阅读说明：本技术 一种莲子芯的微型提取方法 (Miniature extraction method of lotus plumule ) 是由 曹君 石敏珍 朱思晨 余亚玲 于 2021-07-15 设计创作，主要内容包括：本发明涉及天然药物中生物碱的提取制备领域,针对提取生物碱耗时、费溶剂的问题,提供一种莲子芯的微型提取方法,包括以下步骤：将粉碎后的莲子芯样品和分散剂混合,研磨形成均匀的混合粉末,将混合粉末转移至固相萃取柱中利用溶剂进行洗脱,收集洗脱溶液并离心,取上层清液进行高效液相分析。本发明将基质固相分散微萃取与高效液相色谱巧妙结合起来,能快速有效的富集天然药物中的生物碱。(The invention relates to the field of extraction and preparation of alkaloids in natural medicines, and provides a micro extraction method of lotus plumule aiming at the problems of time consumption and solvent consumption in alkaloid extraction, which comprises the following steps: mixing the crushed lotus plumule sample with a dispersing agent, grinding to form uniform mixed powder, transferring the mixed powder to a solid phase extraction column, eluting by using a solvent, collecting an elution solution, centrifuging, and taking supernatant for high performance liquid analysis. The invention skillfully combines matrix solid phase dispersion micro-extraction and high performance liquid chromatography, and can quickly and effectively enrich alkaloid in natural medicines.)

1. A micro extraction method of lotus plumule is characterized by comprising the following steps: mixing the crushed lotus plumule sample with a dispersing agent, grinding to form uniform mixed powder, transferring the mixed powder to a solid phase extraction column, eluting by using a solvent, collecting an elution solution, centrifuging, and taking supernatant for high performance liquid analysis.

2. The micro-extraction method of lotus plumule according to claim 1, wherein the dispersant is selected from boron nitride, silica gel, chitosan, C18 and Flori.

3. The micro extraction method of lotus plumule according to claim 2, wherein the dispersant is boron nitride nanosheet, and the mass ratio of the lotus plumule sample to the boron nitride nanosheet is 4 (1-5).

4. The micro extraction method of lotus plumule according to claim 3, wherein the mass ratio of the lotus plumule sample to the boron nitride nanosheet is 1: 1.

5. The micro extraction method of lotus plumule according to claim 1, wherein the grinding is performed in an agate mortar for 30-150 s.

6. The micro extraction method of lotus plumule as claimed in claim 5, wherein the grinding time is 120 s.

7. The micro-extraction method of lotus plumule according to claim 1, wherein the eluting solvent is selected from methanol, acetonitrile, ethanol, acetone and chloroform.

8. The micro-extraction method of lotus plumule as claimed in claim 7, wherein the eluting solvent is methanol.

Technical Field

The invention relates to the field of extraction and preparation of alkaloids in natural medicines, in particular to a micro extraction method of lotus plumule.

Background

Matrix Solid Phase Dispersion (MSPD) extraction is an efficient sample pretreatment method firstly proposed by Baker and the like. The basic principle of MSPD is to put the sample and the dispersing agent together, grind them into a uniform mixture, facilitate the attachment of the sample powder to the dispersing agent, then introduce the mixture into a solid phase extraction tube, and elute the target compound from the dispersing agent with different elution solvents. In the MSPD process, the extraction and cleaning of the sample are completed in one step, and the time consumption is obviously reduced. With the development of the method, the method is gradually applied to the extraction and analysis of pesticide residues, heavy metals and bioactive compounds. In addition, in recent years, MSPD micro-extraction has attracted much attention because of its advantages such as low cost and environmental protection. The currently used dispersing agents include C18, C8, Florisil, alumina, silica gel and the like. The MSPD process exhibits different extraction efficiencies based on different dispersants, and it is therefore of great value to find new dispersants to improve the selectivity and extraction efficiency of extraction, especially for matrix solid phase dispersion micro-extraction.

