CN115015430A - Method for detecting aflatoxin in soy sauce - Google Patents
Method for detecting aflatoxin in soy sauce Download PDFInfo
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- CN115015430A CN115015430A CN202210708281.0A CN202210708281A CN115015430A CN 115015430 A CN115015430 A CN 115015430A CN 202210708281 A CN202210708281 A CN 202210708281A CN 115015430 A CN115015430 A CN 115015430A
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- 229930195730 Aflatoxin Natural products 0.000 title claims abstract description 63
- 239000005409 aflatoxin Substances 0.000 title claims abstract description 63
- 238000000034 method Methods 0.000 title claims abstract description 47
- 235000013555 soy sauce Nutrition 0.000 title claims abstract description 39
- XWIYFDMXXLINPU-UHFFFAOYSA-N Aflatoxin G Chemical compound O=C1OCCC2=C1C(=O)OC1=C2C(OC)=CC2=C1C1C=COC1O2 XWIYFDMXXLINPU-UHFFFAOYSA-N 0.000 title claims abstract description 27
- 238000005185 salting out Methods 0.000 claims abstract description 64
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- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
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- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 2
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- OQIQSTLJSLGHID-WNWIJWBNSA-N aflatoxin B1 Chemical compound C=1([C@@H]2C=CO[C@@H]2OC=1C=C(C1=2)OC)C=2OC(=O)C2=C1CCC2=O OQIQSTLJSLGHID-WNWIJWBNSA-N 0.000 description 1
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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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- 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
- 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
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
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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
- 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
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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
- G01N30/86—Signal analysis
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/047—Standards external
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/90—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in food processing or handling, e.g. food conservation
Abstract
The invention relates to a food detection method, in particular to a method for detecting aflatoxin in soy sauce. It comprises the following steps: s1, preparation of mixed control solution: formulating AFT B 1 、B 2 、G 1 、G 2 Mixing the standard stock solution; s2, preparation of a test solution: selecting a salting-out agent and an extraction solvent, and preparing a test solution; s3, measurement: precisely absorbing the mixed standard working solution, detecting in a liquid chromatograph-mass spectrometer, measuring the area of a quantitative ion peak, drawing an aflatoxin matrix standard by taking the sample concentration (X) as a horizontal coordinate and the area of the quantitative ion peak (Y) as a vertical coordinateA quasi-solution curve chart; measuring the area of a quantitative ion peak of the test solution under the same condition, and obtaining the concentration of the aflatoxin in the test solution according to the matrix standard curve linear equation; s4, specific requirements for chromatographic and mass spectrometry conditions. The method adopts a salting-out assisted liquid-liquid extraction method combined with an ultra-high performance liquid chromatography-tandem mass spectrometry method, and can perform high-throughput detection on aflatoxin in the soy sauce.
Description
Technical Field
The invention relates to a food detection method, in particular to a method for detecting aflatoxin in soy sauce.
Background
The soy sauce has unique flavor and rich nutrition, is easy to digest and absorb, and is popular with consumers. The main raw materials of the soy sauce are crops such as soybean, wheat and bran, the crops are easily polluted by mycotoxin in the planting and storing process, and in addition, the soy sauce needs the participation of fungi such as saccharomycetes and aspergillus in the manufacturing process and is easily polluted by the mycotoxin to cause the problem of residual mycotoxin. Aflatoxins (AFT) are a group of compounds with dihydrofurocoumarin structures produced by secondary metabolism of aspergillus flavus and aspergillus parasiticus. Aflatoxins are highly toxic substances and have severe carcinogenicity, teratogenicity and mutagenicity. At present, more than 20 aflatoxins are reported and are mainly AFT B 1 、AFT B 2 、AFT G 1 、AFT G 2 Wherein AFT B 1 The compound has the strongest toxicity and is one of the compounds with the strongest toxicity at present. Therefore, in order to ensure the safety of soy sauce food, the national GB 2761-2017 mycotoxin limit in food safety national standard food explicitly stipulates the AFT B in soy sauce 1 The maximum limit value of (2) is not more than 5 mu g/kg.
At present, the quantitative detection of aflatoxin in food is mainly high performance liquid chromatography and high performance liquid chromatography-tandem mass spectrometry. Because food samples are various and the component matrixes are complex, and the concentration of aflatoxin in food is often low, a proper sample pretreatment technology is required to be adopted for enrichment and purification before instrument analysis. Common pretreatment technologies mainly comprise a liquid-liquid extraction method, a solid-phase extraction method, an immunoaffinity chromatography, a magnetic bead immune purification method, a QuEChERS method and the like, and because fermentation seasoning matrixes such as soy sauce and the like are complex, the conventional pretreatment method mainly comprises the immunoaffinity chromatography, but the method is high in detection cost, relatively complex in operation and not suitable for high-throughput detection.
Disclosure of Invention
The invention aims to provide a method for detecting aflatoxin in soy sauce, which has the advantages of quick and simple pretreatment and low cost.
