Separation and identification method for fat-soluble components in sargentgloryvine stem-abortion sauce decoction

Abstract

The invention provides a method for separating and identifying fat-soluble components in a sargentgloryvine stem and abortion sauce decoction, which adopts a comprehensive two-dimensional chromatography HSCCC * HPLC-Q-TOF-MS / MS combined analysis technology, and realizes efficient separation and identification of 207 fat-soluble components in the sargentgloryvine stem and abortion sauce decoction by optimizing a solvent system and chromatographic conditions. The method has high resolution, high sensitivity and high orthogonality, the identification quantity and accuracy of the chemical components of the complex traditional Chinese medicine compound are remarkably improved, and a reliable technical means is provided for fundamental research of pharmacodynamic substances of the sargentgloryvine stem and abortion sauce decoction.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology. The present invention relates to a method for separating and identifying small molecule liposoluble components of the traditional Chinese medicine compound Hongteng Baijiang Decoction, and particularly relates to a method for separating and identifying based on the combined analysis technology of comprehensive two-dimensional chromatography HSCCC×HPLC-Q-TOF-MS / MS. Background Art

[0002] Compound Hongteng Baijiang Decoction is a traditional Chinese medicine compound preparation made from ten medicinal materials including Sargentodoxa cuneata, Patrinia scabiosaefolia, Polygonum cuspidatum, Salvia miltiorrhiza, Olibanum, Myrrha, Cortex Moutan, Angelica sinensis, Sparganium stoloniferum, and Rhizoma Zedoariae. It has the effects of clearing heat and detoxifying, regulating qi and activating blood circulation, removing stasis and dissipating masses, and is mainly used for treating diseases such as damp-heat stasis syndrome. However, the material basis of its efficacy is not yet clear, especially the separation and identification of liposoluble components face technical bottlenecks. However, there is currently no report on the study of the chemical components of the compound decoction of Hongteng Baijiang Decoction. Since the components of the traditional Chinese medicine compound decoction are very complex, usually mainly water-soluble components, and at the same time, the decoction also contains a large amount of liposoluble components. Although the content of these liposoluble components is relatively much lower than that of water-soluble components, they play an important synergistic role in the exertion of the efficacy.

[0003] High speed countercurrent chromatography (HSCCC) is an efficient partition chromatography technology that has emerged in recent years and forms good complementarity with traditional high performance liquid chromatography technology in terms of separation mechanism. HSCCC is based on the liquid-liquid partition principle. Although it avoids sample loss caused by adsorption of the stationary phase (the recovery rate can reach 100%), its separation mechanism depends on the dynamic balance of the two-phase solvents, and the separation efficiency is much lower than that of high performance liquid chromatography (HPLC). In recent years, the quadrupole-time of flight mass spectrometry (Q-TOF-MS / MS) technology has played an important role in the separation and analysis of complex components of traditional Chinese medicine due to its characteristics of high resolution, high sensitivity, and high efficiency. However, its analysis performance is limited by the front-end chromatographic separation ability. Moreover, high-abundance components in traditional Chinese medicine compounds are prone to cause ion suppression effects during the electrospray ionization (ESI) process, suppressing the ionization efficiency of low-content components, resulting in the mass spectrometry signal being possibly completely masked, and causing such key efficacy components to be undetectable.

[0004] The above defects make it difficult for traditional technologies to comprehensively analyze the chemical components of traditional Chinese medicine compounds such as Hongteng Baijiang Decoction, especially the omission of trace liposoluble active components, which seriously hinders the research on the material basis of efficacy and the establishment of quality control standards. Therefore, there is an urgent need for a multi-dimensional separation and identification technology with high orthogonality and high peak capacity to break through the limitations of existing analytical methods and provide new technical means for the research on the material basis of its efficacy. Summary of the Invention

[0005] In view of the above, the object of the present invention is to provide a comprehensive two-dimensional chromatography HSCCC×HPLC-Q-TOF-MS / MS combined analysis and identification technology, and this combined analysis and separation method can be efficiently used for the comprehensive separation, analysis and identification of compounds in traditional Chinese medicine compound prescriptions. By using this combined separation and analysis technology, the separation, analysis and identification of the fat-soluble components of the traditional Chinese medicine compound prescription Hongteng Baijiang Decoction have been completed for the first time. Compared with the one-dimensional chromatography-mass spectrometry combination, the number of identified compounds has been significantly improved.

[0006] The present invention provides a method for separating and identifying the fat-soluble components of the water decoction of Hongteng Baijiang Decoction, which is characterized in that a comprehensive two-dimensional chromatography HSCCC×HPLC-Q-TOF-MS / MS combined analysis technology is adopted, and the method includes the following steps:

[0007] (1) Prepare an ethyl acetate extract of the water decoction of Hongteng Baijiang Decoction;

[0008] (2) Perform the first-dimensional chromatography separation on the extract by high-speed counter-current chromatography (HSCCC) to obtain a separated sample;

[0009] (3) According to the optimized optimal elution conditions and detection wavelengths, perform the second-dimensional HPLC chromatography separation on the separated sample in step (2);

[0010] (4) According to the separation conditions determined in step (3), use the HPLC-Q-TOF-MS / MS technology to perform mass spectrometry detection on the separated sample in step (2);

[0011] (5) Establish a chemical composition database for each medicinal material, and identify the fat-soluble components according to the mass spectrometry data in step (4).

[0012] In some embodiments, the composition of the Hongteng Baijiang Decoction is as follows:

[0013] Sargentodoxa cuneata 30 g, Patrinia scabiosaefolia 30 g, Angelica sinensis 12 g, Salvia miltiorrhiza 30 g, Paeonia suffruticosa 10 g, Sparganium stoloniferum 15 g, Curcuma zedoaria 15 g, Myrrha 6 g, Olibanum 6 g, Polygonum cuspidatum root 30 g.