Boron Nitride (BN) nanosheets are important inorganic compounds consisting of equal amounts of boron and nitrogen, which are arranged alternately to present an infinitely extending hexagonal honeycomb structure. The boron nitride layers are stacked under van der waals forces, and the structure of the boron nitride layers is similar to that of graphene. Thus, boron nitride has a high specific surface area, excellent lubricity, good thermal conductivity, significant thermochemical stability and superior mechanical properties. Due to the characteristics, the hexagonal boron nitride is widely applied to the fields of electronic devices, optical storage, hydrogen storage, photocatalysis and the like. In addition, boron nitride is also widely used for adsorption of grease, heavy metals, proteins, dyes and plant growth regulators. However, it has been found from previous studies that boron nitride nanosheets have not been applied as dispersants to matrix solid phase dispersion microextraction.

The plumula Nelumbinis is green young leaf and radicle of lotus seed of Nymphaeaceae, and can be used for treating arrhythmia, nervous disorder, hypertension and cardiovascular diseases. Nowadays, lotus plumule has been widely used in people's daily life, especially in asia, where it is used as dish, tea and medicine. The pharmacological properties of lotus plumule are closely related to active substances such as flavonoid, alkaloid, volatile oil and the like contained in lotus plumule. The lotus seed contains alkaloids such as liensinine, isoliensinine, neferine, etc., and has high bioactivity and health promotion function. In particular to neferine which is beneficial to oxidation resistance, thrombosis resistance and hypertension resistance. In conventional studies, methods for extracting alkaloids mainly include microwave-assisted extraction, reflux extraction, ultrasonic reflux extraction (pharmacopoeia method), and the like. For example, patent publication No. CN103893296B discloses a microwave-assisted method for extracting aconitum sungpanense alkaloid, which comprises the following steps: (1) adding an ammonia reagent into the aconitum sungpanense hand-mazz medicinal material crushed to 20-30 meshes, wetting the aconitum sungpanense hand-mazz medicinal material, and placing the moistened aconitum sungpanense hand-mazz medicinal material in microwave extraction equipment for 2-4 hours; then adding ethanol, stirring uniformly, and performing microwave extraction to obtain an extracting solution; (2) carrying out suction filtration on the extracting solution to obtain filtrate A; concentrating the filtrate A under reduced pressure until no ethanol is contained to obtain Aconitum sungpanense hand-Mazz alkaloid extract; (3) adding an acidic solution into the alkaloid extract to adjust the pH value to 2-4, and filtering to remove insoluble substances to obtain a filtrate B; adjusting the pH value of the filtrate B to 10-12 by using an ammonia reagent, extracting by using an ether-chloroform solution, and combining the extract liquor; concentrating the extractive solution under reduced pressure to obtain dry paste, and obtaining purified Aconitum sungpanense hand-Mazz alkaloid product. The invention is fast and efficient, but consumes a lot of time and elution solvent, requiring a large amount of sample. Accordingly, an ideal solution is needed.

Disclosure of Invention

The invention aims to solve the problems of time consumption and solvent consumption in extracting alkaloid, and provides a micro extraction method of lotus plumule, which skillfully combines matrix solid phase dispersion micro extraction and high performance liquid chromatography and can quickly and effectively enrich alkaloid in natural medicine.

In order to achieve the purpose, the invention adopts the following technical scheme:

a micro extraction method of lotus plumule comprises the following steps: mixing the crushed lotus plumule sample with a dispersing agent, grinding to form uniform mixed powder, transferring the mixed powder to a solid phase extraction column, eluting by using a solvent, collecting an elution solution, centrifuging, and taking supernatant for high performance liquid analysis. Compared with the traditional alkaloid extraction method, such as reflux extraction, microwave-assisted extraction, reflux extraction, ultrasonic reflux and the like, the method greatly shortens the sample processing time and the sample amount, simultaneously keeps high extraction efficiency, and has great practical value for precious samples or samples with low alkaloid content.

