CN113419022B - Method for measuring residual quantity of iminoctadine in plant-derived food by solid phase extraction-liquid chromatography-tandem mass spectrometry - Google Patents
Method for measuring residual quantity of iminoctadine in plant-derived food by solid phase extraction-liquid chromatography-tandem mass spectrometry Download PDFInfo
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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Abstract
The invention provides a method for measuring the residual quantity of iminoctadine in plant-derived food by solid-phase extraction-liquid chromatography-tandem mass spectrometry, belonging to the technical field of food detection. Before solid phase extraction, the sample is extracted by weighing the processed sample to be detected, placing the sample in a plastic centrifuge tube, adding acetic acid and dichloromethane, performing oscillation extraction, and centrifuging to obtain supernatant; purifying by activating a balance solid phase extraction column, taking supernate to pass through the solid phase extraction column, adding water and methanol forest in sequence to wash the small column, eluting with acetonitrile formate, and collecting into a plastic centrifuge tube; blowing nitrogen to be nearly dry, adding formic acid water to constant volume for instrument analysis; and further separating the compound by adopting a liquid chromatograph, and carrying out qualitative analysis and quantitative analysis on the separated detection liquid by adopting a mass spectrometer. The method is suitable for detecting and quantifying the residual quantity of the iminoctadine in plant-derived foods such as Or, jackfruit, lettuce, cucumber, brown rice, almond, tea and the like, and has the characteristics of rapidness, accuracy, sensitivity and low toxicity.
Description
Technical Field
The invention relates to the technical field of food detection, in particular to a method for measuring residual quantity of iminoctadine and biguanide salts (including iminoctadine trioctylphenylsulfonate and iminoctadine acetate) in plant-derived food by solid phase extraction-liquid chromatography-tandem mass spectrometry.
Background
The iminoctadine is a broad-spectrum fungicide, has contact poisoning and prevention effects, and has a good effect on most fungal diseases caused by ascomycetes and deuteromycetes. Biguanide trioctyl benzene sulfonate (imidazole sulfonate) and biguanide trioctyl acetate (imidazole triacetate) are the most common salt bactericides, and are widely applied to disease control and preservation and fresh keeping of fruits and vegetables such as oranges, apples, tomatoes, lettuce and the like. The iminoctadine belongs to a moderately toxic pesticide, has extremely high toxicity to aquatic organisms, and can generate adverse effects on the environment and human bodies if released into the environment due to large-scale use. Therefore, the national standard GB 2763-2019 stipulates that the maximum residual limit of the iminoctadine in fruits and vegetables is 0.2-3 mg/kg, the European Union stipulates that the residual quantity of iminoctadine in citrus fruits cannot exceed 0.05mg/kg, and the Japanese stipulates that the iminoctadine cannot exceed 6 mu g/L. However, the iminoctadine has strong polarity and poor stability, and is easily adsorbed and interfered by a sample matrix and various experimental material instruments, so that the iminoctadine is difficult to detect. At present, a liquid chromatography-post-column derivatization fluorescence detection method is adopted internationally, and the method is complex in operation, low in sensitivity and poor in reproducibility, and cannot meet the requirements of trace detection and quantification of iminoctadine.
The invention with publication number CN103983723B discloses a method for detecting the residual quantity of iminoctadine in fruits and vegetables, which comprises the steps of extracting a sample by using a chemical reagent, purifying by using a small column of carboxymethyl silylated silica gel to obtain a test solution, and detecting by using a liquid chromatography-tandem mass spectrometer. This method requires a large amount of sample (100 g); complex extraction steps such as pretreatment, repeated extraction, liquid-liquid extraction, reduced pressure concentration and the like are required, and a large amount of flammable, explosive, toxic and harmful hazardous chemical reagents such as guanidine hydrochloride, triethylamine, n-butanol, n-hexane, sulfate and the like are required; meanwhile, the mass spectrometry method collects single-charge parent ions and daughter ions thereof which are unstable and have poor response. Therefore, in practice, the method has complex detection steps, consumes a large amount of dangerous chemicals, has poor detection sensitivity and reproducibility, and is not suitable for popularization and application.
