CN111366652A - Method for determining 16 mycotoxins in tea by using ultra-high performance liquid chromatography-tandem mass spectrometry - Google Patents

Method for determining 16 mycotoxins in tea by using ultra-high performance liquid chromatography-tandem mass spectrometry Download PDF

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CN111366652A
CN111366652A CN202010250908.3A CN202010250908A CN111366652A CN 111366652 A CN111366652 A CN 111366652A CN 202010250908 A CN202010250908 A CN 202010250908A CN 111366652 A CN111366652 A CN 111366652A
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tea
sample
mass spectrometry
mycotoxins
liquid chromatography
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刘文静
傅建炜
黄彪
韦航
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Institute Of Quality Standard And Testing Technology For Agro-Products Fujian Academy Of Agricultural Sciences
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02Column chromatography
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02Column chromatography
    • G01N30/04Preparation or injection of sample to be analysed
    • G01N30/06Preparation
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02Column chromatography
    • G01N30/62Detectors specially adapted therefor
    • G01N30/72Mass spectrometers
    • G01N30/7233Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
    • G01N30/724Nebulising, aerosol formation or ionisation
    • G01N30/7266Nebulising, aerosol formation or ionisation by electric field, e.g. electrospray
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02Column chromatography
    • G01N30/86Signal analysis
    • G01N30/8624Detection of slopes or peaks; baseline correction
    • G01N30/8631Peaks
    • G01N30/8637Peak shape
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02Column chromatography
    • G01N30/86Signal analysis
    • G01N30/8675Evaluation, i.e. decoding of the signal into analytical information
    • G01N30/8679Target compound analysis, i.e. whereby a limited number of peaks is analysed
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02Column chromatography
    • G01N30/04Preparation or injection of sample to be analysed
    • G01N2030/042Standards
    • G01N2030/045Standards internal
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02Column chromatography
    • G01N30/04Preparation or injection of sample to be analysed
    • G01N30/06Preparation
    • G01N2030/062Preparation extracting sample from raw material

Abstract

The invention provides a method for determining 16 mycotoxins in tea by using an ultra-high performance liquid chromatography-tandem mass spectrometry method, belonging to the technical field of analytical chemistry. Extracting with formic acid/acetonitrile (10:90), adding QuEChERS salt bag, shaking, centrifuging, treating the extractive solution with OASIS PRIME HLB column and Dspe purification tube, and purifying with Waters HSS T3Separating chromatographic columns, adopting a multi-reactive ion monitoring (MRM) mode of simultaneously scanning positive ions and negative ions, matching tea substrates with standard solutions, and quantifying by an isotope internal standard method. The method is stable, accurate, sensitive and quick, and can meet the requirement of analyzing various toxin residues in various tea leaves.

Description

Method for determining 16 mycotoxins in tea by using ultra-high performance liquid chromatography-tandem mass spectrometry
Technical Field
The invention relates to the technical field of analytical chemistry, in particular to a method for determining 16 mycotoxins in tea by using an ultra-high performance liquid chromatography-tandem mass spectrometry method.
Background
Mycotoxins (mycotoxins) are secondary toxic metabolites produced by certain fungi under certain environmental conditions that are harmful to humans and animals. Over 400 mycotoxins have been found to date, mainly including aflatoxin, ochratoxin A, patulin, deoxynivalenol and T-2 toxin of trichothecenes, zearalenone, fumonisins, variegated aspergillins, citrinin, etc. These toxins are acute toxic to humans and animals by oral or dermal inhalation, and may also act on the liver, kidney, nervous system, endocrine system and immune system, with chronic toxicities such as teratogenicity, carcinogenesis, mutagenicity, toxic renal damage, hepatotoxicity, immunosuppression and reproductive disorders. China is a major country for tea production, export and consumption, and concerns about mycotoxin pollution in tea, particularly in fermented tea such as Pu 'er tea, Fuzhuan tea and the like, have attracted consumers' attention. Although the drying stage of tea processing may kill most microorganisms in tea, if not properly packaged and stored, especially after the tea leaves are wet, it may be contaminated with fungi and mycotoxins. The detection of mycotoxins such as aflatoxin, deoxynivalenol, zearalenone, fumonisin, ochratoxin, T-2 toxin and the like contaminated by tea has been reported. The aged tea is also called as aged tea, and refers to tea with a health care function after being stored for a period of time, and because the storage process is longer, the tea is likely to go moldy and go bad due to improper storage, and toxin is generated. At present, related aspergillus flavus B in some tea leaves at home and abroad1And ochratoxin A and other single mycotoxin pollution conditions are detected and analyzed more, but research on simultaneous detection of 10 or more than 10 mycotoxins in tea leaves is lacked. Therefore, it is necessary to establish a method for simultaneously detecting a plurality of mycotoxins in tea leaves.
At present, the detection method of mycotoxin in tea mainly comprises a thin-layer chromatography method, an enzyme-linked immunosorbent assay method, a gas chromatography-tandem mass spectrometry combined method, a liquid chromatography-tandem mass spectrometry combined method and the like. Among them, the enzyme-linked immunosorbent assay is easy to generate false positive and can only be used as a screening method; high performance liquid chromatography needs pre-column or post-column derivatization, byproducts formed by pre-column derivatization may cause great difficulty to chromatographic separation, impurities or interference peaks are easily introduced or samples are lost in the derivatization process, post-column derivatization is limited for a certain solvent and limited reaction time, the selected reaction conditions are limited, insufficient reaction is easily caused or reaction byproducts are excessive, and post-column derivatization needs additional equipment, a reactor can cause peak broadening, the resolution ratio is reduced, and finally, not only quantitative results are influenced, but also instruments are polluted; compared with other methods, the liquid chromatography-tandem mass spectrometry combined technology has more accurate quantification and higher selectivity and sensitivity, and has gradually replaced the traditional liquid chromatography method to be applied to the detection of mycotoxin in tea. At present, the pretreatment steps for detecting the tea toxin by adopting a liquid chromatography-tandem mass spectrometry combined technology are complex, and a multifunctional evolutionary column and an immunoaffinity column are required to be used for purifying an extracting solution. The sample type matrix involved in the national standard is relatively simple and can substantially remove impurities by two purifications. However, for tea leaves with complex matrixes, impurities are difficult to remove by two times of simple purification, and the excessive impurities cause obvious matrix effect in mass spectrometry, so that the final quantification is inaccurate. Therefore, the sample extraction liquid must be purified by a more efficient method.
