CN111413420B - Full two-dimensional liquid phase separation method for dendrobium nobile lindl - Google Patents
Full two-dimensional liquid phase separation method for dendrobium nobile lindl Download PDFInfo
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Abstract
The invention relates to the technical field of separation of active ingredients in natural medicines, and provides a full two-dimensional liquid phase separation method for dendrobe, aiming at solving the problems of complicated procedures, toxic solvent use, low extraction efficiency and recovery rate of the traditional dendrobe extraction method, which comprises the following steps: (1) pulverizing herba Dendrobii, and sieving with a third sieve to obtain herba Dendrobii powder; (2) extracting the dendrobium powder by adopting a matrix solid-phase dispersion extraction method to obtain dendrobium extract; (3) and (3) carrying out sample injection analysis and characterization on the dendrobium extract by using two-dimensional ultra-performance liquid chromatography and mass spectrometry. The invention skillfully combines the two-dimensional ultra-high performance liquid chromatography and mass spectrometry combined technology with the matrix solid phase extraction technology, and realizes convenient and efficient separation and extraction of effective components in natural products.
Description
Technical Field
The invention relates to the technical field of separation of active ingredients in natural medicines, in particular to a full two-dimensional liquid phase separation method for dendrobium.
Background
Two-dimensional ultra-high performance liquid chromatography is an ideal and powerful tool, and due to its large peak capacity and high resolution, it has attracted more attention in solving the complexity. In two-dimensional ultra high performance liquid chromatography systems, peaks, parts or whole chromatograms undergo different separation mechanisms with the help of various chromatographic columns. Two-dimensional ultra-high performance liquid chromatography methods can be divided into full two-dimensional and multi-center cutting, which depends on whether all components of the sample are transferred to the second dimension. In the full two-dimensional mode, all of the components of the first dimension are sent to the second dimension. This is obtained by collecting the efficiency of the one-dimensional detector into two cycles, which are alternately shifted into the two-dimensional chromatography column. This combination requires a second dimension of rapid separation (separation time typically less than 1 minute) to increase the sampling frequency and maintain one-dimensional separation. Furthermore, the above protocol can be used for the isolation of complex compounds, combined with mass spectrometry, which have been used for the isolation of active ingredients in proteins, herbal extracts and natural products.
The dendrobium plant is an important tonic and functional food, and belongs to perennial herb plants in orchid. The dendrobium comprises thousands of species including dendrobium officinale, dendrobium huoshanense, dendrobium nobile, dendrobium moniliforme, dendrobium chrysotoxum and the like, wherein the dendrobium nobile is mainly distributed in Zhejiang, Anhui, Guizhou and Yunnan of China. Dendrobe contains various active ingredients, such as polysaccharides, flavones, alkaloids, stilbenes, amino acids, phenanthrenes, bibenzyls, phenols, etc. Furthermore, the dendrobe species have important pharmacological effects and provide a number of beneficial functions, such as heat removal, vision enhancement, maintenance of gastric tone, improvement of humoral production and immunoregulatory effects, etc. Due to the important pharmacological action of dendrobe, it is essential to develop and research a quality control method of dendrobe. However, according to the existing literature reports, the sample pretreatment method is complicated, and a more comprehensive method for analyzing the active ingredients in dendrobium is lacked.
The existing literature has various dendrobium plant extraction methods, such as ultrasonic/microwave-assisted solid-liquid dispersion extraction, pressurized liquid extraction, Soxhlet extraction, metabolic extraction, mechanochemical extraction, DNA extraction and the like. However, most of these extraction methods are cumbersome, time consuming, costly and labor intensive. Moreover, these methods typically involve cumbersome procedures, use of toxic organic solvents, process conditions at high temperatures or exposure to radiation, consumption of large amounts of sample and solvent, resulting in extraction processes with low extraction efficiency and recovery. Therefore, it is essential to analyze, determine and separate trace compounds in dendrobium officinale by using a high-efficiency green sample pretreatment method and a high-precision chromatographic technology.