[0014] In some embodiments, step (1) includes:

[0015] Decoct the medicinal materials of Hongteng Baijiang Decoction with water, concentrate and freeze-dry the water decoction; dissolve the freeze-dried product in water, then extract with ethyl acetate, combine the organic phase extracts, and concentrate and dry.

[0016] In some embodiments, the water decoction in step (1) is obtained by decocting with water 2 - 3 times and combining the filtrates.

[0017] In some embodiments, the dosage ratio of ethyl acetate to the freeze-dried product in step (1) is 50 ml:1 g.

[0018] In some embodiments, the number of extractions in step (1) is 2 - 3 times, preferably 3 times.

[0019] In some embodiments, the specific steps of step (1) are as follows: decoct the medicinal materials of Hongteng Baijiang Decoction with water twice, concentrate and freeze-dry the water decoction; dissolve the freeze-dried product in water, then extract with ethyl acetate three times, and the volume ratio of ethyl acetate to the freeze-dried product is 50 ml: 1 g. Combine the organic phase extracts and concentrate and dry.

[0020] In some embodiments, step (2) adopts a reverse-phase head-to-tail elution mode.

[0021] In some embodiments, the solvent system of the HSCCC is n-hexane, ethyl acetate, methanol and water.

[0022] In some embodiments, step (2) includes the following steps: ① Preparation of the HSCCC system: Mix n-hexane, ethyl acetate, methanol and water respectively according to the volume ratio, then shake vigorously for 1 - 2 min, and then let it stand for phase separation. The upper-phase solution after phase separation is used as the stationary phase, and the lower-phase solution is used as the mobile phase, and ultrasonic degassing is carried out for 10 - 20 min.

[0023] ② Perform the first-dimensional separation by HSCCC: Fill the high-speed counter-current chromatography separation column with the stationary phase, pump in the mobile phase. After the entire stationary phase-mobile phase system (i.e., the two-phase system) reaches hydrodynamic equilibrium, dissolve the extract sample prepared in step (1) in an equal volume of the two-phase system, and then inject the sample; elute; continuously monitor the effluent with a UV detector at 280 nm and collect the effluent.

[0024] In some embodiments, the solvent system of the HSCCC is n-hexane, ethyl acetate, methanol and water, and the volume ratio is 1:9:0.5:9.5 to 1:9:8:2.

[0025] In some embodiments, the volume ratios of n-hexane, ethyl acetate, methanol and water in the solvent system of the HSCCC are 1:9:0.5:9.5, 1:9:2:8, 1:9:4:6, 1:9:6:4, 1:9:8:2 respectively.

[0026] In some embodiments, the elution method of the HSCCC is gradient elution, and the specific program is as follows: 0 - 84 minutes: 1:9:0.5:9.5, v / v; 84 - 124 minutes: 1:9:2:8, v / v; 124 - 164 minutes: 1:9:4:6, v / v; 164 - 204 minutes: 1:9:6:4, v / v; 204 - 246 minutes: 1:9:8:2, v / v; 246 - 340 minutes: push elution; the flow rate is 1 - 2 mL / min, and the rotation speed is about 800 rmp.

[0027] In some embodiments, in step (2), the collection of the effluent is carried out by continuously collecting the effluent every 1 minute using an automatic fraction collector for 80 - 350 minutes of the effluent containing the elution components, with a collection volume of 2 ml per minute, and a total of 270 sample solutions are collected.

[0028] In some embodiments, the second - dimensional HPLC chromatographic separation in step (3) includes the following operations: concentrating the sample solution collected in step (2); separately pipetting each portion of the sample solution, injecting it into HPLC, and measuring and recording the chromatographic data of each sample.

[0029] Preferably, the injection volume is 20 μL.

[0030] In some embodiments, the chromatographic conditions for the second - dimensional HPLC separation are as follows: chromatographic column: C18 column; mobile phase: 0.2% formic acid in water (A) and acetonitrile (B); detection wavelengths are 254 nm and 280 nm; flow rate: 0.8 mL·min -1 ; column temperature: 30 °C; injection volume: 20 μl.

[0031] In some embodiments, the elution method for the second - dimensional HPLC separation is gradient elution, and the specific method of the gradient elution is as follows: 0–10 min, 10%–25% B; 10–30 min, 25% - 40% B; 30–31 min, 40% - 60% B; 31–40 min, 60%–80% B; 40–41 min, 80%–100% B; 41–45 min, 100% B; 45–46 min, 100% - 10% B; 46–55 min, 10% B.

[0032] In some embodiments, the chromatographic column in the second - dimensional HPLC separation is Welch XB - C18.

[0033] In some embodiments, step (3) further includes establishing a comprehensive two - dimensional chromatogram HSCCC and an HPLC contour map, and performing orthogonality evaluation through the space coverage rate and the linear correlation coefficient.

[0034] In some embodiments, the contour map is obtained by importing the chromatographic data of each component collected by HSCCC and HPLC into Origin 2022 and MatLab (R2024a) for processing, and presenting the comprehensive two - dimensional chromatogram in the form of a contour map.

[0035] In some embodiments, the space coverage rate of the chemical components of the extract separated by the comprehensive two - dimensional chromatogram HSCCC×HPLC is obtained by using the convex hull calculation method.

[0036] In some embodiments, the linear correlation is represented by the linear correlation coefficient.

[0037] In some embodiments, the space coverage rate (f coverage ) and the linear correlation coefficient (r) are calculated by the formula:

[0038]

[0039] r = cosθ

[0040] where θ is the peak spreading angle and r is the linear correlation coefficient of the one-dimensional and two-dimensional retention times.

