Hybrid monoliths doped with halloysite nanotubes, methods of making and using the same

Abstract

The application discloses a hybrid monolithic column doped with halloysite nanotubes and a preparation method and application thereof. S After the quartz capillary is activated and dried, the quartz capillary is modified with a mixed solution of gamma-MAPS and methanol to obtain a vinyl-modified quartz capillary; 24-26 parts of GMA, 15-25 parts of amino-PEG-amino, and 350-700 parts of formamide are mixed and reacted at 30-50 DEG C for 0.5-1.5 h, and then the reaction is terminated in an environment of 0-4 DEG C to obtain a mixed solution; 12-13 parts of AM, 25-60 parts of HNT S After the mixed solution is shaken and dissolved, the mixed solution is injected into the vinyl-modified quartz capillary, then both ends of the capillary are sealed with silica gel, and the capillary is placed in a water bath at 70-80 DEG C and reacted for 6-20 h; after the reaction is completed, the capillary is washed with acetonitrile or methanol.

Description

Technical Field

[0001] This disclosure relates to the field of detection technology for polar cationic pesticide residues in agricultural products, and in particular to a hybrid monolithic column doped with halloysite nanotubes, its preparation method and application. Background Technology

[0002] Agricultural products are essential foods in people's daily lives, with huge daily consumption and market supply and demand. To increase yield and improve quality, the use of pesticides is unavoidable. However, the impact of pesticide residues on food safety and the ecological environment cannot be ignored. Therefore, pesticide residues have become one of the major food safety issues. GB2763-2021, the National Food Safety Standard for Maximum Residue Limits of Pesticides in Food, implemented in September 2021, stipulates maximum residue limits for 564 pesticides in 10,092 items across 376 types of food. Faced with such a wide variety of pesticide maximum residue limits, monitoring pesticide residues has become extremely difficult. Currently, multi-residue analysis of pesticides in agricultural products is usually completed through the following steps: based on non-specific extraction, followed by determination of hundreds of pesticides using liquid chromatography-tandem mass spectrometry or gas chromatography-tandem mass spectrometry. However, these methods do not include some special pesticides, namely compounds classified as single-residue methods. Due to their strong polarity, these pesticides exhibit weak retention on standard reversed-phase chromatographic columns and suffer significant decomposition and loss during sample processing, leading to their exclusion from most pesticide multi-residue analysis methods. Therefore, employing appropriate sample pretreatment to avoid interference from complex matrices and achieve accurate detection of highly polar pesticide residues in agricultural products is extremely important to compensate for the shortcomings of existing pesticide multi-residue analysis methods.

[0003] Due to the high water solubility of highly polar pesticides, they are not suitable for extraction using the traditional QuEChERS (Quick easy cheap effective rugged and safe) or ethyl acetate. Currently, the extraction method for highly polar pesticides usually refers to the Quick Polar Pesticides (QuPPe) method provided by the European Union Reference Laboratory [1]. Polar pesticides are extracted with a mixed solution of acidified methanol and water, heated to 80°C, and after 15 min, diluted with the initial mobile phase at a ratio of 1:4 and analyzed directly by HILIC-MS / MS [2]. Nortes-Méndez et al. [3] also referred to the QuPPe method, using a mixed solution of acidified methanol and water for extraction, diluted with acetonitrile containing 0.1% formic acid at a volume ratio of 1:4, centrifuged and filtered, and then determined by LC-MS / MS. López et al. [4] used water and acidified methanol to extract highly polar pesticides from food. Before injecting the extract into LC-MS / MS for analysis, the extract was diluted 12.5 times to reduce matrix interference. The research group further used a 60% acetonitrile aqueous solution containing 0.2% trifluoroacetic acid to extract highly polar anionic pesticides from plant milk, wine, and beer. The extract was diluted 50 times before determination [5]. As with the above method, since the purification step is omitted after the extraction of highly polar pesticides, the extract needs to be diluted to reduce matrix interference, with the maximum dilution factor reaching 50 times. Although diluting the extract can reduce matrix interference, it also limits the detection limit of the method, which is not conducive to the detection of trace highly polar pesticides.

[0004] To reduce matrix interference, Kaczyński[6] improved the QuPPe method by using a 1:1 (v / v) mixture of acidified methanol and water to extract highly polar herbicides from plant-based foods, followed by a purification step: the supernatant was mixed with chitosan or graphene, centrifuged and filtered, and then analyzed by LC-MS / MS. Francesquett et al.[7] used 1:1 acidified methanol and water to extract quaternary ammonium pesticides from barley and wheat, followed by purification with dichloromethane, chitosan and acetonitrile, and finally detection by HILIC-MS / MS. Although adding a purification adsorbent to the extract can reduce matrix interference, it does not enrich the target compound, making it difficult to detect trace amounts of highly polar pesticides.

[0005] References

[0006] [1]M.Anastassiades,DIKolberg,E.Eichhorn,AKWachtler,A.Benkestein,S.Zechmann,D.Mack,C.Wildgrube,A.Barth,I.Sigalov,S. D. G.Cerchia.Quick method for the analysis of numerous highly polar pesticidesin food involving extraction with acidified methanol and LC-MS / MS measurement1.Food and plant origin(QuPPe-PO-Method)-Version 11.In EU ReferenceLaboratory for pesticides requiring Single Residue Methods(EURL-SRM)2020.

[0007] [2]A.Vass,J.Robles-Molina,P.Pérez-Ortega,B.Gilbert-López,M.Dernovics,A.Molina-Díaz,J.F.García-Reyes.Study of different HILIC,mixed-mode,and otheraqueous normal-phase approaches for the liquid chromatography / massspectrometry-based determination of challenging polar pesticides.Anal.Bioanal.Chem.2016,408(18):4857-4869.

[0008] [3]R.Nortes-Méndez,J.Robles-Molina,R.López-Blanco,A.Vass,A.Molina-Díaz,J.F.Garcia-Reyes.Determination of polar pesticides in olive oil and olivesby hydrophilic interaction liquid chromatography coupled to tandem massspectrometry and high resolution mass spectrometry.Talanta 2016,158:222-228.

[0009] [4]S.H.López,J.Scholten,B.Kiedrowska,A.de Kok.Method validation andapplication of a selective multiresidue analysis of highly polar pesticidesin food matrices using hydrophilic interaction liquid chromatography and massspectrometry.J.Chromatogr.A,2019,1594:93-104.

