阅读说明：本技术 一种测定铁皮石斛中多种农药残留量的样品前处理方法及其检测方法 (Sample pretreatment method for determining residual quantity of various pesticides in dendrobium officinale and detection method thereof ) 是由 许振岚 李灵香玉 徐明飞 吴声敢 汤涛 张春荣 赵学平 于 2019-12-19 设计创作，主要内容包括：本发明提供了一种测定铁皮石斛中多种农药残留量的样品前处理方法及其检测方法,属于分析化学技术领域。本发明提供的测定铁皮石斛中多种农药残留量的样品前处理方法,包括如下步骤：将铁皮石斛样品匀浆处理后,与乙腈-水溶液混合,进行提取,得到铁皮石斛提取液；将所述铁皮石斛提取液与石墨化多壁碳纳米管混合后,依次经涡旋和离心,然后取上清液作为待测液,完成样品前处理。本发明以石墨化多壁碳纳米管为吸附剂,能够有效去除复杂基质铁皮石斛样品中的杂质,且对具有平面结构的农药吸附少,能够保证多种农药残留量测试结果的准确性。此外,本发明所提供的样品前处理方法操作简单,易于实施。(The invention provides a sample pretreatment method for measuring the residual quantity of various pesticides in dendrobium officinale and a detection method thereof, belonging to the technical field of analytical chemistry. The invention provides a sample pretreatment method for determining various pesticide residues in dendrobium officinale, which comprises the following steps: homogenizing a dendrobium officinale sample, mixing the homogenate with an acetonitrile-water solution, and extracting to obtain a dendrobium officinale extracting solution; and mixing the dendrobium officinale extracting solution with the graphitized multi-walled carbon nano tube, sequentially performing vortex and centrifugation, taking the supernatant as a solution to be detected, and finishing the pretreatment of the sample. The method takes the graphitized multi-walled carbon nanotube as the adsorbent, can effectively remove impurities in the dendrobium officinale sample with a complex matrix, has less adsorption on pesticides with a planar structure, and can ensure the accuracy of test results of various pesticide residues. In addition, the sample pretreatment method provided by the invention is simple to operate and easy to implement.)

1. A sample pretreatment method for determining various pesticide residues in dendrobium officinale is characterized by comprising the following steps:

homogenizing a dendrobium officinale sample, mixing the homogenate with an acetonitrile-water solution, and extracting to obtain a dendrobium officinale extracting solution;

and mixing the dendrobium officinale extracting solution with the graphitized multi-walled carbon nano tube, sequentially performing vortex and centrifugation, taking the supernatant as a solution to be detected, and finishing the pretreatment of the sample.

2. The sample pretreatment method according to claim 1, wherein a volume ratio of acetonitrile to water in the acetonitrile-water solution is 1.8 to 2.2: 1.

3. The sample pretreatment method according to claim 1, wherein the dosage ratio of the dendrobium officinale sample to the acetonitrile-water solution is 2g: 13-17 mL.

4. The sample pretreatment method according to claim 1, wherein the extraction includes the steps of:

after the first oscillation treatment is carried out on the extract, adding salt, carrying out second oscillation treatment, and then centrifuging to obtain supernatant serving as the dendrobium officinale extract; the extract to be extracted is a mixture of a product obtained after the homogenization treatment of the dendrobium officinale sample and acetonitrile-water solution.

5. The method for pretreating a sample according to claim 4, wherein the first shaking treatment and the second shaking treatment are performed at room temperature; the rotating speed of the first oscillation treatment is 3000-5000 rpm, and the time is 8-12 min; the rotation speed of the second oscillation treatment is 3000-5000 rpm, and the time is 4-6 min.

6. The sample pretreatment method according to claim 4, wherein the salt comprises anhydrous magnesium sulfate and sodium chloride, and the mass ratio of the anhydrous magnesium sulfate to the sodium chloride is 3.8-4.2: 1.

7. The sample pretreatment method according to any one of claims 1 to 6, wherein the dosage ratio of the dendrobium officinale extract to the graphitized multi-walled carbon nanotube is 1.2mL: 4-18 mg.

8. The sample pretreatment method according to claim 7, wherein when the dendrobium officinale sample is a stem of dendrobium officinale, the dosage ratio of the dendrobium officinale extract to the graphitized multi-walled carbon nanotube is 1.2mL: 4-6 mg; when the dendrobium officinale sample is leaves of dendrobium officinale, the dosage ratio of the dendrobium officinale extracting solution to the graphitized multi-walled carbon nano tube is 1.2mL: 13-18 mg.

