Centrifugal-free pretreatment method of plant sample for rapid solid-liquid separation assisted by magnetic particles

Abstract

The invention discloses a plant sample centrifugation-free pretreatment method for magnetic particle-assisted solid-liquid rapid separation, which comprises the following steps: adding magnetic particles into a plant sample extraction system, and fully combining the magnetic particles with insoluble solids under the action of vortex mixing and / or ultrasonic treatment to obtain a plant sample solid-liquid separation solution; then, solid-liquid rapid separation is achieved under the action of an external magnetic field, clear supernate is obtained, and the sample pretreatment process does not need to be completed through traditional centrifugal operation. The method does not depend on high-speed centrifugal equipment, is simple and convenient to operate and short in treatment time, and is particularly suitable for powdery or sticky plant samples. The obtained sample extracting solution can be directly used for gas chromatography-mass spectrometry or liquid chromatography-mass spectrometry analysis, and the analysis result has good consistency with that of a traditional centrifugal method. The method can effectively reduce the dependence on centrifugal equipment, simplify the sample pretreatment process and improve the pretreatment efficiency of complex plant samples, and is suitable for rapid detection and quality control of traditional Chinese medicines, spices, foods and other plant source samples.

Description

Technical Field

[0001] This invention relates to the field of sample pretreatment and analysis technology, specifically to a method for rapid solid-liquid separation of plant samples without centrifugation using magnetic particles, applicable to sample pretreatment of plant-derived samples before gas chromatography, liquid chromatography and their coupled mass spectrometry analysis. Background Technology

[0002] Plant samples have wide applications in the analysis of food, pharmaceuticals, traditional Chinese medicine, spices, and natural products. Their chemical composition is complex, typically containing volatile components, phenolic acids, flavonoids, alkaloids, and high-molecular-weight impurities such as polysaccharides and resins. Before performing gas chromatography, liquid chromatography, or their coupled mass spectrometry analysis, plant samples usually require pretreatment to effectively release the target components, thereby obtaining a clear extract suitable for instrumental analysis.

[0003] Currently, the most common method for plant sample pretreatment is solvent extraction combined with centrifugation. This method typically involves adding an organic solvent to the plant-derived sample (such as a solid sample or a solid-liquid mixture), followed by vortex mixing or ultrasonic extraction, and then separating the solid sample residue from the organic solvent extract by high-speed centrifugation. However, this method generally suffers from numerous operational steps, strong dependence on high-speed centrifugation equipment, and a cumbersome and time-consuming overall process.

[0004] Especially when processing powdery, fibrous, granular, or viscous plant samples, centrifugation efficiency is easily affected by the physicochemical properties of the samples, often resulting in unclear stratification, turbid supernatant, or the need for repeated centrifugation, thus reducing analytical efficiency and affecting the repeatability and stability of the results. Furthermore, the process of transferring the supernatant after centrifugation requires a high level of operational skill and is easily affected by human factors. When processing multiple batches of samples simultaneously, the centrifugation step and manual transfer of the supernatant often become key bottlenecks limiting analytical throughput, further increasing experimental time and labor costs.

[0005] In recent years, magnetic materials have been introduced into the field of sample separation and enrichment due to their rapid response under an applied magnetic field. However, existing magnetic separation techniques are mostly used for the selective adsorption or magnetic solid-phase extraction of target components, and usually require subsequent magnetic separation operations after solvent extraction and centrifugation to obtain the supernatant. For the solid-liquid separation stage in plant sample pretreatment, especially for achieving rapid separation of solid residues and extracts without centrifugation, there is still a lack of reported universal and simplified technical solutions.

[0006] Therefore, there is still an urgent need for a pretreatment method that is simple in structure, easy to operate, and applicable to various types of plant samples. This method should ensure the reliability of analytical results while achieving rapid solid-liquid separation and replacing the traditional centrifugation step, thereby improving the efficiency and applicability of plant sample pretreatment.

[0007] To address the above problems, this invention is proposed. Summary of the Invention

[0008] I. Purpose of the Invention

[0009] To address the problems of existing plant sample pretreatment methods, which generally rely on high-speed centrifugation equipment, involve cumbersome procedures, have low processing efficiency, and exhibit unstable separation effects when processing powdery, fibrous, granular, or viscous samples, this invention aims to provide a centrifugation-free pretreatment method for plant samples with magnetic particle-assisted solid-liquid rapid separation. This method achieves rapid separation of solid residues from the extract during plant sample extraction without centrifugation, thereby simplifying the pretreatment process, reducing equipment dependence, and improving the efficiency and applicability of pretreatment for complex plant samples.