Disclosure of Invention
The invention provides a full two-dimensional liquid phase separation method for dendrobium with high separation degree and high sensitivity, aiming at solving the problems of complicated procedures, toxic solvent use, low extraction efficiency and recovery rate of the traditional dendrobium plant extraction method.
In order to achieve the purpose, the invention adopts the following technical scheme:
a full two-dimensional liquid phase separation method of dendrobe comprises the following steps:
(1) pulverizing herba Dendrobii, and sieving with a third sieve to obtain herba Dendrobii powder;
(2) extracting the dendrobium powder by adopting a matrix solid-phase dispersion extraction method to obtain dendrobium extract;
(3) and (3) carrying out sample injection analysis and characterization on the dendrobium extract by using two-dimensional ultra-performance liquid chromatography and mass spectrometry.
The method has novel originality of separation links and ingenious idea. The method is successfully applied to the field of natural product extraction and separation by adopting a two-dimensional liquid phase-mass spectrometry combined method as an analysis method, and realizes the successful separation and identification of 16 compounds in the dendrobium under a full two-dimensional mode. The separation detection system has a series of excellent and outstanding characteristics: the operation is simple, the sensitivity and stability of the instrument are good, and the operation environment is safe and reliable. The experimental extraction and separation effect provided by the invention is high-efficiency and obvious, the operation environment is safe, and the operation steps are simple and clear.
Preferably, the matrix solid phase dispersion extraction method comprises the following steps: mixing and grinding the dendrobium powder and a dispersing agent, filling the obtained mixture into a solid phase extraction column, eluting and centrifuging, and taking supernatant to obtain dendrobium extract.
Compared with the traditional method, the matrix solid phase dispersion method solves the problem of large consumption of samples and organic solvents and shortens the extraction time. The components of the dendrobium plants extracted by the matrix solid-phase dispersion micro-extraction method are obviously higher than those extracted by the traditional extraction method, and the content of the dendrobium plants is also obviously increased; after the matrix solid phase dispersion extraction method is adopted for treatment, the sensitivity of the instrument is greatly improved, the reproducibility is also improved, and the method is more accurate and effective in measuring 16 components in the dendrobium plant medicinal materials. The simple applicability and convenient repeatability of the technology make the technology become a useful tool in the traditional Chinese medicine extraction research.
Preferably, the dispersant is C18A dispersant; said C is18The mass ratio of the dispersing agent to the dendrobium powder is (1-2): 1. c adopted in the invention18The dispersing agent is cheap and easy to obtain, and has a series of excellent and outstanding properties: good chemical stability and thermal stability, no toxicity to human body, no environmental pollution and no flammability. And the matrix solid phase dispersion method consumes less organic reagent, sample amount and extraction time, and is environment-friendly and green.
Preferably, the eluent used in the elution process is pure methanol.
Preferably, the flow rate of the one-dimensional liquid chromatography column is controlled to be 0.05-0.11 mL/min, and preferably 0.07 mL/min.
The separation rate of vanillic acid and syringic acid is slightly improved with the increase of the one-dimensional flow velocity. The separation rate then drops sharply as the flow rate continues to increase. The optimum one-dimensional flow rate is at 0.07mL/min, which is demonstrated by the improved separation of vanillic acid and syringic acid by varying the flow rate. In addition, the resolution of the other two important pairs has the same trend as that of vanillic acid and syringic acid. The other two key pairs are vitexin and vitexin glucoside, rutin and naringin, respectively. In general, 0.07mL/min was considered the optimal one-dimensional flow rate, since the flow rate was the highest for the separation of three pairs of compounds.
Preferably, the flow rate of the two-dimensional liquid phase chromatographic column is controlled to be 1.5-3.0 mL/min, and preferably 2.0 mL/min.
