Detection Method for Pesticide Residues in Leaves of Pandanus amaryllifolius Roxb.

Abstract

The present invention belongs to the technical field of pesticide residue detection, and particularly relates to a method for detecting the pesticide residue amount in the leaves of Pandanus amaryllifolius Roxb., comprising: crushing the collected Pandanus amaryllifolius Roxb. leaves into leaf fragments, adding an extraction reagent and glass beads to the leaf fragments, and the mass ratio of the leaf fragments, the extraction reagent and the glass beads is 1:10-20:0.3-1; shaking for 20 min-40 min, then centrifuging to remove the glass beads and leaf fragments, collecting the first supernatant, adding a purification and adsorption material to the first supernatant, shaking for 2 min-4 min, centrifuging, collecting the second supernatant, and filtering to obtain a sample injection solution; injecting the sample injection solution into a liquid chromatography-mass spectrometry instrument for pesticide residue detection. The purification and adsorption material and the specific sample treatment method adopted by the method of the present invention can detect more types of pesticide residues, and the detection results have high precision and good repeatability.

Description

Technical Field

[0001] The present invention belongs to the technical field of pesticide residue detection, and particularly relates to a method for detecting pesticide residues in Pandanus amaryllifolius Roxb. leaves. Background Art

[0002] Pandanus amaryllifolius Roxb., commonly known as Pandan leaf, is a perennial herb of the genus Pandanus in the family Pandanaceae. Pandan leaf (powder) is one of the characteristic spice crops in Hainan, belonging to local special food, with a large planting scale and an increasing trend. At present, Pandanus amaryllifolius Roxb. is recorded in the local standard "Local Food Safety Standard of Hainan Province - Pandan Leaf (Powder)" (DBS_46 / 004 - 2022), allowing planting, production and circulation. The pesticide residues are stipulated according to the requirements of leafy vegetables in GB 2763 "National Food Safety Standard - Maximum Residue Limits of Pesticides in Foods", and the recommended daily consumption amount per person is also stipulated. Due to the possible pesticide cross - contamination caused by the mixed planting of different varieties of crops, or the migration of pesticide pollution in the original planting area, or the non - standard use of pesticides, etc., there are pesticide residues in Pandanus amaryllifolius Roxb. leaves. However, the research on the types and contents of pesticide residues and the characteristics of inspection methods in Pandanus amaryllifolius Roxb. leaves is not in - depth. Since Pandanus amaryllifolius Roxb. leaves are not currently included in the food raw material catalog, it is necessary to develop a detection method for pesticide residues in Pandanus amaryllifolius Roxb. leaves that can detect more pesticide residues. Summary of the Invention

[0003] In order to solve the above - mentioned technical problems, the present invention provides a method for detecting pesticide residues in Pandanus amaryllifolius Roxb. leaves.

[0004] The object of the present invention is to provide a method for detecting pesticide residues in Pandanus amaryllifolius Roxb. leaves, including:

[0005] First, sample collection and treatment

[0006] The collected Pandanus amaryllifolius Roxb. leaves are crushed into leaf fragments. An extraction reagent and glass beads are added to the leaf fragments, and the mass ratio of the leaf fragments, the extraction reagent and the glass beads is 1:10 - 20:0.3 - 1; shake for 20 min - 40 min, then centrifuge to remove the glass beads and leaf fragments, collect the first supernatant. A purification and adsorption material is added to the first supernatant, shake for 2 min - 4 min, centrifuge, collect the second supernatant, and filter it to obtain the sample injection solution;

[0007] Among them, the purification and adsorption material is composed of neutral alumina, SCX strong cation exchange chromatography packing and aminated multi - walled carbon nanotubes mixed in a mass ratio of 10 - 15:100:1; the mass ratio of the first supernatant to the purification and adsorption material is 10:1 - 2;

[0008] Second, inject the sample injection solution into a liquid chromatography - mass spectrometry instrument for pesticide residue detection.

[0009] Preferably, for the above method for detecting the pesticide residue in the leaves of *Pandanus amaryllifolius*, the extraction reagent is a mixture of acetonitrile - acetic acid solution, anhydrous magnesium sulfate, and sodium acetate in a mass ratio of 20:6:1 to 2;

[0010] Among them, every 100 L of the acetonitrile - acetic acid solution contains 3 L to 4 L of acetic acid, and the balance is acetonitrile.

[0011] Preferably, for the above method for detecting the pesticide residue in the leaves of *Pandanus amaryllifolius*, the diameter of the glass beads is 1 mm to 3 mm.

[0012] Preferably, for the above method for detecting the pesticide residue in the leaves of *Pandanus amaryllifolius*, the shaking conditions are: placed in a shaker and shaken at 200 rpm to 250 rpm.

[0013] Preferably, for the above method for detecting the pesticide residue in the leaves of *Pandanus amaryllifolius*, the centrifugation conditions are centrifugation at 4200 r / min and 4 °C for 5 min to 10 min.

[0014] Preferably, for the above method for detecting the pesticide residue in the leaves of *Pandanus amaryllifolius*, the filtration is carried out by microfiltration membrane filtration, and the diameter of the micropores is 0.22 μm.

[0015] Preferably, for the above method for detecting the pesticide residue in the leaves of *Pandanus amaryllifolius*, the preparation method of the leaf fragments is: first cut the leaves of *Pandanus amaryllifolius* to be tested into leaf segments, and then stir - crush them to form leaf fragments with a particle size of 1 mm to 2 mm. For example, use a knife - type crusher to stir - crush.

[0016] Preferably, for the above method for detecting the pesticide residue in the leaves of *Pandanus amaryllifolius*, the liquid chromatography conditions of the liquid chromatography - mass spectrometry combined instrument are: chromatographic column C18, inner diameter 2.1 mm × 100 mm, particle size 1.8 μm; mobile phase: phase A is an aqueous solution of ammonium formate - formic acid, phase B is a methanol solution of ammonium formate - formic acid, with a gradient change in the mobile phase; flow rate 0.3 mL / min; column temperature 40 °C; injection volume of the sample solution is 2 μL.

