A method for analyzing chemical components in Huazhuo Sanjie Chubi prescription

Abstract

The invention discloses an analysis method for chemical components in a Huazhuo Sanjie Chubi prescription, and an ultra-high performance liquid chromatography-quadrupole / electrostatic field orbital trap high-resolution mass spectrometer is used to determine the components of the Huazhuo Sanjie Chubi prescription; the chromatographic conditions are: a Thermo Hypersil GOLD AQ chromatographic column is used, acetonitrile is used as mobile phase A, 0.1% formic acid aqueous solution is used as mobile phase B for gradient elution, the column temperature is 40°C, the injection volume is 2μL; the mobile phase flow rate is 0.2 mL / min. The method of the invention can quickly and accurately identify and analyze the chemical components in the Huazhuo Sanjie Chubi prescription, and lays a foundation for the research on its quality evaluation, pharmacological substances and mechanism of action.

Description

Technical Field

[0001] The invention relates to the technical field of drug preparation analysis, and in particular to an analysis method for chemical components in a Huazhuo Sanjie Chubi prescription. Background Art

[0002] "Huazhu Sanjie Chubi Fang" is a traditional Chinese medicine prescription created for the pathogenesis of chronic gouty arthritis with spleen deficiency, damp-heat, phlegm, turbidity and blood stasis. It is composed of multiple Chinese medicines, including Atractylodes, Smilax glabra, Phellodendron chinense, Coix seeds, Sarcandra chinensis, Radix Dioscoreae, Herba Lysimachiae, Herba Lysimachiae, Verbena and Rhizoma Cynoglossi, which have the effects of strengthening the spleen, removing dampness and turbidity, clearing heat, removing phlegm and activating blood circulation. It can significantly reduce the blood uric acid level of patients with chronic gouty arthritis, reduce the degree of inflammation and pain, improve TCM symptoms and protect kidney function. The main ingredients in the prescription are Atractylodes lancea, which is bitter and warm in nature, and enters the spleen, stomach and liver meridians, strengthens the spleen and dries dampness. When the spleen is healthy, dampness and turbidity are removed to remove the source of dampness; Smilax glabra is sweet and mild in nature, enters the liver and stomach meridians, and is good at removing dampness and turbidity, unblocking joints, and strengthening the spleen; the two herbs are used together to strengthen the spleen, remove dampness and turbidity, and are the main medicines. Assisted by Phellodendron chinense, which is bitter and cold in nature, enters the kidney and bladder meridians, and is especially good at clearing heat and drying dampness; Coix seed is sweet, light and cool in nature, enters the spleen, stomach, and lung meridians, and is diuretic and dampness-injecting, removing numbness and resolving nodules, and strengthening the spleen; Radix Atractylodes is pungent and bitter in nature and belongs to the heart and liver meridians, and has the functions of promoting blood circulation, unblocking meridians, reducing swelling and relieving pain. Clinically, it is particularly effective for swelling and pain in muscles and bones, and nodules caused by retention of phlegm, blood stasis, and indigestion. The three medicines are collectively the minister medicines. The herb is bitter and neutral in nature, and enters the kidney and stomach meridians to remove dampness and turbidity, dispel wind and remove numbness; the herb is sweet and salty in nature and slightly cold in nature, and enters the liver, gallbladder, kidney, and bladder meridians. It is good at clearing heat and removing dampness, and leads damp-heat evil to be discharged through urine; the herb is sweet and spicy in nature and warm in nature, and enters the liver and lung meridians to resolve phlegm and soften hard masses, and dissipate nodules and reduce swelling; the herb Verbena is bitter and cool in nature, and enters the liver and spleen meridians to promote blood circulation and dissipate blood stasis, and promote diuresis and remove dampness. The above four herbs play the role of clearing heat and removing dampness, resolving phlegm and activating blood circulation, and unblocking meridians and removing turbidity, and are adjuvants. The herb is sweet, slightly bitter and neutral in nature, and enters the liver and kidney meridians to clear joints, activate blood circulation and remove blood stasis, remove dampness and promote diuresis, and remove damp-heat and blood stasis from below, and is an adjuvant. The whole prescription takes into account both the symptoms and the root cause, uses both cold and warm, and strengthens the body and eliminates evil, and plays the role of strengthening the spleen, removing dampness and turbidity, and clearing heat, removing phlegm and activating blood circulation. Clinical observation trials have shown that the "Huazhuo Sanjie Chubi Prescription" has achieved satisfactory results in the treatment of chronic gouty arthritis, and is superior to Tongfengning in improving patients' blood uric acid and TCM symptoms of chronic gout.

[0003] At present, there is little basic research on this prescription. In terms of material basis research, existing research mainly focuses on single medicinal materials, and there has not been a systematic and comprehensive study on the chemical composition of the Huazhuo Sanjie Chubi prescription. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a rapid and accurate analytical method for identifying the chemical components in the Huazhuo Sanjie Chubi prescription.