The technical purpose of the invention is realized by the following technical scheme:
a method for detecting aflatoxin in soy sauce comprises the following steps:
s1, preparation of mixed control solution: formulating AFT B 1 、B 2 、G 1 、G 2 Mixing the standard stock solution;
s2, preparation of a test solution: selecting a salting-out agent and an extraction solvent, and preparing a test solution;
s3, measurement: precisely absorbing the mixed standard working solution, detecting the mixed standard working solution in a liquid chromatograph-mass spectrometer, determining the area of a quantitative ion peak, and drawing a standard solution curve chart of the aflatoxin matrix by taking the sample concentration (X) as a horizontal coordinate and the area (Y) of the quantitative ion peak as a vertical coordinate; measuring the area of a quantitative ion peak of the test solution under the same condition, and obtaining the concentration of the aflatoxin in the test solution according to the matrix standard curve linear equation;
s4, chromatographic and mass spectrometric conditions:
chromatographic conditions are as follows: a mobile phase A: methanol/acetonitrile mixture (v/v ═ 50/50), mobile phase B: 4-6mmol/L ammonium acetate solution; gradient elution procedure: 0.0-5.0 min, 40% A; 0.5-3.0 min, 40% -60% A; 3.0-4.0 min, 60% A; 4.0-4.2 min, 60% -100% A; 4.2-5 min, 100% A; 5.0-5.5 min, 100% -40% A; 5.5-9.0 min, 40% A; the flow rate is 0.2-0.4 mL/min; the sample injection volume is 4-6 mu L; the column temperature is 30-40 ℃;
mass spectrum conditions: electrospray positive ion (ESI +) mode scanning, Multiple Reaction Monitoring (MRM) mode detection; the capillary voltage is 3-4 kV; the temperature of the drying gas is 270-330 ℃; the flow rate of the drying gas is 6-8L/min; atomizer pressure 40-50 psi; sheath gas flow temperature: 320 ℃ and 380 ℃; the air flow rate of the sheath flow is 10-12L/min.
Salting-out liquid-liquid extraction (SALLE) is an extraction method in which the separation of the extraction solvent from the mixture is promoted by adding a suitable inorganic salt to the mixture of the sample solution and the extraction solvent. The method has the advantages of simple and quick operation, less solvent consumption, high extraction efficiency and the like. However, when a salt difficult to volatilize such as sodium chloride, magnesium sulfate, potassium carbonate and the like is used as a salting-out agent in combination with a mass spectrum, a salt remaining in an extraction solvent causes a strong ion inhibition effect, the sensitivity of an object to be detected is reduced, and the wide use of the method is limited.
According to the method, volatile ammonium salt is screened as a salting-out agent, and a salting-out auxiliary liquid-liquid extraction method is combined with an ultra-high performance liquid chromatography-tandem mass spectrometry method, so that the method for detecting the aflatoxin in the soy sauce with quick and simple pretreatment and low cost is established, and the aflatoxin in the soy sauce can be detected at high flux.
Preferably, in step S2, ammonium acetate is used as the salting-out agent, and acetonitrile is used as the extraction solvent.
Preferably, step S2 includes weighing 3-8 parts of soy sauce by weight, placing in a salting-out auxiliary liquid-liquid extraction device, sequentially adding 1-3 parts of ammonium acetate and 1-3 parts of acetonitrile solution, vortexing until the ammonium acetate solid is completely dissolved, and centrifuging; transferring all upper organic layers, adding water to a constant volume, uniformly mixing, filtering with a membrane, and analyzing with an instrument.
Preferably, the small salting-out auxiliary liquid-liquid extraction device comprises a box body, a working chamber is arranged in the box body, and a feeding pipe and a discharging pipe are respectively arranged at the top and the bottom of the working chamber; the quantitative component is arranged at the top end of the box body, and the stirring component is arranged in the working cavity;
the ration subassembly includes the support, the filling tube, reinforced carousel, axis of rotation and drive assembly, the support mounting is on the box top, the working chamber top is provided with the charge door, regional the installing on the support in filling tube top, the filling tube bottom is installed on the box top, the filling tube bottom is provided with the breach, reinforced carousel and breach sliding connection, reinforced carousel and box top sliding connection, be provided with the multiunit charge door on the reinforced carousel, the support top is provided with first patchhole, the axis of rotation bottom is rotated and is passed first patchhole and be connected with reinforced carousel, drive assembly installs on the support, the drive assembly output is connected with the axis of rotation.
Through adding the agent of salting out in the charging tube, liquid soy sauce through will salting out adds the work intracavity through the inlet pipe, then start the drive assembly, thereby it rotates to drive the axis of rotation, thereby it rotates to drive reinforced carousel, when reinforced carousel rotates the breach that drives the multiunit charge cavity and pass through the charging tube bottom, the agent of salting out enters into in the charge cavity of process, when reinforced carousel rotates and continues to rotate, the agent of salting out in the corresponding charge cavity just can enter into the work intracavity through the charge door, rotate through the drive axis of rotation at the slow uniform velocity of drive assembly, and control drive assembly's operating time, thereby can realize the even interpolation of ration of agent of salting out, thereby accelerate the extraction through starting the stirring subassembly simultaneously, thereby provide one kind can the salt-out auxiliary liquid-liquid extraction device of agent of salting out of quantitative even addition.
