CN111929396A - On-line enrichment method of dendrobium - Google Patents
On-line enrichment method of dendrobium Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/08—Preparation using an enricher
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- Physics & Mathematics (AREA)
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- Life Sciences & Earth Sciences (AREA)
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Abstract
The invention relates to the field of separation and enrichment of natural medicines, in particular to an online enrichment method of dendrobium, which comprises the following steps: (1) adding the dendrobe extracting solution into a phosphoric acid solution containing CD to obtain a sample solution; (2) dissolving methanol and SDS in a phosphoric acid solution to obtain a BGS solution; (3) introducing the sample solution subjected to ultrasonic treatment and filtration treatment through a capillary tube which is activated and washed by a BGS solution; (4) and (4) separating and enriching under high voltage to obtain the separated and enriched active substance. The method adopts a CD-assisted scavenging-MEKC method to enrich active substances in the dendrobium, has good separation effect, and has the advantages of high enrichment efficiency, simple operation, small using amount of organic solvent, short analysis time, no need of additional instruments and the like; meanwhile, the CD is used for increasing the solubility of the effective components in the dendrobium in water, so that the separation and enrichment effects of the effective components in the dendrobium after capillary electrophoresis are improved.
Description
Technical Field
The invention relates to the field of separation and enrichment of natural medicines, in particular to an online enrichment method of dendrobium.
Background
Dendrobium officinale is an endangered precious medicinal material, and is widely used as a traditional Chinese medicinal material and a functional food in China and other countries in Asia for centuries due to the pharmacological and clinical effects of Dendrobium officinale. From Qingdai, the stem of Dendrobium officinale, i.e. Dendrobium officinale bucket, is widely used as a traditional Chinese medicine. It has effects in nourishing yin, invigorating stomach, moistening lung, and prolonging life. Pharmacological research shows that the dendrobium officinale has various pharmacological activities, such as anti-inflammation, anti-tumor, immunoregulation, blood sugar reduction and the like. The main active ingredients of the dendrobium officinale are polysaccharide and phenols.
Until now, the research on dendrobium officinale mainly focuses on polysaccharides, and the research on analyzing other compounds such as phenols is very limited, and the previous research mainly adopts detection methods such as high performance liquid chromatography, near infrared spectroscopy, ultra high performance liquid chromatography-electrospray mass spectrometry, and the like, for example, the publication number CN106680403A discloses a method for detecting dendrophenol in dendrobium officinale, which comprises the following steps: (1) preparing a standard solution; (2) preparing a test solution: weighing a dendrobium officinale sample, extracting the dendrobium officinale sample by using a methanol water solution as an extraction solvent to obtain a dendrobium officinale extracting solution, drying the solvent in the dendrobium officinale extracting solution, redissolving the dried dendrobium officinale extracting solution in chromatographic grade methanol, standing for 12-24 hours, and finally filtering to obtain a filtrate, namely a test solution; (3) and (3) determination of a test solution: and detecting the standard solution and the test solution by using an HPLC-DAD detection method. The method can only extract dendrophenol from dendrobe, but can not perform good enrichment and separation on other substances in dendrobe, and the enrichment and separation effects of the method are not ideal enough.
Disclosure of Invention
The invention provides an online enrichment method of dendrobium for overcoming the defects that only a small part of active substances can be separated and enriched and the enrichment and separation effects are not ideal in the technology, and the method can be used for simultaneously separating and enriching various active substances from dendrobium and has the advantages of good separation effect and high enrichment degree.
In order to achieve the purpose, the invention adopts the following technical scheme:
an online enrichment method of dendrobium nobile comprises the following steps:
(1) adding the dendrobe extracting solution into a phosphoric acid solution containing CD to obtain a sample solution with the CD concentration of 30-130 mM;
(2) dissolving methanol and SDS in a phosphoric acid solution to obtain a BGS solution;
(3) injecting sample solution subjected to ultrasonic treatment and filtration treatment for 50-200s through a capillary tube which is activated and is washed by BGS solution;
(4) and (4) separating and enriching under high voltage to obtain the separated and enriched active substance.
According to the method, the active substances in the dendrobium are enriched by adopting a CD-assisted scavenging-MEKC method, and in the method, the sample solution does not contain PSP (pseudo stationary phase), and compared with background buffer BGS containing PSP, the sample solution has lower, similar or higher conductivity. Upon application of a voltage, the PSPs penetrate the sample region and the analyte in the sample region partitions and interacts with the PSPs in the BGS, allowing the analyte to be collected and accumulated. In the sweeping process, the area of an analyte is continuously reduced, a plurality of effective components in the dendrobium are separated and enriched, and the dendrobium-rich biological sample separating device has the advantages of high enrichment efficiency, simplicity in operation, small using amount of organic solvent, short analysis time, no need of additional instruments and the like while having a good separation effect.
CD is a cyclic oligosaccharide obtained by enzymatic hydrolysis of starch, which has a truncated cone-like structure and has a hydrophilic outer surface and a hydrophobic inner cavity. Numerous hydroxyl groups on the CD structure can form hydrogen bonds with water in aqueous solutions, increasing the solubility of CD in water. And the CD can absorb all or part of the active substances in the dendrobium into the hydrophobic cavities thereof through non-covalent interaction, so that the active substances in the dendrobium and the active substances in the dendrobium form an inclusion compound, the active substances in the dendrobium are dissolved in the CD, the chemical stability, the water solubility and the like of the active substances in the dendrobium are improved, the sensitivity of the capillary electrophoresis online enrichment technology is improved, and the separation and enrichment effects are improved.
Preferably, the CD in the step (1) is one of alpha-CD, methyl-beta-CD, 2-hydroxypropyl-beta-CD and gamma-CD. alpha-CD, beta-CD and gamma-CD are all natural CD, respectively contain 6, 7 and 8 glucose units, and have a truncated cone structure, a hydrophilic outer surface and a hydrophobic inner cavity, active substances in the dendrobium can be wrapped in the hydrophobic cavity and dissolved in water through hydroxyl on the surface, the solubility of the active substances in the dendrobium in the water is increased, and the separation and enrichment effects of effective components in the dendrobium after capillary electrophoresis are further improved.
Preferably, the CD in the step (1) is 2-hydroxypropyl-. beta. -CD, and the concentration thereof is 70-90 mM. In the 2-hydroxypropyl-beta-CD, the introduction of hydroxypropyl breaks the intramolecular cyclic hydrogen bond of the beta-CD, the water solubility of the CD is increased while the CD cavity is kept, so that the solubility of the CD after the CD wraps active substances in dendrobium is further increased, and the final separation and enrichment effects are increased; meanwhile, the toxicity of the 2-hydroxypropyl-beta-CD is lower than that of the parent CD, the 2-hydroxypropyl-beta-CD is non-toxic when being taken orally, the hemolytic property is low, and the safety of the experiment is improved.