[0041] In some embodiments, the step (4) includes the following operations: concentrating the sample solution collected in step (2); precisely sucking each sample solution respectively, injecting it into a liquid chromatography-mass spectrometry instrument, and measuring and recording the mass spectrometry data of each sample.

[0042] Preferably, the liquid chromatography-mass spectrometry method is HPLC-Q-TOF-MS / MS.

[0043] In some embodiments, the chromatographic conditions of the HPLC in step (4) are: chromatographic column: C18 column; mobile phase: 0.2% formic acid water (A) and acetonitrile (B); detection wavelength: 254 nm; flow rate: 0.8 mL·min -1 ; column temperature: 30 °C; injection volume: 20 μl.

[0044] In some embodiments, the elution mode of the HPLC in step (4) is gradient elution, and the specific method of the gradient elution is: 0–10 min, 10%–25% B; 10–30 min, 25% - 40% B; 30–31 min, 40% - 60% B; 31–40 min, 60%–80% B; 40–41 min, 80%–100% B; 41–45 min, 100% B; 45–46 min, 100% - 10% B; 46–55 min, 10% B.

[0045] In some embodiments, the chromatographic column in step (4) is Welch XB-C18.

[0046] In some embodiments, the mass spectrometry conditions in step (4) are: using a high-resolution mass spectrometer, electrospray ionization source (ESI), positive and negative ion mode scanning; spray voltage 5.5 kV (ESI+), -5.5 kV (ESI-); primary mass spectrometry scanning detection range: m / z 60~1500, secondary mass spectrometry scanning range: m / z 50~1000, ion source temperature 600 °C; atomizing gas and auxiliary gas pressures are both 55 psi (1 psi = 6.895 kPa), curtain gas pressure 35 psi; collision energy 10~35 eV.

[0047] In some embodiments, step (5) includes collecting the compound mass spectrometry information of Sargentodoxa cuneata, Patrinia scabiosaefolia, Polygonum cuspidatum, Salvia miltiorrhiza, Olibanum, Myrrha, Cortex Moutan, Angelica sinensis, Sparganium stoloniferum, and Rhizoma Curcumae, and identifying the high-resolution mass spectrometry data of the chemical components of Hongteng Baijiang Decoction; through the mass spectrometry data and the corresponding fragmentation rule matching method, the substances of the peaks containing MS / MS data obtained in step (4) are identified.

[0048] In some embodiments, the fat-soluble components determined by the separation and identification method include 207 components in total, such as amino acids, phthalides, phenylpropanoids, anthraquinones, flavonoids, terpenoids, and organic acids.

[0049] In some embodiments, the 207 components include: 3 amino acid components, 11 phthalide components, 30 phenylpropanoid components, 1 phenol component, 5 anthraquinone components, 4 stilbene components, 3 phenol components, 1 nucleoside component, 6 flavonoid components, 6 lactone components, 1 purine component, 4 aldehyde components, 8 tannin components, 2 alkaloid components, 2 carbohydrate components, 90 terpenoid components, 2 ketone components, 1 amide component, 16 organic acid components, 3 fatty acid components, 4 ester components, and 4 other components.

[0050] The present invention also provides a separation and identification method using comprehensive two-dimensional chromatography HSCCC×HPLC-Q-TOF-MS / MS combined analysis technology for the identification of fat-soluble components of Hongteng Baijiang Decoction, and the separation and identification method is as described above.

[0051] Beneficial effects

[0052] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0053] The present invention adopts the comprehensive two-dimensional chromatography mass spectrometry HSCCC×HPLC-Q-TOF-MS / MS combined analysis technology to separate, analyze, and identify the fat-soluble components of the water decoction of Hongteng Baijiang Decoction. This method has strong specificity and good reproducibility. Compared with the one-dimensional liquid chromatography mass spectrometry coupling technology, the number of fat-soluble components of the water decoction of Hongteng Baijiang Decoction identified by the method provided by the present invention has been significantly improved.

[0054] The present invention also uses comprehensive two-dimensional chromatography HSCCC×HPLC to separate the components of the ethyl acetate extract of Hongteng Baijiang Decoction. By screening and optimizing the high-speed counter-current solvent system, n-hexane, ethyl acetate, methanol, and water were selected as the two-phase system, and the corresponding volume ratio range was established and optimized to make the distribution of the ethyl acetate extract of Hongteng Baijiang Decoction in the two-phase solvent system uniform. Combining with HPLC-Q-TOF-MS / MS technology, the separation, analysis, and identification of the fat-soluble components in the compound Hongteng Baijiang Decoction were completed. During the separation of the fat-soluble components of Hongteng Baijiang Decoction, it was found that the comprehensive two-dimensional chromatography HSCCC×HPLC has strong orthogonality, large chromatographic peak capacity and resolution, and can identify more compound numbers and types compared with one-dimensional chromatography. Description of the Drawings

[0055] Figure 1 : HSCCC chromatogram of the ethyl acetate extract of Hongteng Baijiang Decoction

[0056] Figure 2 : HPLC chromatogram of the ethyl acetate extract of Hongteng Baijiang Decoction

[0057] Figure 3 : Comprehensive two-dimensional chromatogram of the ethyl acetate extract of Hongteng Baijiang Decoction at a detection wavelength of 254 nm

[0058] Figure 4 : Comprehensive two-dimensional chromatogram of the ethyl acetate extract of Hongteng Baijiang Decoction at a detection wavelength of 280 nm

[0059] Figure 5 : Spatial coverage rate of the chemical components of the ethyl acetate extract of the decoction of Hongteng Baijiang Decoction separated by comprehensive two-dimensional chromatography HSCCC×HPLC under the condition of a detection wavelength of 254 nm obtained by the convex hull calculation method

