A method for detecting chemical components in a traditional Chinese medicine composition

Abstract

The application provides a detection method of chemical components in a traditional Chinese medicine composition. The detection method is combined with liquid chromatography and mass spectrometry, and is used for qualitative detection of chemical components in a traditional Chinese medicine composition prepared from red peony root, astragalus root, rehmannia root, safflower, female privet fruit, eclipta, rhubarb, panax notoginseng, giant worm, ginkgo leaf, cassia seed and paeonia suffruticosa. 82 kinds of chemical components are detected and confirmed from the traditional Chinese medicine composition, so that the detection method can be used as a discrimination method for quality control of the traditional Chinese medicine composition, and can provide analysis technical support for mechanism research and effective component research of the traditional Chinese medicine composition.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, and specifically relates to a method for detecting chemical components in a traditional Chinese medicine composition. Background Technology

[0002] Traditional Chinese medicine has a long history and wide application, and is accepted by most patients in China. However, due to its complex composition and unclear components, it is limited in many aspects such as mechanism research and quality control, and has always faced many difficulties in going abroad and becoming international.

[0003] In countries without a history of using traditional Chinese medicine (TCM), strict quality standards are typically required for pharmaceuticals to ensure that the active ingredients and their content are clearly defined and controllable. This applies to TCM as well. For example, the U.S. *Industrial Guidelines for Herbal Drug Research and Development* (2016 edition) stipulates that for drugs produced by mixing several medicinal herbs, optimal analytical techniques should be used to identify (qualitatively) and quantify each active ingredient or chemical component. Currently, the standards for qualitative and quantitative identification of active ingredients in Chinese patent medicines in the *Chinese Pharmacopoeia* are relatively low and cannot meet the requirements of foreign drug standards. Therefore, most Chinese patent medicines produced according to Chinese drug quality standards cannot be exported. In response to this situation, researchers have been continuously working to develop quality control methods for Chinese patent medicines in order to solve the domestic quality control problems and support their export.

[0004] Chinese patent CN1833698A discloses a drug for treating diabetic retinopathy and its preparation method. The drug uses red peony root as the principal ingredient, astragalus root, rehmannia root, and cattail pollen as assistant ingredients, and is supplemented with privet fruit, eclipta herb, rhubarb, notoginseng, earthworm, ginkgo leaf, and cassia seed, with kudzu root as the guiding ingredient. The combined effects of these herbs are to promote blood circulation, invigorate qi and nourish yin, stop bleeding, and improve eyesight. This drug is an innovative traditional Chinese medicine with broad market prospects; therefore, reliable testing methods are needed to ensure its safe and controllable quality.

[0005] Chinese Patent 202410912823.5 discloses a method for determining the fingerprint spectrum of a traditional Chinese medicine composition. The composition is made from Paeonia lactiflora, Astragalus membranaceus, Rehmannia glutinosa, Typha orientalis, Ligustrum lucidum, Eclipta prostrata, Ginkgo biloba, Rheum palmatum, Panax notoginseng, Pheretima aspergillum, Cassia tora, and Pueraria lobata. The fingerprint spectrum determination method uses puerarin, paeoniflorin, ligustrum lucidum, aloe-emodin, rhein, emodin, rhein, and rhein methyl ether as reference standards. This method only provides qualitative and quantitative detection of eight active ingredients in the traditional Chinese medicine composition. While it can provide a certain basis for quality control of the composition, it is not perfect and falls far short of meeting international pharmaceutical quality testing standards. Summary of the Invention

[0006] To address the problem of imperfect identification methods for chemical components in the aforementioned traditional Chinese medicine compositions containing Paeonia lactiflora, the inventors have provided the following technical solution.

[0007] A method for detecting chemical components in a traditional Chinese medicine composition, wherein the traditional Chinese medicine composition is made from Paeonia lactiflora, Astragalus membranaceus, Rehmannia glutinosa, Typha orientalis, Ligustrum lucidum, Eclipta prostrata, Rheum palmatum, Panax notoginseng, Pheretima aspergillum, Ginkgo biloba, Cassia tora, and Pueraria lobata, and the method employs an ultra-high performance liquid chromatography-mass spectrometry system. The method includes the preparation of the test solution, liquid chromatography separation, and mass spectrometry detection.

[0008] The solvent used in the preparation of the test solution is methanol or ethanol;

[0009] In liquid chromatography analysis, mobile phase A is an aqueous formic acid solution and mobile phase B is an acetonitrile formic acid solution, using gradient elution.

[0010] Mass spectrometry analysis uses an ESI ionization source and two detection modes: positive ion and negative ion.

[0011] In the above detection method, the solvent used to prepare the test solution is 50% methanol.

[0012] In the above detection method, the mobile phase A for liquid chromatography analysis is a 0.1% formic acid aqueous solution, and the mobile phase B is a 0.1% formic acid acetonitrile solution. The specific operation of the gradient elution of the mobile phase is as follows:

[0013] .

[0015] In the above detection method, the chromatographic column used in the liquid chromatography analysis is preferably a C18 reversed-phase column with a particle size of 1.8 μm.

[0016] In the above detection method, the flow rate of the mobile phase in the liquid chromatography analysis is 0.25-0.35 mL / min, preferably 0.30 mL / min, and the column temperature is 30-40℃, preferably 35℃.

[0017] In the above detection method, the preferred detection modes for positive and negative ions in the mass spectrometry analysis are:

[0018] Positive ion mode detection: m / z range 50-1200, capillary voltage 2.0kV, cone voltage 40kV, ion source temperature 100℃, desolvation temperature 450℃, desolvation gas flow rate 800L / h, cone gas flow rate 50L / h, mass-corrected mass-to-nucleus ratio m / z 556.2771;

[0019] Negative ion mode detection: m / z range is 50-1200, capillary voltage is 2.0kV, cone voltage is 40kV, ion source temperature is 100℃, desolvation temperature is 450℃, desolvation gas flow rate is 800L / h, cone gas flow rate is 50L / h, and mass-corrected mass-to-nucleus ratio m / z is 554.2615.

