CN115343390B - Method for extracting effective and harmful components in traditional Chinese medicine by matrix solid-phase dispersion - Google Patents

Abstract

The invention discloses a method for extracting effective and harmful components in traditional Chinese medicine by matrix solid-phase dispersion micro-extraction, which comprises the steps of shaking off most of surface soil of galangal with cultivated soil, grinding into 80-mesh powder by a grinder, grinding with nano graphite powder, uniformly dispersing and adsorbing the powder in the nano graphite powder, loading the powder into a solid-phase extraction column, desorbing by using 2-hydroxypropyl-beta-cyclodextrin solution, and eluting target compounds dispersed in the nano graphite powder to realize extraction. The invention takes the nano graphite powder and the cyclodextrin as the dispersing agent and the eluting solvent for the first time, provides a novel method which is simple, quick, green and pollution-free, can realize the simultaneous extraction of the effective and harmful components in the galangal, has wide application range, and can be used for the extraction and detection of flavonoid compounds in various medicinal materials and the extraction and detection of toxic components in various medicinal materials.

Description

Method for extracting effective and harmful components in traditional Chinese medicine by matrix solid-phase dispersion

Technical Field

The invention belongs to the field of synchronous extraction of effective and harmful components in complex food matrixes, and relates to a method for extracting the effective and harmful components in traditional Chinese medicines by matrix solid-phase dispersion and micro-extraction, so as to realize simultaneous extraction of the effective and harmful components in galangal.

Background

Previous studies have demonstrated the feasibility of matrix solid phase dispersion extraction (MSPD) techniques that can meet the requirement for simultaneous extraction of multiple naturally occurring compounds from solid samples. In recent years, matrix Solid Phase Dispersion Microextraction (MSPDM) has gained popularity due to simplicity of operation, low sample and dispersant consumption, and less subsequent clean-up work.

It is well known that the dispersant/eluting solvent combination used has a significant impact on the selectivity of the MSPD process. The solid carriers commonly used in the current research are C18 bonded silica, florisil and the like. For example, mansur et al developed a C18-assisted MSPD method for extracting and determining flavonoids in tartary buckwheat sprouts;and the MSPD method is established, wherein florisil is used as an adsorbent, pyrethrin is extracted from plant samples, and the recovery rate of an analyte is higher than 70%. Notably, the nano graphite powder is due toThe volume is small, the specific surface area is large, and the potential of the dispersing agent is also provided, but no related research report exists at present.

The nature of the eluting solvent also plays an important role in the MSPD process, as the target compounds should be efficiently desorbed into the eluent, while the majority of non-target components should remain on the extraction column. For extracting natural compounds, common eluting solvents are methanol and ethanol; whereas pesticides are typically eluted with acetonitrile, chloroform, hexane and acetone; therefore, the active and toxic components in the complex matrix are extracted by adopting organic solvents, so that the environmental injury is large. Cyclodextrin (CD) is a cyclic oligosaccharide consisting of a typical tapered tail structure, which can form supramolecular host guest complexes by selectively incorporating various inorganic, organic and biological molecules into its hydrophobic cavity. However, to our knowledge, currently in the field of extraction, there is no research on the use of cyclodextrin as an eluent.

In addition, in previous studies, MSPD was used to extract active ingredients such as flavonoids, phenolic acids and terpenes from plants, or toxic ingredients such as organic pesticides, nitrotoluene, hormones and amine compounds from environmental samples. However, very little research has been conducted on extraction of active and toxic ingredients from the same matrix. Thus, there is a need to create a novel, environmental, more comprehensive method of MSPD to address more complex situations.