[0060] Figure 6 : Spatial coverage rate of the chemical components of the ethyl acetate extract of the decoction of Hongteng Baijiang Decoction separated by comprehensive two-dimensional chromatography HSCCC×HPLC under the condition of a detection wavelength of 280 nm obtained by the convex hull calculation method

[0061] Figure 7 : Linear correlation of the chemical components of the ethyl acetate extract of the decoction of Hongteng Baijiang Decoction separated by comprehensive two-dimensional chromatography HSCCC×HPLC under the condition of a detection wavelength of 254 nm

[0062] Figure 8 : Linear correlation of the chemical components of the ethyl acetate extract of the decoction of Hongteng Baijiang Decoction separated by comprehensive two-dimensional chromatography HSCCC×HPLC under the condition of a detection wavelength of 280 nm

[0063] Figure 9: HSCCC chromatograms of the ethyl acetate extract of Hongteng Baijiang Decoction in Example 3, A. HSCCC chromatogram under screening condition A; B. HSCCC chromatogram under screening condition B; C. HSCCC chromatogram under screening condition C; D. HSCCC chromatogram under screening condition D; E. HSCCC chromatogram under screening condition E; F. HSCCC chromatogram under screening condition F; H. HSCCC chromatogram under screening condition H; G. HSCCC chromatogram under screening condition G.

[0064] Figure 10 : Total ion chromatogram of the ethyl acetate extract of Hongteng Baijiang Decoction in positive ion mode

[0065] Figure 11 : Total ion chromatogram of the ethyl acetate extract of Hongteng Baijiang Decoction in negative ion mode Specific embodiments

[0066] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0067] In the following examples, the instruments and reagents are as follows:

[0068] Instruments: Shimadzu analytical LC-20A high performance liquid chromatograph (Shimadzu); Shanghai Tongtian TBE-300A high speed countercurrent chromatography; SCIEX ZenoTOF TM 7600 high resolution mass spectrometer.

[0069] Samples: Chinese medicinal materials (Ningbo Yinzhou Medical Herbs Co., Ltd.)

[0070] Example 1

[0071] Step (1) Preparation of the ethyl acetate extract of the water decoction of Hongteng Baijiang Decoction: Take one dose of the prescription herbs of Hongteng Baijiang Decoction (30 g of Sargentodoxa cuneata, 30 g of Patrinia scabiosaefolia, 12 g of Angelica sinensis, 30 g of Salvia miltiorrhiza, 10 g of Paeonia suffruticosa, 15 g of Sparganium stoloniferum, 15 g of Curcuma zedoaria, 6 g of Myrrha, 6 g of Olibanum, 30 g of Polygonum cuspidatum root), decoct with water twice. First decoction: Add pure water to cover the herbs by 2 - 3 cm, soak for 30 min, then bring to a boil over high heat and turn to low heat, start timing from the boiling point, decoct for 30 min, and filter with gauze. Second decoction: Add pure water to cover the herbs by 1 - 2 cm, bring to a boil over high heat and turn to low heat, start timing from the boiling point, decoct for 20 min, and filter with gauze. Combine the filtrates. Concentrate the filtrates by rotary evaporation and freeze-dry. Dissolve 6.0 g of the water decoction extract in 300 mL of water, add an equal volume of ethyl acetate and extract 3 times. Combine the organic phase extracts and rotary evaporate to obtain 214 mg of the ethyl acetate extract.

[0072] Step (2) First-dimensional chromatographic HSCCC separation: Use reverse-phase head and tail washing and demoulding.

[0073] ① Preparation of the solvent system for high-speed counter-current chromatography: Mix n-hexane, ethyl acetate, methanol and water according to the volume ratio respectively, then shake vigorously for 2 min, and then let it stand for phase separation. After phase separation, the upper-phase solution is used as the stationary phase and the lower-phase solution is used as the mobile phase, and ultrasonic degassing is carried out for 15 min. Dissolve 62.01 mg of ethyl acetate extract in 10 mL of the upper and lower phase solvent system with equal volume. After reaching equilibrium, inject the sample solution.

[0074] ② First-dimensional separation by HSCCC: Fill the high-speed counter-current chromatography separation column with the stationary phase, pump in the mobile phase. When the entire stationary phase-mobile phase system (i.e., the two-phase system) reaches hydrodynamic equilibrium, that is, when no stationary phase flows out of the column outlet, dissolve 62.01 mg of ethyl acetate extract in 10 mL of the upper and lower phase solvent system with equal volume, and then inject the sample; elute; continuously monitor the eluate at 280 nm with a UV detector (see Figure 1 ), and use an automatic fraction collector to continuously collect the eluate every 1 min. Continuously collect the eluate containing the eluted components for 80 - 350 min, with a collection volume of 2 ml per minute, and a total of 270 sample solutions are collected.

[0075] Elution program: 0 - 84 minutes: (1:9:0.5:9.5, v / v); 84 - 124 minutes: (1:9:2:8, v / v); 124 - 164 minutes: (1:9:4:6, v / v); 164 - 204 minutes (1:9:6:4, v / v); 204 - 246 minutes (1:9:8:2, v / v); 246 - 340 minutes: push elution. The flow rate is 2 mL / min, the retention rate of the stationary phase is 80.68%, and the rotation speed is 802.5 rpm.

[0076] Step (3) Second-dimensional HPLC chromatography separation: Concentrate the sample solutions collected in step (2) to a final volume of 1.4 ml; precisely pipette 20 μL of each sample solution and inject it into HPLC to measure and record the chromatographic data of each sample.

