CN113671084B - Method for detecting neonicotinoid insecticide and metabolite thereof in cerebrospinal fluid - Google Patents

Abstract

The invention provides a method for detecting neonicotinoid insecticides and metabolites thereof in cerebrospinal fluid, which comprises the following steps: adding an internal standard substance marked by a stable isotope into cerebrospinal fluid, and mixing with a formic acid aqueous solution to obtain a mixed solution; passing the mixed solution through a solid phase extraction small column, then eluting by using methanol, and redissolving the eluent by using methanol aqueous solution; and (3) carrying out high performance liquid triple quadrupole tandem mass spectrometry detection on the redissolved solution. The method for detecting the neonicotinoid insecticides and the metabolites thereof in the cerebrospinal fluid adopts a high-efficiency and good-repeatability solid-phase extraction method combined with a high-efficiency liquid chromatography triple quadrupole tandem mass spectrometry technology to determine the neonicotinoid insecticides and the metabolites thereof in the cerebrospinal fluid, and utilizes an isotope marker method to realize qualitative and quantitative determination of the neonicotinoid insecticides and the metabolites thereof in a sample, and the detection method has high sensitivity and reliable data.

Description

A method for the detection of neonicotinoid pesticides and their metabolites in cerebrospinal fluid

technical field

The invention relates to the field of pesticide detection, in particular to a method for detecting neonicotinoid pesticides and their metabolites in cerebrospinal fluid.

Background technique

Neonicotinoids are a new type of broad-spectrum insecticides with high efficiency, safety, good selection and low toxicity to mammals. The target of these insecticides is nicotinic postsynaptic membrane acetylcholine receptors. Mainly acts on the central nervous system of insects. Since its development in the 1990s, neonicotinoid insecticides have been widely used in 140 countries around the world and currently account for more than 25% of the global pesticide market. Based on its superior insecticidal properties and selective toxicity, this type of insecticide is not only widely used in agricultural production, but also has a large number of applications in household life, such as lawn, home garden maintenance and management, livestock and pet pest control, etc. The global application of neonicotinoid insecticides has resulted in their residues in a variety of environmental samples, such as soil, indoor dust, water environment and food, etc. There are also neonicotinoid insecticides in human urine and blood. Metabolite residues have been reported with detection rates as high as 63-100%. Epidemiological reports have pointed out that long-term human exposure to neonicotinoid pesticides may induce diseases such as congenital heart disease, anencephaly and autism.

At present, the number of detections of neonicotinoids and their metabolites disclosed in the prior art is limited (6-8), the operations are cumbersome, and the quantitative accuracy needs to be further improved. Based on the target receptor properties of neonicotinoid insecticides, these substances are likely to enter the brain and affect the nervous system. Animal experiments have verified that exposure to high concentrations of acetamiprid and clothianidin can cause them to remain in the brain tissue. . In order to accurately characterize and quantify the residues of neonicotinoid insecticides in human cerebrospinal fluid, a simple and convenient detection method of neonicotinoid insecticides in cerebrospinal fluid is urgently needed.

SUMMARY OF THE INVENTION

In view of this, the present invention proposes a method for detecting neonicotinoid insecticides and their metabolites in cerebrospinal fluid to solve or partially solve the technical problems existing in the prior art.

In a first aspect, the present invention provides a method for detecting neonicotinoid pesticides and their metabolites in cerebrospinal fluid, comprising the following steps:

Adding stable isotope-labeled internal standard to the cerebrospinal fluid and then mixing with formic acid aqueous solution to obtain a mixed solution;

The mixture was passed through a solid phase extraction cartridge, then eluted with methanol, and the eluate was reconstituted with methanol aqueous solution;

The reconstituted solution was detected by high performance liquid phase triple quadrupole tandem mass spectrometry.