When the concentration of 2-hydroxypropyl-beta-CD is low, the interaction between the active substance in the dendrobium and the 2-hydroxypropyl-beta-CD is weak, so that the release process of the active substance is lacked, and the accumulation efficiency is negatively influenced; when the concentration of the 2-hydroxypropyl-beta-CD is too high, the conductivity of the solution is reduced, so that the electric field intensity of a sample area is increased and even exceeds that of a BGS area, and the concentration of SDS permeating the sample area is lower than that of SDS in the BGS, so that the retention factor between an analyte and an SDS micelle is reduced, and the enrichment efficiency is reduced. Thus, the optimal concentration was selected to be a 90mM concentration of 2-hydroxypropyl-. beta. -CD that provides both satisfactory baseline separation and the highest enrichment efficiency.
Preferably, the concentration of the phosphoric acid solution in the step (1) is 80-120mM, and/or
The concentration of the phosphoric acid solution in the step (2) is 40-60 mM.
Preferably, the volume of methanol in the BGS solution in step (2) is 10-40%, and/or the concentration of SDS is 25-125 mM.
Preferably, the methanol volume ratio of the BGS solution in the step (2) is 30-40%. Along with the increase of the concentration of the methanol, the dissolving amount of the active substances in the dendrobium in the methanol is gradually increased, so that the extraction efficiency of the active substances is also increased; however, too high a methanol concentration results in too long a separation time and reduced separation efficiency, and therefore 30% by volume of methanol in the BGS solution is selected.
Preferably, the concentration of SDS in said step (2) is 75-100 mM. As the concentration of SDS increases, more active species is partitioned into the micellar phase and migrates through the SDS micelles towards the positive electrode with faster electrophoretic mobility; as the concentration of SDS decreases, the conductivity of BGS decreases and the electric field intensity increases, resulting in the deposition of SDS at the boundary between BGS and the sample region, and the deposited SDS micelles accumulate the active substance to be analyzed, resulting in the narrowing of the sample region. In view of separation efficiency, analysis time, and enrichment effect, it is preferable that the concentration of SDS is 100 mM.
Preferably, the sample injection time in the step (3) is 50-150s, and/or the BGS solution washing time is 3-8 min. The increase of the sample injection time can increase the enrichment degree of the active substances, but when the sample injection time is too long, the enrichment degree of the impurities is also increased, the separation efficiency of the active substances and the impurities is low, and therefore 100s is selected as the optimal sample injection time.
Preferably, the separation and enrichment in step (4) are carried out at a voltage of 10-20kv and a temperature of 25-35 ℃.
The online enrichment method is applied to separating and enriching one or more of erianin, dendrophenol, naringenin and scoparone in the dendrobium. By the online enrichment method, erianin, dendrophenol, naringenin and scoparone in the dendrobium can be efficiently separated, and the separation and enrichment effects are good.
In conclusion, the invention has the following beneficial effects: (1) the CD-assisted scavenging-MEKC method is adopted to enrich the active substances in the dendrobium, and the method has the advantages of good separation effect, high enrichment efficiency, simplicity in operation, small organic solvent consumption, short analysis time, no need of additional instruments and the like; (2) the CD is used for increasing the solubility of the effective components in the dendrobium in water, so that the separation and enrichment effects of the effective components in the dendrobium after capillary electrophoresis are improved.
Drawings
In FIG. 1, A is a line graph showing the effect of different kinds of CD on the enrichment efficiency of target analytes, and B is an electropherogram showing the effect of different kinds of CD on the separation efficiency of target analytes, wherein a is alpha-CD, B is methyl-beta-CD, c is 2-hydroxypropyl-beta-CD, and d is gamma-CD. Analytes 1 were erianin, 2 were dendrophenol, 3 was naringenin and 4 was scoparone.
FIG. 2 is a line graph showing the effect of different concentrations of CD on the enrichment efficiency of target analytes, and B is an electropherogram showing the effect of different concentrations of CD on the separation efficiency of target analytes, wherein a is 30mM, B is 50mM, c is 70mM, d is 90mM, e is 110mM, and f is 130 mM; analytes 1 were erianin, 2 were dendrophenol, 3 was naringenin and 4 was scoparone.
In FIG. 3, A is a line graph showing the effect of methanol ratio on the enrichment efficiency of target analytes, and B is an electropherogram showing the effect of different methanol ratios on the separation efficiency of target analytes, wherein a is 10%, B is 20%, c is 30%, and d is 40%; analytes 1 were erianin, 2 were dendrophenol, 3 was naringenin and 4 was scoparone.
FIG. 4 is a line graph showing the effect of different concentrations of SDS on the enrichment efficiency of a target analyte, and B is an electropherogram showing the effect of different concentrations of SDS on the separation of a target analyte, wherein a is 25mM, B is 50mM, c is 75mM, d is 100mM, and e is 125 mM; analytes 1 were erianin, 2 were dendrophenol, 3 was naringenin and 4 was scoparone.
In FIG. 5, A is a line graph showing the effect of different sample injection times on the enrichment efficiency of target analytes, and B is an electropherogram showing the effect of different sample injection times on the separation efficiency of target analytes, wherein a is 3s, B is 50s, c is 100s, d is 150s, and e is 200 s; analytes 1 were erianin, 2 were dendrophenol, 3 was naringenin and 4 was scoparone.
FIG. 6 is an electropherogram of herba Dendrobii sample extractive solution obtained by different sample injection time, wherein a is 100s, and b is 3 s; analytes 1 were erianin, 2 were dendrophenol, 3 was naringenin and 4 was scoparone.
FIG. 7 is an electropherogram of a target analyte obtained from CD-assisted scan-MEKC, BGS-assisted conventional scan-MEKC, water-assisted conventional scan-MEKC and MEKC, respectively, wherein a is CD-assisted scan-MEKC, b is BGS-assisted conventional scan-MEKC, c is water-assisted conventional scan-MEKC, and d is MEKC; analytes 1 were erianin, 2 were dendrophenol, 3 was naringenin and 4 was scoparone.
Detailed Description
The invention is further described with reference to the following detailed description and accompanying drawings.
General examples
An online enrichment method of dendrobium nobile comprises the following steps:
(1) precisely weighing 1g of dendrobium officinale, mixing with 20 ml of methanol, standing for 25 minutes, then carrying out ultrasonic treatment for 40 minutes, filtering by using a 0.22-micron nylon filter, taking supernatant, diluting by 10 times to obtain dendrobium extract, and adding the dendrobium extract into 100mM phosphoric acid solution containing CD to obtain a sample solution with the CD concentration of 30-130 mM;
(2) dissolving methanol and SDS in 50mM phosphoric acid solution to obtain BGS solution, wherein the volume of the methanol accounts for 10-40%, and the concentration of the SDS is 25-125 mM;
(3) activating the capillary, washing with BGS solution for 5min, and introducing sample solution subjected to ultrasonic treatment and filtration treatment through the capillary for 50-200 s;
(4) separating and enriching at a voltage of 20kv and a temperature of 30 ℃ to obtain separated and enriched active substances, wherein the active substances are erianin, dendrophenol, naringenin and scoparone.