[0010] II. Technical Solution

[0011] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0012] This invention provides a method for rapid solid-liquid separation of plant samples without centrifugation, using magnetic particles as an aid, comprising the following steps:

[0013] (1) Add extraction solvent to plant samples to form a plant sample extraction system that is a mixture of liquid and insoluble solid;

[0014] (2) Add magnetic particles to the plant sample extraction system, and under the action of vortex mixing and / or ultrasonic treatment, make the magnetic particles fully contact and combine with the insoluble solids in the plant sample extraction system;

[0015] (3) Under the action of an external magnetic field, the magnetic particles and the insoluble solids they bind rapidly aggregate and separate from the liquid phase to obtain a clear supernatant;

[0016] The method achieves solid-liquid separation of the plant sample extraction system without centrifugation.

[0017] Preferably, the plant sample is a powdery, fibrous, granular, or viscous plant source sample.

[0018] Preferably, the magnetic particles are magnetic metal oxide particles or magnetic composite particles; more preferably, the magnetic particles are Fe3O4 magnetic particles.

[0019] Preferably, the magnetic particles have a particle size of nanometers or micrometers. More preferably, the magnetic particles are nanometers in size, with a particle size of 20nm-100nm.

[0020] The mass ratio of the plant sample to the magnetic particles is 1:5 to 1:15. More preferably, when the plant sample is in powder form, the mass ratio of the plant sample to the magnetic particles is 1:15; when the plant sample is in viscous form, the mass ratio of the plant sample to the magnetic particles is 1:5.

[0021] Preferably, the extraction solvent is methanol, ethanol, acetonitrile, dichloromethane, water, or a mixture of several of these solvents in any proportion.

[0022] Preferably, the ultrasonic treatment time is 10-30 min.

[0023] Preferably, the external magnetic field is provided by a permanent magnet or an electromagnet.

[0024] Preferably, the obtained supernatant can be directly used for analysis by gas chromatography, liquid chromatography, or their coupling with mass spectrometry.

[0025] Preferably, the method is applicable to the rapid detection and quality control of traditional Chinese medicine, spices, food or other plant-derived samples.

[0026] III. Beneficial Effects

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) This invention introduces magnetic particles and combines them with an external magnetic field to achieve rapid solid-liquid separation during the extraction of plant samples without the need for high-speed centrifugation, thus effectively reducing the dependence on centrifugation equipment.

[0029] (2) The method of the present invention is simple, convenient to operate and has a short processing time. It is applicable to various types of plant samples such as powder, fibrous and viscous, and significantly improves the stability and repeatability of pretreatment of complex plant samples.

[0030] (3) The supernatant obtained by the present invention has high clarity and can be directly used for gas chromatography, liquid chromatography and their coupled mass spectrometry analysis. The analysis results have good consistency with traditional centrifugation methods.

[0031] (4) The method of the present invention is conducive to the rapid processing of multiple batches of samples, reduces the intensity of manual operation and human error, and is suitable for rapid detection and quality control of traditional Chinese medicine, spices, food and other plant-derived samples. It has good promotion and application value.

[0032] In summary, the method of this invention involves adding magnetic particles to the plant sample extraction system. Under ultrasonic treatment, the magnetic particles fully bind with the insoluble solids in the sample. Subsequently, rapid solid-liquid separation is achieved under the action of an external magnetic field, resulting in a clear supernatant. This eliminates the need for traditional centrifugation to complete the sample pretreatment process. The method does not rely on high-speed centrifugation equipment, is simple to operate, and has a short processing time, making it particularly suitable for powdery or viscous plant samples. Verification has shown that the sample extract obtained using this method can be directly used for gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS) analysis, and the analytical results show good consistency with traditional centrifugation methods. This invention effectively reduces reliance on centrifugation equipment, simplifies the sample pretreatment process, and improves the pretreatment efficiency of complex plant samples. It is suitable for the rapid detection and quality control of traditional Chinese medicine, spices, food, and other plant-derived samples. Attached Figure Description

[0033] Figure 1 This is a flowchart illustrating the method for rapid solid-liquid separation of plant samples without centrifugation using magnetic particles, as described in this invention. It shows the entire process of adding plant samples, introducing magnetic particles, ultrasonic treatment, applying an external magnetic field, and obtaining a clear supernatant, corresponding to the general centrifugation-free pretreatment method described in Example 1.