Preferably, the dispersant is selected from boron nitride, silica gel, chitosan, C18, and flory. More preferably, the dispersing agent is a boron nitride nanosheet, and the mass ratio of the lotus plumule sample to the boron nitride nanosheet is 4 (1-5). More preferably, the mass ratio of the lotus plumule sample to the boron nitride nanosheets is 1: 1. The invention adopts the boron nitride nanosheet as the matrix solid-phase dispersant for the enrichment technology for the first time, and is applied to the field of alkaloid extraction and enrichment in natural medicines.

Preferably, the grinding is carried out in an agate mortar for a grinding time of 30 to 150 s. More preferably, the grinding time is 120 s.

Preferably, the eluting solvent is selected from the group consisting of methanol, acetonitrile, ethanol, acetone, and chloroform. Preferably, the solvent for elution is methanol, the amount of the methanol is only 200 mu L, the consumption of the organic solvent is greatly reduced, and the principle of environmental protection is followed.

The invention provides an efficient, simple and convenient matrix solid-phase dispersion extraction method assisted by boron nitride nanosheets, which is used for enriching and extracting alkaloids in natural medicines. A series of parameters such as the type of a dispersing agent, the using amount of the dispersing agent, the grinding time, the type of an elution solvent and the like which influence the extraction efficiency are systematically discussed and optimized through a single-factor experiment. Verification experiments of linearity, daily and daytime precision (RSD%), detection Limit (LOD), quantification Limit (LOQ), repeatability, recovery rate, etc. were performed under optimal conditions. The method is successfully applied to qualitative and quantitative analysis of alkaloids (liensinine, isoliensinine and neferine) in lotus plumule, can be used for detecting the alkaloids in various medicinal materials, and has wide application potential in microanalysis for extracting the alkaloids from natural medicinal materials.

Drawings

FIG. 1 is a flow chart of a matrix solid phase dispersion extraction process.

Fig. 2 is a scanning electron microscope image of the boron nitride nanosheet dispersant.

Fig. 3 is a transmission electron microscope image of the boron nitride nanosheet dispersant.

FIG. 4 is a bar chart for examining the enrichment effect of different dispersants, wherein a, b, c, d, e represent the types of the dispersants respectively and are as follows: a-silica gel; b-flory; c-chitosan; d-XB-C18, e-boron nitride nanosheet.

Fig. 5 is a line graph for examining the enrichment effect of different amounts of boron nitride nanosheets, wherein 12.5mg, 25mg, 37.5mg, 50mg and 62.5mg represent the peak areas of the three target compounds at different dispersion doses respectively.

FIG. 6 is a line graph of the enrichment effect in different polishing times, wherein 30s, 60s, 90s, 120s and 150s represent the peak areas of the three target compounds in different polishing times.

Fig. 7 is a bar chart for examining the enrichment effect of different eluent types, wherein 1, 2, 3, 4 represent the types of elution solvents respectively, and are respectively: 1-acetonitrile; 2-trichloromethane; 3-propanone; 4-ethanol; 5-methanol.

Fig. 8 is a schematic view of interaction of a boron nitride nanosheet dispersant with a target compound.

In fig. 9, a is a mixed standard chromatogram of three target compounds, and B is a sample chromatogram extracted by an enrichment method.

Detailed Description

The technical solution of the present invention is further illustrated by the following specific examples.

In the present invention, unless otherwise specified, all the raw materials and equipment used are commercially available or commonly used in the art, and the methods in the examples are conventional in the art unless otherwise specified.

First, an embodiment

General examples

Sample preparation and enrichment: firstly, the lotus plumule sample is dried in an oven at 50 ℃, then is ground into powder by a grinder and is sieved by a 60-mesh sieve. Next, 0.05g of the sample powder and 0.0125 g to 0.0625g of the dispersant were put into an agate mortar and ground with a pestle to obtain a uniform mixture. The mixture was then transferred to a 1mL solid phase extraction column with a sieve plate at the bottom, another sieve plate placed on top, and gently compacted with a plunger. Then the solid phase extraction column is fixed on the branch pipe of the vacuum pump. The mixture was finally eluted with an elution solvent (200. mu.L). Taking the enriched extracting solution to a 1mL centrifuge tube, centrifuging for 5min at 13000rpm, and detecting the supernatant by HPLC. The flow chart of the matrix solid phase dispersion extraction process is shown in figure 1.