Different mycotoxins have different requirements on solvents, for example, aflatoxin is insoluble in water, and some carboxyl group-containing toxins such as fumonisin, ochratoxin and the like, which contain carboxyl group-containing toxins, need to be extracted in a meta-acid environment. Meanwhile, some extraction solvents can more easily extract some impurities such as acid, alcohol, phenols and the like in the tea leaves into the extraction liquid, and some extraction solvents have fewer impurities in the extraction liquid, so that the selection of the optimal extraction solvent has great influence on subsequent purification, detection, sample recovery rate and the like.
In the process of detecting the toxin by adopting the liquid chromatography-tandem mass spectrometry combined technology, the selection of the mobile phase influences the peak type of a detected object, the time required by detection and the like, so that a better mobile phase and gradient elution program is established, the detection sensitivity can be improved, the detection time can be shortened, and the detection efficiency is improved.
At present, the detection method for various mycotoxins in tea at home and abroad is unstable, has low sensitivity, can only detect one or more toxins at the same time, has low detection efficiency, does not detect more than 10 mycotoxins at the same time, and cannot detect various mycotoxins in different tea varieties, namely different degrees of fermented tea at the same time. The invention is researched and proposed aiming at the 3 problems of the efficient solvent extraction, the purification link, the efficient mobile phase elution gradient program and the like.
Disclosure of Invention
The invention aims to provide a method for determining 16 mycotoxins in tea by using an ultra-high performance liquid chromatography-tandem mass spectrometry method, the pretreatment is simple, the determination method is stable, accurate, sensitive and quick, and the requirement of multi-toxin analysis in various types of tea can be met.
In order to achieve the above object, the present invention provides the following technical solutions:
a method for determining 16 mycotoxins in tea by using an ultra-high performance liquid chromatography-tandem mass spectrometry method is characterized by comprising the steps of preparing a sample to be tested and determining by using the ultra-high performance liquid chromatography-tandem mass spectrometry method;
wherein the extracting solution for preparing the sample is formic acid/acetonitrile with water and 1: 5-1: 10 in a volume ratio of 3: 1-1: 3 and then is added with a QuEChERS extraction salt bag;
preparing a sample extracting solution to be tested, and purifying the sample extracting solution by adopting a dSPE purifying tube and an Oasis PRIME HLB small column;
the flow term A in the chromatographic condition in the high performance liquid chromatography-tandem mass spectrometry is 5-10 mmol.L-1Ammonium acetate aqueous solution and 0.1 to 1 volume percent formic acid aqueous solution; mobile phase B: acetonitrile; under the mass spectrum condition, the ion source for mass spectrum analysis is used for removing zearalenone and the internal standard is an electrospray negative ion source ESI-The rest toxins are ESI (electrospray ionization) positive ion source+And the detection mode is as follows: multiple reactive ion monitoring MRM mode, capillary voltage: 1.5 kV; taper hole airflow: nitrogen gas at a flow rate of 50 L.h-1(ii) a Ion source temperature: 120 ℃; desolventizing aerosol gas temperature: at 500 deg.C, the flow rate is 1000L/hr; collision gas: high-purity argon gas;
the 16 mycotoxins tested were: aflatoxin B1Aflatoxins B2Aflatoxin G1Aflatoxin G2Zearalenone, citrinin, deoxynivalenol, fusarenone, nivalenol, ochratoxin, T-2 toxin, HT-2 toxin, ochratoxin, fumonisin B1Fumonisins B2Fumonisins B3
Further, preparing the test sample comprises the following steps:
sample treatment: taking 0.5g of crushed tea to be detected, adding 10ml of water and 10ml of formic acid/acetonitrile solution with the volume ratio of 1: 5-1: 10 into a tea sample, mixing, and then placing the mixture in an automatic oscillator to shake for 10 min; adding QuEChERS extraction salt bag, shaking for 1min, centrifuging at 4000r/min for 5min, and collecting supernatant for purification;
purifying: the HLB column was installed, the column activation step was not performed, about 0.4mL of the supernatant was passed through the HLB column, the filtrate was discarded, and then 1mL of the supernatant was again passed through the column again and the filtrate was collected. Transferring the filtrate into dSPE purification tube containing mixed adsorbent, centrifuging at 10000r/min for 10min, and filtering the supernatant with 0.22 μm filter membrane; and (3) sucking 190 mu L of the stable isotope solution into a 300 mu L inner inserting tube, adding 10 mu L of the stable isotope solution, and uniformly mixing by vortex for sample injection.
Further, in the sample treatment, the crushed tea to be detected is obtained by sieving through a 60-mesh sieve.