9. The method for sample pretreatment according to any one of claims 1 to 6, wherein the vortex is performed at room temperature; the rotation speed of the vortex is 4000-6000 rpm, and the time is 0.8-1.2 min; the rotating speed of the centrifugation is 4500-5500 rpm, and the time is 8-12 min.

10. A method for measuring the residual quantity of various pesticides in dendrobium officinale is characterized by comprising the following steps:

treating a dendrobium officinale sample according to the sample pretreatment method of any one of claims 1 to 9 to obtain a solution to be detected;

performing high performance liquid chromatography-mass spectrometry on the solution to be detected to obtain a spectrogram;

wherein, the conditions of the high performance liquid chromatography are as follows:

a chromatographic column: a Luna Omega C18 LC column, wherein the inner diameter of the column is 2.1mm, the length of the column is 100mm, and the grain diameter of the filler is 1.6 mu m;

column temperature: 38-42 ℃;

mobile phase: the mobile phase A is 0.2% of formic acid aqueous solution containing 2mM of ammonium acetate; the mobile phase B is 0.2 percent of formic acid methanol solution containing 2mM of ammonium acetate;

the elution procedure is shown in table 1:

TABLE 1 elution procedure

Time/min Mobile phase A (v%) Mobile phase B (v%) 0.50 90 10 5.00 50 50 10.00 30 70 15.00 10 90 20.00 10 90 20.10 90 10 22.00 90 10

The conditions of mass spectrum are: electrospray ionization, multiple reaction monitoring mode, capillary voltage of 3.0kV, desolventizing temperature of 400 ℃, desolventizing gas flow of 800L/h;

and calculating to obtain various pesticide residues according to the spectrogram.

Technical Field

The invention relates to the technical field of analytical chemistry, in particular to a sample pretreatment method for determining various pesticide residues in dendrobium officinale and a detection method thereof.

Background

In the artificial planting process of the dendrobium officinale, pesticides are frequently applied when diseases and insect pests occur, and the phenomenon of more pesticide residues is serious, so that an efficient and high-flux method is urgently needed to be established for detecting the pesticide residues in the dendrobium officinale, so that the quality of the artificially planted dendrobium officinale is ensured. The dendrobium officinale is a complex substrate and is rich in substances such as pigments, amino acids, polysaccharides and the like. The method for detecting the pesticide residue is established, wherein the method is high in flux and low in organic solvent usage amount, and the key point is to remove impurities such as pigments in the dendrobium officinale and eliminate the interference of the impurities on detection.

The traditional matrix dispersion solid phase extraction method has the characteristics of rapidness, simplicity, cheapness, effectiveness, reliability, safety and the like, and is widely applied to detection of multiple pesticide residues. The method uses the combination of various adsorbents to achieve the aim of removing impurities. Classical adsorbents commonly used are PSA (primary secondary amine, ethylenediamine-N-propylsilane), C18 and GCB (graphitized carbon). Among them, GCB has a good effect of removing impurities such as pigments, but it has a strong adsorption effect on pesticides having a planar structure, thereby reducing the recovery rate of these pesticides, thus limiting their use. Meanwhile, for dendrobium officinale with high content of impurities such as pigments, the traditional matrix dispersion solid-phase extraction method has the problems of too large dosage of the adsorbent, unsatisfactory impurity removal effect and the like, so that the method has great limitation in treatment of the dendrobium officinale.

Disclosure of Invention

The invention aims to provide a sample pretreatment method for measuring the residual quantity of various pesticides in dendrobium officinale and a detection method thereof.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a sample pretreatment method for measuring residual quantity of various pesticides in dendrobium officinale, which comprises the following steps:

homogenizing a dendrobium officinale sample, mixing the homogenate with an acetonitrile-water solution, and extracting to obtain a dendrobium officinale extracting solution;

and mixing the dendrobium officinale extracting solution with the graphitized multi-walled carbon nano tube, sequentially performing vortex and centrifugation, taking the supernatant as a solution to be detected, and finishing the pretreatment of the sample.

Preferably, the volume ratio of acetonitrile to water in the acetonitrile-water solution is 1.8-2.2: 1.