Disclosure of Invention
The invention aims to: aiming at the existing problems, the invention provides a method for measuring the residual quantity of iminoctadine in plant-derived food by solid phase extraction-liquid chromatography-tandem mass spectrometry, which is suitable for detecting and quantifying the residual quantities of iminoctadine, iminoctadine and iminoctadine in plant-derived food such as fruits, vegetables, cereals, nuts, tea leaves and the like, and has the characteristics of simplicity, rapidness, accuracy, sensitivity and low toxicity.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the method for measuring the residual quantity of the iminoctadine in the plant-derived food by the solid phase extraction-liquid chromatography-tandem mass spectrometry comprises the following steps:
(1) Sample processing
Sample preparation: taking a plant source food sample, removing non-edible part peel or shell, and grinding the sample into uniform and fine samples by using a homogenizer or a grinder for later use; the plant-derived food samples include, but are not limited to, fruits, vegetables, grains, nuts, and tea;
extraction: weighing a proper amount of well treated sample, placing the sample in a plastic centrifuge tube, adding a weakly acidic aqueous solution and dichloromethane, oscillating and extracting, and then carrying out centrifugal treatment to obtain a supernatant for later use;
purifying: activating a balance WCX solid phase extraction column, and taking part of supernatant to pass through the solid phase extraction column; adding water and methanol successively to wash the small column; eluting with 10Vol% acetonitrile formate, and collecting into a plastic centrifuge tube; blowing nitrogen to be nearly dry, adding 0.2Vol% formic acid water to a constant volume of 1mL, uniformly mixing by vortex, filtering through a 0.22 mu m needle filter, and filling into a plastic sampling bottle for subsequent instrument analysis;
(2) Detection and quantification
Separating the compounds in the detection liquid by adopting a liquid chromatograph, and carrying out qualitative and quantitative analysis on the separated target compound, namely, the iminoctadine, by adopting a tandem mass spectrometer; wherein:
chromatographic column conditions: selection C 18 Chromatographic column, the column temperature is 40 ℃; the sample injection amount is 5 mu L; the mobile phase A is 0.2 percent formic acid water; the mobile phase B is methanol; performing gradient elution;
the mass spectrum conditions are as follows: tandem mass spectrometer with electrospray ion source selection in positive ion mode (ESI) + ) Multiple Reaction Monitoring (MRM) scans were performed with the following biguanide octylamine ion pair parameters:
the quantitative method comprises the following steps: selecting a blank matrix consistent with a sample to be detected, adding biguanide octylamine standard solutions with different concentrations, treating the blank matrix and the sample to be detected according to the same extraction and purification mode, detecting the treated solution by an instrument, and drawing a standard curve for quantitative analysis. (ii) a The indexes of the quantitative analysis comprise the residual quantity of the iminoctadine and the residual quantity of biguanide salts of the iminoctadine, wherein the biguanide salts include but are not limited to iminoctadine and iminoctadine hydrochloride; the residual amount of biguanide salt is obtained by converting the residual amount of iminoctadine.
In the present invention, preferably, a proper amount of the ground samples is weighed, wherein the proper amount refers to 5g of the fruit and vegetable samples and 1g of the grain, nut and tea samples.
In the present invention, preferably, the weakly acidic aqueous solution in the extraction step is 0.1vol% to 0.3vol% acetic acid solution.
In the invention, preferably, the WCX solid phase extraction column is a carboxyl modified mixed weak cation exchange column with the specification of 60mg,3cc or 150mg,6cc.
In the present invention, preferably, the plastic centrifuge tube and the plastic sample injection bottle are both polypropylene centrifuge tube and sample injection bottle.
In the present invention, preferably, the pin filter is a hydrophobic polytetrafluoroethylene PTFE pin filter.
In the present invention, preferably, said C 18 The chromatographic column is Eclipse Plus C 18 Column, 2.1X 100mm,1.8 μm.
In the invention, preferably, in the extraction step, the shaking extraction time is 20-40min, and the centrifugation is carried out for 4-10min at 3000-10000 r/min.
In the present invention, preferably, the procedure of the gradient elution is: 0-2.0 min, 95% A; 2.0-3.0 min, by 95% A down to 15% A; 3.0-5.8 min by 15% A to 5%; 5.8 to 6.0min, from 5% by volume A to 95% by volume A; 6.0-8 min, 95% A.
In the present invention, preferably, the tandem mass spectrometer is an AB SCIEX 4500 mass spectrometer, and the mass spectrometry conditions further include air curtain gas (CUR): 20psi; collision gas (CAD): a Medium; ion source voltage (IS): 4500V; ion source Temperature (TEM): 500 ℃; atomizing gas (GS 1): 55psi; auxiliary heating gas (GS 2): 55psi.
The principle that the invention can successfully detect the residual quantity of iminoctadine is as follows:
the method comprises the steps of firstly extracting and purifying a sample to be detected by using 0.1-0.3 vol% acetic acid water, extracting impurities by using dichloromethane, extracting the impurity by using organic reagents such as methanol and acetonitrile with strong polarity of the iminoctadine and high solubility in water to obtain an ideal effect, and extracting the weak-polar substance by using acetic acid water and adding dichloromethane to obtain good purifying and layering effects. And the iminoctadine is alkaline and is easy to combine with carboxyl, so that a carboxyl modified mixed weak cation exchange column WCX is selected for extraction purification, and better retention and recovery can be obtained. In the instrument detection process, the co-effluent components in the sample matrix can influence the chromatographic separation and ionization of a target compound, and easily cause the weakening or enhancement of a detection signal, after the extraction and purification method is adopted to extract and purify the plant source samples such as fruits, vegetables, grains, nuts, tea leaves and the like, the co-effluent components in each sample matrix have small influence on the detection signal, and the matrix effect is very low (< 5%).