Further, in the high performance liquid chromatography-tandem mass spectrometry determination, 16 mycotoxin standard stock solutions are prepared and isotopes are adopted13C17--Aflatoxin B1(0.5μg/mL),D6-Zearalenone(100μg/mL),13C15-Deoxynivalenol(25μg/mL),13C34--Fumonisin B1(25μg/mL),13C24--T-2Toxin(30μg/mL),D5-Ochratoxin A (50. mu.g/mL) internal standard solution;
respectively transferring 6 isotope internal standard solutions with certain volumes into a10 mL volumetric flask, diluting with acetonitrile to a constant volume, fully and uniformly mixing, and storing at-20 ℃ in a dark place for later use;
and (3) sample determination: and (3) carrying out ultra performance liquid chromatography-tandem mass spectrometry determination on the purified sample, wherein:
the chromatographic conditions are as follows: waters ACQUITY HSST3Column, column temperature: 40 ℃; sample temperature: 15 ℃, injection volume: 2 μ L, assay time: flow rate for 8 min: 0.4 mL/min-1Flow term a: 5 mmol. L-1Ammonium acetate aqueous solution +0.1% formic acid aqueous solution, mobile phase B: acetonitrile; gradient elution procedure: 0-2 min, 80% -30% A; 2-3 min, 30% -5% A; 3-5.8 min, 5% A; 5.8-6 min, 5% -80% A; for 6-8 min, 80% of A;
drawing a standard curve: preparing mixed standard solutions with different concentrations according to the toxin response strength, diluting the standard mother solution with different kinds of tea blank matrix extracting solutions, performing the ultra performance liquid chromatography-tandem mass spectrometry detection, and respectively drawing standard working curves by taking the peak area of each component as a vertical coordinate and the concentration as a horizontal coordinate;
and (3) according to the liquid phase and mass spectrum condition sample injection analysis, adding the prepared standard solution with the lowest concentration into the tea sample for processing sample injection according to different responses in a mass spectrum MRM mode, calculating the detection limit LOD of the method according to the signal peak area corresponding to 3 times of signal-to-noise ratio S/N, and calculating the quantification limit LOQ of the method according to the signal peak area corresponding to 10 times of signal-to-noise ratio S/N.
Further, the preparation method of the standard stock solution of 16 mycotoxins comprises the following steps: aflatoxin B1、B2、G1、G2All are 2.5. mu.g.mL-1Zearalenone 100.0. mu.g/mL-1100.0. mu.g/mL of citrinin-1Diluting with methanol, and storing at-20 deg.C; deoxynivalenol 100.0 microgram mL-1Fusarium ketene 10.0 microgram/mL-1Nivalenol 10.0 mug/mL-1100.0 mu g/mL ochratoxin A-1100.0. mu.g/mL of T-2 toxin-1HT-2 toxin 10.0. mu.g/mL-1100.0. mu.g/mL of the variegated-color-toxin-1Diluting with acetonitrile, and storing at-20 deg.C; fumonisins B1、B2、B3Are 5.0. mu.g/mL in each case-1Diluting with 50% acetonitrile water, storing at-20 deg.C,and (5) standby.
The invention has the following beneficial effects:
some detection methods for detecting mycotoxins in tea leaves by using QuEChERS-high performance liquid chromatography-tandem mass spectrometry have been established. The invention is different from the similar technology in that:
1. the method can simultaneously detect 16 mycotoxins in different types of tea, has low cost and short extraction time compared with the traditional method, can be finished within 20min, and has high recovery rate. The lower Limit (LOQ) of the determination of 16 mycotoxins in different kinds of tea reaches 0.1-15 mu g/kg-1The method is far lower than the limit indexes of similar toxins in the national standard GB2761-2017, which shows that the method has higher sensitivity and can meet the requirements of tea quality supervision.
2. Different tea types have different matrix effects, and complex tea components have great interference on detection results. The pretreatment is carried out quickly by using a QuEChERS method, and the comprehensive purification is realized; and the OASIS PRIME HLB small column and the Dspe purification tube are preferably purified by comparing a plurality of purification columns, and different tea substrates are adopted to match with standard solution for calibration, so that the interference of the tea substrates on toxin detection is reduced to the maximum extent.
3. The 16 mycotoxins detected by the invention belong to metabolites of different fungi, and the differences of the compound structures, the physicochemical properties and the like are large. In order to achieve the purpose of simultaneously detecting and considering the recovery rates of different types of mycotoxins, the formic acid with a certain proportion is added into the extraction solvent, so that the recovery rate of the acidity sensitive toxin of the extraction solvent can be obviously improved, and the extraction of other types of toxins is not interfered.
4. The selection of the mobile phase influences the chromatographic peak pattern and the detection time of a detection target, thereby influencing the detection efficiency. Since the present invention detects multiple toxins simultaneously, the optimal mobile phase for one type of toxin may not be compatible with another toxin, or even with interference. Therefore, a mobile phase must be selected that can simultaneously account for 16 mycotoxins. The invention finally selects a solution of acetonitrile, ammonium acetate aqueous solution and formic acid aqueous solution which are mixed according to a certain proportion as a mobile phase, and the chromatographic peak type and the response intensity of 16 toxins are both ideal, the retention time is stable, and the separation degree is good.