In order to keep the cycle duration as short as possible, a relatively high two-dimensional flow rate should be prioritized. And the proper high flow rate is set, so that the influence of the standard solution and the sample solvent can be reduced, and the volume of the one-dimensional eluent is not more than that of the two-dimensional quantitative ring. Considering the specifications of Poroshell 120Bonus column and the pressure resistance of two-dimensional liquid phase system, the flow rate has a direct influence on the separation of vanillic acid/syringic acid from rutin/naringin, since the higher the flow rate, the higher the separation rate. However, the separation rate of vitexin/vitexin glucoside slightly increased with the increase of the two-dimensional flow rate. In contrast, the flow rate was 2.0mL/min, allowing sufficient time intervals, and the two-dimensional separation was good at distinguishing the three pairs of the main active ingredient compounds.
Preferably, the chromatographic conditions of the one-dimensional liquid phase are as follows:
and (3) chromatographic column: SB C18(2.1X 100mm,1.8 μm), column temperature: 50 ℃, sample introduction: 2.0 μ L, detection wavelength: 270nm, sampling frequency: 40Hz, flow rate: 0.07mL/min, mobile phase: a: 0.05% formic acid water, B: methanol. Gradient elution: 0min, 20% B; 3min, 40% B; 22min, 50% B; 25min, 90% B; 29min, 100% B; 35min, 100% B; 40min,100％B。
SB C18The column can effectively separate standard solution, Eclipse Plus C18、SB C18And Amide C16Analytical time ratio of column Chemica C18WpH and Poroshell 120EC-C18The column is short. In general, the better the orthogonality of the separation system, the higher the resolution. In addition, there were significant differences between the columns obtained. Chemical product C18 WpH、Amide C16、Poroshell 120EC-C18The SB C is preferred in the present invention because of poor response, poor orthogonality and poor separability18(2.1X 100mm,1.8 μm) as the stationary phase in the first dimension.
Preferably, the chromatographic conditions of the two-dimensional liquid phase are:
a chromatographic column: poroshell 120Bonus (3.0X 50mm, 1.8 μm), column temperature: 50 ℃, sample introduction: 2.0 μ L, detection wavelength 270nm, sampling frequency, 80Hz, mobile phase: a: 0.05% formic acid water, B: acetonitrile; in the full two-dimensional mode, the gradient stop time is 0.2min, the gradient period is 0.35min, and the two-dimensional flow rate is 2.0 mL/min.
Eclipse Plus C18The column showed more severe point spread of target analyte, Extend C18The column showed that some compounds were not detectable or responded poorly. However, the performance of Poroshell 120Bonus columns was similar to Poroshell 120 phenylhexyl columns, keeping the mobile phase, flow rate and gradient constant. With optimized 1D-LC conditions, both Poroshell 120Bonus columns and Poroshell 120 phenylhexyl columns had better resolution, but Poroshell 120Bonus columns had higher column efficiency and lower background noise. Therefore, 1.8 μm Poroshell 120Bonus (3.0X 50mm) is preferred as the second dimension stationary phase in the present invention.
Preferably, the gradient mode of the two-dimensional liquid phase is: initial gradient: 0min, 15% B; 0.2min, 50% B; gradient at 0 min: 0-5min, 15% B; 5-40min, 15-50% B; gradient at 0.2 min: 0-5min, 50% B; 5-40min, 50-75% B.
The existing two gradient modes, A isocratic elution and gradient and B isocratic elution are integrated, and have the following defects: and performing two-dimensional separation by adopting the same gradient method in the whole analysis process of the mode A. Mode B integrates and combines both gradient and isocratic elution procedures. In the two-dimensional separation process, compared with the flow of the two-dimensional initial gradient adjusted according to the elution capacity of the one-dimensional mobile phase, the gradient elution is obviously improved by the orthogonal distribution of 16 standard products compared with isocratic elution (mode A) because the elution program of the two-dimensional mobile phase is narrower and the mode B consumes less column equilibrium time. The mode of the invention requires a gradient elution, a gradient program is used in the whole analysis process, and the proper gradient elution is beneficial to improving the capability of chromatographic separation of various standard products and complex samples, and the samples have the problem of irregular peak distribution in a separation space. Therefore, the above-described mode of the present invention was selected as the optimum elution gradient.