Preferably, in the method for detecting neonicotinoid insecticides and their metabolites in the cerebrospinal fluid, the high-performance liquid phase triple quadrupole tandem mass spectrometry detection conditions are: Waters ACQUITY BEH C18 is selected as the high-performance liquid chromatography column, The length of the high-performance liquid chromatography column is 100 mm, the inner diameter is 2.1 mm, and the diameter of the filler particles is 1.7 μm; the data is collected in a positive and negative ion mode, the tandem mass spectrometry conditions are electrospray ionization, and the detection conditions in the positive ion mode are: collision gas 10 psi, Curtain gas 35psi, ion spray voltage 4000V, ion source temperature 650℃, ion source gas 1 and 2 are both 70psi; negative ion mode detection conditions are: collision gas 8psi, curtain gas 30psi, ion spray voltage -4500V, ion source temperature 600℃ , ion source gases 1 and 2 are 70 psi and 50 psi, respectively.

Preferably, the method for detecting neonicotinoid insecticides and their metabolites in the cerebrospinal fluid adopts a gradient elution procedure for elution, and the gradient elution procedure is specifically:

Time 0min: use the mixed solution composed of acetonitrile and formic acid aqueous solution with a volume ratio of 5:95 as the mobile phase, and the flow rate is 0.35mL/min;

Time 0.6min: use a mixture of acetonitrile and formic acid aqueous solution with a volume ratio of 5:95 as the mobile phase, and the flow rate is 0.35mL/min;

Time 4min: use a mixture of acetonitrile and formic acid aqueous solution with a volume ratio of 99:1 as the mobile phase, and the flow rate is 0.35mL/min;

Time 5min: use a mixture of acetonitrile and formic acid aqueous solution with a volume ratio of 99:1 as the mobile phase, and the flow rate is 0.35mL/min;

Time 5.5min: use a mixture of acetonitrile and formic acid aqueous solution with a volume ratio of 5:95 as the mobile phase, and the flow rate is 0.35mL/min;

Time 6.5min: use a mixture of acetonitrile and formic acid aqueous solution with a volume ratio of 5:95 as the mobile phase, and the flow rate is 0.35mL/min.

Preferably, in the method for detecting neonicotinoid pesticides and their metabolites in cerebrospinal fluid, the volume concentration of the formic acid aqueous solution is 0.003-0.008%.

Preferably, the method for detecting neonicotinoid insecticides and their metabolites in the cerebrospinal fluid, the neonicotinoid insecticides and their metabolites include acetamiprid, desmethylacetamiprid, imidacloprid , 5-hydroxy imidacloprid, alkene imidacloprid, thiamethoxam, N-desmethylthiamethoxam, thiacloprid, thiacloprid, nitenpyram, clothianidin, flonicamid, chlorothiline, fluorine Acetamiprid and 6-chloronicotinic acid.

Preferably, the method for detecting neonicotinoid pesticides and their metabolites in cerebrospinal fluid, acetamiprid, desmethylacetamiprid, imidacloprid, 5-hydroxyimidacloprid, thiamethoxam, N-nor Data were collected in positive ion mode for thiamethoxam, thiacloprid, thiacloprid, nitenpyram, clothianidin, flonicamid, and chlorothiline; Niacin was collected in negative ion mode.

Preferably, in the method for detecting neonicotinoid insecticides and their metabolites in cerebrospinal fluid, a stable isotope-labeled internal standard is added to the cerebrospinal fluid and then mixed with an aqueous formic acid solution to obtain a mixed solution. 30-45 ng of stable isotope-labeled internal standard is added to 300 μL of cerebrospinal fluid, and then mixed with 1-2 ml of formic acid aqueous solution with a volume concentration of 1-3% to obtain a mixed solution.

Preferably, in the method for detecting neonicotinoid pesticides and their metabolites in cerebrospinal fluid, the stable isotope-labeled internal standard comprises ¹³ C ₃ - ¹⁵ N ₂ -nitenpyram, ¹³ C ₄ - ¹⁵ N-Thiamethoxam, D ₄ -Imidacloprid, ¹³ C ₆ -Acetamiprid, ¹³ C ₆ -Thiacloprid, ¹³ C ₄ - ¹⁵ N- clothianidin, ¹⁸ O- ¹⁵ N-Floracetamid , D ₄ -N-desmethylthiamethoxam, ¹³ C ₆ -thiacloprid, D ₄ -chlorothiline, ¹³ C ₂ - ¹⁵ N-desmethyl acetamiprid, ¹³ C ₆ -6-chloro Niacin, a mixture of D ₃ - ¹³ C ₂ - ¹⁵ N ₂ -flufenapyr, ¹³ C- ¹⁵ N ₂ -alkene imidacloprid, ¹³ C- ¹⁵ N ₂ -5-hydroxyimidacloprid.

