WO2022036780A1 - 化湿败毒组合物的检测方法 - Google Patents

化湿败毒组合物的检测方法 Download PDF

Info

Publication number
WO2022036780A1
WO2022036780A1 PCT/CN2020/114894 CN2020114894W WO2022036780A1 WO 2022036780 A1 WO2022036780 A1 WO 2022036780A1 CN 2020114894 W CN2020114894 W CN 2020114894W WO 2022036780 A1 WO2022036780 A1 WO 2022036780A1
Authority
WO
WIPO (PCT)
Prior art keywords
anthraquinone
emodin
composition
content
solution
Prior art date
Application number
PCT/CN2020/114894
Other languages
English (en)
French (fr)
Inventor
魏梅
程学仁
陈向东
李国卫
何广铭
霍文杰
马瑞瑞
杨小龙
邱韵静
曾荟
胡琦萍
朱德全
李振雨
邓淙友
梁慧
王闽予
Original Assignee
广东一方制药有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 广东一方制药有限公司 filed Critical 广东一方制药有限公司
Publication of WO2022036780A1 publication Critical patent/WO2022036780A1/zh

Links

Images

Classifications

    • 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

Definitions

  • the invention relates to the technical field of quality detection of traditional Chinese medicines, in particular to a detection method of a Huashibaidu composition.
  • Huashibaidu recipe consists of 14 herbs, including raw ephedra, almond, raw gypsum, licorice, Huoxiang, Magnolia, Atractylodes, Caoguo, Pinellia, Poria, Raw Rhubarb, Raw Astragalus, Tinglizi and Red Peony .
  • Clinical experiments have shown that Huashi Baidu Fang has a prominent effect on improving the symptoms of patients and increasing the negative rate of nucleic acid.
  • the current researches on Huashi Baidu Fang are mostly focused on pharmacology and efficacy research, and there is no research on its quality standards. And the existing production is also limited to a small area, there is no large-scale industrial production, and the requirements for quality control are relatively low.
  • the technical problem to be solved by the present invention is to provide a detection method for the composition of dampness and sepsis, which has strong specificity and durability, and can effectively ensure the stability and controllability of product quality.
  • the present invention provides a method for detecting a dampness-removing poison composition, which mainly includes the following components: ephedra, fried bitter almond, gypsum, licorice, patchouli, Magnolia, bran fried Atractylodes, fried grass nuts, Pinellia, Poria, Rhubarb, Astragalus, Tinglizi, red peony;
  • the detection method of the Huashibaidu composition includes using high performance liquid chromatography to measure the total anthraquinone content and the free anthraquinone content in the Huashibaidu composition, and calculate the combined anthraquinone content;
  • bound anthraquinone content total anthraquinone content - free anthraquinone content.
  • the assay method of described total anthraquinone content comprises:
  • the liquid chromatograph uses octadecylsilane-bonded silica gel as a filler, acetonitrile as mobile phase A, and phosphoric acid aqueous solution. Carry out gradient elution for mobile phase B, and determine the total anthraquinone content in the Huashibaidu composition.
  • the assay method of described free anthraquinone content comprises:
  • the liquid chromatograph uses octadecylsilane-bonded silica gel as filler, acetonitrile as mobile phase A, and phosphoric acid aqueous solution. Carry out gradient elution for mobile phase B, and measure the free anthraquinone content in the Huashibaidu composition.
  • the measuring method of described total anthraquinone content and the measuring method of free anthraquinone content all adopt the following elution procedure to carry out:
  • mobile phase A from 35% ⁇ 40%, mobile phase B from 65% ⁇ 60%;
  • mobile phase A is 60%
  • mobile phase B is 40%.
  • Huashibaidu composition Take 0.2-0.5 g of Huashibaidu composition, put it in a stoppered conical flask, add 25-30 mL of methanol, weigh it, heat under reflux for 20-60 minutes, take it out, let it cool, weigh it again, and make up with methanol The lost weight is shaken, filtered, and the filtrate is obtained.
  • octadecylsilane-bonded silica gel is used as a filler
  • acetonitrile is used as mobile phase A
  • a 0.1 vol% aqueous phosphoric acid solution is used.
  • octadecylsilane-bonded silica gel is used as filler
  • acetonitrile is used as mobile phase A
  • 0.1 vol% phosphoric acid is used.
  • the aqueous solution is the mobile phase B for gradient elution
  • the flow rate is 0.8 mL/min
  • the detection wavelength is 254 nm
  • the column temperature is 30 °C.
  • the injection volume of the reference solution and the total anthraquinone test solution is 10 ⁇ L.
  • the injection volume of the reference solution and the free anthraquinone test solution is 10 ⁇ L.
  • the composition for eliminating dampness and baidu mainly includes the following components: 3-60 parts of ephedra, 4.5-90 parts of fried bitter almond, 7.5-150 parts of gypsum, 1.5-30 parts of licorice, patchouli 5-100 servings of incense, 5-100 servings of Magnolia officinalis, 7.5-150 servings of fried Atractylodes bran, 5-100 servings of fried grass nuts, 4.5-90 servings of Pinellia, 7.5-150 servings of Poria, 2.5-50 servings of rhubarb, Astragalus 5-100 parts, Tinglizi 5-100 parts, Red peony root 5-100 parts, appropriate amount of accessories;
  • the dampness-removing and poisoning composition is made into a traditional Chinese medicine preparation, and the traditional Chinese medicine preparation is a granule, a decoction, a powder, a capsule, an oral liquid, a tablet or a pill.
