CN115452997B - Simultaneous detection method for forbidden pesticides, mycotoxins and functional components in traditional Chinese medicinal materials - Google Patents

Simultaneous detection method for forbidden pesticides, mycotoxins and functional components in traditional Chinese medicinal materials Download PDF

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CN115452997B
CN115452997B CN202211173007.4A CN202211173007A CN115452997B CN 115452997 B CN115452997 B CN 115452997B CN 202211173007 A CN202211173007 A CN 202211173007A CN 115452997 B CN115452997 B CN 115452997B
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常巧英
白若镔
盖丽娟
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Zhongyuan Food Laboratory
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • G01N30/04—Preparation or injection of sample to be analysed
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    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
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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 functional components in traditional Chinese medicinal materials. According to the invention, sample preparation is carried out through the HLB solid phase extraction column, and the combination of LC-Q-TOF/MS and GC-Q-TOF/MS is combined with high-resolution mass spectrometry, so that the effective separation of exogenous pollutants such as pesticide residues and mycotoxins is realized by one-time sample injection, the use efficiency of an instrument is improved, and the data acquisition and analysis time is shortened; meanwhile, through a comparison purification technology, pretreatment conditions such as ice bath and oscillation sequence, acidity of an extracting solution, constant volume liquid and the like are optimized, compatibility of sample pretreatment is improved, simultaneous detection of 33 pesticides (54 monomers), 11 mycotoxins and functional components which are forbidden in traditional Chinese medicinal materials is realized, simultaneous detection of cross-category exogenous pollutants and functional components is possible, experimental cost is effectively reduced, detection time is shortened, and high practical application value is realized.

Description

Simultaneous detection method for forbidden pesticides, mycotoxins and functional components in traditional Chinese medicinal materials
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 functional components in traditional Chinese medicinal materials.
Background
The Chinese medicinal materials are the material basis for the inheritance and development of traditional Chinese medicine industry, and are strategic resources for the relationship of national folk life. The unique principle of administration and the overall therapeutic characteristics of traditional Chinese medicine lead to the continuous expansion of the worldwide influence. In order to better complete the connection of the traditional Chinese medicine and the international, the quality safety of the traditional Chinese medicine is necessary and powerful guarantee. In addition to controlling the quality of traditional Chinese medicine by establishing scientific quality standards, exogenous pollutants of traditional Chinese medicine have seriously affected the quality safety of traditional Chinese medicine. The exogenous harmful pollutants of the traditional Chinese medicine are adsorbed or accumulated in the growth process of animals and plants from the contacted soil, water, atmosphere, feed and other environments, and residues after disease and pest control in the cultivation and cultivation processes, and can also be caused by pollution caused by improper processing, processing and storage of decoction pieces, and mainly comprise pesticide and veterinary drug residues, heavy metal and harmful element residues, mycotoxin residues, organic pollutant residues, pathogenic microorganism pollution and the like. In addition, the content of the functional components is a key index for evaluating the medicinal quality of the functional components, but a broad-spectrum detection technology and an evaluation grading standard are not available at present.
The traditional Chinese medicine materials are improperly treated in the process of growing, storing or processing in the field, and can pollute various fungi and generate mycotoxins. The contaminated fungi include not only aspergillus, penicillium, but also fusarium and the like. The toxins produced include a variety of mycotoxins closely related to human health and safety, such as aflatoxins, ochratoxins, fumonisins, zearalenone, and the like. In order to ensure the medication safety of people, the Chinese pharmacopoeia (2020 edition, one part) prescribes that aflatoxin B1 (AFB 1) in 24 traditional Chinese medicinal materials such as coix seeds and the like is not higher than 5 mug/kg, and the total amount of aflatoxins (AFB1+AFB2+AFG1+AFG2) is not higher than 10 mug/kg; the content of zearalenone in Coicis semen should not exceed 500 μg per 1000 g.
According to the Chinese pharmacopoeia (2020), pesticide residues, mycotoxins and functional components in traditional Chinese medicinal materials are divided into 3 independent experiments from pretreatment to instrumental analysis. The pretreatment of pesticide residue adopts a QuEChERS method, the determination of mycotoxin adopts an HLB solid phase extraction method, the functional components are mainly extracted by adopting a heating reflux method, an ultrasonic extraction method and the like, and different sample preparation processes and different analysis instruments are needed, so that experimental consumables and manpower and time are wasted; in addition, the traditional Chinese medicine has complex components and different medicinal parts, and if a rapid and effective mode is not adopted in the pretreatment, the loss of target substances can be caused, and the experimental result is influenced.
The invention comprises the following steps:
aiming at the defects of the prior art, the invention aims to solve one of the problems, provides a method for simultaneously detecting pesticide, mycotoxin and functional components (shown in table 1) in Chinese medicinal materials, combines the chemical properties of different compounds of various pesticide residues, mycotoxin and functional components, and designs an integrated scheme compatible with different technologies of different classes of compounds by optimizing purification technology, acidity of extracting solution, constant volume solution and the like.
In order to achieve the above object, the present invention comprises the following specific steps:
TABLE 1 preparation of samples simultaneously assayed pesticide, mycotoxin and functional ingredient species
Step one: sample preparation:
(1) Weighing traditional Chinese medicine powder, adding formic acid aqueous solution, mixing, swirling to enable the medicinal powder to be fully soaked, standing for a period of time, adding acetonitrile, swirling to mix uniformly, adding anhydrous magnesium sulfate and anhydrous sodium acetate mixed powder after shaking treatment, shaking to mix uniformly, cooling in an ice water bath for a period of time, performing second shaking treatment after cooling, centrifuging after treatment to obtain a supernatant, recording the supernatant as a sample solution A, concentrating the sample solution A of VmL to V/5 by using a parallel concentrator or a rotary evaporator, and obtaining a concentrated solution;
the dosage relationship of the medicinal material powder, the formic acid aqueous solution, the acetonitrile, the anhydrous magnesium sulfate and the anhydrous sodium acetate mixed powder is 3g:15mL:30mL: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 4:1;
(2) The PRiME HLB column is not required to be activated before being used, the concentrated solution prepared in the step (1) is moved into the column, a separation pipe is connected below until air passes through, methanol is used for eluting for a plurality of times after all the liquid is collected, the eluents of the plurality of times are combined, and a centrifuge tube is taken down for blow drying on a nitrogen blowing instrument; adding methanol for redissolution, taking out 1/2 liquid, sub-packaging into another small test tube, drying both small test tubes by nitrogen, and respectively marking the two small test tubes as a test tube I and a test tube II; adding constant volume liquid I containing 200ng/mL atrazine D5 into a test tube I, shaking after ultrasonic redissolution, filtering by an organic film, marking the obtained solution as sample liquid B, and placing the sample liquid B into a sample injection small bottle for measurement by using LC-Q-TOF/MS;
Adding constant volume liquid II into a test tube II, wherein the constant volume liquid II contains 200ng/mL ethyl acetate of epoxy heptachloride; shaking after ultrasonic redissolution, filtering by an organic film, marking the obtained solution as sample liquid C, and placing the sample liquid C in a sample injection small bottle for GC-Q-TOF/MS measurement;
preferably, in step (1), the aqueous formic acid solution has a volume concentration of 5%.