Preparing and enriching a labeled sample: the difference from the steps is that when HPLC is carried out, the supernatant is taken and added with the liensinine, isoliensinine and neferine standard samples, vortex mixing is carried out evenly, the supernatant is taken to a sample injection vial, and then high performance liquid phase analysis is carried out.

In fig. 9, a is a mixed standard chromatogram of three target compounds, and B is a sample chromatogram extracted by an enrichment method.

1. Examples 1 to 5 (examination of the influence of the adsorbent type on the enrichment Effect)

1.1 accurately weighing 50mg of lotus plumule sample powder and 50mg of dispersing agent, and placing the lotus plumule sample powder and the dispersing agent into an agate mortar for mixing and grinding for 2 minutes to form uniformly mixed powder; 5 clean 1mL solid phase extraction columns, numbered 1, 2, 3, 4, 5, were taken and corresponded to examples 1-5, respectively, and the dispersants were: silica gel, flory, chitosan, XB-C18 and Boron Nitride Nanosheets (BNNSs); compacting an upper sieve plate and a lower sieve plate of 5 solid-phase extraction columns;

1.2 leaching the solid phase extraction column by using 200 mu L of methanol to elute the target compound;

1.3 collecting the elution solution by using a centrifugal tube;

1.4 placing the centrifugal tube in a centrifuge, and centrifuging for 5min at 13000 rpm;

1.5 taking supernatant liquid, filling the supernatant liquid into a sample injection vial, and analyzing the result by using HPLC;

1.6 the experiment was repeated three times and the data averaged;

the HPLC chromatographic conditions are as follows:

detection wavelength: 280 nm; column temperature: 30 ℃; a chromatographic column: Extend-C18,5 μm,150mm × 4.6 mm; the mobile phase is acetonitrile (B) and 0.06% ammonia water (A), and the elution gradient is as follows: 0-3 min, 20-45% of B; 3-5min, B45-80%; 5-10 min, wherein the proportion of B is kept at 80%; the flow rate is 0.8mL/min, and the sample injection amount is 2 mu L;

the dispersing agent plays an important role in the MSPD extraction process, not only can be used as a solid carrier of sample powder, but also can disperse and adsorb target compounds. Therefore, it is important to know the nature of the adsorbent and to select the best adsorbent to improve the extraction efficiency. This study performed on dispersions such as BNNSs, silica gel, Flori, chitosan, and XB-C18, and the results are shown in FIG. 4. As can be seen from the histogram, the peak area of the target compound using BNNSs as the dispersant is much higher than other dispersants, about three times that of silica gel. This phenomenon may be caused because BNNSs are composed of an infinite number of six-atom rings periodically and repeatedly arranged, and all target compounds have benzene rings, so that pi-pi conjugated bonds exist between the adsorbent and the target compounds, increasing the adsorption capacity of BNNSs. In addition, the presence of an empty orbital in the boron atom may appear as a Lewis acid, while the target component may provide an-OH group as a Lewis base, and thus a coordination bond may be present, further enhancing the interaction force between the dispersant and the target analyte. This result may also be attributed to the high specific surface area and hydrophobic nature of BNNSs, which allows BNNSs to effectively retain the target compound by van der waals forces. The mechanism of action of these forces is shown in figure 8. Scanning electron micrographs and transmission electron micrographs of BNNSs are shown in fig. 2 and 3. In contrast, the peak area of the target compound using silica gel is significantly lower than other adsorbents. This may be due to the large porosity of the silica gel, leaving a large amount of target compound, making it difficult to elute the analyte with the extraction solution. In summary, the dispersant is optimally chosen to be BNNSs.