According to the method, a C18 chromatographic column is used for further separating compounds in a liquid to be detected, during gradient elution, 95% of water phase is kept at the initial stage to enable iminoctadine to obtain a good retention effect on the C18 chromatographic column, then the proportion of methanol is rapidly increased to 85% within 1min to rapidly elute iminoctadine, so that peak shape tailing can be effectively avoided, then the high proportion of methanol is continuously used for washing the column for 2.8min to effectively reduce residue of iminoctadine in a sample injection system, and finally the initial equilibrium state is adjusted for 2 min.
In the invention, when mass spectrometry is carried out, the electrospray ion source forms adduct ions (M + 2H)] 2+ And Multiple Reaction Monitoring (MRM) scans were performed. Separately collecting the [ M + H ] of iminoctadine] + And [ M +2H] 2+ The addition ions were optimized for mass spectrometry parameters, and as a result, the best parent ion of iminoctadine was 178.8 (m/z) ion with double charge, and the iminoctadine mass spectrum is shown in fig. 1, while the 356.6 (m/z) parent ion with single charge had a poor response and was extremely unstable. Probably because the two ends of the chemical structure of the iminoctadine contain C = N double bonds, 2H are easily obtained in the ionization process + Form a double-charge ion addition mode [ M +2H ]] 2+ . The parent ions form characteristic fragment ions, namely daughter ions after being fragmented in the collision chamber, wherein the ion response abundance is better to be 69.0 (m/z), 100.0 (m/z), 157.7 (m/z) and 1870 (m/z) (as shown in fig. 1), and finally 100.0 (m/z) and 187.0 (m/z) daughter ions with better response and less interference peaks are selected through the verification of actual sample detection.
In the aspect of selecting the constant volume solution, the response abundance of the iminoctadine is compared when 0.01% ammonia water (v/v), pure water and 0.1% (v/v), 0.2% (v/v), 0.4% (v/v) and 0.8% (v/v) formic acid water are taken as the constant volume solution, and as a result, the response values of the formic acid water groups have no obvious difference, but are all obviously higher than those of the 0.01% ammonia water and the pure water group. The acidic condition can promote the hydrolysis of the iminoctadine hydrochloride into iminoctadine, promote the ionization effect of the iminoctadine hydrochloride in an electrospray ion source, and reduce the adsorption and interference of instrument materials such as pipelines and spray needles on the iminoctadine hydrochloride.
In addition, the biguanide octylamine is easily adsorbed by glass, so that the stability and reproducibility of the experimental method are poor when a glass instrument or a sample injection bottle is used. The prior art requires the addition of triethylamine solutions during pretreatment to mask silanol groups of glassware to reduce adsorption of iminoctadine onto glassware. In the pretreatment process, a glass appliance is not used, and a polypropylene (PP) material is selected as a sample injection bottle, so that good linearity and reproducibility can be obtained without adding triethylamine for treatment.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
1. the method is suitable for detecting and quantifying the residual quantity of the iminoctadine, the iminoctadine trioctylacetate and the iminoctadine trioctylphenylsulfonate in plant samples such as fruits, vegetables, grains, nuts, tea leaves and the like, wherein the iminoctadine is decomposed into the iminoctadine during use, the content of the iminoctadine is quantitatively analyzed during testing, and the residual quantity of the iminoctadine can be obtained according to the residual quantity of the iminoctadine and the molecular weight relationship between the iminoctadine and the iminoctadine. When the method is used for detecting fruits and vegetables, the linear correlation of the diguanidinooctylamine concentration in the range of 0.005-0.080 mg/kg standard addition range is good, the correlation coefficient (r) is more than or equal to 0.993, and the quantitative detection limit of the diguanidinooctylamine is 0.010mg/kg; when grains, nuts and tea leaves are detected, the linear correlation of the diguanidino octylamine concentration in the range of 0.020-0.320 mg/kg standard addition is good, the correlation coefficient (r) is more than or equal to 0.998, the accuracy is high, and the quantitative detection limit of the diguanidino octylamine is 0.050mg/kg. The method of the invention has the advantages that the quantitative detection limit of all the plant-derived foods is lower than the national limit standard and the European Union limit standard, and the sensitivity is higher. The standard recovery rate and the relative standard deviation of different concentrations of each sample matrix meet the relevant requirements of national food physicochemical detection-laboratory quality control standard (GB/T27404-2008) and European Union food and feed pesticide residue and analysis quality control and method verification program guide (SANTE 11813/2017).
2. The invention can obtain good linearity and reproducibility without adding triethylamine. Therefore, the reagent used in the invention has lower toxicity, less harm to operators, better environmental protection and safety and is more suitable for popularization and use.
Drawings
FIG. 1 is the diagram of the obtained diguanide octylamine double-charge ionization mass spectrum;
FIG. 2 is a chromatogram of iminoctadine (0.010 mg/kg) from Von.1 of example 1.
Detailed Description
The present invention is further described in detail below with reference to specific examples so that those skilled in the art can more clearly understand the present invention. The following examples are given for the purpose of illustration only and are not intended to limit the scope of the invention. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments of the present invention without any creative effort belong to the protection scope of the present invention.