Preferably, the step one (1) is placed for a period of time of 20-30min; the conditions of the oscillation treatment are as follows: 300r/min,10-15min; the cooling period is 10-15min; the conditions of the second oscillation treatment are as follows: 300r/min,5-8min; the centrifugation conditions are as follows: 4200rpm, 5-8 min.
Preferably, in the step (1), the Chinese medicinal material is preferably angelica.
Preferably, in the step (2), the volume ratio of the concentrated solution, the methanol for eluting, the methanol for redissolving, the constant volume solution I and the constant volume solution II is 2:3:1:0.5:0.5; the constant volume liquid I and the constant volume liquid II are formed by mixing methanol and water in a volume ratio of 3:2;
preferably, the organic films in step (2) are each 0.22 μm in size.
Step two, pesticide and mycotoxin detection conditions:
taking the sample liquid B prepared in the step (2) for detection through LC-Q-TOF/MS; detecting the sample liquid C through GC-Q-TOF/MS;
LC-Q-TOF/MS detection conditions:
chromatographic column: SB-C18:100 mm. Times.2.1 mm. Times.3.5. Mu.m, column temperature: 40 ℃; mobile phase: phase A is 0.1% (v/v) formic acid aqueous solution (5 mmol/L ammonium acetate in solution), and phase B is acetonitrile; the flow rate is 0.4mL/min;
gradient elution procedure: 0-3 min, 1-30% B; 3-6 min, 30-40% B; 6-9 min,40% B; 9-15 min, 40-60% B; 15-19 min,60% -90% B; 19-23 min,90% B;23 to 23.01min,90 to 1 percent of B; 23.01-27.01 min,1% B; sample injection volume: 5. Mu.L;
dual AJS ESI source; scanning mode: positive ion full scanning; full scan range: m/z is 50-1000; capillary voltage: 4000V; atomizing gas: nitrogen gas; atomization gas pressure: 0.14Mpa; sheath temperature: 375 deg.c; sheath air flow rate: 11.0L/min; drying gas flow rate: 12.0L/min; drying gas temperature: 325 deg.c; fragmentation voltage: 145V;
all Ions 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:
chromatographic column: HP-5MS UI:30 m.times.0.25 mm.times.0.25. Mu.m, column temperature: keeping the temperature at 40 ℃ for 1min, programming the temperature to 130 ℃ at 30 ℃/min, heating to 250 ℃ at 5 ℃/min, heating to 300 ℃ at 10 ℃/min, and keeping the temperature for 7min; carrier gas: helium purity is greater than or equal to 99.999%, flow rate: 1.2mL/min; sample inlet temperature: 270 ℃; sample injection amount: 1.0. Mu.L; sample injection mode: sampling without diversion, and opening a purge valve for 1 min;
Ionization mode: electron bombardment (EI); ion source voltage: 70eV; ion source temperature: 280 ℃; quadrupole temperature: 180 ℃; solvent delay: 4min; ion monitoring mode: full scan (TOF MS); scanning range: m/z 45-550; scanning rate: 5Hz.
Step three, detecting conditions of the functional components:
taking the sample liquid A prepared in the step (1) for machine detection; the conditions of the functional components are the same as those of LC-Q-TOF/MS in the second step, and the method is as follows:
chromatographic column: SB-C18:100 mm. Times.2.1 mm. Times.3.5. Mu.m, column temperature: 40 ℃; mobile phase: phase A is 0.1% (v/v) formic acid aqueous solution (5 mmol/L ammonium acetate in solution), and phase B is acetonitrile; the flow rate is 0.4mL/min;
gradient elution procedure: 0-3 min, 1-30% B; 3-6 min, 30-40% B; 6-9 min,40% B; 9-15 min, 40-60% B; 15-19 min,60% -90% B; 19-23 min,90% B;23 to 23.01min,90 to 1 percent of B; 23.01-27.01 min,1% B; sample injection volume: 5. Mu.L;
dual AJS ESI source; scanning mode: positive ion full scanning; full scan range: m/z is 50-1000; capillary voltage: 4000V; atomizing gas: nitrogen gas; atomization gas pressure: 0.14Mpa; sheath temperature: 375 deg.c; sheath air flow rate: 11.0L/min; drying gas flow rate: 12.0L/min; drying gas temperature: 325 deg.c; fragmentation voltage: 145V;
All Ions 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 traditional Chinese medicinal materials, carrying out sample pretreatment according to the first step, adding mixed standard working solutions with different concentrations into the pretreated sample solution B and sample solution C based on the difference of compound quantitative limits, and establishing a series of matrix matching standard curves with different concentrations to obtain a linear regression equation;
the preparation of the mixed standard working solution comprises the following steps: a mixed standard solution prepared by methanol and water according to a volume ratio of 3:2;
(b) Quantitative calculation
Quantification was performed using internal standard: according to the quantitative ion pair peak area of the corresponding compound in the sample, calculating to obtain the concentration of the corresponding compound by adopting a linear regression equation in the step (a) and using MassHunter quantitative analysis software;
(2) And (3) detecting the efficacy components:
(a) Standard working curve configuration: absorbing the sample liquid A in the first step, adding an efficacy component standard solution, diluting the sample liquid A to a series of gradient standard solutions by acetonitrile, and entering a mass spectrum detector; drawing a standard curve by taking the concentration (X) of the standard substance as an abscissa and the peak area (Y) as an ordinate to obtain a linear regression equation;
(b) Quantitative calculation: and (3) calculating the concentration of the effective component of the corresponding compound by adopting a linear regression equation in the step (a) of the step (2) through the corresponding peak area in the sample.
Based on the steps, the simultaneous detection of pesticide and mycotoxin and functional components in Chinese medicinal material angelica can be realized.
The invention has the following beneficial effects:
(1) According to the invention, sample preparation is carried out through the HLB solid phase extraction column, and the combination of LC-Q-TOF and GC-Q-TOF is combined, and the compatibility of sample pretreatment is improved through comparison of a purification method and optimization of conditions such as ice bath and oscillation sequence, acidity of an extracting solution, constant volume liquid and the like, so that 33 pesticides (54 monomers) forbidden in traditional Chinese medicinal materials can be detected simultaneously, 11 mycotoxins and functional components can be detected simultaneously, the experiment cost is effectively reduced, the experiment time is shortened, and the method has a relatively high practical application value.
(2) According to the invention, by optimizing the instrument analysis method, LC-Q-TOF and GC-Q-TOF are combined, and effective separation of exogenous pollutants such as pesticide residues and mycotoxins is realized by one-time sample injection, so that the instrument use efficiency is improved, the data acquisition and data analysis time is shortened, and the experimental efficiency is improved to a great extent.
