Analysis method of chemical components of Tibetan medicine rgyal-ba mang-rgyal based on UPLC-Q-TOF MS

Abstract

The application belongs to the technical field of traditional Chinese medicine component analysis and detection, and particularly relates to a chemical component analysis method of Tibetan medicine Renqing Mangjue based on UPLC-Q-TOF MS. The analysis method comprises the following steps: (1) preparing a test sample solution; (2) preparing a reference sample solution; (3) precisely pipetting the mixed reference sample solution and the test sample solution respectively, injecting the solutions into an ultra-high performance liquid chromatograph, recording a chromatogram, and determining chromatographic conditions; (4) determining mass spectrometric conditions; and analyzing the test sample solution of Renqing Mangjue, and combining the reference sample solution of Renqing Mangjue to analyze and identify the chemical components. The analysis method is reliable and accurate in detection, provides a comprehensive chemical profile for Renqing Mangjue, is convenient for improving a quality evaluation system of Renqing Mangjue, and lays a foundation for clinical reasonable application.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese medicine component detection, and particularly relates to a chemical component analysis method of Tibetan medicine Rangnyimaqie based on UPLC-Q-TOF MS. BACKGROUND

[0002] The Tibetan medicine Rangnyimaqie is first recorded in the Tibetan medical book Four Medical Classics, and detailed description can be found in Pandenqionaini compiled by SiTu · Rangnyimaqie is prepared from several natural and precious medicinal materials such as Fructus Terminaliae Chebulae, Syzygium cumini, Crocus sativus, ox gall, musk, etc. under the guidance of Tibetan medical theory, and has the effects of clearing heat and resolving toxin, benefiting liver and nourishing stomach, brightening eyesight, waking up, healing sore, tonifying and invigorating the body, etc. and is praised as "Tibetan medicine penicillin". In clinical application, Rangnyimaqie is commonly used for treating inflammatory gastrointestinal diseases such as chronic gastroenteritis, chronic atrophic gastritis, digestive tract ulcer, metabolic diseases and various toxicosis including natural toxicosis, food poisoning and prepared toxicosis. Modern research shows that Rangnyimaqie has obvious inhibitory effect on Staphylococcus aureus, Escherichia coli and Salmonella, and also has effects of anti-inflammation and anti-fibrosis. At present, the research on Rangnyimaqie by domestic and foreign scholars is mostly focused on its clinical application, and it is crucial to determine the chemical components of Rangnyimaqie for the safety and effectiveness of the clinical use of the medicine. Therefore, it is of great significance to analyze the chemical components of Rangnyimaqie.

[0003] Ultra performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-Q-TOF MS) is widely used in qualitative analysis of chemical components of traditional Chinese medicine due to its high sensitivity and high separation capacity. There is no report on the analysis of chemical components in Rangnyimaqie by using the method. In order to comprehensively characterize the chemical components in Rangnyimaqie, the application optimizes and explores the mass spectrometry analysis conditions based on the method, and provides a comprehensive chemical profile, so as to lay a foundation for improving the quality evaluation system, explaining the pharmacodynamic material basis and metabolic characteristics, and reasonably applying in clinic. SUMMARY

[0004] In order to solve the above problems, the application provides a chemical component analysis method of Tibetan medicine Rangnyimaqie based on UPLC-Q-TOF MS, which is reliable in result and accurate in detection, provides a comprehensive chemical profile for Rangnyimaqie, lays a foundation for reasonably applying in clinic, and solves the problems in the prior art.

[0005] The chemical component analysis method of Tibetan medicine Rangnyimaqie based on UPLC-Q-TOF MS provided by the application comprises the following steps:

[0006] (1) Preparation of test sample solution

[0007] Precisely weigh the Renshenmangjue powder, add ethanol for heating extraction, continue to add ethanol to make up the weight loss after extraction, take the supernatant after centrifugation;

[0008] (2) Preparation of reference solution

[0009] Respectively, accurately weigh the reference 1-linoleic acid glyceride, alpha-linoleic acid, myristyl ether, obtuse leaf, curcumin, bisdemethoxycurcumin, eugenol, dodecasponin A, naringenin, fomesin, rubrofusarin-6-O-beta-D-gentisidin, rutin, pinocembrin, magnoflorine, chlorogenic acid, hydroxysafflor yellow A, arecoline, dehydrodiisoeugenol, into the volumetric flask, add methanol to dissolve and constant volume, shake well, get mixed reference solution;

[0010] (3) Respectively, accurately take the mixed reference solution and the test solution, inject into the ultra-high performance liquid chromatograph, record the chromatogram, and determine the chromatographic conditions;

[0011] (4) Determine the mass spectrometry conditions; analyze the Renshenmangjue test solution, and identify the chemical components combined with the Renshenmangjue reference solution.

[0012] Further, the chromatographic conditions of the ultra-high performance liquid chromatograph in step (3) are: Agilent ZOBRAX SB-C18 chromatographic column, 4.6mmx250mm, 5μm; mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is 0.1% formic acid acetonitrile; flow rate 1.0mL·min -1 , column temperature 35℃;

[0013] The gradient elution program is: 0-5min, 1%-5% B; 5-10min, 5%-10% B; 10-40min, 10%-30% B; 40-65min, 30%-65% B; 65-95min, 65%-95% B; 95-100min, 95%-100% B; 100-110min, 1%-1%.

[0014] Further, the injection amount is 10μL.