[0034] Figure 2 The images are scanning electron microscope images of the magnetic particles before and after interaction with the plant sample. (A) shows the morphology of the Fe3O4 magnetic particles; (B) shows the morphology of the plant powder sample; (C) and (D) show the microscopic morphology of the Fe3O4 magnetic particles and the insoluble solid composite structure of the plant sample formed after treatment by the method of Example 1 at different magnifications, which are used to illustrate the structural basis of the combination of magnetic particles and plant sample, corresponding to Example 2.

[0035] Figure 3 The diagram shows the optimization results of key experimental parameters of the method of the present invention, wherein: (A) is the effect of the mass ratio of sample to magnetic particles on the solid-liquid separation effect; (B) is the effect of magnetic particles of different sizes on the separation effect; (C) is the comparison of the separation effect under different extraction solvent conditions; and (D) is the effect of ultrasonic treatment time on the separation effect, corresponding to the experimental verification of key parameters in Example 3.

[0036] Figure 4The total ion current chromatograms (TIC) obtained by GC-MS analysis of plant samples treated with the method of the present invention and those treated with conventional centrifugation are compared, corresponding to Example 4. Wherein: (A) is a malt extract sample; (B) is a jujube extract sample; (C) is a licorice powder sample; (D) is a cinnamon powder sample; where the black curve (CSE) represents the analytical result after treatment with conventional centrifugation, and the red curve (MPAS) represents the analytical result after treatment with the magnetic particle-assisted centrifugation-free pretreatment method of the present invention.

[0037] Figure 5 The total ion chromatograms obtained by GC-MS analysis of plant samples treated with the method of this invention and the traditional centrifugation method are compared, corresponding to the analysis results of the powdered plant samples in Example 4. Wherein: (A) is dandelion extract; (B) is grape extract; (C) is salvia miltiorrhiza powder; (D) is cyperus rotundus powder; (E) is scutellaria baicalensis powder; (F) is tobacco powder; where the black curve (CSE) represents the analysis result after treatment with the traditional centrifugation method, and the red curve (MPAS) represents the analysis result after treatment with the magnetic particle-assisted centrifugation-free pretreatment method of this invention.

[0038] Figure 6 The total ion current chromatograms (TIC) obtained in positive ion mode by LC-MS after processing plant samples using the method of this invention and the conventional centrifugation method are compared, corresponding to Example 5. Wherein: (A) is a malt extract sample; (B) is a jujube extract sample; (C) is a licorice powder sample; (D) is a cinnamon powder sample; where the black curve (CSE) represents the analytical result after processing using the conventional centrifugation method, and the red curve (MPAS) represents the analytical result after processing using the magnetic particle-assisted centrifugation-free pretreatment method of this invention.

[0039] Figure 7 The total ion current chromatograms (TIC) obtained by LC-MS in positive ion mode after processing plant samples using the method of this invention and the conventional centrifugation method are compared, corresponding to Example 5. Wherein: (A) is dandelion extract; (B) is grape extract; (C) is salvia miltiorrhiza powder; (D) is cyperus rotundus powder; (E) is scutellaria baicalensis powder; (F) is tobacco powder. The black curve (CSE) represents the analytical result after processing using the conventional centrifugation method, and the red curve (MPAS) represents the analytical result after processing using the magnetic particle-assisted centrifugation-free pretreatment method of this invention.

[0040] Figure 8This is a heatmap showing the relative abundance changes of metabolites obtained by GC-MS analysis of different plant samples under different temperatures and treatment times, after treatment with the method of this invention. It is used to illustrate the application effect of the method of this invention in monitoring the processing of plant samples, corresponding to Example 6. Wherein: (A) is a cinnamon powder sample; (B) is a malt extract sample; (C) is a cyperus rotundus powder sample; (D) is a dandelion extract sample; (E) is a tobacco powder sample; the horizontal axis represents the treatment time (0h, 2h, 4h, 6h, 8h), the vertical axis represents the representative metabolites detected, and the color intensity represents the relative abundance change of metabolites.

[0041] Figure 9 This is a heatmap showing the relative abundance changes of metabolites obtained by LC-MS analysis of different plant samples under different temperatures and treatment times, after treatment with the method of this invention. The results correspond to the LC-MS analysis in Example 6. (A) is a cinnamon powder sample; (B) is a malt extract sample; (C) is a cyperus rotundus powder sample; (D) is a dandelion extract sample; and (E) is a tobacco powder sample. The horizontal axis represents the treatment time (0h, 2h, 4h, 6h, 8h), and the vertical axis represents the representative metabolites detected. The color intensity indicates the change in the relative abundance of metabolites. Detailed Implementation

[0042] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but is not limited to these embodiments. Experimental methods not specifically described in the embodiments generally use conventional conditions, conditions described in manuals, or conditions recommended by the manufacturer. The general equipment, materials, reagents, etc., used are all commercially available unless otherwise specified.