[0077] HPLC separation conditions: The chromatographic column is Welch XB-C18 (4.6 mm × 250 mm, 5 μm); wavelengths 254 nm and 280 nm; flow rate 0.8 ml / min; column temperature 30 °C; use 0.2% formic acid aqueous solution (A) - acetonitrile (B) as the mobile phase, gradient 0–10 min, 10%–25% B; 10–30 min, 25% - 40% B; 30–31 min, 40% - 60% B; 31–40 min, 60%–80% B; 40–41 min, 80%–100% B; 41–45 min, 100% B; 45–46 min, 100% - 10% B; 46–55 min, 10% B.

[0078] Establishment and orthogonality evaluation of the contour map of comprehensive two-dimensional chromatography HSCCC×HPLC: Import the data of 270 components collected by high-speed counter-current chromatography and high-performance liquid chromatography into Origin 2022 and MatLab (R2024a) for data processing, and display the comprehensive two-dimensional chromatogram in the form of a contour map (2D contourplot). The results are shown in Figure 3 and Figure 4 .

[0079] Spatial coverage rate of the chemical components of the separated extract by comprehensive two-dimensional chromatography HSCCC×HPLC obtained by the convex hull calculation method;

[0080] Orthogonality evaluation is carried out by calculating the spatial coverage rate and linear correlation of the separated components of comprehensive two-dimensional chromatography HSCCC×HPLC. The formula is as follows:

[0081]

[0082] r = cosθ

[0083] where θ is the peak spreading angle and r is the linear correlation coefficient of the retention times in one dimension and two dimensions.

[0084] The results of the spatial coverage rate are shown in Figure 5 and Figure 6 . The spatial coverage rate is 90.16% under the detection wavelength of 254 nm and 88.49% under the detection wavelength of 280 nm. The results of the linear correlation are shown in Figure 7 and Figure 8 . Under the detection wavelength of 254 nm, r = 0.110, and under the detection wavelength of 254 nm, r = 0.498.

[0085] It can be seen that the spatial coverage rate is higher and the linear correlation coefficient is smaller under the detection wavelength of 254 nm. This wavelength is used as the separation and detection wavelength in the second dimension.

[0086] Step (4) HPLC-Q-TOF-MS / MS mass spectrometry detection:

[0087] Sample: Precisely pipette 20 μL of each concentrated sample solution collected in step (2) and inject it into the HPLC-Q-TOF-MS / MS liquid chromatography-mass spectrometry instrument to measure and record the chromatographic data of each sample.

[0088] HPLC liquid phase conditions: The chromatographic column is Welch XB-C18 (4.6 mm × 250 mm, 5 μm); wavelength 254 nm; flow rate 0.8 ml / min; column temperature 30 °C; using 0.2% formic acid aqueous solution (A) - acetonitrile (B) as the mobile phase, gradient 0–10 min, 10%–25% B; 10–30 min, 25% - 40% B; 30–31 min, 40% - 60% B; 31–40 min, 60%–80% B; 40–41 min, 80%–100% B; 41–45 min, 100% B; 45–46 min, 100% - 10% B; 46–55 min, 10% B.

[0089] Mass spectrometry conditions: Using SCIEX ZenoTOF TM 7600 high-resolution mass spectrometer, electrospray ionization source (ESI), positive and negative ion mode scanning. Spray voltage 5.5 kV (ESI+), -5.5 kV (ESI-); primary mass spectrometry scan detection range: m / z 60–1500, secondary mass spectrometry scan range: m / z 50–1000, ion source temperature 600 °C; atomizing gas and auxiliary gas pressures are both 55 psi (379.225 kPa, 1 psi = 6.895 kPa), curtain gas pressure 35 psi (241.325 kPa); collision energy 10–35 eV. Data acquisition and analysis are performed using AB SCIEX software.

[0090] Step (5) Find the mass spectrometry data of the chemical components of each medicinal material according to the literature, and identify the fat-soluble components according to the mass spectrometry data:

[0091] Collect the mass spectrometry information of the compounds of Sargentodoxa cuneata, Patrinia scabiosaefolia, Polygonum cuspidatum, Salvia miltiorrhiza, Olibanum, Myrrha, Paeonia suffruticosa, Angelica sinensis, Sparganium stoloniferum, and Curcuma zedoaria, and identify the chemical components in the Sargentodoxa cuneata and Patrinia scabiosaefolia decoction; through the above-mentioned mass spectrometry database and the corresponding fragmentation rule matching method, identify the substances of the peaks obtained in step (4) containing MS / MS data. The identification results are shown in Table 1.

[0092] Table 1. Identification of fat-soluble components in Sargentodoxa cuneata and Patrinia scabiosaefolia decoction by HSCCC×HPLC-Q-TOF-MS / MS method

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107] The results showed that a total of 207 components were identified by HSCCC×HPLC-Q-TOF-MS / MS, including: 3 amino acid components, 11 phthalide components, 30 phenylpropanoid components, 1 phenol component, 5 anthraquinone components, 4 stilbene components, 3 phenolic components, 1 nucleoside component, 6 flavonoid components, 6 lactone components, 1 purine component, 4 aldehyde components, 8 tannin components, 2 alkaloid components, 2 carbohydrate components, 90 terpene components, 2 ketone components, 1 amide component, 16 organic acid components, 3 fatty acid components, 4 ester components, and 4 other components.

[0108] Example 2: Screening of HSCCC Solvent System

[0109] The solvents were mixed according to the ratios in Table 2, and the high-speed counter-current solvent system was screened by colorimetry. The results are shown in Table 2.

[0110] It can be seen that the mixed system of n-hexane, ethyl acetate, methanol and water can be used as the high-speed counter-current solvent.