2. Examples 6 to 10 (examination of the influence of the adsorbent amount on the enrichment effect)

2.1 accurately weighing 50mg of lotus plumule sample powder and a certain amount of BNNSs, and placing the lotus plumule sample powder and the BNNSs into an agate mortar for mixing and grinding for 2 minutes to form uniformly mixed powder; 5 clean 1mL solid phase extraction columns, numbered 1, 2, 3, 4, 5, were used in accordance with examples 6-10, respectively, and the amounts of BNNSs used were: 12.5mg, 25mg, 37.5mg, 50mg, 62.5 mg; compacting an upper sieve plate and a lower sieve plate of 5 solid-phase extraction columns;

2.2 leaching the solid phase extraction column by using 200 mu L of methanol to elute the target compound;

2.3 collecting the elution solution by using a centrifugal tube;

2.4 placing the centrifuge tube in a centrifuge, and centrifuging for 5min at 13000 rpm;

2.5 taking supernatant liquid, filling the supernatant liquid into a sample injection vial, and analyzing the result by using HPLC;

2.6 the experiment was repeated three times and the data averaged;

the HPLC chromatographic conditions are as follows:

detection wavelength: 280 nm; column temperature: 30 ℃; a chromatographic column: Extend-C18,5 μm,150mm × 4.6 mm; the mobile phase is acetonitrile (B) and 0.06% ammonia water (A), and the elution gradient is 0-3 min and 20-45% of B; 3-5min, B45-80%; 5-10 min, wherein the proportion of B is kept at 80%; the flow rate is 0.8mL/min, and the sample injection amount is 2 mu L;

the ratio of the dispersant to the sample is another key parameter in the MSPD extraction process. The proper amount of dispersant can improve the extraction efficiency and help the target compound to be successfully eluted. In order to obtain the best extraction effect, the extraction effect of the dispersing agent with the dosage of 12.5-62.5 mg is researched, and the dosage of the sample powder is kept unchanged at 50mg in the process. The results of the detection are shown in FIG. 5. The line graphs compare peak areas of target analytes using different dispersion doses. The peak area of the target compound steadily increased as the dispersant dosage increased from 12.5mg to 37.5 mg. Meanwhile, when the dispersing agent is added into 50mg, the extraction efficiency of the compound is obviously improved, and the peak area is the largest. This phenomenon is probably due to the fact that the contact area increases significantly with increasing dispersant dosage, with a consequent increase in the absorption force between the analyte and the adsorbent. When the amount of the dispersant was increased from 50mg to 62.5mg, the peak area of the target compound showed a downward trend. The reason may be that the adsorption force between the analyte and the adsorbent is too strong, so that the target cannot be successfully eluted. In addition, a large amount of sample remains in the pores of the adsorbent due to the excessively high ratio of the dispersant to the sample. Therefore, 50mg of BNNSs was chosen as the optimum amount.

3. Examples 11 to 15 (examination of the influence of grinding time on the enrichment effect)

3.1 accurately weighing 50mg of lotus plumule sample powder and 50mg of BNNSs, and placing the lotus plumule sample powder and the BNNSs into an agate mortar for mixing and grinding to form uniformly mixed powder; 5 clean 1mL solid phase extraction columns, numbered 1, 2, 3, 4, 5, were used for examples 11-15, respectively, and the milling times for the mixtures were: 30s, 60s, 90s, 120s, 150 s; compacting an upper sieve plate and a lower sieve plate of 5 solid-phase extraction columns;

3.2. leaching the solid phase extraction column by using 200 mu L of methanol, and eluting the target compound;

3.3 collecting the elution solution by using a centrifugal tube;

3.4 placing the centrifuge tube in a centrifuge, and centrifuging for 5min at 13000 rpm;

3.5 taking supernatant liquid, filling the supernatant liquid into a sample injection vial, and analyzing the result by using HPLC;