  • the present invention establishes a method for determining the binding anthraquinone in the Huashibaidu composition for the first time, and the method has good specificity, accuracy, reliability and good stability.
  • the content of total anthraquinone and free anthraquinone in the Huashibaidu composition can be measured by the above determination method, and then the content of the combined anthraquinone can be calculated, so as to ensure the product quality stability and controllability of the Huashibaidu composition. Provide a good foundation for mass production.
  • Fig. 1 is the HPLC figure of reference substance solution; Wherein, peak 1 is aloe-emodin peak, peak 2 is rhein peak, peak 3 is emodin peak, peak 4 is chrysophanol peak, and peak 5 is emodin methyl ether peak;
  • Fig. 2 is the HPLC chart of free anthraquinone negative sample solution
  • Fig. 3 is the HPLC chart of total anthraquinone negative sample solution
  • Fig. 4 is the HPLC chart of free anthraquinone test solution; wherein, peak 1 is aloe-emodin peak, peak 2 is rhein peak, peak 3 is emodin peak, peak 4 is chrysophanol peak, and peak 5 is emodin methyl ether peak;
  • Fig. 5 is the HPLC figure of total anthraquinone test solution; wherein, peak 1 is aloe-emodin peak, peak 2 is rhein peak, peak 3 is emodin peak, peak 4 is chrysophanol peak, and peak 5 is emodin methyl ether peak
  • Fig. 6 is the standard curve diagram of aloe-emodin reference substance in the determination of free anthraquinone content
  • Fig. 7 is the standard curve diagram of rhein reference substance in the determination of free anthraquinone content
  • Fig. 8 is the standard curve diagram of emodin reference substance in the determination of free anthraquinone content
  • Fig. 9 is the standard curve diagram of chrysophanol reference substance in the determination of free anthraquinone content
  • Fig. 10 is the standard curve diagram of emodin methyl ether reference substance in the determination of free anthraquinone content
  • Figure 11 is a standard curve diagram of aloe-emodin in the determination of total anthraquinone content
  • Figure 12 is the standard curve diagram of rhein reference substance in the determination of total anthraquinone content
  • Figure 13 is the standard curve diagram of emodin reference substance in the determination of total anthraquinone content
  • Figure 14 is the standard curve diagram of chrysophanol reference substance in the determination of total anthraquinone content
  • Figure 15 is the standard curve diagram of emodin methyl ether reference substance in the determination of total anthraquinone content.
  • the composition for eliminating dampness and eliminating toxic substances mainly includes the following components: ephedra, fried bitter almond, gypsum, licorice, patchouli, Magnolia, bran fried Atractylodes, fried grass nuts, Pinellia, Poria, Rhubarb, Astragalus, Tinglizi, red peony.
  • composition of the invention for eliminating dampness and poisoning, in the prescription, ephedra, patchouli, and gypsum are used as king medicines, ephedra, patchouli, acrid, bitter, warm and fragrant in odor, relieve skin and relieve asthma, dispel dampness and neutralize; gypsum, Ponnet, sweet, cold, clearing the lungs and stomach, stagnant heat and promoting body fluid, the three medicines complement each other to achieve the effects of relieving the exterior and dispelling cold, aromatizing dampness, clearing heat and relieving asthma.
  • Dampness strengthens the spleen, circulates qi and unclogs the orifices, dredges the bowels, and helps the evil to go out.
  • astragalus red peony root, tincture seed, rhubarb as adjuvant, astragalus, sweet, warm and invigorating the lung and spleen, red peony root, bitter, slightly cold, cooling blood and dissipating blood stasis, used for the treatment of blood stasis caused by damage to the right qi in the later stage of the epidemic and qi stagnation.
  • Tinglizi is acrid and cold, assists the monarch medicine gypsum to clear lung heat, and also benefits water, preventing or treating "damp lung (pulmonary edema) lesions"; rhubarb, bitter cold enters the large intestine meridian and clears the fu-organs, and the lungs and the large intestine are related to each other.
  • the monarch medicine gypsum plaster is used as an adjuvant to clear the lung heat, and the red peony root is used to cool the blood and activate the blood.
  • licorice as the medicine, licorice ganping, reconciling various medicines, with red peony root and shaoyao licorice decoction to relieve emergency.
  • the whole recipe has the functions of clearing away dampness, clearing away heat and relieving asthma, nourishing qi and dissipating blood stasis.
  • Fever is the main manifestation, which is mostly low body heat, which is difficult to heal, but it can also be moderate to low fever, or even no fever;
  • wheezing and fatigue are the main symptoms;
  • Digestive system symptoms such as anorexia, loose stools, and diarrhea are often combined; 4
  • Most of the patients have thick and greasy tongue coating. Judging from the above characteristics, it is in line with the pathogenic characteristics of dampness: heavy turbidity, hindering qi and injuring yang, sticking, and descending.