[0020] The detection method provided by this invention can be used to detect the chemical components in a traditional Chinese medicine composition (TLMMC) made from red peony root, astragalus root, rehmannia root, cattail pollen, privet fruit, eclipta prostrata, rhubarb, notoginseng, earthworm, ginkgo leaf, cassia seed, and kudzu root. The traditional Chinese medicine composition can be in various dosage forms such as capsules, tablets, granules, pills, and powders.

[0021] The composition and preparation method of the traditional Chinese medicine composition (TLMMC) described in this invention are the same as those in Chinese patent CN1833698A, in which the molian grass mentioned is molian hanlian, kudzu root is fen ge, and rehmannia root is rehmannia root.

[0022] The traditional Chinese medicine composition of the present invention comprises the following parts by weight: 150-250 parts of Paeonia lactiflora, 250-350 parts of Astragalus membranaceus, 150-250 parts of Rehmannia glutinosa, 100-200 parts of Typha orientalis, 150-250 parts of Ligustrum lucidum, 150-250 parts of Eclipta prostrata, 250-350 parts of Ginkgo biloba, 50-150 parts of Rheum palmatum, 50-150 parts of Panax notoginseng, 50-150 parts of Pheretima aspergillum, 250-350 parts of Cassia tora, and 150-250 parts of Pueraria lobata.

[0023] The preferred weight composition of the traditional Chinese medicine composition is as follows: 188 parts of Paeonia lactiflora, 282 parts of Astragalus membranaceus, 188 parts of Rehmannia glutinosa, 141 parts of Typha orientalis, 188 parts of Ligustrum lucidum, 188 parts of Eclipta prostrata, 282 parts of Ginkgo biloba, 94 parts of Rheum palmatum, 94 parts of Panax notoginseng, 94 parts of Pheretima aspergillum, 282 parts of Cassia tora, and 188 parts of Pueraria lobata.

[0024] Alternatively, the following ingredients may be preferred: 150 parts red peony root, 250 parts astragalus root, 150 parts rehmannia root, 100 parts cattail pollen, 150 parts privet fruit, 150 parts eclipta prostrata, 250 parts ginkgo leaf, 50 parts rhubarb, 50 parts notoginseng, 50 parts earthworm, 250 parts cassia seed, and 150 parts kudzu root.

[0025] Alternatively, the following ingredients may be preferred: 250 parts red peony root, 350 parts astragalus root, 250 parts rehmannia root, 200 parts cattail pollen, 250 parts privet fruit, 250 parts eclipta prostrata, 350 parts ginkgo leaf, 150 parts rhubarb, 150 parts notoginseng, 150 parts earthworm, 350 parts cassia seed, and 250 parts kudzu root.

[0026] Alternatively, the following ingredients may be preferred: 150 parts red peony root, 250 parts astragalus root, 150 parts rehmannia root, 100 parts cattail pollen, 150 parts privet fruit, 150 parts eclipta prostrata, 350 parts ginkgo leaves, 150 parts rhubarb, 150 parts notoginseng, 150 parts earthworm, 350 parts cassia seed, and 250 parts kudzu root.

[0027] The preparation method of the capsule of the traditional Chinese medicine composition includes the following steps:

[0028] (1) Weigh out the amount of cattail pollen according to the prescription, use 4-8 times the amount of 60-80% ethanol as solvent, first soak the medicinal material with an appropriate amount of solvent, seal for 10-30 minutes, then pack it into a percolation column, add the remaining solvent to soak for 20-36 hours, percolate at a rate of 2-4 mL / kg, collect the percolate for later use.

[0029] (2) Weigh out the raw Rehmannia glutinosa and earthworm according to the prescription. Add 8-10 times the amount of water for the first time, soak for 10-30 minutes, heat and decoct for 1-2 hours, and filter. Add 6-9 times the amount of water for the second time, decoct for 1-2 hours, filter, combine the filtrates, concentrate to a relative density of 1.15-1.20, test at 60℃, cool, add an appropriate amount of ethanol to make the alcohol content reach 50-70%, stir thoroughly, refrigerate at below 4℃ for 20-36 hours, filter, and keep the filtrate for later use.

[0030] (3) Weigh out Astragalus membranaceus and Paeonia lactiflora according to the prescription, add 6-10 times the amount of 40-60% ethanol, soak for 10-30 minutes, reflux extract 2-3 times, 1-3 hours each time, filter, combine the filtrates and set aside.

[0031] (4) Weigh out the following ingredients according to the prescription: Ligustrum lucidum, Eclipta prostrata, Pueraria lobata, Ginkgo biloba leaves, Rheum palmatum, and Cassia tora. Add 6-9 times the amount of 70-

[0032] Soak in 90% ethanol for 10-30 minutes, reflux and extract 1-3 times, 1-3 hours each time, filter, combine the filtrates and set aside;

[0033] (5) Combine the four extracts obtained in the above steps, recover the ethanol under reduced pressure, and continue to concentrate to a clear extract with a relative density of 1.25-1.30 and a heat test at 60℃;

[0034] (6) Weigh out the amount of Panax notoginseng according to the prescription, grind it into 100-120 mesh powder, add it to the clear extract obtained in step (5), stir evenly, dry it under vacuum of 0.07-0.09 MPa and temperature of 65-70℃, and grind the resulting dry extract into 80-120 mesh powder for later use.

[0035] (7) Take the dry powder obtained in step (6) and starch, mix them evenly, use 70-90% ethanol as a binder to make soft material, granulate with a 20-mesh sieve, dry at 60-70℃, granulate with an 18-20 mesh sieve, and fill into capsules to obtain the product.