Drawings
FIG. 1 is a total ion flux (TIC) chromatogram of a mycotoxin and an internal standard;
FIG. 2.1 shows aflatoxin B1A standard graph;
FIG. 2.2 shows aflatoxin B2A standard graph;
FIG. 2.3 shows aflatoxin G1A standard graph;
FIG. 2.4 shows aflatoxin G2A standard graph;
FIG. 2.5 shows Aspergillus flavus B1Standard map of toxin internal standard;
FIG. 2.6 is a variegated aspergillotoxin;
FIG. 2.7 shows fumonisin B1A standard graph;
FIG. 2.8 shows fumonisin B2A standard graph;
FIG. 2.9 shows fumonisin B3A standard graph;
FIG. 2.10 shows fumonisin B1An internal standard plot;
FIG. 2.11 is a standard diagram of HT-2 toxin;
FIG. 2.12 is a standard graph of T-2 toxin;
FIG. 2.13 is a standard graph of an internal standard T-2 toxin;
FIG. 2.14 is a standard chart of citrinin;
FIG. 2.15 is a deoxynivalenol standard diagram;
FIG. 2.16 is a diagram of a standard diagram of fusarenone;
FIG. 2.17 is nivalenol;
FIG. 2.18 is a standard map of internal standard of deoxynivalenol toxin;
FIG. 2.19 is a standard graph of ochratoxin A;
FIG. 2.20 is a standard chart of ochratoxin A internal standard;
FIG. 2.21 is a standard graph of zearalenone toxin;
FIG. 2.22 is a standard chart of an internal standard of zearalenone toxin;
FIG. 2.23 is a matrix effect plot of 3 toxins in 3 tea leaves;
reference numerals in FIG. 11. Nivalenol; deoxynivalenol; fusarenon X; aflatoxin G2;5.Aflatoxin G1;6.Aflatoxin B2;7.Aflatoxin B1;8.Fumonisin B1;9.Fumonisin B2;10.Citrinin;11.HT-2Toxin;12.Fumonisin B3;13.T-2Toxin;14.Ochratoxin A;15.Zearalenone;16.Sterigimatocystin
b:1.13C15--Deoxynivalenol;2.13C34--Fumonisin B1;3.13C17--Aflatoxin B1;4D5-Ochratoxin A;513C24--T-2Toxin;6.D6 Zearalenone.
Detailed Description
The following embodiment is combined to determine aflatoxin B in Pu' er tea by liquid chromatography-mass spectrometry1The sample pretreatment method and the detection method of the content are described in detail, but they should not be construed as limiting the scope of the present invention.
Example 1
1 materials and methods
1.1 instruments and reagents
UPLC H-Class ultra performance liquid chromatography-tandem mass spectrometer (Waters corporation, USA), Waters HSS T3 column (100.0mm × 2.1.1 mm, particle size 1.7 μm), TDL-5-A type low speed large capacity centrifuge (Shanghai's Tinggtin scientific instrument), KQ-500DE type numerical control ultrasonic cleaner (Kunshan ultrasonic instruments Co., Ltd.),
Figure BDA0002435455260000051
a type water purifier (Mitigo, Inc. of China) and an HD-2500 multi-tube vortex mixer (Yongzhou Yongning instruments, Inc.).
QuEChERS extract salt package (4g magnesium sulfate, 1g sodium chloride, 1g sodium citrate, 0.5g disodium citrate hydrate, Waters, USA), dSPE purification tube (150mg magnesium sulfate, 25mg PSA, 25mg C18,7mg GCB, Waters, USA), Oasis PRIME HLB column (3cc,150mg, Waters, USA), GHP filter membrane (GHP, USA).
Methanol, acetonitrile (LC-MS grade, Merck, Germany), ammonium acetate, formic acid (LC-MS grade, Waters, USA), ultrapure water (Millipore ultrapure water machine preparation).
Standard (purity > 98%): aflatoxins B1, B2, G1 and G2, aflatoxins, fumonisins B1, B2 and B3, HT-2 toxins, T-2 toxins, citrinin, deoxynivalenol, nivalenol, fusarenone, ochratoxin A and zearalenone, wherein the 16 standard products are all purchased from Sigma company in the United states.
Internal standard (purity > 98%):13C17--Aflatoxin B1(0.5μg·mL-1),D6-Zearalenone(100μg·mL-1),13C15-Deoxynivalenol(25μg·mL-1),13C34--Fumonisin B1(25μg·mL-1),13C24--T-2Toxin(30μg·mL-1),D5-Ochratoxin A(50μg·mL-1) The 6 standards were purchased from sigma, usa.
Standard stock solutions: aflatoxin B1、B2、G1、G2All are 2.5. mu.g.mL-1Zearalenone 100.0. mu.g/mL-1100.0. mu.g/mL of citrinin-1Diluted with methanol and stored at-20 ℃. Deoxynivalenol 100.0 microgram mL-1Fusarium ketene 10.0 microgram/mL-1Nivalenol 10.0 mug/mL-1100.0 mu g/mL ochratoxin A-1100.0. mu.g/mL of T-2 toxin-1HT-2 toxin 10.0. mu.g/mL-1100.0. mu.g/mL of the variegated-color-toxin-1Diluted with acetonitrile and stored at-20 ℃. Fumonisins B1、B2、B3Are 5.0. mu.g/mL in each case-1Diluted with 50% acetonitrile water and stored at-20 ℃ for later use.
Respectively transferring 6 isotope internal standard solutions with certain volumes into a10 mL volumetric flask, diluting with acetonitrile to constant volume, fully mixing uniformly, and storing at-20 ℃ in the dark for later use.
According to the experimental requirements, a blank tea substrate is prepared into a proper toxin standard working solution which is used for preparing mixed standard solutions with different concentrations of a standard working curve and is prepared for use.
1.2 sample pretreatment
After the tea leaves are ground, the tea leaves are sieved by a 60-mesh sieve, 0.5g of tea powder sample is weighed and put into a 50mL centrifuge tube, 10mL of water and 10mL of formic acid/acetonitrile (v/v, 10/90) solution are added, and the mixture is placed into an automatic oscillator to shake for 10 min. Adding QuEChERS extraction salt bag, shaking for 1min, and centrifuging (4000 r/min)\5min), taking supernatant for purification. The HLB pillars were installed without performing the pillar activation step. Approximately 0.4mL of the supernatant was passed through the HLB column, the filtrate was discarded, and then 1mL of the supernatant was again passed through the column again and the filtrate was collected. Transferring the filtrate to dSPE purification tube containing mixed adsorbent at 10000r/min\The mixture was centrifuged for 10min, and the supernatant was filtered through a 0.22 μm filter. And (3) sucking 190 mu L of the stable isotope solution into a 300 mu L inner inserting tube, adding 10 mu L of the stable isotope solution, and uniformly mixing by vortex for sample injection.