  • Dampness pathogens can cause disease alone, and can also be cold and hot, manifesting as cold-dampness, dampness-heat, of which heat can be caused by dryness, or it can be transformed by dampness for a long time. Damp pathogens, cold-dampness, and damp-heat can be combined with epidemic toxins to cause disease, which can be seen in mild cold-damp stagnation of the lung, damp-heat accumulation in the lung, or common damp-toxin stagnation in the lung, and cold-dampness in the lung. The disease develops, injures the blood, reverses the pericardium, and evolves into severe new coronary pneumonia.
  • the main disease site is in the lungs, followed by the spleen and stomach.
  • the core pathogenesis is dampness and toxin stagnation. It can be divided into four stages: the initial stage, the middle stage, the critical stage and the recovery stage.
  • the lungs resolve phlegm, promote blood circulation and remove blood stasis, clear the internal organs, and nourish the righteousness. Therefore, the composition of the present invention is based on the core pathogenesis, and should be based on the core treatment method of relieving surface dampness, clearing heat and relieving asthma, and eliminating toxins, and simultaneously removing blood stasis and dredging collaterals, nourishing qi and nourishing yin.
  • the combination of epidemic toxin and cold-dampness, aversion to cold and fever, the treatment is appropriate to relieve the exterior and remove dampness and toxin; the combination of epidemic toxin and dampness and heat can cause loose stools, fatigue and fatigue, and it is suitable for clearing away heat, removing dampness and eliminating toxin, and simultaneously nourishing qi and nourishing yin; Chest and lung, wheezing, chest tightness, shortness of breath.
  • composition for dissolving dampness and eliminating toxic substances of the present invention integrates the core pathogenesis of traditional Chinese medicine treatment in the "Diagnosis and Treatment Plan for Pneumonia Infected by Novel Coronavirus (Trial Version 5)", which is a combination of warmth and dampness, which causes the lungs to lose the ability to disperse and descend, and the lung qi is blocked.” , mainly based on dampness and turbidity and heat stagnation of the lung, highlighting the effect of dampness and qi, dispelling lung and relieving asthma, clearing heat and resolving phlegm, benefiting qi and activating blood.
  • the clinical observation in the early stage shows that the present invention can improve the clinical symptoms of severe novel coronavirus infection pneumonia ,
  • it can obviously relieve the main symptoms such as cough, fatigue, dry mouth or vomiting.
  • the healing time is shortened.
  • the respiratory function of the patient is obviously improved, and the time from oxygen inhalation is shortened.
  • the symptoms are significantly relieved, and the symptoms of loss of appetite and chest tightness can also be improved.
  • the clinical symptoms such as cough, fatigue, dry mouth or vomiting of severe and common novel coronavirus pneumonia have been significantly improved, supplementing the severe and common novel coronavirus pneumonia urgently needed by the current epidemic situation. Corona Virus Infection Pneumonia Treatment Drugs.
  • the dehumidification and sepsis composition includes the following components:
  • the dampness-removing and poisoning composition is made into a traditional Chinese medicine preparation, and the traditional Chinese medicine preparation is a granule, a decoction, a powder, a capsule, an oral liquid, a tablet or a pill.
  • the present invention provides a method for detecting a Huashibaidu composition, which can have a good detection effect on any dosage form of the Huashibaidu composition.
  • the inventors used molecular docking technology to identify the key targets of the invasion, replication, assembly, shedding and transfer of various traditional Chinese medicines in the formula of Huashibaidu composition and COVID-19, as well as to the host. Key role targets in generating lung injury and inflammatory responses were analyzed.
  • rhubarb is the key target of TMPRSS2, which inhibits virus invasion and shedding, and the key targets of tissue damage after virus invades the host: AMPK (oxidative stress, inflammation), VEGFR2 (vascular permeability) and ALK5 (vascular permeability, pulmonary fibrosis). ) in response to COVID-19. Further research showed that rhein, emodin methyl ether, 6-hydroxyrumin-8-O- ⁇ -D-glucoside, protocyanidin B1-3'-O-gallate, Five components including aloe-emodin have effects on the above targets. It can be seen that rhubarb belongs to the core drug in the Huashibaidu composition, and the detection of rhubarb-related components is one of the key means to realize the quality control of the Huashibaidu composition.
  • the present invention chooses to monitor rhein, emodin methyl ether, and aloe-emodin, and the content of rhubarb in the Huashibaidu recipe can be reflected through the monitoring of the above three components.
  • rhubarb in the composition for eliminating dampness and sepsis is to clear lung heat, activate blood and cool blood. methyl ether) achieved.
  • the combined anthraquinone is easily decomposed into free anthraquinone when heated, resulting in a weakened drug effect. To this end, the content of bound anthraquinone needs to be monitored.
  • the present invention selects to measure and monitor the content of the combined anthraquinone in the finished medicine when formulating the detection method of the Huashibaidu composition, so as to realize the quality detection of the Huashibaidu composition.
  • the content of the controlled bound anthraquinone shall not be less than 0.016 wt%.