[0036] The detection method provided by this invention can be used as an identification method for the quality control of traditional Chinese medicine composition (TLMMC) preparations. In addition to the steps of the detection method described above, this identification method also includes the preparation of standard solutions. The preparation of the standard solutions is as follows: ① Accurately weigh appropriate amounts of the standard or reference substances of the chemical components contained in the traditional Chinese medicine composition, dissolve them in an appropriate amount of DMSO, and prepare individual standard stock solutions with a concentration of 1.00 mg / mL; ② Accurately pipette an appropriate amount of the individual standard stock solution, dilute it with 50% methanol, and prepare a mixed standard solution with a concentration of 20 μg / mL for each individual standard or reference substance.

[0037] In the above identification method, the chemical components are astragaloside A, and / or verrucoside glucoside, and / or verrucoside, and / or gentianin, and / or gentianin, and / or paeoniflorin, and / or paeoniflorin lactone, and / or benzoyl paeoniflorin, and / or catechin, and / or gallic acid, and / or privetin, and / or rhodioloside, and / or privetin, and / or ursolic acid, and / or catalpol, and / or rehmannia glycoside D, and / or wedelia lactone, and / or ecliptaside A, and / or puerarin, and / or Daidaidzein, and / or daidzeinogen, and / or isorhamnetin-3-O-neohesperidin, and / or typhain, and / or quercetin, and / or ginkgolide A, and / or ginkgolide B, and / or ginkgolide C, and / or ginkgolide, and / or kaempferol, and / or isorhamnetin, and / or rutin, and / or luteolin, and / or emodin, and / or rhein, and / or emodin methyl ether, and / or aloe-emodin, and / or chrysophanol, and / or cassia seed extract, and / or ferulic acid, and / or ginsenoside R g1 and / or ginsenoside R b1And / or notoginsenoside R1, and / or geniposide, and / or adenosine, and / or protocatechuic acid, and / or paeoniflorin, and / or phenylalanine, and / or puerarin apigenin, and / or echinacoside, and / or genistein, and / or quercetin 3-O-sophoroside, and / or luteolin, and / or isochlorogenic acid B, and / or isorauscoside, and / or isochlorogenic acid C, and / or isochlorogenic acid A, and / or trefoilin-7-O-β-D-glucopyranoside, and / or quercetin-3-O-(2G-α-L-rhamnosyl)-rutin, and / or ginsenoside Re, and / or leonurin, and / or 3-hydroxypuerarin, and / or cassia seed extract. And / or naringenin, and / or ecliptaside IV, and / or phloroglucin A, and / or styracin, and / or ginsenoside Rd, and / or diosmin, and / or cassiaside, and / or astragaloside I, and / or ginkgo biloba flavonoids, and / or astragaloside II, and / or isoginkgo biloba flavonoids, and / or cassiaside C, and / or chlorogenic acid, and / or pine biloba flavonoids, and / or leucamide phenylalanine, and / or guanosine, and / or rehmannia glutinosa glycoside, and / or iso-micro-salicylate, and / or [2,2,4-trimethyl-(3-pentanyl)oxy]-4-oxo-2-butenoate, and / or Pratensein 7-O-glucoside.

[0038] The detection method provided by this invention uses ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) to detect and analyze the chemical components in the traditional Chinese medicine composition TLMMC. Through precise molecular weight and fragment ion comparison, a total of 82 chemical components were qualitatively identified, and 42 of these chemical components were further confirmed using existing reference standards. These 82 chemical components include 36 flavonoids, 11 triterpenoid saponins, 11 monoterpenes, 8 anthraquinones, and 16 other compounds; among them, 13 chemical components are from Astragalus membranaceus, 13 from Ginkgo biloba leaves, 8 from Paeonia lactiflora, 8 from Eclipta prostrata, 6 from Rehmannia glutinosa, 6 from Pueraria lobata, 5 from Cassia tora seeds, 5 from Rheum palmatum, 5 from Panax notoginseng, 5 from Typha pollen, 4 from Ligustrum lucidum, and 4 from Pheretima aspergillum, covering all 12 traditional Chinese medicines in TLMMC.

[0039] The 82 chemical components identified by the test are: astragaloside A, verbascoside, verbascoside, gentianin, gentianin, paeoniflorin, paeoniflorin lactone, benzoylpaeoniflorin, catechin, gallic acid, privetin, rhodioloside, privetin, ursolic acid, catalpol, rehmannia glycoside D, wedelia lactone, ecliptaside A, puerarin, daidzein, daidzein, isorhamnetin-3-O-neohesperidin, typhain, quercetin, ginkgolide A, ginkgolide B, ginkgolide C, ginkgol, kaempferol, isorhamnetin, rutin, luteolin, emodin, rhein, emodin methyl ether, aloe-emodin, rhein, cassia seed extract, ferulic acid, and ginsenoside R. g1 Ginsenoside R b1 Notoginsenoside R1, geniposide, adenosine, protocatechuic acid, paeoniflorin, phenylalanine, puerarin apigenin, echinacoside, genistein, quercetin 3-O-locoloside, luteolin, isochlorogenic acid B, isoverbascoside, isochlorogenic acid C, isochlorogenic acid A, red cloverin-7-O-β-D-glucopyranoside, quercetin-3-O-(2G-α-L-rhamnosyl) - Rutin, Ginsenoside Re, Leonurusin, 3-Hydroxypusin, Cassiatoside, Naringenin, Ecliptaside IV, Cinnamomum cassiaside A, Araliacetin, Ginsenoside Rd, Diosmin, Cassiatoside, Astragaloside I, Ginkgo Biflavonoids, Astragaloside II, Isoginkgo Biflavonoids, Cassiatoside C, Chlorogenic Acid, Psoralen Biflavonoids, Leucamide Phenylalanine, Guanosin, Rehmannia glutinosa, Iso-micro-codonopsis pilosula

[0040] [2,2,4-trimethyl-(3-pentanyl)oxy]-4-oxo-2-butenoate, Pratensein 7-O-glucoside.