Galangal (Alpinia officinarum) is a plant of the genus Alpinia of the family Zingiberaceae, and is widely used worldwide as an inexpensive seasoning, spice or herbal medicine due to its unique flavor and pharmacological effects. In galangal, the content of flavonoids such as galangin, pinocembrin, chrysin, etc. is rich, and the composition has various biological activities such as sterilization, anti-tumor, antioxidation, anti-inflammatory, anti-fibrosis, etc. Common extraction methods for these compounds are Soxhlet extraction and organic solvent assisted ultrasonic extraction, but these methods are time consuming and labor intensive and are contrary to the concept of green development. On the other hand, phenoxyacetic acid herbicides are widely used in crop planting due to the need for plant growth, but their impact on human health and ecological environment has attracted considerable attention. The current common extraction method of the toxic compounds is a solid phase extraction method, which has the defects of low extraction efficiency and the need of using an organic solvent. At present, the simultaneous extraction of active ingredients and toxic ingredients in the galangal through a one-step method is not researched, so that a simple, efficient and green invention is developed, and the simultaneous extraction of the active ingredients and the toxic ingredients in the galangal is very significant.

Disclosure of Invention

The invention aims at establishing a novel, efficient and comprehensive nano graphite powder auxiliary matrix solid-phase dispersion micro-extraction (NG-MSPDM) and ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) technology, and simultaneously extracting 5 effective active compounds including chrysin, pinocembrin, galangin, farnesin and kaempferol from galangal and 5 toxic compounds including 2,4-D propionic acid (2, 4-DP), 2,4, 5-nasal discharge propionic acid (2, 4, 5-TP), 4-phenoxyacetic acid (4-CP), 2-methyl-4-chlorophenoxyacetic acid (2M-4C) and 2, 4-dichlorophenoxyacetic acid (2, 4-D), wherein the toxic compounds are derived from benzoic acid herbicides in the galangal cultivation soil. The adoption of the nano graphite powder as a dispersing agent and the beta-cyclodextrin solution as an eluting solvent is a beneficial attempt, so that the use of an organic solvent is avoided while the efficient extraction is ensured. In order to verify the effectiveness and practicability of the invention, a single factor test is adopted to optimize relevant factors in the process. The result shows that the novel NG-MSPDM-UHPLC-Q-TOF/MS method has wide application prospect in the aspect of simultaneously extracting effective components and toxic components in complex matrixes.

The invention adopts the following technical scheme:

step (1), obtaining galangal carrying cultivation soil; shaking most of the surface soil, and grinding into 80 mesh powder by a grinder;

step (2), extraction of a target compound: mixing the powdery sample and the dispersing agent for co-grinding treatment to obtain a co-grinding product; loading the co-ground product into a solid phase extraction column, and desorbing with a certain volume of eluting solvent; wherein the concentration of the mixed methanol solution of the harmful substance standard substance is 500 micrograms/milliliter, the dispersing agent is nano graphite powder, and the eluting solvent is 2-hydroxypropyl-beta-cyclodextrin;

preferably, the mass ratio of the powdery sample to the dispersing agent in the step (1) is 4:1 to 10, more preferably 4:7, preparing a base material;

preferably, the co-milling time is 30 to 60 seconds, more preferably 60 seconds;

preferably, the volume to mass ratio of the eluting solvent to the powdered sample of step (1) is 500 microliters: 20 mg;

preferably, the eluting solvent concentration is 10 to 30 mmol/l, more preferably 20 mmol/l.

The invention has the advantages that:

1. compared with the traditional method for extracting the compounds in the galangal, the method has the advantages of rapidness, simplicity, greenness and no pollution.

2. The method has wide application range, can be used for extracting and detecting flavonoid compounds in various medicinal materials, can also be used for extracting and detecting toxic components in various medicinal materials, and has wide application potential in extracting natural medicinal materials.

3. The method takes the nano graphite powder and the cyclodextrin as the dispersing agent and the eluting solvent for the first time, and has the advantages of novelty and high efficiency.

Namely, the invention systematically discusses and optimizes a series of parameters such as the type of the dispersing agent, the dosage of the dispersing agent, the grinding time, the type of the eluting solvent, the concentration of the eluting solvent and the like which influence the extraction efficiency through a single factor experiment. Experiments on the daily and daytime precision (RSD), repeatability, recovery rate and the like were performed under the optimal conditions. The invention can be successfully applied to qualitative and quantitative analysis of the activity (chrysin, pinocembrin, galangin, farnesol and kaempferol) and toxic compounds (2, 4-DP,2,4,5-TP, 4-CP,2M-4C and 2, 4-D) in galangal.