Preferably, in the method for detecting neonicotinoid insecticides and their metabolites in cerebrospinal fluid, the mixed solution is passed through a solid-phase extraction cartridge, then eluted with methanol, and then the eluate is reconstituted with an aqueous methanol solution. The solution is specifically:

After mixing methanol and ammonium hydroxide, an activation solution is obtained, and then the solid phase extraction cartridge is activated by using the activation solution;

Rinse the activated solid phase extraction cartridge with water, and then pass the mixture through the solid phase extraction cartridge;

After mixing methanol, formic acid and water, the solid phase extraction cartridge was washed, dried under negative pressure, eluted with methanol, the eluate was blown dry with nitrogen, and reconstituted with methanol aqueous solution.

Preferably, in the method for detecting neonicotinoid pesticides and their metabolites in cerebrospinal fluid, the volume ratio of methanol to ammonium hydroxide is (15-20):1; the volume ratio of methanol, formic acid and water is (4.5 to 5.5): (0.5 to 1.5): (42 to 46).

The method for detecting neonicotinoid pesticides and their metabolites in cerebrospinal fluid of the present invention has the following beneficial effects relative to the prior art:

(1) The method for detecting neonicotinoid insecticides and their metabolites in cerebrospinal fluid of the present invention adopts a high-efficiency and repeatable solid-phase extraction method combined with high-performance liquid chromatography triple quadrupole tandem mass spectrometry technology to measure neonicotinoids in cerebrospinal fluid. Nicotinoid pesticides and their metabolites, using the isotope label method, to achieve qualitative and quantitative determination of neonicotinoid pesticides and their metabolites in samples, the detection method has high sensitivity and reliable data;

(2) The method for detecting neonicotinoid insecticides and their metabolites in cerebrospinal fluid of the present invention can control the recovery rate of 15 kinds of neonicotinoids and their metabolites in cerebrospinal fluid within the range of 80-120%, The quality control matrix is human cerebrospinal fluid, which proves that this method has high accuracy and reliable data for the detection of 15 neonicotinoids and their metabolites in cerebrospinal fluid.

Description of drawings

In order to illustrate the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that are required to be used in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention, and for those of ordinary skill in the art, other drawings can also be obtained from these drawings without creative effort.

Fig. 1 is the chromatogram of 12 kinds of neonicotinoid compounds that adopt positive ion mode to collect data in the embodiment of the present invention 1;

2 is a chromatogram of three neonicotinoid compounds for which data was collected in a negative ion mode in Example 1 of the present invention.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

The embodiments of the present application provide a method for detecting neonicotinoid insecticides and their metabolites in cerebrospinal fluid, comprising the following steps:

S1, adding a stable isotope-labeled internal standard to the cerebrospinal fluid and then mixing it with an aqueous formic acid solution to obtain a mixed solution;

S2, passing the mixed solution through the solid phase extraction cartridge, then using methanol to elute, and then redissolving the eluent using methanol aqueous solution;

S3, performing high-performance liquid phase triple quadrupole tandem mass spectrometry detection on the reconstituted solution.

In some embodiments, the detection method of neonicotinoid pesticides and their metabolites in cerebrospinal fluid, the detection conditions of high performance liquid phase triple quadrupole tandem mass spectrometry are: select Waters ACQUITY BEH C18 as high performance liquid chromatography column, so The length of the high performance liquid chromatography column is 100 mm, the inner diameter is 2.1 mm, and the diameter of the filler particles is 1.7 μm; the data was collected in positive and negative ion mode, the tandem mass spectrometry conditions were electrospray ionization, and the detection conditions in positive ion mode were: collision gas 10 psi, gas curtain Gas 35psi, ion spray voltage 4000V, ion source temperature 650℃, ion source gas 1 and 2 are both 70psi; negative ion mode detection conditions are: collision gas 8psi, curtain gas 30psi, ion spray voltage -4500V, ion source temperature 600℃, Ion source gases 1 and 2 were 70 psi and 50 psi, respectively.