[0015] Further, the mass spectrometry conditions in step (4) are:

[0016] Electrospray ion source is used, positive and negative ion mode detection, dry nitrogen flow rate is 10.0L·min -1 , drying temperature is 350℃, atomization gas pressure is 45psi, sheath gas flow is 12.0L·min -1 , temperature is 400℃, capillary voltage is 3500V, atomizer voltage is 65V, bombardment voltage is 120V, scan range m / z100-1500, collision energy is set to 35eV.

[0017] Further, in the mass spectrometry condition, the scanning range is m / z 100-1000.

[0018] Further, the step (4) of analyzing and identifying the chemical components of the test solution of Rincangmangjue includes:

[0019] S1: Analyzing the mass spectrometry data of the test solution and the control solution of Rincangmangjue according to the determined mass spectrometry condition;

[0020] S2: Extracting all the compound information from the total ion chromatogram, identifying the chromatographic peaks according to the fragmentation rules of the control solution, combining the MS database fragment information matching and the compound mass spectrometry information data, and identifying 166 compounds, including 52 flavonoid components, 32 phenolic acid components, 22 terpene components, 18 alkaloid components, 13 anthraquinone components and 29 other components.

[0021] Further, the other components are 10 triterpenoid saponins, 10 aliphatic compounds, 4 phenylpropanoids and 5 other types of compounds.

[0022] Further, the step of analyzing and identifying the chemical components is to first establish the chemical component database of Rincangmangjue, and then use the molecular feature extractor in the MassHunter Workstation software package to process the data; the extracted compound data is exported from MassHunter in CEF file, and then imported into Mass Profiler Professional software for analysis. The MPP software automatically standardizes, visualizes or filters all the molecular feature data. Through single factor variance analysis, P=0.05 is used as the screening standard to gradually filter out the molecular feature number. Through the MFE algorithm, according to the isotope matching and the relationship between the adduct peaks of the compounds, all the compound information is extracted from the total ion chromatogram, and the number of compounds is reduced through single factor variance analysis with P=0.05 as the screening standard. According to the fragmentation rules of the control solution, combining the MS database fragment information matching and the compound mass spectrometry information data.

[0023] Further, in step (1), 50% ethanol is used as the extraction solvent, the extraction time is 30 min, and the extraction temperature is 50°C.

[0024] Further, the preparation of the test solution in step (1) is as follows: 1.5 g of Rincangmangjue is precisely weighed and placed in a conical flask with a plug, 10 mL of 50% ethanol is precisely added, the mass is determined, ultrasonic extraction is performed at 50°C for 30 min, the mass is determined after cooling to room temperature, 50% ethanol is added to make up for the lost mass, and the mixture is shaken uniformly; the extraction solution is centrifuged at 12000 r·min -1Centrifugal 10 min, take supernatant, filter with microporous filter membrane, place in sample bottle, and obtain.

[0025] Further, the ultrasonic frequency of ultrasonic extraction is 50 kHz, and the power is 250 W.

[0026] Further, the centrifugal radius of centrifugal operation of the extraction solution is 10 mm.

[0027] Further, the preparation of the control solution in step (2) is as follows: 4 mg of control 1-linoleic acid glyceride, 4 mg of alpha-linoleic acid, 1.21 mg of myristic ether, 0.8 mg of obtusifoliol, 0.98 mg of curcumin, 1.31 mg of bisdemethoxycurcumin, 4.3 mg of eugenol, 1.25 mg of pokerranine A, 0.98 mg of naringenin, 1.27 mg of fumarine, 0.53 mg of rubrofusarin-6-O-beta-D-gentiobioside, 1.41 mg of rutin, 0.91 mg of pinocembrin, 1.03 mg of magnoflorine, 1.3 mg of strychnine, 1.27 mg of chlorogenic acid, 1.31 mg of hydroxysafflor yellow A, and 0.92 mg of arecoline, 0.91 mg of dehydrodiisoeugenol are precisely weighed respectively, added to a 10 mL volumetric flask, dissolved with methanol and constant volume, shaken uniformly, and the mixed control solution with a mass concentration of 3-4 g / L is obtained. -1

[0028] Further, the mixed control solution is stored at 4 DEG C, and is filtered through a 0.22 mu m microporous filter membrane when sampling.

[0029] The beneficial effects of the present application include but are not limited to:

[0030] The chemical component analysis method of the present application comprehensively characterizes the chemical components in Rincengmangjue, optimizes the mass spectrum analysis conditions by systematically investigating the chromatographic conditions, the results are reliable, the total ion chromatogram has good repeatability, the instrument stability is verified, and the detection results are accurate.

[0031] The chemical component analysis method of the present application is a preliminary exploration of the chemical composition of the Tibetan medicine Rincengmangjue, which provides a theoretical basis for the identification of the efficacy material basis and the improvement of the quality control standard. BRIEF DESCRIPTION OF DRAWINGS

[0032] The drawings described herein are used to provide further understanding of the present application, and form a part of the present application. The schematic embodiments of the present application and their descriptions are used to explain the present application, and do not constitute an improper limitation on the present application. In the drawings:

[0033] Figure 1 It is a total ion chromatogram of the Rincengmangjue sample of the present application;

[0034] Figure 2 It is a naringenin cleavage pathway analysis in the flavonoid compounds of the present application;​

[0035] Figure 3 The application is to analyze the cleavage pathway of bisdemethoxycurcumin in the phenolic acid component of the application;

[0036] Figure 4 The application is to analyze the cleavage pathway of dehydrodiisoeugenol in the terpenoid component of the application;