Wherein: the mother liquors of phosphoric acid and SDS were prepared with purified water to concentrations of 0.5M and 0.2M, respectively. The pH of the 0.5M phosphoric acid mother liquor was adjusted to 2.5 with 10M NaOH. Standard stock solutions for the four analytes were prepared as 1000. mu.g/mL solutions by weighing the appropriate amount of each standard and dissolving in methanol. The sample solution for the scavenger MEKC assay was prepared by diluting the standard stock solution in 30-130mM CD and 100mM phosphoric acid. BGS for scavenging and separation was prepared by mixing 0.5M phosphoric acid, 0.2M SDS, methanol, and purified water in a certain ratio. All solutions were sonicated and filtered through a 0.22 μm disposable nylon membrane prior to injection into the MEKC system.
Preparing a dendrobium officinale sample solution: precisely weighing 1g of dendrobium officinale, mixing with 20 ml of methanol, standing for 25 minutes, and then carrying out ultrasonic treatment for 40 minutes. The extract was filtered through a 0.22 μm nylon filter. The supernatant in the extract was diluted 10-fold in 90mM CD and 100mM phosphoric acid.
The MEKC analysis was performed on an agilent CE system (7100, palo alto, ca, usa) equipped with a diode array detector. Control and analysis were performed using HP ChemStation (Agilent) software. The detection wavelength was set at 205nm and the reference wavelength was set at 360 nm. The stacking and separation of analytes is performed at a voltage of 20kv and a temperature of 30 ℃. The uncoated fused silica capillary (50 μm inner diameter x 360 μm outer diameter) used in this study had a total length of 50 cm and an effective length of 41.5 cm. Before the primary capillary tube is used for the first time, the primary capillary tube needs to be washed by 1.0M sodium hydroxide for 10 minutes, purified water for 10 minutes, methanol for 5 minutes and purified water for 5 minutes in sequence. Between two continuous runs, the capillary tube was washed with 1.0M sodium hydroxide for 1 minute, purified water for 2 minutes, methanol for 1 minute, and BGS for 5 minutes in sequence before sample injection. The sample solution was injected into the capillary at a constant pressure of 50 mbar.
Example 1
An online enrichment method of dendrobium nobile comprises the following steps:
(1) precisely weighing 1g of dendrobium officinale, mixing with 20 ml of methanol, standing for 25 minutes, then carrying out ultrasonic treatment for 40 minutes, filtering by using a 0.22-micron nylon filter, taking supernatant, diluting by 10 times to obtain dendrobium extract, and adding the dendrobium extract into 100mM phosphoric acid solution containing 2-hydroxypropyl-beta-CD to obtain a sample solution with the concentration of the 2-hydroxypropyl-beta-CD being 90 mM;
(2) dissolving methanol and SDS in 50mM phosphoric acid solution to obtain BGS solution, wherein the volume of the methanol accounts for 30%, and the concentration of the SDS is 100 mM;
(3) activating the capillary, washing with BGS solution for 5min, and introducing sample solution subjected to ultrasonic treatment and filtration treatment through the capillary for 100 s;
(4) separating and enriching at a voltage of 20kv and a temperature of 30 ℃ to obtain separated and enriched active substances, wherein the active substances are erianin, dendrophenol, naringenin and scoparone.
Example 2
An online enrichment method of dendrobium nobile comprises the following steps:
(1) precisely weighing 1g of dendrobium officinale, mixing with 20 ml of methanol, standing for 25 minutes, then carrying out ultrasonic treatment for 40 minutes, filtering by using a 0.22-micron nylon filter, taking supernatant, diluting by 10 times to obtain dendrobium extract, and adding the dendrobium extract into 100mM phosphoric acid solution containing alpha-CD to obtain a sample solution with the alpha-CD concentration of 90 mM;
(2) dissolving methanol and SDS in 50mM phosphoric acid solution to obtain BGS solution, wherein the volume of the methanol accounts for 30%, and the concentration of the SDS is 100 mM;
(3) activating the capillary, washing with BGS solution for 5min, and introducing sample solution subjected to ultrasonic treatment and filtration treatment through the capillary for 100 s;
(4) separating and enriching at a voltage of 20kv and a temperature of 30 ℃ to obtain separated and enriched active substances, wherein the active substances are erianin, dendrophenol, naringenin and scoparone.
Example 3
An online enrichment method of dendrobium nobile comprises the following steps:
(1) precisely weighing 1g of dendrobium officinale, mixing with 20 ml of methanol, standing for 25 minutes, then carrying out ultrasonic treatment for 40 minutes, filtering by using a 0.22-micron nylon filter, taking supernatant, diluting by 10 times to obtain dendrobium extract, and adding the dendrobium extract into 100mM phosphoric acid solution containing methyl-beta-CD to obtain a sample solution with the concentration of the methyl-beta-CD being 90 mM;
(2) dissolving methanol and SDS in 50mM phosphoric acid solution to obtain BGS solution, wherein the volume of the methanol accounts for 30%, and the concentration of the SDS is 100 mM;
(3) activating the capillary, washing with BGS solution for 5min, and introducing sample solution subjected to ultrasonic treatment and filtration treatment through the capillary for 100 s;
(4) separating and enriching at a voltage of 20kv and a temperature of 30 ℃ to obtain separated and enriched active substances, wherein the active substances are erianin, dendrophenol, naringenin and scoparone.
Example 4
An online enrichment method of dendrobium nobile comprises the following steps:
(1) precisely weighing 1g of dendrobium officinale, mixing with 20 ml of methanol, standing for 25 minutes, then carrying out ultrasonic treatment for 40 minutes, filtering by using a 0.22-micron nylon filter, taking supernatant, diluting by 10 times to obtain dendrobium extract, and adding the dendrobium extract into 100mM phosphoric acid solution containing gamma-CD to obtain a sample solution with the gamma-CD concentration of 90 mM;
(2) dissolving methanol and SDS in 50mM phosphoric acid solution to obtain BGS solution, wherein the volume of the methanol accounts for 30%, and the concentration of the SDS is 100 mM;
(3) activating the capillary, washing with BGS solution for 5min, and introducing sample solution subjected to ultrasonic treatment and filtration treatment through the capillary for 100 s;
(4) separating and enriching at a voltage of 20kv and a temperature of 30 ℃ to obtain separated and enriched active substances, wherein the active substances are erianin, dendrophenol, naringenin and scoparone.
