Preparation method of sporopollenin purification material and application thereof

Abstract

The application provides a preparation method of sporopollenin purification material and application thereof. The sporopollenin purification material is prepared by adding plant spores and pollen into an extraction tank and using an accelerated solvent extraction instrument, using methanol as an extraction solvent, an extraction temperature of 70 DEG C, an extraction pressure of 1500 psi, an extraction time of 5 min, and 2 cycles, washing the plant spores and pollen with a solvent acetonitrile / water / formic acid (70:28:2, v / v / v) after extraction, and drying at 60 DEG C for 12 hours, so that a hydrophilic solid-phase extraction adsorbent is successfully prepared. The sporopollenin purification material is rich in source, good in biocompatibility, non-toxic and non-polluted. The prepared sporopollenin purification material is complete in appearance, stable in property, stronger in binding force, cleaner in spore cavity, and has a hydrophilic adsorption effect due to the functional groups of multiple hydroxyl groups on the surface, so that the polar compounds can be well adsorbed.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of functional materials and analytical techniques, in particular to a preparation method of sporopollenin purification material and application thereof. BACKGROUND

[0002] In the process of modern agricultural planting, in addition to strong polar pesticides (glyphosate, glufosinate ammonium, etc.), the high-throughput mass spectrometry method established in the early stage has realized the monitoring. The wide application of these strong polar pesticides in agricultural and non-agricultural fields has caused the residues of the parent compounds and metabolites in water, soil, crops, food and even organisms. It is urgent to find a simple and rapid purification material for strong polar pesticides.

[0003] At present, the typical treatment methods (such as solid phase extraction, QuEChERS, mini-Luke and dispersive solid phase extraction) have very limited recovery rate for strong polar pesticides. These strong polar pesticides have the characteristics of not easy to volatilize, difficult to dissolve in organic solvents, strong hydrophilicity, etc., and need to be purified by using specific purification materials. The existing commercial purification materials (CAX, MAX, HLB, etc.) are based on ion exchange or non-polar adsorption to purify strong polar pesticides, but for large batch sample monitoring, there are disadvantages such as high price and poor stability. All these adsorbents are synthesized by using traditional chemical reagents, so it is urgent to prepare high-efficiency and green purification materials for polar pesticides.

[0004] Plant spores and pollen are the total name of spores and pollen scattered by plants during the breeding period. The shell (sporopollenin) of plant spores and pollen has excellent chemical stability, high strength and unique microstructure, and can be used as a biorenewable polymer filler. As a kind of natural green material, plant spores and pollen have been applied to drug delivery, carbon materials and support materials. In addition, sporopollenin has strong adsorption capacity due to its porous structure and hydroxyl groups, so that sporopollenin becomes a promising hydrophilic solid phase extraction adsorbent. At present, the method for preparing sporopollenin is Soxhlet extraction method, but the preparation method has the problems of large consumption of organic solvents, long time and low impurity removal efficiency. SUMMARY

[0005] The purpose of the present application is to provide a preparation method of sporopollenin purification material and application thereof, to develop a new preparation method of green and efficient sporopollenin purification material. The prepared sporopollenin purification material can be used as a hydrophilic solid phase extraction adsorbent to realize the enrichment and purification of polar pesticides, and meet the urgent need of rapid detection of polar pesticide residues in current agricultural products.

[0006] The present application is realized as follows:

[0007] A preparation method of sporopollenin purification material is prepared according to the following steps:

[0008] Step 1, screen the plant spore powder, remove impurities, and store in a dryer;

[0009] Step 2, soak the plant spore powder with methanol according to a certain plant spore powder mass to methanol volume ratio (g / mL);

[0010] Step 3, attach a gasket (diameter 30.0 mm) to the bottom of the 10-34 mL extraction cell, and tighten the extraction cell;

[0011] Step 4, accurately weigh the plant spore powder sample soaked with methanol 5-20 g, and add it to the extraction cell from the top of the extraction cell, and tighten the cell cap after adding the gasket on the upper layer;

[0012] Step 5, select an organic solvent (methanol, acetonitrile or a mixed solution of a certain proportion) as the extraction solvent, and extract statically at a temperature of 60-80℃ and a pressure of 1200-1500 psi for 5-10 min, with 2-4 extraction cycles;

[0013] Step 6, the elution volume is 40%-60% of the cell volume of the extraction solvent, and the nitrogen gas is blown to collect all the extract in a 100-250 mL collection bottle, and all the extract is discarded;

[0014] Step 7, further, collect the plant spore powder sample after extraction;

[0015] Step 8, wash the crude spore powder according to the following steps, and prepare the washing solvent: acetonitrile / water / formic acid mixed solution, wash 2-4 times;

[0016] Step 9, finally, dry the spore powder sample prepared above at 60-80℃ for 12-24 hours to obtain the purified spore powder material.