[0041] The 42 chemical components compared with existing reference standards are astragaloside A, verbascoside glucoside, verbascoside, gentianin, gentianin, paeoniflorin, paeoniflorin lactone, benzoylpaeoniflorin, catechin, gallic acid, privetin, rhodioloside, privetin, ursolic acid, catalpol, rehmannia glycoside D, wedelia lactone, ecliptaside A, puerarin, daidzein, daidzein, isorhamnetin-3-O-neohesperidin, typhain, quercetin, ginkgolide A, ginkgolide B, ginkgolide C, ginkgol, kaempferol, isorhamnetin, rutin, luteolin, emodin, rhein, emodin methyl ether, aloe-emodin, rhein, cassia seed extract, ferulic acid, ginsenoside R g1 Ginsenoside R b1 Panax notoginsenoside R1.

[0042] The detection method provided by this invention is rapid and reliable, comprehensively analyzes the chemical profile of TLMMC, and systematically elucidates the pharmacodynamic material basis of TLMMC, providing a technical foundation for its quality control, effective component analysis, toxic component research, mechanism of action research, and rational clinical application. Attached Figure Description

[0043] Figure 1 This is the extraction chromatogram of the traditional Chinese medicine composition TLMMC in positive ion mode.

[0044] Figure 2 This is the extraction chromatogram of the traditional Chinese medicine composition TLMMC under negative ion mode. Detailed Implementation

[0045] The present invention will be further described in detail below with reference to specific embodiments.

[0046] Example

[0047] 1. Materials

[0048] 1.1 Instruments and Equipment

[0049] Waters Acquity UPLC system and Q-TOF SYNAPT G2 mass spectrometer (Waters, USA); IKA Vortex2 vortex oscillator (Aika (Guangzhou) Instrument Equipment Co., Ltd.); KQ-500E ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); DV215CD electronic balance (Ohaus Instruments (Shanghai) Co., Ltd.); CP224C electronic balance (Ohaus Instruments (Shanghai) Co., Ltd.); Sigma 3k-15 low-temperature benchtop centrifuge (Beijing Wuzhou Dongfang Technology Development Co., Ltd.); SECURA3102-1CN electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.).

[0050] 1.2 Reference Standards and Reagents

[0051] Astragaloside A (batch number PS012327, Chengdu Pusi Biotechnology Co., Ltd., mass fraction ≥98.00% as determined by HPLC), verbenafil isoflavone glucoside (batch number PS000687, Chengdu Pusi Biotechnology Co., Ltd., mass fraction ≥98.00% as determined by HPLC), verbenafil isoflavone (batch number PS010251, Chengdu Pusi Biotechnology Co., Ltd., mass fraction ≥98.00% as determined by HPLC), styracin (batch number PS000674, Chengdu Pusi Biotechnology Co., Ltd., mass fraction ≥98.00% as determined by HPLC), styracin glycoside (batch number PS000671, Chengdu Pusi Biotechnology Co., Ltd., mass fraction ≥98.00% as determined by HPLC), and styracin glycoside (batch number PS000671, Chengdu Pusi Biotechnology Co., Ltd., mass fraction ≥98.00% as determined by HPLC). The following compounds were found to contain the following components: paeoniflorin (batch number 110736-201438, China National Institutes for Food and Drug Control, HPLC score ≥96.40%), paeoniflorin lactone (batch number PS011455, Chengdu Pusi Biotechnology Co., Ltd., HPLC score ≥95.00%), benzoylpaeoniflorin (batch number PS13011802, Chengdu Pusi Biotechnology Co., Ltd., HPLC score ≥99.81%), catechin (batch number PS012798, Chengdu Pusi Biotechnology Co., Ltd., HPLC score ≥98.00%), and gallic acid (batch number PS000688, Chengdu Pusi Biotechnology Co., Ltd.). The company's products, with a mass fraction ≥98.00% as determined by HPLC, include: purslane (batch number A04HB190321, Shanghai Yuanye Biotechnology Co., Ltd., with a mass fraction ≥98.00% as determined by HPLC), rhodioloside (batch number J25HB186356, Shanghai Yuanye Biotechnology Co., Ltd., with a mass fraction ≥98.00% as determined by HPLC), privetin (batch number P26D9F78576, Shanghai Yuanye Biotechnology Co., Ltd., with a mass fraction ≥98.00% as determined by HPLC), ursolic acid (batch number AF21020553, Chengdu Efa Biotechnology Co., Ltd., with a mass fraction ≥98.00% as determined by HPLC), and citronellol (batch number AF20070). 652, Chengdu Efa Biotechnology Co., Ltd., with a mass fraction ≥98.00% as determined by HPLC; Rehmannia glutinosa D (batch number AF20120402, Chengdu Efa Biotechnology Co., Ltd., with a mass fraction ≥98.00% as determined by HPLC); Wedelia trifoliata lactone (batch number Z28D11S135868, Shanghai Yuanye Biotechnology Co., Ltd., with a mass fraction ≥98.00% as determined by HPLC); Eclipta prostrata A (batch number A25HB192084, Shanghai Yuanye Biotechnology Co., Ltd., with a mass fraction ≥98.00% as determined by HPLC); Puerarin (batch number JOT-10054, Chengdu Pufeide Biotechnology Co., Ltd., with a mass fraction ≥98.00% as determined by HPLC).61%), daidzein (batch number 111738-20160, China National Institutes for Food and Drug Control, mass fraction ≥93.30% by HPLC), daidzein (batch number 111502-200402, China National Institutes for Food and Drug Control, mass fraction ≥99.90% by HPLC), isorhamnetin-3-O-neohesperidin (batch number 55033-90-4, Shanghai Yuanye Biotechnology Co., Ltd., mass fraction ≥98.00% by HPLC), typhain (batch number 104472-68-6, Shanghai Yuanye Biotechnology Co., Ltd., mass fraction ≥98.00% by HPLC), quercetin (batch number PS010462, Chengdu Pusi Biotechnology Co., Ltd.) The following compounds were found to contain ≥98.00% ginkgolide (batch number D04D11G133061, Shanghai Yuanye Biotechnology Co., Ltd.), Ginkgolide B (batch number D24J11G119518, Shanghai Yuanye Biotechnology Co., Ltd.), Ginkgolide C (batch number A05GB157055, Shanghai Yuanye Biotechnology Co., Ltd.), Ginkgolide (batch number J19HB179213, Shanghai Yuanye Biotechnology Co., Ltd.), and Kaempferol (batch number JOT-1). 0060, Chengdu Pufeide Biotechnology Co., Ltd., with a mass fraction ≥99.10% as determined by HPLC), isorhamnetin (batch number J30GB154008, Shanghai Yuanye Biotechnology Co., Ltd., with a mass fraction ≥98.00% as determined by HPLC), rutin (batch number PS020288, Chengdu Pusi Biotechnology Co., Ltd., with a mass fraction ≥98.00% as determined by HPLC), luteolin (batch number PU0033-0025MG, Chengdu Pusi Biotechnology Co., Ltd., with a mass fraction ≥98.00% as determined by HPLC), emodin (batch number A26IB224130, Shanghai Yuanye Biotechnology Co., Ltd., with a mass fraction ≥98.00% as determined by HPLC), rhein (batch number...). The following products were found to contain: A26IB224130 (Shanghai Yuanye Biotechnology Co., Ltd., HPLC mass fraction ≥98.00%), emodin methyl ether (Batch No. M08IB214534, Shanghai Yuanye Biotechnology Co., Ltd., HPLC mass fraction ≥98.00%), aloe-emodin (Batch No. A17GB145379, Shanghai Yuanye Biotechnology Co., Ltd., HPLC mass fraction ≥98.00%), rhein (Batch No. JOT-10022, Chengdu Pufeide Biotechnology Co., Ltd., HPLC mass fraction ≥99.12%), and cassia seed extract (Batch No. JOT-11440, Shanghai Yuanye Biotechnology Co., Ltd., HPLC mass fraction ≥98.00%).69%), ferulic acid (batch number G13S11L124423, Shanghai Yuanye Biotechnology Co., Ltd., mass fraction ≥98.00% as determined by HPLC), ginsenoside R. g1 (Batch number J08IB205578, Shanghai Yuanye Biotechnology Co., Ltd., mass fraction ≥98.00% as determined by HPLC), Ginsenoside R b1 (Batch No. J191B217937, Shanghai Yuanye Biotechnology Co., Ltd., mass fraction ≥98.00% as determined by HPLC), Panax notoginseng saponin R1 (Batch No. A21IB213074, Shanghai Yuanye Biotechnology Co., Ltd., mass fraction ≥98.00% as determined by HPLC).