For example: in the extraction step, the following examples define the sampling amount of the sample to be tested as 5.00g or 1.00g, but in actual practice, 5.00g or 1.00g is only a reference value, and those skilled in the art can adapt the scheme according to the invention, and can adapt the amounts of acetic acid and dichloromethane, or add them proportionally. The operation of the activated equilibrium WCX solid phase extraction column is the conventional operation in the field, and the amount of methanol and water used for activating the equilibrium function, the amount of water and methanol used for washing the small column, the amount of acetonitrile formate used for elution, the speed of the supernatant passing through the solid phase extraction column and the like can be carried out according to the conventional operation rule in the field. The limitation of the amount of the sample or reagent in the examples of the present invention is not intended to limit the scope of the present invention, and it is not intended that the present invention cannot be carried out with a slight change in the amount.
Example 1
In the method for determining the residual quantity of iminoctadine in plant-derived food by solid phase extraction-liquid chromatography-tandem mass spectrometry, in the embodiment, vorkan is used as a to-be-detected product for detection:
(1) Sample processing
Sample preparation: taking a Wo citrus sample, removing non-edible part of peel, and mincing the sample into a uniform and fine sample by a homogenizer for later use;
extraction: weighing 5.00g of homogenized Wookan sample to be detected, placing the sample in a 50mL polypropylene centrifuge tube, adding 10mL0.2Vol% acetic acid water and 10mL dichloromethane, oscillating and extracting for 30min, and centrifuging at 4000r/min for 5min to obtain a supernatant for later use;
purifying: respectively activating and balancing a WCX solid phase extraction column by using 3mL of methanol and 3mL of water, wherein the WCX solid phase extraction column is a carboxyl modified mixed weak cation exchange column with the specification of 60mg and 3cc; taking 3ml of supernatant obtained by extraction, and allowing the supernatant to pass through the solid phase extraction column at a rate of 1 drop/s; adding 1mL of water and 1mL of methanol forest successively to wash the small column; then eluting with 3mL of 10Vol% acetonitrile formate and collecting the eluate into a 15mL polypropylene centrifuge tube; blowing nitrogen to be nearly dry under the condition of 42 ℃ water bath, adding 0.2Vol% formic acid water to a constant volume of 1mL, mixing uniformly by vortex, and filling the mixture into a PP sample injection bottle through a 0.22 mu m hydrophobic PTFE needle type filter for subsequent instrument analysis;
(2) Detection and quantification
Separating the compound in the detection solution by adopting a liquid chromatograph, and carrying out qualitative and quantitative analysis on the separated target compound, namely, the iminoctadine, by adopting a tandem mass spectrometer, wherein:
chromatographic column conditions: using Eclipse Plus C 18 Chromatography column, 2.1 × 100mm,1.8 μm; column temperature: 40 ℃; sample introduction amount: 5 mu L of the solution; a mobile phase A:0.2% (v/v) formic acid water; mobile phaseB: methanol; mobile phase elution procedure: 0-2.0 min, 95% A; 2.0-3.0 min, by 95% A down to 15% A; from 15% to 5% in 3.0-5.8 min; 5.8 to 6.0min, from 5% by volume A to 95% by volume A; 95% is maintained for 6.0-8 min;
mass spectrum conditions:
an ion source: AB SCIEX 4500 Mass Spectrometry, electrospray ion Source, positive ion mode (ESI) + ) (ii) a The scanning mode is as follows: multiple Reaction Monitoring (MRM); air curtain gas (CUR): 20psi; collision gas (CAD): a Medium; ion source voltage (IS): 4500V; ion source Temperature (TEM): 500 ℃; atomizing gas (GS 1): 55psi; auxiliary heating gas (GS 2): 55psi; ion pair parameters for the biguanide octylamine scanned were as follows:
the chromatogram of iminoctadine (0.010 mg/kg) in the Or is shown in FIG. 1, and the peak shape, peak width and abundance of the chromatographic peak are better.
The quantitative detection method comprises the steps of selecting a blank matrix consistent with a sample to be detected, adopting an Or citrus unshielded or undetected iminoctadine as the blank matrix, adding 0, 0.005, 0.010, 0.020, 0.040 and 0.080mg/kg iminoctadine standard substance for testing, treating the blank matrix and the sample to be detected according to the same extraction and purification modes, detecting the processed sample by an instrument, analyzing by liquid chromatography and mass spectrometry, and drawing a standard curve for quantitative analysis. In addition, biguanide octylamine standards were added to the blank matrices at 0.010, 0.020 and 0.050mg/kg, with 6 replicates per concentration set for spiking experiments for recovery and precision analysis.