[0017] The volume ratio of the acetonitrile / water / formic acid mixed solution is (60-80):(20-40):(1-10).

[0018] The method of the present application is based on plant spore powder to prepare spore powder material, and the prepared spore powder material can be applied to the pretreatment of pesticide residue detection samples, and can realize the enrichment and purification of various polar pesticide residues.

[0019] Compared with the prior art, the present application has the following advantages:

[0020] (1) The present application first prepares a pretreatment and purification material for pesticide residue detection based on plant spore powder, develops a new type of green and efficient purification material, and the plant spore powder used is a natural component, which is rich in source, has good biocompatibility, is non-toxic and non-polluting.

[0021] (2) The method is simple to prepare, and the sporopollens purification material can be obtained in one step reaction; the sporopollens purification material prepared by the method has complete morphology, stable property, stronger binding force, and cleaner sporopollens cavity, and the interference of impurities is avoided.

[0022] (3) The sporopollens material prepared by the method has uniform morphology and micron size, fully meets the requirements of solid phase extraction purification adsorbent, and the functional groups of the surface multiple hydroxyl groups have hydrophilic adsorption effect, so that the polar compounds can be well adsorbed; the sporopollens purification material can simply, quickly and efficiently separate and enrich the polar pesticides. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 It is a scanning electron microscope photograph of different treatment stages of the lycopodium sporos in the embodiment 1 of the present application.

[0024] Figure 2 It is an infrared spectrum diagram of different treatment stages of the lycopodium sporos in the embodiment 1 of the present application.

[0025] Figure 3 It is a structure schematic diagram of the needle type solid phase extraction filter in the embodiment 2 of the present application. DETAILED DESCRIPTION

[0026] The present application will be further described in conjunction with specific embodiments, and the embodiments given below are only for illustrating the present application, and are not intended to limit the scope of the present application. The embodiments provided below can be used as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the present application in any way.

[0027] In the following examples, the experimental methods are conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents and the like used in the following examples can be obtained from commercial channels, unless otherwise specified.

[0028] Embodiment 1, a method for preparing sporopollens purification material based on lycopodium sporos.

[0029] Step 1, screen the lycopodium sporos, remove impurities, and store in a desiccator.

[0030] Step 2, immerse 100g of lycopodium sporos in 167mL of methanol.

[0031] Step 3, attach a gasket (diameter 30.0mm) to the bottom of the 34mL extraction cell, and tighten the extraction cell.

[0032] Step 4: Accurately weigh 16.5g of methanol-soaked lycophyte spore sample and add it from the top of the extraction cell to a 34mL extraction cell. After adding a gasket to the top, tighten the cell cap. The remaining lycophyte spores can be used to fill other extraction cells for batch processing.

[0033] Step 5: Select methanol as the extraction solvent and perform static extraction for 5 minutes at a temperature of 70℃ and a pressure of 1500psi, followed by two extraction cycles.

[0034] Step 6: Rinse with 60% of the extraction solvent volume of the pool, purge with nitrogen to collect all the extract into a 250mL collection bottle, and discard all the extract.

[0035] Step 7, further, collect the crude extract of sporophytin.

[0036] Step 8: Wash the crude sporopyram product as follows: Prepare a washing solvent, which is a mixed solution of 1000 mL acetonitrile / water / formic acid (70:28:2, v / v / v), and wash it 4 times.

[0037] Step 9: Finally, the prepared sporophytin sample is dried at 60°C for 12 hours to obtain sporophytin purification material (hereinafter referred to as sporophytin material).