[0037] Figure 5 The application is to analyze the cleavage pathway of fumaritcine in the alkaloid component of the application;

[0038] Figure 6 The application is to analyze the cleavage pathway of emodin in the anthraquinone component of the application;

[0039] Figure 7 The application is to analyze the cleavage pathway of emodin in the anthraquinone component of the application;

[0040] Figure 8 The application is to analyze the cleavage pathway of emodin in the anthraquinone component of the application;

[0041] Figure 9 The application is to analyze the cleavage pathway of emodin in the anthraquinone component of the application;

[0042] Figure 10 The application is to analyze the cleavage pathway of emodin in the anthraquinone component of the application;

[0043] Figure 11 The application is to analyze the cleavage pathway of emodin in the anthraquinone component of the application;

[0044] Figure 12 The application is to analyze the cleavage pathway of emodin in the anthraquinone component of the application;

[0045] Figure 13 The application is to analyze the cleavage pathway of emodin in the anthraquinone component of the application;

[0046] Figure 14 The application is to analyze the cleavage pathway of emodin in the anthraquinone component of the application;

[0047] Figure 15 The application is to analyze the cleavage pathway of emodin in the anthraquinone component of the application;

[0048] Figure 16 The total ion current chromatogram (TIC chromatogram) collected in positive ion mode for the mixed reference standard of this invention;

[0049] Figure 17 The total ion current chromatogram (TIC chromatogram) was collected in negative ion mode for the mixed reference standard of this invention. Detailed Implementation

[0050] The present invention will be further described below with reference to specific embodiments, but the scope of protection of this application is not limited to these embodiments.

[0051] The chemical composition analysis method of Tibetan medicine Renqing Mangjue based on UPLC-Q-TOF MS provided in this embodiment is described in detail below:

[0052] I. Instruments, Equipment and Materials

[0053] 1730R miniature centrifuge (Hong Kong Gene Technology Co., Ltd.), Alliance e2695 ultra-high performance liquid chromatograph (Waters Instruments, USA), Agilent 6520QTOF high resolution mass spectrometer (Agilent Technologies, USA), XSR105DU 1 / 100,000 electronic analytical balance (Shanghai Mettler Toledo Instruments Co., Ltd.), Milli-Q ultrapure water system (Merck, Inc.); small high-speed grinder [Zhongke Netzchi Technology (Beijing) Co., Ltd., model: ZN-08L]; electric thermostatic water bath (Tianjin Test Instruments Co., Ltd., model: DK-98-ⅡA); drum drying oven (Shanghai Yiheng Scientific Instruments Co., Ltd., model: DHG-9055A); KQ-100DE CNC ultrasonic cleaner, Kunshan Ultrasonic Instruments Co., Ltd.

[0054] 1-Linoleic acid glyceride (batch number 24012238), α-linoleic acid (batch number 23110625), myristole (batch number 23081850), obtusifolin (batch number 23111956), curcumin (batch number 23052344), bisdemethoxycurcumin (batch number 23112987), eugenol (batch number 23021142), phytolaccaside A (batch number 23121252), naringenin (batch number 23030812), corydaline A (batch number Y-158-1) Reference standards for 60823), rutin (batch number 23021510), jugazosin (batch number 23080920), magnoflorine (batch number 23020631), strychnine (batch number 23081945), chlorogenic acid (batch number 23011507), hydroxysaffron yellow A (batch number 23030601), arecoline (batch number 23071152), and dehydrodiisoeugenol (batch number 23070452) were all purchased from Beijing Better Renkang Biomedical Technology Co., Ltd., and all had a purity ≥98%.

[0055] Renzengmangjue (Jinhe Tibetan Medicine Co., Ltd., batch number 01231213). Water was ultrapure water, methanol, formic acid, acetonitrile were mass spectrometry pure, and other reagents were analytical pure.

[0056] II. Methods and Results

[0057] 2.1 Preparation of solution

[0058] 2.1.1 Preparation of test solution

[0059] Renzengmangjue was ground and passed through a 100 mesh sieve, 1.5 g was accurately weighed and placed in a conical flask with a stopper, 10 mL of 50% ethanol was accurately added, tightly stoppered, the mass was determined, ultrasonic extraction was performed at 50°C for 30 min (frequency 50 kHz, power 250 W), and the mass was determined after cooling to room temperature. 50% ethanol was added to make up for the loss of mass, and shaken well. The extract was centrifuged at 12000 r·min -1 for 10 min (centrifugal radius 10 mm), the supernatant was taken and filtered with a 0.22 μm microporous filter, and placed in a sample bottle to obtain the Renzengmangjue test solution.

[0060] 2.1.2 Preparation of control solution

[0061] 1- Linoleic acid glyceride 4 mg, α-linoleic acid 4 mg, myristic ether 1.21 mg, obtusifoliol 0.8 mg, curcumin 0.98 mg, bisdemethoxycurcumin 1.31 mg, eugenol 4.3 mg, polyadione A 1.25 mg, naringenin 0.98 mg, fumaritine 1.27 mg, rubrofusarin-6-O-β-D-gentisidin 0.53 mg, rutin 1.41 mg, pinocembrin 0.91 mg, magnoflorine 1.03 mg, strychnine 1.3 mg, chlorogenic acid 1.27 mg, hydroxysafflor yellow A 1.31 mg, arecoline 0.92 mg, dehydrodiisoeugenol 0.91 mg were accurately weighed respectively, added to a 10 mL volumetric flask, dissolved and diluted with methanol, shaken well, and a mixed control solution with a mass concentration of 3-4 g·L -1 was obtained. It was stored at 4°C and used as needed. When sampling, it was filtered through a 0.22 μm microporous filter.