Example 2
In the method for measuring the residual quantity of iminoctadine in plant-derived food by solid-phase extraction-liquid chromatography-tandem mass spectrometry, in the embodiment, jack fruit is used as a to-be-detected product for detection:
(1) Sample processing
Sample preparation: taking a jack fruit sample, removing a non-edible shell, and mincing the sample into uniform and finely-crushed samples by using a pulp homogenizer for later use;
extraction: weighing 5.00g of homogenized jack fruit sample, placing the jack fruit sample in a 50mL polypropylene centrifuge tube, adding 10mL of 0.2Vol% acetic acid water and 10mL of dichloromethane, oscillating and extracting for 30min, and centrifuging at 4000r/min for 5min to obtain a supernatant for later use;
purifying: respectively activating and balancing a WCX solid phase extraction column by using 3mL of methanol and 3mL of water, wherein the solid phase extraction column is a carboxyl modified mixed weak cation exchange column with the specification of 60mg and 3cc; taking 3ml of supernatant obtained by extraction, and allowing the supernatant to pass through the solid phase extraction column at a rate of 1 drop/s; adding 1mL of water and 1mL of methanol forest successively to wash the small column; then eluting with 3mL of 10Vol% acetonitrile formate and collecting the eluate into a 15mL polypropylene centrifuge tube; blowing nitrogen to be nearly dry under the condition of water bath at 45 ℃, adding 0.2Vol% formic acid water to fix the volume to 1mL, mixing uniformly by vortex, putting the mixture into a PP sample injection bottle through a 0.22 mu m hydrophobic PTFE needle type filter for subsequent instrument analysis;
(2) Detection and quantification
Separating the compound in the detection solution by adopting a liquid chromatograph, and carrying out qualitative and quantitative analysis on the separated target compound, namely, the iminoctadine, by adopting a tandem mass spectrometer, wherein:
chromatographic column conditions: using Eclipse Plus C 18 Chromatography column, 2.1X 100mm,1.8 μm; column temperature: at 40 ℃; sample introduction amount: 5 mu L of the solution; a mobile phase A:0.2% (v/v) formic acid water; mobile phase B: methanol; mobile phase elution procedure: 0-2.0 min, 95% A; 2.0-3.0 min, from 95% to 15% A; from 15% to 5% in 3.0-5.8 min; 5.8-6.0 min, by 5% A to 95%; 95% is maintained for 6.0-8 min;
mass spectrum conditions:
an ion source: AB SCIEX 4500 Mass Spectroscopy, electrospray ionization Source, positive ion mode (ESI) + ) (ii) a The scanning mode comprises the following steps: multiple Reaction Monitoring (MRM); air curtain gas (CUR): 20psi; collision gas (CAD): a Medium; ion source voltage (IS): 4500V; ion source Temperature (TEM): 500 ℃; atomizing gas (GS 1): 55psi; auxiliary heating gas (GS 2): 55psi; ion pair parameters of the biguanide octylamine scanned were as follows:
the quantitative detection method comprises the steps of selecting a blank matrix consistent with a sample to be detected, adopting jackfruit not sprayed with or detected from iminoctadine as the blank matrix, adding 0, 0.005, 0.010, 0.020, 0.040 and 0.080mg/kg iminoctadine standard substance for testing, treating the blank matrix and the sample to be detected according to the same extraction and purification modes, detecting the processed blank matrix and the sample to be detected by an instrument, analyzing the sample by using liquid chromatography and mass spectrometry, and drawing a standard curve for quantitative analysis. In addition, biguanide octylamine standards were added to the blank matrices at 0.010, 0.020 and 0.050mg/kg, with 6 replicates per concentration set for spiking experiments for recovery and precision analysis.
Example 3
In the method for determining the residual amount of iminoctadine in plant-derived food by solid-phase extraction-liquid chromatography-tandem mass spectrometry, lettuce is used as a sample to be detected to perform detection:
(1) Sample processing
Sample preparation: taking a lettuce sample, cleaning, and mincing the lettuce sample into a uniform and fine sample by using a grinder for later use;
extraction: weighing 5.00g of homogenized lettuce sample, placing the lettuce sample in a 50mL polypropylene centrifuge tube, adding 10mL of 0.1Vol% acetic acid water and 10mL of dichloromethane, oscillating and extracting for 20min, and centrifuging at 4000r/min for 10min to obtain supernatant for later use;
purifying: respectively activating and balancing a WCX solid phase extraction column by using 3mL of methanol and 3mL of water, wherein the solid phase extraction column is a carboxyl modified mixed weak cation exchange column with the specification of 60mg and 3cc; taking 3ml of supernatant obtained by extraction, and allowing the supernatant to pass through the solid phase extraction column at a rate of 1 drop/s; adding 1mL of water and 1mL of methanol forest successively to wash the small column; then eluting with 3mL of 10Vol% acetonitrile formate and collecting the eluate into a 15mL polypropylene centrifuge tube; blowing nitrogen to be nearly dry under the condition of water bath at 48 ℃, adding 0.2Vol% formic acid water to fix the volume to 1mL, mixing uniformly by vortex, putting the mixture into a PP sample injection bottle through a 0.22 mu m hydrophobic PTFE needle type filter for subsequent instrument analysis;
(2) Detection