Preferably, in the step (1), the dendrobium raw materials comprise dendrobium officinale, dendrobium nobile and dendrobium chrysotoxum. The method has wide application range, and the dendrobium officinale, the dendrobium nobile and the dendrobium chrysotoxum can be separated and enriched by the method. The application of the method for measuring dendrobium officinale, dendrobium nobile and dendrobium chrysotoxum also belongs to the first example.
Preferably, the effective components in the dendrobium comprise vanillic acid, syringic acid, apigenin-6, 8-di-c-beta-d-glucoside, p-hydroxycinnamic acid, ferulic acid, vitexin glucoside, vitexin rhamnoside, vitexin, rutin, naringin, naringenin, luteolin, erianin, dendrophenol, dendrobine and withanolide.
Therefore, the invention has the following beneficial effects: the invention skillfully combines the two-dimensional ultra-high performance liquid chromatography and mass spectrometry combined technology with the matrix solid phase extraction technology, and realizes convenient and efficient separation and extraction of effective components in natural products.
Drawings
FIG. 1 is a schematic process flow diagram of the present invention.
FIG. 2 is a graph showing the comparison of the separation effect of the two-dimensional gradients of comparative example 1(Mode A), comparative example 2(Mode B) and example 1(Mode C).
FIG. 3 is a graph comparing the extraction effects of comparative example 3(A1) and example 1(A2) with different extraction methods.
FIG. 4 is a graph showing the comparison of the extraction effects between the different extraction methods of comparative example 4(B1) and example 2 (B2).
FIG. 5 is a graph showing the comparison of the extraction effects of the different extraction methods of comparative example 5(C1) and example 3 (C2).
Detailed Description
The technical solution of the present invention is further specifically described below by using specific embodiments and with reference to the accompanying drawings.
In the present invention, all equipment and materials are commercially available or commonly used in the art, and the methods in the following examples are conventional in the art unless otherwise specified.
Example 1
(1) According to the flow chart shown in fig. 1, the dendrobium officinale raw material is crushed and sieved by a third sieve to obtain dendrobium officinale powder;
(2) mixing 25g herba Dendrobii powder with 25g C18Mixing and grinding the dispersing agents, transferring the obtained mixture into a 1mL solid phase extraction column, eluting the mixture in the solid phase extraction column by using 200 mu L methanol, centrifuging for 5 minutes in a 13000rpm centrifugal machine, and taking supernatant to obtain dendrobium officinale extracting solution;
(3) carrying out two-dimensional ultra-high performance liquid chromatography and mass spectrometry combined sample injection analysis on the dendrobium officinale extract to characterize the separation effect:
the two-dimensional ultra-high performance liquid chromatography comprises a one-dimensional liquid phase and a two-dimensional liquid phase,
the chromatographic conditions of the one-dimensional liquid phase are as follows:
a chromatographic column: SB C18(2.1X 100mm,1.8 μm), column temperature: 50 ℃, sample introduction: 2.0 μ L, detection wavelength: 270nm, sampling frequency: 40Hz, flow rate: 0.07mL/min, mobile phase: a: 0.05% formic acid water, B: methanol. Gradient elution: 0min, 20% B; 3min, 40% B; 22min, 50% B; 25min, 90% B; 29min, 100% B; 35min, 100% B; 40min, 100% B;
the chromatographic conditions of the two-dimensional liquid phase are as follows:
a chromatographic column: poroshell 120Bonus (3.0X 50mm, 1.8 μm), column temperature: 50 ℃, sample introduction: 2.0 μ L, detection wavelength 270nm, sampling frequency, 80Hz, mobile phase: a: 0.05% formic acid water, B: acetonitrile; in the full two-dimensional mode, the gradient stopping time is 0.2min, the gradient period is 0.35min, and the two-dimensional flow rate is 2.0 mL/min;
the gradient mode of the two-dimensional liquid phase is: initial gradient: 0min, 15% B; 0.2min, 50% B; gradient at 0 min: 0-5min, 15% B; 5-40min, 15-50% B; gradient at 0.2 min: 0-5min, 50% B; 5-40min, 50-75% B.
