130 deg.C at 30 deg.C/min, heating to 250 deg.C at 5 deg.C/min, heating to 300 deg.C at 10 deg.C/min, and maintaining for 7min; carrier gas: helium purity is more than or equal to 99.999%, flow rate: 1.2mL/min; sample inlet temperature: 270 ℃; sample introduction amount: 1.0 μ L; and (3) sample introduction mode: sampling without flow splitting, and opening the purge valve for 1 min. Ionization mode: electron bombardment (EI); ion source voltage: 70eV; ion source temperature: 280 ℃; quadrupole rod temperature: 180 ℃; solvent retardation: 4min; ion monitoring mode: full scan (TOF MS); scanning range: m/z is 45-550; scanning rate: 5Hz.
Step three, preparing a coix seed functional component sample:
weighing 0.2g of sample powder, precisely adding 30mL of acetonitrile, weighing, soaking for 2 hours, carrying out ultrasonic treatment (power 300W and frequency 50 kHz) for 30 minutes, placing in a centrifuge, and centrifuging at the centrifuge rotation speed of 4200rpm for 5 minutes. Taking 1mL of supernatant, directly passing through a 0.22 μm organic membrane to obtain a solution, marking as sample solution D, diluting by 10 3 Double, used for verifying the effective components.
Step four, instrument conditions of coix seed functional components are as follows:
and (3) chromatographic column: SB-C18 (100 mm. Times.2.1 mm (i.d.). Times.3.5 μm), column temperature: 45 ℃; flow rate: 0.3mL/min; the mobile phase (A) is methanol-acetonitrile-0.1% formic acid solution (19: 2) containing 5mmol of ammonium acetate by volume, B is isopropanol, A-B (55: 45); the column temperature is 45 ℃; the sample size was 5. Mu.L.
The detection mode is the multiple reaction monitoring mode (MRM), and the ion reaction for quantitative analysis is m/z 902.8 → m/z 603.6 (cone aperture voltage 80eV, collision energy 29 eV), m/z 902.8 → m/z 265.2 (cone aperture voltage 80eV, collision energy 40 eV).
Method for verifying pesticide and mycotoxin of semen coicis
1 linear range
Selecting 6 parts of blank coix seeds, carrying out sample pretreatment according to the first step, adding mixed standard working solutions with different concentrations into the pretreated sample liquid B and the sample liquid C based on the difference of compound quantification limits, and establishing matrix matching standard curves of 0.5LOQ, 1LOQ, 2LOQ, 4LOQ, 10LOQ and 20 LOQ; wherein the mixed standard working solution is a mixed standard solution prepared from methanol and water in a volume ratio of 3.
The sample liquid B prepared in the first step is detected by LC-Q-TOF/MS, the sample liquid C is detected by GC-Q-TOF/MS, the results are shown in Table 4, and R is in the linear range of 65 compounds detected in total, 40 compounds detected by LC-Q-TOF/MS and 41 compounds detected by GC-Q-TOF/MS 2 ＞0.9801。
2 limit of quantification
All compounds satisfy the minimum addition level of recovery 70-120% and RSD ≦ 20% at different addition levels as quantitative limits, with reference to the requirements specified in the European Union directive document SANTE/12682/2021.
LC-Q-TOF/MS and GC-Q-TOF/MS jointly detect 65 compounds, wherein the limit of quantification of 1 pesticide (cloxaphos) and 8 mycotoxins (aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, T-2 toxin, fumonisin B1, fumonisin B2 and patulin) is the same as that of Chinese pharmacopoeia (2020 edition), and the rest 56 compounds can reach the limit of quantification lower than that of the Chinese pharmacopoeia (2020 edition).
In the LC-Q-TOF/MS and GC-Q-TOF/MS combined detection of 65 compounds, 16 compounds are co-detected compounds, wherein the LC-Q-TOF/MS detection ratio of 10 compounds can reach lower limit of quantitation than that of GC-Q-TOF/MS, the GC-Q-TOF/MS detection ratio of 1 compound can reach lower limit of quantitation than that of LC-Q-TOF/MS, and the LC-Q-TOF/MS and the GC-Q-TOF/MS quantification limit of 5 compounds are the same. The results are shown in Table 4.
Table 4 shows the linear ranges, linear equations, correlation coefficients and quantitative limits for 65 compounds
3 recovery and relative standard deviation
LC/GC-Q-TOF/MS determined the recovery and relative standard deviation of 65 compounds at LOQ, 2LOQ, and 10 LOQ. At each addition level 6 replicates were taken and blanks were run to subtract interference from endogenous substances in the matrix. 40 compounds measured by LC-Q-TOF/MS and 41 compounds measured by GC-Q-TOF/MS can meet the recovery rate of 70-120% and the RSD is less than or equal to 20% under three addition levels. The results are shown in Table 5 (including tables 5-1 and 5-2), and Table 5 shows the matrix effect, recovery rate and precision results for 65 compounds.