Separating the compound in the detection liquid by adopting a liquid chromatograph, and carrying out qualitative and quantitative analysis on the separated target compound, namely, the iminoctadine, by adopting a tandem mass spectrometer, wherein:
chromatographic column conditions: using Eclipse Plus C 18 Chromatography column, 2.1 × 100mm,1.8 μm; column temperature: 40 ℃; sample introduction amount: 5 mu L of the solution; mobile phase A:0.1% (v/v) formic acid water; and (3) mobile phase B: methanol; mobile phase elution procedure: 0-2.0 min, 95% A; 2.0-3.0 min, from 95% to 15% A; from 15% to 5% in 3.0-5.8 min; 5.8 to 6.0min, from 5% by volume A to 95% by volume A; 95% is maintained for 6.0-8 min;
mass spectrum conditions:
an ion source: electrospray ion source, AB SCIEX 4500 Mass Spectroscopy, positive ion mode (ESI) + ) (ii) a The scanning mode is as follows: multiple Reaction Monitoring (MRM); air curtain gas (CUR): 20psi; collision gas (CAD): medium; ion source voltage (IS): 4500V; ion source Temperature (TEM): 500 ℃; atomizing gas (GS 1): 55psi; auxiliary heating gas (GS 2): 55psi; the ion pair parameters of iminoctadine are as follows:
the quantitative detection method comprises the steps of selecting a blank matrix consistent with a sample to be detected, adopting lettuce which is not sprayed or detected with diguanidine octylamine as the blank matrix, adding 0, 0.005, 0.010, 0.020, 0.040 and 0.080mg/kg diguanidine octylamine standard substance for testing, treating the blank matrix and the sample to be detected according to the same extraction and purification modes, detecting the processed product by an instrument, analyzing the product by liquid chromatography and mass spectrometry, drawing a standard curve and carrying out quantitative analysis. In addition, biguanide octylamine standards were added to the blank matrices at 0.010, 0.020 and 0.050mg/kg, with 6 replicates per concentration set for spiking experiments for recovery and precision analysis.
Example 4
In the method for measuring the residual quantity of iminoctadine in plant-derived food by solid phase extraction-liquid chromatography-tandem mass spectrometry, cucumber is used as a to-be-detected product to perform detection:
(1) Sample processing
Sample preparation: taking a cucumber sample, removing non-edible part of cucumber peel, and mincing the sample into a uniform and fine sample by using a homogenizer for later use;
extraction: weighing 5.00g of homogenized cucumber sample to be detected, placing the cucumber sample in a 50mL polypropylene centrifuge tube, adding 10mL0.3Vol% acetic acid water and 10mL of dichloromethane, oscillating and extracting for 30min, and centrifuging at 4000r/min for 5min to obtain a supernatant for later use;
purifying: respectively activating and balancing a WCX solid phase extraction column by using 3mL of methanol and 3mL of water, wherein the solid phase extraction column is a carboxyl modified mixed weak cation exchange column with the specification of 60mg and 3cc; taking 3ml of supernatant obtained by extraction, and allowing the supernatant to pass through the solid phase extraction column at a rate of 1 drop/s; adding 1mL of water and 1mL of methanol forest successively to wash the small column; then eluting with 3mL of 10Vol% acetonitrile formate and collecting the eluate into a 15mL polypropylene centrifuge tube; blowing nitrogen to be nearly dry under the condition of water bath at 45 ℃, adding 0.2Vol% formic acid water to be constant volume to 1mL, vortex mixing uniformly, putting into a PP sample injection bottle through a 0.22 mu m hydrophobic PTFE needle type filter for subsequent instrument analysis;
(2) Detection and quantification
Separating the compound in the detection liquid by adopting a liquid chromatograph, and carrying out qualitative and quantitative analysis on the separated target compound, namely, the iminoctadine, by adopting a tandem mass spectrometer, wherein:
chromatographic column conditions: using Eclipse Plus C 18 Chromatography column, 2.1X 100mm,1.8 μm; column temperature: 40 ℃; sample introduction amount: 5 mu L of the solution; mobile phase A:0.1% (v/v) formic acid water; mobile phase B: methanol; mobile phase elution procedure: 0-2.0 min, 95% A; 2.0-3.0 min, by 95% A down to 15% A; from 15% to 5% in 3.0-5.8 min; 5.8-6.0 min, by 5% A to 95%; 95% is maintained for 6.0-8 min;
mass spectrum conditions:
an ion source: AB SCIEX 4500 Mass Spectrometry, electrospray ion Source, positive ion mode (ESI) + ) (ii) a The scanning mode is as follows: multiple Reaction Monitoring (MRM); air curtain gas (CUR): 20psi; collision gas (CAD): medium; ion source voltage (IS): 4500V; ion source Temperature (TEM): 500 ℃; atomizing gas (GS 1): 55psi; auxiliary heating gas (GS 2): 55psi; the ion pair parameters of iminoctadine are as follows:
the quantitative detection method comprises the steps of selecting a blank matrix consistent with a sample to be detected, adopting cucumbers not sprayed with or detecting iminoctadine, adding 0, 0.005, 0.010, 0.020, 0.040 and 0.080mg/kg iminoctadine standard substance for testing, treating the blank matrix and the sample to be detected according to the same extraction and purification modes, detecting the processed sample by an instrument, analyzing by liquid chromatography and mass spectrometry, drawing a standard curve and carrying out quantitative analysis. In addition, biguanide octylamine standard substances are added to the blank matrix according to 0.010, 0.020 and 0.050mg/kg, and 6 standard adding tests are set in each concentration for parallel analysis of recovery rate and precision.