[0038] The materials were characterized using field emission scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (IR), and organic elemental analysis (EA), as follows:

[0039] (1) Particle size and morphology characterization

[0040] The morphology and particle size of the prepared sporophytin material were observed using an EDS X-MAX50 field emission scanning electron microscope (FEI, USA). Figure 1 In this context, AC represents plant pollen (or natural pollen), and DF represents the synthesized sporophytin material. Figure 1 As can be seen, the prepared sporophytin material has a particle size of about 29 μm and a loose and porous surface; both natural sporophytin and sporophytin materials show complete structural morphology (A and D); both natural sporophytin and sporophytin materials show complete honeycomb structure (B and E); after grinding, the cytoplasmic contents are present in the core of the natural sporophytin (C); after grinding, the sporophytin material shows clean cavities, indicating that the cytoplasm has been removed (F).

[0041] (2) Functional group characterization

[0042] The functional group changes of natural pollen and sporophytin materials were characterized using a Nicolet-iS50 Fourier transform infrared spectrometer (Thermo Fisher Scientific, USA), such as... Figure 2 As shown. From Figure 2 It can be seen that the natural pollen is 3007cm.-1 vibrations at 2926 cm -1 and 2855 cm -1 : These two sharp vibrations are attributed to asymmetric and symmetric C-H stretching in methylene (-CH 2 -) groups, respectively. Chemical treatment does not seem to significantly affect the intensity of these vibrations; 1745 cm -1 : This strong vibration is C=0 stretching of aliphatic ester groups, mainly from fatty acids or lipids, which disappears after accelerated solvent extraction treatment; 1713 cm -1 : This is a carbonyl (C=0) stretching vibration, possibly from alkyl esters or carboxylic acids. 1657 cm -1 : The vibration at 1607 cm -1 : This vibration appears in all Lycopodium spore spectra. 997 cm -1 : This vibration is attributed to C-O-C stretching in carbohydrates, which completely disappears after accelerated solvent extraction treatment. The results indicate that a unique combination of chemical functionalities is formed during the preparation of spores.

[0043] (3) Organic elemental analysis test

[0044] The UNICUBE organic elemental analysis test (Germany-elementar company) was used to estimate the content of carbon, hydrogen, nitrogen and oxygen. The elemental analysis of natural spores and spores material was carried out, and the results are shown in Table 1. As can be seen from Table 1, after accelerated solvent treatment, the content of nitrogen, carbon and hydrogen is significantly reduced, and the content of oxygen is slightly increased. The elemental analysis results show that some substances such as proteins are removed.

[0045] Table 1 Elemental composition content of natural spores and spores material (wt%).

[0046] Name C element N element H element O element Natural spore powder 65.22 1.76 8.86 22.69 Spore powder material 58.39 1.45 7.67 25.41

[0047] Example 2, preparation of needle type solid phase extraction filter based on spores purification material prepared in example 1.

[0048] As Figure 3 shown, the needle type solid phase extraction filter includes a 1 mL column tube, and the upper and lower sides of the column tube are respectively connected with a syringe nipple interface and a needle plug interface; an upper sieve plate, 400 mg of spores purification material prepared in example 1, and a lower sieve plate are sequentially arranged in the inner cavity of the column tube from top to bottom; and hydrophobic holes with a pore size of 20 μm are arranged on the upper and lower sieve plates, respectively.

[0049] Example 3, the use of sporopollenin purification material in Example 1 for purifying silage extract.

[0050] The sporopollenin material prepared by the method of the present application is used for sample pretreatment of polar pesticide residue detection, and the specific steps of the sample pretreatment technology are as follows:

[0051] ①Cut the silage sample into small pieces, mix well, seal, and mark.

[0052] ②Put 2 g of homogenized silage sample into a 50 mL polypropylene centrifuge tube, add 2 mL of water, and vortex for 30 s. Then, add 10 mL of 1% formic acid methanol solution to the tube. Shake on a shaker for 10 minutes at a shaking frequency of 250 rpm, and then centrifuge at 4500 rpm for 5 minutes. Take 2.5 mL of supernatant, dry with nitrogen, and re-dissolve with 1 mL of acetonitrile.