In some embodiments, a gradient elution program is used for elution, and the gradient elution program is specifically:

Time 0min: use the mixed solution composed of acetonitrile and formic acid aqueous solution with a volume ratio of 5:95 as the mobile phase, and the flow rate is 0.35mL/min;

Time 0.6min: use a mixture of acetonitrile and formic acid aqueous solution with a volume ratio of 5:95 as the mobile phase, and the flow rate is 0.35mL/min;

Time 4min: use a mixture of acetonitrile and formic acid aqueous solution with a volume ratio of 99:1 as the mobile phase, and the flow rate is 0.35mL/min;

Time 5min: use a mixture of acetonitrile and formic acid aqueous solution with a volume ratio of 99:1 as the mobile phase, and the flow rate is 0.35mL/min;

Time 5.5min: use a mixture of acetonitrile and formic acid aqueous solution with a volume ratio of 5:95 as the mobile phase, and the flow rate is 0.35mL/min;

Time 6.5min: use a mixture of acetonitrile and formic acid aqueous solution with a volume ratio of 5:95 as the mobile phase, and the flow rate is 0.35mL/min

In some embodiments, the volume concentration of the aqueous formic acid used in the mobile phase is 0.003-0.008%.

In some embodiments, the neonicotinoid insecticides and their metabolites include acetamiprid, desmethylacetamiprid, imidacloprid, 5-hydroxy imidacloprid, alkene imidacloprid, thiamethoxam, N-desmethylthiazinon oxazine, thiacloprid, thiacloprid, nitenpyram, clothianidin, flonicamid, chlorothiline, sflufenamid, and 6-chloronicotinic acid.

In some embodiments, acetamiprid, desmethylacetamiprid, imidacloprid, 5-hydroxyimidacloprid, thiamethoxam, N-desmethylthiamethoxam, thiacloprid, thiacloprid, nitenpyram , clothianidin, flonicamid, and chlorothiline were collected in positive ion mode.

In some embodiments, the alkene imidacloprid, sulfenalazine, and 6-chloronicotinic acid are used to collect data in negative ion mode.

In some embodiments, the stable isotope-labeled internal standard ^is13C3-15N2 _- ^nitenpyram , _{13C4-15N -} ^thiamethoxam , D4 _- imidacloprid, ^13C6 _- ^acetamiprid , ¹³ C ₆ -thiacloprid, ¹³ C ₄ - ¹⁵ N-thiacloprid, ¹⁸ O- ¹⁵ N-fluacetamid, D ₄ -N-desmethylthiamethoxam, ¹³ C ₆ -thiacloprid amide, D ₄ -chlorothiline, ¹³ C _2-15 N-desmethylacetamiprid, ¹³ C _6-6 -chloronicotinic acid, D _3- ¹³ C _2- ¹⁵ N ² _- flufenamid, Mixtures of ^13C - ^15N2 _- alkene imidacloprid, ^13C - ^15N2-5 _- hydroxyimidacloprid. That is, the stable isotope-labeled internal standard is composed of the above-mentioned 15 substances.

In some embodiments, adding a stable isotope-labeled internal standard to the cerebrospinal fluid and then mixing it with an aqueous formic acid solution to obtain a mixed solution is specifically: adding 30-45 ng of 15 stable isotope-labeled internal standards to 250-300 μL of the cerebrospinal fluid It is then mixed with 1-2 ml of an aqueous formic acid solution with a volume concentration of 1-3% to obtain a mixed solution.

It should be noted that, in the actual detection process, the added amount of each specific substance in the internal standard was 2.5 ng.