Preferably, the dosage ratio of the dendrobium officinale sample to the acetonitrile-water solution is 2g: 13-17 mL.

Preferably, the extraction comprises the steps of:

after the first oscillation treatment is carried out on the extract, adding salt, carrying out second oscillation treatment, and then centrifuging to obtain supernatant serving as the dendrobium officinale extract; the extract to be extracted is a mixture of a product obtained after the homogenization treatment of the dendrobium officinale sample and acetonitrile-water solution.

Preferably, the first shaking treatment and the second shaking treatment are performed at room temperature; the rotating speed of the first oscillation treatment is 3000-5000 rpm, and the time is 8-12 min; the rotation speed of the second oscillation treatment is 3000-5000 rpm, and the time is 4-6 min.

Preferably, the salt comprises anhydrous magnesium sulfate and sodium chloride, and the mass ratio of the anhydrous magnesium sulfate to the sodium chloride is 3.8-4.2: 1.

Preferably, the dosage ratio of the dendrobium officinale extracting solution to the graphitized multi-walled carbon nano tube is 1.2mL to 4-18 mg.

Preferably, when the dendrobium officinale sample is a stem of dendrobium officinale, the dosage ratio of the dendrobium officinale extracting solution to the graphitized multi-walled carbon nanotube is 1.2mL: 4-6 mg; when the dendrobium officinale sample is leaves of dendrobium officinale, the dosage ratio of the dendrobium officinale extracting solution to the graphitized multi-walled carbon nano tube is 1.2mL: 13-18 mg.

Preferably, the vortexing is performed at room temperature; the rotation speed of the vortex is 4000-6000 rpm, and the time is 0.8-1.2 min; the rotating speed of the centrifugation is 4500-5500 rpm, and the time is 8-12 min.

The invention also provides a method for measuring the residual quantity of various pesticides in dendrobium officinale, which comprises the following steps:

treating the dendrobium officinale sample according to the sample pretreatment method of the technical scheme to obtain a solution to be detected;

performing high performance liquid chromatography-mass spectrometry on the solution to be detected to obtain a spectrogram;

wherein, the conditions of the high performance liquid chromatography are as follows:

a chromatographic column: a Luna Omega C18 LC column, wherein the inner diameter of the column is 2.1mm, the length of the column is 100mm, and the grain diameter of the filler is 1.6 mu m;

column temperature: 38-42 ℃;

mobile phase: the mobile phase A is 0.2% of formic acid aqueous solution containing 2mM of ammonium acetate; the mobile phase B is 0.2 percent of formic acid methanol solution containing 2mM of ammonium acetate;

the elution procedure is shown in table 1:

TABLE 1 elution procedure

Time/min	Mobile phase A (v%)	Mobile phase B (v%)
			0.50	90	10
5.00	50	50
			10.00	30	70
15.00	10	90
			20.00	10	90
20.10	90	10
			22.00	90	10

The conditions of mass spectrum are: electrospray ionization, multiple reaction monitoring mode, capillary voltage of 3.0kV, desolventizing temperature of 400 ℃, desolventizing gas flow of 800L/h;

and calculating to obtain various pesticide residues according to the spectrogram.

The invention provides a sample pretreatment method for measuring residual quantity of various pesticides in dendrobium officinale, which comprises the following steps: homogenizing a dendrobium officinale sample, mixing the homogenate with an acetonitrile-water solution, and extracting to obtain a dendrobium officinale extracting solution; and mixing the dendrobium officinale extracting solution with the graphitized multi-walled carbon nano tube, sequentially performing vortex and centrifugation, taking the supernatant as a solution to be detected, and finishing the pretreatment of the sample. The method takes the graphitized multi-walled carbon nanotube as the adsorbent, can effectively remove impurities in the dendrobium officinale sample with a complex matrix, has less adsorption on pesticides with a planar structure, and can ensure the accuracy of test results of various pesticide residues. In addition, the sample pretreatment method provided by the invention is simple to operate and easy to implement.

Detailed Description

The invention provides a sample pretreatment method for measuring residual quantity of various pesticides in dendrobium officinale, which comprises the following steps:

homogenizing a dendrobium officinale sample, mixing the homogenate with an acetonitrile-water solution, and extracting to obtain a dendrobium officinale extracting solution;

mixing the dendrobium officinale extracting solution with graphitized multi-walled carbon nanotubes (namely the multi-walled carbon nanotubes carbonized in a high-temperature carbonization furnace), sequentially performing vortex and centrifugation, and taking supernate as a liquid to be detected to finish sample pretreatment.