[0005] The present invention is achieved through the following technical solutions:

[0006] A method for analyzing chemical components in a Huazhuo Sanjie Chubi prescription comprises the following steps: preparing a Huazhuo Sanjie Chubi prescription test solution, and determining the components by using an ultra-high performance liquid chromatography-quadrupole / electrostatic field orbital trap high-resolution mass spectrometer;

[0007] The chromatographic conditions were as follows: using a Thermo Hypersil GOLD AQ column, acetonitrile as mobile phase A, 0.1% formic acid aqueous solution as mobile phase B for gradient elution, column temperature of 40°C, injection volume of 2 μL; mobile phase flow rate of 0.2 mL / min;

[0008] The mass spectrometry conditions were as follows: heated electrospray ion source; spray voltage was +3.2 kV in positive ion mode and -3.0 kV in negative ion mode; ion transfer tube temperature was 325 °C; sheath gas flow rate was 45 arb; auxiliary gas flow rate was 10 arb; S-Lens voltage was 60 V; auxiliary gas heating temperature was 350 °C; Full MS resolution was 70000, dd-MS 2 Resolution 17500; TopN is 8; normalized collision energy gradient is 20-80 eV; scanning range m / z 100-1500.

[0009] Preferably, the gradient elution parameters are: 0-0.1 min, the volume fraction of mobile phase A is 10%; 0.1-3.8 min, the volume fraction of mobile phase A is 10%-10.5%; 3.8-4.2 min, the volume fraction of mobile phase A is 10.5%-17%; 4.2-9.4 min, the volume fraction of mobile phase A is 17%; 9.4-9.8 min, the volume fraction of mobile phase A is 17%-20%; 9.8-11.2 min, the volume fraction of mobile phase A is 20%; 11.2-12.2 min, the volume fraction of mobile phase A is 20%-90%; 12.2-13 min, the volume fraction of mobile phase A is 90%; 13-13.4 min, the volume fraction of mobile phase A is 90%-10%; 13.4-16 min, the volume fraction of mobile phase A is 10%.

[0010] Preferably, the specifications of the chromatographic column are: column length 100 mm, inner diameter 2.1 mm, and particle size 1.9 μm.

[0011] Furthermore, the test solution of Huazhuo Sanjie Chubi recipe was injected into an ultra-high performance liquid chromatography-quadrupole / electrostatic field orbital trap high-resolution mass spectrometer, and the total ion current spectrum data in positive ion mode and negative ion mode were collected respectively. Based on the precise relative molecular weight information of the spectrum, the compound molecules whose mass deviation from the measured value of the mass-to-charge ratio of the primary quasi-molecular ion peak was within 5 ppm were calculated, and they were preliminarily speculated to be the target compounds. The chromatographic peaks of the target compounds and their secondary fragment ion information were further extracted, and their cleavage pathways were inferred based on the secondary fragments. The target compounds were identified and confirmed by combining the literature information and the chromatographic behavior and mass spectrometry information of the reference substances for comparison and verification, and finally the chemical components in Huazhuo Sanjie Chubi recipe were analyzed and determined.

[0012] Furthermore, the method for extracting the secondary fragment ion information is to perform high-energy collision dissociation on the precursor ion peak of the target compound in HCD mode to obtain its fragment ion spectrum.

[0013] Furthermore, after the preliminary speculation of the target compounds, the target compounds were matched and screened by using Thermo TraceFinder and CompoundDiscoverer software: the spectra were imported into the Thermo TraceFinder and CompoundDiscoverer databases, the threshold parameters of the primary mass deviation were set to 5ppm, the threshold parameters of the secondary mass deviation were set to 10ppm, the threshold parameters of the isotope matching fit were set to 70%, and the retention time window was set to 60s. Matching was performed in the above databases respectively, and then high-precision screening was performed based on the retention time, chemical structure, precise molecular mass and fragment ion information of the compounds.

[0014] The invention provides a method for preparing the test solution, comprising the following steps: weighing the raw material medicine of Huazhuo Sanjie Chubi prescription according to the prescription amount, adding purified water, heating and refluxing extraction, filtering, concentrating the filtrate, centrifuging, taking the supernatant, filtering through a microporous filter membrane, adding a mixed solution of 0.1% formic acid water and acetonitrile in a volume ratio of 9:1 to dilute, and obtaining the Huazhuo Sanjie Chubi prescription test solution.

[0015] Preferably, the reflux extraction step is repeated 2-4 times, each time for 1.5-3 hours. After the extraction is completed, the filtrate is filtered while hot and the filtrate is combined.

[0016] Preferably, the pore size of the microporous filter membrane is ≤0.22 μm.