In some embodiments, the mixture is passed through a solid phase extraction cartridge, then eluted with methanol, and the eluent is reconstituted with an aqueous methanol solution, specifically:

After mixing methanol and ammonium hydroxide, an activation solution is obtained, and then the solid phase extraction cartridge is activated by using the activation solution;

Rinse the activated solid phase extraction cartridge with water, and then pass the mixture through the solid phase extraction cartridge;

After mixing methanol, formic acid and water, the solid phase extraction cartridge was rinsed, dried under negative pressure, eluted with methanol, the eluate was blown dry with nitrogen, and reconstituted with methanol aqueous solution.

In some embodiments, the volume ratio of methanol to ammonium hydroxide is (15-20): 1; wherein, the volume concentration of the ammonium hydroxide used is 28-30%; the volume ratio of methanol, formic acid, and water is (4.5 ~5.5):(0.5~1.5):(42~46).

The method for detecting neonicotinoid insecticides and their metabolites in cerebrospinal fluid of the present invention adopts the solid phase extraction method with high efficiency and good repeatability combined with high performance liquid chromatography triple quadrupole tandem mass spectrometry technology to determine the neonicotinoid in cerebrospinal fluid. Insecticides and their metabolites, by using the isotope label method, the qualitative and quantitative determination of neonicotinoid insecticides and their metabolites in the sample is realized, the detection method has high sensitivity and reliable data; the detection method of the present invention can The pretreatment process of the invention is quick and easy, batch processing can be realized, and the work efficiency is high; the detection method of the invention can simultaneously measure 9 kinds of neonicotinoid insecticides and 6 kinds of metabolites, which greatly improves the detection efficiency.

Specifically, the solid phase extraction cartridge used was Bond Elut Plexa 3cc, 60 mg (Agilent, MA, USA).

The method for detecting neonicotinoid pesticides and their metabolites in cerebrospinal fluid of the present application is further described below with specific examples.

The liquid phase instrument used in the following examples is the 1290INFINITY II ultra-high performance liquid chromatography system of Shimadzu Corporation of Japan, the mass spectrometer is the electrospray triple quadrupole mass spectrometer of the 6500 model of ABSSCIEX Corporation of the United States, and the chromatographic column is selected from Waters ACQUITY BEH C18 high-efficiency liquid The phase chromatography column has a length of 100 mm, an inner diameter of 2.1 mm, a filler particle diameter of 1.7 μm, and a solid phase extraction cartridge of Bond Elut Plexa 3cc, 60 mg (Agilent, MA, USA).

Example 1

The embodiments of the present application provide a method for detecting neonicotinoid insecticides and their metabolites in cerebrospinal fluid, comprising the following steps:

S1. Cerebrospinal fluid should be stored in -80℃ refrigerator after sampling;

S2. Add stable isotope-labeled internal standard to 250 μL of cerebrospinal fluid sample and mix with 1.5 mL of 2% formic acid aqueous solution by volume to obtain a mixed solution;

S3, mix methanol and ammonium hydroxide solution (volume concentration 30%) with a volume ratio of 19:1 to obtain an activation solution; activate the solid-phase extraction cartridge with 3 ml of activation solution, and then use water to rinse the solid-phase extraction cartridge, Then pass the mixed solution through the solid phase extraction cartridge; use 2 ml of a mixed solution of methanol, formic acid, and water in a volume ratio of 5:1:44 to rinse the solid phase extraction cartridge, dry under negative pressure for 1 min, and elute the target with 3 mL of methanol. The eluate was blown dry with nitrogen, and reconstituted with 250 μL of a solution consisting of methanol and water with a volume ratio of 1:3;

S4. Perform high-performance liquid phase triple quadrupole tandem mass spectrometry detection on the reconstituted solution, specifically:

The data was collected in positive and negative ion mode, and the tandem mass spectrometry conditions were electrospray ionization. The positive ion mode (ESI+) was used to detect 12 neonicotinoids. The positive ion mode detection conditions were: collision gas 10psi, curtain gas 35psi, ion spray voltage 4000V, The source temperature is 650°C, and the ion source gases 1 and 2 are both 70 psi; the negative ion mode (ESI-) detects 3 neonicotinoids. The source temperature is 600 °C, and the ion source gases 1 and 2 are 70 psi and 50 psi, respectively;