  • the total anthraquinone content and the free anthraquinone content in the Huashibaidu composition are determined by high performance liquid chromatography, and then the bound anthraquinone content is calculated.
  • bound anthraquinone content total anthraquinone content - free anthraquinone content.
  • the assay method of total anthraquinone content includes:
  • Chromatographic conditions use octadecylsilane-bonded silica gel chromatographic column as the chromatographic column, use acetonitrile-0.1 vol% phosphoric acid aqueous solution as the mobile phase, the flow rate is 0.8 mL/min, the detection wavelength is 254 nm, and the column temperature is 30 °C ; The injection volume is 10 ⁇ L.
  • the mobile phase is eluted in the following elution sequence:
  • the assay method of free anthraquinone content is as follows:
  • Chromatographic conditions use octadecylsilane-bonded silica gel chromatographic column as the chromatographic column, use acetonitrile-0.1 vol% phosphoric acid aqueous solution as the mobile phase, the mobile phase flow rate is 0.8 mL/min, the detection wavelength is 254 nm, and the column temperature is 30°C, and the injection volume was 10 ⁇ L.
  • the mobile phase is eluted in the following elution sequence:
  • the method for measuring the total anthraquinone content and the method for measuring the content of free anthraquinone all adopt the same elution procedure. the same reference solution.
  • the present invention redesigns the gradient elution procedure on the basis of considering the accuracy, durability and specificity of the test method. Specifically, the study found that: in 10min ⁇ 38min, the ratio of acetonitrile is 40% ⁇ 60%, and the ratio of 0.1vol% phosphoric acid is 60% ⁇ 40%, which can effectively separate the aloe-emodin, rhein, rhubarb in the Huashibaidu composition. Vinegar, chrysophanol, no need to set too many gradient changes.
  • test substance chromatogram had the same chromatographic peak at the retention time corresponding to the reference substance chromatogram, the free anthraquinone negative sample had no interference, and the total anthraquinone negative sample had no interference.
  • the specificity of the method was good.
  • Each 1mL contains 0.411 ⁇ g, 1.028 ⁇ g, 2.057 ⁇ g, 6.170 ⁇ g, 10.283 ⁇ g of rhein, each 1mL contains 0.055 ⁇ g, 0.138 ⁇ g, 0.275 ⁇ g, 0.825 ⁇ g, 1.375 ⁇ g of emodin, each 1mL contains 0.033 ⁇ g, 0.082 ⁇ g, 0.163 ⁇ g, 0.489 ⁇ g, 0.816 ⁇ g of chrysophanol, each 1mL contains 0.012 ⁇ g, 0.030 ⁇ g, 0.061 ⁇ g, 0.183 ⁇ g, 0.305 ⁇ g of emodin methyl ether reference solution.
  • anthraquinone reference solution a solution containing 15.807 ⁇ g of aloe-emodin, 30.167 ⁇ g of rhein, 16.039 ⁇ g of emodin, 26.563 ⁇ g of chrysophanol, and 5.221 ⁇ g of emodin methyl ether per 1 mL was prepared as the mother liquor of total anthraquinone reference substance.
  • aloe rhubarb containing 0.316 ⁇ g, 0.790 ⁇ g, 1.581 ⁇ g, 4.742 ⁇ g, and 7.903 ⁇ g per 1mL.
  • Each 1mL contains 0.603 ⁇ g, 1.508 ⁇ g, 3.017 ⁇ g, 9.050 ⁇ g, 15.084 ⁇ g of rhein, each 1mL contains 0.321 ⁇ g, 0.802 ⁇ g, 1.604 ⁇ g, 4.812 ⁇ g, 8.109 ⁇ g of emodin, each 1mL contains 0.531 ⁇ g, 1.328 ⁇ g, 2.656 ⁇ g, 7.969 ⁇ g, 13.282 ⁇ g of chrysophanol, each 1mL contains 0.104 ⁇ g, 0.261 ⁇ g, 0.522 ⁇ g, 1.566 ⁇ g, 2.611 ⁇ g of emodin methyl ether reference solution.
  • the same total anthraquinone test solution was injected and measured at 0.0, 2.5, 6.0, 9.5, 13.0, 17.0, 20.5, and 25.5 hours respectively. Except for the RSD value of the peak area of emodin methyl ether, the other components were less than 3.0%. All are greater than 3%, it is recommended that the total anthraquinone test solution be measured within 13 hours.
  • aloe-emodin reference substance 2.497mg, rhein reference substance 3.960mg, emodin reference substance 2.707mg, chrysophanol reference substance 2.313mg, emodin methyl ether reference substance 1.995mg put them in 50mL measuring bottles respectively, add methanol A solution containing 0.04994 mg of aloe-emodin, 0.07920 mg of rhein, 0.05414 mg of emodin, 0.04625 mg of chrysophanol, and 0.03990 mg of emodin methyl ether per 1 mL was prepared as a sample addition and recovery mother liquor.
  • Pheromon Kinetex-EVO C18 chromatographic column (4.6mm ⁇ 150mm, 5 ⁇ m; serial number: JS-091)
  • Waters HSS T3 chromatographic column (4.6mm ⁇ 150mm, 5 ⁇ m; serial number: JS-136 )
  • Waters X-bridge C18 chromatographic column (4.6mm ⁇ 150mm, 5 ⁇ m; serial number: JS-154).
  • the column temperature was set at 28°C, 30°C and 32°C, respectively, and the influence of column temperature on the determination of free anthraquinone and total anthraquinone in Huashibaidu composition was investigated.
  • the flow rates were set to 0.7mL/min, 0.8mL/min and 0.9mL/min respectively, and the influence of flow rate on the determination of free anthraquinone and total anthraquinone in Huashibaidu composition was investigated.
  • the present invention establishes a method for determining the binding of anthraquinone in a Huashibaidu composition, and the method has good specificity, accuracy and reliability, and good stability.
  • the content of total anthraquinone and free anthraquinone in the Huashibaidu composition can be measured, and then the content of the combined anthraquinone can be calculated, so as to ensure the product quality stability and controllability of the Huashibaidu composition, Provide a good foundation for large-scale production.