More preferably, the driving assembly comprises a first motor, a driving shaft, a first bevel gear and a second bevel gear, the first motor is mounted on the support, the first bevel gear is connected with the output end of the first motor through the driving shaft, the second bevel gear is connected with the rotating shaft, and the first bevel gear is meshed with the second bevel gear.
More preferably, the stirring assembly comprises a second motor and a stirring shaft, a second insertion hole is formed in the working cavity, the stirring shaft penetrates through the second insertion hole in a rotating mode to be connected with the working cavity in a rotating mode, a plurality of groups of stirring blades are arranged on the stirring shaft, the second motor is installed on the box body, and the output end of the second motor is connected with the stirring shaft.
More preferably, the small-size supplementary liquid-liquid extraction device of salting out still includes the dispersion piece, and the dispersion piece is installed on the charge door, and the fast top of feeding in raw material is provided with the collecting vat, and the collecting vat intercommunication is provided with the multiunit dispersion guide hole.
More preferably, the small salting-out auxiliary liquid-liquid extraction device further comprises a sealing ring and a flow guide pipe, wherein the sealing ring is arranged on the working cavity and sleeved with the stirring shaft; the honeycomb duct is installed in row material socle.
More preferably, step S2 is to weigh 5.00g of soy sauce (to the nearest 0.01g) into a small salting-out assisted liquid-liquid extraction device, add 2.00g of ammonium acetate and 2.00mL of acetonitrile solution in sequence, vortex until the ammonium acetate solid is completely dissolved (about 1min), and then centrifuge at 10000r/min for 5 min; transferring all upper organic layer (about 1.4mL) into volumetric flask, adding water to constant volume to scale, mixing, passing through 0.22 μm nylon filter membrane, and analyzing with instrument.
Preferably, the chromatographic and mass spectrometric conditions of step S4 are:
a chromatographic column: ZoRBAX SB-C 18 (100 mm. times.2.1 mm, 1.8 μm); mobile phase A: methanol/acetonitrile mixed solution (v/v-50/50), mobile phase B: 5mmol/L ammonium acetate solution; gradient elution procedure: 0.0-5.0 min, 40% A; 0.5-3.0 min, 40% -60% A; 3.0-4.0 min, 60% A; 4.0-4.2 min, 60% -100% A; 4.2-5 min, 100% A; 5.0-5.5 min, 100% -40% A; 5.5-9.0 min, 40% A; the flow rate is 0.3 mL/min; the sample injection volume is 5 mu L; the column temperature is 35 ℃;
mass spectrum conditions: electrospray positive ion (ESI +) mode scanning, Multiple Reaction Monitoring (MRM) mode detection; the capillary voltage is 3.5 kV; the temperature of the drying gas is 300 ℃; the flow rate of the drying gas is 7L/min; atomizer pressure 45 psi; sheath gas flow temperature: 350 ℃; the sheath flow rate was 11L/min.
In conclusion, the invention has the following beneficial effects:
1. a specific small salting-out auxiliary liquid-liquid extraction device is used, so that the quantitative and uniform addition of a salting-out agent is facilitated, and the extraction is accelerated by starting a stirring component;
2. the invention establishes a detection method for detecting four aflatoxins in soy sauce by combining a volatile ammonium salt salting-out auxiliary liquid-liquid extraction technology with a liquid chromatography-mass spectrometry technology; the method adopts a specific extraction solvent and a salting-out auxiliary agent, adopts an ultra-efficient liquid chromatograph-mass spectrometer to detect in a positive ion mode, and adopts a matrix labeling external standard method for quantification, thereby solving the problem of ion signal inhibition caused by a salting-out agent which is difficult to volatilize in mass spectrometry.
Drawings
FIG. 1 is a schematic diagram of the salting-out agent type screening of the present invention;
FIG. 2 is a schematic of the solvent type screening of the present invention;
FIG. 3 is a schematic illustration of a mass screening of ammonium acetate according to the present invention;
FIG. 4 is a schematic diagram of acetonitrile volume condition screening according to the present invention;
FIG. 5 is a standard solution chromatogram of AFTB1, B2, G1 and G2 mixed matrix according to the present invention;
FIG. 6 is a schematic structural view of the present invention;
FIG. 7 is a schematic top view of the feed turntable of the present invention;
FIG. 8 is a schematic top view of a discrete block of the present invention;
FIG. 9 is a partially enlarged schematic view of A of the present invention;
in the drawings, the reference numbers: 1. a box body; 2. a discharge pipe; 3. a support; 4. a feed tube; 5. a charging turntable; 6. a rotating shaft; 7. a plurality of groups of feed holes; 8. a first motor; 9. a drive shaft; 10. a first bevel gear; 11. a second bevel gear; 12. a second motor; 13. a stirring shaft; 14. a plurality of groups of stirring blades; 15. dispersing blocks; 16. a plurality of groups of dispersed guide holes; 17. a seal ring; 18. a flow guide pipe; 19. a transparent viewing window; 20. and a plurality of groups of adjusting threaded rods.