Drawings

FIG. 1 is a flow chart of the extraction and separation of target compounds according to the present invention.

FIG. 2 is a bar graph of the extraction effect for examining the different dispersant species. In the figure, 1 is chrysin, 2 is pinocembrin, 3 is galangin, 4 is farnesol, 5 is kaempferol, 6 is 4-CP,7 is 2M-4C,8 is 2,4-D,9 is 2,4-DP, and 10 is 2,4,5-TP.

FIG. 3 is a bar graph of the extraction effect for different dispersant amounts. In the figure, 1 is chrysin, 2 is pinocembrin, 3 is galangin, 4 is farnesol, 5 is kaempferol, 6 is 4-CP,7 is 2M-4C,8 is 2,4-D,9 is 2,4-DP, and 10 is 2,4,5-TP.

Fig. 4 is a bar graph for examining the extraction effect at different grinding times. In the figure, 1 is chrysin, 2 is pinocembrin, 3 is galangin, 4 is farnesol, 5 is kaempferol, 6 is 4-CP,7 is 2M-4C,8 is 2,4-D,9 is 2,4-DP, and 10 is 2,4,5-TP.

FIG. 5 is a bar graph of the extraction effect for examining different eluting solvent species. In the figure, 1 is chrysin, 2 is pinocembrin, 3 is galangin, 4 is farnesol, 5 is kaempferol, 6 is 4-CP,7 is 2M-4C,8 is 2,4-D,9 is 2,4-DP, and 10 is 2,4,5-TP.

FIG. 6 is a bar graph of the extraction effect for examining the concentration of different elution solvents. In the figure, 1 is chrysin, 2 is pinocembrin, 3 is galangin, 4 is farnesol, 5 is kaempferol, 6 is 4-CP,7 is 2M-4C,8 is 2,4-D,9 is 2,4-DP, and 10 is 2,4,5-TP.

Detailed Description

As described above, in view of the shortcomings of the prior art, the present inventors have long studied and practiced a lot and have proposed a method for matrix solid phase dispersion and microextraction of active and harmful ingredients in traditional Chinese medicines.

In the method, after the mixed methanol solution of galangal powder and harmful substance standard substances and the nano graphite powder are co-ground, the mixture is uniformly dispersed and adsorbed in the nano graphite powder, the powder is put into a solid phase extraction column to be desorbed by using a 2-hydroxypropyl-beta-cyclodextrin solution, and the target compound dispersed in the nano graphite powder is eluted to realize extraction. And taking an eluted upper layer sample solution, centrifugally filtering, and carrying out UHPLC-Q-TOF/MS analysis, wherein the content of the target compound is measured by Agilent Q-TOF/MS to show the effectiveness of the extraction method. The invention provides a simple, quick and environment-friendly novel method capable of simultaneously extracting and detecting the effective and harmful components in the galangal.

In the practical application scene, the harmful components come from benzoic acid herbicides in soil. However, the content of harmful ingredients in the soil is low, so that the inventor adopts a harmful substance standard substance as a source of the harmful ingredients in the process of the invention, so that the extraction result is more remarkable, and related factors are better optimized, thereby being convenient for being put into practical application.

The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent.

The invention discloses an extraction and separation of effective and toxic components in galangal by nano graphite powder-assisted matrix solid-phase dispersion microextraction (NG-MSPDM), which comprises the following steps:

the galangal carrying the cultivation soil is obtained, most of the surface soil is shaken off, and then crushed into 80-mesh powder by a crusher. Mixing the powder sample and the dispersing agent, performing co-grinding treatment to obtain a mixture, pouring the mixture into a solid phase extraction column with a sieve plate at the bottom, adding the powder, adding the same sieve plate, and carefully compacting by using a glass rod. The effective and toxic compounds are then eluted with 500. Mu.l of elution solvent at a concentration of 10 to 40 mM. No additional force is applied during elution. The eluate was collected and diluted 5-fold, centrifuged at 15000 rpm for 3 minutes, filtered through a microporous membrane (50 mm. Times.0.45 μm), and injected into UHPLC-Q-TOF/MS for analysis after collection. The above steps were repeated 3 times.