The injection volume was 3 μL, and the gradient elution procedure for the 15 compounds was as follows:

Time 0min: use the mixed solution composed of acetonitrile and formic acid aqueous solution with a volume ratio of 5:95 as the mobile phase, and the flow rate is 0.35mL/min;

Time 0.6min: use a mixture of acetonitrile and formic acid aqueous solution with a volume ratio of 5:95 as the mobile phase, and the flow rate is 0.35mL/min;

Time 4min: use a mixture of acetonitrile and formic acid aqueous solution with a volume ratio of 99:1 as the mobile phase, and the flow rate is 0.35mL/min;

Time 5min: use a mixture of acetonitrile and formic acid aqueous solution with a volume ratio of 99:1 as the mobile phase, and the flow rate is 0.35mL/min;

Time 5.5min: use a mixture of acetonitrile and formic acid aqueous solution with a volume ratio of 5:95 as the mobile phase, and the flow rate is 0.35mL/min;

Time 6.5min: use a mixture of acetonitrile and formic acid aqueous solution with a volume ratio of 5:95 as the mobile phase, and the flow rate is 0.35mL/min.

Among them, the volume concentration of formic acid aqueous solution in the mobile phase is 0.005%; the internal standards labeled with stable isotopes are ¹³ C ₃ - ¹⁵ N ₂ -nitenpyram, ¹³ C ₄ - ¹⁵ N-thiamethoxam, D ₄ -imidacloprid , ¹³ C ₆ -acetamiprid, ¹³ C ₆ -thiacloprid, ¹³ C ₄ - ¹⁵ N- clothianidin, ¹⁸ O- ¹⁵ N-fluacetamid, D ₄ -N-desmethylthiamethoxam , ¹³ C ₆ -thiacloprid, D ₄ -chlorothiline, ¹³ C ₂ - ¹⁵ N-desmethylacetamiprid, ¹³ C ₆ -6-chloronicotinic acid, D ₃ - ¹³ C ₂ - ¹⁵ N A mixture consisting of ₂ -flufenapyr, ¹³ C- ¹⁵ N ₂ -alkene imidacloprid, and ¹³ C- ¹⁵ N ₂ -5-hydroxy imidacloprid, wherein the amount of each component added in the internal standard was 2.5 ng.

The detection method of neonicotinoid insecticides and their metabolites in cerebrospinal fluid in Example 1, the Chinese and English names of the detected compounds, their abbreviations and mass spectrometry accurate mass numbers are shown in Table 1 below.

Table 1 - Chinese and English names of different compounds and their abbreviations and mass spectrometry accurate mass numbers

Chinese name English name short name Q1/Q3(m/z) Acetamiprid Acetamiprid ACE 222.8/126 Demethylacetamiprid N-desmethyl-acetamiprid N-dm-ACE 209/126 Imidacloprid Imidacloprid IMI 256/209 5-Hydroxyimidacloprid 5-Hydroxy-imidacloprid 5-OH-IMI 272/225 Olefin imidacloprid Olefin-imidaclprid Of-IMI 251.97/205 Thiamethoxam Thiamethoxam THX 291.8/211.1 N-Demethylthiamethoxam N-desmethyl thiamethoxam N-DMT 278/132 Thiacloprid Thiacloprid THI 253/126 Thiacloprid Thiacloprid-amide TA 271.1/126.1 Nitenpyram Nitenpyram NIT 271.1/126 clothianidin Clothianidin CLO 250/169.1 Flonicamid Flonicamid FLO 230/203 Chlorothialine Imidaclothiz IMZ 262/181 sulfenalazine Sulfoxaflor SUF 275.9/213 6-Chloronicotinic acid 6-Chloronicotinic acid 6-CN 155.9/111.9

According to the detection method in Example 1, the chromatograms of 12 neonicotinoid compounds whose data were collected in positive ion mode are shown in FIG. 1 . Chromatograms of the three neonicotinoid compounds with data collected in negative ion mode are shown in Figure 2.