Example 5
In the method for determining the residual amount of iminoctadine in plant-derived food by solid-phase extraction-liquid chromatography-tandem mass spectrometry, brown rice is used as a sample to be detected to perform detection:
(1) Sample processing
Sample preparation: taking a brown rice sample, removing husks, and grinding the sample into a uniform and fine sample by using a grinding instrument for later use;
extraction: weighing 1.00g of ground brown rice sample, placing in a 50mL polypropylene centrifuge tube, adding 10mL0.2Vol% acetic acid water and 10mL dichloromethane, oscillating and extracting for 40min, and centrifuging at 8000r/min for 5min to obtain supernatant for later use;
the other steps are basically the same as example 2, except that brown rice is adopted as blank matrix to replace jackfruit correspondingly in quantitative analysis, biguanide octylamine standard substance is added to the blank matrix according to 0.050 and 0.100mg/kg, and labeling tests are carried out in parallel by setting 6 per concentration for recovery rate and precision analysis.
Example 6
In the method for measuring the residual quantity of iminoctadine in plant-derived food by solid phase extraction-liquid chromatography-tandem mass spectrometry, the present embodiment uses almond as a to-be-detected product for detection:
(1) Sample processing
Sample preparation: taking an almond sample, shelling, and grinding the almond sample into a uniform and fine sample by using a grinding instrument for later use;
extraction: weighing 1.00g of ground almond sample, placing the almond sample in a 50mL polypropylene centrifuge tube, adding 10mL of 0.2Vol% acetic acid water and 10mL of dichloromethane, oscillating and extracting for 40min, and then centrifuging for 5min at 8000r/min to obtain supernatant for later use;
the other steps are substantially the same as example 2, except that almond is adopted as a blank substrate for quantitative analysis instead of jack fruit correspondingly, biguanide octylamine standard substances are added into the blank substrate at 0.050 and 0.100mg/kg, and 6 standard addition tests are carried out in parallel at each concentration setting for recovery rate and precision analysis.
Example 7
In the method for determining the residual amount of iminoctadine in plant-derived food by solid phase extraction-liquid chromatography-tandem mass spectrometry, green tea is used as a to-be-detected product to perform detection:
(1) Sample processing
Sample preparation: taking a green tea sample, and grinding the green tea sample into a uniform and fine sample by using a grinding instrument for later use;
extraction: weighing 1.00g of ground green tea sample, placing the green tea sample in a 50mL polypropylene centrifuge tube, adding 10mL0.2Vol% acetic acid water and 10mL dichloromethane, oscillating and extracting for 40min, and centrifuging at 10000r/min for 4min to obtain a supernatant for later use;
purifying: activating and balancing WCX solid phase extraction column with 6mL of methanol and 6mL of water respectively, wherein the solid phase extraction column is a carboxyl modified mixed weak cation exchange column with the specification of 150mg,6cc; taking 3ml of supernatant obtained by extraction, and allowing the supernatant to pass through the solid phase extraction column at a rate of 1 drop/s; adding 2mL of water and 2mL of methanol forest successively to wash the small column; eluting with 4mL of 10Vol% acetonitrile formate, and collecting into a 15mL polypropylene centrifuge tube; blowing nitrogen to be nearly dry under the condition of water bath at 45 ℃, adding 0.2Vol% formic acid water to be constant volume to 1mL, vortex mixing uniformly, putting into a PP sample injection bottle through a 0.22 mu m hydrophobic PTFE needle type filter for subsequent instrument analysis;
the other steps are substantially the same as example 2 except that brown rice was used as the blank matrix for quantitative analysis instead of green tea, and that 6 standards of iminoctadine were added to the blank matrix at 0.050 and 0.100mg/kg, and each concentration was set in parallel to perform the labeling test for recovery and precision analysis.
The results of quantitative analysis, recovery rate and precision analysis of examples 1 to 7 are shown in Table 1.
TABLE 1 Linear correlation, recovery and relative standard deviation of the spiking test
The table shows that the linear correlations of the addition of iminoctadine to Or, jack fruit, lettuce and cucumber are all good, the correlation coefficient (r) is more than or equal to 0.993, and the limit of quantitative detection is 0.010mg/kg; the linear correlations of the concentrations of the added iminoctadine in the brown rice, the almond and the green tea in the range of 0.020-0.320 mg/kg are good, the correlation coefficients (r) are all more than or equal to 0.998, and the limit of quantitative detection is 0.050mg/kg; the standard recovery rate and the relative standard deviation of different concentrations of each sample matrix meet the related requirements of national food physicochemical detection-laboratory quality control standard (GB/T27404-2008) and the quality control and method verification program guide (SANTE 11813/2017) of pesticide residue and analysis in European food and feed.
