WO2024007538A1 - Procédé de mesure de contenu pour six composants alcaloïdes dans une petite pilule activant le méridien - Google Patents

Procédé de mesure de contenu pour six composants alcaloïdes dans une petite pilule activant le méridien Download PDF

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WO2024007538A1
WO2024007538A1 PCT/CN2022/139273 CN2022139273W WO2024007538A1 WO 2024007538 A1 WO2024007538 A1 WO 2024007538A1 CN 2022139273 W CN2022139273 W CN 2022139273W WO 2024007538 A1 WO2024007538 A1 WO 2024007538A1
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solution
content
aconitine
xiaohuoluo
acetonitrile
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PCT/CN2022/139273
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English (en)
Chinese (zh)
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赵振霞
刘永利
段吉平
雷蓉
王璐
朱靖
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河北省药品医疗器械检验研究院(河北省化妆品检验研究中心)
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Publication of WO2024007538A1 publication Critical patent/WO2024007538A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
    • G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N2030/062—Preparation extracting sample from raw material
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the technical field of traditional Chinese medicine detection, and is specifically a method for determining the content of six alkaloid components in Xiaohuoluo Pills.
  • Xiaohuoluo Pill is a traditional Chinese patent medicine, formerly known as Huoluo Dan, also known as Xiaohuoluo Dan.
  • Huoluo Dan also known as Xiaohuoluo Dan.
  • the 1977 edition of "Chinese Pharmacopoeia” was revised to "Xiaohuoluo Pills", and subsequent editions of the Pharmacopoeia were all recorded as "Xiaohuoluo Pills”.
  • Xiaohuoluo Pill is composed of six traditional Chinese medicines: Dannanxing, Zhichuanwu, Zhicaowu, Dilong, Frankincense (made), and Myrrh (made).
  • symptoms include pain in limb joints, or cold pain, or tingling, or pain that is severe at night, difficulty in joint flexion and extension, numbness and spasm, etc.
  • Zhichuanwu and Zhicaowu warm the meridians, dispel cold, expel wind and dampness, relieve numbness and relieve pain, and are the king medicines;
  • Dannanxing removes dampness and resolves phlegm, removes wind, phlegm and dampness in the meridians, and can relieve pain, and is the prescription.
  • the Aconite Sichuan and Aconite Radix in the prescription are traditional Chinese medicines from the Ranunculaceae family.
  • the diterpenoid diester alkaloids (MA, HA, AC) contained in them are highly toxic, mainly causing damage to the nervous and cardiovascular systems. Serious injuries. When Aconitum plants are used as clinical medicines, poisoning or even death often occurs due to improper processing or medication. After processing, these medicinal materials can be hydrolyzed into the corresponding diterpene monoester alkaloids (BMA, BAC, BHA), which have active activity, reduced toxicity, and improved therapeutic index. They are often used in clinical applications as the main medicinal ingredients. In order to reduce raw material quality risks, ensure efficacy and prevent low-limit feeding, it is necessary to control the content of finished products.
  • the 2020 version of the "Chinese Pharmacopoeia" quality standard for Xiaohuoluo Pills only contains the TLC limit inspection item for aconitine. It is difficult to fully control the quality of the toxic medicinal flavor in the prescription, and the control limit for the limit of aconitine is relatively high. May cause risk of poisoning.
  • the present invention provides a method for determining the content of 6 alkaloid components in Xiaohuoluo Pills, which includes the following steps:
  • test solution Take an appropriate amount of small Huoluo pills, cut into pieces, take 3g, weigh it, place it in a stoppered Erlenmeyer flask, add 25ml of 0.2mol/L hydrochloric acid solution, stopper it tightly, weigh it, and conduct ultrasonic treatment for 30 minutes, let cool, weigh again, use 0.2mol/L hydrochloric acid solution to make up for the lost weight, shake well, centrifuge for 20 minutes, measure 15ml of the additional filtrate, place it on the processed solid phase extraction column, and in turn use 0.05 Elute with 10 ml each of mol/L hydrochloric acid solution and acetonitrile. Discard the eluent and leave it for 5 minutes.
  • the preparation method of the mixed reference solution is: taking benzoyl aconitine, benzoyl aconitine, benzoyl aconitine reference substance and aconitine diester alkaloids Weigh the control extract accurately, add acetonitrile to make a mixed solution containing 300 ⁇ g, 100 ⁇ g, 100 ⁇ g, and 300ug per 1 ml respectively.
  • As the reference substance stock solution measure 5 ml of the reference substance stock solution and place it in a 50 ml measuring bottle. Add the volume ratio Dilute 90:10 acetonitrile-concentrated ammonia solution to the mark, shake well, and it is ready.
  • the ultrasonic treatment in step B power 400W, frequency 40kHz, water temperature below 40°C.
  • the centrifugal speed in step B is 6000 rpm.
  • the solid-phase extraction column in step B is a solid-phase extraction column filled with a mixed cation exchange reversed-phase adsorbent, 500 mg, 6 ml, and is eluted with 6 ml of acetonitrile and 6 ml of water in sequence before use.
  • a mixed cation exchange reversed-phase adsorbent 500 mg, 6 ml, and is eluted with 6 ml of acetonitrile and 6 ml of water in sequence before use.