[0062] 2.2 Detection conditions

[0063] 2.2.1 Chromatographic conditions

[0064] Agilent ZOBRAX SB-C18 column (4.6 mm x 250 mm, 5 μm) was used with mobile phase of 0.1% formic acid in water (A) - 0.1% formic acid in acetonitrile (B) with gradient elution (0-5 min, 1%-5% B; 5-10 min, 5%-10% B; 10-40 min, 10%-30% B; 40-65 min, 30%-65% B; 65-95 min, 65%-95% B; 95-100 min, 95%-100% B; 100-110 min, 1%-1% B) at a flow rate of 1.0 mL·min -1 , column temperature 35 °C, and injection volume 10 μL.

[0065] 2.2.2 Mass spectrometry conditions

[0066] Electrospray ion source (ESI) was used with positive and negative ion mode detection, dry nitrogen gas flow rate 10.0 L·min -1 , dry temperature 350 °C, nebulization gas pressure 45 psi, sheath gas flow rate 12.0 L·min -1 , temperature 400 °C, capillary voltage 3500 V, atomizer voltage 65 V, bombardment voltage 120 V, scan range m / z 100-1500, and collision energy set at 35 eV.

[0067] 2.3 Mass spectrometry data analysis

[0068] Based on the information of compounds in PubChem, MassBank and other databases, and the mass spectrometry information of single medicinal materials in Rangnyingmangjue, and referring to the literature reports at home and abroad, the chemical component database of Rangnyingmangjue was established. The mass spectrometry data were analyzed by MassHunter Workstation (B 05.00 version, Agilent Technologies, Santa Clara, CA, USA) software, and the data were processed by the molecular feature extractor (MFE) in the software. The peak height of ion was not less than 500 counts, and the ion charge state was monovalent. The parameters of molecular features included retention time, abundance and accurate mass. The threshold of complex ion count was set to two or more ions, and the isotope algorithm was selected as the normal organic ion mode. The extracted compound data were exported from MassHunter in CEF file format, and then imported into Mass Profiler Professional (MPP) software (B.12.05 version, Agilent Technologies, Santa Clara, CA, USA) for further analysis. MPP software automatically standardized, visualized or screened all molecular feature data. Through one-way ANOVA analysis, P=0.05 was used as the screening standard to gradually eliminate molecular feature numbers. To avoid cross interference between samples, blank solvent was added between different samples during analysis. The quality control sample was a mixed control solution containing 19 standard samples, which was used to confirm the stability and accuracy of the instrument during analysis. The quality control sample was injected before and after sample analysis. Through MFE algorithm, all compound information was automatically and quickly extracted from the total ion chromatogram, including the accurate mass-to-charge ratio, molecular formula, response intensity, retention time and other information of the compound. Through one-way ANOVA analysis, P=0.05 was used as the screening standard to reduce the number of compounds. According to the fragmentation rules of the reference substance, combined with the MS database fragment information matching and compound mass spectrometry information data, the chromatographic peaks were identified.

[0069] 2.4 Mass spectrometry analysis

[0070] The chemical components of the test solution and the reference solution were analyzed by UPLC-Q-TOF MS, and the total ion chromatograms (TIC) of positive and negative ion modes were obtained, as shown in Figure 1 . The results showed that a total of 166 compounds were identified from Rangnyingmangjue, as shown in Table 4; among them, 19 components were confirmed by comparing the retention time, primary and secondary fragment information with the reference substance.

[0071] 2.4.1 Flavonoids

[0072] Flavonoids are the main compounds in Rengmangjue, a total of 52 were identified, mainly from short-budded rabbit ear grass, Cassia seed, mango kernel and other medicinal materials. 2-phenyl chromone is the basic mother nucleus of flavonoids, and flavonoid glycosides are substituted with glycosyl. Flavonoid aglycone often has hydroxyl, methyl, methoxyl and other substituents on the A and B rings. Therefore, flavonoids are generally first glycosidic bond broken to generate flavonoid aglycone, and flavone mother nucleus undergoes reverse Diels-Alder (RDA) cleavage to generate a series of characteristic ion peaks, and energy collision loses CO and side chain substituents and other neutral fragments.

[0073] Take compound 109 as an example to illustrate its possible fragmentation rules. In negative ion mode, the quasi-molecular ion of compound 109 is m / z 271.0636 [M-H] - , the retention time of the compound is 47.99 min, the molecular formula is C 15 H 12 O5. In the fragmentation process, the quasi-molecular ion undergoes RDA cleavage to form fragment ions m / z 151.0056 [M-H-C8H8O] - and fragment ions m / z 119.0529 [M-H-C7H4O4] - . On the other hand, the parent ion loses C9H6O4 to obtain fragment ion m / z 93.03772 [M-H-C9H6O4] - . The fragment ions m / z 107.0165 and 83.0164 are speculated to be fragment ions m / z 151.0056 [M-H-C8H8O] - losing one molecule of CO2 to obtain fragment ion m / z 107.0165 [M-H-C8H8O-CO2] - , and then losing one molecule of CO to obtain fragment ion m / z 107.0165 [M-H-C8H8O-CO2-CO] - . According to the mass spectrum information of the reference substance and the data in the literature, it is speculated that the compound is naringenin, and the possible fragmentation pathway of the fragment ion is shown in Figure 2 .