Figures 1 and 2 are positive and negative ion mode chromatograms of 18 neonicotinoid insecticides and their metabolites. In Figure 1, the retention time of dinotefuran (DIN) is 2.57min, the retention time of two degradation products of dinotefuran (DIN) DIN-U and DIN-G are 2.65min, 2.57min; the retention time of NIT is 2.9 min, THX retention time is 3.13 min, 5-OH-IMI retention time is 3.16 min, FLO retention time is 3.20 min, TA retention time is 3.24 min, N-dm-ACE retention time is 3.28 min, CLO The retention time was 3.29 min for IMI, 3.36 min for IMI, 3.42 min for ACE, 3.42 min for IMZ, 3.44 min for N-DMT and 3.58 min for THI. In Figure 2, the retention time of Of-IMI was 3.11 min, the retention time of 6-CN was 3.24 min, and the retention time of SUF was 3.63 min. However, the controlled recovery rate of dinotefuran (DIN) and its two degradation products is not between 80-120%, so this method excludes the detection of dinotefuran and its two degradation products. The retention time of the chromatographic peaks of the 15 nicotinic pesticides and their metabolites that have passed the quality control is basically the same as the retention time of the isotope standard, and the difference is not more than ±2%. Using the mass spectrometry multiple reaction detection technology, the data is selected for mass spectrometry. Signal acquisition, collision induction, signal recording of ions that conform to the rules, and removal of interference from ions that do not conform to the rules. The method of the invention is accurate, reliable and high in precision for the detection of neonicotinoids in cerebrospinal fluid.

Table 2 shows the recovery rates and relative standard deviations of different compounds using the detection method in Example 1. Specifically, the cerebrospinal fluid samples were spiked with three concentration gradients, 1 ng/mL, 10 ng/mL and 20 ng/mL, respectively, and the spiked substances were the external standard substances of all 15 neonicotinoids and their metabolites in this application.

Table 2 - Spike recoveries and relative standard deviations for different compounds

It can be proved from the data in Table 2 that by using the detection method of the present invention, the recovery rate of 15 kinds of neonicotinoids and their metabolites in cerebrospinal fluid can be controlled within the range of 80-120%, and the quality control matrix is human cerebrospinal fluid. The method was used to detect 15 neonicotinoids and their metabolites in cerebrospinal fluid with high accuracy and reliable data.

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the present invention. within the scope of protection.

Claims (1)