  • the chromatographic conditions are:
  • Mobile phase A methanol
  • mobile phase B acetonitrile
  • mobile phase C 0.1% phosphoric acid
  • the gradient elution procedure is as follows:
  • the HPLC method of the present invention has the advantages of high efficiency, sensitivity, easy operation, and high accuracy.
  • the present invention establishes an HPLC method for simultaneous determination of the contents of six alkaloids in Xiaohuoluo pills.
  • the method is simple, highly specific, and can simultaneously measure single
  • the control of ester and diester alkaloids is more conducive to the quality control of Aconitum medicinal flavor, ensuring the safety and effectiveness of clinical medication, and providing a reference for product quality control and quality evaluation.
  • Figure 1 is the chromatogram of the mixed reference solution, including: 1-benzoylaconitine; 2-benzoylaconitine; 3-benzoylaconitine; 4-benzoylaconitine Aconitine; 5-aconitine; 6-aconitine;
  • Figure 2 is the chromatogram of the test solution, including: 1-benzoyl aconitine; 2-benzoyl aconitine; 3-benzoyl aconitine; 4-benzoyl aconitine Alkali; 5-aconitine; 6-aconitine;
  • Figure 3 is the chromatogram of the negative sample solution.
  • any reference to “one embodiment” means that the specific features, structures or parameters, steps, etc. described in the embodiment are included in at least one embodiment according to the present invention. Therefore, in the description of the present invention, if terms such as “according to one embodiment of the present invention” and “in an embodiment” are used, they are not used to specifically refer to the same embodiment. If terms such as “in an embodiment” are used, Terms such as “in other embodiments”, “according to different embodiments of the present invention”, “according to other embodiments of the present invention” are not used to specifically indicate that the mentioned features can only be included in specific different embodiments. . Those skilled in the art should understand that each specific feature, structure or parameter, step, etc. disclosed in one or more embodiments of the present invention may be combined in any suitable manner.
  • the object of the present invention is to provide a method for simultaneously determining the content of six alkaloid components in Xiaohuoluo Pills.
  • the present invention uses HPLC method to determine benzoyl aconitine (BMA), benzoyl aconitine aconitine (BAC), benzoyl aconitine (BHA), aconitine (MA), aconitine (HA) and aconitine (AC) content.
  • the content determination method of the present invention adopts HPLC method, and the method is as follows:
  • Chromatographic conditions Chromatographic column: PICKERING C18 column (4.6 ⁇ 250mm, 5 ⁇ m); use: methanol as mobile phase A, acetonitrile as mobile phase B, 0.1% phosphoric acid as mobile phase C, the gradient elution procedure is shown in the table below; column temperature :30°C, flow rate: 1ml/min; detection wavelength: 232nm; injection volume: 15 ⁇ l.
  • Preparation of mixed reference solution Take appropriate amounts of benzoyl aconitine, benzoyl aconitine, benzoyl aconitine reference substance and aconitine diester alkaloid control extract, and weigh them accurately. Determine, add acetonitrile to make a mixed solution containing 300 ⁇ g, 100 ⁇ g, 100 ⁇ g, and 300ug per 1 ml respectively.
  • As the reference substance stock solution accurately measure 5 ml of the reference substance stock solution and place it in a 50 ml measuring bottle.
  • test solution Take an appropriate amount of this product, cut into pieces, take about 3g, weigh it accurately, place it in a stoppered conical flask, add 25ml of 0.2mol/L hydrochloric acid solution accurately, stopper it tightly, weigh it, and ultrasonicate it.
  • Assay method Precisely draw 15 ⁇ l each of the mixed reference solution and the test solution, inject them into the liquid chromatograph, measure, and calculate the content using the external standard method.
  • High performance liquid chromatograph equipped with quaternary pump, DAD detector, Waters e2695; analytical balance: Mettler XPE26 (parts per million) (Shanghai Mettler Instrument Co., Ltd.); Mettler XS105 (parts per hundred thousand) (Shanghai Mettler Instrument Co., Ltd.); KQ-400KDE ultrasonic cleaning instrument (Kunshan Ultrasonic Instrument Co., Ltd.); ultrapure water instrument (American Millipore Company).
  • Aconitine diester alkaloid control extract (batch number: 112029-201601, for content determination, content is based on neo-aconitine 31.7%, hypo-aconitine 30.0%, aconitine 31.8%)
  • Benzoylaconitine (batch number: 111794-201705, content based on 99.1%)
  • Reagents methanol, acetonitrile (chromatographically pure, Merck, Germany), water is ultrapure water; other reagents are of analytical grade.
  • Chromatographic column PICKERING C18 column (4.6 ⁇ 250mm, 5 ⁇ m); use: methanol as mobile phase A, acetonitrile as mobile phase B, 0.1% phosphoric acid as mobile phase C, the gradient elution program is shown in Table 2; column temperature: 30°C , flow rate: 1ml/min; detection wavelength: 232nm; injection volume: 15 ⁇ l.
  • the method of the present invention is simple, highly specific, and simultaneously controls monoester and diester alkaloids, which is more conducive to the quality control of Aconitum medicinal flavor and ensures the safety and effectiveness of clinical medication. , which can provide reference for product quality control and quality evaluation.