(3) The invention uses high-resolution mass spectrum 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. While the number of compounds that can be monitored in a single run of conventional low resolution mass spectrometry severely limits its screening capabilities.
(4) In the traditional method, the detection of the triolein in the coix seeds needs liquid chromatography equipped with an evaporative light scattering detector, and the preparation of the sample during the measurement of the ferulic acid in the angelica sinensis needs a heating reflux device and the detection is carried out by using the liquid chromatography. The experiment realizes 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 shows chromatograms of 4 mycotoxins in different constant volume liquids, wherein FIG. A shows deoxynivalenol (Don) chromatograms, FIG. B shows Patulin chromatograms (Patulin), FIG. C shows fumonisin B1 chromatograms (FB 1), and FIG. D shows fumonisin B2 chromatograms (FB 2); wherein (3:2) represents methanol and water are mixed in a volume ratio of 3:2 to be used as constant volume liquid, ACN represents acetonitrile to be used as constant volume liquid, and MeOH represents methanol to be used as constant volume liquid.
Fig. 2 is a graph comparing chromatograms of deoxynivalenol and patulin in constant volume methanol aqueous solution (methanol: water=3:2) and acetonitrile aqueous solution (acetonitrile: water=3:2), respectively;
wherein figures a and B are deoxynivalenol (Don) chromatograms, a being acetonitrile: water (3:2) is used as constant volume liquid; b is methanol: water (3:2) is used as constant volume liquid; panels C and D are patulin chromatograms, panel C is acetonitrile: water (3:2) is used as constant volume liquid; d is methanol: and water (3:2) is used as constant volume liquid.
The specific embodiment is as follows:
the invention will be further illustrated with reference to specific examples;
sample preparation:
3g of traditional Chinese medicine powder is weighed, 15mL of formic acid water solution with volume concentration of 5% is added into a 80mL polystyrene centrifuge tube with a plug, vortex is carried out to fully infiltrate the medicine powder, and the mixture is placed for 30min. Adding 30mL of acetonitrile, mixing uniformly by vortex, putting on an oscillator (300 r/min) to oscillate for 10min, adding 7.5g of anhydrous magnesium sulfate and anhydrous sodium acetate mixed powder (mass ratio 4:1), covering a cover, immediately shaking and mixing uniformly, cooling in an ice water bath for 10min, putting on the oscillator (300 r/min) to oscillate for 5min, putting in a centrifugal machine to centrifuge at 4200rpm for 5min, obtaining supernatant, marking the supernatant as sample liquid A, taking 10mL of sample liquid A, adding into a large test tube, and concentrating to 2mL by a parallel concentrator or a rotary evaporator.
Before the PRiME HLB column is used, the concentrated solution is moved into the column without activation, the separation tube is connected down until air passes through, after all the liquid is collected, 3mL of methanol is used for eluting for 2 times, the 2 times of eluents are combined, and the centrifuge tube is taken down for blow drying on a nitrogen blowing instrument. Adding 1mL of methanol for redissolution, taking out 0.5mL of the solution, subpackaging the solution into another small test tube, blowing the two test tubes with nitrogen, adding 0.5mL of constant volume liquid (methanol: water=3:2) containing 200ng/mL atrazine D5 into one test tube, shaking the test tube after ultrasonic redissolution, passing through an organic film of 0.22 mu m, marking the obtained solution as sample liquid B, and placing the sample liquid B into a sample injection small bottle for measurement by using LC-Q-TOF/MS. And adding 0.5mL of ethyl acetate constant volume solution containing 200ng/mL of epoxy heptachloride into the other test tube, shaking after ultrasonic redissolution, passing through a 0.22 mu m organic film, marking the obtained solution as sample solution C, and placing the sample solution C into a sample injection small bottle for GC-Q-TOF/MS measurement.
1 pretreatment method optimization (Chinese medicinal materials selected from Chinese angelica and coix seed)
1.1 purification method optimization
Chinese angelica and coix seed, each of which is Carb/NH 2 The HLB (200 mg,6 mL) column purification mode and 2 QuEChERS purification modes, the addition level is 10 mug/kg, 3 parallels are examined, namely 3 matrixes are added with the standard before and 1 matrix is added with the standard after; the pesticide quantity required by the recovery rate of more than or equal to 70 percent and less than or equal to 120 percent and the RSD of less than or equal to 20 percent is inspected by different pretreatment methods.
As a result, it was found that HLB is more effective in recovering most of the compounds, particularly the sulfosulfuron-type agricultural chemicals and aflatoxins B1 and B2, and HLB was finally selected as a pretreatment purification method.
1.2HLB method ice bath oscillation sequence optimization
The angelica is subjected to two pretreatment processes of oscillation firstly and then ice bath and oscillation firstly and then ice bath, and the difference between the number of detected compounds and the number of qualified compounds is verified to be small. The number of the compounds is not greatly different in the two ways of LC detection of the coix seeds, but the number of the compounds qualified by GC verification is greatly different, 26 compounds are qualified by ice bath firstly, 16 compounds are qualified by oscillation firstly, the oscillation is presumed to be carried out after ice bath firstly, the oscillation heat release can be relieved in a low-temperature environment, and the extraction of thermally unstable compounds is facilitated.
1.3 optimization of different acidity extracts
The different acidity of the sample liquid A has a 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 and 2% formic acid aqueous solution, taking 3% formic acid aqueous solution, 5% formic acid aqueous solution and 10% formic acid aqueous solution as extraction solvents, and examining the influence of different acidity sample liquid A on extraction efficiency.
Experiments show that the recovery rate of fumonisins B1 and B2 is obviously improved along with the increase of the concentration of formic acid in the sample liquid A. Sample liquid A has too small acidity, the recovery rates of fumonisins B1 and B2 are poor, the acidity is too large, and the time of rotary evaporation and parallel concentration can be prolonged, so that the stability of thermosensitive compounds is affected. When the concentration of formic acid in the angelica is more than 5%, the recovery rate of Fu Ma toxins B1 and B2 is qualified; when the formic acid concentration in coix seed is more than 3%, the recovery rate of Fu Ma toxin B1 and B2 is qualified.
And comprehensively considering the influences of different experimental factors and different acidity on other objects to be detected, and finally selecting 5% formic acid solution as an extraction solution.
1.4 optimization of different constant volume liquids (the ratio of the solutions involved is the volume ratio, v/v)
(1) Methanol, acetonitrile, methanol: comparison of Water (3:2)
Methanol, acetonitrile and methanol respectively: water (3:2) was formulated as a 100ng/mL mixed standard solution, and mass spectrometry was performed to compare the peak areas to determine the overall response level of the liquid test compound. It was found that the peak areas of the different constant volume liquids were not greatly different for most compounds.