[0017] Preferably, for the above method for detecting the pesticide residue in the leaves of *Pandanus amaryllifolius*, the mass spectrometry conditions of the liquid chromatography - mass spectrometry combined instrument are: electrospray ionization source; ion source temperature 350 °C; simultaneous scanning of positive and negative ions; electrospray voltage: 5500 V for positive ions, - 4500 V for negative ions; nebulizing gas: 0.345 MPa; auxiliary heating gas: 0.345 MPa 。

[0018] Preferably, in the above method for detecting pesticide residues in the leaves of Pandanus amaryllifolius Roxb., the pesticides include one or more of isoprocarb, oxadiargyl, toxaphene, atratone, prometon, simazine, atrazine, propazine, glyphosate, terbuthylazine, parathion-ethyl, carbaryl, carbofuran, diazinon, butralin, azoxystrobin, iprobenfos, isoprothiolane, parathion-methyl, cyflufenamid, fenpyrazamine, cloethocarb, simetryn, ametryn, oxadixyl, cyproconazole, imidazolinone, prometryn, terbutryn, fenitrothion, malathion, dichlorvos, dimethomorph, phenthoate, quinalphos, p, p'-DDE, buprofezin, p, p'-DDD, triazophos, chlorantraniliprole, tolfenpyrad, abamectin, phosalone, procymidone, metalaxyl, fipronil, iprovalicarb, thiabendazole, thiophanate-methyl, carbendazim, phoxim, butachlor, dinotefuran, clothianidin, imidacloprid, tebuconazole, and thiamethoxam.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] Sample treatment is the most crucial step affecting the detection results, which determines the types and contents of pesticide residues in the sample to be tested, so it also affects the detection accuracy. Therefore, the present invention focuses on discussing the influence of different sample treatment methods on the detection results, and finally provides an optimal sample treatment method to improve the detection accuracy.

[0021] The method of the present invention replaces the type of purification and adsorption material compared with the prior art. The purification and adsorption material is composed of neutral alumina, SCX strong cation exchange chromatography packing material and amino-functionalized multi-walled carbon nanotubes mixed in a mass ratio of 10-15:100:1. The present invention also defines a specific sample treatment method. Compared with the prior art, more types of pesticides can be detected in the sample solution obtained by the treatment of the present invention. A standardized operation and highly accurate method for detecting pesticide residues in the leaves of Pandanus amaryllifolius Roxb. is established, and a liquid chromatography-mass spectrometry instrument is used for detection, data analysis and statistics, and the varieties and content characteristics of pesticide residues in the leaves of Pandanus amaryllifolius Roxb. are analyzed. Detailed implementation manners

[0022] In order to enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below with reference to specific embodiments.

[0023] In the description of the present invention, unless otherwise specified, the reagents used are commercially available, and the methods used are conventional techniques in the art.

[0024] In the description of the present invention, introduction of relevant materials

[0025] (1) Neutral alumina: AL-N, is neutral Al2O3 particles with a particle size of 125 μm, pH 7.5, retention mechanism: Lewis acid-base interaction, polar interaction and ion exchange interaction, from DiKMA.

[0026] (2) SCX strong cation exchange chromatography packing material: average particle size 40 μm to 60 μm, from Hangzhou Micropie Technology Co., Ltd.

[0027] (3) Amino-functionalized multi-walled carbon nanotubes, from Shanghai Gaibang Industrial Co., Ltd.

[0028] (4) Graphitized carbon black (GCB), 40 μm to 120 μm.

[0029] (5) N-propylethylenediamine adsorbent (PSA), 40 μm to 60 μm, Qiyuan Pharmaceutical and Chemical Industry.

[0030] (6) Microporous filter membrane (organic phase): 13 mm × 0.22 μm.

[0031] (7) 2 mmol / L ammonium formate-formic acid aqueous solution: Weigh 0.1261 g of ammonium formate, dissolve and dilute it to 1000 mL with 0.01% (v / v) formic acid aqueous solution, and shake well.

[0032] (8) 2 mmol / L ammonium formate-formic acid methanol solution: Weigh 0.1261 g of ammonium formate, dissolve and dilute it to 1000 mL with 0.01% (v / v) formic acid methanol solution, and shake well.

[0033] Example 1

[0034] A method for detecting pesticide residues in leaves of Pandanus amaryllifolius Roxb., comprising:

[0035] First, sample collection and treatment

[0036] The collected leaves of Pandanus amaryllifolius Roxb. are crushed into leaf fragments. Add extraction reagent and glass beads to the leaf fragments, and the mass ratio of leaf fragments, extraction reagent and glass beads is 1:10:0.3; shake on a shaker at 200 rpm for 20 min, then centrifuge to remove the glass beads and leaf fragments, quantitatively collect the first supernatant, add purification and adsorption material to the first supernatant, shake on a vortex mixer for 2 min, centrifuge, quantitatively collect the second supernatant, and filter it to obtain the sample loading solution.

[0037] Among them, the purification and adsorption material is composed of neutral alumina, SCX strong cation exchange chromatography packing material and amino-functionalized multi-walled carbon nanotubes mixed in a mass ratio of 10:100:1; the mass ratio of the first supernatant to the purification and adsorption material is 10:1. It should be noted that when processing the sample, the key attention is paid to the mass ratio of the first supernatant to the purification and adsorption material. The amounts of the first supernatant and the second supernatant taken can be more or less. If a larger volume of the sample loading solution is needed, a certain volume of the first supernatant and the second supernatant are taken more. The volume values of the two do not affect the final detected amount of pesticide types. The same is true for other embodiments of the present invention.

[0038] The extraction reagent is a mixture of acetonitrile-acetic acid solution, anhydrous magnesium sulfate, and sodium acetate in a mass ratio of 20:6:1; wherein the acetonitrile-acetic acid solution is a mixture of acetonitrile and acetic acid in a volume ratio of 97:3.

[0039] The method for preparing leaf pieces is as follows: firstly, the leaves of the tested bergamot are cut into leaf segments, and then crushed with a knife crusher to form leaf pieces with a particle size of 1 mm.

[0040] The diameter of the glass beads is 3 mm.

[0041] Second, liquid chromatography-mass spectrometry was used for detection

[0042] The liquid chromatography conditions were as follows: chromatographic column C18, inner diameter 2.1 mm × 100 mm, particle size 1.8 μm; mobile phase: phase A was ammonium formate-formic acid aqueous solution, phase B was ammonium formate-formic acid methanol solution, mobile phase gradient change; flow rate 0.3 mL / min; column temperature 40°C; injection volume of the sample solution was 2 μL.