[0074] 2.4.2 Phenolic acid components

[0075] Phenolic acid compounds are mainly derived from areca nuts, a total of 32 were identified. This kind of compound is easy to lose H2O in the fragmentation process, and the carbonyl group is easy to break and lose -O, -CO, -CO2 and other small molecule groups. Take compound 135 as an example, which has good response in negative ion mode, with a retention time of 60.64 min, a quasi-molecular ion of m / z 307.1005 [M-H] - , and a molecular formula of C 19 H16 O4. During the fragmentation process, the parent ion loses one molecule of C9H6O2 to produce fragment ion m / z 161.0600 [M-H-C9H6O2] - , which loses one molecule of O, H2O and CO2 respectively to produce fragment ions m / z 161.0600 [M-H-C9H6O2-O] - , m / z 143.0520 [M-H-C9H6O2-H2O] - and m / z 117.0373 [M-H-C9H6O2-H2O] - . The parent ion can also lose one molecule of C 11 H8O3 to produce fragment ion m / z 117.0373 [M-H-C 11 H8O3] - . The retention time and fragment ions of the compound were consistent with those of the reference substance of bisdemethoxycurcumin, so it was identified as bisdemethoxycurcumin, see Figure 3 .

[0076] 2.4.3 Terpenoids

[0077] The terpenoids in R. tanguticum were mainly derived from Terminalia chebula Retz., including iridoid and phenolic terpenoids, and 22 compounds were identified, of which 3 were identified by comparison with reference substances. These compounds were prone to lose fragments such as CH3, C2H4 and OH during the fragmentation process.

[0078] For example, compound 56 responded well in the positive ion mode, with a parent ion m / z of 327.1598 [M+H] + , and a molecular formula of C 20 H 22 O4. In the secondary mass spectrum, the parent ion lost one molecule of C2H5 and one molecule of CH3 to produce fragment ion m / z 283.0944 [M+H-C3H8] + ; in addition, the parent ion lost one molecule of C7H8O2 and one molecule of CH3 to produce fragment ion m / z 188.0814 [M+H-C7H8O2-CH3] + , and then lost one molecule of CH3 to produce fragment ion m / z 173.0584 [M+H-C7H8O2-2CH3] + . The parent ion peak 327.1598 [M+H] + further lost one molecule of C2H4 on the basis of furan ring cleavage to produce fragment ion m / z 137.0587 [M+H-C 10 H 10 O2-C2H4] +Continuously lose 2 OH, respectively, to generate fragment ions m / z 117.0684[M+H-C 10 H 10 O2-C2H4-OH] + and m / z 103.0536[M+H-C 10 H 10 O2-C2H4-2OH] + The retention time, fragment ions of this compound are consistent with the dehydrodiisoeugenol reference substance, and its fragmentation rule is also consistent with the literature reported, so it is identified as dehydrodiisoeugenol, and its fragmentation pathway is shown in Figure 4 .

[0079] 2.4.4 Alkaloid components

[0080] A total of 18 alkaloid components were identified from the prescription, mainly from Cassiae Semen and Terminalia chebula Retz, etc. Alkaloids are prone to lose N, CH3, H2O and other neutral molecules during mass spectrometry fragmentation. Tetrahydroprotoberberine alkaloids can undergo RDA fragmentation to produce complementary ions.

[0081] Compound 94 is taken as an example to illustrate the fragmentation process. Compound 94 has a good response in the positive ion mode, with a quasi-molecular ion of m / z 370.2022[M+H] + , and a molecular formula of C 22 H 27 NO4. During fragmentation, the quasi-molecular ion loses CH4 to produce a fragment ion of m / z 354.1695[M+H-CH4] + . The quasi-molecular ion can undergo RDA fragmentation to produce fragment ions of m / z 192.1008[M+H-C 11 H 14 O2] + and m / z 165.0902[M+H-C 12 H 15 O2] + , and then, these two fragment ions both lose a molecule of CH3 to produce fragment ions of m / z 177.0789[M+H-C 11 H 14 O2-CH3] + and m / z 150.0668[M+H-C 12 H 15 O2-CH3] + The retention time and fragment ions of this compound are consistent with the literature

[17] and reference substance of fumariline, so it is identified as fumariline, see Figure 5 .

[0082] 2.4.5 Anthraquinone components

[0083] Thirteen anthraquinones were co-identified, including eight anthraquinone glycosides and five free anthraquinones, mainly from Cassia obtusifolia. Anthraquinone glycosides can be de-sugared to form anthraquinone aglycone; the anthraquinone parent nucleus group can be sequentially de-sugared to lose CO neutral fragments, and the side chain substituent group is easy to lose CO, H2O, CH3, CO2, and other neutral small molecule fragments.

[0084] For example, compound 82, in negative ion mode, has a retention time of 34.104 min, a molecular formula of C 15 H 10 O5, and a quasi-molecular ion of m / z 269.0482 [M-H] - , a fragment ion of m / z 241.0472 [M-H-CO] - , a further loss of -O to form a fragment ion of m / z 224.0483 [M-H-CO-O] - ; or a fragment ion of m / z 241.0472 [M-H-CO] - , a continuous loss of -CO to obtain fragment ions of m / z 185.0627 [M-H-2CO] - and m / z 241.0472 217.7245 [M-H-3CO] - . By comparison with the reference, this compound is identified as emodin.