[0017] The invention provides a method for preparing the reference substance, comprising the following steps: accurately weighing the reference substance and placing it in a solution preparation container, dissolving and diluting the reference substance mother solution with methanol, and then diluting the mother solution with 0.1% formic acid water and acetonitrile in a volume ratio of 9:1 to obtain a reference substance solution with a required concentration.

[0018] Preferably, the concentration of the reference substance mother solution is 100 μg / mL; the concentration of the reference substance solution is 500 ng / mL

[0019] The document is a prior art document that records the chromatographic behavior and mass spectrum information of the target compound.

[0020] The prescription for removing turbidity, dispersing knots and removing arthralgia of the present invention is a traditional Chinese medicine prescription composed of atractylodes, smilax glabra, phellodendron, coix seeds, scutellaria baicalensis, radix polygoni multiflori, herba schizonepetae, herba catnip, verbena and cyathulae bidentatae.

[0021] The chemical components of the Huazhuo Sanjie Chubi prescription include 51 flavonoid compounds, 35 phenylpropanoid compounds, 26 organic acid compounds, 20 terpenoid compounds, 17 alkaloid compounds, 16 phenolic compounds, 6 steroid compounds and 13 other compounds.

[0022] The present invention has the following beneficial effects:

[0023] The present invention uses ultra-high performance liquid chromatography-quadrupole / electrostatic field orbital trap high-resolution mass spectrometry (UPLC-Q-Orbitrap HRMS) to analyze the components of the Huazhuo Sanjie Chubi recipe, and establishes a high-resolution mass spectrometry analysis method for quickly identifying the chemical components of the Huazhuo Sanjie Chubi recipe. Through the method of the present invention, 184 chemical components were finally identified from the Huazhuo Sanjie Chubi recipe, including 51 flavonoids, 35 phenylpropanoids, 26 organic acids, 20 terpenes, 17 alkaloids, 16 phenols, 6 steroids and 13 other components, of which 64 compounds were confirmed by comparison with reference substances. The method can quickly and accurately identify and analyze the chemical components in the Huazhuo Sanjie Chubi recipe, laying a foundation for its quality evaluation, pharmacological substances and mechanism of action. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the total ion current spectrum of the test solution of Huazhuo Sanjie Chubi Recipe in positive ion mode and negative ion mode;

[0025] Figure 2 This is the mass spectrometry fragmentation pathway analysis diagram of luteolin;

[0026] Figure 3 This is a mass spectrometry fragmentation pathway analysis diagram of verbascoside;

[0027] Figure 4 This is a mass spectrometry fragmentation pathway analysis diagram of dextrorotatory quinic acid;

[0028] Figure 5 This is a diagram for the mass spectrometry fragmentation pathway analysis of geniposide;

[0029] Figure 6This is the mass spectrometry fragmentation pathway analysis diagram of berberine;

[0030] Figure 7 This is the mass spectrometry fragmentation pathway analysis diagram of paeonol;

[0031] Figure 8 This is the mass spectrometry fragmentation pathway of pseudo-protodioscin. DETAILED DESCRIPTION

[0032] In order to elaborate on the technical content, purpose and effect of the present invention, the technical solution of the present invention is clearly and completely described in combination with the implementation mode and the accompanying drawings below, but the described embodiments are only part of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] The instruments and reagents used in the embodiments of the present invention are described as follows:

[0034] 1. Instrument

[0035] Vanquish Flex UHPLC ultra-high performance liquid chromatography system (Thermo Fisher Scientific, USA); Q-Exactive Plus Obitrap-MS quadrupole / electrostatic field orbital trap high-resolution mass spectrometer (Thermo Fisher Scientific, USA); MILLI-Q Direct16 ultrapure water preparation instrument (Millipore, USA); CPA225D 1 / 100,000 electronic balance (Sartorius, USA); DZTW constant temperature electric heating mantle (Shanghai Lichen Instrument Technology Co., Ltd.); Heraeus Fresco17 centrifuge (Thermo Fisher Scientific, USA).

[0036] 2. Drug testing

[0037] Reagents: Acetonitrile, methanol and pure water were of mass spectrometry grade and purchased from Merck, Germany; formic acid was of HPLC grade and purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; the remaining reagents were of analytical grade.