[0043] Among them, when the retention time is 0 to 1 min, the volume fraction of phase A is 97%, and the volume fraction of phase B is 3%; when the retention time is 1 min to 1.5 min, the volume fraction of phase A is 85%, and the volume fraction of phase B is 15%; when the retention time is 1.5 min to 2.5 min, the volume fraction of phase A is 50%, and the volume fraction of phase B is 50%; when the retention time is 2.5 min to 18 min, the volume fraction of phase A is 30%, and the volume fraction of phase B is 70%; when the retention time is 18 min to 27 min, the volume fraction of phase A is 2%, and the volume fraction of phase B is 98%; when the retention time is 27 min to 30 min, the volume fraction of phase A is 97%, and the volume fraction of phase B is 3%.

[0044] The mass spectrometry conditions were as follows: electrospray ion source; ion source temperature 350°C; positive ion and negative ion scanning simultaneously; electrospray voltage: positive ion 5500V, negative ion -4500V; nebulizer gas: 0.345MPa; auxiliary heating gas: 0.345MPa.

[0045] A total of 57 pesticides were detected, including isoprocarb, oxadiazon, toxaphene, atratone, prometon, simazine, atrazine, propazine, glyphosate, terbuthylazine, parathion-ethyl, carbaryl, carbofuran, diazinon, secbumeton, azoxystrobin, iprobenfos, isoprothiolane, parathion-methyl, cyflufenamid, fenpyrazamine, cloquintocet-mexyl, simetryn, ametryn, oxathiapiprolin, cyproconazole-imidazole, imazethapyr, prometryn, terbutryn, fenitrothion, malathion, dichlorvos, dimethomorph, phenthoate, quinalphos, p, p'-DDE, buprofezin, p, p'-DDD, triazophos, chlorantraniliprole, tolfenpyrad, abamectin, phosalone, procymidone, metalaxyl, fipronil, iprovalicarb, thiabendazole, thiophanate-methyl, carbendazim, phoxim, butachlor, dinotefuran, clothianidin, imidacloprid, tebuconazole, and thiamethoxam.

[0046] Example 2

[0047] A method for detecting pesticide residues in leaves of Pandanus amaryllifolius Roxb., comprising:

[0048] First, sample collection and treatment

[0049] The collected leaves of Pandanus amaryllifolius Roxb. are crushed into leaf fragments, an extraction reagent and glass beads are added to the leaf fragments, and the mass ratio of the leaf fragments, the extraction reagent, and the glass beads is 1:20:0.3; shaken on a shaker at 200 rpm for 20 min, then centrifuged to remove the glass beads and leaf fragments, the first supernatant is quantitatively collected, a purification and adsorption material is added to the first supernatant, vortexed on a vortex mixer for 2 min, centrifuged, the second supernatant is quantitatively collected, and filtered to obtain the sample loading solution.

[0050] Among them, the purification and adsorption material is composed of neutral alumina, SCX strong cation exchange chromatography packing, and amino-functionalized multi-walled carbon nanotubes mixed in a mass ratio of 10:100:1; the mass ratio of the first supernatant to the purification and adsorption material is 10:1.

[0051] The extraction reagent is a mixture of acetonitrile-acetic acid solution, anhydrous magnesium sulfate, and sodium acetate in a mass ratio of 20:6:1; among them, the acetonitrile-acetic acid solution is a mixture of acetonitrile and acetic acid in a volume ratio of 97:3.

[0052] The preparation method of the leaf fragments is: first cut the leaves of Pandanus amaryllifolius Roxb. to be tested into leaf segments, and then crush them with a knife crusher to form leaf fragments with a particle size of 1 mm.

[0053] The diameter of the glass beads is 3 mm.

[0054] Second, detect using a liquid chromatography-mass spectrometry

[0055] The liquid chromatography conditions were as follows: chromatographic column C18, inner diameter 2.1 mm × 100 mm, particle size 1.8 μm; mobile phase: phase A was an aqueous solution of ammonium formate - formic acid, phase B was a methanol solution of ammonium formate - formic acid, with a gradient change in the mobile phase; flow rate 0.3 mL / min; column temperature 40°C; injection volume of the sample solution was 2 μL.

[0056] Among them, for the retention time of 0 - 1 min, the volume fraction of phase A was 97%, and that of phase B was 3%; for the retention time of 1 min - 1.5 min, the volume fraction of phase A was 85%, and that of phase B was 15%; for the retention time of 1.5 min - 2.5 min, the volume fraction of phase A was 50%, and that of phase B was 50%; for the retention time of 2.5 min - 18 min, the volume fraction of phase A was 30%, and that of phase B was 70%; for the retention time of 18 min - 27 min, the volume fraction of phase A was 2%, and that of phase B was 98%; for the retention time of 27 min - 30 min, the volume fraction of phase A was 97%, and that of phase B was 3%.

[0057] The mass spectrometry conditions were as follows: electrospray ionization source; ion source temperature 350°C; simultaneous scanning of positive and negative ions; electrospray voltage: 5500 V for positive ions and -4500 V for negative ions; nebulizing gas: 0.345 MPa; auxiliary heating gas: 0.345 MPa.

[0058] A total of 57 pesticides were detected, including isoprocarb, oxadiargyl, toxaphene, atratone, prometon, simazine, atrazine, propazine, glyphosate, terbuthylazine, parathion - ethyl, carbaryl, carbofuran, diazinon, sec - butylazine, azoxystrobin, isoprothiolane, isoprobenfos, parathion - methyl, cyflufenamid, pyridinitril, fenitropan, simetryn, ametryn, oxadixyl, cyproconazole, imidazolinone, prometryn, terbutryn, fenitrothion, malathion, dichlorvos, dimethomorph, phenthoate, quinalphos, p, p'-DDE, buprofezin, p, p'-DDD, triazophos, chlorantraniliprole, tolfenpyrad, abamectin, phosalone, procymidone, metalaxyl, fipronil, iprobenfos, thiabendazole, thiophanate - methyl, carbendazim, phoxim, butachlor, dinotefuran, clothianidin, imidacloprid, tebuconazole, and thiamethoxam.