Example 4
An online enrichment method of dendrobium nobile comprises the following steps:
(1) precisely weighing 1g of dendrobium officinale, mixing with 20 ml of methanol, standing for 25 minutes, then carrying out ultrasonic treatment for 40 minutes, filtering by using a 0.22-micron nylon filter, taking supernatant, diluting by 10 times to obtain dendrobium extract, and adding the dendrobium extract into 100mM phosphoric acid solution containing gamma-CD to obtain a sample solution with the gamma-CD concentration of 90 mM;
(2) dissolving methanol and SDS in 50mM phosphoric acid solution to obtain BGS solution, wherein the volume of the methanol accounts for 30%, and the concentration of the SDS is 100 mM;
(3) activating the capillary, washing with BGS solution for 5min, and introducing sample solution subjected to ultrasonic treatment and filtration treatment through the capillary for 100 s;
(4) separating and enriching at a voltage of 20kv and a temperature of 30 ℃ to obtain separated and enriched active substances, wherein the active substances are erianin, dendrophenol, naringenin and scoparone.
Examples 1-4 the enrichment efficiency line graph and electropherogram are shown in fig. 1, where fig. 1A shows the effect of different kinds of CD on the target analyte enrichment efficiency. It can be seen that the peak areas of the four analytes reached the highest values when 2-hydroxypropyl-. beta. -CD was used as a sample solvent, and at the same time, the degree of separation of the target analyte also reached satisfactory results as can be seen from FIG. 1B. The reason for this phenomenon may be that the interaction between 2-hydroxypropyl-. beta. -CD and SDS micelles is stronger than that of other CDs, and the probability of reaction with other substances increases due to breakage of intramolecular ring hydrogen bonds thereof, thereby causing the narrowing of the stacking region. In addition, 2-hydroxypropyl-. beta.CD has better water solubility than the other three CDs, and therefore, 2-hydroxypropyl-. beta.CD is preferable as the CD species.
Example 5
An online enrichment method of dendrobium nobile comprises the following steps:
(1) precisely weighing 1g of dendrobium officinale, mixing with 20 ml of methanol, standing for 25 minutes, then carrying out ultrasonic treatment for 40 minutes, filtering by using a 0.22-micron nylon filter, taking supernatant, diluting by 10 times to obtain dendrobium extract, and adding the dendrobium extract into 100mM phosphoric acid solution containing 2-hydroxypropyl-beta-CD to obtain a sample solution with the concentration of 2-hydroxypropyl-beta-CD being 30 mM;
(2) dissolving methanol and SDS in 50mM phosphoric acid solution to obtain BGS solution, wherein the volume of the methanol accounts for 30%, and the concentration of the SDS is 100 mM;
(3) activating the capillary, washing with BGS solution for 5min, and introducing sample solution subjected to ultrasonic treatment and filtration treatment through the capillary for 100 s;
(4) separating and enriching at a voltage of 20kv and a temperature of 30 ℃ to obtain separated and enriched active substances, wherein the active substances are erianin, dendrophenol, naringenin and scoparone.
Example 6
An online enrichment method of dendrobium nobile comprises the following steps:
(1) precisely weighing 1g of dendrobium officinale, mixing with 20 ml of methanol, standing for 25 minutes, then carrying out ultrasonic treatment for 40 minutes, filtering by using a 0.22-micron nylon filter, taking supernatant, diluting by 10 times to obtain dendrobium extract, and adding the dendrobium extract into 100mM phosphoric acid solution containing 2-hydroxypropyl-beta-CD to obtain a sample solution with the concentration of the 2-hydroxypropyl-beta-CD being 50 mM;
(2) dissolving methanol and SDS in 50mM phosphoric acid solution to obtain BGS solution, wherein the volume of the methanol accounts for 30%, and the concentration of the SDS is 100 mM;
(3) activating the capillary, washing with BGS solution for 5min, and introducing sample solution subjected to ultrasonic treatment and filtration treatment through the capillary for 100 s;
(4) separating and enriching at a voltage of 20kv and a temperature of 30 ℃ to obtain separated and enriched active substances, wherein the active substances are erianin, dendrophenol, naringenin and scoparone.
Example 7
An online enrichment method of dendrobium nobile comprises the following steps:
(1) precisely weighing 1g of dendrobium officinale, mixing with 20 ml of methanol, standing for 25 minutes, then carrying out ultrasonic treatment for 40 minutes, filtering by using a 0.22-micron nylon filter, taking supernatant, diluting by 10 times to obtain dendrobium extract, and adding the dendrobium extract into 100mM phosphoric acid solution containing 2-hydroxypropyl-beta-CD to obtain a sample solution with the concentration of the 2-hydroxypropyl-beta-CD being 70 mM;
(2) dissolving methanol and SDS in 50mM phosphoric acid solution to obtain BGS solution, wherein the volume of the methanol accounts for 30%, and the concentration of the SDS is 100 mM;
(3) activating the capillary, washing with BGS solution for 5min, and introducing sample solution subjected to ultrasonic treatment and filtration treatment through the capillary for 100 s;
(4) separating and enriching at a voltage of 20kv and a temperature of 30 ℃ to obtain separated and enriched active substances, wherein the active substances are erianin, dendrophenol, naringenin and scoparone.
Example 8
An online enrichment method of dendrobium nobile comprises the following steps:
(1) precisely weighing 1g of dendrobium officinale, mixing with 20 ml of methanol, standing for 25 minutes, then carrying out ultrasonic treatment for 40 minutes, filtering by using a 0.22-micron nylon filter, taking supernatant, diluting by 10 times to obtain dendrobium extract, and adding the dendrobium extract into 100mM phosphoric acid solution containing 2-hydroxypropyl-beta-CD to obtain a sample solution with the concentration of 2-hydroxypropyl-beta-CD being 110 mM;
(2) dissolving methanol and SDS in 50mM phosphoric acid solution to obtain BGS solution, wherein the volume of the methanol accounts for 30%, and the concentration of the SDS is 100 mM;
(3) activating the capillary, washing with BGS solution for 5min, and introducing sample solution subjected to ultrasonic treatment and filtration treatment through the capillary for 100 s;
(4) separating and enriching at a voltage of 20kv and a temperature of 30 ℃ to obtain separated and enriched active substances, wherein the active substances are erianin, dendrophenol, naringenin and scoparone.