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  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
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  • Engineering & Computer Science (AREA)
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  • Investigating Or Analysing Biological Materials (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
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Abstract

La présente invention concerne le domaine technique de la détection de médecine chinoise traditionnelle, et concerne spécifiquement un procédé de mesure de contenu pour six composants alcaloïdes dans une petite pilule d'activation méridienne, comprenant : la préparation d'une solution de référence mixte ; la préparation d'une solution de test ; l'aspiration respective et précise de 15 µl de la solution de référence mixte et de 15 µl de la solution de test, l'injection de la solution de référence mixte et de la solution de test dans un chromatographe en phase liquide, la mesure et le calcul du contenu au moyen d'un procédé standard externe. Dans la présente invention, un procédé HPLC est établi pour mesurer simultanément la teneur de six composants alcaloïdes dans la petite pilule d'activation de méridien, le procédé est simple et pratique, et a une spécificité élevée, et des alcaloïdes de monoester et de diester sont commandés en même temps, de telle sorte que le contrôle de qualité de l'Aconitum de médicament à base d'herbes est facilité, la sécurité et l'efficacité du médicament clinique sont assurées, et une référence est fournie pour un contrôle de qualité et une évaluation de qualité de produits.
PCT/CN2022/139273 2022-07-06 2022-12-15 Procédé de mesure de contenu pour six composants alcaloïdes dans une petite pilule activant le méridien WO2024007538A1 (fr)

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