[0059] Example 3

[0060] A method for detecting pesticide residues in the leaves of Pandanus amaryllifolius Roxb. includes:

[0061] First, sample collection and treatment

[0062] The collected Elaeagnus odoratus leaves were crushed into leaf pieces, and extraction reagents and glass beads were added to the leaf pieces, with the mass ratio of leaf pieces, extraction reagents and glass beads being 1:10:1; the mixture was shaken on a shaker at 200 rpm for 20 min, and then the glass beads and leaf pieces were removed by centrifugation, the first supernatant was quantitatively collected, purification adsorption materials were added to the first supernatant, the mixture was shaken on a vortex machine for 2 min, and centrifuged, the second supernatant was quantitatively collected, and filtered as the sample solution.

[0063] The purification adsorption material is a mixture of neutral alumina, SCX strong cationic chromatography filler and amino multi-walled carbon nanotubes in a mass ratio of 10:100:1; the mass ratio of the first supernatant to the purification adsorption material is 10:1.

[0064] The extraction reagent is a mixture of acetonitrile-acetic acid solution, anhydrous magnesium sulfate, and sodium acetate in a mass ratio of 20:6:1; wherein the acetonitrile-acetic acid solution is a mixture of acetonitrile and acetic acid in a volume ratio of 97:3.

[0065] The method for preparing leaf pieces is as follows: firstly, the leaves of the tested bergamot are cut into leaf segments, and then crushed with a knife crusher to form leaf pieces with a particle size of 1 mm.

[0066] The diameter of the glass beads is 3 mm.

[0067] Second, liquid chromatography-mass spectrometry was used for detection

[0068] The liquid chromatography conditions were as follows: chromatographic column C18, inner diameter 2.1 mm × 100 mm, particle size 1.8 μm; mobile phase: phase A was ammonium formate-formic acid aqueous solution, phase B was ammonium formate-formic acid methanol solution, mobile phase gradient change; flow rate 0.3 mL / min; column temperature 40°C; injection volume of the sample solution was 2 μL.

[0069] Among them, when the retention time is 0 to 1 min, the volume fraction of phase A is 97%, and the volume fraction of phase B is 3%; when the retention time is 1 min to 1.5 min, the volume fraction of phase A is 85%, and the volume fraction of phase B is 15%; when the retention time is 1.5 min to 2.5 min, the volume fraction of phase A is 50%, and the volume fraction of phase B is 50%; when the retention time is 2.5 min to 18 min, the volume fraction of phase A is 30%, and the volume fraction of phase B is 70%; when the retention time is 18 min to 27 min, the volume fraction of phase A is 2%, and the volume fraction of phase B is 98%; when the retention time is 27 min to 30 min, the volume fraction of phase A is 97%, and the volume fraction of phase B is 3%.

[0070] The mass spectrometry conditions were as follows: electrospray ion source; ion source temperature 350°C; positive ion and negative ion scanning simultaneously; electrospray voltage: positive ion 5500V, negative ion -4500V; nebulizer gas: 0.345MPa; auxiliary heating gas: 0.345MPa.

[0071] A total of 57 kinds of pesticides were detected, including isoprocarb, oxadiazon, toxaphene, atratone, prometon, simazine, atrazine, propazine, glyphosate, terbuthylazine, parathion-ethyl, carbaryl, carbofuran, diazinon, secbumeton, azoxystrobin, isoprothiolane, isoprothiolane, parathion-methyl, cyflufenamid, fenpyrazamine, carboxin, simetryn, ametryn, oxathiapiprolin, cyproconazole-imidazole, imidazolinone, prometryn, terbutryn, fenitrothion, malathion, dichlorvos, dimethomorph, phenthoate, quinalphos, p, p'-DDE, buprofezin, p, p'-DDD, triazophos, chlorantraniliprole, tolfenpyrad, abamectin, phosalone, procymidone, metalaxyl, fipronil, iprobenfos, thiabendazole, thiophanate-methyl, carbendazim, phoxim, butachlor, dinotefuran, clothianidin, imidacloprid, tebuconazole and thiamethoxam.

[0072] Example 4

[0073] A method for detecting pesticide residues in leaves of Pandanus amaryllifolius Roxb., comprising:

[0074] First, sample collection and treatment

[0075] The collected leaves of Pandanus amaryllifolius Roxb. are crushed into leaf fragments, an extraction reagent and glass beads are added to the leaf fragments, and the mass ratio of the leaf fragments, the extraction reagent and the glass beads is 1:10:0.3; shaken on a shaker at 200 r / min for 20 min, then centrifuged to remove the glass beads and leaf fragments, the first supernatant is quantitatively collected, a purification and adsorption material is added to the first supernatant, shaken on a vortex mixer for 2 min, centrifuged, the second supernatant is quantitatively collected, and filtered to obtain a sample loading solution.

[0076] Among them, the purification and adsorption material is composed of neutral alumina, SCX strong cation chromatography packing and amino-functionalized multi-walled carbon nanotubes mixed in a mass ratio of 10:100:1; the mass ratio of the first supernatant to the purification and adsorption material is 10:1.

[0077] The extraction reagent is a mixture of acetonitrile-acetic acid solution, anhydrous magnesium sulfate and sodium acetate in a mass ratio of 20:6:2; among them, the acetonitrile-acetic acid solution is a mixture of acetonitrile and acetic acid in a volume ratio of 97:3.

[0078] The preparation method of the leaf fragments is: first cut the leaves of Pandanus amaryllifolius Roxb. to be measured into leaf segments, and then crush them with a knife crusher to form leaf fragments with a particle size of 1 mm.

[0079] The diameter of the glass beads is 3 mm.

[0080] Second, detect by liquid chromatography-mass spectrometry

[0081] The liquid chromatography conditions are as follows: chromatographic column C18, inner diameter 2.1 mm × 100 mm, particle size 1.8 μm; mobile phase: phase A is an aqueous solution of ammonium formate - formic acid, phase B is a methanol solution of ammonium formate - formic acid, with a gradient change in the mobile phase; flow rate 0.3 mL / min; column temperature 40 °C; injection volume of the sample solution is 2 μL.

[0082] Among them, retention time 0 - 1 min, volume fraction of phase A 97%, volume fraction of phase B 3%; retention time 1 min - 1.5 min, volume fraction of phase A 85%, volume fraction of phase B 15%; retention time 1.5 min - 2.5 min, volume fraction of phase A 50%, volume fraction of phase B 50%; retention time 2.5 min - 18 min, volume fraction of phase A 30%, volume fraction of phase B 70%; retention time 18 min - 27 min, volume fraction of phase A 2%, volume fraction of phase B 98%; retention time 27 min - 30 min, volume fraction of phase A 97%, volume fraction of phase B 3%.