[0010] [5]S.H.López,J.Dias,H.Mol,A.de Kok.Selective multiresiduedetermination of highly polar anionic pesticides in plant-based milk,wine andbeer using hydrophilic interaction liquid chromatography combined with tandemmass spectrometry.J.Chromatogr.A 2020,1625:461226.

[0011] [6]P.Kaczyński.Clean-up and matrix effect in LC-MS / MS analysis offood of plant origin for high polar herbicides.Food Chem.2017,230:524-531.

[0012] [7]JZFrancesquett,T.Rizzetti,TRSCadaval Jr.,ODPrestes,MBAdaime,R.Zanella.Simultaneous determination of the quaternary ammoniumpesticides paraquat,diquat,chlormequat,and mepiquat in barley and wheat using amodified quick polar pesticides method,diluted standard addition calibrationand hydrophilic interaction liquid chromatography coupled to tandem massspectrometry.J.Chromatogr.A 2019,1592:101-111. Summary of the Invention

[0013] This disclosure provides a hybrid monolithic column doped with halloysite nanotubes, its preparation method, and its application, in order to at least solve one of the technical problems existing in the prior art.

[0014] According to a first aspect of this disclosure, a method for preparing a hybrid monolithic column doped with halloysite nanotubes is provided, comprising the following steps: Step 1): purifying halloysite using sodium hexametaphosphate to obtain sodium hexametaphosphate purified halloysite nanotubes;

[0015] Step 2): Modify halloysite nanotubes purified from sodium hexametaphosphate with anhydrous toluene containing 3-(methacryloyloxy)propyltrimethoxysilane to obtain vinyl-modified halloysite;

[0016] Step 3): After activating and drying the quartz capillary, it is modified with a mixture of 3-(methacryloyloxy)propyltrimethoxysilane and methanol to obtain a vinyl-modified quartz capillary.

[0017] Step 4): Including Step 4-1): Mix 24-26 parts of glycidyl methacrylate, 15-25 parts of amino-PEG-amino and 350-700 parts of formamide and react at 30-50°C for 0.5-1.5 h, then terminate the reaction at 0-4°C to obtain a mixed solution;

[0018] Step 4-2): Add 12-13 parts acrylamide, 25-60 parts vinyl-modified halloysite, 24-26 parts N,N′-methylenebisacrylamide, 70-180 parts PEG and 1-2 parts azobisisobutyronitrile to the mixed solution, shake to dissolve, and then inject into the vinyl-modified quartz capillary. Then seal both ends of the capillary with silicone and place it in a water bath at 70-80℃ for 6-20 hours. After the reaction is completed, rinse the capillary with acetonitrile or methanol to obtain the hybrid monolithic column of the halloysite nanotubes.

[0019] In one embodiment, in step 4-1), the average molecular weight of amino-PEG-amino is 2000; in step 4-2), PEG is PEG10000.

[0020] In one embodiment, in step 4-1), 25 parts of glycidyl methacrylate, 25 parts of amino-PEG-amino, and 350 parts of formamide are used.

[0021] In step 4-2), there are 12.5 parts of acrylamide, 60 parts of vinyl-modified halloysite, 25 parts of N,N′-methylenebisacrylamide, 70 parts of PEG, and 1.5 parts of azobisisobutyronitrile.

[0022] In one embodiment, the sodium hexametaphosphate purified halloysite nanotubes obtained in step 1) are prepared by the following method: Halloysite is taken and added to an aqueous solution containing sodium hexametaphosphate at a mass concentration of 0.05% to 0.25%. After stirring for 30 to 60 minutes, the mixture is allowed to stand, the precipitate is discarded, and the dispersed halloysite is washed with water several times, centrifuged, and dried at 60 to 105°C for 5 to 10 hours to obtain the sodium hexametaphosphate purified halloysite; wherein the mass-volume ratio of halloysite to aqueous solution is 1 g: 8 to 20 mL.

[0023] In one embodiment, the vinyl-modified halloysite obtained in step 2) is prepared by the following method: halloysite nanotubes purified by sodium hexametaphosphate and 3-(methacryloyloxy)propyltrimethoxysilane are added to anhydrous toluene, stirred with a magnetic stirrer and refluxed at 110°C for 8–12 h; after the reaction is completed, the mixture is washed several times with anhydrous ethanol and dried at 70–85°C for 6–8 h to obtain the vinyl-modified halloysite; wherein the mass-to-volume ratio of halloysite nanotubes purified by sodium hexametaphosphate to 3-(methacryloyloxy)propyltrimethoxysilane is 3 g: 5–15 mL.

[0024] In one embodiment, the vinyl-modified quartz capillary obtained in step 3) is prepared by the following method: the quartz capillary is first rinsed sequentially with 0.5-1.0 mol / L sodium hydroxide, water, 0.5-1.0 mol / L hydrochloric acid, water, and methanol for 0.5-2 hours each, wherein the sodium hydroxide treatment time is at least 10 hours. After drying, a mixed solution of 50% 3-(methacryloyloxy)propyltrimethoxysilane and methanol is added, and the capillary is rinsed clean with methanol and dried for later use to obtain the vinyl-modified quartz capillary.

[0025] According to a second aspect of this disclosure, this application provides a hybrid monolithic column doped with halloysite nanotubes, characterized in that it is prepared by the preparation method in any of the embodiments of the first aspect.

[0026] According to a third aspect of this disclosure, this application provides the application of hybrid monolithic columns doped with halloysite nanotubes in the detection of polar cationic pesticide residues.

[0027] In one embodiment, the polar cationic pesticide includes any one or more of maleic hydrazine, glyphosate, and cyproheptadine.

[0028] According to a fourth aspect of this disclosure, this application provides a method for detecting polar cationic pesticide residues using a hybrid monolithic column of doped halloysite nanotubes obtained in the second aspect, comprising the following steps:

[0029] Step 1): After crushing the sample, weigh an appropriate amount into a container, and then add a standard solution of polar cationic pesticide;

[0030] Step 2): Then add acetonitrile and sodium chloride, shake, centrifuge at 5000-8000 r / min for 5-10 min, take the supernatant of the upper layer, and repeat the extraction of the lower precipitate with acetonitrile at least once to obtain the supernatant. Combine the supernatants, filter, and obtain the pesticide extract.