However, the response values of some mycotoxins such as fumonisins B1, fumonisins B2, ochratoxins a, deoxynivalenol and spanwise toxin in ACN are significantly lower than those of methanol: water (3:2). Comparing the peak-out time, peak area and peak shape of chromatographic peaks of fumonisin B1, fumonisin B2, deoxynivalenol and spanish toxin in different constant volume liquids, it can be seen from figure 1 that the deoxynivalenol and spanish toxin have double peaks in acetonitrile constant volume liquid, and the ratio of methanol to methanol: the water (3:2) was unimodal. Fumonisins B1, B2 differ little from peak to peak, fumonisins B1 in methanol: peak areas in water (3:2), acetonitrile and methanol are 19232, 952 and 8208 respectively; fumonisin B2 in methanol: peak areas in water (3:2), acetonitrile and methanol were 19257, 282, 13174, respectively. Thus, for fumonisins B1, B2, deoxynivalenol, and spanwise toxin, methanol was selected: water (3:2) is of significant advantage as a response value or peak shape for the constant volume liquid.
(2) Methanol: water (3:2) and acetonitrile: comparison of Water (3:2)
The experiment compares acetonitrile: water (3:2) and methanol: the effect of water (3:2) as constant volume liquid on instrument response values and peak shape. Aiming at four mycotoxins of fumonisin B1, fumonisin B2, vomitoxin and patulin, acetonitrile is used at the concentration of 1 ppm: water (3:2) and methanol: water (3:2) is respectively used as constant volume solution, and the response values and peak shapes of fumonisin B1 and fumonisin B2 (positive ion mode) in the two constant volume solutions are not obviously different, but for vomitoxin and patulin in negative ion mode, methanol: the chromatographic peak shape of water (3:2) as constant volume liquid is obviously narrower and sharper, acetonitrile: water (3:2) is prone to leading peaks for both compounds, as shown in figure 2, so methanol is chosen for these 4 mycotoxins: water (3:2) was used as constant volume solution.
1.5 Experimental Condition optimization of functional Components
The Chinese pharmacopoeia (2020 edition) prescribes that the effective component glycerol trioleate represented by coix seed is measured by using a high performance liquid chromatography (general rule 0512), a sample is extracted by acetonitrile-dichloromethane (65:35) by ultrasonic, and octadecylsilane chemically bonded silica is used as a chromatographic column; acetonitrile-dichloromethane (65:35) as mobile phase; the evaporative light scattering detector detects.
According to the invention, acetonitrile, dichloromethane, isopropanol and water are used as extraction solvents, and the extraction efficiency of acetonitrile, dichloromethane and isopropanol on triolein is found to be not very different, but water is used as the extraction solvents or water in the extraction solvents can influence the effective extraction of triolein. Therefore, acetonitrile is used as an extraction solvent, and the detection is carried out by combining a liquid chromatograph-mass spectrometer.
The ferulic acid which is a representative effective component of angelica in Chinese pharmacopoeia (2020 edition) is measured by using a high performance liquid chromatography (general rule 0512) and octadecylsilane chemically bonded silica is used as a filler; acetonitrile-0.085% phosphoric acid solution (17:83) is used as a mobile phase; the detection wavelength is 316nm; the column temperature is 35 ℃, and the sample needs to be heated and refluxed for 30min.
According to the invention, only the extraction step operation in the sample pretreatment process is needed, and the liquid chromatography-mass spectrometer is directly used for detection, so that the detection sensitivity of the traditional method is improved, and meanwhile, the integrated pretreatment operation with pesticide residues and mycotoxins can be carried out, the reasonable configuration of the instrument is realized, and the labor cost, the time cost and the economic cost are saved.
Implementation of a specific scheme based on the optimized conditions:
Example 1: the Chinese medicinal materials are angelica, 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, swirling to fully infiltrate the powder, standing for 30min, adding 30mL of acetonitrile, swirling to mix uniformly, putting on an oscillator (300 r/min) to oscillate for 10min, adding 7.5g of anhydrous magnesium sulfate and anhydrous sodium acetate mixed powder (mass ratio 4:1), covering a cover to shake and mix uniformly immediately, cooling in an ice water bath for 10min, putting on the oscillator (300 r/min) to oscillate for 5min, putting in a centrifuge to centrifuge, and rotating at 4200rpm for 5min; the obtained supernatant was recorded as sample solution A, 10mL of sample solution A was taken and added to a large test tube, and concentrated to 2mL with a parallel concentrator or rotary evaporator.
Before the PRiME HLB column is used, the concentrated solution is moved into the column without activation, a small test tube is connected below until air passes through, after all the liquid is collected, 3mL of methanol is used for eluting, 2 times of eluents are combined, and a centrifuge tube is taken down for drying on a nitrogen blowing instrument. Adding 1mL of methanol for redissolution, taking out 0.5mL of the solution, subpackaging the solution into another small test tube, blowing the two test tubes with nitrogen, adding 0.5mL of constant volume liquid (methanol: water=3:2) containing 200ng/mL atrazine D5 into one test tube, shaking the test tube after ultrasonic redissolution, passing through an organic film of 0.22 mu m, marking the obtained solution as sample liquid B, and placing the sample liquid B into a sample injection small bottle for measurement by using LC-Q-TOF/MS.
And adding 0.5mL of ethyl acetate constant volume solution containing 200ng/mL of epoxy heptachloride into the other test tube, shaking after ultrasonic redissolution, passing through a 0.22 mu m organic film, marking the obtained solution as sample solution C, and placing the sample solution C into a sample injection small bottle for GC-Q-TOF/MS measurement.
Step two, pesticide and mycotoxin sample detection conditions:
the sample liquid B prepared in the first step is detected by LC-Q-TOF/MS; detecting the sample liquid C through GC-Q-TOF/MS;
(1) LC-Q-TOF/MS instrument detection conditions:
chromatographic column: SB-C18:100mm×2.1mm (i.d.) ×3.5 μm, column temperature: 40 ℃; mobile phase: phase A is 0.1% (v/v) formic acid aqueous solution (5 mmol/L ammonium acetate in solution), and phase B is acetonitrile; the flow rate was 0.4mL/min.
Gradient elution procedure: 0-3 min, 1-30% B; 3-6 min, 30-40% B; 6-9 min,40% B; 9-15 min, 40-60% B; 15-19 min,60% -90% B; 19-23 min,90% B;23 to 23.01min,90 to 1 percent of B; 23.01-27.01 min,1% B. Sample injection volume: 5. Mu.L.
Dual AJS ESI source; scanning mode: positive ion full scanning; full scan range: m/z is 50-1000; capillary voltage: 4000V; atomizing gas: nitrogen gas; atomization gas pressure: 0.14Mpa; sheath temperature: 375 deg.c; sheath air flow rate: 11.0L/min; drying gas flow rate: 12.0L/min; drying gas temperature: 325 deg.c; fragmentation voltage: 145V. All Ions MS/MS mode conditions: at 0min, the collision energy is 0V; at 0.5min, the collision energies were 0, 15 and 35V, respectively.