[0083] The mass spectrometry conditions are as follows: electrospray ionization source; ion source temperature 350 °C; positive and negative ions are scanned simultaneously; electrospray voltage: 5500 V for positive ions, -4500 V for negative ions; nebulizing gas: 0.345 MPa; auxiliary heating gas: 0.345 MPa.

[0084] A total of 57 pesticides were detected, including isoprocarb, oxadiargyl, toxaphene, atraton, prometon, simazine, atrazine, propazine, glyphosate, terbuthylazine, parathion - ethyl, carbaryl, carbofuran, diazinon, butralin, azoxystrobin, isoprothiolane, isoprobenfos, parathion - methyl, cyflufenamid, pyridinitril, fenclorim, simetryn, ametryn, famoxadone, cyproconazole, imazethapyr, prometryn, terbutryn, fenitrothion, malathion, dichlorvos, dimethomorph, phenthoate, quinalphos, p, p'-DDE, buprofezin, p, p'-DDD, triazophos, chlorantraniliprole, tolfenpyrad, avermectin, phosalone, procymidone, metalaxyl, fipronil, iprovalicarb, thiabendazole, thiophanate - methyl, carbendazim, phoxim, butachlor, dinotefuran, clothianidin, imidacloprid, tebuconazole, and thiamethoxam.

[0085] Example 5

[0086] A method for detecting pesticide residues in leaves of Pandanus amaryllifolius Roxb., comprising:

[0087] First, sample collection and treatment

[0088] The collected Elaeagnus odoratus leaves were crushed into leaf pieces, and extraction reagents and glass beads were added to the leaf pieces, with the mass ratio of leaf pieces, extraction reagents and glass beads being 1:10:0.3; the mixture was shaken on a shaker at 200 rpm for 20 min, and then the glass beads and leaf pieces were removed by centrifugation, the first supernatant was quantitatively collected, purification adsorption materials were added to the first supernatant, the mixture was shaken on a vortex machine for 2 min, and centrifuged, the second supernatant was quantitatively collected, and filtered as the sample solution.

[0089] The purification adsorption material is a mixture of neutral alumina, SCX strong cationic chromatography filler and amino multi-walled carbon nanotubes in a mass ratio of 15:100:1; the mass ratio of the first supernatant to the purification adsorption material is 10:1.

[0090] The extraction reagent is a mixture of acetonitrile-acetic acid solution, anhydrous magnesium sulfate, and sodium acetate in a mass ratio of 20:6:1; wherein the acetonitrile-acetic acid solution is a mixture of acetonitrile and acetic acid in a volume ratio of 97:3.

[0091] The method for preparing leaf pieces is as follows: firstly, the leaves of the tested bergamot are cut into leaf segments, and then crushed with a knife crusher to form leaf pieces with a particle size of 1 mm.

[0092] The diameter of the glass beads is 3 mm.

[0093] Second, liquid chromatography-mass spectrometry was used for detection

[0094] The liquid chromatography conditions were as follows: chromatographic column C18, inner diameter 2.1 mm × 100 mm, particle size 1.8 μm; mobile phase: phase A was ammonium formate-formic acid aqueous solution, phase B was ammonium formate-formic acid methanol solution, mobile phase gradient change; flow rate 0.3 mL / min; column temperature 40°C; injection volume of the sample solution was 2 μL.

[0095] Among them, when the retention time is 0 to 1 min, the volume fraction of phase A is 97%, and the volume fraction of phase B is 3%; when the retention time is 1 min to 1.5 min, the volume fraction of phase A is 85%, and the volume fraction of phase B is 15%; when the retention time is 1.5 min to 2.5 min, the volume fraction of phase A is 50%, and the volume fraction of phase B is 50%; when the retention time is 2.5 min to 18 min, the volume fraction of phase A is 30%, and the volume fraction of phase B is 70%; when the retention time is 18 min to 27 min, the volume fraction of phase A is 2%, and the volume fraction of phase B is 98%; when the retention time is 27 min to 30 min, the volume fraction of phase A is 97%, and the volume fraction of phase B is 3%.

[0096] The mass spectrometry conditions were as follows: electrospray ion source; ion source temperature 350°C; positive ion and negative ion scanning simultaneously; electrospray voltage: positive ion 5500V, negative ion -4500V; nebulizer gas: 0.345MPa; auxiliary heating gas: 0.345MPa.

[0097] A total of 57 kinds of pesticides were detected, including isoprocarb, oxadiargyl, toxaphene, atratone, prometon, simazine, atrazine, propazine, glyphosate, terbuthylazine, parathion-ethyl, carbaryl, carbofuran, diazinon, secbumeton, azoxystrobin, isoprothiolane, isoprothiolane, parathion-methyl, cyflufenamid, fenpyrazamine, cloquintocet-mexyl, simetryn, ametryn, oxadixyl, cyproconazole, imidazolinone, prometryn, terbutryn, fenitrothion, malathion, dichlorvos, dimethomorph, phenthoate, quinalphos, p, p'-DDE, buprofezin, p, p'-DDD, triazophos, chlorantraniliprole, tolfenpyrad, abamectin, phosalone, procymidone, metalaxyl, fipronil, iprobenfos, thiabendazole, thiophanate-methyl, carbendazim, phoxim, butachlor, dinotefuran, clothianidin, imidacloprid, tebuconazole and thiamethoxam.

[0098] Example 6

[0099] A method for detecting pesticide residues in leaves of Pandanus amaryllifolius Roxb., comprising:

[0100] First, sample collection and treatment

[0101] The collected leaves of Pandanus amaryllifolius Roxb. are crushed into leaf fragments, an extraction reagent and glass beads are added to the leaf fragments, and the mass ratio of the leaf fragments, the extraction reagent and the glass beads is 1:10:0.3; shaken on a shaker at 200 r / min for 20 min, then centrifuged to remove the glass beads and leaf fragments, the first supernatant is quantitatively collected, a purification and adsorption material is added to the first supernatant, vortexed on a vortex mixer for 2 min, centrifuged, the second supernatant is quantitatively collected, and filtered to obtain a sample loading solution.