[0031] Step 3): Equilibrate the 5-20 cm hybrid monolithic column of doped halloysite nanotubes with 150-500 μL of acetonitrile (volume fraction at least 98%) at a flow rate of 3-20 μL / min; Load 20-150 μL of the pesticide extract obtained in Step 2) at a flow rate of 3-20 μL / min, and then elute with 30-100 μL of a mixture of acetonitrile (pure acetonitrile) and ammonium formate at a flow rate of 3-15 μL / min to obtain the eluent;

[0032] Step 4): Perform liquid chromatography-mass spectrometry detection on the eluent.

[0033] In one embodiment, in step 1), the sample is an agricultural product; the polar cationic pesticide is any one or more of maleic hydrazine, glyphosate, and cyproheptadine.

[0034] In one embodiment, the acetonitrile-ammonium formate mixture in step 3) is obtained by mixing 30% acetonitrile and 70% ammonium formate by volume, wherein the concentration of ammonium formate is 50-200 mmol / L.

[0035] Compared with the prior art, the advantages of this application are: 1) The adsorption capacity of the hybrid monolithic column doped with halloysite nanotubes prepared in this application is greatly increased, thereby improving its adsorption of polar pesticides, which is beneficial for the enrichment and detection of trace polar pesticides; 2) The PEG groups on the surface of the hybrid monolithic column doped with halloysite nanotubes prepared in this application and the residual silanol groups on the surface of the halloysite nanotubes both make it hydrophilic, which easily forms a hydrophilic interaction with polar cationic pesticides, so that the polar cationic pesticides are retained on the hybrid monolithic column doped with halloysite nanotubes. Thus, trace amounts of polar cationic pesticide residues in agricultural products are enriched, with an adsorption capacity of 2.16 μg / cm; 3) The hybrid monolithic column doped with halloysite nanotubes in this application increases the loading volume of polar cationic pesticide extracts; 4) The limit of detection for the detection of trace amounts of polar cationic pesticide residues in agricultural products using the hybrid monolithic column doped with halloysite nanotubes in this application is significantly reduced, improving the sensitivity for the detection of polar cationic pesticides, especially reducing the limit of detection for cyproheptadine to 0.06 ng / mL.

[0036] It should be understood that the description in this section is not intended to identify key or essential features of the embodiments of this disclosure, nor is it intended to limit the scope of this disclosure. Other features of this disclosure will become readily apparent from the following description. Attached Figure Description

[0037] The above and other objects, features, and advantages of this disclosure will become readily apparent from the following detailed description of exemplary embodiments, taken in conjunction with the accompanying drawings. Several embodiments of this disclosure are illustrated in the drawings by way of example and not limitation, in which:

[0038] In the accompanying drawings, the same or corresponding reference numerals indicate the same or corresponding parts.

[0039] Figure 1 A fabrication route diagram of a hybrid monolithic column with doped halloysite nanotubes according to an embodiment of this disclosure is shown;

[0040] Figure 2 A scanning electron microscope image of a hybrid monolithic column of doped halloysite nanotubes according to an embodiment of the present disclosure is shown;

[0041] Figure 3 The X-ray photoelectron spectrum of a hybrid monolithic column of doped halloysite nanotubes according to an embodiment of the present disclosure is shown.

[0042] Figure 4 Thermogravimetric analysis (TGA) plot of a hybrid monolithic column of doped halloysite nanotubes according to an embodiment of this disclosure is shown.

[0043] Figure 5 The infrared spectrum of a hybrid monolithic column of doped halloysite nanotubes according to an embodiment of the present disclosure is shown. Detailed Implementation

[0044] To make the objectives, features, and advantages of this disclosure more apparent and understandable, the technical solutions in the embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this disclosure, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this disclosure without creative effort are within the scope of protection of this disclosure.

[0045] This invention relates to in-tube solid-phase microextraction (SPME) based on a hydrophilic interaction mechanism, applied to the sample pretreatment of highly polar pesticides (i.e., polar cationic pesticides) in agricultural products. It utilizes an adsorbent modified with hydrophilic groups to interact with the highly polar pesticide, adsorbing trace amounts of the pesticide onto halloysite nanotubes (HNT). S On organic-inorganic hybrid monolithic materials (i.e., hybrid monolithic columns), interference from hydrophobic impurities can be effectively reduced. While removing matrix effects, it can also enrich trace amounts of highly polar pesticides. When coupled with hydrophilic interaction chromatography (HILIC)-tandem mass spectrometry (MS / MS), it can achieve ultrasensitive detection of trace amounts of highly polar pesticides in agricultural products, thus improving the method for analyzing highly polar pesticide residues in the multi-residue pesticide analysis system.

[0046] Based on this, in a first aspect, this application provides a method for preparing a hybrid monolithic column doped with halloysite nanotubes, comprising the following steps: Step 1): Halloysite is purified using sodium hexametaphosphate to obtain halloysite nanotubes purified with sodium hexametaphosphate.

[0047] Step 2): Halloysite nanotubes purified from sodium hexametaphosphate were modified with anhydrous toluene containing 3-(methacryloyloxy)propyltrimethoxysilane to obtain vinyl-modified halloysite (HNT).S -vinyl);

[0048] Step 3): After activating and drying the quartz capillary, it is modified with a mixture of 3-(methacryloyloxy)propyltrimethoxysilane and methanol, and then washed with methanol to obtain vinyl-modified quartz capillary.

[0049] Step 4): Including Step 4-1): Mix 24-26 parts of glycidyl methacrylate, 15-25 parts of amino-PEG-amino and 350-700 parts of formamide and react at 30-50°C for 0.5-1.5 h, then cool at 0-4°C to terminate the reaction and obtain a mixed solution;

[0050] Step 4-2): Add 12-13 parts acrylamide, 25-60 parts vinyl-modified halloysite, 24-26 parts N,N′-methylenebisacrylamide, 70-180 parts PEG and 1-2 parts azobisisobutyronitrile to the mixed solution, shake to dissolve, and then inject into the vinyl-modified quartz capillary. Then seal both ends of the capillary with silicone and place it in a water bath at 70-80℃ for 6-20 hours. After the reaction is completed, rinse the capillary with acetonitrile or methanol to obtain a hybrid monolithic column of halloysite nanotubes.

[0051] Preferably, in step 4-1), the average molecular weight of the amino-PEG-amino group is 2000 and the purity is 95%; in step 4-2), the PEG (polyethylene glycol) used is PEG10000.