Detailed Description
Example one
1 materials and methods
1.1 materials and reagents
Soy sauce samples were purchased from a lake city supermarket.
And (3) standard substance: aflatoxin B 1 、B 2 、G 1 、G 2 Standard solutions (both 100. mu.g/mL, Qingdao Pop bioengineering, Inc.) were mixed. Reagent: methanol, ethanol, n-propanol, acetonitrile, acetone (chromatographically pure, merck, germany); ammonium formate, ammonium acetate, ammonium chloride, ammonium sulfate (analytical purity, Shanghai Michelin Biochemical technologies, Ltd.); the test water was ultrapure water.
1.2 instruments and devices
Model 1260 high performance liquid chromatograph (agilent, usa); model 6470 triple quadrupole tandem mass spectrometer (agilent, usa); an electronic analytical balance model ME204E (mettler, switzerland); genius 3 vortex mixer (aka germany); KQ-500DB ultrasonic cleaning instrument (ultrasonic instruments of Kunshan, China); an Allegra X-12R model bench refrigerated centrifuge (Beckmann Kort, USA); silicon type ultrapure water meter (millipore corporation, usa). .
1.3 Experimental methods
1.3.1 preparation of Mixed control solutions
AFT B 1 、B 2 、G 1 、G 2 Preparing a mixed standard stock solution: absorb purchased AFT B 1 、B 2 、G 1 、G 2 Mixing with appropriate amount of standard solution, diluting with acetonitrile, and preparing AFT B with concentration of 1 μ g/mL 1 、B 2 、G 1 、G 2 Mix standard stock solutions and store at-20 ℃ in the dark. Suction of AFT B 1 、B 2 、G 1 、G 2 Mixing with appropriate amount of standard stock solution, diluting with blank matrix solution, and making into AFT B with concentration of 0.5, 1, 5, 10, 20, 50ng/mL 1 、B 2 、G 1 、G 2 The standard working solution was mixed. 1mL of the standard solution was passed through a 0.22 μm nylon filter and analyzed by an instrument.
1.3.2 preparation of test solutions
Weighing 5.00g soy sauce (accurate to 0.01g) into a 15mL centrifuge tube, adding 2.00g ammonium acetate and 2.00mL acetonitrile solution, vortexing until the ammonium acetate solid is completely dissolved (about 1min), and centrifuging at 10000r/min for 5 min. All the upper organic layer (about 1.4mL) was removed and placed in a 2.0mL volumetric flask, water was added to a constant volume to a desired volume, mixed well, filtered through a 0.22 μm nylon filter and analyzed by an instrument.
1.3 assay method
Precisely absorbing each 5 mu L of the mixed standard working solution, detecting by a liquid chromatograph-mass spectrometer, determining the area of a quantitative ion peak, and drawing a curve chart of 4 aflatoxin matrix standard solutions by taking the sample concentration (X) as a horizontal coordinate and the area (Y) of the quantitative ion peak as a vertical coordinate. And (4) measuring the area of a quantitative ion peak of the test solution under the same condition, and obtaining the concentration of the aflatoxin in the test solution according to the matrix standard curve linear equation.
1.4 chromatographic and Mass Spectrometry conditions
And (3) chromatographic column: ZoRBAX SB-C 18 (100 mm. times.2.1 mm, 1.8 μm); mobile phase A: methanol/acetonitrile mixture (v/v ═ 50/50), mobile phase B: 5mmol/L ammonium acetate solution; gradient elution procedure: 0.0-5.0 min, 40% A; 0.5-3.0 min, 40% -60% A; 3.0-4.0 min, 60% A; 4.0-4.2 min, 60% -100% A; 4.2-5 min, 100% A; 5.0-5.5 min, 100% 40% A; 5.5-9.0 min, 40% A; the flow rate is 0.3 mL/min; the sample injection volume is 5 mu L; the column temperature was 35 ℃.
Mass spectrum conditions: electrospray positive ion (ESI +) mode scanning, Multiple Reaction Monitoring (MRM) mode detection; the capillary voltage is 3.5 kV; the temperature of the drying gas is 300 ℃ 2; the flow rate of the drying gas is 7L/min; atomizer pressure 45 psi; sheath flow gas temperature: 350 ℃; the flow rate of the sheath flow is 11L/min; AFTB 1 、B 2 、G 1 、G 2 The relevant mass spectral parameters are detailed in table 1.
TABLE 1 monitoring ion pairs and optimal Mass Spectrometry parameters for analytes
2 results and analysis
2.1 selection of the type of salting-out agent
The selection of the salting-out agent directly influences the extraction efficiency of the target substance. In the salting-out liquid-liquid extraction, excessive salt is used to cause the salting-out agent to be carried in an extraction solvent, and the residual salt which is difficult to volatilize can reduce the abundance of target ions in a mass spectrum and cause corrosion and pollution to instrument hardware. Therefore, the research selects the volatile ammonium salt as the salting-out agent, and the extraction efficiency of ammonium formate, ammonium acetate, ammonium chloride and ammonium sulfate on the aflatoxin is respectively investigated. The results are shown in fig. 1(n ═ 3), and show that the four aflatoxins respond the highest when ammonium acetate is used as the salting-out agent, so ammonium acetate is selected as the salting-out agent.