[0038] Reference substances: citric acid (batch number: 111679-201602), chlorogenic acid (batch number: 110753-202119), phellodendron hydrochloride (batch number: 111895-201805), baicalin (batch number: 111084-202010), verbascoside (batch number: 11530-201914), jatrorrhizine hydrochloride (batch number: 110733-202110), resveratrol (batch number: 111535-201703), palmatine hydrochloride (batch number: 110732-201913), luteolin (batch number: 111520-202107), diosgenin (batch number: 111707-201703), and atractylodes lactone II (batch number: 111976-201501) were purchased from the China Food and Drug Administration; geniposide (batch number: J16HB188675), neochlorogenic acid (batch number: J16HB188675), and No.: D23GB172337), protocatechuic acid (batch number: O23HB197783), catechin (batch number: S01HB191501), cryptochlorogenic acid (batch number: J01GB147635), protocatechuic aldehyde (batch number: J09HB184599), quercetin (batch number: Y13N11Q130724), 5-hydroxyverbengin (batch number: C19A10G86190), caffeic acid (batch number: No.: M28HB183194), vanillic acid (batch number: H11J9Z65318), syringic acid (batch number: S18J10K93270), verbena glycoside (batch number: Z30A10S587300), magnolamine (batch number: A31HB193420), vanillin (batch number: J24A6R2696), schaftoside (batch number: P07J12F136614), isoschaftoside (batch number:

[0039] P13D11S134210), vitexin rhamnoside (batch number: Y27A9H60174), ferulic acid (batch number: G13S11L124423), vitexin (batch number: M27IB210737), rutin (batch number: D13HB202526), ​​calycosin isoflavone glycosides (batch number: J19HB174062), taxifolin (batch number: C15J11Q107526), ​​isoflavone (batch number: M27GB141526), ​​quinacridone (batch number: M27GB141526), ​​apiosyl liquiritifolia (batch number: J14HB188614), isoquercitrin (Batch number: X29O11Y128970), luteolin (Batch number: A22GB141264), astilbin (Batch number: D26GB172582), kaempferol (Batch number: Y11N11H130735), forsythiaside A (Batch number: A15GB145520), demethyleneberberine (Batch number: M19GB148924), astragaloside (Batch number: F18HB175834), quercetin (Batch number: M23IB210619), wild sumac glycoside (Batch number: N12GB167810), rosmarinic acid (Batch number: Y06A9K67402), cup amaranth Sterol (batch number: A27GB158923), africanine (batch number: Y23D11W128338), berberine (batch number: J08HB187720), formononetin (batch number: O18GB164369), dihydroflavonoids (batch number: J27HB189795), berberine hydrochloride (batch number: S01A10K94340), eriodictyol (batch number: J15HB188611), baicalein (batch number: Z21J9X66217), quercetin (batch number: O29HB199514), irisin (batch number: Y17N9L75483), kaempferol Phenol (batch number: A01HB190000), isoetzol (batch number: G29A11L122927), limonin (batch number: H23J9K65962), hydroxygenkanthin (batch number: R16D7F26926), melaleucatin A (batch number: F22HB175829), atractylodesin (batch number: O12GB163570), and trigonelline hydrochloride (batch number: Z19A11H121866) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; mulberry yellow (batch number: I2223417) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; all the above reference substances were HPLC grade.

[0040] Medicinal materials: Atractylodis Rhizoma (AR), Smilacis Glabrae (SG), Phellodendri Chinensis (PC), Coix Semen (CS), Sarcandrae Herba (SH), Dioscorea Septemloba (DS), Lysimachia Christiniae (LC), Ranunculi Ternati (RT), Verbena Herba (VH), Cyathulae Radix, CR) were purchased from Zhicheng Pharmacy, Shangjie, Minhou County, Fuzhou, with batch numbers 221018003, 221014, 221004, 22112601, 21062307, 2202018119, 2209019005, 22031208, 221001, and 211201. The above ten medicinal materials were identified by Professor Huang Zehao of the School of Pharmacy of Fujian University of Chinese Medicine and all met the standards of the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0041] Embodiment 1 A method for analyzing the chemical components in the Huazhuo Sanjie Chubi prescription, the specific steps are as follows:

[0042] Preparation of the test solution of Huazhuo Sanjie Chubi prescription: weigh 10 raw medicinal materials of Huazhuo Sanjie Chubi prescription according to the prescription amount (weight ratio of Atractylodes: Smilax glabra: Phellodendron chinense: Coix seed: Sarcandra chinensis: Radix Dioscoreae: Lysimachia chinensis: Cat's claw: Verbena officinalis: Cyathula officinalis = 1:3:1:3:3:2:3:3:2, a total of 115 g / prescription), put in a round-bottom flask, add 12 times the amount of water, shake well, and soak for 30 min. Heat under reflux in an electric heating mantle for extraction 3 times, each time for 2 hours, filter while hot, combine the filtrate, let it stand and cool, then concentrate the extract to 1L, accurately pipette an appropriate amount of the extract, centrifuge at 10000rpm / min for 10min, take the supernatant, filter through a 0.22μm microporous filter membrane, and dilute 5 times with 0.1% formic acid water-acetonitrile (v / v, 9:1) solution to obtain the test solution of Huazhuo Sanjie Chubi recipe.