[0052] Preferably, in step 4-1), there are 25 parts of glycidyl methacrylate, 25 parts of amino-PEG-amino, and 350 parts of formamide.

[0053] In step 4-2), there are 12.5 parts of acrylamide, 60 parts of vinyl-modified halloysite, 25 parts of N,N′-methylenebisacrylamide, 70 parts of PEG, and 1.5 parts of azobisisobutyronitrile.

[0054] Preferably, the halloysite nanotubes purified by sodium hexametaphosphate obtained in step 1) are prepared by the following method: Halloysite is added to an aqueous solution containing 0.05%–0.25% sodium hexametaphosphate by mass, stirred for 30–60 min, allowed to stand, the precipitate is discarded, the dispersed halloysite is washed several times with water, centrifuged, and dried at 60–105 °C for 5–10 h to obtain halloysite purified by sodium hexametaphosphate; wherein the mass-to-volume ratio of halloysite to aqueous solution is 1 g: 8–20 mL. More preferably, the mass-to-volume ratio of halloysite to aqueous solution is 1 g: 10 mL.

[0055] In step 1, sodium hexametaphosphate is used to purify halloysite. An excess of sodium hexametaphosphate is used, as more sodium hexametaphosphate facilitates dispersion of halloysite. A mass-to-volume ratio of 1g:8–20mL is recommended.

[0056] Preferably, the vinyl-modified halloysite (HNT) obtained in step 2) S Vinyl-modified halloysite was prepared as follows: Halloysite nanotubes purified with sodium hexametaphosphate and 3-(methacryloyloxy)propyltrimethoxysilane (γ-MAPS) were added to anhydrous toluene, stirred with a magnetic stirrer, and refluxed at 110°C for 8–12 h. After the reaction was complete, the mixture was washed several times with anhydrous ethanol and dried at 70–85°C for 6–8 h to obtain vinyl-modified halloysite. The mass-to-volume ratio of sodium hexametaphosphate purified halloysite nanotubes to γ-MAPS was 3 g: 5–15 mL. γ-MAPS was required in excess; a mass-to-volume ratio of 3 g: 5–15 mL was preferred, as it yielded vinyl-modified halloysite with good performance while conserving γ-MAPS.

[0057] Preferably, the mass-to-volume ratio of sodium hexametaphosphate-purified halloysite nanotubes, γ-MAPs, and anhydrous toluene is 3 g: 5–15 mL: 50–150 mL. More preferably, the mass-to-volume ratio of sodium hexametaphosphate-purified halloysite nanotubes, γ-MAPs, and anhydrous toluene is 3 g: 5 mL: 50 mL.

[0058] Preferably, the vinyl-modified quartz capillary obtained in step 3) is prepared by the following method: The quartz capillary is first rinsed sequentially with 0.5–1.0 mol / L sodium hydroxide, water, 0.5–1.0 mol / L hydrochloric acid, water, and methanol for 0.5–2 hours each, with the sodium hydroxide treatment time being at least 10 hours. After drying, a 50% (v / v) mixture of γ-MAPS and methanol is added, and the capillary is rinsed clean with methanol and dried for later use to obtain the vinyl-modified quartz capillary. In the γ-MAPS and methanol mixture, the volume percentage of γ-MAPS is 50% and the volume percentage of methanol is 50%.

[0059] In this application, the preparation route of the hybrid monolithic pillar doped with halloysite nanotubes is as follows: Figure 1 As shown. Figure 1 (a) is vinyl-modified halloysite (HNT) S Preparation of -vinyl), Figure 1 (b) and Figure 1 (c) shows the preparation of a hybrid monolithic column doped with halloysite nanotubes, in which... Figure 1(b) is the preparation of vinyl-PEG-Vinyl from step 4-1) using glycidyl methacrylate (GMA), amine-PEG-amine, and formamide. Figure 1 (c) is the preparation route of step 4-2), namely, vinyl-PEG-Vinyl, acrylamide (AM), HNT S A hybrid monolith based on halloysite nanotubes was prepared by reacting -vinyl, N,N′-methylenebisacrylamide (MBA), and azobisisobutyronitrile (AIBN).

[0060] Secondly, this application provides a hybrid monolithic column doped with halloysite nanotubes, which is prepared by the preparation method described in the first aspect above.

[0061] Thirdly, based on the hydrophilic interaction of the hybrid monolithic column doped with halloysite nanotubes using IT-SPME (in-tube solid-phase microextraction) technology, this application also provides the application of the hybrid monolithic column doped with halloysite nanotubes in the detection of polar cationic pesticide residues.

[0062] Among them, polar cationic pesticides include any one or more of maleic hydrazide, glyphosate, and cyproheptadine.

[0063] Fourthly, based on the hydrophilic interaction IT-SPME technique using a hybrid monolithic column doped with halloysite nanotubes, this application also provides a method for detecting polar cationic pesticide residues using the aforementioned hybrid monolithic column doped with halloysite nanotubes, comprising the following steps:

[0064] Step 1): After crushing the sample, weigh an appropriate amount into a container, and then add a standard solution of polar cationic pesticide;

[0065] Step 2): Then add acetonitrile and sodium chloride, shake, centrifuge at 5000-8000 r / min for 5-10 min, take the supernatant of the upper layer, and repeat the extraction of the lower precipitate with acetonitrile at least once to obtain the supernatant. Combine the supernatants, filter, and obtain the pesticide extract.

[0066] Step 3): Equilibrate the 5–20 cm hybrid monolithic column doped with halloysite nanotubes with 150–500 μL of acetonitrile (volume fraction at least 98%) at a flow rate of 3–20 μL / min; Load 20–150 μL of the pesticide extract obtained in Step 2) at a flow rate of 3–20 μL / min, and then elute with 30–100 μL of a mixture of acetonitrile (pure acetonitrile) and ammonium formate at a flow rate of 3–15 μL / min to obtain the eluent;

[0067] Step 4): Analyze the eluent by liquid chromatography-mass spectrometry.

[0068] Preferably, in step 1), the polar cationic pesticide is any one or more of maleic hydrazine, glyphosate, and cyproheptadine.

[0069] The mass-to-volume ratio of acetonitrile and sodium chloride added in step 2) to the sample added in the container in step 1) is 10 mL: 5 g: 10 g.