In a specific embodiment, the method adopted in the experiment is as follows: pulverizing rhizoma Alpiniae Officinarum sample into 80 mesh powder with pulverizer; a standard solution of a mixture of 20 mg of sample, 5-50 mg of nano-graphite powder and 5. Mu.l of toxic analyte with a concentration of 500. Mu.g/ml was ground in an agate mortar for 30-120 seconds. The mixture was then poured into a solid phase extraction column with a sieve plate at the bottom, and after adding the powder, the same sieve plate was added and carefully compacted with a glass rod. The effective and toxic compounds are then eluted with 500. Mu.l of elution solvent at a concentration of 10 to 40 mM. No additional force is applied during elution. The eluate was collected and diluted 5-fold, centrifuged at 15000 rpm for 3 minutes, filtered through a microporous membrane (50 mm. Times.0.45 μm), and injected into UHPLC-Q-TOF/MS for analysis after collection. The above steps were repeated 3 times. The above process and schematic diagram are shown in fig. 1.

UHPLC conditions:

the target analyte was separated by means of an Agilent Agilent Eclipse Plus C chromatographic column (2.1X100 mm,3.5 μm) with a column temperature set at 30 ℃. The mobile phase was 0.1% formic acid in water (solvent a) and methanol (solvent B), the mobile phase gradient was set as follows: 0-3 minutes, 10-75% B;3-10 minutes, 75-83% B;10-11 minutes, 83-100% B. The flow rate was 0.4 ml/min and the injection volume was 5 μl.

The mass spectrometer adopts an electrospray ionization source, works in a negative ionization mode, and has a mass spectrum range of m/z:100-1000, the acquisition rate is 1 spectrum per second. Other parameters were set as follows: drying gas temperature, 300 ℃; drying gas flow, 10L/min; a nebulizer, 50psig; sheath temperature: 350 ℃; sheath air flow: 11L/min; capillary voltage, 40000V; nozzle voltage, 1000V; the fragmentation voltage, 400V. The processing and analysis of the collected data was performed by Mass Hunter software Version 10.00 (Agilent Technologies, santa Clara, CA).

The following description of the present invention is further provided with reference to several preferred embodiments, but the experimental conditions and setting parameters should not be construed as limiting the basic technical scheme of the present invention. And the scope of the present invention is not limited to the following examples. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.

Example 1 examination of the Effect of dispersant species on extraction Effect

1.1 taking 4 clean agate mortars, numbering 1, 2, 3 and 4, and sequentially adding 20 mg of galangal powder;

1.2 adding 20 mg of C18, nano graphite powder, silica gel and florisil into 4 agate mortars correspondingly, and then adding 5 microliter of toxic substance mixed standard methanol solution with the concentration of 500 micrograms/milliliter respectively;

1.3 grinding each group with the same force for 60 seconds;

1.4, taking out the ground product, sequentially loading the ground product into 4 solid-phase extraction columns with sieve plates at the bottoms, then loading the same sieve plates, carefully compacting the powder by using a glass rod, and then placing the powder into a solid-phase extraction device;

1.5 elution with 500. Mu.l of 2-hydroxypropyl-beta-cyclodextrin solution at a concentration of 20 mmol/l, without applying additional force;

1.6, respectively diluting the eluent in a centrifuge tube for 5 times, and centrifuging at 15000 r for 3 min;

1.7 aspiration of intermediate liquid and injection into UHPLC-Q-TOF/MS.

The chromatographic conditions are as follows:

column temperature: 30 DEG C

The experimental results are shown in FIG. 2. Fig. 2 is a bar graph of extraction effect for examining different dispersant species.