(2) GC-Q-TOF/MS instrument detection conditions:
chromatographic column: HP-5MS UI:30m×0.25mm (i.d.) ×0.25 μm, column temperature: keeping the temperature at 40 ℃ for 1min, programming the temperature to 130 ℃ at 30 ℃/min, heating to 250 ℃ at 5 ℃/min, heating to 300 ℃ at 10 ℃/min, and keeping the temperature for 7min; carrier gas: helium purity is greater than or equal to 99.999%, flow rate: 1.2mL/min; sample inlet temperature: 270 ℃; sample injection amount: 1.0. Mu.L; sample injection mode: sampling without diversion, and opening a purge valve for 1 min;
ionization mode: electron bombardment (EI); ion source voltage: 70eV; ion source temperature: 280 ℃; quadrupole temperature: 180 ℃; solvent delay: 4min; ion monitoring mode: full scan (TOF MS); scanning range: m/z 45-550; scanning rate: 5Hz.
Step three, detecting the effective components of the angelica sinensis:
taking the sample liquid A obtained in the step one, and performing machine detection;
LC-Q-TOF/MS instrument detection conditions:
chromatographic column: SB-C18:100mm×2.1mm (i.d.) ×3.5 μm, column temperature: 40 ℃; mobile phase: phase A is 0.1% (v/v) formic acid aqueous solution (5 mmol/L ammonium acetate in solution), and phase B is acetonitrile; the flow rate was 0.4mL/min.
Gradient elution procedure: 0-3 min, 1-30% B; 3-6 min, 30-40% B; 6-9 min,40% B; 9-15 min, 40-60% B; 15-19 min,60% -90% B; 19-23 min,90% B;23 to 23.01min,90 to 1 percent of B; 23.01-27.01 min,1% B. The sample injection volume was 5. Mu.L.
Dual AJS ESI source; scanning mode: positive ion full scanning; full scan range: m/z is 50-1000; capillary voltage: 4000V; atomizing gas: nitrogen gas; atomization gas pressure: 0.14Mpa; sheath temperature: 375 deg.c; sheath air flow rate: 11.0L/min; drying gas flow rate: 12.0L/min; drying gas temperature: 325 deg.c; fragmentation voltage: 145V. All Ions MS/MS mode conditions: at 0min, the collision energy is 0V; at 0.5min, the collision energies were 0, 15 and 35V, respectively.
Chinese angelica methodology validation
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 the pretreated extracting solution based on the difference of compound quantitative limits, and establishing a matrix matching standard curve 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 according to a volume ratio of 3:2;
the sample liquid B prepared in the first step is subjected to LC-Q-TOF/MS and the sample liquid C is subjected to GC-Q-TOF/MS to detect 65 compounds, and the results are shown in the table 1, wherein 38 compounds detected by LC-Q-TOF/MS and 41 compounds detected by GC-Q-TOF/MS are in the respective linear ranges, and R2 is more than 0.9801.
Table 1 shows the linear range, linear equation, correlation coefficient and quantitative limit of 65 compounds
2 quantitative limit
With reference to the requirements specified in the EU guidelines SANTE/12682/2021, the lowest addition level at which all compounds meet recovery rates of 70-120% and RSD.ltoreq.20% at different addition levels is the quantitative limit.
LC-Q-TOF/MS and GC-Q-TOF/MS are combined to detect 65 compounds, wherein the quantitative limits of 1 pesticide (cloxazophos) and 8 mycotoxins (aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, T-2 toxin, fumonisin B1, fumonisin B2 and patulin) are the same as those of Chinese pharmacopoeia (2020 edition), and the rest 55 compounds can reach lower quantitative limits than those of Chinese pharmacopoeia (2020 edition).
Of 65 compounds detected by LC-Q-TOF/MS and GC-Q-TOF/MS, 16 compounds are co-detection compounds, wherein 10 compounds can reach lower quantitative limit than GC-Q-TOF/MS, 1 compound can reach lower quantitative limit than LC-Q-TOF/MS, and 5 compounds can reach the same quantitative limit as GC-Q-TOF/MS.
3 recovery and relative standard deviation
The recovery and relative standard deviation of 65 compounds at LOQ, 2LOQ and 10LOQ were determined by LC/GC-Q-TOF/MS. Each addition level was 6 replicates, while blank experiments were performed to subtract the interference of 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 is less than or equal to 20% at three addition levels. The results are shown in Table 2 (including Table 2-1 and Table 2-2), and Table 2 shows the results of matrix effect, recovery and precision for 65 compounds.
TABLE 2 detection results of LC-Q-TOF/MS at three addition levels of LOQ, 2LOQ and 10LOQ
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TABLE 2 detection results of GC-Q-TOF/MS at three addition levels of LOQ, 2LOQ and 10LOQ
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4 methodological verification of Chinese angelica effective components
4.1 quantitative limit
And (3) gradually diluting the sample liquid A prepared in the step (A) according to a certain dilution multiple until the signal to noise ratio of the ferulic acid can be detected to be 10, wherein the signal to noise ratio is used as the lowest quantitative limit (S/N > 10), and the quantitative limit of the ferulic acid is detected to be 50ng/mL.
4.2 matrix matching Standard Curve
Accurately sucking a proper amount of standard solution, adding the standard solution into the sample solution A for sample preparation, obtaining matrix matching standard curve solutions with the ferulic acid concentration of 0.5, 1, 2, 3, 5 and 10 mug/mL respectively, and entering a mass spectrum detector. Taking the detected concentration minus the ferulic acid background concentration (X) as an abscissa and the peak area (Y) as an ordinate, and drawing a standard curve to obtain a linear regression equation: y=66005x+7362.7 (R 2 = 0.9988), showing good linearity of ferulic acid within 0.5-10 μg/mL.
4.3 precision of instrument
Taking 5 mu L of ferulic acid matrix labeled sample with the labeled concentration of 3 mu g/mL, continuously injecting sample for 6 times, recording the peak area of the matrix labeled sample, and calculating the RSD value of the peak area to be 0.92%, wherein the RSD value indicates that the instrument precision is good.
4.4 stability
Sample liquid A is taken and is respectively sampled and measured at 0, 2, 4, 8, 12 and 24 hours, peak areas are recorded, and RSD value is calculated to be 1.9%, so that the sample liquid A has good stability in 24 hours.
4.5 repeatability
6 parts of angelica sample powder of the same batch are taken, each part is 0.2g, the operation is carried out according to the sample preparation method, the peak area is recorded, the average content of ferulic acid is 0.056% by an external standard method, and the RSD value is 3.2% (n=6), which indicates that the method repeatability is good.
4.6 recovery rate
Precisely weighing 0.2g of angelica powder with known content in the same batch, precisely adding 100 mu L of 1.229mg/mL mother liquor respectively, and calculating recovery rate by operating according to a sample preparation method at the same adding level of 6 parts. 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 rate experiments.