[0070] Step 3) involves mixing acetonitrile-ammonium formate in a solution with a volume fraction of 30% acetonitrile and 70% ammonium formate, wherein the concentration of ammonium formate is 50–200 mmol / L. The specific preparation method is as follows: ammonium formate is dissolved in water to obtain an ammonium formate aqueous solution, wherein the molar concentration of ammonium formate in the aqueous solution is 50–200 mmol / L. Then, acetonitrile (100% volume fraction) and the ammonium formate aqueous solution are measured separately, such that the volume percentage of acetonitrile in the acetonitrile-ammonium formate mixture is 30%, and the volume percentage of ammonium formate in the acetonitrile-ammonium formate mixture is 70%.

[0071] The present application will be further described in detail below with reference to specific embodiments:

[0072] Example 1

[0073] (I): A method for preparing a hybrid monolithic column doped with halloysite nanotubes (hereinafter referred to as "hybrid monolithic column"), specifically including the following:

[0074] Step 1): Purification of halloysite nanotubes (HNT) with sodium hexametaphosphate S ):

[0075] Take 10g of halloysite and add it to 100mL of an aqueous solution containing 0.05% sodium hexametaphosphate. Stir for 30min, let stand for 20min, discard the precipitate, wash the dispersed halloysite several times with water, centrifuge, and dry at 105℃ for 5h to obtain sodium hexametaphosphate purified halloysite nanotubes.

[0076] Step 2), vinyl-modified halloysite (HNT) S Preparation of -vinyl):

[0077] Take 3g of sodium hexametaphosphate to purify halloysite nanotubes (HNT). S ) and 5 mL of 3-(methacryloyloxy)propyltrimethoxysilane (γ-MAPS, γ-Methacryloxypropyltrimethoxysilane) were added to 50 mL of anhydrous toluene, stirred with a magnetic stirrer and refluxed at 110 °C for 12 h; after the reaction was completed, the mixture was washed three times with anhydrous ethanol and dried at 70 °C for 8 h.

[0078] Step 3), Preparation of vinyl-modified quartz capillaries:

[0079] First, the quartz capillary was rinsed sequentially with 0.5 mol / L sodium hydroxide, water, 0.5 mol / L hydrochloric acid, water, and methanol for 2 hours each, with the sodium hydroxide treatment time being 10 hours. After drying, a mixed solution of 50% γ-MAPS and methanol was added to react with the silanol groups on the capillary wall. Then, it was rinsed clean with methanol and dried for later use.

[0080] Step 4): Includes:

[0081] Step 4-1): Preparation of hybrid monolithic pillars based on halloysite nanotubes (HNTs):

[0082] 25 mg of glycidyl methacrylate (GMA), 25 mg of amino-PEG-amine (average molecular weight 2000, 95%) and 350 mg of formamide were mixed and reacted at 40 °C for 1 h. The reaction was then terminated at 4 °C for 10 min to obtain a mixed solution.

[0083] Step 4-2): Add 12.5 mg of acrylamide (AM) and 60 mg of HNT to the mixed solution obtained in step 4-1). S Vinyl, 25 mg N,N′-methylenebisacrylamide (MBA), 70 mg PEG 10000 and 1.5 mg azobisisobutyronitrile (AIBN) were dissolved by shaking and injected into a vinyl-modified quartz capillary. The two ends of the capillary were then sealed with silicone and placed in a 75°C water bath for 20 h. After the reaction was completed, the unreacted reagents were flushed out of the capillary with acetonitrile and set aside for later use, resulting in a hybrid monolithic column doped with halloysite nanotubes.

[0084] The permeability and adsorption capacity of the hybrid monolithic column prepared in Example 1 are listed in Table 1.

[0085] The hybrid monolith prepared in Example 1 was detected by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), thermogravimetric analysis (TGA), and Fourier transform infrared spectroscopy (FTIR). The results are as follows: Figure 2-5 As shown.

[0086] Figure 2 This is a scanning electron microscope image. From Figure 2 As can be seen, the fiber skeleton is covered with a layer of organic polymer, forming a uniform network structure. Spherical cross-links with a diameter of approximately 500 nm form macropores of 1-3 μm, as well as micropores ranging from tens to hundreds of nanometers. The flow channels formed by the micron-sized macropores reduce the back pressure (i.e., counter-pressure) when the mobile phase passes through the monolithic column.

[0087] Figure 3 This is an X-ray photoelectron spectrum. Figure 3 a, Figure 3 b and Figure 3 c represents HNTs and HNT-free items, respectively. S Organic monolithic materials and doped HNT S The hybrid monolithic material (i.e., the hybrid monolithic column prepared in Example 1). Wherein, Figure 3 In a, HNTs are sodium hexametaphosphate purified halloysite nanotubes (HNTs) prepared in step 1) of Example 1. S ).

[0088] Figure 3 b does not contain HNT S The preparation of the organic monolithic material is largely the same as in Example 1, except that HNT is not added in step 4-2). S -vinyl (vinyl-modified halloysite).

[0089] from Figure 3 From this, we can see that HNTs have characteristic peaks of O1s, C1s, Si2p, and Al2p. Figure 3 b shows that organic monolithic materials without HNTs exhibit characteristic peaks of O1s, C1s, and N1s; from Figure 3 c indicates that characteristic peaks of both HNTs were detected on the hybrid monolithic material doped with HNTs, namely characteristic peaks of O1s, C1, N1s, Si2p and Al2p. The results show that the hybrid monolithic material based on HNTs was successfully prepared, that is, the hybrid monolithic column doped with halloysite nanotubes in Example 1 was successfully prepared.

[0090] Figure 4 This is a thermogravimetric analysis (TGA) chart. From... Figure 4It can be observed that within the temperature range of 40-120℃, the first weight loss is approximately 4.82%, related to the evaporation of residual porogens and template molecules (i.e., formamide and PEG 10000). The second stage occurs between 200-400℃, attributed to intrapolymer chain breakage, with a weight loss of approximately 28.92%. Furthermore, the third stage occurs between 400-600℃, attributed to the decomposition of the monomer backbone and the dehydration of silanol groups present in the HNTs. Finally, at temperatures above 600℃, approximately 37.5% of the residual weight represents the content of HNTs in the HNT-doped hybrid monolithic material, consistent with its content in the polymerization solution (40.7%). The discrepancy may be due to incomplete drying of the HNTs.