The type of dispersed adsorbent is an important parameter in the MSPDM process, involving the dispersed state of the adsorbent in the matrix by co-milling, as well as the ability of the adsorbent to disrupt the solid structure and extract the target analyte. From the graph, the extraction rate of the nano graphite powder on the active component and the toxic component is the highest in the 4 dispersing agents. The extraction efficiency of florisil, silica gel and C18 for target analytes is significantly weaker than that of nano-graphite powder, probably due to the hydrophobic nature of flavonoid active ingredients, while these adsorbents also have a significant hydrophobic surface, resulting in a strong adsorption capacity for active ingredients and the inability of the eluting solvent to overcome the strength of this interaction. In addition, the nano graphite powder has small particle size, can be well mixed with a sample with destroyed structure after grinding, and is extracted in an elution step. In addition, the nano graphite powder has stronger affinity to planar molecules, and can be used as a purifying agent to remove some pigments in a sample matrix. Therefore, the nano graphite powder was chosen as the best adsorbent for further investigation.

EXAMPLE 2 investigation of the Effect of dispersant usage on extraction Effect

2.1 taking 4 clean agate mortars with the numbers of 1, 2, 3 and 4, and sequentially adding 20 milligrams of galangal powder;

2.2 adding 5, 20, 35 and 50 milligrams of nano graphite powder into 4 agate mortars correspondingly, and then adding 5 microliters of toxic substance mixed standard methanol solution with the concentration of 500 micrograms/milliliter respectively;

2.3 grinding each group with the same force for 60 seconds;

2.4, taking out the ground product, sequentially loading the ground product into 4 solid-phase extraction columns with sieve plates at the bottoms, then loading the same sieve plates, carefully compacting the powder by using a glass rod, and then placing the powder into a solid-phase extraction device;

2.5 elution with 500. Mu.l of 2-hydroxypropyl-beta-cyclodextrin solution at a concentration of 20 mmol/l, without applying additional force;

2.6, respectively diluting the eluent in a centrifuge tube for 5 times, and centrifuging at 15000 r for 3 min;

2.7 aspiration of intermediate liquid and injection into UHPLC-Q-TOF/MS.

The chromatographic conditions are as follows:

column temperature: 30 DEG C

The experimental results are shown in FIG. 3. FIG. 3 is a bar graph of extraction results for examining the amounts of different dispersants.

Optimizing the amount of nano-graphite powder is a critical step in the MSPDM process because the proper amount of dispersed adsorbent allows the target analyte in the sample to be fully adsorbed. From the graph, the extraction efficiency of active analytes (except chrysin) and toxic analytes showed a trend of increasing and then decreasing with increasing dispersant usage, and peaked at 35 mg. This phenomenon may occur because, when the amount of the dispersant is less than 35 mg, it is not sufficiently dispersed on the surface of the sample powder, resulting in incomplete extraction; when the dosage of the dispersing agent reaches 35 mg, the dispersing agent is fully contacted with the sample matrix through surface grinding treatment, and is combined to form intermolecular forces, and the intermolecular forces are isolated by the analysis effect of the eluent, so that the target analyte is fully extracted. When the amount is more than 35 mg, the extraction rate does not rise any more, indicating that the adsorption has reached a saturated state, and the increase in the amount causes difficulty in analysis and waste of reagents. Thus, 35 mg of nano graphite powder was selected as the optimal amount for the following experiment.

Example 3 investigation of the Effect of milling time on extraction

3.1 taking 4 clean agate mortars with the numbers of 1, 2, 3 and 4, and sequentially adding 20 milligrams of galangal powder;

3.2, respectively adding 35 mg of nano graphite powder, and then respectively adding 5 microliters of toxic substance mixed standard methanol solution with the concentration of 500 micrograms/milliliter;

3.3 grinding each group with the same force for 30, 60, 90 and 120 seconds;

3.4, taking out the ground product, sequentially loading the ground product into 4 solid-phase extraction columns with sieve plates at the bottoms, then loading the same sieve plates, carefully compacting the powder by using a glass rod, and then placing the powder into a solid-phase extraction device;

3.5 elution with 500. Mu.l of 2-hydroxypropyl-beta-cyclodextrin solution at a concentration of 20 mmol/l, no additional force being applied during this procedure;

3.6, respectively diluting the eluent in a centrifuge tube for 5 times, and centrifuging at 15000 r for 3 min;

3.7 sucking the intermediate liquid and injecting UHPLC-Q-TOF/MS.