[0052] Mass spectrometry-grade methanol (MEOH) was purchased from Fisher Scientific, lot number: 178335; mass spectrometry-grade acetonitrile (ACN) was purchased from Fisher Scientific, lot number: 172473; dimethyl sulfoxide (DMSO) was purchased from Amresco Scientific, lot number: 2543C551; formic acid (HCOOH) was purchased from Sinopharm Chemical Reagent Beijing Co., Ltd.; and purified water was purchased from Hangzhou Wahaha Co., Ltd.

[0053] 1.3 Test Sample

[0054] The test sample is a capsule of the traditional Chinese medicine composition (TLMMC) described in this invention, provided by Shijiazhuang Yiling Pharmaceutical Co., Ltd., batch number 20230911, with a specification of 0.4g per capsule (equivalent to 2.21g of decoction pieces).

[0055] 2 methods

[0056] 2.1 Preparation of Standard Solutions and Sample Solutions

[0057] 2.1.1 Preparation of standard solutions

[0058] Standard stock solutions: Take appropriate amounts of each of the 42 compound reference standards, weigh them accurately, dissolve them in an appropriate amount of DMSO, and prepare individual standard stock solutions with a concentration of 1.00 mg / mL.

[0059] Mixed standard solution: Accurately pipette appropriate amounts of the individual standard stock solutions of 42 compounds with a concentration of 1.00 mg / mL, place them in the same volumetric flask, dilute to the mark with 50% methanol, and prepare a mixed standard solution with a concentration of 20 μg / mL for each compound.

[0060] 2.1.2 Preparation of the test solution

[0061] Take an appropriate amount of TLMMC contents, accurately weigh it, dissolve it in 50% methanol, sonicate it (power 400W, frequency 40kHz, room temperature 25℃) for 30 minutes, add 50% methanol to make up the volume, and prepare a test solution with a concentration of 1 mg / mL based on the contents of the capsule. Analyze it by UHPLC-Q-TOF-MS.

[0062] 2.2 Establishment of TLMMC-related component database

[0063] To ensure the reliability of the identified spectral peaks, an internal database of TLMMC was established by integrating TCMSP (Database of Systems Pharmacology of Traditional Chinese Medicine), TCMID (Comprehensive Database of Traditional Chinese Medicine), ChemSpider (http: / / www.ChemSpider.com / ), Chemical Book (http: / / www.chemicalbook.com / ), and relevant literature. The compound name, molecular formula, molecular weight, structural formula, and source information were listed in an Excel spreadsheet.