Landscapes

  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Engineering & Computer Science (AREA)
  • Library & Information Science (AREA)
  • Medicines Containing Plant Substances (AREA)

Abstract

本发明公开了一种化湿败毒组合物的检测方法，其中，化湿败毒组合物主要包括以下组分：麻黄，炒苦杏仁，生石膏，甘草，广藿香，厚朴，麸炒苍术，炒草果仁，法半夏，茯苓，大黄，黄芪，葶苈子，赤芍；化湿败毒组合物的质量检测方法包括采用高效液相色谱法测定化湿败毒组合物中总蒽醌含量和游离蒽醌含量，并计算结合蒽醌含量；其中，结合蒽醌含量＝总蒽醌含量-游离蒽醌含量。本发明中的测定方法专属性和耐用性较强，稳定性高，能够有效保证产品质量的稳定性和可控性。

Description

化湿败毒组合物的检测方法 技术领域
本发明涉及中药质量检测技术领域，尤其涉及一种化湿败毒组合物的检测方法。
背景技术
2019新型冠状病毒(COVID-19)感染引起的肺炎疫情，由于传染性强，传播迅速，人群普遍易感，且特效药缺乏，已在全球范围内形成大流行，成为了全球性的超重大公共卫生突发事件。中医药在抗击新冠肺炎疫情的过程中，发挥了独特的、重要的作用。中国中医药管理局指出，经研究筛选，“三药三方”中的金花清感颗粒、连花清瘟颗粒、血必净注射液、清肺排毒汤、化湿败毒方、宣肺败毒方在此次抗击疫情中发挥了良好的作用。
化湿败毒方由14味中药组成，包括生麻黄、杏仁、生石膏、甘草、藿香、厚朴、苍术、草果、法半夏、茯苓、生大黄、生黄芪、葶苈子和赤芍。临床实验表明，化湿败毒方对改善患者症状、提高核酸转阴率具有突出的作用。但目前对于化湿败毒方的研究多集中在药理、疗效的研究，尚无对于其质量标准的研究。且现有的生产也仅限于小范围内进行，无大规模工业化生产，对质量监控的要求也相对较低。
文献《抗新型冠状病毒肺炎(COVID-19)的化湿败毒颗粒药味的物质基础研究》(中国现代中药，2020年第3期)研究了各味药物的物质基础。但并未对药品的具体质量控制方法进行研究。
发明内容
本发明所要解决的技术问题在于，提供一种化湿败毒组合物的检测方法，该方法专属性和耐用性强，能够有效保证产品质量的稳定性和可控性。
为了解决上述技术问题，本发明提供了一种化湿败毒组合物的检测方法，所述化湿败毒组合物主要包括以下组分：麻黄，炒苦杏仁，生石膏，甘草，广 藿香，厚朴，麸炒苍术，炒草果仁，法半夏，茯苓，大黄，黄芪，葶苈子，赤芍；
所述化湿败毒组合物的检测方法包括采用高效液相色谱法测定化湿败毒组合物中总蒽醌含量和游离蒽醌含量，并计算结合蒽醌含量；
其中，结合蒽醌含量＝总蒽醌含量-游离蒽醌含量。
作为上述技术方案的改进，所述总蒽醌含量的测定方法包括：
(1)取芦荟大黄素、大黄酸、大黄素、大黄酚、大黄素甲醚适量，加甲醇制成混合溶液，制得对照品溶液；
(2)取化湿败毒组合物，利用甲醇提取，制得总蒽醌供试品溶液；
(3)吸取对照品溶液和总蒽醌供试品溶液，注入液相色谱仪，所述液相色谱仪以十八烷基硅烷键合硅胶为填充剂，以乙腈为流动相A，磷酸水溶液为流动相B进行梯度洗脱，测定得到化湿败毒组合物中总蒽醌含量。
作为上述技术方案的改进，所述游离蒽醌含量的测定方法包括：
(一)取芦荟大黄素、大黄酸、大黄素、大黄酚、大黄素甲醚适量，加甲醇制成混合溶液，制得对照品溶液；
(二)取化湿败毒组合物，利用甲醇提取，制得游离蒽醌供试品溶液；
(三)吸取对照品溶液和游离蒽醌供试品溶液，注入液相色谱仪，所述液相色谱仪以十八烷基硅烷键合硅胶为填充剂，以乙腈为流动相A，磷酸水溶液为流动相B进行梯度洗脱，测定得到化湿败毒组合物中游离蒽醌含量。
作为上述技术方案的改进，所述总蒽醌含量的测定方法和游离蒽醌含量的测定方法均采用以下洗脱程序进行：
0～10min，流动相A从35％→40％，流动相B从65％→60％；
10～38min，流动相A从40％→60％，流动相B从60％→40％；
38～48min，流动相A为60％，流动相B为40％。
作为上述技术方案的改进，所述总蒽醌供试品溶液由下述方法制得：
取化湿败毒组合物0.2～0.5g，置具塞锥形瓶中，加入甲醇20～30mL，加热回流提取20～30分钟，取出，放冷，再称定重量，用甲醇补足减失的重量，摇匀，滤过，取续滤液10～15mL，减压回收溶剂至干，加8％盐酸溶液10～15mL，超声处理2～5分钟，再加三氯甲烷10～20mL，加热回流1～3小时，放冷，置分液漏斗中，用少量三氯甲烷洗涤容器，并入分液漏斗中，分取三氯甲烷层，酸 液再用三氯甲烷提取2～5次，每次10～15mL，合并三氯甲烷液，减压回收溶剂至干，残渣加甲醇使溶解，转移至10mL量瓶中，加甲醇至刻度，摇匀，滤过，取续滤液，即得。