[0111] Table 2. Results of Screening HSCCC Solvent Systems with Different Ratios by Colorimetry

[0112]

[0113]

[0114] Example 3: High-Speed Counter-Current Chromatography Separation under Different Conditions

[0115] The first - dimension HSCCC separation was the same as in Example 1. Screening condition A: The selected biphasic solvent system consisted of n - hexane / ethyl acetate / methanol / water. The mobile phase was the lower aqueous phase, and the stationary phase was the upper organic phase. Elution program: 0 - 111 minutes: (1:9:0.5:9.5, v / v); 111 - 152 minutes: (1:9:2:8, v / v); 152 - 192 minutes: (1:9:4:6, v / v); 192 - 233 minutes (1:9:6:4, v / v); 233 - 274 minutes (1:9:8:2, v / v); 274 - 400 minutes: push - through elution. The flow rate was 2 mL min⁻¹, the stationary - phase retention rate was 79.6%, and the rotation speed was 802.5 rpm. 56.00 mg of the ethyl - acetate extract was dissolved in 10 mL of an equal - volume upper - and - lower - phase solvent system. After reaching equilibrium, the sample solution was injected. The eluate was continuously monitored at 280 nm with a UV detector to obtain the HSCCC chromatogram, as shown in Figure 9 A.

[0116] The first - dimension HSCCC separation was the same as in Example 1. Screening condition B: The selected biphasic solvent system consisted of n - hexane / ethyl acetate / methanol / water. The mobile phase was the lower aqueous phase, and the stationary phase was the upper organic phase. Elution program: 0 - 124 minutes: (1.5:8.5:0.5:9.5, v / v); 124 - 172 minutes: (1.5:8.5:1:9, v / v); 172 - 230 minutes: (1.5:8.5:2:8, v / v); 230 - 290 minutes (1.5:8.5:3.5:6.5, v / v); 290 - 340 minutes: (1.5:8.5:5:5, v / v); 340 - 480 min: push - through elution. The flow rate was 2 mL min⁻¹, the stationary - phase retention rate was 74.2%, and the rotation speed was 802.5 rpm. 55.00 mg of the ethyl - acetate extract was dissolved in 10 mL of an equal - volume upper - and - lower - phase solvent system. After reaching equilibrium, the sample solution was injected. The eluate was continuously monitored at 280 nm with a UV detector to obtain the HSCCC chromatogram, as shown in Figure 9 B.

[0117] The first - dimension HSCCC separation was the same as in Example 1. Screening condition C: The selected biphasic solvent system consisted of n - hexane / ethyl acetate / methanol / water. The mobile phase was the lower aqueous phase, and the stationary phase was the upper organic phase. Isocratic elution program: 0 - 300 minutes: (2:8:2:8, v / v). The flow rate was 2 mL min⁻¹, the stationary - phase retention rate was 33.3%, and the rotation speed was 550 rpm. 59.65 mg of the ethyl - acetate extract was dissolved in 10 mL of an equal - volume upper - and - lower - phase solvent system. After reaching equilibrium, the sample solution was injected. The eluate was continuously monitored at 280 nm with a UV detector to obtain the HSCCC chromatogram, as shown in Figure 9 C.

[0118] The first - dimensional HSCCC separation was the same as in Example 1, screening condition D: flow rate: 2 mL min-1; rotation speed: 800 rpm; wavelength: 280 nm. The ethyl acetate extract was dissolved in 10 mL of an equal - volume upper - and - lower - phase solvent system; solvent system: n - hexane: ethyl acetate: methanol: water; injection volume: 49 mg; elution program: 0 - 109 minutes: (1:9:0.5:9.5, v / v); 109 - 159 minutes: (1:9:1:9, v / v); 159 - 225 minutes: (1:9:2:8, v / v); 225 - 300 minutes (1:9:3.5:6.5, v / v); 300 - 440 minutes: push - through elution. The eluate was continuously monitored at 280 nm with a UV detector to obtain the HSCCC chromatogram, as Figure 9 D.

[0119] The first - dimensional HSCCC separation was the same as in Example 1, screening condition E: flow rate: 2 mL min-1; rotation speed: 800 rpm; wavelength: 280 nm. The ethyl acetate extract was dissolved in 10 mL of an equal - volume upper - and - lower - phase solvent system; solvent system: n - hexane: ethyl acetate: methanol: water; elution program: 0 - 69 minutes: (1.5:8.5:0.5:9.5, v / v); 69 - 116 minutes: (1.5:8.5:1:9, v / v); 116 - 162 minutes: (1.5:8.5:2:8, v / v); 162 - 195 minutes (1.5:8.5:3.5:6.5, v / v); 195 - 212 minutes: (1.5:8.5:5:5, v / v); 212 - 350 min: push - through elution. Injection volume 40 mg. The eluate was continuously monitored at 280 nm with a UV detector to obtain the HSCCC chromatogram, as Figure 9 E.

[0120] The first - dimensional HSCCC separation was the same as in Example 1, screening condition F: flow rate: 2 mL min-1; rotation speed: 800 rpm; wavelength: 280 nm. The ethyl acetate extract was dissolved in 10 mL of an equal - volume upper - and - lower - phase solvent system; solvent system: n - hexane: ethyl acetate: methanol: water; elution program: 0 - 94 minutes: (1:9:1:9, v / v); 94 - 158 minutes: (1:9:2:8, v / v); 158 - 201 minutes (1:9:3.5:6.5, v / v); 201 - 270 minutes: (1:9:5:5, v / v); 270 - 330 min: push - through elution. Injection volume 33 mg. The eluate was continuously monitored at 280 nm with a UV detector to obtain the HSCCC chromatogram, as Figure 9 F.