[0064] 2.3U HPLC–Q–TOF–MS analysis

[0065] 2.3.1 Liquid Chromatography Conditions

[0066] The chromatographic column was an Acquity UPlc HSS T3 column (100 mm × 2.1 mm, 1.8 μm). Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution. The UHPLC elution gradient is shown in Table 1. The injection volume was 5 μL, and the run time for each sample was 80 min.

[0067] Table 1. Elution gradient of mobile phase used in UHPLC for qualitative analysis of TLMMC chemical composition.

[0068]

[0069] 2.3.2 Mass Spectrometry Conditions

[0070] An ESI ionization source is used.

[0071] Positive ion mode detection: m / z range 50-1200, capillary voltage 2.0kV, cone voltage 40kV, ion source temperature 100℃, desolvation temperature 450℃, desolvation gas flow rate 800L / h, cone gas flow rate 50L / h, mass-corrected mass-to-nucleus ratio m / z 556.2771.

[0072] Negative ion mode detection: m / z range is 50-1200, capillary voltage is 2.0kV, cone voltage is 40kV, ion source temperature is 100℃, desolvation temperature is 450℃, desolvation gas flow rate is 800L / h, cone gas flow rate is 50L / h, and mass-corrected mass-to-nucleus ratio m / z is 554.2615.

[0073] 2.4 Chemical Composition Analysis

[0074] An automated screening process was conducted using the previously established TLMMC internal database to identify compounds with matching molecular formulas as potential candidates. A list of compounds with a matching score higher than 85.00 was manually created, including accurate precursor ion mass and retention time information. The score was calculated by software, considering not only the precise mass number but also isotopic distribution: a higher score indicates a more reliable elemental composition. Based on this list, target MS / MS analysis was performed. Candidate compounds were identified by their retention time, accurate mass with an error within 10 ppm, and reasonable fragmentation pathways, and were confirmed using available standards.

[0075] 2.5 Data Processing and Analysis

[0076] Mass spectrometry peak data of all identified compounds in the sample were extracted and analyzed using Masslynx software.

[0077] 3 Results

[0078] 3.1 Identification of the main components of TLMMC

[0079] The TLMMC test solution was analyzed using UHPLC-Q-TOF-MS, and the resulting extraction chromatogram was obtained. Figure 1 This is the extraction chromatogram of TLMMC in positive ion mode. Figure 2 This is the extraction chromatogram of TLMMC in negative ion mode.

[0080] This invention qualitatively identified 82 chemical components in TLMMC. By comparing the retention time, precise molecular weight, and fragment ion composition of existing reference standards, 42 of these components were identified; another 40 components were inferred through comparison of precise molecular weight and fragment ion composition. These chemical components are distributed among the 12 medicinal ingredients used in the preparation of TLMMC, and are mainly classified into flavonoids, triterpenoid saponins, monoterpenoids, anthraquinones, organic acids, and other categories. The identification and classification results are shown in Table 2.

[0081] Table 2 Identification and Attribution of Chemical Components of TLMMC

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090] Note: # * indicates a compound that has been compared with a reference standard; * indicates a compound confirmed by mass spectrometry peak data analysis using Masslynx software.

[0091] ① The corresponding Chinese name for [2,2,4-trimethyl-(3-pentanyl)oxy]-4-oxo-2-butenoate could not be found.

[0092] ② is Pratensein 7-O-glucoside, but no corresponding Chinese name was found.

[0093] 3.1.1 Flavonoids

[0094] Based on the basic skeleton and substituents of the compounds, this study identified 36 flavonoid components, including gentianin, gentianoside, verbascoside, and catechins, mainly derived from Astragalus membranaceus and Ginkgo biloba leaves. They exhibited good responses in both positive and negative ion modes, with their molecular ion peaks mostly ending in [M+H]. + and [MH] - It exists in the form of [specific compound name - likely a reference standard]. By comparing the retention time and precise molecular weight with those of the reference standard, compound 46 was identified as luteolin, with a molecular ion peak at m / z 285.0 [MH]. - MS 2 The spectrum shows a fragment ion peak at m / z 268.1 indicating the loss of one neutral H₂O molecule; simultaneously, there is significant RDA breakage, forming a fragment ion at m / z 151.0; further neutral loss of one H₂O molecule forms a fragment ion at m / z 133.0; the molecular ion peak of compound 25 is at m / z 447.1 [MH]. - It has 162.1 Da more than compound 46 (luteolin), and its fragment ions m / z 285.1, m / z 268.1, m / z 151.1, and m / z 133.1 are all similar to luteolin, so the compound is identified as luteolinoglycoside.

[0095] Compound 19 was detected with a molecular ion peak at m / z 447.1 in positive ion mode. The loss of one glucose molecule resulted in a fragment ion at m / z 285.1, followed by the loss of another CO molecule to m / z 257.1. The characteristic ions at m / z 149.0 and m / z 137.0 are formed by the RDA breakage of the C-ring of its flavonoid core. Comparison of retention time and molecular weight with the reference standard confirmed that this component is verbascoside glucoside. Compound 43 has one less Glc molecule than verbascoside glucoside, and its secondary mass spectrum shows a similar fragmentation pattern. Comparison of retention time with the reference standard confirmed that this component is verbascoside.

[0096] Analysis results show that flavonoids are the main active ingredients in TLMMC.

[0097] 3.1.2 Triterpenoid saponins

[0098] This invention identified 11 triterpenoid saponins from TLMMC, mainly tetracyclic triterpenoids from Astragalus membranaceus and Panax notoginseng, such as astragaloside A, astragaloside II, astragaloside I, and notoginsenoside R1. They are mostly [M+H] compounds. + [MH] - or[M+Na] + It exists in the form of a quasi-molecular ion peak. It is ginsenoside R. g1 For example, its secondary mass spectrum shows continuous breaking of oxyglycosidic bonds, with fragment ions appearing at m / z 637.5 and m / z 475.4, presumably formed after the continuous loss of two molecules of glucuronide; compound 63 has a molecular ion peak at m / z 807.5 [M+Na] in positive ion mode. + The precursor ion loses glucose to form m / z 623.4, and further loses xylose to form m / z 491.3. After comparison with the astragaloside A reference standard, its chromatographic retention behavior and mass spectrometric fragmentation mode are basically consistent, so the compound is identified as astragaloside A.