[0091] Figure 5 This is an infrared spectrum. Specifically, Figure 5 Infrared spectra of the hybrid monolith prepared in Example 1 and the sodium hexametaphosphate purified halloysite nanotubes (HNTs) prepared in step 1) of Example 1.

[0092] from Figure 5 From this, we can know that 3695cm -1 and 3620cm -1 The peak at 1035 cm⁻¹ is attributed to the -OH stretching vibration of the inner surface of HNTs. -1 and 912cm -1 The peak at 537 cm⁻¹ is attributed to the stretching vibrations of the Si-O-Si framework and Al-OH. -1 and 468cm -1 The peak at 3436 cm⁻¹ is attributed to the bending vibrations of Si-O-Si and Al-O-Si. These characteristic peaks were observed in both the spectra of HNTs and the HNT-doped monolithic hybrid materials, indicating that HNTs were successfully incorporated into the monolithic material. Furthermore, in the infrared spectrum of the HNT-doped monolithic material, a peak at 3436 cm⁻¹ is observed. -1 2945cm -1 2872cm -1 1723cm -1 1660cm -1 1297cm -1 and 1080cm -1 A new characteristic peak was observed at 3436 cm⁻¹. -1 The broad peak at 2945 cm⁻¹ is attributed to the NH stretching vibration and the OH stretching vibration, indicating the presence of -NH₂ and -OH groups. The peak at 2945 cm⁻¹ is also attributed to these vibrations. -1 and 2872cm -1Two peaks appeared at 1723 cm⁻, representing both asymmetric and symmetric stretching vibrations of -CH₂-. The C=O stretching vibration peaks were located at 1723 cm⁻¹. -1 This indicates that AM (acrylamide), GMA (glycidyl methacrylate), and MBA (N,N′-methylenebisacrylamide) participated in the reaction. 1660cm -1 The peak represents the stretching vibration of the amide I band. 1297 cm⁻¹ -1 The peak at 1080 cm⁻¹ is attributed to the stretching vibration of CN. -1 The peak at that location is caused by the COC groups in PEG. These results confirm the successful preparation of hybrid monolithic materials doped with HNTs.

[0093] Example 2

[0094] Similar to Example 1, except that in step 4), during the preparation of the hybrid monolithic column doped with halloysite nanotubes, the permeability and adsorption capacity of the hybrid monolithic columns prepared with different proportions of raw materials (glycidyl methacrylate, amino-PEG-amino, formamide, acrylamide, vinyl-modified halloysite, N,N′-methylenebisacrylamide and PEG10000) were tested, and the results are listed in Table 1.

[0095] Comparative Example

[0096] This comparative example is largely the same as Example 1, except that in step 4), different proportions of raw materials (glycidyl methacrylate, amino-PEG-amino, formamide, acrylamide, vinyl-modified halloysite, N,N′-methylenebisacrylamide and PEG10000) are used. The test results of the prepared hybrid monolithic column are listed in Table 1.

[0097] Table 1. Permeability and Adsorption Capacity of Hybrid Monolithic Columns with Different Proportions

[0098]

[0099] Table 1 shows that the hybrid monolithic column prepared in Example 1 has the highest adsorption capacity and best performance, reaching 2.16 μg / cm. The monolithic columns 12-13 prepared in Comparative Examples 2 and 3 have excessive back pressure, preventing methanol from escaping the capillary. The monolithic column 11 prepared in Comparative Example 1 does not form a homogeneous solution due to insufficient formamide solvent, resulting in an incomplete homogeneous column. In contrast, the remaining proportions in Examples 1-10 all yielded homogeneous columns, indicating that the hybrid monolithic columns prepared according to the proportions of each component exhibit different permeabilities and adsorption capacities.

[0100] Therefore, the hybrid monolithic column doped with halloysite nanotubes prepared in this application can improve its adsorption of polar pesticides, which is beneficial for the enrichment and detection of trace polar pesticides.

[0101] Example 3

[0102] Application of a hybrid monolithic column doped with halloysite nanotubes in the detection of polar cationic pesticide residues. Specifically:

[0103] A method for detecting polar cationic pesticide residues using a hybrid monolithic column doped with halloysite nanotubes includes the following steps:

[0104] Step 1): Purchase cucumbers, carrots, apples, green beans, and eggplants from a local supermarket in Ningbo. First, use a Joyoung juicer / blender (JYL-C012) to evenly crush each sample. Weigh 10g of each crushed sample into a 50mL plastic centrifuge tube, and simultaneously add standard solutions of three cationic pesticides—maleic hydrazine, glyphosate, and cyprodinium—at different concentrations to achieve the spiking concentrations shown in Table 2. Specifically, the spiking concentrations for maleic hydrazine were 20μg / kg, 40μg / kg, and 200μg / kg; for glyphosate, they were 20μg / kg, 40μg / kg, and 200μg / kg; and for cyprodinium, they were 0.8μg / kg, 2μg / kg, and 8μg / kg.

[0105] Step 2): Then add 10 mL of acetonitrile and 5 g of sodium chloride, shake for 5 min, centrifuge at 5000 r / min for 10 min, take the supernatant into a clean plastic centrifuge tube, add another 10 mL of acetonitrile to the precipitate, repeat the extraction once, combine the supernatants obtained from the two centrifugations, filter and obtain the extract.

[0106] Step 3): Equilibrate the 10 cm long hybrid monolithic column doped with halloysite nanotubes (the hybrid monolithic column prepared in Example 1) with 200 μL of 98% acetonitrile aqueous solution at a flow rate of 20 μL / min; Load 150 μL of the extract obtained in Step 2) at a flow rate of 20 μL / min, and then elute with 30 μL of acetonitrile-ammonium formate mixture (30% acetonitrile + 70% 100 mmol / L ammonium formate) at a flow rate of 15 μL / min to obtain the eluent.

[0107] Step 4): Directly inject the eluent into liquid chromatography-mass spectrometry for detection: Specifically,

[0108] Liquid chromatography-mass spectrometry detection methods:

[0109] A) Liquid chromatography separation

[0110] Chromatographic column: Shim-pack GIS HILIC column (4.6×150mm, 5.0μm); column temperature: 30℃; mobile phase: 10mmol / L ammonium formate containing 0.1% formic acid and acetonitrile in a volume ratio of 60:40 (i.e., the ammonium formate aqueous solution contains 0.1% formic acid and 10mmol / L ammonium formate; the volume ratio of ammonium formate aqueous solution to acetonitrile is 60:40); flow rate: 0.8mL / min; injection volume: 10μL.

