CN118566390A - Analysis method for chemical components in prescription for eliminating turbid pathogen, resolving masses and removing arthralgia - Google Patents

Abstract

The invention discloses an analytical 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

An analytical method for chemical components in Huazhuo Sanjie Chubi prescription

Technical Field

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

"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 has the function of 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 particularly 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 enters 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, and can 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 can lead 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, and can resolve phlegm, soften hard masses, and relieve swelling; the herb Verbena is bitter and cool in nature, and enters the liver and spleen meridians, and can promote blood circulation and dissipate blood stasis, and can promote diuresis and remove dampness. The above four herbs can play the role of clearing heat and removing dampness, resolving phlegm and activating blood circulation, and dredge meridians and remove turbidity, and they are all adjuvants. The herb is sweet, slightly bitter and neutral in nature, and enters the liver and kidney meridians, and can clear joints, activate blood circulation and remove blood stasis, remove dampness and promote diuresis, and remove damp-heat and blood stasis from below, and it 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 can play the role of strengthening the spleen, removing dampness and turbidity, and clearing heat, removing phlegm and activating blood circulation. Clinical observation and 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 traditional Chinese medicine symptoms of chronic gout.

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 been no systematic and comprehensive research analyzing the chemical composition of the Huazhuo Sanjie Chubi prescription.

Summary of the invention

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.

The present invention is achieved through the following technical solutions:

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;

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 mass spectrometry conditions were as follows: a heated electrospray ion source; 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 Full MS resolution was 70000, the dd-MS ² resolution was 17500; the TopN was 8; the normalized collision energy gradient was 20 to 80 eV; and the scanning range was m/z 100 to 1500.

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%.

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

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.

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.

Furthermore, after preliminary speculation on the target compounds, the target compounds were matched and screened using the 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.

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 test solution of Huazhuo Sanjie Chubi prescription.

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.

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

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.

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

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

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.

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.

The present invention has the following beneficial effects:

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

FIG1 is a total ion current spectrum of the Huazhuo Sanjie Chubi prescription test solution in positive ion mode and negative ion mode;

FIG2 is a diagram showing the mass spectrometry fragmentation pathway analysis of luteolin;

FIG3 is a diagram showing the mass spectrometry fragmentation pathway analysis of verbascoside;

Fig. 4 is a mass spectrometry fragmentation pathway analysis diagram of dextrorotatory quinic acid;

FIG5 is a diagram showing the mass spectrometry fragmentation pathway analysis of geniposide;

FIG6 is a diagram showing the mass spectrometry fragmentation pathway analysis of berberine;

FIG7 is a diagram showing the mass spectrometry fragmentation pathway analysis of paeonol;

FIG8 shows the mass spectrometry fragmentation pathway of pseudo-protodioscin.

DETAILED DESCRIPTION

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.

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

1. Instrument

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).

2. Drug testing

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.

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:

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 liquiritifolioside (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.

Medicinal materials: Atractylodis Rhizoma (AR), Smilacis Glabrae (SG), Phellodendri Chinensis (PC), Coicis 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.

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

Preparation of 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 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.

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.

Set the measurement conditions:

(1) Chromatographic conditions

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%.

(2) Mass spectrometry conditions

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 was 70000, dd-MS ² resolution was 17500; Loop count (TopN): 8; the normalized collision energy gradient was 20-80 eV; and the scanning range was m/z 100-1500.

Analysis and identification of ingredients in Huazhuo Sanjie Chubi prescription:

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 the negative ion mode were collected respectively (see Figure 1). The collected data were processed by Xcalibur 4.0 workstation, and the possible molecular formulas of each compound within the mass deviation range of 5 ppm were calculated according to the accurate 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 TraceFinderV 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.

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

Note: “*” means verified by reference substance.

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

(1) Flavonoids

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 scanned. Among them, m/z 287 was caused 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 cleavage pathway is shown in Figure 2.

(2) Phenylpropanoid compounds

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-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.

(4) Organic acid compounds

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.

(5) Terpenoids

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-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.

(6) Alkaloid compounds

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 by reference substances and combined with relevant literature [Wang Yongli, Huang Guangjian, Liu Congjin, et al. UHPLC-Q-Exactive Orbitrap HRMS analysis of the chemical composition and tissue distribution of Huanglian Jiedu Decoction 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.

(7) Phenolic compounds

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 HPLC-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.

(8) Steroid compounds

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.

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.

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 (10)

1. An analysis method of chemical components in a prescription for resolving turbidity, resolving masses and removing arthralgia is characterized by comprising the following steps: preparing a sample solution for resolving turbidity, resolving masses and removing arthralgia, and measuring components by adopting an ultra-high performance liquid chromatography-quadrupole/electrostatic field orbit trap high-resolution mass spectrometer;

The chromatographic conditions are as follows: gradient elution is carried out by adopting Thermo Hypersil GOLD AQ chromatographic columns, acetonitrile is taken as a mobile phase A phase, 0.1% formic acid aqueous solution is taken as a mobile phase B phase, the column temperature is 40 ℃, and the sample injection amount is 2 mu L; the flow rate of the mobile phase is 0.2 mL/min;

The mass spectrum conditions are as follows: heating an electrospray ion source; the spraying voltage is positive ion mode +3.2kV and negative ion mode-3.0 kV; the temperature of the ion transmission tube is 325 ℃; sheath gas flow rate 45arb; an auxiliary air flow rate of 10arb; S-Lens voltage is 60V; the heating temperature of the auxiliary gas is 350 ℃; full MS resolution 70000, dd-MS ² resolution 17500; topN is 8; normalizing the collision energy gradient to 20-80 eV; the scanning range m/z is 100-1500.