2.2 selection of extraction solvent type
The extraction solvent is one of the important factors affecting the extraction efficiency. In salting-out liquid-liquid extraction, an extraction solvent is generally required to have a density smaller than that of water and to be miscible with water, and is required to be separated from water after salt is added, so that five common extraction solvents, namely acetonitrile, methanol, ethanol, n-propanol and acetone, are studied in the research. After adding ammonium acetate serving as a salting-out agent, only acetonitrile, n-propanol and acetone are separated, and the response of four targets is measured, the result is shown in figure 2 (n-3), and acetonitrile is larger than acetone and larger than n-propanol, so that acetonitrile is selected as an extraction solvent.
2.3 optimization of salting-out agent quality
The quality of the salting-out agent is of great importance to the salting-out effect. The present study further examined the effect of the amount of ammonium acetate (0.50g, 1.00g, 1.50g, 2.00g, 2.50g) on the effect of salting out liquid-liquid extraction enrichment and extraction efficiency. The results are shown in fig. 3(n ═ 3), and studies indicate: the volume of the organic phase after salting out is slightly influenced by different ammonium acetate dosage, but the response is reduced after the ammonium acetate dosage is increased, and when the ammonium acetate dosage is 2.00g, the four aflatoxins have the highest response. Therefore, 2.00g was determined as the optimum salting-out quality.
2.4 optimization of extraction solvent volume
Too low volume of extraction solvent results in incomplete extraction and reduced extraction efficiency, while too high volume of extraction solvent reduces the enrichment factor and wastes organic reagents. Therefore, the research comparatively examines the influence of the dosage (0.8 mL-2.4 mL) of the extraction solvent acetonitrile on the layering effect of the extraction solvent and the sample solution and the extraction efficiency of the target object. The results are shown in FIG. 4(n is 3), when the acetonitrile dosage is 0.8mL, the volume of the organic layer after salting out is small, the absorption error is large, when the acetonitrile dosage is 1.2 mL-2.0 mL, the response of the aflatoxin is gradually increased along with the increase of the acetonitrile volume, and when the acetonitrile dosage is more than 2.0mL, the response of the aflatoxin is almost unchanged along with the increase of the acetonitrile volume. Therefore, to save organic solvent and shorten extraction time, 2.0mL was determined as the optimal extraction volume.
2.5 Standard Curve, Linear Range and detection Limit
The 5 aflatoxins with the concentration are mixed with the contrast matrix solution and are sequentially put into a mass spectrometer for measurement, 4 aflatoxin standard curve graphs are drawn, a linear equation and a correlation coefficient are automatically obtained through linear fitting of instrument software, and the result is shown in table 2. As can be seen from Table 2, 4 targetsGood fit of the object to determine the coefficient (R) 2 ) Are all greater than 0.999. AFT B 1 、B 2 、G 1 、G 2 The total ion current chromatogram of the control (5.0ng/mL) is shown in FIG. 5. As can be seen from FIG. 5, the 4 aflatoxins are completely separated, the peak shapes are symmetrical, and the interference of miscellaneous peaks does not exist around the aflatoxins, so that the quantitative detection requirement can be met. The detection limits for 4 aflatoxins were obtained by decreasing the feed concentration according to the signal-to-noise ratio (S/N) ═ 3, and the results are shown in table 2.
TABLE 2 regression equation and Linear Range
2.6 recovery and precision experiments
And (3) adding the aflatoxin mixed standard solution with high, medium and low concentrations into the soy sauce negative blank sample, repeatedly measuring for 6 times according to the established method, and calculating the standard addition recovery rate and the Relative Standard Deviation (RSD), wherein the results are shown in Table 3. As can be seen from Table 3, the blank substrate spiking recovery rate of 4 aflatoxins is 73.2-94.5%, and the RSD is 0.9-5.9%. The precision and recovery rate of the 4 aflatoxins can meet the requirement of quantitative detection.
Table 3 precision and recovery test results (n ═ 6)
2.7 sample detection and analysis
In order to verify the practicability and reliability of the established method, the method is used for screening the aflatoxin in the actual sample. The method is used for detecting the content of 4 aflatoxins in each batch of soy sauce samples, and the result shows that 4 aflatoxins are not detected, thus the soy sauce safety condition on the market is generally good.
Example two
The same as the first embodiment, except that the step S2 includes weighing 5 parts of soy sauce by weight, placing in a small salting-out auxiliary liquid-liquid extraction device, adding 2 parts of ammonium acetate and 2 parts of acetonitrile solution in sequence, vortexing until the ammonium acetate solid is completely dissolved, and then centrifuging; transferring all upper organic layers, adding water to a constant volume, uniformly mixing, filtering with a membrane, and analyzing with an instrument.