[0111] B) Mass spectrometry detection

[0112] Positive ion scanning was used for quantitative analysis of the target analytes in multiple reaction monitoring (MRM) mode. The electrospray ionization source conditions were as follows:

[0113] Spray voltage: 4.5kV; Ion source temperature: 500℃; Collision gas CAD: medium; Air curtain gas CUR: 25psi; Atomizing gas GS1: 40psi; Auxiliary heating gas GS2: 40psi.

[0114] The quantitative and confirmatory parameters of the MRM mode for the three cationic pesticides are shown in Table 2 below:

[0115] Table 2. Quantitative and confirmatory parameters of MRM patterns for three cationic pesticides.

[0116]

[0117] In this Example 3, the results of detecting polar cationic pesticide residues in different vegetables and fruits using the hybrid monolithic column prepared in Example 1 are listed in Table 3.

[0118] Table 3. Spiking recoveries of cationic pesticides in vegetables and fruits at different spiking concentrations.

[0119]

[0120]

[0121] As shown in Table 3, the recoveries of cationic pesticides in the above-mentioned vegetables and fruits ranged from 80.2% to 100.8%, with RSD values ​​less than 10.7%. Furthermore, using liquid chromatography-mass spectrometry (LC-MS / MS) and the hydrophilic interaction of the hybrid monolithic column doped with halloysite nanotubes, the limits of detection (LODs) for cyproheptadine, maleic hydrazine, and glyphosate were 0.06 ng / mL, 1.2 ng / mL, and 1.2 ng / mL, respectively, equivalent to mass concentrations of 0.12 μg / kg, 2.4 μg / kg, and 2.4 μg / kg. That is, the hybrid monolithic column 1 prepared in this application has LODs for cyproheptadine, maleic hydrazine, and glyphosate of 0.06 ng / mL, 1.2 ng / mL, and 1.2 ng / mL, respectively, significantly reducing the LODs for polar cationic pesticide residues and improving the sensitivity for their detection.

[0122] Example 4

[0123] Following the method for detecting polar cationic pesticide residues using the hybrid monolithic column in Example 3, the hybrid monolithic column with the lowest adsorption capacity listed in Table 1 (i.e., hybrid monolithic column 5 prepared in Example 5) was used to detect polar cationic pesticide residues in vegetables. Specifically, step 1): Cucumbers, carrots, apples, green beans, and eggplants purchased from a local supermarket in Ningbo were first uniformly crushed using a Joyoung juicer / blender (JYL-C012). 10g of the crushed sample was weighed into 50mL plastic centrifuge tubes, and standard solutions of three cationic pesticides—maleic hydrazine, glyphosate, and cyproheptadine—at different concentrations were added to achieve the spiking concentrations shown in Table 3.

[0124] Step 2): Add 10 mL of acetonitrile and 5 g of sodium chloride to the centrifuge tube after the treatment in Step 1), shake for 5 min, centrifuge at 8000 r / min for 5 min, take the supernatant of the upper layer into a clean plastic centrifuge tube, and add acetonitrile to the precipitate of the lower layer to repeat the extraction once to obtain the supernatant. Combine the supernatants, filter, and obtain the pesticide extract.

[0125] Step 3): The 20 cm hybrid monolithic column doped with halloysite nanotubes (the hybrid monolithic column prepared in Example 5) was equilibrated with 300 μL of 98% acetonitrile aqueous solution at a flow rate of 15 μL / min; 20 μL of the pesticide extract obtained in Step 2) was loaded at a flow rate of 4 μL / min, and then eluted with 30 μL of acetonitrile-ammonium formate mixture (30% acetonitrile + 70% 50 mmol / L ammonium formate) at a flow rate of 3 μL / min to obtain the eluent;

[0126] Step 4): Directly inject the eluent into liquid chromatography-mass spectrometry for detection.

[0127] The test results are listed in Table 4.

[0128] Table 4. Detection of polar cationic pesticide residues in vegetables using hybrid monolithic columns with the lowest adsorption capacity.

[0129]

[0130] As shown in Table 4, the recoveries of cationic pesticides in the above-mentioned vegetables and fruits ranged from 81.1% to 102.4%, with RSD values ​​less than 11.2%. Furthermore, using liquid chromatography-mass spectrometry (LC-MS), the limits of detection (LODs) for cyproheptadine, maleic hydrazine, and glyphosate using the hydrophilic in-tube solid-phase microextraction method with a hybrid monolithic column doped with halloysite nanotubes were 0.5 ng / mL, 10.0 ng / mL, and 10.0 ng / mL, respectively, equivalent to mass concentrations of 1.0 μg / kg, 20.0 μg / kg, and 20.0 μg / kg. That is, using LC-MS, the LODs for cyproheptadine, maleic hydrazine, and glyphosate prepared in this application were 0.5 ng / mL, 10.0 ng / mL, and 10.0 ng / mL, respectively, significantly reducing the LODs for polar cationic pesticide residues and improving the sensitivity for their detection.

[0131] In summary, due to the strong polarity of the selected pesticides, they are difficult to retain on traditional adsorbents based on hydrophobic interactions, i.e., enrichment is difficult. This invention utilizes an IT-SPME technique for polar pesticides based on a hydrophilic mechanism. The PEG groups on the surface of the hybrid monolithic column doped with halloysite nanotubes, as well as the residual silanol groups on the surface of the halloysite nanotubes, both contribute to its hydrophilicity, readily interacting with the strong polar pesticides. This allows the strong polar pesticides to be retained on the hybrid monolithic column doped with halloysite nanotubes, achieving an adsorption capacity of 2.16 μg / cm. The 10 cm long hybrid monolithic column doped with halloysite nanotubes prepared in Example 1 achieved a sample loading volume as high as 150 μL, which is 30 times higher than the 5 μL sample loading volume of the polyacrylamide monolithic column doped with attapulgite (Anal. Chem. 2016, 88, 1535-1541). Furthermore, this invention reduces the limit of detection for cyproheptadine from 100 ng / mL to 0.06 ng / mL. Therefore, the IT-SPME technology using a hybrid monolithic column doped with halloysite nanotubes developed in this invention, combined with liquid chromatography-mass spectrometry, significantly reduces the limit of detection for polar pesticides and improves the sensitivity of polar pesticide detection.