Example 9
An online enrichment method of dendrobium nobile comprises the following steps:
(1) precisely weighing 1g of dendrobium officinale, mixing with 20 ml of methanol, standing for 25 minutes, then carrying out ultrasonic treatment for 40 minutes, filtering by using a 0.22-micron nylon filter, taking supernatant, diluting by 10 times to obtain dendrobium extract, and adding the dendrobium extract into 100mM phosphoric acid solution containing 2-hydroxypropyl-beta-CD to obtain a sample solution with the concentration of the 2-hydroxypropyl-beta-CD being 130 mM;
(2) dissolving methanol and SDS in 50mM phosphoric acid solution to obtain BGS solution, wherein the volume of the methanol accounts for 30%, and the concentration of the SDS is 100 mM;
(3) activating the capillary, washing with BGS solution for 5min, and introducing sample solution subjected to ultrasonic treatment and filtration treatment through the capillary for 100 s;
(4) separating and enriching at a voltage of 20kv and a temperature of 30 ℃ to obtain separated and enriched active substances, wherein the active substances are erianin, dendrophenol, naringenin and scoparone.
The enrichment efficiency line graphs and electropherograms of example 1 and examples 5 to 9 are shown in fig. 2, in which fig. 2A is a line graph showing the effect of different concentrations of CD on the enrichment efficiency of target analytes, and fig. 2B is an electropherogram showing the effect of different concentrations of CD on the separation of target analytes, from which it can be seen that the migration time and peak area of target analytes gradually increase as the CD concentration increases from 30mM to 90mM, possibly due to the fact that the interaction of the analytes with smaller amounts of CD is weak, resulting in lack of release process, thereby negatively affecting the stacking efficiency; however, when the CD concentration is increased from 90mM to 130mM, the peak area of the target analyte is slightly decreased, which is probably because the higher the concentration of CD in the sample solution, the lower the conductivity, the higher the electric field intensity of the sample region, and thus the higher the electric field intensity of the BGS region, and the lower the concentration of SDS that permeates into the sample region than the concentration of SDS in BGS, resulting in a decrease in the retention factor between the analyte and the SDS micelles, leading to a decrease in the enrichment efficiency, and thus 90mM is selected as the optimum CD concentration.
Example 10
An online enrichment method of dendrobium nobile comprises the following steps:
(1) precisely weighing 1g of dendrobium officinale, mixing with 20 ml of methanol, standing for 25 minutes, then carrying out ultrasonic treatment for 40 minutes, filtering by using a 0.22-micron nylon filter, taking supernatant, diluting by 10 times to obtain dendrobium extract, and adding the dendrobium extract into 100mM phosphoric acid solution containing 2-hydroxypropyl-beta-CD to obtain a sample solution with the concentration of the 2-hydroxypropyl-beta-CD being 90 mM;
(2) dissolving methanol and SDS in 50mM phosphoric acid solution to obtain BGS solution, wherein the volume of the methanol accounts for 10%, and the concentration of the SDS is 100 mM;
(3) activating the capillary, washing with BGS solution for 5min, and introducing sample solution subjected to ultrasonic treatment and filtration treatment through the capillary for 100 s;
(4) separating and enriching at a voltage of 20kv and a temperature of 30 ℃ to obtain separated and enriched active substances, wherein the active substances are erianin, dendrophenol, naringenin and scoparone.
Example 11
An online enrichment method of dendrobium nobile comprises the following steps:
(1) precisely weighing 1g of dendrobium officinale, mixing with 20 ml of methanol, standing for 25 minutes, then carrying out ultrasonic treatment for 40 minutes, filtering by using a 0.22-micron nylon filter, taking supernatant, diluting by 10 times to obtain dendrobium extract, and adding the dendrobium extract into 100mM phosphoric acid solution containing 2-hydroxypropyl-beta-CD to obtain a sample solution with the concentration of the 2-hydroxypropyl-beta-CD being 90 mM;
(2) dissolving methanol and SDS in 50mM phosphoric acid solution to obtain BGS solution, wherein the volume of the methanol accounts for 20%, and the concentration of the SDS is 100 mM;
(3) activating the capillary, washing with BGS solution for 5min, and introducing sample solution subjected to ultrasonic treatment and filtration treatment through the capillary for 100 s;
(4) separating and enriching at a voltage of 20kv and a temperature of 30 ℃ to obtain separated and enriched active substances, wherein the active substances are erianin, dendrophenol, naringenin and scoparone.
Example 12
An online enrichment method of dendrobium nobile comprises the following steps:
(1) precisely weighing 1g of dendrobium officinale, mixing with 20 ml of methanol, standing for 25 minutes, then carrying out ultrasonic treatment for 40 minutes, filtering by using a 0.22-micron nylon filter, taking supernatant, diluting by 10 times to obtain dendrobium extract, and adding the dendrobium extract into 100mM phosphoric acid solution containing 2-hydroxypropyl-beta-CD to obtain a sample solution with the concentration of the 2-hydroxypropyl-beta-CD being 90 mM;
(2) dissolving methanol and SDS in 50mM phosphoric acid solution to obtain BGS solution, wherein the volume of the methanol accounts for 40%, and the concentration of the SDS is 100 mM;
(3) activating the capillary, washing with BGS solution for 5min, and introducing sample solution subjected to ultrasonic treatment and filtration treatment through the capillary for 100 s;
(4) separating and enriching at a voltage of 20kv and a temperature of 30 ℃ to obtain separated and enriched active substances, wherein the active substances are erianin, dendrophenol, naringenin and scoparone.
The line graphs and electropherograms of the enrichment efficiency of example 1 and examples 10 to 12 are shown in fig. 3, wherein fig. 3A is a line graph showing the influence of different methanol ratios on the enrichment efficiency of a target analyte, and B is an electropherogram showing the influence of different methanol ratios on the separation effect of the target analyte, and it can be seen from the graph that the peak area, the resolution and the migration time of the target analyte are increased as the concentration of methanol is increased. Satisfactory baseline separation and enrichment efficiency was obtained when 30% by volume methanol was used. The reason for the increase in migration time with increasing methanol concentration is due to the increased partitioning of the analyte in the solution phase. However, as the methanol volume ratio was further increased to 40%, the migration time of the analyte became longer and the peak became broader. And combining the conditions, and selecting 30% methanol by volume as the optimal concentration.
Example 13
An online enrichment method of dendrobium nobile comprises the following steps:
(1) precisely weighing 1g of dendrobium officinale, mixing with 20 ml of methanol, standing for 25 minutes, then carrying out ultrasonic treatment for 40 minutes, filtering by using a 0.22-micron nylon filter, taking supernatant, diluting by 10 times to obtain dendrobium extract, and adding the dendrobium extract into 100mM phosphoric acid solution containing 2-hydroxypropyl-beta-CD to obtain a sample solution with the concentration of the 2-hydroxypropyl-beta-CD being 90 mM;
(2) dissolving methanol and SDS in 50mM phosphoric acid solution to obtain BGS solution, wherein the volume of the methanol accounts for 30%, and the concentration of the SDS is 25 mM;
(3) activating the capillary, washing with BGS solution for 5min, and introducing sample solution subjected to ultrasonic treatment and filtration treatment through the capillary for 100 s;
(4) separating and enriching at a voltage of 20kv and a temperature of 30 ℃ to obtain separated and enriched active substances, wherein the active substances are erianin, dendrophenol, naringenin and scoparone.