Example 2
(1) Pulverizing herba Dendrobii raw material according to the flow chart shown in FIG. 1, and sieving with a third sieve to obtain herba Dendrobii powder;
(2) mixing 25g herba Dendrobii powder with 25g C18Mixing and grinding the dispersing agents, transferring the obtained mixture into a 1mL solid phase extraction column, eluting the mixture in the solid phase extraction column by using 200 mu L methanol, centrifuging for 5 minutes in a 13000rpm centrifugal machine, and taking supernatant to obtain dendrobium nobile extract;
(3) performing two-dimensional ultra-high performance liquid chromatography and mass spectrometry combined sample injection analysis on the dendrobe extract to characterize the separation effect:
the two-dimensional ultra-high performance liquid chromatography comprises a one-dimensional liquid phase and a two-dimensional liquid phase,
the chromatographic conditions of the one-dimensional liquid phase are as follows:
and (3) chromatographic column: SB C18(2.1X 100mm,1.8 μm), column temperature: 50 ℃, sample introduction: 2.0 μ L, detection wavelength: 270nm, sampling frequency: 40Hz, flow rate: 0.05mL/min, mobile phase: a: 0.05% formic acid water, B: methanol. Gradient elution: 0min, 20% B; 3min, 40% B; 22min, 50% B; 25min, 90% B; 29min, 100% B; 35min, 100% B; 40min, 100% B;
the chromatographic conditions of the two-dimensional liquid phase are as follows:
a chromatographic column: poroshell 120Bonus (3.0X 50mm, 1.8 μm), column temperature: 50 ℃, sample introduction: 2.0 μ L, detection wavelength 270nm, sampling frequency, 80Hz, mobile phase: a: 0.05% formic acid water, B: acetonitrile; in the full two-dimensional mode, the gradient stopping time is 0.2min, the gradient period is 0.35min, and the two-dimensional flow rate is 1.5 mL/min;
the gradient mode of the two-dimensional liquid phase is: initial gradient: 0min, 15% B; 0.2min, 50% B; gradient at 0 min: 0-5min, 15% B; 5-40min, 15-50% B; gradient at 0.2 min: 0-5min, 50% B; 5-40min, 50-75% B.
Example 3
(1) Pulverizing herba Dendrobii, and sieving with a third sieve to obtain herba Dendrobii powder according to the flow chart shown in FIG. 1;
(2) mixing 25g of herba Dendrobii powder with 25g C18Mixing and grinding the dispersing agents, transferring the obtained mixture into a 1mL solid phase extraction column, eluting the mixture in the solid phase extraction column by using 200 mu L methanol, centrifuging for 5 minutes in a 13000rpm centrifugal machine, and taking supernatant to obtain dendrobium chrysotoxum extracting solution;
(3) performing two-dimensional ultra-high performance liquid chromatography and mass spectrometry combined sample injection analysis on the dendrobe extract to characterize the separation effect:
the two-dimensional ultra-high performance liquid chromatography comprises a one-dimensional liquid phase and a two-dimensional liquid phase,
the chromatographic conditions of the one-dimensional liquid phase are as follows:
and (3) chromatographic column: SB C18(2.1X 100mm,1.8 μm), column temperature: 50 ℃, sample introduction: 2.0 μ L, detection wavelength: 270nm, sampling frequency: 40Hz, flow rate: 0.11mL/min, mobile phase: a: 0.05% formic acid water, B: methanol. Gradient elution: 0min, 20% B; 3min, 40% B; 22min, 50% B; 25min, 90% B; 29min, 100% B; 35min, 100% B; 40min, 100% B;
the chromatographic conditions of the two-dimensional liquid phase are as follows:
a chromatographic column: poroshell 120Bonus (3.0X 50mm, 1.8 μm), column temperature: 50 ℃, sample introduction: 2.0 μ L, detection wavelength 270nm, sampling frequency, 80Hz, mobile phase: a: 0.05% formic acid water, B: acetonitrile; in a full two-dimensional mode, the gradient stopping time is 0.2min, the gradient period is 0.35min, and the two-dimensional flow rate is 3.0 mL/min;
the gradient mode of the two-dimensional liquid phase is mode C: initial gradient: 0min, 15% B; 0.2min, 50% B; gradient at 0 min: 0-5min, 15% B; 5-40min, 15-50% B; gradient at 0.2 min: 0-5min, 50% B; 5-40min, 50-75% B.