[0043] Example 1: A general method for rapid solid-liquid separation of plant samples without centrifugation using magnetic particles for pretreatment.

[0044] like Figure 1 As shown in the figure, this embodiment provides a method for rapid solid-liquid separation of plant samples without centrifugation.

[0045] Weigh 50 mg of plant sample and place it in a 10 mL centrifuge tube. Add 1.0 mL of extraction solvent to form the plant sample extraction system. Then add Fe3O4 magnetic nanoparticles to make the mass ratio of plant sample to magnetic particles 1:15.

[0046] The above system was directly subjected to ultrasonic treatment for 10 min. During the ultrasonic process, the magnetic particles were fully dispersed under the action of ultrasonic cavitation and came into full contact with the insoluble solids in the plant sample, thereby promoting the release of the target components and forming a "magnetic particle-solid residue" composite system.

[0047] After sonication, an external magnetic field is applied to the outside of the centrifuge tube. The magnetic particles and their bound insoluble solids rapidly aggregate within 30–60 seconds, and the liquid phase becomes clear. The supernatant can then be directly transferred for subsequent analysis.

[0048] No centrifugation was performed during the entire process.

[0049] Example 2: Microstructural verification of the binding morphology of magnetic particles with plant samples

[0050] To verify the binding mechanism of magnetic particles with the insoluble solids of plant samples in the method of this invention, scanning electron microscopy was used to observe the magnetic particles, plant powder samples, and the treated composite system. The results are as follows: Figure 2 As shown.

[0051] like Figure 2 As shown in Figure A, the Fe3O4 magnetic particles exhibit a regular granular structure with a particle size at the nanoscale; as... Figure 2 As shown in B, the plant powder sample has a large particle size and an irregular surface structure.

[0052] After ultrasonic treatment using the method described in Example 1, as Figure 2 C and Figure 2 As shown in Figure D, Fe3O4 magnetic particles are clearly attached to the surface of the plant sample particles and form a stable composite structure. This composite structure can migrate as a whole under the action of an external magnetic field, thereby achieving rapid separation of plant solid residues from the liquid phase.

[0053] The above results show that magnetic particles can effectively bind insoluble solids in plant samples through physical adsorption, which is the structural basis for the present invention to achieve centrifugation-free solid-liquid separation.

[0054] Example 3: Optimization of sample-to-magnetic particle ratio, particle size, and solvent conditions

[0055] To determine the applicable parameters of the method of the present invention, a systematic experimental verification of the key conditions was conducted, and the results are as follows: Figure 3 As shown.

[0056] (1) Mass ratio of sample to magnetic particles

[0057] Taking powdered plant samples as an example, with a fixed sample amount of 50 mg, the amount of magnetic particles was gradually increased. For example... Figure 3 As shown in Figure A, the results indicate that when the mass ratio of the sample to the magnetic particles is less than 1:10, the supernatant still exhibits significant turbidity; when the mass ratio reaches 1:15, the turbidity of the supernatant decreases significantly and tends to stabilize, and further increasing the amount of magnetic particles does not significantly improve the separation effect.

[0058] For viscous samples, a stable and clear supernatant can be obtained when the mass ratio reaches 1:5.

[0059] (2) Particle size of magnetic particles

[0060] Experiments were conducted using Fe3O4 magnetic particles with average particle sizes of approximately 20 nm, 50 nm, and 100 nm, respectively. Figure 3 As shown in Figure B, the results indicate that, under the same mass ratio conditions, the clarity of the supernatant obtained from magnetic particles of different sizes is not significantly different, and effective solid-liquid separation can be achieved in all cases.

[0061] (3) Extraction solvent type

[0062] Methanol, ethanol, acetonitrile, and dichloromethane were selected as extraction solvents for the experiments. Figure 3 As shown in Figure C, the results indicate that the clarification effect of the supernatant is basically the same under different solvent conditions, which shows that the method of the present invention has good adaptability to extraction solvents.

[0063] (4) Ultrasound time

[0064] Experiments were conducted within the range of 10-30 minutes of ultrasound administration. For example... Figure 3 As shown in D, after 10 minutes of sonication, the clarity of the supernatant stabilized, and further extending the sonication time did not significantly improve the separation effect.