[0099] 3.1.3 Monoterpenes

[0100] This invention identified 11 monoterpenoids from TLMMC, mainly derived from Paeonia lactiflora. These components are mostly pinane-type monoterpenes, including paeoniflorin, paeoniflorin lactone, benzoylpaeoniflorin, and oxypaeoniflorin, etc., and exhibit good response in negative ion mode, with [MH] - [M-H+HCOOH] - It exists in the form of a quasi-molecular ion peak. Taking paeoniflorin as an example, in MS... 2 In the spectrum, paeoniflorin is represented by [M-H+HCOOH]. -The compound exists in the form of an ion (m / z 525.2). The loss of one benzoic acid molecule and one formaldehyde molecule produces a fragment ion with m / z 327.1. The fragment with m / z 121.0 is presumed to be benzoic acid. Compound 12 has the same molecular weight as paeoniflorin and should be an isomer of paeoniflorin. Using Masslynx, its molecular formula is estimated to be C1. 23 H 28 O 11 However, no ion peak at m / z 449.2 was observed in the secondary mass spectrum. The remaining fragmentation patterns were similar to those of paeoniflorin, indicating that the component was paeoniflorin lactone. By comparing the retention time with its reference standard, it was finally identified as paeoniflorin lactone.

[0101] 3.1.4 Anthraquinones

[0102] This invention identified eight anthraquinone compounds, mainly derived from rhubarb, including rhein, emodin, aloe-emodin, emodin methyl ether, and chrysophanol. These compounds showed good response in negative ion mode, with [MH] - These compounds exist in the form of quasi-molecular ion peaks, and continuously lose CO during mass spectrometry fragmentation. Taking emodin as an example, in MS... 2 In the spectrum, rhein is represented by [MH]. - The compound 44 exists in the form of (m / z 269.0) ions. The successive loss of three CO molecules generates fragment ion peaks at m / z 241.1, m / z 225.9, and m / z 197.8, respectively. Compound 44 was detected in negative ion mode with a molecular ion peak at m / z 283.0. The successive loss of COOH and CO molecules produces fragment ions at m / z 239.0 and m / z 211.1. Comparison with the retention time and molecular weight of the reference standard confirms that this component is rhein.

[0103] 3.2 Distribution and Classification of Major Components in TLMMC

[0104] This invention qualitatively identified 82 components in TLMMC, of ​​which 42 components have been compared with existing reference standards. These 82 compounds include 36 flavonoids, 11 triterpenoid saponins, 11 monoterpenes, 8 anthraquinones, and 16 other compounds; among them, 13 chemical components are from Astragalus membranaceus, 13 compounds are from Ginkgo biloba leaves, 8 chemical components are from Paeonia lactiflora, 8 chemical components are from Eclipta prostrata, 6 chemical components are from Rehmannia glutinosa, 6 chemical components are from Pueraria lobata, 5 chemical components are from Cassia tora, 5 chemical components are from Rheum palmatum, 5 chemical components are from Panax notoginseng, 5 chemical components are from Typha pollen, 4 chemical components are from Ligustrum lucidum, and 4 chemical components are from Pheretima aspergillum.

[0105] 4 Results Analysis

[0106] As a traditional Chinese medicine compound, TLMMC has a complex and diverse chemical composition, making separation difficult. In addition, it contains multiple pairs of isomers such as luteolin and kaempferol, paeoniflorin and paeoniflorin lactone, emodin and aloe-emodin, which greatly increases the difficulty of separation.

[0107] During the research process, the present invention examined multiple conditions of the detection method.

[0108] First, the extraction of TLMMC components was investigated using 50% methanol, 100% methanol, and 50% ethanol as solvents. It was found that the TLMMC component dissolution rate was highest when 50% methanol was added and ultrasonically extracted for 30 minutes.

[0109] Secondly, the flow rates of the detection method were tested at 0.20, 0.30, and 0.40 mL / min. The results showed that a flow rate of 0.20 mL / min resulted in poor column resolution and asymmetrical broad peaks, which was not conducive to component identification. A flow rate of 0.40 mL / min was too high, leading to increased column pressure and a large accumulation of chromatographic peaks. A flow rate of 0.30 mL / min resulted in good peak shape and good chromatographic peak resolution. Therefore, a flow rate of 0.30 mL / min was ultimately selected.

[0110] In addition, this invention tested three mobile phases: methanol:water, acetonitrile:water, and acetonitrile:0.1% formic acid water. It was found that for the complex traditional Chinese medicine compound described in this invention, acetonitrile has better elution ability than methanol, while the addition of formic acid improves the baseline resolution to a certain extent and optimizes the peak shape.

[0111] In addition, the column temperature was also investigated during the research process. Column temperatures of 20, 35 and 45°C were tried, and it was found that the method had the best adaptability at 35°C.

[0112] Finally, this invention experimented with adjusting the mobile phase gradient and chromatographic separation time based on the types and polarities of components in TLMMC. At a time of 60 min, a large number of chromatographic peaks accumulated, resulting in poor resolution. Therefore, adjusting the time to 80 min yielded better peak resolution and better peak shape, and this method was determined to be the final optimized method.

[0113] The UHPLC-Q-TOF-MS method established in this invention can rapidly and comprehensively analyze the chemical profile of TLMMC and systematically elucidate its chemical components.