Comparative example 1
Comparative example 2 differs from example 1 in that in step (3), the gradient Mode of the two-dimensional liquid phase is Mode a: 0min, 15% B; 0.2min, 100% B; the rest of the process is completely the same.
Comparative example 2
Comparative example 2 differs from example 1 in that in step (3), the gradient Mode of the two-dimensional liquid phase is Mode B: 0min, 15% B; 0.2min, 45% B. At 0 min: 0-3min, 15-15% B; 3-3.1min, 15-35% B,3.1-25min, 35-35% B,25-25.1min, 35-80% B,25.1-40min, 80-80% B. At 0.2 min: 0-3min, 45-45% B; 3-3.1min, 45-90% B; 3.1-25min, 90-90% B,25-25.1min, 90-100% B; 25.1-40min, 100-; the rest of the process is completely the same.
The separation effect of different two-dimensional gradients of example 1 and comparative examples 1 and 2 was examined, and the results are shown in fig. 2, and fig. 2 shows a graph of the separation effect of different two-dimensional gradients. As can be seen from FIG. 2, mode A is isocratic elution, and the same gradient method is used for two-dimensional separation throughout the analysis. Mode C requires a gradient elution and a gradient program is used throughout the analysis. In addition, mode B integrates and combines both gradient and isocratic elution procedures. During the two-dimensional separation process, the flow ratio of the two-dimensional initial gradient is adjusted according to the elution capacity of the one-dimensional mobile phase. Gradient elution significantly improved the orthogonal distribution of 16 standards compared to isocratic elution (mode a). In addition, mode B consumes less column equilibration time due to the narrower elution program of the two-dimensional mobile phase. It is noted that proper gradient elution is advantageous to improve the ability of chromatography to separate various standards and complex samples that have problems with irregular peak distributions in the separation space. Thus, mode C was selected as the optimal elution gradient.
Comparative example 3
The difference between the comparative example 3 and the example 1 is that in the step (2), the dendrobium officinale is extracted by adopting a traditional method: sieving herba Dendrobii powder with a third sieve 1.0g, placing in a conical flask with a plug, adding 50.0mL methanol, soaking for 20 min, performing ultrasonic treatment for 30 min, shaking, centrifuging, and filtering to obtain filtrate to obtain herba Dendrobii extract; the rest of the process is completely the same.
Comparative example 4
The difference between the comparative example 4 and the example 2 is that in the step (2), dendrobium nobile lindl is extracted by adopting a traditional method: sieving herba Dendrobii powder with a third sieve 1.0g, placing into a conical flask with a plug, adding 50.0mL methanol, soaking for 20 min, performing ultrasonic treatment for 30 min, shaking, centrifuging, and filtering to obtain filtrate to obtain herba Dendrobii extractive solution; the rest of the process is completely the same.
Comparative example 5
Comparative example 5 differs from example 3 in that, in step (2), dendrobium chrysotoxum was extracted by a conventional method: sieving herba Dendrobii powder with a No. three sieve 1.0g, placing into a conical flask with a plug, adding 50.0mL methanol, soaking for 20 min, performing ultrasound for 30 min, shaking, centrifuging, and filtering to obtain filtrate to obtain herba Dendrobii extract; the rest of the process is completely the same.