The chromatographic conditions are as follows:

column temperature: 30 DEG C

The experimental results are shown in FIG. 4. Fig. 4 is a bar graph of extraction effect for examining different milling times.

The milling time can significantly affect the interaction of the dispersed adsorbent with the sample matrix, thereby significantly affecting the extraction efficiency of the target compound. As can be seen from the figure, the extraction efficiency of the target compound was improved with the increase of the polishing time (from 30 seconds to 60 seconds). And as the milling time increases to 90 seconds, the extraction efficiency of all five analytes drops dramatically. It is presumed that the grinding time of 30 seconds only allows a small amount of sample to be adsorbed on the surface of the nano graphite powder, and the adsorption force is weak, so that only a small amount of analyte is eluted; with increasing milling time, the sample and dispersant are thoroughly and homogeneously mixed, and particularly for high levels of compounds, better dispersion is achieved; however, lengthening the milling time can result in too strong an adsorption of the analyte on the graphite nanopowder, greatly increasing the difficulty of the elution step, resulting in a certain amount of compounds not being eluted. Thus, a 60 second milling time was chosen for further investigation.

Example 4 investigation of the Effect of eluting solvent species on extraction Effect

4.1 taking 4 clean agate mortars with the numbers of 1, 2, 3 and 4, and sequentially adding 20 milligrams of galangal powder;

4.2, respectively adding 35 mg of nano graphite powder, and then respectively adding 5 microliters of toxic substance mixed standard methanol solution with the concentration of 500 micrograms/milliliter;

4.3 grinding each group with the same force for 60 seconds;

4.4, taking out the ground product, sequentially loading the ground product into 4 solid-phase extraction columns with sieve plates at the bottoms, then loading the same sieve plates, carefully compacting the powder by using a glass rod, and then placing the powder into a solid-phase extraction device;

4.5 The 4 solid phase extraction columns were eluted with 500 μl of α -cyclodextrin, β -cyclodextrin, γ -cyclodextrin and 2-hydroxypropyl- β -cyclodextrin solutions, respectively, at a concentration of 20 mmol/l, without applying additional force during the process;

4.6, respectively diluting the eluent in a centrifuge tube for 5 times, and centrifuging at 15000 r for 3 min;

4.7 aspiration of intermediate liquid and injection into UHPLC-Q-TOF/MS.

The chromatographic conditions are as follows:

column temperature: 30 DEG C

The experimental results are shown in FIG. 5. FIG. 5 is a bar graph of extraction results for examining different eluting solvent species.

It is known from the prior art that the choice of eluent is determined by the strength of interaction between the analyte and the dispersant and is critical to the removal of interfering components and to achieve higher recovery. As can be seen from the figure, on the one hand, the highest efficiency of using 2-hydroxypropyl-beta-cyclodextrin is achieved, and especially for chrysin and kaempferol, the elution capacity of 2-hydroxypropyl-beta-cyclodextrin is about 50 and 100 times that of alpha-CD. This phenomenon may be due to the fact that after the nano graphite powder is combined with the active ingredients of the flavonoid compounds through interaction, a solution with strong desorption capacity is required to elute them, while the desorption capacity of α -cyclodextrin, β -cyclodextrin, γ -cyclodextrin is obviously too weak to elute sufficiently. On the other hand, the four eluting solvents have little difference in extraction efficiency of five toxic components, and the desorption capacity of 2-hydroxypropyl-beta-cyclodextrin is slightly better. Considering that 2-hydroxypropyl-beta-cyclodextrin has a relatively good extraction effect on both components, it is selected as the optimal eluent.