The above description is intended to describe in detail the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the claims of the present invention, and all equivalent changes and modifications made within the technical spirit of the present invention should fall within the scope of the claims of the present invention.
Claims (8)
1. The method for measuring the residual quantity of the iminoctadine in the plant-derived food by the solid phase extraction-liquid chromatography-tandem mass spectrometry is characterized by comprising the following steps of:
(1) Sample processing
Sample preparation: taking a plant source food sample, removing peel or shell of non-edible part, and grinding the sample into a uniform and fine sample by a homogenizer or a grinder for standby; the plant source food sample comprises fruits, vegetables, grains, nuts and tea leaves;
extraction: weighing a proper amount of well-treated sample, placing the sample in a plastic centrifuge tube, adding a weakly acidic aqueous solution and dichloromethane, oscillating and extracting, and then carrying out centrifugal treatment to obtain a supernatant for later use;
purifying: activating a balance WCX solid phase extraction column, and taking part of supernatant to pass through the solid phase extraction column; adding water and methanol successively to wash the small column; eluting with 10Vol% acetonitrile formate and collecting into a plastic centrifuge tube; blowing nitrogen to be nearly dry, adding 0.2Vol% formic acid water to a constant volume of 1mL, uniformly mixing by vortex, filtering through a 0.22 mu m needle filter, and filling into a plastic sampling bottle for subsequent instrument analysis;
(2) Detection and quantification
Separating the compound in the detection liquid by adopting a liquid chromatograph, and carrying out qualitative and quantitative analysis on the separated target compound, namely, the iminoctadine, by adopting a tandem mass spectrometer, wherein:
chromatographic column conditions: selection C 18 A chromatographic column with a column temperature of 40 ℃; the sample injection amount is 5 mu L; the mobile phase A is 0.2 percent formic acid water; the mobile phase B is methanol; performing gradient elution; said C is 18 The chromatographic column is Eclipse Plus C 18 A chromatographic column with the specification of 2.1 multiplied by 100mm and 1.8 mu m; the procedure for the gradient elution was: 0-2.0 min, 95% A; 2.0-3.0 min, from 95% to 15% A; from 15% to 5% in 3.0-5.8 min; 5.8-6.0 min, by 5% A to 95%; 95% is maintained for 6.0-8 min;
the mass spectrum conditions are as follows: tandem mass spectrometer with electrospray ion source selection in positive ion mode (ESI) + ) Multiple Reaction Monitoring (MRM) scans were performed with the following biguanide octylamine ion pair parameters:
the quantitative method comprises the following steps: selecting a blank matrix consistent with a sample to be detected, adding biguanide octylamine standard solutions with different concentrations, treating the blank matrix and the sample to be detected according to the same extraction and purification mode, detecting the treated solution by an instrument, and drawing a standard curve for quantitative analysis; the quantitative analysis indexes comprise the residual quantity of the iminoctadine and the residual quantity of biguanide salts of the iminoctadine, wherein the biguanide salts comprise iminoctadine and iminoctadine hydrochloride; the residual amount of biguanide salt is obtained by converting the residual amount of iminoctadine.
2. The method for determining the residual quantity of iminoctadine in plant-derived food of claim 1, wherein the method comprises the following steps: weighing a proper amount of ground samples, wherein the proper amount refers to 5g of fruit and vegetable samples and 1g of grain, nut and tea samples.
3. The method for determining the residual quantity of iminoctadine in plant-derived food of claim 1, wherein the method comprises the following steps: the weakly acidic aqueous solution in the extraction step is 0.1-0.3 vol% acetic acid water.
4. The method for determining the residual amount of iminoctadine in plant-derived food according to claim 1, wherein the method comprises the following steps: the WCX solid phase extraction column is a carboxyl modified mixed weak cation exchange column with specification of 60mg,3cc or 150mg,6cc.
5. The method for determining the residual quantity of iminoctadine in plant-derived food of claim 1, wherein the method comprises the following steps: the plastic centrifuge tube and the plastic sample injection bottle are both made of polypropylene materials.
6. The method for determining the residual quantity of iminoctadine in plant-derived food of claim 1, wherein the method comprises the following steps: the needle type filter is a hydrophobic Polytetrafluoroethylene (PTFE) needle type filter.
7. The method for determining the residual quantity of iminoctadine in plant-derived food of claim 1, wherein the method comprises the following steps: in the extraction step, the shaking extraction time is 20-40min, and the centrifugation is carried out for 4-10min at 3000-10000 r/min.
8. The method for determining the residual quantity of iminoctadine in plant-derived food of claim 1, wherein the method comprises the following steps: the tandem mass spectrometer is an AB SCIEX 4500 mass spectrometer, and mass spectrum conditions of the tandem mass spectrometer further comprise air curtain gas (CUR): 20psi; collision gas (CAD): a Medium; ion source voltage (IS): 4500V; ion source Temperature (TEM): 500 ℃; atomizing gas (GS 1): 55psi; auxiliary heating gas (GS 2): 55psi.
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