Example 14
An online enrichment method of dendrobium nobile comprises the following steps:
(1) precisely weighing 1g of dendrobium officinale, mixing with 20 ml of methanol, standing for 25 minutes, then carrying out ultrasonic treatment for 40 minutes, filtering by using a 0.22-micron nylon filter, taking supernatant, diluting by 10 times to obtain dendrobium extract, and adding the dendrobium extract into 100mM phosphoric acid solution containing 2-hydroxypropyl-beta-CD to obtain a sample solution with the concentration of the 2-hydroxypropyl-beta-CD being 90 mM;
(2) dissolving methanol and SDS in 50mM phosphoric acid solution to obtain BGS solution, wherein the volume of the methanol accounts for 30%, and the concentration of the SDS is 50 mM;
(3) activating the capillary, washing with BGS solution for 5min, and introducing sample solution subjected to ultrasonic treatment and filtration treatment through the capillary for 100 s;
(4) separating and enriching at a voltage of 20kv and a temperature of 30 ℃ to obtain separated and enriched active substances, wherein the active substances are erianin, dendrophenol, naringenin and scoparone.
Example 15
An online enrichment method of dendrobium nobile comprises the following steps:
(1) precisely weighing 1g of dendrobium officinale, mixing with 20 ml of methanol, standing for 25 minutes, then carrying out ultrasonic treatment for 40 minutes, filtering by using a 0.22-micron nylon filter, taking supernatant, diluting by 10 times to obtain dendrobium extract, and adding the dendrobium extract into 100mM phosphoric acid solution containing 2-hydroxypropyl-beta-CD to obtain a sample solution with the concentration of the 2-hydroxypropyl-beta-CD being 90 mM;
(2) dissolving methanol and SDS in 50mM phosphoric acid solution to obtain BGS solution, wherein the volume of the methanol accounts for 30%, and the concentration of the SDS is 75 mM;
(3) activating the capillary, washing with BGS solution for 5min, and introducing sample solution subjected to ultrasonic treatment and filtration treatment through the capillary for 100 s;
(4) separating and enriching at a voltage of 20kv and a temperature of 30 ℃ to obtain separated and enriched active substances, wherein the active substances are erianin, dendrophenol, naringenin and scoparone.
Example 16
An online enrichment method of dendrobium nobile comprises the following steps:
(1) precisely weighing 1g of dendrobium officinale, mixing with 20 ml of methanol, standing for 25 minutes, then carrying out ultrasonic treatment for 40 minutes, filtering by using a 0.22-micron nylon filter, taking supernatant, diluting by 10 times to obtain dendrobium extract, and adding the dendrobium extract into 100mM phosphoric acid solution containing 2-hydroxypropyl-beta-CD to obtain a sample solution with the concentration of the 2-hydroxypropyl-beta-CD being 90 mM;
(2) dissolving methanol and SDS in 50mM phosphoric acid solution to obtain BGS solution, wherein the volume of the methanol accounts for 30%, and the concentration of the SDS is 125 mM;
(3) activating the capillary, washing with BGS solution for 5min, and introducing sample solution subjected to ultrasonic treatment and filtration treatment through the capillary for 100 s;
(4) separating and enriching at a voltage of 20kv and a temperature of 30 ℃ to obtain separated and enriched active substances, wherein the active substances are erianin, dendrophenol, naringenin and scoparone.
The line graphs and electropherograms of the enrichment efficiency of example 1 and examples 13 to 16 are shown in fig. 4, in which fig. 4A is a line graph showing the effect of different SDS concentrations on the enrichment efficiency of target analytes, and fig. 4B is an electropherogram showing the effect of different SDS concentrations on the separation of target analytes, and it can be seen from the graph that the migration time and peak area of analytes decrease as the SDS concentration increases. As SDS content increases, the migration time of the target analyte decreases, probably because more analyte is partitioned into the micellar phase and migrates through the SDS micelles towards the positive electrode with faster electrophoretic mobility. The peak area of the analyte increases with decreasing SDS concentration, which may be due to lower SDS content resulting in decreased conductivity of the BGS and increased electric field strength, resulting in SDS stacking at the boundary between the BGS and the sample region. Thus, the stacked SDS micelles accumulate the target analyte, resulting in narrowing of the sample region. When the SDS concentration is 25mM, the area of the highest peak of the analyte appears, but the analysis time is significantly prolonged and the peak shape becomes broad. When the SDS content was set to 50mM, 75mM, and 125mM, respectively, baseline separation of the target analyte from the impurity peak could not be achieved. When the SDS concentration is 100mM, the analysis time is short, the separation efficiency is high and the peak shape is good. The optimum concentration of SDS was selected at 100mM in consideration of the separation efficiency, analysis time and enrichment effect.
Example 17
An online enrichment method of dendrobium nobile comprises the following steps:
(1) precisely weighing 1g of dendrobium officinale, mixing with 20 ml of methanol, standing for 25 minutes, then carrying out ultrasonic treatment for 40 minutes, filtering by using a 0.22-micron nylon filter, taking supernatant, diluting by 10 times to obtain dendrobium extract, and adding the dendrobium extract into 100mM phosphoric acid solution containing 2-hydroxypropyl-beta-CD to obtain a sample solution with the concentration of the 2-hydroxypropyl-beta-CD being 90 mM;
(2) dissolving methanol and SDS in 50mM phosphoric acid solution to obtain BGS solution, wherein the volume of the methanol accounts for 30%, and the concentration of the SDS is 100 mM;
(3) activating the capillary, washing with BGS solution for 5min, and introducing sample solution subjected to ultrasonic treatment and filtration treatment through the capillary for 3 s;
(4) separating and enriching at a voltage of 20kv and a temperature of 30 ℃ to obtain separated and enriched active substances, wherein the active substances are erianin, dendrophenol, naringenin and scoparone.
Example 18
An online enrichment method of dendrobium nobile comprises the following steps:
(1) precisely weighing 1g of dendrobium officinale, mixing with 20 ml of methanol, standing for 25 minutes, then carrying out ultrasonic treatment for 40 minutes, filtering by using a 0.22-micron nylon filter, taking supernatant, diluting by 10 times to obtain dendrobium extract, and adding the dendrobium extract into 100mM phosphoric acid solution containing 2-hydroxypropyl-beta-CD to obtain a sample solution with the concentration of the 2-hydroxypropyl-beta-CD being 90 mM;
(2) dissolving methanol and SDS in 50mM phosphoric acid solution to obtain BGS solution, wherein the volume of the methanol accounts for 30%, and the concentration of the SDS is 100 mM;
(3) activating the capillary, washing with BGS solution for 5min, and introducing sample solution subjected to ultrasonic treatment and filtration treatment through the capillary for 50 s;
(4) separating and enriching at a voltage of 20kv and a temperature of 30 ℃ to obtain separated and enriched active substances, wherein the active substances are erianin, dendrophenol, naringenin and scoparone.