[0043] Prepare reference substance solution: accurately weigh each reference substance, place it in a volumetric flask, dissolve it with methanol and make up to volume to obtain a reference substance mother solution of about 100 μg / mL; accurately aspirate the reference substance mother solution and dilute it with 0.1% formic acid water-acetonitrile (v / v, 9:1) solution to a reference substance solution of 500 ng / mL.

[0044] Set the measurement conditions:

[0045] (1) Chromatographic conditions

[0046] Chromatographic column: Thermo Hypersil GOLD AQ column, specification: 2.1×100 mm, 1.9 μm; mobile phase: acetonitrile (phase A)-0.1% formic acid aqueous solution (phase B); flow rate: 0.2 mL / min; column temperature: 40°C; injection volume: 2 μL; gradient elution parameters: 0-0.1 min, the volume fraction of mobile phase A is 10%; 0.1-3.8 min, the volume fraction of mobile phase A is 10%-10.5%; 3.8-4.2 min, the volume fraction of mobile phase A is 10.5%-17%; 4.2-9.4 min, mobile phase A is 10%-20%; The volume fraction of phase A is 17%; from 9.4 to 9.8 min, the volume fraction of mobile phase A is 17% to 20%; from 9.8 to 11.2 min, the volume fraction of mobile phase A is 20%; from 11.2 to 12.2 min, the volume fraction of mobile phase A is 20% to 90%; from 12.2 to 13 min, the volume fraction of mobile phase A is 90%; from 13 to 13.4 min, the volume fraction of mobile phase A is 90% to 10%; from 13.4 to 16 min, the volume fraction of mobile phase A is 10%.

[0047] (2) Mass spectrometry conditions

[0048] A heated electrospray ion source (HESI) was used with positive and negative ion detection modes; the spray voltage was +3.2 kV in positive ion mode and -3.0 kV in negative ion mode; the ion transfer tube temperature was 325 °C; the sheath gas flow rate was 45 arb; the auxiliary gas flow rate was 10 arb; the S-Lens voltage was 60 V; the auxiliary gas heating temperature was 350 °C; the scanning mode was Full MS / dd-MS 2 , Full MS resolution 70000, dd-MS 2 Resolution: 17500; Loop count (TopN): 8; Normalized collision energy gradient: 20-80 eV; Scanning range: m / z 100-1500.

[0049] Analysis and identification of ingredients in Huazhuo Sanjie Chubi prescription:

[0050] The sample solution of Huazhuo Sanjie Chubi Recipe was injected into the ultra-high performance liquid chromatography-quadrupole / electrostatic field orbital trap high-resolution mass spectrometry system, and the total ion current spectra in the positive ion mode and negative ion mode were collected respectively (see Figure 1), the collected data were processed using Xcalibur 4.0 workstation, and the possible molecular formulas of each compound within the mass deviation range of 5 ppm were calculated based on the precise molecular mass information of the compounds obtained on the total ion current chromatographic peak. Then, the target precursor ion peak was selected for high-energy collision dissociation (HCD mode) to obtain its fragment ions, and then the spectrum was imported into Thermo TraceFinder V 5.0 and Compound Discoverer V 3.3 database, set the threshold parameter of the primary mass deviation to 5ppm, the threshold parameter of the secondary mass deviation to 10ppm, the threshold parameter of the isotope matching fitting degree to 70%, and the retention time window to 60s, and matched them in the above databases respectively, and then carried out high-precision screening according to the retention time, chemical structure, accurate molecular mass and fragment ion information of the compound, and combined with references, compared and verified by comparing the chromatographic behavior and mass spectrometry information with the reference substances, and finally identified 184 components from the Huazhuo Sanjie Chubi prescription, including 51 flavonoids, 35 phenylpropanoids, 26 organic acids, 20 terpenes, 17 alkaloids, 16 phenols, 6 steroids and 13 other components, of which 64 compounds were identified by the reference substances. The results are shown in Table 1.

[0051] Table 1 Mass spectrometry information of identified components in Huazhuo Sanjie Chubi prescription

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064] Note: “*” means verified by reference substance.

[0065] Mass spectrometry fragmentation pathway analysis of the main components in Huazhuo Sanjie Chubi prescription:

[0066] (1) Flavonoids

[0067] The identified flavonoid compounds include C-glycoside flavonoids, O-glycoside flavonoids and flavonoid aglycones, and their characteristic fragments are often obtained by cleavage of the sugar group or rearrangement and cleavage of the chromone nucleus. Taking compound 116 as an example, in the positive ion mode, its quasi-molecular ion peak is 449.1081, and its precise molecular formula calculated by the Xcalibur workstation is C21H20O11. It is preliminarily speculated that the component is luteolin and its isomers. After HCD fragmentation, four stable fragment ions, m / z 287, 269, 241, and 153, were detected. m / z 287 was formed by the breakage of a glucose molecule from the precursor ion, which was further dehydrated to obtain m / z 269. Fragment m / z 241 was formed by the loss of a carbon monoxide molecule from m / z 269. When the parent B-ring was completely cleaved, fragment m / z 153 was obtained. Finally, after verification by reference substances and combined with relevant literature [Liang Yuting, Wang Jingyu, Su Weiwei, et al. Chemical composition analysis of Zhuang medicine Zhan Gu based on UFLC-Triple TOF-MS / MS [J]. Central South Pharmacy, 2018, 16(10): 1369-1373.], compound 116 was determined to be luteolin, and its fragmentation pathway is shown in Figure 2 .