[0102] Among them, the purification and adsorption material is composed of neutral alumina, SCX strong cation chromatography packing and amino-functionalized multi-walled carbon nanotubes mixed according to a mass ratio of 10:100:1; the mass ratio of the first supernatant to the purification and adsorption material is 10:2.

[0103] The extraction reagent is a mixture of acetonitrile-acetic acid solution, anhydrous magnesium sulfate and sodium acetate according to a mass ratio of 20:6:1; wherein the acetonitrile-acetic acid solution is a mixture of acetonitrile and acetic acid according to a volume ratio of 97:3.

[0104] The preparation method of the leaf fragments is: first cut the leaves of Pandanus amaryllifolius Roxb. to be tested into leaf segments, and then crush them with a knife crusher to form leaf fragments with a particle size of 1 mm.

[0105] The diameter of the glass beads is 3 mm.

[0106] Second, detect by liquid chromatography-mass spectrometry

[0107] The liquid chromatography conditions were as follows: chromatographic column C18, inner diameter 2.1 mm × 100 mm, particle size 1.8 μm; mobile phase: phase A was an aqueous solution of ammonium formate - formic acid, phase B was a methanol solution of ammonium formate - formic acid, with a gradient change in the mobile phase; flow rate 0.3 mL / min; column temperature 40 °C; injection volume of the sample solution was 2 μL.

[0108] Among them, for retention time 0 - 1 min, the volume fraction of phase A was 97% and that of phase B was 3%; for retention time 1 min - 1.5 min, the volume fraction of phase A was 85% and that of phase B was 15%; for retention time 1.5 min - 2.5 min, the volume fraction of phase A was 50% and that of phase B was 50%; for retention time 2.5 min - 18 min, the volume fraction of phase A was 30% and that of phase B was 70%; for retention time 18 min - 27 min, the volume fraction of phase A was 2% and that of phase B was 98%; for retention time 27 min - 30 min, the volume fraction of phase A was 97% and that of phase B was 3%.

[0109] The mass spectrometry conditions were as follows: electrospray ionization source; ion source temperature 350 °C; simultaneous scanning of positive and negative ions; electrospray voltage: 5500 V for positive ions and -4500 V for negative ions; nebulizing gas: 0.345 MPa; auxiliary heating gas: 0.345 MPa.

[0110] A total of 57 pesticides were detected, including isoprocarb, oxadiazon, toxaphene, atratone, prometon, simazine, atrazine, propazine, glyphosate, terbuthylazine, parathion - ethyl, carbaryl, carbofuran, diazinon, sec - butylazine, azoxystrobin, isoprothiolane, isoprothiolane, methyl parathion, cyflufenamid, fenpyrazamine, cloethocarb, simetryn, ametryn, famoxadone, cyproconazole, imidazolinone, prometryn, terbutryn, fenitrothion, malathion, dichlorvos, dimethomorph, phenthoate, quinalphos, p, p'-DDE, buprofezin, p, p'-DDD, triazophos, chlorantraniliprole, tolfenpyrad, abamectin, phosalone, procymidone, metalaxyl, fipronil, iprobenfos, thiabendazole, thiophanate - methyl, carbendazim, phoxim, butachlor, dinotefuran, clothianidin, imidacloprid, tebuconazole, and thiamethoxam.

[0111] In addition, we also set up Examples 7 to 11 in Table 1, and 57 kinds of pesticides can be detected, including isoprocarb, oxadiargyl, toxaphene, atratone, prometon, simazine, atrazine, propazine, glyphosate, terbutryn, parathion-ethyl, carbaryl, carbofuran, diazinon, secbumeton, azoxystrobin, iprobenfos, isoprothiolane, parathion-methyl, cyflufenamid, fenpyrazamine, cloquintocet-mexyl, simetryn, ametryn, oxadixyl, cycloxydim, imidazolinone, prometryn, terbuthylazine, fenitrothion, malathion, dichlorvos, dimethomorph, phenthoate, quinalphos, p, p'-DDE, buprofezin, p, p'-DDD, triazophos, chlorantraniliprole, tolfenpyrad, abamectin, phosalone, procymidone, metalaxyl, fipronil, iprovalicarb, thiabendazole, thiophanate-methyl, carbendazim, phoxim, butachlor, dinotefuran, clothianidin, imidacloprid, tebuconazole, and thiamethoxam.

[0112] Table 1 Settings of Different Examples

[0113] Example Condition Example 7 The leaves of *Pandanus amaryllifolius* were crushed into leaf fragments with a size of 2 mm, and the rest was the same as in Example 1 Example 8 The times of the two oscillations were changed to 40 min and 4 min respectively Example 9 The diameter of the glass beads was 1 mm, and the rest was the same as in Example 1 Example 10 Shake on a shaker at 250 r / min, and the rest was the same as in Example 1 Example 11 Centrifuge at 4200 r / min and 4 °C for 10 min, and the rest was the same as in Example 1

[0114] Table 2 Detection Effects of Different Purification and Adsorption Materials

[0115]

[0116] Specifically, the key points and results of the detection methods for each control are as follows.

[0117] Control 1

[0118] A method for detecting pesticide residues in the leaves of Pandanus amaryllifolius Roxb., and the purification and adsorption material is a mixture of neutral alumina and SCX strong cation chromatography packing material in a mass ratio of 1:10.

[0119] All other operating conditions are the same as those in Example 1.

[0120] A total of 36 kinds of pesticides were detected, including isoprocarb, oxadiargyl, toxaphene, atratone, prometon, simazine, atrazine, propazine, terbutryn, parathion-ethyl, carbaryl, carbofuran, diazinon, secbumeton, isoprothiolane, cyflufenamid, fenpyrazamine, cloquintocet-mexyl, oxadixyl, cycloxydim, imidazolinone, prometryn, fenitrothion, malathion, quinalphos, triazophos, abamectin, phosalone, procymidone, metalaxyl, iprovalicarb, thiabendazole, thiophanate-methyl, carbendazim, butachlor, dinotefuran, and thiamethoxam.

[0121] Control 2

[0122] A method for detecting pesticide residues in the leaves of Pandanus amaryllifolius Roxb., and the purification and adsorption material is a mixture of neutral alumina and amino-functionalized multi-walled carbon nanotubes in a mass ratio of 10:1.

[0123] All other operating conditions are the same as those in Example 1.