As shown in fig. 6 to 9, the small salting-out auxiliary liquid-liquid extraction device of the present invention comprises a box body 1, a working chamber is arranged in the box body 1, a feeding pipe and a discharging pipe 2 are respectively arranged at the top and the bottom of the working chamber, and a plurality of groups of supporting legs are arranged at the bottom of the box body 1; the quantitative component is arranged at the top end of the box body 1, and the stirring component is arranged in the working cavity;
the quantitative assembly comprises a support 3, a feeding pipe 4, a feeding turntable 5, a rotating shaft 6 and a driving assembly, the support 3 is installed at the top end of a box body 1, a feeding port is formed in the top end of a working cavity, the top area of the feeding pipe 4 is installed on the support 3, the bottom end of the feeding pipe 4 is installed at the top end of the box body 1, a notch is formed in the bottom end of the feeding pipe 4, the feeding turntable 5 is in sliding connection with the notch, the feeding turntable 5 is in sliding connection with the top end of the box body 1, a plurality of groups of feeding holes 7 are formed in the feeding turntable 5, a first inserting hole is formed in the top end of the support 3, the bottom end of the rotating shaft 6 rotates to penetrate through the first inserting hole to be connected with the feeding turntable 5, the driving assembly is installed on the support 3, and the output end of the driving assembly is connected with the rotating shaft 6; by adding the salting-out agent and the extraction solvent into the feeding pipe, by adding the liquid to be salted out into the working cavity through the feeding pipe, then the driving component is started to drive the rotating shaft to rotate, thereby driving the charging turntable to rotate, when the charging turntable rotates to drive a plurality of groups of charging holes to pass through the gap at the bottom end of the charging tube, the salting-out agent and the extraction solvent enter the feeding holes, when the feeding turntable rotates to continue rotating, the salting-out agent and the extraction solvent in the corresponding feed holes can enter the working cavity through the feed inlet, the driving component drives the rotating shaft to rotate slowly and uniformly and controls the working time of the driving component, thereby realizing the quantitative and uniform addition of the salting-out agent and the extraction solvent, simultaneously accelerating the reaction by starting the stirring component, thereby providing a salting-out auxiliary liquid-liquid extraction device which can quantitatively and uniformly add a salting-out agent and an extraction solvent.
The driving assembly comprises a first motor 8, a driving shaft 9, a first bevel gear 10 and a second bevel gear 11, the first motor 8 is installed on the support 3, the first bevel gear 10 is connected with the output end of the first motor 8 through the driving shaft 9, the second bevel gear 11 is connected with the rotating shaft 6, and the first bevel gear 10 is meshed with the second bevel gear 11; thereby drive first bevel gear through the drive shaft and rotate through starting first motor to drive second bevel gear and rotate, thereby accomplish the drive, first bevel gear rotates several rings after, second bevel gear just can rotate the round, thereby accomplish the deceleration process.
The stirring assembly comprises a second motor 12 and a stirring shaft 13, a second insertion hole is formed in the working cavity, the stirring shaft 13 rotatably penetrates through the second insertion hole to be rotatably connected with the working cavity, a plurality of groups of stirring blades 14 are arranged on the stirring shaft 13, the second motor 12 is installed on the box body 1, and the output end of the second motor 12 is connected with the stirring shaft 13; thereby drive the (mixing) shaft through starting the second motor and rotate to drive multiunit stirring vane and stir liquid, thereby accelerate extraction and reaction rate.
The small salting-out auxiliary liquid-liquid extraction device also comprises dispersing blocks 15, wherein the dispersing blocks 15 are arranged on a feeding opening, a collecting tank is arranged at the top end of the feeding block, and the collecting tank is communicated with a plurality of groups of dispersing guide holes 16; the salting-out agent and the extraction solvent in the feeding holes enter the collecting groove, and then are poured into different positions in the working cavity through a plurality of groups of dispersing guide holes in different directions, so that the salting-out agent and the extraction solvent are reduced from being excessively and intensively put in.
The small salting-out auxiliary liquid-liquid extraction device also comprises a sealing ring 17, a flow guide pipe 18 and a plurality of groups of adjusting threaded rods 20, wherein the sealing ring 17 is arranged on the working cavity, and the sealing ring 17 is sleeved with the stirring shaft 13; the sealing performance of the stirring shaft and the second insertion hole is improved through the sealing ring; the draft tube 18 is arranged at the bottom end of the discharge tube 2; thereby conveniently follow the collection of arranging the liquid that the material pipe flows through the honeycomb duct. A transparent observation window 19 is arranged at the top end of the box body 1; the condition in the working cavity is facilitated through the transparent observation window. The bottom ends of the multiple groups of supporting legs are respectively provided with multiple groups of adjusting threaded holes, and the multiple groups of adjusting threaded rods 20 are respectively in threaded connection with the multiple groups of adjusting threaded holes; thereby conveniently adjust the stability of multiunit landing leg through multiunit adjusting screw.