[0085] 2.4.6 Other types of components

[0086] In addition to the above five types of compounds, some other components were identified from Rincengangjue, including ten triterpene saponins, mainly from Terminalia chebula; ten aliphatic components, mainly from Phyllanthus emblica and Humulus scandens; and nine phenylpropanoids, stilbene glycosides, and aromatic compounds. Triterpenoids are connected by several isoprene structural units, and in secondary mass spectrometry cracking, the triterpenoid fused skeleton is not easy to break, but the hydroxyl, carboxyl, or sugar group on the ring is easy to be lost. The main cracking characteristics of fatty acid compounds in mass spectrometry are the loss of CH3, CO, CO2, H2O, COOH, CH3, and other neutral molecules. Phenylpropanoids, stilbene glycosides, and aromatic compounds: a total of nine were identified. The mass spectrometry cracking characteristics of these compounds usually include: phenylpropanoids may lose hydroxyl, methyl, or other substituents on the ring; stilbene glycosides often lose sugar groups, hydroxyl groups, and methyl groups; aromatic compounds may lose hydrogen atoms, hydroxyl groups, or other small molecule fragments on the aromatic ring.

[0087] III. Optimization of sample preparation and detection conditions

[0088] 3.1 Optimization of sample extraction method

[0089] 3.1.1 Optimization of extraction solvent

[0090] The seven solvents, water, methanol, ethanol, acetonitrile, 50% methanol, 50% acetonitrile, and 50% ethanol, were investigated respectively, and the chromatograms of the samples extracted by different solvents were compared. The results are shown in Table 1. Figure 7 .

[0091] The research results show that the chromatographic peaks of the sample are significantly less when the pure organic phase is used for extraction, and the chromatographic peaks of the sample are more when the 50% proportion of the organic phase is used for extraction. By comparing the chromatograms of the samples extracted by 50% ethanol, 50% methanol, and 50% acetonitrile, it is suggested that the chromatographic peaks of the sample are more and the baseline is more stable when 50% ethanol is used as the extraction solvent, and the extraction solvent of the sample is determined to be 50% ethanol.

[0092] 3.1.2 Optimization of extraction time

[0093] The three time points of ultrasonic extraction for 15 min, 30 min, and 45 min were investigated respectively, and the chromatograms of the samples under different extraction times were compared. The results are shown in Table 2. Figure 8 The research results show that there is no obvious difference in peak height and peak number in the chromatogram of the sample with the increase of the extraction time, and therefore the extraction time is finally determined to be 30 min.

[0094] 3.1.3 Optimization of extraction temperature

[0095] The three extraction temperatures of room temperature, 35°C, and 50°C were investigated, and the chromatograms of the samples under different extraction temperatures were compared. The results are shown in Table 3. Figure 9 .

[0096] The research results show that the chromatographic peaks of the sample slightly increase with the increase of the extraction temperature, and the peak height of each chromatographic peak is slightly higher than that of the other extraction temperatures when the extraction temperature is 50°C. Therefore, the extraction temperature is finally determined to be 50°C.

[0097] 3.1.4 Optimization results of extraction method

[0098] By investigating the solvent, temperature, and time of the sample extraction method, the sample analysis extraction method is finally determined as follows: about 0.5 g of sample is weighed, 10 mL of 50% ethanol is used as the extraction solvent, the extraction time is 30 min, and the extraction temperature is 50°C.

[0099] 3.2 Optimization of chromatographic method

[0100] 3.2.1 Selection of detection wavelength

[0101] After the sample analysis, the chromatographic peak conditions under different collection wavelengths were compared, and the results are shown in Table 4. Figure 10 .

[0102] The results of the study show that when the wavelength of 240 nm is collected, the number of chromatographic peaks in the chromatogram is more, and the chromatographic peak is slightly high, so the wavelength of the ultraviolet detection of the sample is set to 240 nm.

[0103] 3.2.2 Investigation of the mobile phase system

[0104] The sample was analyzed by high performance liquid chromatography, and the mobile phase system was optimized. The chromatograms of different mobile phase systems of methanol, acetonitrile, 0.1% formic acid water, 0.2% formic acid water, 0.1% formic acid acetonitrile, and 0.2% formic acid acetonitrile were compared. The results are as follows Figure 11 .

[0105] The chromatogram results of the sample show that when methanol is used as the eluent of the chromatographic column, the chromatographic peak is obviously less, and it is speculated that the sample is not completely eluted. Therefore, acetonitrile is determined as the mobile phase of the chromatographic system. The results are as follows Figure 12 .

[0106] The chromatogram results of the sample show that when formic acid is added to the chromatographic system, it has the effect of improving the chromatographic peak shape, but with the increase of the proportion of added formic acid, the baseline drift of the chromatogram is obvious, therefore, 0.1% formic acid acetonitrile-0.1% formic acid water is determined as the mobile phase of the chromatographic system.

[0107] 3.2.3 Investigation of the gradient elution conditions of the mobile phase

[0108] The sample was analyzed by high performance liquid chromatography, and the gradient elution conditions of the mobile phase were optimized, different proportions of the mobile phase gradient were used, and the gradient elution optimization is listed in the following tables 1 and 2.

[0109] Table 1 Gradient elution optimization 1

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[0111] Table 2 Gradient elution optimization 2

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[0113] The final chromatographic gradient elution program and sample chromatogram are as follows Figure 13 .