[0068] (2) Phenylpropanoid compounds

[0069] The identified phenylpropanoid compounds include phenylpropanoids, coumarin, phenylethanoid glycosides, etc., and their fragments are usually formed by the fragmentation of the polymer nucleus or the removal of the sugar group. Taking compound 120 as an example, in the negative ion mode, its primary ion peak is 623.1991, and its accurate molecular formula is calculated to be C29H36O15. It is preliminarily speculated that the component is verbascoside and its isomers. After collision dissociation, the ion peak can stably scan three high-abundance secondary ions of m / z 461, 161, and 133, among which m / z 461 is formed by the precursor ion removing a molecule of rhamnose and an oxygen atom, and the fragments m / z 161 and 133 are both caused by the breakage of a molecule of glucose from m / z 461, and then the loss of C8H10O3 and C8H6O5 groups respectively. After verification with reference substances and combined with relevant literature [Nan Haipeng, Liu Jinlian, Chen Yawen, et al. Analysis of chemical components of Qingli Shire granules based on ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry [J / OL]. World Traditional Chinese Medicine: 1-8.], compound 120 was determined to be verbascoside, and its cleavage pathway is shown in Figure 3 .

[0070] (4) Organic acid compounds

[0071] Organic acid compounds often remove neutral molecules such as CO2, CO and H2O to form characteristic fragments. Taking compound 2 as an example, in the negative ion mode, its quasi-molecular ion peak is 191.0553, and its exact molecular formula is calculated to be C7H12O6. It is preliminarily speculated that the component is dextrorotatory quinic acid and its isomers. After the fragmentation of the ion peak, three fragment ions m / z 129, 111, and 87 can be stably formed. Among them, m / z 129 is generated by the precursor ion removing a molecule of carbon dioxide and water in turn, and the fragments m / z 111 and 87 are caused by the precursor ion losing 5 oxygen atoms and C3H4O4 groups, respectively. By comparing with relevant literature [Sun Yan, Feng Feng, Huang Tehui, etc. Rapid analysis of the effective components of seabuckthorn based on UPLC-Q-Exactive technology combined with OTCML database [J]. Natural Product Research and Development, 2019, 31(07): 1192-1202.], compound 2 was determined to be dextrorotatory quinic acid, and its cleavage pathway is shown in Figure 4 .

[0072] (5) Terpenoids

[0073] The identified terpenoid compounds include iridoid glycosides, triterpenes, etc., and their fragments are usually generated by the loss of sugar groups or the fragmentation of the parent nucleus. Taking compound 9 as an example, in the negative ion mode, its quasi-molecular ion peak is 373.1142, and its accurate molecular formula is calculated to be C16H22O10. It is preliminarily speculated that the component is geniposide acid and its isomers. This ion peak can stably generate four fragment ions of m / z 211, 167, 149, and 123 after collision dissociation. Among them, m / z 211 is obtained by removing a molecule of glucose from the precursor ion, and after further removing a molecule of CO2 and H2O, fragments m / z 167 and 149 are obtained in turn, while m / z 123 is due to the fragmentation of the parent nucleus structure and the further loss of the C2H2 group from m / z 149. After verification with reference substances and combined with relevant literature [Nan Haipeng, Liu Jinlian, Chen Yawen, et al. Analysis of chemical components of Qingli Shire granules based on ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry [J / OL]. World Traditional Chinese Medicine: 1-8.], compound 9 was finally determined to be geniposide acid, and its cleavage pathway is shown in Figure 5 .

[0074] (6) Alkaloid compounds

[0075] Alkaloid compounds are mostly alkaline, and because of the positive charge center in their structure, they can usually stably form positive ion peaks. Taking compound 149 as an example, in the positive ion mode, its molecular ion peak is 322.1075, and its precise molecular formula is calculated to be C19H15NO4. It is preliminarily speculated that the component is berberine and its isomers. The ion peak dissociates through high-energy collision to generate four fragment ions of m / z307, 279, 264, and 250. Among them, m / z 307 is formed by the precursor ion removing a methyl molecule, and it further removes a molecule of carbon monoxide to obtain m / z 279, while the fragment m / z 264 is caused by the loss of two molecules of aldehyde groups from the precursor ion, and m / z 250 is generated by the precursor ion removing a molecule of CO2 and CO in turn. After verification with reference substances and combined with relevant literature [Wang Yongli, Huang Guangjian, Liu Congjin, et al. Analysis of chemical components of Huanglian Jiedu Decoction by UHPLC-Q-Exactive Orbitrap HRMS and their tissue distribution in rats [J]. Chinese Herbal Medicine, 2022, 53(22): 6985-7000.], compound 149 was finally determined to be berberine, and its cleavage pathway is shown in Figure 6 .