[0065] Example 4: Comparison of the method of the present invention and the traditional centrifugation method in GC-MS analysis

[0066] The viscous plant samples and powdered plant samples were processed using the method in Example 1, and compared with the traditional method of "solvent extraction + centrifugation at 10,000 rpm for 10 min". The obtained samples were analyzed by GC-MS.

[0067] like Figure 4 and Figure 5 As shown, the total ion chromatograms obtained by the two methods are highly consistent in terms of the retention time and overall distribution of the main peaks. In some samples, the signal-to-noise ratio of the chromatographic peaks treated by the method of this invention is slightly improved, indicating that the magnetic particles help reduce matrix interference while removing solid impurities.

[0068] The results show that the method of the present invention can obtain GC-MS analysis results comparable to those of the traditional centrifugation method without relying on centrifugation.

[0069] Example 5: Comparison of the method of the present invention and the traditional centrifugation method in LC-MS analysis

[0070] The supernatant obtained by the method in Example 1 was analyzed by LC-MS in positive ion mode and compared with the results of traditional centrifugation.

[0071] like Figure 6 and Figure 7 As shown, the two methods maintain a high degree of consistency in chromatographic retention time, peak shape, and overall signal distribution of the main chemical components. In a few samples, the chromatographic baseline treated by the method of this invention is more stable, indicating that this method is beneficial in reducing interference from solid impurities in the detection.

[0072] The above results further verify the reliability and versatility of the method of the present invention in LC-MS analysis.

[0073] Example 6: Application of the method of the present invention in monitoring the processing of plant samples

[0074] Plant samples such as cinnamon powder, cyperus powder and malt extract were selected and treated at 40 ℃, 60 ℃ and 80 ℃ for 0 h, 2 h, 4 h, 6 h and 8 h, respectively.

[0075] Samples at each time point were pretreated using the method of this invention and analyzed by GC-MS and LC-MS, respectively. The results of the relative abundance changes of metabolites are presented in the form of a heatmap, as shown below. Figure 8 and Figure 9 As shown.

[0076] The results showed that the volatile and polar components in different plant samples exhibited significant changing trends with temperature and treatment time, indicating that the method of the present invention can stably and sensitively reflect the component changes in plant samples during processing, and is suitable for quality control and process monitoring.

[0077] The present invention has been described above by way of example. It should be noted that any simple modifications, alterations or other equivalent substitutions that can be made by those skilled in the art without creative effort without departing from the core of the present invention fall within the protection scope of the present invention.

Claims

1. A method for rapid solid-liquid separation of plant samples without centrifugation using magnetic particles, characterized in that, It includes the following steps: (1) Add extraction solvent to plant sample to obtain plant sample extraction system of liquid and insoluble solid mixture; (2) Add magnetic particles to the plant sample extraction system, and under the action of vortex mixing and / or ultrasonic treatment, make the magnetic particles contact and combine with the insoluble solids in the plant sample extraction system; (3) Under the action of an external magnetic field, the magnetic particles and the insoluble solids they bind aggregate and separate from the liquid phase to obtain a clear supernatant; The method achieves solid-liquid separation of the plant sample extraction system without centrifugation.

2. The method according to claim 1, characterized in that, The plant samples are powdery, fibrous, granular, or viscous plant source samples.

3. The method according to claim 1 or 2, characterized in that, The magnetic particles are magnetic metal oxide particles or magnetic composite particles; The magnetic particles have a particle size of nanometers or micrometers. The mass ratio of the plant sample to the magnetic particles is 1:5 to 1:

15.

4. The method according to claim 3, characterized in that, The magnetic particles are Fe3O4 magnetic particles; The magnetic particles are nanoscale, with a particle size of 20nm-100nm. When the plant sample is in powder form, the mass ratio of the plant sample to the magnetic particles is 1:15; when the plant sample is in viscous form, the mass ratio of the plant sample to the magnetic particles is 1:

5.

5. The method according to claim 1, characterized in that, The extraction solvent is methanol, ethanol, acetonitrile, dichloromethane, water, or a mixture of several of these solvents in any proportion.

6. The method according to claim 1, characterized in that, The duration of the ultrasonic treatment is 10–30 min.

7. The method according to claim 1, characterized in that, The external magnetic field is provided by a permanent magnet or an electromagnet.

8. The method according to claim 1, characterized in that, The supernatant is used directly for analysis by gas chromatography, liquid chromatography, or a combination of gas chromatography and mass spectrometry.

9. The method according to claim 1, characterized in that, The method is applicable to the rapid detection and quality control of traditional Chinese medicine, spices, and food plant-derived samples.

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