CN108645926B

CN108645926B - A kind of detection method of pesticide residues in bee pollen

Info

Publication number: CN108645926B
Application number: CN201810373005.7A
Authority: CN
Inventors: 赵柳微; 梁馨文; 吴黎明; 薛晓锋; 万正瑞
Original assignee: Institute of Apiculture Research
Current assignee: Institute of Apiculture Research
Priority date: 2018-04-24
Filing date: 2018-04-24
Publication date: 2021-06-18
Anticipated expiration: 2038-04-24
Also published as: CN108645926A

Abstract

The invention relates to a method for detecting pesticide residues in bee pollen. The main improvement is that the following methods are used to pretreat the bee pollen to be tested: 1) acetonitrile is used to fully extract the pesticide residues in the bee pollen to be tested, and the The acetonitrile solution of pesticide residues; 2) placing the acetonitrile solution dissolved with pesticide residues at -75～-80°C for 3～8min to remove lipids therein; 3) removing lipids through a purification column; The acetonitrile solution was purified to obtain the sample to be tested. By optimizing the extraction method of pesticide residues in bee pollen, the present invention can fully extract the pesticide residues in bee pollen for extraction, which lays a foundation for subsequent accurate detection. The present invention further optimizes the detection method and can realize accurate detection.

Description

Method for detecting pesticide residue in bee pollen

Technical Field

The invention relates to the technical field of liquid chromatography detection, in particular to detection of common pesticide residues in bee pollen.

Background

At present, the analysis methods of various pesticide residues in bee pollen are few, and most of the reported methods are used for detecting the neonicotinoid pesticide residues. However, since the beehives are exposed to the environment of the crops with much residue, other pesticides such as acaricides and bactericides are also used in the hive to reduce the damage of mites and microsporidia. The data indicate that pesticide, fungicide, and herbicide residues are present at various concentration levels within the hive. Therefore, there is a need to develop a simple and sensitive method for analyzing pesticide residues in common use.

Because the lipid (10-20%) and protein (30-40%) in bee pollen can interfere the determination of residual analysis, if the pesticide residue in bee pollen is to be determined accurately, a pretreatment method capable of removing the internal interferents (lipid and protein) in bee pollen with high efficiency is found as a premise, and the determination of proper chromatographic detection conditions is the key point of successful detection.

Disclosure of Invention

The invention aims to provide a method for detecting pesticide residues in bee pollen, which is characterized in that the bee pollen to be detected is pretreated as follows before being detected:

1) fully extracting the pesticide residue in the bee pollen to be detected by using acetonitrile to obtain acetonitrile solution in which the pesticide residue is dissolved;

2) placing the acetonitrile solution dissolved with the pesticide residues at-75 to-80 ℃ for 3 to 8min to remove lipid substances in the acetonitrile solution;

3) purifying the acetonitrile solution without the lipid substances through a purification column to obtain a sample to be detected.

The technical scheme of extracting the pesticide residues in the bee pollen by adopting acetonitrile also exists in the prior art, but only one or two kinds of pesticide residues can be usually extracted, the 9 kinds of common pesticide residues in the bee pollen, namely carbendazim, thiamethoxam, clothianidin, imidacloprid, acetamiprid, triadimenol, azoxystrobin, coumaphos and chlorpyrifos, can be simultaneously and fully extracted by adopting the pretreatment method, the loss of the pesticide residues is small, the interference impurities of lipids and proteins in the extracting solution are few, and a foundation is laid for accurately detecting the 9 kinds of common pesticide residues.

Preferably, the step 1) is specifically: dissolving the bee pollen in water to obtain a bee pollen aqueous solution, then adding acetonitrile into the bee pollen aqueous solution, and fully extracting to obtain an acetonitrile solution dissolved with pesticide residues;

further preferably, the method comprises the following steps:

A. mixing the bee pollen to be detected with water according to the mass-volume ratio of 1: 2-3, and then fully stirring to fully dissolve the bee pollen in the water to obtain a bee pollen water solution;

B. adding acetonitrile into an aqueous solution of bee pollen according to the mass-to-volume ratio of the bee pollen to be detected to acetonitrile of 1: 2.5-10 to obtain a mixed solution, carrying out ultrasonic treatment on the mixed solution, then carrying out layering treatment, and taking an acetonitrile layer to obtain an acetonitrile solution dissolved with pesticide residues.

Preferably, the freezing condition in the step 2) is freezing for 3-5min at-80 ℃.

Preferably, the scavenger is PSA, C18, GCB or Cleanert NANO CARB (m-PFC).