[0076] (7) Phenolic compounds

[0077] Phenolic compounds can usually generate fragments by fragmentation of the parent nucleus or removal of functional groups such as phenolic hydroxyl groups. Taking compound 11 as an example, in the positive ion mode, its quasi-molecular ion peak is 167.0704, and its exact molecular formula is calculated to be C9H10O3. It is preliminarily speculated that the component is paeonol and its isomers. After the ion peak is fragmented, four fragment ions of m / z 134, 123, 106, and 78 are generated. Among them, m / z 134 is generated by the precursor ion after the methoxyl group and hydrogen are removed, and it further loses a molecule of carbon dioxide and methylene to obtain m / z 78, while the fragment m / z 123 is caused by the precursor ion losing the C2H4O group, and it continues to remove the hydroxyl group to generate m / z 106. By comparing with relevant literature [Xu Dongchuan, Sui Zaiyun, Yang Qing, et al. Analysis of chemical components of Xiebaisan by high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry [J]. Chinese Journal of Traditional Chinese Medicine, 2022, 40(09): 251-258+287.], compound 11 was determined to be paeonol, and its cleavage pathway is shown in Figure 7 .

[0078] (8) Steroid compounds

[0079] The identified steroidal compounds include sterones and saponins. Due to their complex cyclic core structure, they are difficult to fragment, and their fragments are usually formed by the breakage of side chain groups. Taking compound 146 as an example, in the positive ion mode, its quasi-molecular ion peak is 1031.5418, and its accurate molecular formula is calculated to be C51H82O21. It is preliminarily speculated that this component is pseudo-protodioscin and its isomers. After the ion peak is fragmented, five fragment ions of m / z 870, 725, 577, 415, and 253 are stably formed. Among them, m / z 870 is caused by the loss of a molecule of rhamnose and an oxygen atom from the precursor ion, and m / z 725 is produced by the loss of two molecules of rhamnose and a methylene from the precursor ion; while m / z 577 and 415 are formed by the sequential removal of several rhamnose and glucose from the precursor ion, and m / z 253 is caused by the loss of a molecule of C8H18O3 group from the fragment ion m / z 415. By comparing with relevant literature [Wei Rui, Yang Linjiao, Qin Xuemei, et al. Rapid identification of chemical components in asparagus stem peel based on UPLC-Q-TOF-MS / MS and molecular network technology [J]. Acta Pharmaceutica Sinica, 2022, 57(09): 2839-2850.], compound 146 was determined to be pseudo-protodioscin, and its cleavage pathway is shown in Figure 8 .

[0080] The present invention adopts UPLC-Q-Orbitrap HRMS technology to realize the rapid identification of chemical components in Huazhuo Sanjie Chubi prescription, and uses acetonitrile-0.1% formic acid aqueous solution to elute the system, obtains obvious characteristic peaks and good separation effect; adopts positive and negative ion modes to inject and scan respectively, and finds that the base peak ion flow charts in the two modes have good responses. By comparing the retention time, accurate quasi-molecular ion peak and secondary fragments of the compounds with reference literature reports and reference substances, and inferring the mass spectrometry fragmentation rules of the secondary fragments of the main types of chemical components, 184 compounds are preliminarily identified and inferred, including flavonoids, phenylpropanoids, organic acids, terpenes, alkaloids, phenols, steroids, etc., which can obtain the mass spectrometry information of the components in Huazhuo Sanjie Chubi prescription to the greatest extent possible, and more comprehensively reveal the chemical substance basis of Huazhuo Sanjie Chubi prescription.