TABLE 3 Experimental results on recovery of ferulic acid
Example 2:
the detection method is not limited to angelica, can be applied to other medicinal materials, and can be used for directly detecting pesticides and mycotoxins; the selected Chinese medicinal material is coix seed; the detection steps are as follows:
step one, preparing pesticide and mycotoxin samples:
3g of coix seed powder is weighed, 15mL of 5% formic acid aqueous solution is added into an 80mL polystyrene centrifuge tube with a plug, vortex is carried out to fully infiltrate the powder, and the mixture is placed for 30min. Then 30mL of acetonitrile is added, vortex mixing is carried out, the mixture is put on an oscillator (300 r/min) to oscillate for 10min, 7.5g of anhydrous magnesium sulfate and anhydrous sodium acetate mixed powder (mass ratio 4:1) is added, the mixture is covered with a cover to immediately shake and disperse, mixing is carried out, cooling is carried out for 10min in ice water bath, the mixture is put on the oscillator (300 r/min) to oscillate for 5min, the mixture is put on a centrifuge to centrifuge, and the rotational speed of the centrifuge is 4200rpm for 5min. The obtained supernatant was recorded as sample solution A, 10mL of sample solution A was taken and added to a large test tube, and concentrated to 2mL with a parallel concentrator or rotary evaporator.
Before the PRiME HLB column is used, the concentrated solution is moved into the column without activation, a small test tube is connected below until air passes through, after all the liquid is collected, 3mL of methanol is used for eluting, 2 times of eluents are combined, and a centrifuge tube is taken down for drying on a nitrogen blowing instrument. Adding 1mL of methanol for redissolution, taking out 0.5mL of the solution, subpackaging the solution into another small test tube, blowing the two test tubes with nitrogen, adding 0.5mL of constant volume liquid (methanol: water=3:2) containing 200ng/mL atrazine D5 into one test tube, shaking the test tube after ultrasonic redissolution, passing through an organic film of 0.22 mu m, marking the obtained solution as sample liquid B, and placing the sample liquid B into a sample injection small bottle for measurement by using LC-Q-TOF/MS.
And adding 0.5mL of ethyl acetate constant volume solution containing 200ng/mL of epoxy heptachloride into the other test tube, shaking after ultrasonic redissolution, passing through a 0.22 mu m organic film, marking the obtained solution as sample solution C, and placing the sample solution C into a sample injection small bottle for GC-Q-TOF/MS measurement.
Step two, pesticide and mycotoxin sample detection conditions:
(1) LC-Q-TOF/MS instrument detection conditions:
chromatographic column: SB-C18:100mm×2.1mm (i.d.) ×3.5 μm, column temperature: 40 ℃; mobile phase: phase A is 0.1% (v/v) formic acid aqueous solution (5 mmol/L ammonium acetate in solution), and phase B is acetonitrile; the flow rate was 0.4mL/min.
Gradient elution procedure: 0-3 min, 1-30% B; 3-6 min, 30-40% B; 6-9 min,40% B; 9-15 min, 40-60% B; 15-19 min,60% -90% B; 19-23 min,90% B;23 to 23.01min,90 to 1 percent of B; 23.01-27.01 min,1% B. The sample injection volume was 5. Mu.L.
Dual AJS ESI source; scanning mode: positive ion full scanning; full scan range: m/z is 50-1000; capillary voltage: 4000V; atomizing gas: nitrogen gas; atomization gas pressure: 0.14Mpa; sheath temperature: 375 deg.c; sheath air flow rate: 11.0L/min; drying gas flow rate: 12.0L/min; drying gas temperature: 325 deg.c; fragmentation voltage: 145V. All Ions MS/MS mode conditions: at 0min, the collision energy is 0V; at 0.5min, the collision energies were 0, 15 and 35V, respectively.
(2) GC-Q-TOF/MS instrument detection conditions:
chromatographic column: HP-5MS UI 30m x 0.25mm (i.d.) x 0.25 μm, column temperature: keeping the temperature at 40 ℃ for 1min, programming the temperature to 130 ℃ at 30 ℃/min, heating to 250 ℃ at 5 ℃/min, heating to 300 ℃ at 10 ℃/min, and keeping the temperature for 7min; carrier gas: helium purity is greater than or equal to 99.999%, flow rate: 1.2mL/min; sample inlet temperature: 270 ℃; sample injection amount: 1.0. Mu.L; sample injection mode: and (5) sampling without split flow, and opening a purge valve for 1 min. Ionization mode: electron bombardment (EI); ion source voltage: 70eV; ion source temperature: 280 ℃; quadrupole temperature: 180 ℃; solvent delay: 4min; ion monitoring mode: full scan (TOF MS); scanning range: m/z 45-550; scanning rate: 5Hz.
Step three, preparation of a coix seed functional component sample:
0.2g of sample powder is weighed, 30mL of acetonitrile is precisely added, the mixture is weighed, soaked for 2 hours, subjected to ultrasonic treatment (power 300W and frequency 50 kHz) for 30 minutes, and placed into a centrifugal machine for centrifugation, and the rotation speed of the centrifugal machine is 4200rpm for 5 minutes. Taking 1mL of supernatant, directly passing through 0.22 mu m organic film, recording the obtained solution as sample solution D, diluting 10 3 The product is used for verifying the effective components.
Fourth, instrument conditions of the coix seed effective components:
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-formic acid solution (19:19:2) with the volume concentration of 5mmol of ammonium acetate being 0.1%, B is isopropanol, and A-B (55:45); column temperature 45 ℃; the sample injection amount was 5. Mu.L.
The detection mode is a multi-reaction monitoring mode (MRM), and the ion reaction for quantitative analysis is m/z 902.8- & gt m/z 603.6 (cone hole voltage 80eV, collision energy 29 eV), and m/z 902.8- & gt m/z 265.2 (cone hole voltage 80eV, collision energy 40 eV).
Coix seed pesticide and mycotoxin methodology validation
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 a sample solution B and a sample solution C after pretreatment based on the difference of compound quantitative limits, and establishing a matrix matching standard curve of 0.5LOQ, 1LOQ, 2LOQ, 4LOQ, 10LOQ and 20 LOQ; wherein the mixed standard working solution is a mixed standard solution prepared by mixing methanol and water according to a volume ratio of 3:2 to prepare 100 ng/mL.
Passing the sample solution B prepared in the step one through LC-Q-TOF/MS and sample solution C were detected by GC-Q-TOF/MS, and the results are shown in Table 4, in which 65 compounds were detected in total, 40 compounds detected by LC-Q-TOF/MS and 41 compounds detected by GC-Q-TOF/MS were R in the respective linear ranges 2 ＞0.9801。
2 quantitative limit
With reference to the requirements specified in the EU guidelines SANTE/12682/2021, the lowest addition level at which all compounds meet recovery rates of 70-120% and RSD.ltoreq.20% at different addition levels is the quantitative limit.