[0124] A total of 35 pesticides were detected, including isoprocarb, toxaphene, atratone, prometon, simazine, atrazine, propazine, parathion-ethyl, carbaryl, diazinon, isoprothiolane, cyflufenamid, simetryn, ametryn, oxfendazole, cyproconazole, imidazolinone, prometryn, terbutryn, malathion, phenthoate, p, p'-DDE, buprofezin, triazophos, tolfenpyrad, abamectin, metalaxyl, fipronil, iprobenfos, thiabendazole, phoxim, butachlor, dinotefuran, clothianidin, and thiamethoxam.

[0125] Control 3

[0126] A method for detecting pesticide residues in the leaves of Pandanus amaryllifolius Roxb., wherein the purification and adsorption material is prepared by mixing SCX strong cation exchange chromatography packing and aminated multi-walled carbon nanotubes at a mass ratio of 100:1.

[0127] All other operating conditions are the same as those in Example 1.

[0128] A total of 36 pesticides were detected, including isoprocarb, oxazole oxalic acid, toxaphene, atratone, prometon, simazine, atrazine, propazine, carbaryl, carbofuran, diazinon, secbumeton, azoxystrobin, isoprothiolane, cyflufenamid, oxfendazole, imidazolinone, prometryn, terbutryn, fenitrothion, malathion, dichlorvos, p, p'-DDD, triazophos, chlorantraniliprole, phosalone, procymidone, fipronil, iprobenfos, phoxim, butachlor, dinotefuran, clothianidin, imidacloprid, tebuconazole, and thiamethoxam.

[0129] Control 4

[0130] A method for detecting pesticide residues in the leaves of Pandanus amaryllifolius Roxb., wherein the purification and adsorption material is prepared by mixing neutral alumina, SCX strong cation exchange chromatography packing and GCB at a mass ratio of 10:100:1.

[0131] All other operating conditions are the same as those in Example 1.

[0132] A total of 34 pesticides were detected, including isoprocarb, oxazole oxalic acid, toxaphene, atratone, prometon, simazine, carbofuran, diazinon, secbumeton, azoxystrobin, isoprothiolane, isoprothiolane, methyl parathion, imidazolinone, prometryn, terbutryn, fenitrothion, malathion, dichlorvos, quinalphos, p, p'-DDE, buprofezin, p, p'-DDD, triazophos, metalaxyl, fipronil, iprobenfos, thiabendazole, thiophanate-methyl, carbendazim, phoxim, butachlor, tebuconazole, and thiamethoxam.

[0133] Control 5

[0134] A method for detecting pesticide residues in leaves of Pandanus amaryllifolius Roxb., wherein the purification and adsorption material is neutral alumina, SCX strong cation chromatography packing and GCB in a mass ratio of 10:100:1.

[0135] All other operating conditions are the same as those in Example 1.

[0136] A total of 29 pesticides were detected, including butralin, azoxystrobin, isoprothiolane, isoprobenfos, methyl parathion, pyridinitril, cloethocarb, simetryn, ametryn, oxadixyl, cyproconazole, prometryn, terbutryn, fenitrothion, malathion, dichlorvos, dimethomorph, phenthoate, quinalphos, iprovalicarb, thiabendazole, thiophanate-methyl, carbendazim, phoxim, butachlor, dinotefuran, imidacloprid, tebuconazole and thiamethoxam.

[0137] Control 6

[0138] A method for detecting pesticide residues in leaves of Pandanus amaryllifolius Roxb., wherein the purification and adsorption material is neutral alumina.

[0139] All other operating conditions are the same as those in Example 1.

[0140] A total of 30 pesticides were detected, including isoprocarb, oxazole oxalic acid, propazine, simazine, glyphosate, terbuthylazine, ethyl parathion, carbaryl, carbofuran, diazinon, butralin, cyproconazole, imidazolinone, prometryn, terbutryn, fenitrothion, phenthoate, quinalphos, p, p'-DDE, buprofezin, p, p'-DDD, triazophos, chlorantraniliprole, carbendazim, tolfenpyrad, abamectin, phosalone, procymidone, imidacloprid and tebuconazole.

[0141] Control 7

[0142] A method for detecting pesticide residues in leaves of Pandanus amaryllifolius Roxb., wherein the purification and adsorption material is SCX strong cation chromatography packing.

[0143] All other operating conditions are the same as those in Example 1.

[0144] A total of 32 pesticides were detected, including isoprocarb, oxazole oxalic acid, toxaphene, atratone, propazine, simazine, atrazine, propazine, glyphosate, terbuthylazine, ethyl parathion, carbaryl, carbofuran, diazinon, butralin, azoxystrobin, terbutryn, fenitrothion, malathion, dichlorvos, dimethomorph, phenthoate, quinalphos, triazophos, chlorantraniliprole, tolfenpyrad, carbendazim, phoxim, butachlor, dinotefuran, thiamethoxam and imidacloprid.

[0145] Control 8

[0146] A method for detecting pesticide residues in leaves of Pandanus amaryllifolius Roxb., wherein the purification and adsorption material is amino-functionalized multi-walled carbon nanotubes.

[0147] All other operating conditions are the same as in Example 1.

[0148] A total of 30 kinds of pesticides were detected, including isoprocarb, oxadiargyl, prometon, simazine, atrazine, propazine, carbofuran, diazinon, butralin, cycloanilide, imidazolinone, prometryn, terbutryn, fenitrothion, malathion, dichlorvos, phosalone, procymidone, metalaxyl, fipronil, iprovalicarb, thiabendazole, thiophanate-methyl, carbendazim, phoxim, butachlor, dinotefuran, imidacloprid, tebuconazole, and thiamethoxam.

[0149] Table 2 compares the single-factor purification adsorption materials and different combinations of purification adsorption materials. The results show that the method of the embodiment of the present invention detects the largest number of pesticide species. And it can be seen from Table 2 that even though they are all purification adsorption materials, when different combinations are used, there are still significant differences in the detected pesticide species, and there is no rule to predict which combination may have better effects. The present invention first discovers that for the detection of pesticide residues in Pandanus amaryllifolius leaves, when the purification adsorption material is composed of neutral alumina, SCX strong cation chromatography packing, and amino-functionalized multi-walled carbon nanotubes in a mass ratio of 10-15:100:1, the largest number of pesticide species can be detected, reaching 57 kinds.