1.3 chromatographic and Mass Spectrometry conditions
Chromatographic conditions are as follows: waters ACQUITY HSST3Column, column temperature: 40 ℃; sample temperature: 15 ℃, injection volume: 2 μ L, assay time: flow rate for 8 min: 0.4 mL/min-1Flow term a: 5 mmol. L-1Ammonium acetate aqueous solution +0.1% (v/v) formic acid aqueous solution, mobile phase B: acetonitrile; gradient elution procedure: 0-2 min, 80% -30% A; 2-3 min, 30% -5% A; 3-5.8 min, 5% A; 5.8-6 min, 5% -80% A; 6-8 min, 80% A.
Mass spectrum conditions: ion source for mass spectrometry to remove zearalenone and internal standard thereof is electrospray ionization (ESI)-) The rest toxins are electrospray positive ion sources (ESI)+) And the detection mode is as follows: multiple reactive ion monitoring (MRM) mode, capillary voltage: 1.5 kV; taper hole airflow: nitrogen gas at a flow rate of 50 L.h-1(ii) a Ion source temperature: 120 ℃; desolventizing aerosol gas temperature: at 500 deg.C, the flow rate is 1000L/hr; collision gas: high purity argon 2 results.
The following tests were carried out using the above conditions for the measurement by hplc-tandem mass spectrometry:
2.1 optimized test results for extraction solvent
In the course of the experiment, the choice of extraction solvent directly affects the level of recovery, and in order to obtain the highest possible recovery, it is necessary to choose a solvent with high extraction efficiency. Compared with acetonitrile and methanol as extraction solvents, experiments show that methanol as a protonation solvent has better solubility than acetonitrile, but acid, alcohol and phenols in tea leaves are easily extracted, so that more impurities exist in the extracting solution; compared with the methanol extract, the acetonitrile extract has less fat and pigment, which is beneficial to salting out and purification in the subsequent steps, and acetonitrile is finally selected as the extraction solvent.
Part of toxin contains carboxyl group and is sensitive to acidity in the extraction solvent, so that the stability and recovery rate of carboxyl group-containing toxins such as fumonisin, ochratoxin and the like can be enhanced by adding formic acid into the extraction solvent. The recovery rate of each mycotoxin is respectively determined by setting the extraction reagent of the formic acid acetonitrile with the formic acid content of 1%, 2%, 5%, 10% and 15%,
table 116 recovery of mycotoxins at different formic acid content extractants
Table 1 Recoveries of 16 mycotoxins extracted by acetonitrile withdifferent formic acid content
Figure BDA0002435455260000071
As can be seen from Table 1, the formic acid content in the extraction solvent has different effects on the recovery rates of different types of toxins, wherein 4 types of aflatoxins, aflatoxins and fusarenols are less affected by the change of the formic acid concentration; the remaining 10 toxins increased with increasing formic acid concentration in the extractant, but when the formic acid content in the extractant reached 15%, the recovery rates of nivalenol, citrinin, zearalenone, HT-2 toxin, T-2 toxin decreased and reached a significant level of difference (P <0.05) compared to 10% formic acid content. From the average value of the recovery rates of the formic acid added with different concentrations of the 16 toxins, the average value of the recovery rates is in an ascending trend within the range of 1-10% of the formic acid added concentration, and slightly decreases when the recovery rate exceeds 10%.
The tea sample is dried powder after being crushed and sieved, water needs to be added for complete infiltration before extraction, and meanwhile, based on the extraction principle of the QuEDhERS method, the sample needs to be added with water which is equal to the extraction reagent, so that the tea fiber tissue cell expanding agent is soaked in 10mL of distilled water, and the permeability of the tea fiber tissue cell expanding agent is increased, so that the tea fiber tissue cell expanding agent is beneficial to full extraction. The QuEDhERS salt bag is adopted, so that the effect of a buffer solution can be realized, the extraction efficiency of the mycotoxin is improved, and the full separation of a water phase and an organic phase can be promoted. The mycotoxin extraction solvent has many kinds, including methanol, acetonitrile, ethyl acetate and the like, and the selection of the extraction solvent needs to consider the factors of the polarity, the extraction efficiency and the like of a target object. Methanol is a polar protic solvent, and organic acids with strong polarity in tea leaves and phenolic substances are easily extracted together, so that the subsequent purification process is complicated. Acetonitrile can be used as an extraction solvent to fully extract the toxin components in the matrix, and in addition, the experiment result compares the addition extraction effects of formic acid with different proportions in consideration of a group containing more carboxyl groups in fumonisins, as shown in the table above, the recovery rate of the toxins is not in positive correlation with the concentration of methanol, the extraction effect of 16 toxins with 10% formic acid content is the best, and the recovery rate achieves the satisfactory effect. According to the invention, 10% formic acid/acetonitrile and QuEChERS extraction salt packets are used as extraction solvents, so that the toxin can be fully extracted, and impurities in the extracting solution can be reduced; calibrating by adopting different tea matrix matching standard solutions; meanwhile, isotope internal standard substances and analytes are similar in structural properties and similar in chromatographic mass spectrum behavior, so that interference on the substances to be detected is avoided, an instrument sample injection volume error isotope internal standard method can be corrected, a satisfactory extraction effect is obtained, pretreatment is performed by using a QuEChERS method for rapid treatment, comprehensive purification is performed, toxins in the tea leaves are fully extracted, various biological toxins in the tea leaves can be fully extracted through simple and rapid pretreatment, the extraction time is short, the cost is low, and pigment interference in the tea leaves can be effectively removed; therefore, the addition of 10% formic acid is more beneficial to fully extracting the toxin, so that the invention selects 10% formic acid acetonitrile as an extraction solvent.