The small salting-out auxiliary liquid-liquid extraction device can accelerate the batch preparation of the sample solution and reduce the consumption of organic reagents.
To summarize: the invention establishes a detection method for detecting 4 aflatoxins in soy sauce by combining a volatile ammonium salt salting-out auxiliary liquid-liquid extraction technology with a liquid chromatography-mass spectrometry technology; the method adopts acetonitrile as an extraction solvent, volatile ammonium acetate as a salting-out aid, adopts an ultra-high performance liquid chromatography-mass spectrometer for detection in a positive ion mode, adopts a matrix labeling external standard method for quantification, and solves the problem of ion signal inhibition caused by a salting-out agent which is difficult to volatilize in mass spectrometry. Compared with the national food safety standard GB5009.22-2016, the method has the advantages that the detection limit reaches the current national standard, but the consumption of organic reagents is low, the cost is greatly reduced compared with that of the national standard method, the operation is simple and rapid, the practicability is high, and the method is very suitable for high-throughput detection of aflatoxin pollution in soy sauce.
The present embodiment is only for explaining the present invention, and it is not limited to the present invention, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present invention.
Claims (10)
1. A method for detecting aflatoxin in soy sauce is characterized by comprising the following steps:
s1, preparation of mixed control solution: formulating AFT B 1 、B 2 、G 1 、G 2 Mixing the standard stock solution;
s2, preparation of a test solution: selecting a salting-out agent and an extraction solvent, and preparing a test solution;
s3, measurement: precisely absorbing the mixed standard working solution, detecting the mixed standard working solution in a liquid chromatograph-mass spectrometer, determining the area of a quantitative ion peak, and drawing a standard solution curve chart of the aflatoxin matrix by taking the sample concentration (X) as a horizontal coordinate and the area (Y) of the quantitative ion peak as a vertical coordinate; measuring the area of a quantitative ion peak of the test solution under the same condition, and obtaining the concentration of the aflatoxin in the test solution according to the matrix standard curve linear equation;
s4, chromatographic and mass spectrometric conditions:
chromatographic conditions are as follows: mobile phase A: methanol/acetonitrile mixture (v/v ═ 50/50), mobile phase B: 4-6mmol/L ammonium acetate solution; gradient elution procedure: 0.0-5.0 min, 40% A; 0.5-3.0 min, 40% -60% A; 3.0-4.0 min, 60% A; 4.0-4.2 min, 60% -100% A; 4.2-5 min, 100% A; 5.0-5.5 min, 100% -40% A; 5.5-9.0 min, 40% A; the flow rate is 0.2-0.4 mL/min; the sample injection volume is 4-6 mu L; the column temperature is 30-40 ℃;
mass spectrum conditions: scanning in an electrospray positive ion mode and detecting in a multi-reaction monitoring mode; the capillary voltage is 3-4 kV; the temperature of the drying gas is 270-330 ℃; the flow rate of the drying gas is 6-8L/min; atomizer pressure 40-50 psi; sheath gas flow temperature: 320 ℃ and 380 ℃; the air flow rate of the sheath flow is 10-12L/min.
2. The method for detecting aflatoxins in soy sauce according to claim 1, which comprises the following steps: and step S2, ammonium acetate is selected as a salting-out agent, and acetonitrile is selected as an extraction solvent.
3. The method for detecting aflatoxins in soy sauce according to claim 2, which comprises the following steps: step S2 includes weighing 3-8 parts of soy sauce by weight, placing in a salting-out auxiliary liquid-liquid extraction device, sequentially adding 1-3 parts of ammonium acetate and 1-3 parts of acetonitrile solution, vortexing until ammonium acetate solid is completely dissolved, and centrifuging; transferring all upper organic layers, adding water to a constant volume, uniformly mixing, filtering with a membrane, and analyzing with an instrument.
4. The method for detecting aflatoxins in soy sauce according to claim 3, which comprises the following steps: the small salting-out auxiliary liquid-liquid extraction device comprises a box body, wherein a working chamber is arranged in the box body, and the top and the bottom of the working chamber are respectively provided with a feeding pipe and a discharging pipe; the quantitative component is arranged at the top end of the box body, and the stirring component is arranged in the working cavity; the ration subassembly includes the support, the filling tube, reinforced carousel, axis of rotation and drive assembly, the support mounting is on the box top, the working chamber top is provided with the charge door, regional the installing on the support in filling tube top, the filling tube bottom is installed on the box top, the filling tube bottom is provided with the breach, reinforced carousel and breach sliding connection, reinforced carousel and box top sliding connection, be provided with the multiunit charge door on the reinforced carousel, the support top is provided with first patchhole, the axis of rotation bottom is rotated and is passed first patchhole and be connected with reinforced carousel, drive assembly installs on the support, the drive assembly output is connected with the axis of rotation.
5. The method for detecting aflatoxins in soy sauce as claimed in claim 4, wherein: the driving assembly comprises a first motor, a driving shaft, a first bevel gear and a second bevel gear, the first motor is installed on the support, the first bevel gear is connected with the output end of the first motor through the driving shaft, the second bevel gear is connected with the rotating shaft, and the first bevel gear is meshed with the second bevel gear.