LC-Q-TOF/MS and GC-Q-TOF/MS are combined to detect 65 compounds, wherein the quantitative limits of 1 pesticide (cloxazophos) and 8 mycotoxins (aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, T-2 toxin, fumonisin B1, fumonisin B2 and patulin) are the same as those of Chinese pharmacopoeia (2020 edition), and the rest 56 compounds can reach lower quantitative limits than those of Chinese pharmacopoeia (2020 edition).
Of 65 compounds detected by LC-Q-TOF/MS and GC-Q-TOF/MS, 16 compounds are co-detection compounds, wherein 10 compounds can reach lower quantitative limit than GC-Q-TOF/MS, 1 compound can reach lower quantitative limit than LC-Q-TOF/MS, and 5 compounds can reach the same quantitative limit as GC-Q-TOF/MS. The results are shown in Table 4.
Table 4 shows the linear range, linear equation, correlation coefficient and quantitative limit of 65 compounds
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3 recovery and relative standard deviation
The recovery and relative standard deviation of 65 compounds at LOQ, 2LOQ and 10LOQ were determined by LC/GC-Q-TOF/MS. Each addition level was 6 replicates, while blank experiments were performed to subtract the interference of endogenous substances in the matrix. 40 compounds determined by LC-Q-TOF/MS and 41 compounds determined by GC-Q-TOF/MS can meet the recovery rate of 70-120% and the RSD is less than or equal to 20% at three addition levels. The results are shown in Table 5 (including Table 5-1 and Table 5-2), and Table 5 shows the matrix effect, recovery and precision results for 65 compounds.
TABLE 5-1 LC-Q-TOF/MS detection results at three addition levels of LOQ, 2LOQ and 10LOQ
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TABLE 5-2 GC-Q-TOF/MS detection results at three addition levels of LOQ, 2LOQ and 10LOQ
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4 methodological verification of coix seed functional components
4.1 quantitative limit
And (3) gradually diluting the sample solution D according to a certain dilution multiple until the signal to noise ratio of the triolein can be detected to be 10, wherein the minimum quantitative limit (S/N > 10) is used, and the result shows that the quantitative limit of the triolein is 0.2ng/mL.
4.2 Standard Curve
Precisely absorbing a proper amount of triolein standard solution, and diluting with acetonitrile to obtain the triolein with the concentration of 0.5745 respectively Standard solutions of 1.149, 5.745, 11.49, 22.98, 57.45, 114.9ng/mL, enter the mass spectrum detector. And drawing a standard curve by taking the concentration (X) of the standard substance as an abscissa and the peak area (Y) as an ordinate to obtain a linear regression equation: y=9335.004831x+3009.203884 (R 2 =0.9998). The linear relationship of the triolein is good within 0.5745-114.9 ng/mL.
4.3 precision of instrument
And taking 5 mu L of a triolein standard substance solution with the concentration of 22.98ng/mL, continuously injecting the sample for 6 times, recording the peak area of the triolein, and calculating the RSD value of the peak area to be 2.14%, wherein the instrument precision is good.
4.4 stability
Sample liquid D is taken and is respectively sampled and measured at 0, 2, 4, 8, 12 and 24 hours, peak areas are recorded, and RSD value is calculated to be 3.37%, so that the stability of the sample solution is good within 24 hours.
4.5 repeatability
6 parts of coix seed sample powder in the same batch are taken, each part is 0.2g, the operation is carried out according to the methods of 4.1 and 4.2, the peak area is recorded, the average content of the triolein is 0.56% by calculation of an external standard method, and the RSD value is 2.76% (n=6), so that the method has good repeatability.
4.6 recovery rate
Precisely weighing 0.2g of coix seed powder with known content in the same batch, precisely adding 100 mu L of 11.49mg/mL mother liquor respectively, and operating with the same adding level of 6 parts according to the methods of 4.1 and 4.2, and calculating the recovery rate. 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 of triolein recovery).
TABLE 6 results of recovery of triolein
Description: the above embodiments are only for illustrating the present invention and not for limiting the technical solution described in the present invention; thus, while the invention has been described in detail with reference to the various embodiments described above, it will be understood by those skilled in the art that the invention may be modified or equivalents; all technical solutions and modifications thereof that do not depart from the spirit and scope of the present invention are intended to be included in the scope of the appended claims.

Claims (6)

1. A method for simultaneously detecting pesticide, mycotoxin and functional components in Chinese medicinal materials is characterized by comprising the following steps of:
step one: preparing a sample;
(1) Weighing traditional Chinese medicine powder, adding formic acid aqueous solution, mixing, swirling to enable the medicinal powder to be fully soaked, standing for a period of time, adding acetonitrile, swirling to mix uniformly, adding anhydrous magnesium sulfate and anhydrous sodium acetate mixed powder after shaking treatment, shaking to mix uniformly, cooling in an ice water bath for a period of time, performing secondary shaking treatment after cooling, centrifuging after treatment to obtain a supernatant, recording the supernatant as a sample solution A, and concentrating V mL of the sample solution A to V/5 by using a parallel concentrator or a rotary evaporator to obtain a concentrated solution;
The dosage relationship of the medicinal material powder, the formic acid aqueous solution, the acetonitrile, the anhydrous magnesium sulfate and the anhydrous sodium acetate mixed powder is 3g:15mL:30 mL:7.5 g; 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 4:1;
(2) The PRiME HLB column is not required to be activated before being used, the concentrated solution prepared in the step (1) is moved into the column, a separation pipe is connected below until air passes through, methanol is used for eluting for a plurality of times after all the liquid is collected, the eluents of the plurality of times are combined, and a centrifuge tube is taken down for blow drying on a nitrogen blowing instrument; adding methanol for redissolution, taking out 1/2 liquid, sub-packaging into another small test tube, drying both small test tubes by nitrogen, and respectively marking the two small test tubes as a test tube I and a test tube II; adding constant volume liquid I containing 200ng/mL atrazine D5 into a test tube I, shaking after ultrasonic redissolution, filtering by an organic film, marking the obtained solution as sample liquid B, and placing the sample liquid B into a sample injection small bottle for measurement by using LC-Q-TOF/MS;
adding constant volume liquid II into a test tube II, wherein the constant volume liquid II contains 200ng/mL ethyl acetate of epoxy heptachloride; shaking after ultrasonic redissolution, filtering by an organic film, marking the obtained solution as sample liquid C, and placing the sample liquid C in a sample injection small bottle for GC-Q-TOF/MS measurement;
Step two, detecting conditions of pesticides and mycotoxins;
detecting the sample liquid B prepared in the step (2) through LC-Q-TOF/MS; detecting the sample liquid C through GC-Q-TOF/MS; the instrument detection conditions were as follows:
LC-Q-TOF/MS detection conditions:
chromatographic column: SB-C18:100 mm×2.1mm×3.5 μm, column temperature: 40. the temperature is lower than the temperature; mobile phase: the phase A is formic acid aqueous solution with volume concentration of 0.1%, ammonium acetate is also dissolved in the formic acid aqueous solution, the concentration of the ammonium acetate is 5 mmol/L, and the phase B is acetonitrile; the flow rate is 0.4 mL/min;
gradient elution procedure: 0-3 min, 1-30% B; 3-6 min, 30-40% of B; 6-9 min,40% B; 9-15 min, 40-60% of B; 15-19 min, 60-90% of B; 19-23 min,90% B;23 to 23.01 min,90 to 1 percent of B; 23.01 to 27.01 min,1 percent of B; sample injection volume: 5. mu L;
dual AJS ESI source; scanning mode: positive ion full scanning; full scan range: m/z is 50-1000; capillary voltage: 4000 V, V; atomizing gas: nitrogen gas; atomization gas pressure: 0.14 Mpa; sheath temperature: 375 deg.c; sheath air flow rate: 11.0 L/min; drying gas flow rate: 12.0 L/min; drying gas temperature: 325 deg.c; fragmentation voltage: 145 V, V;
all Ions MS/MS mode conditions: at 0 min, the collision energy is 0V; 0.5 At min, the collision energy is 0, 15 and 35V respectively;
GC-Q-TOF/MS detection conditions:
chromatographic column: HP-5 MS UI:30 m.times.0.25 mm.times.0.25. Mu.m, column temperature: keeping the temperature at 40 ℃ for 1min, programming the temperature to 130 ℃ at 30 ℃/min, heating to 250 ℃ at 5 ℃/min, heating to 300 ℃ at 10 ℃/min, and keeping the temperature for 7min; carrier gas: helium purity is greater than or equal to 99.999%, flow rate: 1.2 mL/min; sample inlet temperature: 270 ℃; sample injection amount: 1.0 2, L; sample injection mode: sampling without diversion, and opening a purge valve for 1 min;
ionization mode: electron bombardment; ion source voltage: 70 eV; ion source temperature: 280 ℃; quadrupole temperature: 180 ℃; solvent delay: 4 min; ion monitoring mode: full scanning; scanning range: m/z 45-550; scanning rate: 5 Hz;
step three, detecting the functional components;
taking the sample liquid A prepared in the step one, and performing machine detection; the conditions of the functional components are the same as those of LC-Q-TOF/MS in the second step, and the method is as follows:
chromatographic column: SB-C18:100 mm×2.1mm×3.5 μm, column temperature: 40. the temperature is lower than the temperature; mobile phase: the phase A is formic acid aqueous solution with volume concentration of 0.1%, ammonium acetate is also dissolved in the formic acid aqueous solution, the concentration of the ammonium acetate is 5 mmol/L, and the phase B is acetonitrile; the flow rate is 0.4 mL/min;
gradient elution procedure: 0-3 min, 1-30% B; 3-6 min, 30-40% of B; 6-9 min,40% B; 9-15 min, 40-60% of B; 15-19 min, 60-90% of B; 19-23 min,90% B;23 to 23.01 min,90 to 1 percent of B; 23.01 to 27.01 min,1 percent of B; sample injection volume: 5. mu L;
Dual AJS ESI source; scanning mode: positive ion full scanning; full scan range: m/z is 50-1000; capillary voltage: 4000 V, V; atomizing gas: nitrogen gas; atomization gas pressure: 0.14 Mpa; sheath temperature: 375 deg.c; sheath air flow rate: 11.0 L/min; drying gas flow rate: 12.0 L/min; drying gas temperature: 325 deg.c; fragmentation voltage: 145 V, V;
all Ions MS/MS mode conditions: at 0 min, the collision energy is 0V; 0.5 At min, 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 traditional 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 quantitative limits, and establishing a series of matrix matching standard curves with different concentrations; the preparation of the mixed standard working solution comprises the following steps: a mixed standard solution prepared by methanol and water according to a volume ratio of 3:2;
(b) Quantitative calculation
Quantification was performed using internal standard: according to the quantitative ion pair peak area of the corresponding compound in the sample, calculating to obtain the concentration of the corresponding compound by adopting a linear regression equation in the step (a) and using MassHunter quantitative analysis software;
(2) And (3) detecting the efficacy components:
(a) Standard working curve configuration: absorbing the sample liquid A in the first step, adding an efficacy component standard solution, diluting the sample liquid A to a series of gradient standard solutions by acetonitrile, and entering a mass spectrum detector; drawing a standard curve by taking the concentration (X) of the standard substance as an abscissa and the peak area (Y) as an ordinate to obtain a linear regression equation;
(b) Quantitative calculation: calculating the concentration of the effective components of the corresponding compound by adopting a linear regression equation in the step (a) through the corresponding peak area in the sample;
the Chinese medicinal material is radix Angelicae sinensis or Coicis semen; the pesticides are 65 kinds, respectively, methamidophos, methyl parathion, monocrotophos, phosphamide, alpha-hexa, beta-hexa, lindane, delta-hexa and p, p ' -drop, o, p ' -drop, p, p ' -drop, insecticidal amidine, herbicidal ether, ainsliac, di's agent, phenthoate sulfone, phenthoate sulfoxide, dinotefuran, thiotepa, coumaphos, terbutafos sulfone, terbutafos sulfoxide, chlorsulfuron, amicarbazone, metsulfuron, methamphetamine sulfone, methamphetamine sulfoxide, methylisoprophos, the composition comprises the components of benfophos, carbofuran, tri-hydroxy carbofuran, aldicarb sulfoxide, methofos, chlorzophos, fenpyrad, endosulfan, alpha-endosulfan, beta-endosulfan, fipronil sulfone, fipronil sulfoxide, 2,4' -trichloroacaricidal, trichlorfon, thiocyclam and methiphos;
The mycotoxins are 11 types, namely 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.
2. The method for simultaneous detection of pesticides, mycotoxins and functional components in a Chinese medicinal material according to claim 1, wherein the step (1) is carried out for a period of time of 20-30min; the conditions of the oscillation treatment are as follows: 300r/min,10-15min; the cooling period is 10-15min; the conditions of the second oscillation treatment are as follows: 300r/min,5-8min; the centrifugation conditions are as follows: 4200 rpm, 5-8 min.
3. The method for simultaneous detection of pesticide, mycotoxin and functional components in Chinese medicinal materials according to claim 1, wherein the concentration of the formic acid aqueous solution in the step (1) is 5%.
4. The method for simultaneously detecting pesticide, mycotoxin and functional components in Chinese herbal medicines according to claim 1, wherein the volume ratio of the concentrated solution, the methanol for elution, the methanol for reconstitution, the constant volume solution I and the constant volume solution II in the step (2) is 2:3:1:0.5:0.5; the constant volume liquid I and the constant volume liquid II are both formed by mixing methanol and water in a volume ratio of 3:2.
5. The method for simultaneous detection of pesticide, mycotoxin and functional components in Chinese medicinal materials according to claim 1, wherein the organic membranes in the step (2) are 0.22 μm in size.
6. The method according to any one of claims 1 to 5 for simultaneous detection of pesticides, mycotoxins and functional components.
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