TABLE 5-1 LC-Q-TOF/MS measurements at three addition levels of LOQ, 2LOQ, and 10LOQ
TABLE 5-2 GC-Q-TOF/MS results of LOQ, 2LOQ and 10LOQ additions
4 functional ingredient methodology verification of coix seeds
4.1 limit of quantitation
And diluting the sample solution D step by step according to a certain dilution multiple until the signal-to-noise ratio of the triolein is detected to be 10, and taking the signal-to-noise ratio as the lowest quantitative limit (S/N is more than 10) of the triolein, wherein the quantitative limit of the triolein is detected to be 0.2ng/mL.
4.2 Standard Curve
Precisely sucking a proper amount of the glycerol trioleate standard solution, diluting the glycerol trioleate standard solution with acetonitrile to obtain standard solutions with the concentrations of the glycerol trioleate of 0.5745, 1.149, 5.745, 11.49, 22.98, 57.45 and 114.9ng/mL respectively, and feeding the standard solutions into a mass spectrum detector. And (3) drawing a standard curve by taking the concentration (X) of the standard substance as a horizontal coordinate and the peak area (Y) as a vertical coordinate to obtain a linear regression equation: y =9335.004831X+3009.203884 (R) 2 = 0.9998). The results showed that the glycerol trioleate had a good linear relationship between 0.5745 and 114.9 ng/mL.
4.3 precision of the Instrument
And (3) continuously sampling 5 mu L of a glycerol trioleate standard substance solution with the concentration of 22.98ng/mL for 6 times, recording the peak area of the glycerol trioleate, and calculating the RSD value of the peak area to be 2.14%, thereby indicating that the precision of the instrument is good.
4.4 stability
And sampling sample solution D, respectively carrying out sample injection measurement for 0, 2,4, 8, 12 and 24 hours, recording peak areas, and calculating an RSD value to be 3.37%, which indicates that the stability of the sample solution in 24 hours is good.
4.5 repeatability
Taking 6 parts of coix seed sample powder of the same batch, wherein each part is 0.2g, operating according to methods of '4.1' and '4.2', recording peak areas, and calculating by an external standard method to obtain the glycerol trioleate with the average content of 0.56% and the RSD value of 2.76% (n = 6), which indicates that the method has good repeatability.
4.6 recovery
Precisely weighing 0.2g of Coicis semen powder with known content in the same batch, precisely adding 11.49mg/mL mother liquor 100 μ L, respectively, adding 6 parts of the same amount, and calculating recovery rate by "4.1" and "4.2" method. The content of the triolein in the sample is 5.43-5.81 mg/g, the addition amount of the standard substance is 5.74mg/g, the average recovery rate is 103.7%, and the RSD value is 4.70%. The results are shown in Table 6 (results on the recovery of triolein).
TABLE 6 triolein recovery results
Description of the drawings: the above embodiments are only used to illustrate the present invention and do not limit the technical solutions described in the present invention; thus, while the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted; all such modifications and variations are intended to be included herein within the scope of this disclosure and the present invention and within the scope of the following claims.