3.6 the experiment was repeated three times and the data averaged;

the HPLC chromatographic conditions are as follows:

detection wavelength: 280 nm; column temperature: 30 ℃; a chromatographic column: Extend-C18,5 μm,150mm × 4.6 mm; the mobile phase is acetonitrile (B) and 0.06% ammonia water (A), and the elution gradient is 0-3 min and 20-45% of B; 3-5min, B45-80%; 5-10 min, wherein the proportion of B is kept at 80%; the flow rate is 0.8mL/min, and the sample injection amount is 2 mu L;

grinding time is a key optimization parameter that affects the contact force and dispersion degree of the sample powder and the adsorbent. Under the condition that other extraction parameters are not changed, the optimal extraction efficiency of different grinding times within the range of 30-150s is researched. As can be seen from fig. 6, the peak area of the target compound steadily increased as the milling time was increased from 30 seconds to 90 seconds. When the time is prolonged to 120s, the peak area of the measured object is obviously increased, and the extraction efficiency is highest. The reasons may be: the mixing of the sample and the dispersing agent is insufficient, so that the force therebetween is weak, and when the grinding time is too short, the target compound cannot be sufficiently extracted. Therefore, sufficient milling time is required to obtain good extraction efficiency. However, the line graph also shows that the peak area of the target compound decreases sharply when the time is increased from 120 seconds to 150 seconds. This may be due to the fact that the force between the dispersing agent and the sample is too great and the test substance cannot be successfully eluted over time. Therefore, 120s is considered as the optimum grinding time.

4. Examples 16 to 20 (examination of the influence of the kind of elution solvent on the enrichment effect)

4.1 accurately weighing 50mg of lotus plumule sample powder and 50mg of BNNSs, and placing the lotus plumule sample powder and the BNNSs into an agate mortar for mixing and grinding for 120s to form uniformly mixed powder; 5 clean 1mL solid phase extraction columns, numbered 1, 2, 3, 4, 5, were taken, and correspond to examples 16-20, respectively, and the types of elution solvents were: acetonitrile, chloroform, acetone, ethanol, methanol; compacting an upper sieve plate and a lower sieve plate of 5 solid-phase extraction columns;

4.2. leaching the solid phase extraction column by using 200 mu L of elution solvent, and eluting the target compound;

4.3 collecting the elution solution by using a centrifugal tube;

4.4 placing the centrifuge tube in a centrifuge, and centrifuging for 5min at 13000 rpm;

4.5 taking supernatant liquid, filling the supernatant liquid into a sample injection vial, and analyzing the result by using HPLC;

4.6 the experiment was repeated three times and the data averaged;

the HPLC chromatographic conditions are as follows:

detection wavelength: 280 nm; column temperature: 30 ℃; a chromatographic column: Extend-C18,5 μm,150mm × 4.6 mm; the mobile phase is acetonitrile (B) and 0.06% ammonia water (A), and the elution gradient is 0-3 min and 20-45% of B; 3-5min, B45-80%; 5-10 min, wherein the proportion of B is kept at 80%; the flow rate is 0.8mL/min, and the sample injection amount is 2 mu L;

during the extraction process, a suitable elution solvent is required to elute the target compound while ensuring that the matrix remains in the solid phase extraction column to achieve higher extraction of the analyte. In the experiment, 6 elution solvents such as methanol, acetonitrile, ethanol, acetone, trichloromethane and the like are adopted, and the influence of the elution solvents on enrichment is researched by keeping other conditions unchanged. As a result, as shown in FIG. 7, it can be seen that the peak area of the target compound eluted with methanol was the highest as compared with the elution with other elution solvents. This phenomenon is related to the polarity of the eluent and the analyte, and to some extent determines the degree of elution. According to the principle of similar compatibility, the polarity of the target substance is similar to that of methanol, which is beneficial to dissolution. In addition, the tested compound and the methanol both contain-OH groups, so that hydrogen bonds exist between the tested compound and the methanol, the interaction is increased, and the elution efficiency of the target compound is improved. In contrast, acetonitrile, acetone and chloroform are much less efficient at extraction than methanol and ethanol. The reason may be that the force between them is too weak to form hydrogen bond with the object to be measured, resulting in insufficient elution amount and low extraction efficiency. Methanol is therefore the best elution solvent.