作为上述技术方案的改进，所述游离蒽醌供试品溶液由下述方法制得：
取化湿败毒组合物0.2～0.5g，置具塞锥形瓶中，加入甲醇25～30mL，称定重量，加热回流20～60分钟，取出，放冷，再称定重量，用甲醇补足减失的重量，摇匀，滤过，取续滤液，即得。
作为上述技术方案的改进，所述对照品溶液由下述方法制得：
称定芦荟大黄素对照品1.581mg、大黄酸对照品3.017mg、大黄素对照品1.604mg、大黄酚对照品2.656mg、大黄素甲醚对照品5.221mg，置100mL量瓶中，加甲醇分别制成每1mL含芦荟大黄素15.807μg、大黄酸30.167μg、大黄素16.039μg、大黄酚26.563μg、大黄素甲醚52.213μg的母液；分别精密吸取上述芦荟大黄素、大黄酸、大黄素、大黄酚母液各1mL，大黄素甲醚母液0.1mL，置10mL量瓶中，加甲醇制成每1mL含芦荟大黄素1.581μg、大黄酸3.017μg、大黄素1.604μg、大黄酚2.656μg、大黄素甲醚0.522μg的混合溶液，即得。
作为上述技术方案的改进，所述总蒽醌含量测定方法和游离蒽醌含量的测定方法中，以十八烷基硅烷键合硅胶为填充剂，以乙腈为流动相A，0.1vol％磷酸水溶液为流动相B进行梯度洗脱，流速为0.6～1mL/min，检测波长为253～256nm，柱温为25～35℃。
作为上述技术方案的改进，所述总蒽醌含量的测定方法和游离蒽醌含量的测定方法中，以十八烷基硅烷键合硅胶为填充剂，以乙腈为流动相A，0.1vol％磷酸水溶液为流动相B进行梯度洗脱，流速为0.8mL/min，检测波长为254nm，柱温为30℃。
作为上述技术方案的改进，所述总蒽醌含量的测定方法中，所述对照品溶液、总蒽醌供试品溶液的进样量为10μL。
作为上述技术方案的改进，所述游离蒽醌含量的测定方法中，所述对照品溶液、游离蒽醌供试品溶液的进样量为10μL。
作为上述技术方案的改进，所述化湿败毒组合物主要包括下述组分：麻黄3-60份，炒苦杏仁4.5-90份，生石膏7.5-150份，甘草1.5-30份，广藿香5-100份，厚朴5-100份，麸炒苍术7.5-150份，炒草果仁5-100份，法半夏4.5-90份， 茯苓7.5-150份，大黄2.5-50份，黄芪5-100份，葶苈子5-100份，赤芍5-100份，辅料适量；
所述化湿败毒组合物被制成中药制剂，所述中药制剂为颗粒剂、汤剂、散剂、胶囊剂、口服液、片剂或丸剂。
实施本发明，具有如下有益效果：
本发明首次建立了化湿败毒组合物中结合蒽醌的测定方法，该检测方法的专属性良好，准确可靠，稳定性好。通过上述测定方法可对化湿败毒组合物中总蒽醌和游离蒽醌的含量进行测定，进而计算出结合蒽醌含量，从而保证化湿败毒组合物产品质量稳定性和可控性，为大生产提供良好的基础。
附图说明
图1是对照品溶液的HPLC图；其中，峰1为芦荟大黄素峰，峰2为大黄酸峰，峰3为大黄素峰，峰4为大黄酚峰，峰5为大黄素甲醚峰；
图2是游离蒽醌阴性样品溶液的HPLC图；
图3是总蒽醌阴性样品溶液的HPLC图；
图4是游离蒽醌供试品溶液的HPLC图；其中，峰1为芦荟大黄素峰，峰2为大黄酸峰，峰3为大黄素峰，峰4为大黄酚峰，峰5为大黄素甲醚峰；
图5是总蒽醌供试品溶液的HPLC图；其中，峰1为芦荟大黄素峰，峰2为大黄酸峰，峰3为大黄素峰，峰4为大黄酚峰，峰5为大黄素甲醚峰
图6是游离蒽醌含量测定中芦荟大黄素对照品标准曲线图；
图7是游离蒽醌含量测定中大黄酸对照品标准曲线图；
图8是游离蒽醌含量测定中大黄素对照品标准曲线图；
图9是游离蒽醌含量测定中大黄酚对照品标准曲线图；
图10是游离蒽醌含量测定中大黄素甲醚对照品标准曲线图；
图11是总蒽醌含量测定中芦荟大黄素标准曲线图；
图12是总蒽醌含量测定中大黄酸对照品标准曲线图；
图13是总蒽醌含量测定中大黄素对照品标准曲线图；
图14是总蒽醌含量测定中大黄酚对照品标准曲线图；
图15是总蒽醌含量测定中大黄素甲醚对照品标准曲线图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将结合附图及具体实施方式对本发明作进一步地详细描述。
本发明中，化湿败毒组合物主要包括以下组分：麻黄，炒苦杏仁，生石膏，甘草，广藿香，厚朴，麸炒苍术，炒草果仁，法半夏，茯苓，大黄，黄芪，葶苈子，赤芍。