1. a detection method for neonicotinoid insecticides and metabolites thereof in cerebrospinal fluid, is characterized in that, comprises the following steps: Adding stable isotope-labeled internal standard to the cerebrospinal fluid and then mixing with formic acid aqueous solution to obtain a mixed solution; The mixed solution was passed through a solid phase extraction cartridge, then eluted with methanol, and the eluate was reconstituted with an aqueous methanol solution; the solid phase extraction cartridge was Bond Elut Plexa 3 cc; The reconstituted solution was detected by high performance liquid phase triple quadrupole tandem mass spectrometry; The detection conditions of high performance liquid phase triple quadrupole tandem mass spectrometry were as follows: Waters ACQUITY BEH C18 was selected as the high performance liquid chromatography column, the length of the high performance liquid chromatography column was 100 mm, the inner diameter was 2.1 mm, and the diameter of the filler particles was 1.7 μm ; The data were collected in positive and negative ion mode, and the tandem mass spectrometry conditions were electrospray ionization. The detection conditions in positive ion mode were: collision gas 10 psi, curtain gas 35 psi, ion spray voltage 4000 V, ion source temperature 650 ℃, ion source gas 1 and 2 are 70 psi; negative ion mode detection conditions are: collision gas 8 psi, curtain gas 30 psi, ion spray voltage -4500 V, ion source temperature 600 ℃, ion source gas 1 and 2 are 70 psi and 50 psi respectively; The gradient elution procedure is used for elution, and the gradient elution procedure is as follows: Time 0min: use the mixed solution composed of acetonitrile and formic acid aqueous solution with a volume ratio of 5:95 as the mobile phase, and the flow rate is 0.35mL/min; Time 0.6min: use a mixture of acetonitrile and formic acid aqueous solution with a volume ratio of 5:95 for flow phase, the flow rate is 0.35mL/min; Time 4min: use a mixture of acetonitrile and formic acid aqueous solution with a volume ratio of 99:1 as the mobile phase, and the flow rate is 0.35mL/min; Time 5min: use a mixture of acetonitrile and formic acid aqueous solution with a volume ratio of 99:1 as the mobile phase, The flow rate is 0.35mL/min; Time 5.5min: use a mixture of acetonitrile and formic acid aqueous solution with a volume ratio of 5:95 as the mobile phase, and the flow rate is 0.35mL/min; Time 6.5min: use a mixture of acetonitrile and formic acid aqueous solution with a volume ratio of 5:95 for flow phase, the flow rate is 0.35mL/min; The volume concentration of the formic acid aqueous solution is 0.003 to 0.008%; The neonicotinoid insecticides and their metabolites include acetamiprid, desmethylacetamiprid, imidacloprid, 5-hydroxy imidacloprid, alkene imidacloprid, thiamethoxam, N-desmethylthiamethoxam, thiamethoxam chlorfenapyr, thiacloprid, nitenpyram, clothianidin, flonicamid, chlorothiline, flonicamid, and 6-chloronicotinic acid; Acetamiprid, Demethylacetamiprid, Imidacloprid, 5-Hydroxyimidacloprid, Thiamethoxam, N-Demethylthiamethoxam, Thiacloprid, Thiacloprid, Nitenpyram, Clothianidin, Fluorine Acetamiprid and chlorothiline were collected in positive ion mode; alkene imidacloprid, sulfenalazine and 6-chloronicotinic acid were collected in negative ion mode; The stable isotope-labeled internal standard is added to the cerebrospinal fluid, and then mixed with the aqueous formic acid solution to obtain a mixed solution. Specifically, 30-45 ng of the stable-isotope-labeled internal standard is added to 250-300 μL of the cerebrospinal fluid, and then the volume concentration of 1-2 ml is added. It is 1~3% formic acid aqueous solution mixed to obtain a mixed solution; The stable isotope-labeled internal standard includes ¹³ C ₃ - ¹⁵ N ₂ -nitenpyram, ¹³ C ₄ - ¹⁵ N-thiamethoxam, D ₄ -imidacloprid, ¹³ C ₆ -acetamiprid, ¹³ C ₆ - Thiacloprid, ¹³ C ₄ - ¹⁵ N- clothianidin, ¹⁸ O- ¹⁵ N-fluacetamid, D ₄ -N-desmethylthiamethoxam, ¹³ C ₆ -thiacloprid, D ₄ - Chlorothialine, ¹³ C ₂ - ¹⁵ N-desmethylacetamiprid, ¹³ C ₆ -6-chloronicotinic acid, D ₃ - ¹³ C ₂ - ¹⁵ N ₂ -flufenapyr, ¹³ C- ¹⁵ Mixtures of N ₂ -alkene imidacloprid, ¹³ C- ¹⁵ N ₂ -5-hydroxy imidacloprid; The mixture was passed through a solid-phase extraction cartridge, then eluted with methanol, and the eluate was reconstituted with an aqueous methanol solution. Specifically: After mixing methanol and ammonium hydroxide, an activation solution is obtained, and then the solid phase extraction cartridge is activated by using the activation solution; Rinse the activated solid phase extraction cartridge with water, and then pass the mixture through the solid phase extraction cartridge; After mixing methanol, formic acid and water, the solid phase extraction cartridge was rinsed, dried under negative pressure, eluted with methanol, the eluent was blown dry with nitrogen, and reconstituted with methanol aqueous solution; The volume ratio of methanol and ammonium hydroxide is (15~20):1; the volume ratio of methanol, formic acid and water is (4.5~5.5):(0.5~1.5):(42~46).

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