2.2 test results for purification column selection
The invention adopts Oasis PRIME HLB purifying column for first-step purification, can effectively remove macromolecular lipid impurities in tea leaves, and then purifies the tea leaves by dSPEThe chemical tube and the purification tube contain magnesium sulfate component capable of removing water in the matrix, PSA removes sugar and acid component in the tea matrix, C18The residual lipid component is mainly absorbed, the pigment component in the tea is mainly removed by GCB, and the satisfactory recovery rate can be achieved by applying the purification method. Compared with a multi-toxin immunoaffinity column, a Mycosep 226 column, an Oasis HLB purification column and an Oasis PRIME HLB purification column, the result shows that the types of toxins measurable by the multi-toxin immunoaffinity column can not cover all 16 toxins to be measured, the pretreatment is more complex, and the recovery rate is low; the Mycosep 226 column purification has lower recovery rate of HT-2 toxin and zearalenone, and the pigment removing effect is not ideal; the Oasis HLB column has a non-ideal purification effect and has an adsorption effect on toxins, so that the recovery rate is low and the experimental requirements cannot be met. Therefore, the method of combining the Oasis PRIME HLB purifying column with the dSPE purifying tube can reduce the interference of the complex matrix of the tea on the extraction of the toxin, separate the mycotoxin from the matrix of the tea and achieve the purifying effect.
2.3 results of selection test of mobile phase
The invention selects methanol-pure water and acetonitrile-pure water as elution mobile phases respectively, inspects the influence of two mobile phase systems on the separation effect, and experiments show that: AFB with methanol-pure water as mobile phase1Fumonisins B1The response value of (a) is significantly reduced; when acetonitrile-pure water is in mobile phase, fumonisin B2Fumonisins B3Cannot be completely separated. Therefore, when pure water is used as the mobile phase, the ionization efficiency is poor and the sensitivity is low. In order to enhance the ionization strength of each component substance, the invention adds formic acid and ammonium acetate (the final concentration is 0.1 percent and 5 mmol.L respectively) into the aqueous solution-1). The result shows that the addition of 0.1% formic acid is beneficial to inhibiting the electrostatic interaction between the mycotoxin and a chromatographic column, the tailing phenomenon is avoided, and the symmetry of the fumonisin peak type is obviously improved; 5 mmol. L-1The ammonium acetate solution can enhance the response value of the aflatoxin. Therefore, when acetonitrile, 5mmol/L ammonium acetate aqueous solution and 0.1% (v/v) formic acid aqueous solution are finally selected as the mobile phase, the chromatographic peak type and the response intensity of the 16 toxins are ideal, the retention time is stable, and the separation degree is good. Chromatographic condition optimizationThe chromatogram of the final mycotoxin and Total Ion Current (TIC) is shown in FIG. 1.
2.4 Mass Spectrometry Condition optimization test results
According to the chemical ionization property of 16 mycotoxin molecules, electrospray ESI is respectively selected+And ESI-In the mode, a parent ion is selected in an ms-scan mode, a daughter-scan mode is adopted to optimize the mass spectrum parameters such as the child ions, collision energy, taper hole voltage and the like, and zearalenone easily loses protons due to a hydroxyl-containing structure to generate [ M-H ]]-Ion, therefore the experimental zearalenone and internal standard employed ESI-Mode, ESI is selected as the rest toxin+In the mode, optimized parameters are shown in the following table 2.16, mycotoxins and internal standards thereof are well separated, the requirements of the European Union No. 2002/657/EC resolution of mass spectrometry method identification points on the qualitative and quantitative of the object to be detected are met, and the toxin spectrograms obtained in the test are shown in the figures 2.1-2.22.
TABLE 2 mycotoxin detection Mass Spectrometry parameters
Table 2 Mass spectrometric parameters of mycotoxins
Figure BDA0002435455260000091
Figure BDA0002435455260000101
Note: expressed as quantitative ions
2.5 substrate benefit test results
The tea components are complex, the matrix effect is difficult to completely eliminate, in order to improve the accuracy and reliability of the experiment, the experiment screens oolong tea, black tea and white tea without toxins as matrix blanks, the ratio of the slope of a prepared toxin mixed standard solution to the slope of a standard curve prepared by a flowing phase is added by the same processing method to evaluate the matrix effect of different tea matrixes on the toxins, the matrix effect is generally determined to be not obvious when the ratio is 0.8-1.2, when the ratio is more than 1.2, the matrix enhancement effect is considered, the ratio is less than 0.8, the matrix inhibition effect is considered, a determination result graph is 2.23, and the result shows that the aspergillus flavus has the matrix inhibition effectToxin B1Aflatoxins B2Aflatoxin G1Aflatoxin G2The matrix effect of the hybrid aflatoxin, the HT-2 toxin, the T-2 toxin, the citrinin, the nivalenol and the fusarenol is 0.813-1.166%; fumonisins B1Fumonisins B2Fumonisins B3The substrate effect of the deoxynivalenol is more than 1.2; the ochratoxin A and zearalenone are both less than 0.8, and the standard solution matched with the matrix is adopted for calibration and quantification, so that the matrix effect is compensated according to components, and the recovery rate of the test is improved.
2.6 Standard Curve, detection Limit, quantification Limit results
According to the response strength of 16 toxins in a sample, mixed standard solutions with different concentrations are prepared, and different tea blank matrix extracting solutions are used for diluting a standard mother solution to obtain a standard curve working solution. And (3) according to the liquid phase and mass spectrum conditions, sampling and analyzing, and respectively drawing a standard working curve by taking the peak area of each component as a vertical coordinate and the concentration as a horizontal coordinate. According to different responses in a mass spectrum MRM mode, the prepared standard solution with the lowest concentration is added into a tea sample for processing and sample injection, the detection Limit (LOD) of the method can be calculated according to the signal peak area corresponding to 3 times of signal-to-noise ratio (S/N), and the quantification Limit (LOQ) of the method can be calculated according to the signal peak area corresponding to 10 times of signal-to-noise ratio (S/N). The results obtained are shown in table 3 below.