2. The method for analyzing chemical components in a prescription for resolving turbidity, resolving masses and removing arthralgia according to claim 1, wherein gradient elution parameters are as follows: 0-0.1 min, wherein the volume fraction of the mobile phase A is 10%; 0.1-3.8 min, wherein the volume fraction of the mobile phase A is 10% -10.5%; 3.8-4.2 min, wherein the volume fraction of the mobile phase A is 10.5% -17%; 4.2-9.4 min, wherein the volume fraction of the mobile phase A is 17%; 9.4-9.8 min, wherein the volume fraction of the mobile phase A is 17% -20%; 9.8-11.2 min, wherein the volume fraction of the mobile phase A is 20%; 11.2-12.2 min, wherein the volume fraction of the mobile phase A is 20% -90%; 12.2-13 min, wherein the volume fraction of the mobile phase A is 90%; 13-13.4 min, wherein the volume fraction of the mobile phase A is 90% -10%; 13.4-16 min, and the volume fraction of the mobile phase A is 10%.

3. The method for analyzing chemical components in a prescription for resolving turbidity, resolving masses and removing arthralgia according to claim 1, which is characterized in that the specification of a chromatographic column is as follows: the column length was 100mm, the inner diameter was 2.1mm, and the particle size was 1.9. Mu.m.

4. The method for analyzing chemical components in a prescription for resolving turbidity, resolving masses and removing arthralgia according to claim 1, which is characterized in that the preparation method of the sample solution comprises the following steps: weighing the bulk drugs of the prescription according to the prescription, adding purified water, heating and refluxing for extraction, filtering, concentrating filtrate, centrifuging, taking supernatant, filtering by a microporous filter membrane, adding 0.1% of formic acid water and acetonitrile to dilute by a volume ratio of 9:1, and obtaining the prescription sample solution for removing the obstruction.

5. The analysis method of chemical components in a turbidity-resolving, stagnation-resolving and arthralgia-removing formula according to claim 1, which is characterized in that a sample solution of the turbidity-resolving, stagnation-resolving and arthralgia-removing formula is injected into an ultra-high performance liquid chromatography-quadrupole/electrostatic field orbit trap high resolution mass spectrometer, total ion flow chart data in a positive ion mode and a negative ion mode are respectively collected, compound molecules with mass deviation within a range of 5 ppm from a first-level excimer ion peak-to-mass charge ratio actual measurement value are calculated according to accurate relative molecular weight information of the spectrograms, and the compound molecules are primarily presumed to be a target compound; extracting chromatographic peaks of the target compound and secondary fragment ion information thereof, deducing a cracking path according to the secondary fragment, combining literature information and comparing and verifying chromatographic behavior and mass spectrum information of a reference substance, identifying and confirming the target compound, and finally analyzing and determining chemical components in the prescription for resolving turbidity, resolving hard mass and removing arthralgia.

6. The method for analyzing chemical components in a prescription for resolving turbidity, resolving hard mass and removing arthralgia according to claim 5, wherein the extraction method of the secondary fragment ion information is to perform high-energy collision dissociation on a precursor ion peak of a target compound in an HCD mode to obtain a fragment ion spectrogram of the target compound.

7. The method for analyzing chemical components in a prescription for resolving turbidity, resolving masses and removing arthralgia according to claim 5, which is characterized in that after the target compound is primarily presumed, the target compound is screened by matching Thermo TraceFinder with Compound Discoverer software: the spectrograms are imported Thermo TraceFinder and Compound Discoverer into a database, the threshold value parameter of the primary mass deviation is set to be 5 ppm, the threshold value parameter of the secondary mass deviation is set to be 10 ppm, the threshold value parameter of the isotope matching fitting degree is set to be 70%, the retention time window is set to be 60 s, the spectrograms are respectively matched in the database, and then high-precision screening is carried out according to the retention time, the chemical structure, the accurate molecular mass and the fragment ion information of the compound.

8. The method for analyzing chemical components in a prescription for resolving turbidity, resolving masses and removing arthralgia according to claim 5, which is characterized in that the preparation method of the reference substance comprises the following steps: precisely weighing the reference substance, placing the reference substance into a solution preparation container, dissolving and diluting the reference substance with methanol to prepare reference substance mother solution, and diluting the reference substance with 0.1% of mixed solution of formic acid and acetonitrile in a volume ratio of 9:1 to prepare the reference substance solution with required concentration.

9. The method for analyzing chemical components in a prescription for resolving turbidity, resolving masses and removing arthralgia according to claim 5, wherein the literature is a prior art literature recording chromatographic behavior and mass spectrum information of a target compound.

10. The method for analyzing chemical components in a prescription for resolving turbidity, resolving masses and removing arthralgia according to claim 1, wherein the chemical components of the prescription comprise 51 flavonoid compounds, 35 phenylpropanoid compounds, 26 organic acid compounds, 20 terpene compounds, 17 alkaloid compounds, 16 phenolic compounds, 6 steroid compounds and 13 other compounds.