[0132] It should be understood that the various forms of processes shown above can be used to rearrange, add, or delete steps. For example, the steps described in this disclosure can be executed in parallel, sequentially, or in different orders, as long as the desired result of the technical solution disclosed in this disclosure can be achieved, and this is not limited herein.

[0133] The above description is merely a specific embodiment of this disclosure, but the scope of protection of this disclosure is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this disclosure should be included within the scope of protection of this disclosure. Therefore, the scope of protection of this disclosure should be determined by the scope of the claims.

Claims

1. A method for preparing a hybrid monolithic column doped with halloysite nanotubes, characterized in that: The process includes the following steps: Step 1): Halloysite is purified using sodium hexametaphosphate to obtain sodium hexametaphosphate purified halloysite nanotubes; Step 2): Modify halloysite nanotubes purified from sodium hexametaphosphate with anhydrous toluene containing 3-(methacryloyloxy)propyltrimethoxysilane to obtain vinyl-modified halloysite; Step 3): After activating and drying the quartz capillary, it is modified with a mixture of 3-(methacryloyloxy)propyltrimethoxysilane and methanol to obtain a vinyl-modified quartz capillary. Step 4): Including Step 4-1): Mix 24-26 parts of glycidyl methacrylate, 15-25 parts of amino-PEG-amino, and 350-700 parts of formamide, and react at 30-50°C for 0.5-1.5 h, then terminate the reaction at 0-4°C to obtain a mixed solution; wherein, the average molecular weight of the amino-PEG-amino is 2000; Step 4-2): Add 12-13 parts acrylamide, 25-60 parts vinyl-modified halloysite, 24-26 parts N,N′-methylenebisacrylamide, 70-180 parts PEG, and 1-2 parts azobisisobutyronitrile to the mixed solution, shake to dissolve, and then inject into a vinyl-modified quartz capillary. Seal both ends of the capillary with silicone, and then place it in a water bath at 70-80°C for 6-20 hours. After the reaction, rinse the capillary with acetonitrile or methanol to obtain the hybrid monolithic column of the halloysite-doped nanotubes; wherein, the PEG used is PEG10000. The hybrid monolithic column doped with halloysite nanotubes is used for the detection of polar cationic pesticide residues; the polar cationic pesticide is any one or more of maleic hydrazine, glyphosate, and cyproheptadine.

2. The preparation method according to claim 1, characterized in that: In step 4-1), there are 25 parts of glycidyl methacrylate, 25 parts of amino-PEG-amino, and 350 parts of formamide. In step 4-2), there are 12.5 parts of acrylamide, 60 parts of vinyl-modified halloysite, 25 parts of N,N′-methylenebisacrylamide, 70 parts of PEG, and 1.5 parts of azobisisobutyronitrile.

3. The preparation method according to claim 1, characterized in that: The sodium hexametaphosphate purified halloysite nanotubes obtained in step 1) are prepared by the following method: Halloysite is added to an aqueous solution containing sodium hexametaphosphate at a mass concentration of 0.05%~0.25%, stirred for 30~60 min, allowed to stand, and the precipitate is discarded. The dispersed halloysite is washed with water several times, centrifuged, and dried at 60~105 ℃ for 5~10 h to obtain the sodium hexametaphosphate purified halloysite; wherein, the mass-volume ratio of halloysite to aqueous solution is 1 g: 8~20 mL.

4. The preparation method according to any one of claims 1-3, characterized in that: The vinyl-modified halloysite obtained in step 2) is prepared by the following method: Halloysite nanotubes purified by sodium hexametaphosphate and 3-(methacryloyloxy)propyltrimethoxysilane are added to anhydrous toluene, stirred with a magnetic stirrer and refluxed at 110°C for 8-12 h; after the reaction is completed, the mixture is washed several times with anhydrous ethanol and dried at 70-85°C for 6-8 h to obtain the vinyl-modified halloysite; wherein, the mass-to-volume ratio of halloysite nanotubes purified by sodium hexametaphosphate to 3-(methacryloyloxy)propyltrimethoxysilane is 3 g: 5-15 mL.

5. The preparation method according to any one of claims 1-3, characterized in that: The vinyl-modified quartz capillary obtained in step 3) is prepared by the following method: First, the quartz capillary is rinsed sequentially with 0.5~1.0 mol / L sodium hydroxide, water, 0.5~1.0 mol / L hydrochloric acid, water, and methanol for 0.5~2 h each, wherein the sodium hydroxide treatment time is at least 10 h. After drying, a mixed solution of 50% 3-(methacryloyloxy)propyltrimethoxysilane and methanol is added to react with the silanol groups on the capillary wall. After rinsing with methanol, the capillary is dried and ready for use to obtain the vinyl-modified quartz capillary.

6. A hybrid monolithic column doped with halloysite nanotubes, characterized in that: It is prepared by the preparation method described in any one of claims 1-5.

7. A method for detecting polar cationic pesticide residues using a hybrid monolithic column doped with halloysite nanotubes as described in claim 6, characterized in that: Includes the following steps: Step 1): After crushing the sample, weigh an appropriate amount into a container, and then add a standard solution of polar cationic pesticide; Step 2): Then add acetonitrile and sodium chloride, shake, centrifuge at 5000~8000 r / min for 5~10 min, take the supernatant of the upper layer, and repeat the extraction of the lower precipitate with acetonitrile at least once to obtain the supernatant. Combine the supernatants, filter, and obtain the pesticide extract. Step 3): Equilibrate the 5-20 cm hybrid monolithic column doped with halloysite nanotubes with 150-500 μL of acetonitrile at a flow rate of 3-20 μL / min; load 20-150 μL of the pesticide extract obtained in Step 2) at a flow rate of 3-20 μL / min, and then elute with 30-100 μL of acetonitrile-ammonium formate mixture at a flow rate of 3-15 μL / min to obtain the eluent; Step 4): Perform liquid chromatography-mass spectrometry detection on the eluent.

8. The method according to claim 7, characterized in that: In step 1), the sample is an agricultural product; the polar cationic pesticide is any one or more of maleic hydrazine, glyphosate, and cyproheptadine.

9. The method according to claim 7, characterized in that: In step 3), the acetonitrile-ammonium formate mixture is obtained by mixing 30% acetonitrile and 70% ammonium formate by volume, wherein the concentration of ammonium formate is 50~200 mmol / L.

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