Example 5 investigation of the Effect of elution solvent concentration on extraction Effect

5.1 taking 4 clean agate mortars with the numbers of 1, 2, 3 and 4, and sequentially adding 20 milligrams of galangal powder;

5.2 adding 35 mg of nano graphite powder respectively, and then adding 5 microliter of toxic substance mixed standard methanol solution with the concentration of 500 micrograms/milliliter respectively;

5.3 grinding each group with the same force for 60 seconds;

5.4, taking out the ground product, sequentially loading the ground product into 4 solid-phase extraction columns with sieve plates at the bottoms, then loading the same sieve plates, carefully compacting the powder by using a glass rod, and then placing the powder into a solid-phase extraction device;

5.5 The 4 solid phase extraction columns were eluted with 500. Mu.l of 2-hydroxypropyl-beta-cyclodextrin solution having a concentration of 10 mM, 20 mM, 30 mM, 40 mM, respectively, without applying additional force during the procedure;

5.6, respectively diluting the eluent in a centrifuge tube for 5 times, and centrifuging at 15000 r for 3 min;

5.7 aspiration of intermediate liquid and injection into UHPLC-Q-TOF/MS.

The chromatographic conditions are as follows:

column temperature: 30 DEG C

The experimental results are shown in FIG. 6. FIG. 6 is a bar graph of extraction results for examining different eluting solvent concentrations.

The appropriate concentration of elution solvent facilitates efficient desorption of the target analyte while retaining the remaining matrix components in the MSPD column. From the graph, as the concentration of the eluting solvent increases from 10 mmol/l to 20 mmol/l, the extraction rate of the two components shows an increasing trend, and then decreases significantly as the concentration is higher than 20 mmol/l. This is probably due to the fact that at an eluent concentration of 10 mmole/liter, the polarity of the eluent is too high to elute all the less polar target compounds, whereas at a concentration exceeding 30 mmole/liter, both the target and non-target compounds in the sample matrix are eluted, affecting the signal of the chromatogram. In addition, the viscosity of 2-hydroxypropyl-beta-cyclodextrin increases with increasing concentration, resulting in poor diffusion capacity, and thus, a high concentration of eluting solvent is difficult to penetrate into plant tissues, impeding mass transfer of analytes from sample matrix to eluting phase, thereby reducing extraction efficiency. Thus, depending on the test conditions, exactly 20 mmole/liter of 2-hydroxypropyl-beta-cyclodextrin is sufficient to obtain satisfactory extraction of the active ingredient and of the toxic ingredient.

To further verify the feasibility of the method, methodological examinations were performed including intra-day precision, inter-day precision, reproducibility, and sample recovery.

Precision within the day

1. Taking 3 clean 1.5 ml centrifuge tubes, numbering 1, 2 and 3, and respectively preparing standard solutions of 5 active ingredients and 5 harmful ingredient analytes of 10 micrograms/ml; the 5 active ingredients are chrysin, pinocembrin, galangin, farnesol and kaempferol, and the 5 harmful ingredients are 4-CP,2M-4C,2,4-D,2,4-DP,2,4,5-TP.

2. Centrifuging at 15000 rpm for 3 min;

3. sucking the intermediate liquid and injecting the intermediate liquid into UHPLC-Q-TOF/MS;

4. and (3) sample injection analysis, wherein 6 times of sample injection are performed in different time periods in the same day.

Precision of daytime

1. Taking 3 clean 1.5 ml centrifuge tubes, numbering 1, 2 and 3, and respectively preparing standard solutions of 5 active ingredients and 5 harmful ingredient analytes of 10 micrograms/ml; the 5 active ingredients are chrysin, pinocembrin, galangin, farnesol and kaempferol, and the 5 harmful ingredients are 4-CP,2M-4C,2,4-D,2,4-DP,2,4,5-TP.

2. Centrifuging at 15000 rpm for 3 min;

3. sucking the intermediate liquid and injecting the intermediate liquid into UHPLC-Q-TOF/MS;

4. sample injection analysis, sample injection is performed at the same time point within three days, and the sample injection is performed 2 times a day.

Repeatability of

Referring to the following experimental procedure, 3 groups were made in parallel for investigation

1. Taking 3 clean 1.5 ml centrifuge tubes, with the numbers of 1, 2 and 3, respectively preparing sample extracting solutions under the optimal conditions (35 mg of nano graphite powder as dispersing agent, 60 seconds as grinding time and 20 mmol/L of 2-hydroxypropyl-beta-cyclodextrin solution as eluting solvent);

2. centrifuging at 15000 rpm for 3 min;

3. the intermediate liquid was aspirated, injected into UHPLC-Q-TOF/MS, and analyzed for results.