[0081] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention's specification and drawings, or directly or indirectly applied in related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for analyzing the chemical components in a prescription for removing turbidity, dispersing stagnation and eliminating arthralgia, characterized in that: The following steps are involved: The test solution of Huazhuo Sanjie Chubi Recipe was prepared, and the components were determined by ultra-high performance liquid chromatography-quadrupole / electrostatic field orbital trap high-resolution mass spectrometry; The chromatographic conditions were as follows: using a Thermo Hypersil GOLD AQ column, acetonitrile as mobile phase A, 0.1% formic acid aqueous solution as mobile phase B for gradient elution, column temperature of 40°C, injection volume of 2 μL; mobile phase flow rate of 0.2 mL / min; The gradient elution parameters were as follows: 0-0.1 min, the volume fraction of mobile phase A was 10%; 0.1-3.8 min, the volume fraction of mobile phase A was 10%-10.5%; 3.8-4.2 min, the volume fraction of mobile phase A was 10.5%-17%; 4.2-9.4 min, the volume fraction of mobile phase A was 17%; 9.4-9.8 min, the volume fraction of mobile phase A was 17%-20%; 9.8-11.2 min, the volume fraction of mobile phase A was 20%; 11.2-12.2 min, the volume fraction of mobile phase A is 20%-90%; 12.2-13 min, the volume fraction of mobile phase A is 90%; 13-13.4 min, the volume fraction of mobile phase A is 90%-10%; 13.4-16 min, the volume fraction of mobile phase A is 10%; The specifications of the chromatographic column are: column length 100 mm, inner diameter 2.1 mm, particle size 1.9 μm; The mass spectrometry conditions were as follows: heated electrospray ion source; spray voltage was +3.2 kV in positive ion mode and -3.0 kV in negative ion mode; ion transfer tube temperature was 325 °C; sheath gas flow rate was 45 arb; auxiliary gas flow rate was 10 arb; S-Lens voltage was 60 V; Auxiliary gas heating temperature 350℃; Full MS resolution 70000, dd-MS 2 Resolution: 17500; TopN: 8; Normalized collision energy gradient 20~80 eV; scanning range m / z 100~1500; The preparation method of the test solution comprises the following steps: weighing the raw material of Huazhuo Sanjie Chubi prescription according to the prescription amount, adding purified water, heating reflux extraction, filtering, concentrating the filtrate, centrifuging, taking the supernatant, filtering through a microporous filter membrane, adding a mixed solution of 0.1% formic acid water and acetonitrile in a volume ratio of 9:1 to dilute, and obtain the test solution of Huazhuo Sanjie Chubi prescription.

2. The method for analyzing the chemical components in the Huazhuo Sanjie Chubi prescription according to claim 1, characterized in that: The test solution of Huazhuo Sanjie Chubi recipe was injected into an ultra-high performance liquid chromatography-quadrupole / electrostatic field orbital trap high-resolution mass spectrometer, and the total ion current spectrum data in positive and negative ion modes were collected respectively. According to the precise relative molecular weight information of the spectrum, the compound molecules whose mass deviation from the measured value of the mass-to-charge ratio of the primary quasi-molecular ion peak was within 5 ppm were calculated, and they were preliminarily inferred to be the target compounds. The chromatographic peaks of the target compounds and their secondary fragment ion information were further extracted, and their fragmentation pathways were inferred based on the secondary fragments. Combined with the literature information and the chromatographic behavior and mass spectrometry information of the reference substance, the target compounds were identified and confirmed, and finally the chemical components in Huazhuo Sanjie Chubi recipe were analyzed and determined.

3. The method for analyzing the chemical components in the Huazhuo Sanjie Chubi prescription according to claim 2 is characterized in that: The method for extracting the secondary fragment ion information is to perform high-energy collision dissociation on the precursor ion peak of the target compound in HCD mode to obtain its fragment ion spectrum.

4. The method for analyzing the chemical components in the Huazhuo Sanjie Chubi prescription according to claim 2, characterized in that: After preliminary speculation of the target compounds, the target compounds were matched and screened by using Thermo TraceFinder and Compound Discoverer software: the spectra were imported into the Thermo TraceFinder and Compound Discoverer databases, the threshold parameters of the primary mass deviation were set to 5 ppm, the threshold parameters of the secondary mass deviation were set to 10 ppm, the threshold parameters of the isotope matching fit were set to 70%, and the retention time window was set to 60 s. Matching was performed in the above databases respectively, and then high-precision screening was performed based on the retention time, chemical structure, accurate molecular mass and fragment ion information of the compounds.

5. The method for analyzing the chemical components in the Huazhuo Sanjie Chubi prescription according to claim 2, characterized in that: The preparation method of the reference substance comprises the following steps: accurately weighing the reference substance and placing it in a solution preparation container, dissolving and diluting it with methanol to obtain a reference substance mother solution, and then diluting it with a mixed solution of 0.1% formic acid water and acetonitrile in a volume ratio of 9:1 to obtain a reference substance solution of required concentration.

6. The method for analyzing the chemical components in the Huazhuo Sanjie Chubi prescription according to claim 1, characterized in that: The chemical components of the Huazhuo Sanjie Chubi prescription include 51 flavonoid compounds, 35 phenylpropanoid compounds, 26 organic acid compounds, 20 terpenoid compounds, 17 alkaloid compounds, 16 phenolic compounds, 6 steroid compounds and 13 other compounds.

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