The extraction effects of the different extraction methods of examples 1-3 and comparative examples 3-5 were characterized, and the results are shown in fig. 3-5: compared with the traditional method, the matrix solid phase dispersion method solves the problem of large consumption of samples and organic solvents and shortens the extraction time. In addition, three different dendrobe plants are taken as samples, the types of compounds of the dendrobe plants are determined, and the differences of the types are compared. The three Dendrobium plants are herba Dendrobii, and herba Dendrobii drumstick. The results show that the components of the dendrobium plants extracted by the matrix solid-phase dispersion micro-extraction method are obviously higher than those extracted by the traditional extraction method, and the content of the components is also obviously increased. In summary, the preferred matrix solid phase dispersion micro-extraction method of the present invention is used to extract the dendrobe sample.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and other variations and modifications may be made without departing from the spirit of the invention as set forth in the claims.
Claims (3)
1. A full two-dimensional liquid phase separation method of dendrobium nobile lindl is characterized by comprising the following steps:
(1) pulverizing herba Dendrobii, and sieving with a third sieve to obtain herba Dendrobii powder;
(2) extracting the dendrobium powder by adopting a matrix solid-phase dispersion extraction method to obtain dendrobium extract;
(3) performing two-dimensional ultra-high performance liquid chromatography and mass spectrometry combined sample injection analysis on the dendrobe extract to characterize the separation effect;
in the step (2), the matrix solid phase dispersion extraction method comprises the following steps: mixing and grinding the dendrobium powder and a dispersing agent, filling the obtained mixture into a solid phase extraction column, eluting and centrifuging, and taking supernatant to obtain dendrobium extract;
the dispersant is C18A dispersant; said C is18The mass ratio of the dispersing agent to the dendrobium powder is (1-2): 1;
the effective components in herba Dendrobii comprise vanillic acid, syringic acid, apigenin-6, 8-di-c-beta-d-glucoside, p-hydroxy cinnamic acid, ferulic acid, vitexin glucoside, vitexin rhamnoside, vitexin, rutin, naringin, naringenin, luteolin, erianin, dendrophenol, dendrobine and artanolide;
in the step (3), the two-dimensional ultra-high performance liquid chromatography comprises a one-dimensional liquid phase and a two-dimensional liquid phase, and the chromatographic conditions of the one-dimensional liquid phase are as follows:
a chromatographic column: SB C182.1X 100mm,1.8 μm, column temperature: 50 ℃, sample size: 2.0 μ L, detection wavelength: 270nm, sampling frequency: 40Hz, flow rate: 0.05-0.11 mL/min, mobile phase: a: 0.05% formic acid water, B: methanol; gradient elution: 0min, 20% B, 3min, 40% B, 22min, 50% B,25 min, 90% B, 29min, 100% B, 35min, 100% B, 40min, 100% B;
the chromatographic conditions of the two-dimensional liquid phase are as follows:
a chromatographic column: poroshell 120Bonus, 3.0X 50mm, 1.8 μm, column temperature: 50 ℃, sample introduction: 2.0 μ L, detection wavelength 270nm, sampling frequency, 80Hz, mobile phase: a: 0.05% formic acid water, B: acetonitrile; in the full two-dimensional mode, the gradient stopping time is 0.2min, the gradient period is 0.35min, and the two-dimensional flow rate is 1.5-3.0 mL/min;
the gradient mode of the two-dimensional liquid phase is as follows: initial gradient: 0min, 15% B; 0.2min, 50% B; gradient at 0 min: 0-5min, 15% B; 5-40min, 15-50% B; gradient at 0.2 min: 0-5min, 50% B; 5-40min, 50-75% B.
2. The method of claim 1, wherein the eluent used in the elution process is pure methanol.
3. The method for full-two-dimensional liquid phase separation of dendrobium according to any one of claims 1-2, wherein in step (1), the dendrobium raw materials comprise dendrobium officinale, dendrobium nobile and dendrobium chrysotoxum.
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