Table 3 linear equations, correlation coefficients, detection limits and quantitation limits for mycotoxins (n ═ 6)
Table 3 Linear equation,correlation coefficient,LOD and LOQ ofmycotoxins in white tea(n=6)
Figure BDA0002435455260000102
Figure BDA0002435455260000111
2.7 recovery and precision of the Process
The method has the advantages that the types of tea are various, the influence of the matrix of each tea is different, the interference is generated on the analysis of mycotoxin, and the result accuracy is influenced, so that the recovery rates are measured by adding low, medium and high concentration levels to black tea, white tea and oolong tea respectively, 3 samples are measured at each level, the standard addition recovery rates of different matrixes at 3 levels are measured within 1d within day precision, 3 samples under standard addition water are measured continuously for 6d within day precision, the relative deviation is obtained by measuring 1 time per day, and the result is shown in table 4. The recovery rate of the 16 mycotoxins is 61.5-112.1%, and the Relative Standard Deviation (RSD) is 1.3-11.5%. The detection result is proved to have good precision.
TABLE 4 recovery and precision of different tea species (n ═ 3)
Table 3 Recoveries and precision of different tea(n=3)
Figure BDA0002435455260000112
Figure BDA0002435455260000121
2.8 determination of application results
The invention detects 121 parts of tea samples (38 parts of oolong, 53 parts of white tea and 30 parts of black tea) in total in each region of Fujian province, wherein aflatoxin B is detected from 1 part of hydrangea oolong sample1The content is 34 mug/kg-1Reference is made to the maize standard of GB2761-2017 (20. mu.g.kg)-1) Exceeding a limit value [26]1 portion of kungfu black tea sample detects ochratoxin A with the content of 1.6 mug.kg-1Reference is made to the GB2761-2017 coffee Standard (5.0. mu.g.kg)-1) Value of not exceeding the limit[26]. The effectiveness and the sensitivity of the method are verified.
The invention establishes a method for determining 16 mycotoxins in different tea types by using an ultra-high performance liquid chromatography-tandem mass spectrometry method. Extracting with formic acid/acetonitrile (10:90), adding QuEChERS salt pack, shaking, centrifuging, treating the extractive solution with OASISPRIME HLB column and Dspe purification tube, and purifying with Waters HSS T3Separating chromatographic columns, adopting a multi-reactive ion monitoring (MRM) mode of simultaneously scanning positive ions and negative ions, matching tea substrates with standard solutions, and quantifying by an isotope internal standard method. The 16 mycotoxins are in good linear relationship over the respective concentration ranges.
The QuEChERS technology is divided into 3 steps of extraction, salting out and purification. Extraction is an important component of the QuEChERS technology, and the extraction efficiency of the extracting solution determines the accuracy of the final analysis result. The method has the advantages that the extraction rate of acetonitrile is high, the application range is wide, and the acetonitrile is selected as the extraction solvent, so that the method has less fat and pigment compared with a methanol extracting solution, is favorable for salting out and purification in subsequent steps, and improves the detection efficiency. For extracting target toxin sensitive to polarity range, auxiliary reagents (acetic acid, formic acid, methanol, etc.) are added into the extracting solution to enhance the effect of combined extraction. The detection of the invention finds that the average value of the recovery rates of the 16 toxins is in an ascending trend within the range of 1-10% of the addition concentration of the formic acid, and slightly decreases when the recovery rate exceeds 10%, so that the invention selects 10% of formic acid acetonitrile as an extraction solvent, and the recovery rates of the 16 mycotoxins are higher.
The invention adopts a QuEChERS method to extract a pretreatment method combining double purification of an OASIS PRIME HLB small column and a Dspe purification tube, has the cost far lower than that of the currently generally adopted immunoaffinity column, is simpler to operate, does not need activation and balance steps, can remove most matrix interferents, and has faster solution flow rate, thus ensuring that the whole operation is smoother and faster; the method adopts a high-sensitivity UPLC/MSMS analysis method, simultaneously measures 16 mycotoxins, has short extraction time, can be completed within 20min, and greatly improves the flux of the test; the lower Limit (LOQ) of the determination of 16 mycotoxins in the optimized method reaches 0.1-15 mu g/kg-1The method has the advantages of high sensitivity and capability of meeting the requirements of product quality supervision.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (5)

1. A method for determining 16 mycotoxins in tea by using an ultra-high performance liquid chromatography-tandem mass spectrometry method is characterized by comprising the steps of preparing a sample to be tested and determining by using the ultra-high performance liquid chromatography-tandem mass spectrometry method;
wherein the extracting solution for preparing the sample is formic acid/acetonitrile with water and 1: 5-1: 10 in a volume ratio of 3: 1-1: 3 and then is added with a QuEChERS extraction salt bag;
preparing a sample extracting solution to be tested, and purifying the sample extracting solution by adopting a dSPE purifying tube and an Oasis PRIME HLB small column;
the flow term A in the chromatographic condition in the high performance liquid chromatography-tandem mass spectrometry is 5-10 mmol.L-1Ammonium acetate aqueous solution and formic acid aqueous solution with the volume ratio of 0.1-1%; mobile phase B: acetonitrile; under the mass spectrum condition, the ion source for mass spectrum analysis is used for removing zearalenone and the internal standard is an electrospray negative ion source ESI-The rest toxins are ESI (electrospray ionization) positive ion source+And the detection mode is as follows: multiple reactive ion monitoring MRM mode, capillary voltage: 1.5 kV; taper hole airflow: nitrogen gas at a flow rate of 50 L.h-1(ii) a Ion source temperature: 120 ℃; desolventizing aerosol gas temperature: at 500 deg.C, the flow rate is 1000L/hr; collision gas: high-purity argon gas;
the 16 mycotoxins tested were: aflatoxin B1Aflatoxins B2Aflatoxin G1Aflatoxin G2Zearalenone, citrinin, deoxynivalenol, fusarenone, nivalenol, ochratoxin, T-2 toxin, HT-2 toxin, ochratoxin, fumonisin B1Fumonisins B2Fumonisins B3
2. The method for determining 16 mycotoxins in tea by using ultra-high performance liquid chromatography-tandem mass spectrometry as claimed in claim 1, wherein: the preparation of the test sample comprises the following steps:
sample treatment: taking 0.5g of crushed tea to be detected, adding 10ml of water and 10ml of formic acid/acetonitrile solution with the volume ratio of 1: 5-1: 10 into a tea sample, mixing, and then placing the mixture in an automatic oscillator to shake for 10 min; adding QuEChERS extraction salt bag, shaking for 1min, centrifuging at 4000r/min for 5min, and collecting supernatant for purification;
purifying: installing an HLB column, not executing a column activation step, taking about 0.4mL of supernatant to pass through the HLB column, discarding filtrate, then taking 1mL of supernatant to pass through the column again and collecting the filtrate, transferring the filtrate into a dSPE purification tube containing a mixed adsorbent, centrifuging for 10min at 10000r/min \ and filtering the supernatant through a 0.22 mu m filter membrane; and (3) sucking 190 mu L of the stable isotope solution into a 300 mu L inner inserting tube, adding 10 mu L of the stable isotope solution, and uniformly mixing by vortex for sample injection.