[0114] Table 3 Gradient elution program

[0115]

[0116] The chromatographic method for final determination of sample analysis was as follows: Agilent ZOBRAX SB-C18 column (4.6 mm x 250 mm, 5 μm); mobile phase: water (0.1% formic acid, A), acetonitrile (0.1% formic acid, B); flow rate 1.0 mL / min; column temperature 35 °C; injection volume 10 μL; detection wavelength 240 nm. The gradient elution program is shown in Table 3 above.

[0117] 3.3 Mass spectrometric analysis of chemical components

[0118] Agilent ZOBRAX SB-C18 column (4.6 mm x 250 mm, 5 μm); flow rate 1.0 mL / min; column temperature 35 °C; injection volume 10 μL; mobile phase: water (0.1% formic acid, A), acetonitrile (0.1% formic acid, B); gradient elution program as follows: 0-5 min, 1%-5% B; 5-10 min, 5%-10% B; 10-40 min, 10%-30% B; 40-55 min, 30%-65% B; 55-75 min, 65%-95% B. Agilent 6520 QTOF MS mass spectrometry system; electrospray ion source; dry nitrogen flow rate 10.0 L / min, dry temperature 350 °C; atomizing gas pressure 45 psi; sheath gas flow rate 12.0 L / min; temperature 400 °C; capillary voltage 3500 V; atomizer voltage 65 V; bombardment voltage 120 V; collision energy 40 V; m / z collection range set to 100-1000; positive ion scan mode.

[0119] The total ion chromatograms of Rendong Mangjue (RQ) extract in positive ion mode and negative ion mode, and the total ion chromatograms of mixed reference substance in positive ion mode and negative ion mode are shown in Figures 1-4, respectively. Figures 14-17 wherein Figure 14 and Figure 15 are the color graphs corresponding to the total ion chromatograms of Rendong Mangjue sample. Figure 1 The total ion chromatograms of Rendong Mangjue sample in positive ion mode and negative ion mode are shown in Figures 5 and 6, respectively.

[0120] Mass spectrometry data were processed using the molecular feature extractor in the MassHunter Workstation (B 05.00 version, qualitative analysis, Agilent Technologies, Santa Clara, CA, USA) software suite, extracting ions with peak height no less than 500 counts and ion charge valence of 1. The parameters of molecular features included retention time, abundance, and accurate mass. The composite ion count threshold was set to two or more ions, and the isotopic algorithm selected the normal organic ion mode. The extracted compound data were exported from MassHunter as CEF files and then imported into Mass Profiler Professional (MPP) software (B.12.05 version, Agilent Technologies, Santa Clara, CA, USA) for analysis. The MPP software automatically standardized, visualized, or filtered all molecular feature data. Through one-way ANOVA analysis, P = 0.05 was used as the screening standard to gradually filter out molecular feature numbers. To avoid cross interference between samples, blank solvent analysis was added between different samples.

[0121] The molecular feature extraction algorithm (MFE) can automatically and quickly extract all compound information from the total ion chromatogram, including the accurate mass-to-charge ratio, molecular formula, response intensity, retention time, and other information of the compound, based on the relationship between the isotope matching and adduct peaks of the compound. The entire analysis process can be completed in a few minutes. Through one-way ANOVA analysis, P = 0.05 was used as the screening standard to reduce the number of compounds. According to the fragmentation rules of the reference substance, combined with the MS database fragment information matching and compound mass spectrum information data, part of the compound identification information is shown in Table 4.

[0122] By analyzing the fragmentation rules of 19 reference substances of various types such as aliphatic, phenolic acid, terpenoid, phenylpropanoid, flavonoid, triterpenoid saponin, alkaloid, and other types, combined with online database, literature fragment information matching, and compound mass spectrum information data. A total of 166 chemical components have been identified, including 32 phenolic acid compounds, 18 alkaloid compounds, 4 phenylpropanoid compounds, 22 terpenoid compounds, 52 flavonoid compounds, 13 anthraquinone compounds, 10 triterpenoid saponin compounds, 10 aliphatic compounds, and 5 other types of compounds.

[0123] After optimizing the ultrasonic extraction, chromatographic conditions, and mass spectrometry analysis conditions in the analysis method, the samples showed good sensitivity and mass spectrometry response intensity in positive and negative ion modes. To ensure the reliability of the results, a quality control sample was introduced during the analysis process, and the total ion chromatogram showed good reproducibility, the instrument stability was verified, and the detection results were accurate.

[0124] The invention explores the chemical composition of Renqing Mangjue, which has important value for the development and utilization of its medicine. Renqing Mangjue is a compound composed of dozens of medicinal materials, and the chemical composition of the medicine is complex. After the prescription, new components will be produced, which makes the overall chemical composition of Renqing Mangjue more complex, and the material basis research more difficult. The invention analyzes the chemical composition of Renqing Mangjue by UPLC-Q-TOF MS technology, and identifies and speculates 166 compounds including 52 flavonoids, 22 terpenes, 32 phenolic acids, 18 alkaloids, 13 anthraquinones and 29 others by combining the mass spectrum information of reference substances and literature data. The research provides a theoretical basis for the identification of the material basis of Renqing Mangjue and the improvement of the quality control standard. The results show that among all categories of compounds, flavonoids and terpenes account for a large proportion.

[0125] The above merely describes the embodiments of the present application, and the protection scope of the present application is not limited by these specific embodiments, but is determined by the claims of the present application. The present application can have various changes and variations for those skilled in the art. Any modification, equivalent replacement, improvement, etc. within the technical thought and principle of the present application shall be included in the protection scope of the present application.