[0121] The first - dimension HSCCC separation was the same as in Example 1, with screening condition G: flow rate: 2 mL min-1; rotation speed: 550 rpm; detection wavelength: 254 nm; sample solution: 55 mg of ethyl acetate extract was dissolved in 10 mL of an equal - volume upper - and - lower - phase solvent system; solvent system: n - hexane: ethyl acetate: methanol: water; elution program: 0 - 111 minutes: (1:9:0.5:9.5, v / v); 110 - 180 minutes: (1:9:1:9, v / v); 180 - 225 minutes: (1:9:2:8, v / v); 225 - 282 minutes (1:9:3.5:6.5, v / v); 282 - 315 minutes: (1:9:5:5, v / v); 315 - 400 min: push - through elution. The eluate was continuously monitored at 254 nm with a UV detector to obtain the HSCCC chromatogram, as shown in Figure 9 G.

[0122] The first - dimension HSCCC separation was the same as in Example 1, with screening condition H: flow rate: 2 mL min-1; rotation speed: 800 rpm; wavelength: 280 nm, the ethyl acetate extract was dissolved in 10 mL of an equal - volume upper - and - lower - phase solvent system; solvent system: n - hexane: ethyl acetate: methanol: water; elution program: 0 - 114 minutes: (1:9:0.5:9.5, v / v); 114 - 155 minutes: (1:9:1:9, v / v); 155 - 198 minutes: (1:9:2:8, v / v); 198 - 242 minutes (1:9:3.5:6.5, v / v); 242 - 360 minutes: push - through elution. The injection volume was 62 mg. The eluate was continuously monitored at 280 nm with a UV detector to obtain the HSCCC chromatogram, as shown in Figure 9 H.

[0123] As can be seen from Figure 9 the HSCCC chromatograms under screening conditions A - H could not obtain good chromatographic peaks.

[0124] Example 4: Other parts were the same as in Example 1, and the different part was:

[0125] (4) HPLC - Q - TOF - MS mass spectrometry detection:

[0126] The extract in step (1) was dissolved in chromatographic - grade methanol to 5 mg / ml. 20 μL of each portion was accurately pipetted and injected into an HPLC - Q - TOF - MS liquid chromatography - mass spectrometry instrument to measure and record the sample chromatographic data. The total ion current chromatograms in positive and negative ion modes are shown in Figure 10 、 Figure 11 respectively.

[0127] The results showed that the 137 medium and small polar components identified by one-dimensional chromatography HPLC-Q-TOF-MS / MS included: 1 amino acid component, 7 phthalide components, 18 phenylpropanoid components, 1 phenol component, 3 anthraquinone components, 4 stilbene components, 3 phenol components, 1 nucleoside component, 5 flavonoid components, 3 lactone components, 1 purine component, 1 aldehyde component, 7 tannin components, 2 alkaloid components, 2 carbohydrate components, 62 terpene components, 1 ketone component, 1 amide component, 9 organic acid components, 1 fatty acid component, 1 ester component, and 3 other components.

[0128] It can be seen that the 70 medium and small polar components identified by comprehensive two-dimensional chromatography mass spectrometry HSCCC×HPLC-Q-TOF-MS / MS compared with single HPLC-Q-TOF-MS / MS included: 2 amino acid components, 4 phthalide components, 12 phenylpropanoid components, 2 anthraquinone components, 1 flavonoid component, 1 other component, 3 lactone components, 3 aldehyde components, 1 tannin component, 28 terpene components, 1 ketone component, 7 organic acid components, 2 fatty acid components, and 3 ester components. The specific comparison results are shown in Table 3.

[0129] Table 3. Comparison of the number of results of the analysis and identification of the fat-soluble compounds in Hongteng Baijiang Decoction by the comprehensive two-dimensional chromatography mass spectrometry HSCCC×HPLC-Q-TOF-MS / MS combination analysis technique and one-dimensional chromatography HPLC-Q-TOF-MS / MS

[0130]

[0131]

[0132] Unless otherwise defined, all professional and scientific terms used in this article have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the present invention. The preferred implementation methods and materials described in this article are only for demonstration purposes and cannot limit the content of this application.

Claims

1. A method for separating and identifying the fat-soluble components of the water decoction of Hongteng Baijiang Decoction, characterized in that, The HSCCC×HPLC-Q-TOF-MS / MS combined analysis technique is adopted, including the following steps: (1) Prepare the ethyl acetate extract of the water decoction of Hongteng Baijiang Decoction; (2) Perform the first-dimensional chromatographic separation on the extract by high-speed counter-current chromatography (HSCCC) to obtain the separated sample; (3) Perform the second-dimensional HPLC chromatographic separation on the separated sample in step (2); (4) Determine the separation conditions according to step (3), and perform mass spectrometry detection on the separated sample in step (2) using the HPLC-Q-TOF-MS / MS technique; (5) Establish a chemical composition database for each medicinal material, and identify the fat-soluble components according to the mass spectrometry data in step (4).

2. The separation and identification method according to claim 1, characterized in that The step (1) includes: decocting the medicinal materials of Hongteng Baijiang Decoction with water, concentrating and freeze-drying the water decoction; dissolving the freeze-dried product in water, then extracting with ethyl acetate, combining the organic phase extracts, and concentrating and drying; Preferably, the water decoction is obtained by decocting with water 2-3 times and combining the filtrates; the dosage ratio of ethyl acetate to the freeze-dried product is 50 ml: 1 g; the number of extraction times is 2-3 times.

3. The separation and identification method according to claim 1, characterized in that The step (2) adopts the reverse-phase head-to-tail elution mode; Preferably, the solvent system of the HSCCC is n-hexane, ethyl acetate, methanol and water; more preferably, the volume ratios of n-hexane, ethyl acetate, methanol and water are 1:9:0.5:9.5, 1:9:2:8, 1:9:4:6, 1:9:6:4, 1:9:8:2 respectively; Preferably, the elution method of the HSCCC is gradient elution, and the specific procedure is: 0-84 minutes: 1:9:0.5:9.5, v / v; 84-124 minutes: 1:9:2:8, v / v; 124-164 minutes: 1:9:4:6, v / v; 164-204 minutes 1:9:6:4, v / v; 204-246 minutes: 1:9:8:2, v / v; 246-340 minutes: push elution; the flow rate is 1-2 mL / min, and the rotation speed is about 800 rmp.