Claims (8)
1. A method for simultaneously detecting forbidden pesticides, mycotoxins and effective components in traditional Chinese medicinal materials is characterized by comprising the following steps:
the method comprises the following steps: preparing a sample;
(1) Weighing traditional Chinese medicine powder, adding formic acid water solution, mixing, performing vortex to fully soak the traditional Chinese medicine powder, placing for a period of time, adding acetonitrile, performing vortex mixing, performing oscillation treatment, adding anhydrous magnesium sulfate and anhydrous sodium acetate mixed powder, shaking and mixing uniformly, cooling for a period of time in an ice water bath, performing secondary oscillation treatment after cooling, performing centrifugation after treatment to obtain supernatant, marking the supernatant as sample liquid A, and concentrating the sample liquid A with a parallel concentrator or a rotary evaporator to V/5 to obtain concentrated solution;
the dosage relationship of the mixed powder of the medicinal material powder, the aqueous solution of formic acid, acetonitrile, anhydrous magnesium sulfate and anhydrous sodium acetate is 3g:15mL of: 30mL of: 7.5g; the volume concentration of the formic acid aqueous solution is 5-10%; the mass ratio of the anhydrous magnesium sulfate to the anhydrous sodium acetate in the anhydrous magnesium sulfate and anhydrous sodium acetate mixed powder is 4;
(2) The PRIME HLB column does not need to be activated before use, the concentrated solution prepared in the step (1) is transferred into the column, a centrifugal tube is connected below the column until air passes through the column, methanol is used for eluting for a plurality of times after all the liquid is collected, eluent for a plurality of times is combined, and the centrifugal tube is taken down and dried on a nitrogen blowing instrument; adding methanol for redissolution, taking out 1/2 of the liquid, and respectively filling the liquid into another small test tube, wherein the two small test tubes are dried by nitrogen and respectively marked as a test tube I and a test tube II; adding a constant volume solution I into the test tube I, wherein the constant volume solution I contains 200ng/mL atrazine D5, shaking after ultrasonic redissolution, filtering through an organic membrane to obtain a solution marked as a sample solution B, and placing the sample solution B into a sample injection vial for measurement by LC-Q-TOF/MS;
adding a constant volume liquid II into the test tube II, wherein the constant volume liquid II contains ethyl acetate of epoxy heptachlor with the concentration of 200 ng/mL; shaking after ultrasonic redissolution, filtering by an organic membrane to obtain a solution marked as sample solution C, and placing the sample solution C into a sample injection vial for GC-Q-TOF/MS determination;
step two, detecting conditions of pesticide and mycotoxin;
detecting the sample liquid B prepared in the step one (2) through LC-Q-TOF/MS; detecting the sample liquid C by GC-Q-TOF/MS; the instrument detection conditions were as follows:
LC-Q-TOF/MS detection conditions:
and (3) chromatographic column: SB-C18:100 mm. Times.2.1 mm. Times.3.5. Mu.m, column temperature: 40 ℃; mobile phase: the phase A is a formic acid aqueous solution with the volume concentration of 0.1 percent, ammonium acetate is also dissolved in the formic acid aqueous solution, the concentration of the ammonium acetate is 5mmol/L, and the phase B is acetonitrile; the flow rate is 0.4mL/min;
gradient elution procedure: 0-3min, 1-30 percent of C; 3-6 min, 30-40 percent of B;6 to 9min,40 percent B;9 to 15min,40 to 60% by weight; 15 to 19min,60 to 90 percent of B; 19-23min, 90%; 23 to 23.01min,90 to 1 percent of B; 23.01-27.01min, 1% by weight of B; sample injection volume: 5 mu L of the solution;
dual AJS ESI source; the scanning mode comprises the following steps: positive ion full scanning; full scan range: m/z is 50-1000; capillary voltage: 4000V; atomizing gas: nitrogen gas; atomizing gas pressure: 0.14Mpa; temperature of sheath gas: 375 ℃; flow rate of sheath gas: 11.0L/min; flow rate of drying gas: 12.0L/min; temperature of drying gas: 325 ℃; fragmentation voltage: 145V;
all ion MS/MS mode conditions: at 0min, the collision energy is 0V; at 0.5min, the collision energy is 0, 15 and 35V respectively;
GC-Q-TOF/MS detection conditions:
a chromatographic column: HP-5MS UI:30m × 0.25mm × 0.25 μm, column temperature: maintaining at 40 deg.C for 1min, heating to 130 deg.C at 30 deg.C/min, heating to 250 deg.C at 5 deg.C/min, heating to 300 deg.C at 10 deg.C/min, and maintaining for 7min; carrier gas: helium purity is more than or equal to 99.999%, flow rate: 1.2mL/min; sample inlet temperature: 270 ℃; sample injection amount: 1.0 μ L; and (3) sample introduction mode: sampling without shunting, and opening a purge valve for 1 min;
ionization mode: electron bombardment; ion source voltage: 70eV; ion source temperature: 280 ℃; temperature of the quadrupole rods: 180 ℃; solvent retardation: 4min; ion monitoring mode: full scanning; scanning range: m/z is 45-550; scanning speed: 5Hz;
step three, detecting functional components;
taking the sample liquid A prepared in the step one, and detecting on a machine; and the instrument conditions of the functional components are the same as those of the LC-Q-TOF/MS in the second step, and the specific steps are as follows:
a chromatographic column: SB-C18:100 mm. Times.2.1 mm. Times.3.5. Mu.m, column temperature: at 40 ℃; mobile phase: the phase A is a formic acid aqueous solution with the volume concentration of 0.1 percent, ammonium acetate is also dissolved in the formic acid aqueous solution, the concentration of the ammonium acetate is 5mmol/L, and the phase B is acetonitrile; the flow rate is 0.4mL/min;
gradient elution procedure: 0 to 3min,1 to 30 percent of B; 3-6 min, 30-40 percent of B;6 to 9min,40% by weight; 9 to 15min,40 to 60% by weight of B;15 to 19min,60 to 90 percent of B;19 to 23min,90% by weight B;23 to 23.01min,90 to 1 percent of B;23.01 to 27.01min,1% by weight B; sample introduction volume: 5 mu L of the solution;
dual AJS ESI source; the scanning mode is as follows: positive ion full scanning; full scan range: m/z is 50-1000; capillary voltage: 4000V; atomizing gas: nitrogen gas; atomizing gas pressure: 0.14Mpa; temperature of sheath gas: 375 ℃; flow rate of sheath gas: 11.0L/min; flow rate of drying gas: 12.0L/min; temperature of the drying gas: 325 ℃; fragmentation voltage: 145V;
all ion MS/MS mode conditions: at 0min, the collision energy is 0V; at 0.5min, the collision energy is 0, 15 and 35V respectively;
step four, calculating a result;
(1) Pesticide and mycotoxin detection:
(a) Standard working curve configuration: selecting blank Chinese medicinal materials, carrying out sample pretreatment according to the first step, adding mixed standard working solutions with different concentrations into a sample solution B and a sample solution C after pretreatment based on the difference of compound quantification limits, and establishing a matrix matching standard curve with a series of concentrations; the preparation of the mixed standard working solution comprises the following steps: preparing a mixed standard solution by using methanol and water in a volume ratio of 3;
(b) Quantitative calculation
Quantifying by adopting an internal standard method: according to the quantitative ion pair peak areas of the corresponding compounds in the sample, adopting a linear regression equation in the step (a), and calculating by using MassHunter quantitative analysis software to obtain the concentrations of the corresponding compounds;
(2) And (3) detection of functional components:
(a) Standard working curve configuration: absorbing the sample solution A in the step I, adding an effective component standard solution, diluting the sample solution A with acetonitrile to a series of standard solutions with gradient, and entering a mass spectrum detector; drawing a standard curve by taking the concentration (X) of the standard substance as a horizontal coordinate and the peak area (Y) as a vertical coordinate to obtain a linear regression equation;
(b) And (3) quantitative calculation: calculating the concentration of the effective component of the corresponding compound by adopting the linear regression equation in the step (a) according to the corresponding peak area in the sample.
2. The method for simultaneously detecting forbidden pesticides, mycotoxins and effective components in traditional Chinese medicinal materials according to claim 1, wherein in the step one (1), the traditional Chinese medicinal materials are preferably angelica sinensis.
3. <xnotran> 1 , , , (1) 65 , , , , , , α - , β - , , δ - , p, p ' - , o, p ' - , p, p ' - , p, p ' - , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , α - , β - , , , , , 2,4' - , , , ; </xnotran>
The mycotoxins are 11, and specifically comprise aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, zearalenone, T-2 toxin, fumonisin B1, fumonisin B2, deoxynivalenol, patulin and ochratoxin A;
the effective components comprise ferulic acid and glycerol trioleate.
4. The method for simultaneously detecting forbidden pesticides, mycotoxins and effective components in Chinese herbal medicines according to claim 1, wherein in the step one (1), the Chinese herbal medicines are placed for a period of 20-30min; the conditions of the oscillation treatment are as follows: 300r/min,10-15min; the cooling time is 10-15min; the conditions of the second oscillation treatment are as follows: 300r/min,5-8min; the centrifugation conditions were: 4200rpm, 5-8 min.
5. The method for simultaneously detecting forbidden pesticides, mycotoxins and effective components in traditional Chinese medicinal materials according to claim 1, wherein the volume concentration of the formic acid aqueous solution in the step (1) in the step one is 5%.
6. The method for simultaneously detecting forbidden pesticides, mycotoxins and effective components in traditional Chinese medicinal materials according to claim 1, wherein the volume ratio of the concentrated solution, methanol for elution, methanol for redissolution, constant volume solution I and constant volume solution II in step one (2) is 2: 0.5:0.5; the constant volume liquid I and the constant volume liquid II are both formed by mixing methanol and water according to a volume ratio of 3.
7. The method for simultaneously detecting forbidden pesticides, mycotoxins and effective components in traditional Chinese medicinal materials according to claim 1, wherein the sizes of the organic membranes in the step (2) are all 0.22 μm.
8. Use of the method according to any one of claims 1 to 7 for the simultaneous detection of pesticides, mycotoxins and functional ingredients.
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