本发明化湿败毒组合物，方中以麻黄、广藿香、石膏为君药，麻黄、广藿香，辛、苦、温兼气味芳香，解表平喘，化湿和中；石膏，辛、甘、寒，清泻肺胃郁热兼以生津，三药相辅，以达解表散寒、芳香化湿、清热平喘之效。炒苦杏仁、法半夏、厚朴、麸炒苍术、炒草果仁、茯苓共为臣药，炒苦杏仁、法半夏、厚朴，辛、苦、温，行气降逆，开结平喘；麸炒苍术、炒草果仁，辛烈、苦温，入脾胃经，燥湿健脾、破戾气所结；茯苓，淡渗除湿健脾；六味药共用以达助君药燥湿健脾，行气通窍，疏泄腠理，助邪外出之效。以黄芪、赤芍、葶苈子、大黄为佐药，黄芪，甘温益肺脾气，赤芍，苦、微寒，凉血散瘀，用以治疗疫病后期伤其正气，气机郁闭导致的血瘀等；葶苈子辛寒，辅助君药石膏清泄肺热，兼以利水，预防或治疗“湿肺(肺水肿)病变”；大黄，苦寒入大肠经而通腑，肺与大肠相表里，辅助君药石膏清泄肺热，并配合赤芍凉血活血，四药共为佐药，以达顾护正气、泻热凉血、活血化瘀之效。以甘草为使药，甘草甘平，调和诸药，配赤芍取芍药甘草汤意缓急。全方共奏解表化湿，清热平喘、益气散瘀之功。
临床发现新冠肺炎重型患者具有如下特点：①发热为主要表现多为身热不扬，缠绵难愈，但亦可为中低热，甚至不发热；②喘憋、乏力显著，亦为主要表现；③多合并纳差、便溏、腹泻等消化系统症状；④大多舌苔厚腻。从上述特点来看，符合湿邪致病特点：重浊、碍气伤阳、黏滞、趋下。湿邪既可单独致病，又可兼寒、兼热，表现为寒湿、湿热，其中热可为伏燥所致，亦可为湿邪久郁所化。湿邪、寒湿、湿热均可与疫毒合而致病，可见于轻型的寒湿郁肺、湿热蕴肺，普通型的湿毒郁肺、寒湿阻肺，若失治误治，或疾病发展，伤及营血，逆传心包，演变为新冠肺炎重型。因此，认为新冠肺炎突出表现为“湿毒疫”，病位在肺，与脾密切相关，病理性质为寒热错杂、虚实并见，病理因素为毒、湿、热、寒、瘀、虚，其中疫毒为根本，核心病机为疫毒与湿邪博结，可 兼寒、热侵袭机体，闭阻胸肺，气机升降失常，血脉瘀阻，气阴耗伤。新冠肺炎病理性质复杂，涉及多个病理因素。
主要病位在肺，其次在脾胃，湿毒郁闭是其核心病机，可分为初期、中期、危重期及恢复期四期进行辨证论治，治法有化湿行气，辟秽解毒，清肺化痰，活血化瘀、通腑攻下和补益正气等方法。因此，本发明化湿败毒组合物配伍依据核心病机，当以解表化湿，清热平喘，祛毒为核心治法，兼以化瘀通络，益气养阴。疫毒与寒湿相合，恶寒发热，治宜解表化湿祛毒；疫毒与湿热相合，便溏不爽，倦怠乏力，治宜清热化湿祛毒，兼以益气养阴；闭阻胸肺，喘憋、胸闷、气短，治宜平喘，兼以化瘀通络。
本发明化湿败毒组合物融合了《新型冠状病毒感染的肺炎诊疗方案(试行第五版)》中医治疗的核心病机，属温热夹湿，致肺失宣降，肺气壅闭”，以湿浊化热郁肺为主，突显化湿行气，宣肺平喘，清热化痰，益气活血之功效。前期临床观察显示，本发明能够改善重型新型冠状病毒感染肺炎的临床症状，对于重症患者，可明显缓解咳嗽、乏力、口干或呕吐等主要症状，中西医结合治疗后，治愈时间缩短。明显改善患者的呼吸功能，脱离吸氧时间缩短。对于普通型患者，对发热症状明显缓解，还可改善食欲减退、胸闷症状。对于重型及普通型新型冠状病毒感染肺炎的咳嗽、乏力、口干或呕吐等临床症状改善显著，补充了目前疫情形势急需的重型及普通型新型冠状病毒感染肺炎治疗用药。
优选的，化湿败毒组合物包括以下组分：
麻黄3-60份，炒苦杏仁4.5-90份，生石膏7.5-150份，甘草1.5-30份，广藿香5-100份，厚朴5-100份，麸炒苍术7.5-150份，炒草果仁5-100份，法半夏4.5-90份，茯苓7.5-150份，大黄2.5-50份，黄芪5-100份，葶苈子5-100份，赤芍5-100份，辅料适量。
所述化湿败毒组合物被制成中药制剂，所述中药制剂为颗粒剂、汤剂、散剂、胶囊剂、口服液、片剂或丸剂。
本发明提供了一种化湿败毒组合物的检测方法，其对任意剂型的化湿败毒组合物均可以起到良好的检测效果。而在化湿败毒组合物药方的研究过程中，发明人采用分子对接技术对化湿败毒组合物配方中各味中药与COVID-19侵入、复制、组装、脱落转移的关键靶标以及对宿主产生肺部损伤和炎症反应的关键作用靶标进行分析。结果表明：大黄对于抑制病毒侵入和脱落的靶标TMPRSS2， 病毒侵入宿主后产生的组织损伤关键靶标AMPK(氧化应激、炎症)、VEGFR2(血管通透)和ALK5(血管通透、肺部纤维化)有响应，从而发挥抗COVID-19的作用。进一步的研究表明，大黄中的大黄酸、大黄素甲醚、6-羟基酸模素-8-O-β-D-葡糖糖苷、原矢车菊素B1-3’-O-没食子酸酯、芦荟大黄素等5个成分对上述靶标有作用。由此可见，大黄属于化湿败毒组合物中的核心药，对大黄相关成分的检测是实现化湿败毒组合物质量控制的关键手段之一。
进一步的，6-羟基酸模素-8-O-β-D-葡糖糖苷、原矢车菊素B1-3’-O-没食子酸酯等成分测试困难。因此，本发明选择对大黄酸、大黄素甲醚、芦荟大黄素进行监控，通过以上3个组分的监控即可体现化湿败毒方中大黄的含量。