Cleanert NANO CARB is more preferred. Through the purification treatment of the purification column, impurities in the extracting solution can be effectively removed.

Preferably, the mass volume ratio of the bee pollen to be detected to the acetonitrile is 1: 6-8. The sufficient extraction can be realized under the condition of the dosage ratio and the waste of acetonitrile can not be caused.

Preferably, the ultrasonic treatment is carried out for 15-25 min under the ultrasonic condition with the power of 100W. The ultrasonic treatment under the conditions can fully extract the pesticide residue in the pollen to be detected, and the extraction efficiency of the solvent is obviously improved.

Preferably, the salt is added to the mixed solution after the completion of the ultrasonic treatment to separate the layers.

Further preferably, the salt is sodium chloride or magnesium sulfate. The water in the liquid to be detected can be removed or the water phase and the acetonitrile organic phase can be completely separated by adding salts for layering, so that the target pesticide compound is completely leached into the acetonitrile organic phase, and the recovery rate of the pesticide is obviously improved.

It is further preferred that during operation the sample is purified 2 times through the purification column.

Preferably, the pretreatment of the sample comprises the following steps:

1) mixing the bee pollen to be detected with water according to the mass-volume ratio of 1: 2-3, and then fully stirring to fully dissolve the bee pollen in the water to obtain a bee pollen water solution; adding acetonitrile into an aqueous solution of bee pollen according to the mass-to-volume ratio of the bee pollen to be detected to acetonitrile of 1: 6-8 to obtain a mixed solution, treating the mixed solution for 15-25 min under the ultrasonic condition with the power of 100W, adding sodium chloride or magnesium sulfate to layer the mixed solution, and taking an acetonitrile layer dissolved with pesticide residues;

2) placing the acetonitrile layer dissolved with pesticide residues at-80 deg.C for 4min to remove lipid substances therein;

3) and purifying the acetonitrile solution without the lipid substances for 2 times by using a purifying column Cleanert NANO CARB to obtain a sample to be detected.

Preferably, the steps of detecting the bee pollen to be detected by the liquid chromatography are as follows:

1) pretreating the bee pollen to be detected to obtain a sample to be detected;

2) detecting the sample to be detected by liquid chromatography, selecting aqueous solution of formic acid with volume fraction of 0.1% as a mobile phase A and acetonitrile solution of formic acid with volume fraction of 0.1% as a mobile phase B, and performing gradient elution on the bee pollen to be detected;

in the process of gradient elution, the volume fraction of the mobile phase B is increased from 30% to 50% in 0-3 min; 3-5min, the volume fraction of the mobile phase B is increased from 50% to 75%; 5-6min, the volume fraction of the mobile phase B is increased from 75% to 90%; the volume fraction of mobile phase B is maintained at 90% for 6-11 min.

Under the detection conditions, the 9 common pesticides extracted can be ideally separated and detected, namely carbendazim, thiamethoxam, clothianidin, imidacloprid, acetamiprid, triadimenol, azoxystrobin, coumaphos and chlorpyrifos.

Preferably, the flow rate of the mobile phase is 0.3-0.4 mL/min;

preferably, the detector is a triple quadrupole mass spectrometer detector.

Further preferably, the conditions of mass spectrometry detection are that the gas temperature is 340 ℃, the gas flow rate is 12L/min, the atomizer voltage is 40psi, the capillary voltage is 4000V, the current of the atomizing chamber is 1.1 muA, and the electron multiplier voltage is 400, and the sensitivity of instrument detection and the response value of target monitoring ions can be effectively improved through mass spectrometry detection.

The method of the invention has the following beneficial effects:

1) the method for extracting the pesticide residues in the bee pollen is optimized, so that the 9 common pesticide residues in the bee pollen can be fully extracted, and a foundation is laid for the subsequent accurate detection.

2) The invention can ideally separate the common pesticide residue in the bee pollen by optimizing the liquid chromatogram detection condition and the mass spectrum detection condition, thereby realizing accurate detection.

Drawings

FIG. 1 Effect of extraction solvent volume on recovery of 9 pesticides;

FIG. 2 effect of freezing time on recovery of 9 pesticides;

FIG. 3 effect of scavenger type on recovery of 9 pesticides;

FIG. 4 effect of clean NANO CARB filtration number on recovery of 9 pesticides.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1

The embodiment relates to detection of pesticide residues in bee pollen, which comprises the following steps:

A. pretreatment of samples

1) Weighing 2g of pollen to be detected, placing the pollen into a 50mL centrifuge tube, adding 5mL of pure water into the centrifuge tube, and performing vortex for 1min to obtain an aqueous solution of the bee pollen;

2) adding 15mL of acetonitrile into the bee pollen aqueous solution, extracting for 20min under the ultrasonic condition with the power of 100W, adding a salt bag (sodium chloride or magnesium sulfate) into a 50mL centrifuge tube, carrying out vortex oscillation for 1min, and taking an acetonitrile layer;

3) and (3) placing the acetonitrile layer at-80 ℃ for 4min, centrifuging at 3800rpm and 4 ℃ for 5min, and taking supernatant.