Claims

1. A method for detecting chemical components in a traditional Chinese medicine composition, wherein the traditional Chinese medicine composition is made from Paeonia lactiflora, Astragalus membranaceus, Rehmannia glutinosa, Typha orientalis, Ligustrum lucidum, Eclipta prostrata, Ginkgo biloba, Rheum palmatum, Panax notoginseng, Pheretima aspergillum, Cassia tora, and Pueraria lobata, characterized in that, The detection method employs an ultra-high performance liquid chromatography-mass spectrometry system, including the preparation of the test sample solution, liquid chromatography separation, and mass spectrometry detection; The solvent used in the preparation of the test solution is methanol or ethanol; The mobile phase A for liquid chromatography separation was an aqueous formic acid solution, and the mobile phase B was an aqueous formic acid solution with acetonitrile, using gradient elution. Mass spectrometry detection uses an ESI ionization source and supports both positive and negative ion detection modes.

2. The detection method according to claim 1, characterized in that, The solvent used to prepare the test solution is 50% methanol.

3. The detection method according to claim 1, characterized in that, The mobile phase A for the liquid chromatography separation is a 0.1% formic acid aqueous solution, and the mobile phase B is a 0.1% formic acid acetonitrile solution. The specific operation of the mobile phase gradient elution is as follows:

4. The detection method according to claim 1, characterized in that, The chromatographic column used in the liquid chromatography separation is a C18 reversed-phase column with a particle size of 1.8 μm.

5. The detection method according to claim 1, characterized in that, The flow rate of the mobile phase in the liquid chromatography separation is 0.25-0.35 mL / min, and the column temperature is 30-40℃.

6. The detection method according to claim 1, characterized in that, The two detection modes for positive and negative ions in the mass spectrometry analysis are as follows: Positive ion mode detection: m / z range 50-1200, capillary voltage 2.0kV, cone voltage 40kV, ion source temperature 100℃, desolvation temperature 450℃, desolvation gas flow rate 800L / h, cone gas flow rate 50L / h, mass-corrected mass-to-nucleus ratio m / z 556.2771; Negative ion mode detection: m / z range is 50-1200, capillary voltage is 2.0kV, cone voltage is 40kV, ion source temperature is 100℃, desolvation temperature is 450℃, desolvation gas flow rate is 800L / h, cone gas flow rate is 50L / h, and mass-corrected mass-to-nucleus ratio m / z is 554.2615.

7. The application of the detection method according to any one of claims 1-6 in the quality control method of a preparation of traditional Chinese medicine composition made from red peony root, astragalus root, rehmannia root, cattail pollen, privet fruit, eclipta prostrata, ginkgo leaf, rhubarb, notoginseng, earthworm, cassia seed, and kudzu root.

8. The application according to claim 7, characterized in that... The quality control method also includes the preparation of standard solutions.

9. The application according to claim 7, characterized in that, The standard solution is prepared as follows: ① Take appropriate amounts of the standard or reference substances of the chemical components contained in the traditional Chinese medicine composition, accurately weigh them, dissolve them in an appropriate amount of DMSO, and prepare individual standard stock solutions with a concentration of 1.00 mg / mL; ② Accurately pipette an appropriate amount of the individual standard stock solution, dilute it with 50% methanol, and prepare a mixed standard solution with a concentration of 20 μg / mL for each individual standard or reference substance.

10. The application according to claim 9, characterized in that, The chemical components are astragaloside A, and / or versicolor glucoside, and / or versicolor, and / or gentianin, and / or gentianin, and / or paeoniflorin, and / or paeoniflorin lactone, and / or benzoyl paeoniflorin, and / or catechin, and / or gallic acid, and / or privetin, and / or rhodioloside, and / or privetin, and / or ursolic acid, and / or catalpol, and / or rehmannia glycoside D, and / or wedelia lactone, and / or ecliptaside A, and / or puerarin, and / or daidzein. And / or daidzein, and / or isorhamnetin-3-O-neohesperidin, and / or typhain, and / or quercetin, and / or ginkgolide A, and / or ginkgolide B, and / or ginkgolide C, and / or ginkgolide, and / or kaempferol, and / or isorhamnetin, and / or rutin, and / or luteolin, and / or emodin, and / or rhein, and / or emodin methyl ether, and / or aloe-emodin, and / or chrysophanol, and / or cassia seed extract, and / or ferulic acid, and / or ginsenoside R g1 and / or ginsenoside R b1 And / or notoginsenoside R1, and / or geniposide, and / or adenosine, and / or protocatechuic acid, and / or paeoniflorin, and / or phenylalanine, and / or puerarin apigenin, and / or echinacoside, and / or genistein, and / or quercetin 3-O-sophoroside, and / or luteolin, and / or isochlorogenic acid B, and / or isorauscoside, and / or isochlorogenic acid C, and / or isochlorogenic acid A, and / or trefoilin-7-O-β-D-glucopyranoside, and / or quercetin-3-O-(2G-α-L-rhamnosyl)-rutin, and / or ginsenoside Re, and / or leonurin, and / or 3-hydroxypuerarin, and / or cassia seed extract. And / or naringenin, and / or ecliptaside IV, and / or phloroglucin A, and / or styracin, and / or ginsenoside Rd, and / or diosmin, and / or cassiaside, and / or astragaloside I, and / or ginkgo biloba flavonoids, and / or astragaloside II, and / or isoginkgo biloba flavonoids, and / or cassiaside C, and / or chlorogenic acid, and / or pine biloba flavonoids, and / or leucamide phenylalanine, and / or guanosine, and / or rehmannia glutinosa glycoside, and / or iso-micro-salicylate, and / or [2,2,4-trimethyl-(3-pentanyl)oxy]-4-oxo-2-butenoate, and / or Pratensein 7-O-glucoside.

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