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Claims

1. A method for chemical composition analysis of Tibetan medicine Renqing Mangjue based on UPLC-Q-TOF MS, characterized in that, Includes the following steps: (1) Preparation of test solution Accurately weigh the powder of Rhizoma Rhizoma Amomum villosum, add ethanol and heat to extract, add more ethanol to make up for the lost mass after extraction, centrifuge and take the supernatant; (2) Preparation of reference solution Accurately weigh the following reference standards: 1-linoleic acid glyceride, α-linolenic acid, myristole, obtusifolia extract, curcumin, dimethoxycurcumin, eugenol, phytolaccaside A, naringenin, corydaline A, and erythromycin-6- O-β -D-Gentianobiose, rutin, jugol, magnoflorine, strychnine, chlorogenic acid, hydroxysaffron yellow A, arecoline, dehydrodiisoeugenol, add to volumetric flask, add methanol to dissolve and dilute to volume, shake well to obtain mixed reference solution. (3) Accurately pipette the mixed reference solution and the test solution separately, inject them into the ultra-high performance liquid chromatograph, record the chromatogram, and determine the chromatographic conditions; (4) Determine the mass spectrometry conditions; analyze the Rinchen Mangjue test solution and perform chemical composition analysis and identification in conjunction with the Rinchen Mangjue reference solution; Step (3) The chromatographic conditions of the ultra-high performance liquid chromatograph were as follows: Agilent ZOBRAX SB-C18 column, 4.6 mm × 250 mm, 5 μm; mobile phase A was 0.1% formic acid aqueous solution, mobile phase B was 0.1% formic acid acetonitrile; flow rate was 1.0 mL·min -1 Column temperature 35 °C; The gradient elution program is as follows: 0-5 min, 1%-5% B; 5-10 min, 5%-10% B; 10-40 min, 10%-30% B; 40-65 min, 30%-65% B. 65-95 min, 65%-95% B; 95-100 min, 95%-100% B; 100-110 min, 1%-1% B.

2. The method for chemical composition analysis of Tibetan medicine Renqing Mangjue based on UPLC-Q-TOF MS according to claim 1, characterized in that, The mass spectrometry conditions for step (4) are as follows: An electrospray ionization source was used for detection in both positive and negative ion modes, with a drying nitrogen flow rate of 10.0 L·min. -1 The drying temperature was 350 °C, the atomizing gas pressure was 45 psi, and the sheath gas flow rate was 12.0 L·min. -1 Temperature: 400 °C; Capillary voltage: 3500 V; Atomizer voltage: 65 V; Bombardment voltage: 120 V; Scan range: m / z 100~1500, collision energy set at 35eV.

3. The method for chemical composition analysis of Tibetan medicine Renqing Mangjue based on UPLC-Q-TOF MS according to claim 1, characterized in that, Step (4) involves chemical composition analysis and identification of the Renqing Mangjue test solution, including: S1 analyzed the mass spectrometry data of the Renqing Mangjue test sample and the control sample under the determined mass spectrometry conditions; S2 extracts information on all compounds from total ion chromatography. Based on the fragmentation pattern of the reference standard, combined with fragment information matching from the MS database and compound mass spectrometry data, the chromatographic peaks are identified. A total of 166 compounds are identified, including 52 flavonoids, 32 phenolic acids, 22 terpenoids, 18 alkaloids, 13 anthraquinones, and 29 other compounds.

4. The method for chemical composition analysis of Tibetan medicine Renqing Mangjue based on UPLC-Q-TOF MS according to claim 1, characterized in that, Step (1) uses 50% ethanol as the extraction solvent, extracts for 30 minutes, and extracts at 50°C.

5. The method for chemical composition analysis of Tibetan medicine Renqing Mangjue based on UPLC-Q-TOF MS according to claim 1 or 4, characterized in that, Step (1) Preparation of the test solution: After grinding Renqing Mangjue into a fine powder and sieving, take 1.5 g, accurately weigh it, place it in a stoppered conical flask, accurately add 10 mL of 50% ethanol, seal tightly, weigh, and extract by ultrasonication at 50 °C for 30 min. Let it cool to room temperature, weigh it, add 50% ethanol to make up for the lost mass, and shake well; then, immerse the extract at 12000 r·min. -1 Centrifuge for 10 min, collect the supernatant, filter it through a microporous membrane, and place it in a sample vial.

6. The method for chemical composition analysis of Tibetan medicine Renqing Mangjue based on UPLC-Q-TOF MS according to claim 1, characterized in that, Step (2) Preparation of the reference solution is as follows: Accurately weigh the following reference solutions: 1-linoleic acid glyceride 4 mg, α-linoleic acid 4 mg, myristole ether 1.21 mg, obtusifolin 0.8 mg, curcumin 0.98 mg, dimethoxycurcumin 1.31 mg, eugenol 4.3 mg, phytolaccaside A 1.25 mg, naringenin 0.98 mg, corydalis A 1.27 mg, and erythromycin-6- O- β -D-gentiobiglycoside 0.53 mg, rutin 1.41 mg, geraniol 0.91 mg, magnoflorine 1.03 mg, strychnine 1.3 mg, chlorogenic acid 1.27 mg, hydroxysaffron yellow A 1.31 mg, arecoline 0.92 mg, dehydrodiisoeugenol 0.91 mg were added to a 10 mL volumetric flask, dissolved in methanol and diluted to volume. The solution was shaken well to obtain a concentration of 3–4 g / L. -1 A mixed reference solution.

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