The method comprises the steps of homogenizing a dendrobium officinale sample, mixing the homogenate with an acetonitrile-water solution, and extracting to obtain the dendrobium officinale extracting solution.

In the invention, the dendrobium officinale sample is preferably stem and/or leaf of dendrobium officinale.

The invention has no special limitation on the condition of the homogenization treatment, and the homogenization treatment is carried out according to the conventional homogenization treatment mode; the homogenization treatment is beneficial to completely extracting various pesticides in the dendrobium officinale sample.

In the invention, the volume ratio of acetonitrile to water in the acetonitrile-water solution is preferably 1.8-2.2: 1, and more preferably 2: 1. In the invention, the acetonitrile-water solution with the proportion is beneficial to extracting pesticides with different polarities, so that the pretreatment method is more suitable for extracting various pesticide residues.

In the invention, the dosage ratio of the dendrobium officinale sample to the acetonitrile-water solution is preferably 2g: 13-17 mL, and more preferably 2g:15 mL.

In the present invention, the extraction preferably comprises the steps of:

after the first oscillation treatment is carried out on the extract, adding salt, carrying out second oscillation treatment, and then centrifuging to obtain supernatant serving as the dendrobium officinale extract; the extract to be extracted is a mixture of a product obtained after the homogenization treatment of the dendrobium officinale sample and acetonitrile-water solution.

In the invention, the first oscillation treatment is firstly carried out, which is beneficial to fully and uniformly mixing the extract to be extracted with the acetonitrile-water solution, so as to be beneficial to the extraction of various pesticide residues; the salt plays a role in dewatering and salting out, and is beneficial to the pesticide to enter an acetonitrile organic phase to obtain acetonitrile phase extracting solution, namely the obtained dendrobium officinale extracting solution is the acetonitrile solution of the pesticide.

In the present invention, the first shaking treatment and the second shaking treatment are preferably performed at room temperature; the rotation speed of the first oscillation treatment is preferably 3000-5000 rpm, and more preferably 5000 rpm; the time is preferably 8-12 min, and more preferably 10 min; the rotation speed of the second oscillation treatment is preferably 3000-5000 rpm, and more preferably 5000 rpm; the time is preferably 4-6 min, and more preferably 5 min.

In the present invention, the salt preferably includes anhydrous magnesium sulfate and sodium chloride; the mass ratio of the anhydrous magnesium sulfate to the sodium chloride is preferably 3.8-4.2: 1, and more preferably 4: 1; the dosage ratio of the acetonitrile-water solution to the salt is preferably 1.8-2.2 mL:1g, and more preferably 2mL:1 g.

In the extraction process, the rotation speed of centrifugation is preferably 3500-4500 rpm, more preferably 4000rpm, and the time is preferably 8-12 min, more preferably 10 min.

After the dendrobium officinale extracting solution is obtained, the dendrobium officinale extracting solution and the graphitized multi-walled carbon nano tube are mixed, and then are sequentially subjected to vortex and centrifugation, and then the supernatant is taken as a liquid to be detected, so that the pretreatment of a sample is completed.

The source of the graphitized multi-walled carbon nano-tube is not particularly limited, and the graphitized multi-walled carbon nano-tube sold in the market can be adopted. In the embodiment of the invention, the graphitized multi-walled carbon nanotube is preferably purchased from Nanjing Xiancheng materials science and technology Limited and has a tube diameter of 10-20 nm.

In the invention, the preferable dosage ratio of the dendrobium officinale extract to the graphitized multi-walled carbon nano tube is 1.2mL to 4-18 mg; when the dendrobium officinale sample is a stem of dendrobium officinale, the preferable dosage ratio of the dendrobium officinale extract to the graphitized multi-walled carbon nano tube is 1.2mL to 4-6 mg; when the dendrobium officinale sample is leaves of dendrobium officinale, the preferable dosage ratio of the dendrobium officinale extracting solution to the graphitized multi-walled carbon nano tube is 1.2mL to 13-18 mg; and when the dendrobium officinale sample is a mixture of stems and leaves of the dendrobium officinale, performing pretreatment on the dendrobium officinale sample serving as the leaves.