4) And (3) passing the supernatant through a Cleanert NANO CARB column, and pushing for 2 times in total to finish the pretreatment of the sample.

B. Chromatography analysis

And performing LC-QQQ-MS/MS analysis on the pretreated sample.

The conditions for the liquid chromatography analysis were: the sample injection amount is 0.5 mu L;

flow rate of mobile phase: 0.350 mL/min;

mobile phase: A. 0.1 volume percent formic acid water B, and 0.1 volume percent formic acid acetonitrile;

the procedure for gradient elution was: the volume fraction of the mobile phase B is increased from 30% to 50% in 0-3 min; 3-5min, the volume fraction of the mobile phase B is increased from 50% to 75%; 5-6min, the volume fraction of the mobile phase B is increased from 75% to 90%; the volume fraction of mobile phase B is maintained at 90% for 6-11 min.

The mass spectrum conditions are as follows: the gas temperature is 340 ℃, the gas flow rate is 12L/min, the pressure of the sprayer is 40psi, the capillary voltage is 4000V, the current of the spraying chamber is 1.1 muA, and the voltage of the electron multiplier tube is 400. The mass spectrometric acquisition parameters for each compound are shown in table 1.

C. Linear equation, detection limit and quantification limit

Preparing standard substance of various pesticide components into 100mg L^-1The stock solution of (1). Diluting acetonitrile/matrix solution, and preparing into concentration of 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5mg L^-1And (3) measuring 7 mixed standard solutions with different levels by using HPLC-QQQ, and establishing a standard curve by using the mass concentration as a horizontal coordinate and the peak area as a vertical coordinate. The peak response value of the recovered sample was 3 times the noise addition concentration calculation method detection Limit (LOD), and the noise addition concentration was 10 times the noise addition concentration calculation method quantification Limit (LOQ).

The results are shown in Table 2 at 0.01-5mg L^-1The concentration range is good in linearity, the correlation coefficient range is 0.9922-0.9996, and the mechanism effect range is 0-29.29%. The detection limit and the quantitative limit of the carbendazim are respectively 0.60 and 2.03 mu g/kg, the detection limit and the quantitative limit of the thiamethoxam, the clothianidin, the imidacloprid, the acetamiprid, the triadimenol and the azoxystrobin are respectively 0.75 and 2.63 mu g/kg, and the detection limit and the quantitative limit of the coumaphos and the chlorpyrifos are respectively 45 and 150 mu g/kg.

TABLE 19 Retention time, monitoring ion and Collision Voltage of Compounds

TABLE 29 detection Lines (LOD), limit of quantitation (LOQ), Linear equation, correlation coefficient (R2), and Matrix Effect (ME) for pesticides

Note: matrix effect (%) - (response value of pesticide of the same content added in sample matrix)/(response value of pesticide in pure solvent) × 100

Examples of the experiments

The experimental example verifies the accuracy of the method, and adds 150, 375 and 750 mug/kg 3 mixed standard solutions with different levels into a blank rape pollen sample to carry out a recovery rate experiment, wherein each addition concentration is repeated in parallel for 5 times, and the recovery rate and the Relative Standard Deviation (RSD) are calculated. The results are shown in Table 3, the average spiking recovery rate of 9 pesticides in rape pollen is between 82.5% and 112.9%, the Relative Standard Deviation (RSD) is between 1.6% and 10.8%, and the method has better recovery rate and reproducibility.

Table 39 sample recovery (n ═ 5) and relative deviation (RSD) for the pesticides

Comparative example 1

This comparative example relates to the adjustment of the volume of the extractant, which differs from example 1 only in that, in step 1) of the sample pre-treatment, the volume of the extraction solvent acetonitrile is optimized: 5mL, 10mL, and 20mL, and compared to 15mL in example 1, the results are shown in FIG. 1. As can be seen from FIG. 1, when the volume of the extractant is 15mL, the content of the analyte obtained by extraction is the highest.

Comparative example 2

The comparison example relates to the adjustment of the freezing time in the extraction process, and compared with the example 1, the difference is that in the step 3) of sample pretreatment, the freezing time is respectively adjusted to 0min and 8min, and compared with the freezing time of 4min in the phase of the example 1, the result is shown in figure 2, and as can be seen from figure 2, when the freezing time is 4min, the content of the extracted object to be measured is highest.