6. The method for detecting aflatoxins in soy sauce according to claim 5, which comprises the following steps: the stirring assembly comprises a second motor and a stirring shaft, a second inserting hole is formed in the working cavity, the stirring shaft penetrates through the second inserting hole in a rotating mode to be connected with the working cavity, a plurality of groups of stirring blades are arranged on the stirring shaft, the second motor is installed on the box body, and the output end of the second motor is connected with the stirring shaft.
7. The method for detecting aflatoxins in soy sauce according to claim 6, which comprises the following steps: the small salting-out auxiliary liquid-liquid extraction device further comprises dispersing blocks, the dispersing blocks are installed on the feeding port, a collecting tank is arranged at the top end of the feeding block, and a plurality of groups of dispersing guide holes are formed in the collecting tank in a communicated mode.
8. The method for detecting aflatoxins in soy sauce according to claim 7, which comprises the following steps: the small salting-out auxiliary liquid-liquid extraction device also comprises a sealing ring and a flow guide pipe, wherein the sealing ring is arranged on the working cavity and is sleeved with the stirring shaft; the honeycomb duct is installed in row material socle.
9. The method for detecting aflatoxins in soy sauce according to any one of claims 1 to 8, which comprises: step S2 is specifically that 5.00g of soy sauce (accurate to 0.01g) is weighed and placed in a small salting-out auxiliary liquid-liquid extraction device, 2.00g of ammonium acetate and 2.00mL of acetonitrile solution are added in sequence, vortex is carried out until ammonium acetate solid is completely dissolved (about 1min), and then centrifugation is carried out for 5min at 10000 r/min; all the upper organic layers (about 1.4mL) were removed and placed in a volumetric flask, water was added to a constant volume to a desired volume, mixed well, filtered through a 0.22 μm nylon filter and then analyzed by an instrument.
10. The method for detecting aflatoxins in soy sauce according to claim 9, which comprises the following steps: the chromatographic and mass spectrometric conditions of step S4 are:
a chromatographic column: ZORBAX SB-C 18 (100 mm. times.2.1 mm, 1.8 μm); mobile phase A: methanol/acetonitrile mixture (v/v ═ 50/50), mobile phase B: 5mmol/L ammonium acetate solution; gradient elution procedure: 0.0-5.0 min, 40% A; 0.5-3.0 min, 40% -60% A; 3.0-4.0 min, 60% A; 4.0-4.2 min, 60% -100% A; 4.2-5 min, 100% A; 5.0-5.5 min, 100% -40% A; 5.5-9.0 min, 40% A; the flow rate is 0.3 mL/min; the sample injection volume is 5 mu L; the column temperature is 35 ℃;
mass spectrum conditions: electrospray positive ion (ESI +) mode scanning, Multiple Reaction Monitoring (MRM) mode detection; the capillary voltage is 3.5 kV; the temperature of the drying gas is 300 ℃; the flow rate of the drying gas is 7L/min; atomizer pressure 45 psi; sheath gas flow temperature: 350 ℃; the sheath flow rate was 11L/min.
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| CN107907600A (en) * | 2017-10-25 | 2018-04-13 | 大连理工大学 | It is a kind of that the method for aflatoxin and flavouring agent in vegetable fat is measured based on liquid-liquid extraction Liquid Chromatography-Tandem Mass Spectrometry at the same time |
| CN109568137A (en) * | 2018-12-27 | 2019-04-05 | 湖州市食品药品检验研究院 | A kind of Chinese medicine pilling equipment |
| CN110274984A (en) * | 2019-07-19 | 2019-09-24 | 安徽中创食品检测有限公司 | Aflatoxin B in liquid-liquid extraction purification-high performance liquid chromatography detection food1Method |
| CN113219092A (en) * | 2021-04-29 | 2021-08-06 | 中国烟草总公司湖北省公司 | Method for simultaneously measuring multiple aflatoxins in tobacco leaves by dispersion liquid-liquid microextraction-high performance liquid chromatography-tandem mass spectrometry |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107907600A (en) * | 2017-10-25 | 2018-04-13 | 大连理工大学 | It is a kind of that the method for aflatoxin and flavouring agent in vegetable fat is measured based on liquid-liquid extraction Liquid Chromatography-Tandem Mass Spectrometry at the same time |
| CN109568137A (en) * | 2018-12-27 | 2019-04-05 | 湖州市食品药品检验研究院 | A kind of Chinese medicine pilling equipment |
| CN110274984A (en) * | 2019-07-19 | 2019-09-24 | 安徽中创食品检测有限公司 | Aflatoxin B in liquid-liquid extraction purification-high performance liquid chromatography detection food1Method |
| CN113219092A (en) * | 2021-04-29 | 2021-08-06 | 中国烟草总公司湖北省公司 | Method for simultaneously measuring multiple aflatoxins in tobacco leaves by dispersion liquid-liquid microextraction-high performance liquid chromatography-tandem mass spectrometry |
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