In the present invention, the vortexing is preferably performed at room temperature; the rotation speed of the vortex is preferably 4000-6000 rpm, more preferably 5000rpm, and the time is preferably 0.8-1.2 min, more preferably 1 min; the rotation speed of the centrifugation is preferably 4500-5500 rpm, more preferably 5000rpm, and the time is preferably 8-12 min, more preferably 10 min.

After centrifugation, the obtained supernatant is preferably filtered by a microporous filter membrane to obtain a solution to be detected; the pore size of the microfiltration membrane is preferably 0.22 μm.

In the invention, the sample pretreatment method is suitable for determining the residual quantity of 76 pesticides in a dendrobium officinale sample, and the 76 pesticides are preferably methamidophos, carbendazim, trichlorfon, pirimicarb, imidacloprid, dimethoate, acetamiprid, phosphamidon, dichlorvos, pyrimethanil, imazalil, carbofuran, carbaryl, forchlorfenuron, cyantraniliprole, isoprocarb, cyprodinil, paclobutrazol, methiocarb, prochloraz, diethofencarb, spirotetramat, phosmet, fenamiphos, myclobutanil, fenamiphos, azoxystrobin, triadimefon, fenhexamid, boscalid, tebuconazole, tetraconazole, mandipropamid, penconazole, triflumilast, iprodione, triazophos, emamectin benzoate, isazofos, cyproconazole, azoxystrobin, trifloxystrobin, diazinon, tebufenofos, pyraclostrobin, trifloxystrobin, quinclorac, fluvofos, phoxim, profenofos, cyflufenamid, trifloxystrobin, tetramethrin, hexythiazox, chlorpyrifos, etoxazole, pyridaben, spirodiclofen, 3-hydroxy carbofuran, clothianidin, tricyclazole, fosthiazate, isoprothiolane, flutolamide, cyproconazole, epoxiconazole, flusilazole, hexaconazole, propiconazole, buprofezin and bifenthrin.

The invention also provides a method for measuring the residual quantity of various pesticides in dendrobium officinale, which comprises the following steps:

treating the dendrobium officinale sample according to the sample pretreatment method of the technical scheme to obtain a solution to be detected;

performing high performance liquid chromatography-mass spectrometry on the solution to be detected to obtain a spectrogram;

wherein, the conditions of the high performance liquid chromatography are as follows:

a chromatographic column: a Luna Omega C18 LC column, wherein the inner diameter of the column is 2.1mm, the length of the column is 100mm, and the grain diameter of the filler is 1.6 mu m;

column temperature: 38-42 ℃;

mobile phase: the mobile phase A is 0.2% formic acid aqueous solution with the mass concentration, and contains 2mM (namely mmol/L) of ammonium acetate; the mobile phase B is 0.2 percent of formic acid methanol solution containing 2mM of ammonium acetate;

the elution procedure is shown in table 1:

TABLE 1 elution procedure

Time/min	Mobile phase A (v%)	Mobile phase B (v%)
			0.50	90	10
5.00	50	50
			10.00	30	70
15.00	10	90
			20.00	10	90
20.10	90	10
			22.00	90	10

The conditions of mass spectrum are: electrospray ionization, multiple reaction monitoring mode, capillary voltage of 3.0kV, desolventizing temperature of 400 ℃, desolventizing gas flow of 800L/h;

and calculating to obtain various pesticide residues according to the spectrogram.

In the present invention, the column temperature is preferably 40 ℃.

In the present invention, the flow rate of the mobile phase is preferably 0.2 mL/min; the amount of sample is preferably 5. mu.L.

In the present invention, the mass spectrum parameters of 76 pesticides in the mass spectrum conditions are shown in Table 2,

mass spectrometric conditions for the pesticides in Table 276

The calculation method of the various pesticide residues is not particularly limited, and those skilled in the art can calculate according to conventional methods, such as an external standard method and an internal standard method, in the embodiment of the present invention, the calculation method is preferably an external standard method, more preferably a standard curve method, and the standard curve is preferably a matrix standard curve.

The solution to be tested obtained by the sample pretreatment method provided by the invention can be used for testing 76 pesticide residues in the dendrobium officinale sample, and the test results of 72 pesticide residues are high in accuracy.

The sample pretreatment method and the detection method for determining the residual amount of various pesticides in dendrobium officinale provided by the invention are described in detail with reference to the following examples, but the methods are not to be construed as limiting the scope of the invention.