[0053] ③Use the needle-type solid-phase extraction filter in Example 2 to activate the column tube with 2 mL of acetonitrile + water (9 / 1, v / v) and 2 mL of acetonitrile first, and discard the effluent. Transfer the sample concentrate to the column tube, wash the extraction column with 1 mL of acetonitrile, and discard the wash. Then elute the extraction column with 2 mL of acetonitrile / water / formic acid (70 / 28 / 2, v / v / v), collect the eluate, dry with nitrogen, and re-dissolve with 1 mL of acetonitrile / methanol (1 / 9, v / v) for LC-Q-TOF / MS analysis.

[0054] ④LC-Q-TOF / MS: Chromatographic column: HILIC-Z column (2.1 x 150 mm, 2.7 μm); set column temperature: 35℃; mobile phase: A 0.1% formic acid aqueous solution, select acetonitrile as B phase solvent; injection volume selection 10 μL. Liquid chromatography gradient elution program as follows: 0 min, 95% B; 1 min, 95% B; 2 min, 20% B; 12 min, 20% B; 12.5 min, 95% B; maintain 2 min; flow rate set to 0.5 mL / min.

[0055] ⑤Ion source: Dual AJS ESI+; scanning mode: negative ion full scan; capillary voltage: 4000V; atomizing gas: nitrogen; atomizing gas pressure: 0.14 MPa; drying gas temperature: 325℃; sheath gas flow rate and temperature are 11.0 L / min and 375℃, respectively; drying gas flow rate: 12.0 L / min; drying gas temperature: 325℃; fragmentation voltage: 145V. Acquisition range, m / z 50-300; All Ions MS / MS mode conditions: 0 min, collision energy 0V; 0.5 min, collision energy 10V.

[0056] (5) Pesticides glyphosate, glufosinate and fosetyl-Al in the range of 0.01-2 mg / L, after purification treatment (impurity influence and instrument service life influence from samples without purification treatment), the pesticide response value and peak area have good linear correlation, see Table 2, the quantification limit of the method is 0.01-1 mg / kg.

[0057] Table 2 Method linearity and detection line

[0058] Name Linear regression equation Correlation coefficient R 2 ]] Limit of quantification (mg / kg) Glyphosate Y = 272.4x - 2566 0.998 0.05 Glufosinate Y = 603.94x + 199.2 0.999 0.01 Fosetyl-aluminum Y = 272.4x + 1546 0.999 0.01

[0059] The above detailed description is only a specific description of the feasible implementation of the present application, and is not used to limit the protection scope of the present application. Equivalent implementation or changes without departing from the spirit of the present application should be included in the protection scope of the present application.

Claims

1. Use of a sporopollenin purifying material, characterized in that, The application is the application of the sporopollens purification material in pesticide residue detection sample pretreatment; the pesticide is glyphosate, glufosinate-ammonium or fosetyl-aluminum; The preparation method of the sporopollens purification material comprises the following steps: S1, screening plant sporopollen, removing impurities and storing in a dryer; the plant sporopollen is lycopodium spore; S2, immersing the plant sporopollen in methanol; S3, attaching a gasket at the bottom of a 10-34 mL extraction cell and tightening the extraction cell; S4, weighing 5-20 g of the plant sporopollen immersed in methanol and adding into the extraction cell from the top of the extraction cell, and then tightening the cell cap after adding the gasket on the upper layer; S5, selecting methanol, acetonitrile or a mixed solution of methanol and acetonitrile as the extraction solvent, and performing static extraction under the conditions of temperature 60-80 ℃ and pressure 1200-1500 psi for 5-10 min, and the extraction cycle is 2-4 times; S6, the elution volume is 40%-60% of the volume of the extraction cell, and the extracted liquid is collected in a collection bottle by nitrogen blowing, and then the extracted liquid is discarded; S7, collecting the plant sporopollen after extraction, which is the crude sporopollens product; S8, washing the crude sporopollens product with a washing solvent for 2-4 times; the washing solvent is a mixed solution of acetonitrile, water and formic acid; S9, drying the washed sporopollens at 60-80 ℃ for 12-24 hours to obtain the sporopollens purification material.

2. Use of a sporopollenin-purifying material according to claim 1, characterized in that, In step S2, the mass ratio of the plant sporopollen to methanol is 3 g:5 mL.

3. Use of a sporopollenin scavenging material according to claim 1, characterized in that, In step S8, the volume ratio of acetonitrile, water and formic acid in the mixed solution of acetonitrile, water and formic acid is (60-80):(20-40):(1-10).

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