Example 19
An online enrichment method of dendrobium nobile comprises the following steps:
(1) precisely weighing 1g of dendrobium officinale, mixing with 20 ml of methanol, standing for 25 minutes, then carrying out ultrasonic treatment for 40 minutes, filtering by using a 0.22-micron nylon filter, taking supernatant, diluting by 10 times to obtain dendrobium extract, and adding the dendrobium extract into 100mM phosphoric acid solution containing 2-hydroxypropyl-beta-CD to obtain a sample solution with the concentration of the 2-hydroxypropyl-beta-CD being 90 mM;
(2) dissolving methanol and SDS in 50mM phosphoric acid solution to obtain BGS solution, wherein the volume of the methanol accounts for 30%, and the concentration of the SDS is 100 mM;
(3) activating the capillary, washing with BGS solution for 5min, and introducing sample solution subjected to ultrasonic treatment and filtration treatment through the capillary for 150 s;
(4) separating and enriching at a voltage of 20kv and a temperature of 30 ℃ to obtain separated and enriched active substances, wherein the active substances are erianin, dendrophenol, naringenin and scoparone.
Example 20
An online enrichment method of dendrobium nobile comprises the following steps:
(1) precisely weighing 1g of dendrobium officinale, mixing with 20 ml of methanol, standing for 25 minutes, then carrying out ultrasonic treatment for 40 minutes, filtering by using a 0.22-micron nylon filter, taking supernatant, diluting by 10 times to obtain dendrobium extract, and adding the dendrobium extract into 100mM phosphoric acid solution containing 2-hydroxypropyl-beta-CD to obtain a sample solution with the concentration of the 2-hydroxypropyl-beta-CD being 90 mM;
(2) dissolving methanol and SDS in 50mM phosphoric acid solution to obtain BGS solution, wherein the volume of the methanol accounts for 30%, and the concentration of the SDS is 100 mM;
(3) activating the capillary, washing with BGS solution for 5min, and introducing sample solution subjected to ultrasonic treatment and filtration treatment through the capillary for 200 s;
(4) separating and enriching at a voltage of 20kv and a temperature of 30 ℃ to obtain separated and enriched active substances, wherein the active substances are erianin, dendrophenol, naringenin and scoparone.
The line graphs of enrichment efficiency and electropherograms of example 1 and examples 17 to 20 are shown in fig. 5, in which fig. 5A is a line graph showing the influence of different injection times on the enrichment efficiency of a target analyte, and fig. 5B is an electropherogram showing the influence of different injection times on the separation effect of a target analyte, and it can be seen from the graph that when the injection times are 150 and 200s, the separation efficiency between the peak of the target analyte and the impurity peak is poor and the analysis time is long. When the injection time was 50s, the enrichment efficiency was low and some analyte and impurity peaks overlapped. The peaks of the four analytes were barely detectable when the injection time was 3 s. In terms of peak area, the SEFs at a sampling time of 100s was 28.5 to 46.8, compared with the result at a sampling time of 3 s. In addition, when the injection time exceeds 150s, the peak shape and the current become unstable. When the injection time is 100s, the baseline separation of the target analytes is completed in a shorter time and satisfactory enrichment efficiency is obtained. Therefore, 100s was selected as the optimum injection time in terms of analysis time, enrichment efficiency and resolution.
The standard stock solutions of the four controls were prepared as a series of control solutions of varying concentrations (90 mM of 2-hydroxypropyl-. beta. -CD and 100mM of phosphoric acid in solvent). Wherein the linear concentration range of erianin and scoparone is 0.15-3 μ g/mL, and the linear concentration range of dendrophenol and naringenin is 0.5-10 μ g/mL. Sample injection is carried out according to the conditions of example 1, capillary electrophoresis chromatograms of 4 reference substances are obtained, the concentration of the reference substance is used as the abscissa, and the peak area of the capillary electrophoresis chromatographic peak of the reference substance is used as the ordinate to draw the standard curve of the 4 standard substances. According to the peak areas of the various spectral peaks in the chromatogram of the dendrobium officinale extract and the standard curves of the components, the contents of the erianin, the dendrophenol, the naringenin and the scoparone in the dendrobium officinale extract are calculated and obtained, and then the contents of the substances in the extract relative to the dendrobium officinale sample can be obtained through corresponding conversion.
FIG. 6 is an electropherogram of the extract of Dendrobium officinale sample obtained by different sampling times. The result shows that the method provided by the invention has obvious enrichment efficiency on the target analyte.
The standard curves and detection and quantitation limits for 4 target analytes are shown in table 1 below:
TABLE 1 Linear regression data, LOD and LOQ
Taking the sample injection conditions of example 1, making 2 groups in parallel, adding a group of control solutions of erianin and scoparone to the concentration of 1.5 mu g/mL, adding a group of control solutions of dendrophenol and naringenin to the concentration of 5 mu g/mL, and adding no control to the other group, repeating the steps for three times, and taking an average value. As can be seen from Table 2, the average recovery rate of the analytes is in the range of 80.5-94.47%, and the method has the advantages of good repeatability, high recovery rate and high detection accuracy.
The results of the repeatability, assay, recovery tests are summarized in table 2 below:
TABLE 2 repeatability, assay, recovery
Example 21
An online enrichment method of dendrobium nobile comprises the following steps:
(1) precisely weighing 1g of dendrobium officinale, mixing with 20 ml of methanol, standing for 25 minutes, then carrying out ultrasonic treatment for 40 minutes, filtering by using a 0.22-micron nylon filter, taking supernatant, diluting by 10 times to obtain dendrobium extract, and adding the dendrobium extract into 80mM phosphoric acid solution containing 2-hydroxypropyl-beta-CD to obtain a sample solution with the concentration of the 2-hydroxypropyl-beta-CD being 90 mM;
(2) dissolving methanol and SDS in 40mM phosphoric acid solution to obtain BGS solution, wherein the volume of the methanol accounts for 30%, and the concentration of the SDS is 100 mM;
(3) activating the capillary, washing with BGS solution for 8min, and introducing sample solution subjected to ultrasonic treatment and filtration treatment through the capillary for 100 s;
(4) separating and enriching at a voltage of 15kv and a temperature of 30 ℃ to obtain separated and enriched active substances, wherein the active substances are erianin, dendrophenol, naringenin and scoparone.