[0150] To further verify the effect of the present invention, we also carried out a standard addition recovery experiment. The Pandanus amaryllifolius leaves cultured in the laboratory without any pesticide addition were used as a blank control, processed into leaf fragments according to the method of Example 1, then a series of concentration standard solutions were added to the extraction reagent, and then processed and detected according to the method of Example 1 to draw a standard curve.

[0151] In addition, low, medium, and high amounts of specific pesticides were added to the Pandanus amaryllifolius leaves in the known wild areas of Hainan for the standard addition recovery test, and the results are shown in Table 2. The results show that the standard addition recovery rates are all above 90%.

[0152] Table 2 Results of the standard addition recovery experiment

[0153]

[0154] For 10 independent repeated detection tests with a pesticide addition amount of 0.1 mg / kg, the average value and standard deviation were calculated. The results are shown in Table 3.

[0155] Table 3 Results of the standard addition recovery experiment

[0156] Pesticide name Average value mg / kg Standard deviation Isoprocarb 0.11 0.3％ Oxaziclomefone 0.11 1.4％ Atrizine 0.10 0.1％ Prometon 0.12 0.6％

[0157] It should be noted that when the present invention involves numerical ranges, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the step methods adopted are the same as those in the embodiments, in order to avoid redundancy, the present invention describes preferred embodiments. Although the preferred embodiments of the present invention have been described, once those skilled in the art know the basic inventive concept, additional changes and modifications can be made to these embodiments, and these changes and modifications all fall within the scope of the present invention.

[0158] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. If these modifications and variations of the present invention fall within the scope of equivalent technologies of the present invention, the present invention also intends to include these changes and modifications.

Claims

1. A method for detecting the pesticide residue amount in the leaves of Pandanus amaryllifolius Roxb., characterized in that, include: First, sample collection and processing The collected leaves of the fragrant loquat were crushed into leaf pieces, and the extraction reagent and glass beads were added to the leaf pieces, and the mass ratio of leaf pieces, extraction reagent and glass beads was 1:10-20:0.3-1; the mixture was shaken for 20-40 minutes, and then the glass beads and leaf pieces were removed by centrifugation, and the first supernatant was collected, and the purification adsorption material was added to the first supernatant, and the mixture was shaken for 2-4 minutes, and centrifuged, and the second supernatant was collected and filtered as the sample solution; The purification adsorption material is a mixture of neutral alumina, SCX strong cationic chromatography filler and amino multi-walled carbon nanotubes in a mass ratio of 10-15:100:1; the mass ratio of the first supernatant to the purification adsorption material is 10:1-2; Second, the sample solution is injected into the liquid chromatography-mass spectrometer for pesticide residue detection; The pesticides include isoprocarb, oxadiazine, toxaphene, atrazine, promethazine, simazine, atrazine, promethazine, glyphosate, terbuthion, ethyl parathion, carbaryl, furadan, diazinon, sec-butyl, azoxystrobin, isothiocyanate, pyraclostrobin, methyl parathion, cyfluthrin, pyraclostrobin, chloranil, oxadiazine, cypermethrin, oxadiazine, cypermethrin, pyraclostrobin, cypermethrin, cypermethrin, oxadiazine, cypermethrin, pyraclostrobin, cypermethrin, cypermethrin, cypermethrin, cypermethrin, cypermethrin, cypermethrin, cypermethrin, cypermethrin One or more of phosphorus, malathion, dichlorvos, dimethomorph, fenthion, quinalphos, p,p'-DDE, buprofezin, p,p'-DDD, triazophos, chlorfenapyr, tolfenpyrad, avermectin, phosalone, procymidone, metalaxyl, fipronil, imamectin, thiabendazole, methyl thiophanate, carbendazim, phoxim, butachlor, dinotefuran, clothianidin, imidacloprid, tebuconazole and thiamethoxam.

2. The detection method of the pesticide residue in the leaves of Pandanus amaryllifolius Roxb. according to claim 1, wherein The extraction reagent is a mixture of acetonitrile-acetic acid solution, anhydrous magnesium sulfate, and sodium acetate in a mass ratio of 20:6:1-2; Among them, every 100L of acetonitrile-acetic acid solution contains 3L~4L of acetic acid, and the remainder is acetonitrile.

3. The detection method of pesticide residue in leaves of Pandanus amaryllifolius Roxb. according to claim 1, characterized in that, The diameter of glass beads is 1mm~3mm.

4. The detection method for pesticide residue in leaves of Pandanus amaryllifolius Roxb. according to claim 1, characterized in that, The shaking conditions are: placed in a shaker and shaken at 200 rpm ~ 250 rpm.

5. The detection method of pesticide residue in the leaves of Pandanus amaryllifolius Roxb. according to claim 1, wherein, The centrifugation conditions are 4200r / min and 4℃ for 5min~10min.

6. The detection method for the pesticide residue amount of Pandanus amaryllifolius Roxb. leaves according to claim 1, characterized in that, The filtration is carried out using a microporous membrane filter, wherein the diameter of the micropores is 0.22 μm.

7. The detection method of pesticide residue in the leaves of Pandanus amaryllifolius Roxb. according to claim 1, characterized in that, The method for preparing the leaf pieces is as follows: firstly cut the leaves of the tested bergamot into leaf segments, and then grind them into leaf pieces with a particle size of 1 mm to 2 mm.

8. The detection method of the pesticide residue amount in the leaves of Pandanus amaryllifolius Roxb. according to claim 1, characterized in that, The liquid chromatography conditions of the liquid chromatography-mass spectrometry instrument are: Chromatographic column C18, inner diameter 2.1 mm × 100 mm, particle size 1.8 μm; Mobile phase: Phase A is ammonium formate-formic acid aqueous solution, phase B is ammonium formate-formic acid methanol solution, mobile phase gradient changes; Flow rate: 0.3 mL / min; Column temperature: 40°C; The injection volume of the sample solution was 2 μL.

9. The detection method of pesticide residue in the leaves of Pandanus amaryllifolius Roxb. according to claim 8, characterized in that, The mass spectrometry conditions of the liquid chromatography-mass spectrometry instrument are: Electrospray ion source; ion source temperature 350°C; positive ion and negative ion scanning simultaneously; electrospray voltage: positive ion 5500V, negative ion -4500V; nebulizing gas: 0.345MPa; auxiliary heating gas: 0.345MPa.

Images