Recovery rate of adding mark

Referring to the following experimental procedure, 3 groups were made in parallel for each concentration

1. 20 mg of galangal powder (80 mesh), 5. Mu.l of a toxic substance mixed with a standard methanol solution having a concentration of 500. Mu.g/ml and 35 mg of nano graphite powder were weighed, ground for 60 seconds, and the ground product was taken out, put into a solid-phase extraction column having a sieve plate at the bottom, then mounted with an identical sieve plate, and put into a solid-phase extraction apparatus after carefully compacting the powder with a glass rod. Eluting with 500 microliter of 2-hydroxypropyl-beta-cyclodextrin solution with concentration of 20 millimoles/liter, diluting the collected eluent in a centrifuge tube for 5 times, centrifuging at 15000 r for 3 minutes, and taking out 100 microliter of intermediate liquid;

2. adding a label (the content is 1 mug/mg and 10 mug/mg of mixed label), and standing at room temperature for 2 hours to enable the mixed label to uniformly react with a sample;

3. centrifuging at 15000 rpm for 3 min;

4. the intermediate liquid was aspirated and injected into UHPLC-Q-TOF/MS.

The results of the experiments are summarized in tables 1-2 below.

Table 15 daily precision, daytime precision and reproducible results for the active ingredients and 5 harmful ingredients

Table 2 5 results of the normalized recovery of the active ingredients and 5 harmful ingredients

The result shows that the method has good repeatability, proper recovery rate and accurate separation and extraction result.

Claims (8)

1. A method for extracting effective and harmful components in a traditional Chinese medicine by matrix solid-phase dispersion and micro-extraction is characterized in that the effective components comprise chrysin, pinocembrin, galangin, farnesol and kaempferol, and the harmful components comprise 2, 4-propionic acid, 2,4, 5-nasal discharge propionic acid, 4-phenoxyacetic acid, 2-methyl-4-phenoxyacetic acid and 2, 4-dichlorophenoxyacetic acid; the method comprises the following steps:

step (1), obtaining galangal carrying cultivation soil, shaking off the surface soil, and then grinding into powder through a grinder;

step (2), extraction of a target compound: mixing the powdery sample and the dispersing agent obtained in the step (1) for co-grinding treatment to obtain a co-grinding product; loading the co-ground product into a solid phase extraction column, and desorbing with a certain volume of eluting solvent; wherein the dispersing agent is nano graphite powder, and the eluting solvent is 2-hydroxypropyl-beta-cyclodextrin.

2. The method for matrix solid phase dispersion micro-extraction of active and harmful ingredients in traditional Chinese medicine according to claim 1, wherein the mass ratio of the powdery sample to the dispersing agent in the step (1) is 4:1 to 10.

3. The method for matrix solid phase dispersion micro-extraction of active and harmful ingredients in traditional Chinese medicine according to claim 2, wherein the mass ratio of the powdery sample to the dispersing agent in the step (1) is 4:7.

4. the method for matrix solid phase dispersion micro-extraction of active and harmful ingredients in a traditional Chinese medicine according to claim 1, wherein the co-grinding treatment time is 30-60 seconds.

5. The method for matrix solid phase dispersion micro-extraction of active and harmful ingredients in Chinese medicine according to claim 4, wherein the co-milling treatment time is 60 seconds.

6. The method for matrix solid phase dispersion micro-extraction of active and harmful ingredients in traditional Chinese medicine according to claim 1, wherein the volume mass ratio of the eluting solvent to the powdery sample in the step (1) is 500 microliters: 20 mg.

7. The method for matrix solid phase dispersion micro-extraction of active and harmful ingredients in a traditional Chinese medicine according to claim 1, wherein the concentration of the eluting solvent is 10-30 mmol/l.

8. The method for matrix solid phase dispersion micro-extraction of active and harmful ingredients in a traditional Chinese medicine according to claim 7, wherein the concentration of the eluting solvent is 20 mmol/l.