4. The separation and identification method according to claim 3, wherein, The step (2) includes the following steps: ① Preparation of the HSCCC system: Mix n-hexane, ethyl acetate, methanol and water according to the volume ratio respectively, then shake vigorously for 1-2 min, let stand for layer separation, and use the upper phase solution after layer separation as the stationary phase and the lower phase solution as the mobile phase, and perform ultrasonic degassing for 10-20 min; ② Perform the first-dimensional separation by HSCCC: Fill the high-speed counter-current chromatography separation column with the stationary phase, pump in the mobile phase, and after the entire stationary phase-mobile phase system (i.e., the two-phase system) reaches hydrodynamic equilibrium, dissolve the extract sample prepared in step (1) in an equal volume of the two-phase system, and then inject the sample; elute; continuously monitor the effluent with a UV detector at 280 nm, and collect the effluent; Preferably, in the step (2), the collection of the effluent is to continuously collect the effluent every 1 min using an automatic fraction collector, continuously collect the effluent containing the eluted components for 80-350 min, and the collection volume per minute is 2 ml, and a total of 270 sample solutions are collected.

5. The separation and identification method according to claim 1, characterized in that, The second - dimensional HPLC chromatographic separation described in step (3) includes the following operations: concentrating the sample solution collected in step (2); respectively taking each portion of the sample solution, injecting it into HPLC, and measuring and recording the chromatographic data of each sample.

6. The separation and identification method according to claim 5, wherein The chromatographic conditions for the second - dimension HPLC separation are as follows: chromatographic column: C18 column; mobile phase: 0.2% formic acid in water (A) and acetonitrile (B); detection wavelengths are 254 nm and 280 nm; flow rate: 0.8 mL·min -1 ; Column temperature: 30 °C; Injection volume: 20 μl; The elution mode of the second - dimensional HPLC separation is gradient elution. The specific method of the gradient elution is as follows: 0–10 min, 10%–25% B; 10–30 min, 25% - 40% B; 30–31 min, 40% - 60% B; 31–40 min, 60%–80% B; 40–41 min, 80%–100% B; 41–45 min, 100% B; 45–46 min, 100% - 10% B; 46–55 min, 10% B; Preferably, the chromatographic column in the second-dimensional HPLC separation is Welch XB-C18.

7. The separation and identification method according to claim 5, characterized in that Step (3) also includes establishing a comprehensive two - dimensional chromatogram HSCCC and an HPLC contour map, and evaluating orthogonality through the space coverage rate and the linear correlation coefficient; Preferably, the contour map is obtained by importing the chromatographic data of each component collected by HSCCC and HPLC into analysis software for processing, and displaying the comprehensive two - dimensional chromatogram in the form of a contour map; the space coverage rate of the chemical components of the separated extract of HSCCC×HPLC in the comprehensive two - dimensional chromatography is obtained by the convex hull calculation method; The calculation of the space coverage rate and the linear correlation coefficient is as follows: r = cosθ where θ is the peak spreading angle and r is the linear correlation coefficient between the retention times in one - dimensional and two - dimensional chromatography.

8. The separation and identification method according to claim 1, characterized in that, Step (4) includes the following operations: concentrating the sample solution collected in step (2); respectively precisely taking each portion of the sample solution, injecting it into a liquid chromatography - mass spectrometry instrument, and measuring and recording the mass spectrometry data of each sample; preferably, the liquid chromatography - mass spectrometry method is HPLC - Q - TOF - MS / MS.

9. The separation and identification method according to claim 8, wherein, The HPLC chromatographic conditions described in step (4) are as follows: chromatographic column: C18 column; mobile phase: 0.2% formic acid water (A) and acetonitrile (B); detection wavelength: 254 nm; flow rate: 0.8 mL·min -1 ; Column temperature: 30 °C; Injection volume: 20 μl; In step (4), the elution mode of the HPLC is gradient elution. The specific method of the gradient elution is as follows: 0–10 min, 10%–25% B; 10–30 min, 25% - 40% B; 30–31 min, 40% - 60% B; 31–40 min, 60%–80% B; 40–41 min, 80%–100% B; 41–45 min, 100% B; 45–46 min, 100% - 10% B; 46–55 min, 10% B; And, the mass spectrometry conditions described in step (4) are as follows: using SCIEX ZenoTOF TM 7600 high-resolution mass spectrometer, electrospray ionization source (ESI), positive and negative ion mode scanning; spray voltage 5.5 kV (ESI+), -5.5 kV (ESI-); primary mass spectrometry scanning detection range: m / z 60 - 1500, secondary mass spectrometry scanning range: m / z 50 - 1000, ion source temperature 600 °C; atomizing gas and auxiliary gas pressures are both 55 psi (1 psi = 6.895 kPa), curtain gas pressure 35 psi; collision energy 10 - 35 eV.

10. The separation and identification method according to claim 1, characterized in that, Step (5) includes collecting the mass spectrometry information of the compounds in Sargentodoxa cuneata, Patrinia scabiosaefolia, Polygonum cuspidatum, Salvia miltiorrhiza, Olibanum, Myrrha, Cortex Moutan, Angelica sinensis, Sparganium stoloniferum, and Rhizoma Zedoariae, and identifying the high - resolution mass spectrometry data of the chemical components of Hongteng Baijiang Decoction; through the mass spectrometry data and the corresponding fragmentation rule matching method, the substances of the peaks containing MS / MS data obtained in step (4) are identified.

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