Comparative example 3

The comparison example relates to the adjustment of a multi-wall carbon nano tube filtration type purifying column in the extraction process, and compared with the example 1, the difference is that in the step 4) of pretreatment, the types of purifying agents are respectively selected as follows: the results of comparing PSA, C18, GCB and the clean column Cleanert NANO CARB (m-PFC) of example 1 are shown in FIG. 3, and it can be seen from FIG. 3 that the clean NANO CARB (m-PFC) clean column has the best extraction efficiency.

Comparative example 4

This comparative example relates to the adjustment of the number of times the supernatant passed through the Cleanert NANO CARB solid phase extraction column compared to example 1, except that the supernatant was passed through the Cleanert NANO CARB solid phase extraction column 1 and 3 times, respectively, and compared to the 2-pass procedure of example 1, the results of which are shown in FIG. 4. As can be seen from FIG. 4, the content of the analyte obtained by extraction was the highest when the column was passed 2 times.

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. a detection method for pesticide residues in bee pollen, is characterized in that, comprises the steps:

1) fully extract the pesticide residues in the bee pollen to be tested with acetonitrile to obtain the acetonitrile solution dissolved with the pesticide residues, and the specific operations are as follows;

A. Mix the bee pollen to be tested with water in a mass-to-volume ratio of 1:2 to 3 and then fully stir, so that the bee pollen is fully dissolved in the water to obtain an aqueous solution of bee pollen;

B, according to the mass volume ratio of the bee pollen to be measured and acetonitrile 1:6～8, add acetonitrile to the aqueous solution of the bee pollen to obtain a mixed solution, and the mixed solution is treated under the ultrasonic condition of 100W for 15 ~25min,

After the ultrasonic treatment is completed, add magnesium chloride or magnesium sulfate to the mixed solution to make it layered;

Take the acetonitrile layer to obtain the acetonitrile solution dissolved with pesticide residues;

2) placing the acetonitrile solution dissolved with pesticide residues at -80°C for 3 to 5 minutes to remove lipids therein;

3) Purify the acetonitrile solution for removing lipids through a purification column to obtain a sample to be tested;

4) Detect the sample to be tested by liquid chromatography, select an aqueous solution of formic acid with a volume fraction of 0.1% as mobile phase A, and an acetonitrile solution of formic acid with a volume fraction of 0.1% as mobile phase B. gradient elution;

During gradient elution, the volume fraction of mobile phase B increased from 30% to 50% in 0-3min; the volume fraction of mobile phase B increased from 50% to 75% in 3-5min; 5-6min, mobile phase B The volume fraction of mobile phase B increased from 75% to 90%; 6-11min, the volume fraction of mobile phase B remained at 90%;

5) Detected by mass spectrometry, the detector is a triple quadrupole mass spectrometer detector, the gas temperature is 340°C, the gas flow rate is 12L/min, the spray gas pressure: 40psi, the capillary voltage: 4000V, the spray chamber current: 1.1μA, electron multiplier tube voltage: 400;

The pesticide residues are carbendazim, thiamethoxam, clothianidin, imidacloprid, acetamiprid, triadimefon, azoxystrobin, fenamiphos and chlorpyrifos; the purification column is C18, GCB or Cleanert NANO CARB.

2. detection method according to claim 1 is characterized in that, described purification column is Cleanert NANO CARB.

3. detection method according to claim 1, is characterized in that, before the bee pollen to be measured is detected, it is carried out following pretreatment:

1) Mix the bee pollen to be tested and water in a mass-volume ratio of 1:2 to 3 and fully stir to fully dissolve the bee-pollen in the water to obtain an aqueous solution of bee pollen; according to the mass-to-volume ratio of the bee pollen to be tested and acetonitrile 1:6 ～8, adding acetonitrile to the aqueous solution of the bee pollen to obtain a mixed solution, treating the mixed solution for 15 to 25 min under ultrasonic conditions with a power of 100W, adding sodium chloride or magnesium sulfate to make the mixed solution layered, Take the acetonitrile layer with pesticide residues dissolved;

2) placing the acetonitrile layer in which the pesticide residues are dissolved for 4 min at -80°C to remove lipids therein;

3) Purify the acetonitrile solution for removing lipids twice with a purification column Cleanert NANO CARB to obtain the sample to be tested.

4. The detection method according to claim 1, wherein the flow rate of the mobile phase is 0.3-0.4 mL/min.