Example 22
An online enrichment method of dendrobium nobile comprises the following steps:
(1) precisely weighing 1g of dendrobium officinale, mixing with 20 ml of methanol, standing for 25 minutes, then carrying out ultrasonic treatment for 40 minutes, filtering by using a 0.22-micron nylon filter, taking supernatant, diluting by 10 times to obtain dendrobium extract, and adding the dendrobium extract into 120mM phosphoric acid solution containing 2-hydroxypropyl-beta-CD to obtain a sample solution with the concentration of the 2-hydroxypropyl-beta-CD being 80 mM;
(2) dissolving methanol and SDS in a 60mM phosphoric acid solution to obtain a BGS solution, wherein the volume percentage of the methanol is 30 percent, and the concentration of the SDS is 75 mM;
(3) activating the capillary, washing with BGS solution for 5min, and introducing sample solution subjected to ultrasonic treatment and filtration treatment through the capillary for 100 s;
(4) separating and enriching at a voltage of 10kv and a temperature of 35 ℃ to obtain separated and enriched active substances, wherein the active substances are erianin, dendrophenol, naringenin and scoparone.
Example 23
An online enrichment method of dendrobium nobile comprises the following steps:
(1) precisely weighing 1g of dendrobium officinale, mixing with 20 ml of methanol, standing for 25 minutes, then carrying out ultrasonic treatment for 40 minutes, filtering by using a 0.22-micron nylon filter, taking supernatant, diluting by 10 times to obtain dendrobium extract, and adding the dendrobium extract into 80mM phosphoric acid solution containing 2-hydroxypropyl-beta-CD to obtain a sample solution with the concentration of the 2-hydroxypropyl-beta-CD being 70 mM;
(2) dissolving methanol and SDS in a 60mM phosphoric acid solution to obtain a BGS solution, wherein the volume percentage of the methanol is 20 percent, and the concentration of the SDS is 100 mM;
(3) activating the capillary, washing with BGS solution for 3min, and introducing sample solution subjected to ultrasonic treatment and filtration treatment through the capillary for 150 s;
(4) separating and enriching at 25 deg.C under 10kv to obtain separated and enriched active substances, wherein the active substances are erianin, dendrophenol, naringenin and scoparone.
Comparative example 1
The standard stock solution was diluted in BGS solution without SDS to prepare a standard control solution for analysis, i.e., BGS-assisted conventional scavenger-MEKC method, in which erianin and scoparone were 3 μ g/mL, and dendrophenol and naringenin were 10 μ g/mL, and the sample injection, enrichment, and separation detection were performed under the conditions of example 1.
Comparative example 2
The standard stock solution was diluted in an aqueous solution without SDS to prepare a standard control solution for analysis, i.e., a water-assisted conventional scavenger-MEKC method in which erianin and scoparone were 3. mu.g/mL and dendrophenol and naringenin were 10. mu.g/mL, and the samples were injected, enriched, and separated for detection under the conditions of example 1.
Comparative example 3
MEKC method of diluting standard stock solution in BGS, wherein erianin and scoparone are 3 μ g/mL, and dendrophenol and naringenin are 10 μ g/mL, and sample injection, enrichment and separation detection are performed under the conditions of example 1.
The electropherograms obtained in example 1, comparative examples 1 to 3, i.e. the electropherograms of the target analyte obtained by subjecting the target analyte to CD-assisted scan-MEKC, water-assisted conventional scan-MEKC, BGS-assisted conventional scan-MEKC and MEKC, respectively, are shown in fig. 7, wherein a is CD-assisted scan-MEKC, b is BGS-assisted conventional scan-MEKC, c is water-assisted conventional scan-MEKC, and d is MEKC; analytes 1 were erianin, 2 were dendrophenol, 3 was naringenin and 4 was scoparone. As can be seen from FIG. 7, the CD-assisted scavenger-MEKC process of the present invention has significantly higher enrichment efficiency than the other three processes.
Claims (10)
1. An online enrichment method of dendrobium nobile lindl is characterized by comprising the following steps:
(1) adding the dendrobe extracting solution into a phosphoric acid solution containing CD to obtain a sample solution with the CD concentration of 30-130 mM;
(2) dissolving methanol and SDS in a phosphoric acid solution to obtain a BGS solution;
(3) injecting sample solution subjected to ultrasonic treatment and filtration treatment for 50-200s through a capillary tube which is activated and is washed by BGS solution;
(4) and (4) carrying out separation and enrichment under electrification to obtain the separated and enriched active substance.
2. The on-line enrichment method of dendrobium according to claim 1, wherein the type of CD in step (1) is one of alpha-CD, methyl-beta-CD, 2-hydroxypropyl-beta-CD and gamma-CD.
3. The on-line enrichment method of dendrobium according to claim 2, wherein the CD in the step (1) is 2-hydroxypropyl-beta-CD, and the concentration of the CD is 70-90 mM.
4. The on-line enrichment method of dendrobium according to claim 4, wherein the concentration of the phosphoric acid solution in the step (1) is 80-120mM, and/or
The concentration of the phosphoric acid solution in the step (2) is 40-60 mM.
5. The on-line enrichment method of dendrobium nobile as claimed in claim 4, wherein the methanol volume ratio in the BGS solution in the step (2) is 10-40%, and/or the concentration of SDS is 25-125 mM.
6. The online enrichment method of dendrobium nobile as claimed in claim 5, wherein the methanol volume ratio in the BGS solution in step (2) is 20-40%.
7. The on-line enrichment method of dendrobium according to claim 5, wherein the concentration of SDS in the step (2) is 50-100 mM.
8. The online enrichment method of dendrobium according to claim 1, wherein the sample injection time in step (3) is 50-150s, and/or the BGS solution washing time is 3-8 min.
9. The on-line enrichment method of dendrobium nobile as claimed in claim 1, wherein the separation and enrichment in step (4) are performed at a voltage of 10-20kv and a temperature of 25-35 ℃.
10. The on-line enrichment method of any one of claims 1-9, applied to the separation and enrichment of one or more of erianin, dendrophenol, naringenin, and scoparone in dendrobe.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103254045A (en) * | 2013-05-23 | 2013-08-21 | 南京泽朗医药科技有限公司 | Preparation method of high-purity Erianin |
| CN106680403A (en) * | 2016-12-30 | 2017-05-17 | 安徽农业大学 | Method for detecting dendrophenol in Dendrobium officinale |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103254045A (en) * | 2013-05-23 | 2013-08-21 | 南京泽朗医药科技有限公司 | Preparation method of high-purity Erianin |
| CN106680403A (en) * | 2016-12-30 | 2017-05-17 | 安徽农业大学 | Method for detecting dendrophenol in Dendrobium officinale |
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Application publication date: 20201113 |