3. The method for determining 16 mycotoxins in tea by using ultra-high performance liquid chromatography-tandem mass spectrometry as claimed in claim 1, wherein: in the high performance liquid chromatography-tandem mass spectrometry determination, 16 mycotoxin standard stock solutions are prepared and isotope of 0.5 mu g/mL is adopted-1Is/are as follows13C17--Aflatoxin B1,100μg·mL-1D of (A)6-Zearalenone,25μg·mL-1Is/are as follows13C15-Deoxynivalenol,25 μg·mL-1Is/are as follows13C34--Fumonisin B1,30μg·mL-1Is/are as follows13C24--T-2 Toxin,50μg·mL-1D of (A)5-Ochratoxin a internal standard solution;
respectively transferring 6 isotope internal standard solutions with certain volumes into a10 mL volumetric flask, diluting with acetonitrile to a constant volume, fully and uniformly mixing, and storing at-20 ℃ in a dark place for later use;
and (3) sample determination: and (3) carrying out ultra performance liquid chromatography-tandem mass spectrometry determination on the purified sample, wherein:
the chromatographic conditions are as follows: waters ACQUITY HSST3Column, column temperature: 40 ℃; sample temperature: 15 ℃, injection volume: 2 μ L, assay time: flow rate for 8 min: 0.4 mL/min-1Flow term a: 5 mmol. L-1Ammonium acetate aqueous solution +0.1% formic acid aqueous solution, mobile phase B: acetonitrile; gradient elution procedure: 0-2 min, 80% -30% A; 2-3 min, 30% -5% A; 3-5.8 min, 5% A; 5.8-6 min, 5% -80% A; for 6-8 min, 80% of A;
drawing a standard curve: preparing mixed standard solutions with different concentrations according to the toxin response strength, diluting the standard mother solution with different kinds of tea blank matrix extracting solutions, performing the ultra performance liquid chromatography-tandem mass spectrometry detection, and respectively drawing standard working curves by taking the peak area of each component as a vertical coordinate and the concentration as a horizontal coordinate;
and (3) according to the liquid phase and mass spectrum condition sample injection analysis, adding the prepared standard solution with the lowest concentration into the tea sample for processing sample injection according to different responses in a mass spectrum MRM mode, calculating the detection limit LOD of the method according to the signal peak area corresponding to 3 times of signal-to-noise ratio S/N, and calculating the quantification limit LOQ of the method according to the signal peak area corresponding to 10 times of signal-to-noise ratio S/N.
4. The method for determining 16 mycotoxins in tea by using ultra-high performance liquid chromatography-tandem mass spectrometry as claimed in claim 2, wherein: in the sample treatment, the crushed tea to be detected is screened by a 60-mesh sieve to obtain the tea.
5. The method for determining 16 mycotoxins in tea by using ultra high performance liquid chromatography-tandem mass spectrometry as claimed in claim 3, wherein: the preparation method of the standard stock solution of 16 mycotoxins comprises the following steps: aflatoxin B1、B2、G1、G2All are 2.5. mu.g.mL-1Zearalenone 100.0. mu.g/mL-1100.0. mu.g/mL of citrinin-1Diluting with methanol, and storing at-20 deg.C; deoxynivalenol 100.0 microgram mL-1Fusarium ketene 10.0 microgram/mL-1Nivalenol 10.0 mug/mL-1100.0 mu g/mL ochratoxin A-1100.0. mu.g/mL of T-2 toxin-1HT-2 toxin 10.0. mu.g/mL-1100.0. mu.g/mL of the variegated-color-toxin-1Diluting with acetonitrile, and storing at-20 deg.C; fumonisins B1、B2、B3Are 5.0. mu.g/mL in each case-1Diluted with 50% acetonitrile water and stored at-20 ℃ for later use.
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CN115047112A (en) * 2022-07-06 2022-09-13 贵州省产品质量检验检测院 Detection method for determining mycotoxin in cereal grains by QuEChERS pretreatment combined with LC-MS/MS
CN115452997A (en) * 2022-09-26 2022-12-09 北京合众恒星检测科技有限公司 Method for simultaneously detecting forbidden pesticides, mycotoxins and effective components in traditional Chinese medicinal materials
CN115452997B (en) * 2022-09-26 2023-07-25 中原食品实验室 Simultaneous detection method for forbidden pesticides, mycotoxins and functional components in traditional Chinese medicinal materials

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