Second, methodology investigation

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

1. Precision within a day

1.1 accurately weighing 50mg of lotus plumule sample powder and 50mg of BNNSs, and placing the lotus plumule sample powder and the BNNSs into an agate mortar for mixing and grinding for 120s to form uniformly mixed powder; 1, filling 1mL solid phase extraction column with the mixed powder, and compacting an upper sieve plate and a lower sieve plate;

1.2 leaching the solid phase extraction column by using 200 mu L of methanol to elute the target compound;

1.3 collecting the elution solution by using a centrifugal tube;

1.4 placing the centrifugal tube in a centrifuge, and centrifuging for 5min at 13000 rpm;

1.5 taking supernatant liquid, filling the supernatant liquid into a sample injection vial, and analyzing the result by using HPLC;

1.6 sample injection analysis, and sample injection is carried out for 6 times in different time periods in the same day.

2. Precision of day

2.1 accurately weighing 50mg of lotus plumule sample powder and 50mg of BNNSs, and placing the lotus plumule sample powder and the BNNSs into an agate mortar for mixing and grinding for 120s to form uniformly mixed powder; 1, filling 1mL solid phase extraction column with the mixed powder, and compacting an upper sieve plate and a lower sieve plate;

2.2 leaching the solid phase extraction column by using 200 mu L of methanol to elute the target compound;

2.3 collecting the elution solution by using a centrifugal tube;

2.4 placing the centrifuge tube in a centrifuge, and centrifuging for 5min at 13000 rpm;

2.5 taking supernatant liquid, filling the supernatant liquid into a sample injection vial, and analyzing the result by using HPLC;

2.6 injection analysis, injection at the same time point within three days, 2 times per day.

3. Repeatability of

Refer to the following experimental procedure, run 3 groups in parallel as a survey

3.1 accurately weighing 50mg of lotus plumule sample powder and 50mg of BNNSs, and placing the lotus plumule sample powder and the BNNSs into an agate mortar for mixing and grinding for 120s to form uniformly mixed powder; 1, filling 1mL solid phase extraction column with the mixed powder, and compacting an upper sieve plate and a lower sieve plate;

3.2 leaching the solid phase extraction column by using 200 mu L of methanol to elute the target compound;

3.3 collecting the elution solution by using a centrifugal tube;

3.4 placing the centrifuge tube in a centrifuge, and centrifuging for 5min at 13000 rpm;

3.5 supernatant was taken and filled into injection vials, and the results were analyzed by HPLC.

4. Sample recovery rate

With reference to the following experimental procedure, 3 groups were run in parallel for each concentration

4.1 crushing the lotus plumule sample, accurately weighing 50mg of lotus plumule sample powder and 50mg of BNNSs, and placing the lotus plumule sample powder and the 50mg of BNNSs into an agate mortar for mixing and grinding for 120s to form uniformly mixed powder;

4.2 preparing a part according to the step 1;

4.3 taking 2 clean 1mL solid-phase extraction columns, numbering 1 and 2, filling the prepared mixed powder into the columns, and compacting an upper sieve plate and a lower sieve plate of the 2 solid-phase extraction columns;

4.4 leaching the solid phase extraction column by using 200 mu L of methanol to elute the target compound;

4.5 collecting the elution solution by using a centrifugal tube;

4.6 adding mixed standard samples with the concentration of 1 mu g/mL and 10 mu g/mL into the two centrifuge tubes respectively; swirling to fully and uniformly mix the sample solution and the mixing standard;

4.7 placing the centrifuge tube in a centrifuge, and centrifuging for 5min at 13000 rpm;

4.8 supernatant was taken and filled into injection vials, and the results were analyzed by HPLC.

The experimental results are summarized in the following tables 1 and 2, and the results show that the method has good repeatability, high recovery rate and accurate detection.

Table 1.

Table 2.

Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.