此外，化湿败毒组合物中大黄主要的作用是清泻肺热，活血凉血；其主要是通过大黄所引入的结合蒽醌(芦荟大黄素、大黄酸、大黄素、大黄酚、大黄素甲醚)实现的。但结合蒽醌在受热时容易分解成游离蒽醌，导致药效减弱。为此，需要对结合蒽醌的含量进行监控。
综合以上几个方面，本发明在制定化湿败毒组合物的检测方法时，选择对成品药物中结合蒽醌的含量进行测定、监控，以实现对化湿败毒组合物的质量检测。具体的，在本发明的化湿败毒组合物中，控制结合蒽醌的含量不得少于0.016wt％。
本发明通过高效液相色谱法测定化湿败毒组合物中的总蒽醌含量和游离蒽醌含量，进而计算结合蒽醌含量。其中，结合蒽醌含量＝总蒽醌含量-游离蒽醌含量。
具体的，在本发明中，总蒽醌含量的测定方法包括：
(1)色谱条件：以十八烷基硅烷键合硅胶色谱柱为色谱柱，以乙腈-0.1vol％磷酸水溶液为流动相，流速为0.8mL/min，检测波长为254nm，柱温为30℃；进样量为10μL。
流动相按如下洗脱顺序进行梯度洗脱：
Figure PCTCN2020114894-appb-000001
(2)总蒽醌供试品溶液制备：取化湿败毒组合物约0.5g，精密称定，置具塞锥形瓶中，精密加入甲醇25mL，称定重量，加热回流提取30分钟，取出，放冷，再称定重量，用甲醇补足减失的重量，摇匀，滤过，取续滤液10mL，减压回收溶剂至干，加8％盐酸溶液10mL，超声处理2分钟，再加三氯甲烷10mL，加热回流1小时，放冷，置分液漏斗中，用少量三氯甲烷洗涤容器，并入分液漏斗中，分取三氯甲烷层，酸液再用三氯甲烷提取3次，每次10mL，合并三氯甲烷液，减压回收溶剂至干，残渣加甲醇使溶解，转移至10mL量瓶中，加甲醇至刻度，摇匀，滤过，取续滤液，即得。
(3)对照品溶液制备：称定芦荟大黄素对照品1.581mg、大黄酸对照品3.017mg、大黄素对照品1.604mg、大黄酚对照品2.656mg、大黄素甲醚对照品5.221mg，置100mL量瓶中，加甲醇分别制成每1mL含芦荟大黄素15.807μg、大黄酸30.167μg、大黄素16.039μg、大黄酚26.563μg、大黄素甲醚52.213μg的母液；分别精密吸取上述芦荟大黄素、大黄酸、大黄素、大黄酚母液各1mL，大黄素甲醚母液0.1mL，置10mL量瓶中，加甲醇制成每1mL含芦荟大黄素1.581μg、大黄酸3.017μg、大黄素1.604μg、大黄酚2.656μg、大黄素甲醚0.522μg的混合溶液，即得。
(4)精密吸取总蒽醌供试品溶液、对照品溶液各10μL，注入液相色谱仪，测定，即得化湿败毒组合物中总蒽醌含量。
在本发明中，游离蒽醌含量的测定方法如下：
(1)色谱条件：以十八烷基硅烷键合硅胶色谱柱为色谱柱，以乙腈-0.1vol％磷酸水溶液为流动相，流动相流速为0.8mL/min，检测波长为254nm，柱温为30℃，进样量为10μL。
流动相按如下洗脱顺序进行梯度洗脱：
Figure PCTCN2020114894-appb-000002
(2)游离蒽醌供试品溶液制备：取化湿败毒组合物约0.5g，精密称定，置具塞锥形瓶中，精密加入甲醇25mL，称定重量，加热回流30分钟，取出，放 冷，再称定重量，用甲醇补足减失的重量，摇匀，滤过，取续滤液，即得。
(3)对照品溶液制备：称定芦荟大黄素对照品1.581mg、大黄酸对照品3.017mg、大黄素对照品1.604mg、大黄酚对照品2.656mg、大黄素甲醚对照品5.221mg，置100mL量瓶中，加甲醇分别制成每1mL含芦荟大黄素15.807μg、大黄酸30.167μg、大黄素16.039μg、大黄酚26.563μg、大黄素甲醚52.213μg的母液；分别精密吸取上述芦荟大黄素、大黄酸、大黄素、大黄酚母液各1mL，大黄素甲醚母液0.1mL，置10mL量瓶中，加甲醇制成每1mL含芦荟大黄素1.581μg、大黄酸3.017μg、大黄素1.604μg、大黄酚2.656μg、大黄素甲醚0.522μg的混合溶液，即得。
(4)精密吸取游离蒽醌供试品溶液、对照品溶液各10μL，注入液相色谱仪，测定得到化湿败毒组合物中游离蒽醌含量。
需要说明的是，所述总蒽醌含量的测定方法和游离蒽醌含量的测定方法均采用相同的洗脱程序进行，所述总蒽醌含量的测定方法和游离蒽醌含量的测定方法中采用相同的对照品溶液。
还需要说明的是，由于化湿败毒组合物总共含有14味原料药，其成品成分复杂，对于总蒽醌和游离蒽醌的测试准确度有较大的影响。为此，本发明在考虑测试方法准确性、耐用性以及专属性的基础上，重新设计了梯度洗脱程序。具体的，研究发现：在10min～38min，乙腈比例40％→60％，0.1vol％磷酸比例60％→40％，可有效地分离化湿败毒组合物中的芦荟大黄素、大黄酸、大黄素、大黄酚，无需设置太多梯度变化。此外，在38min～48min，大黄素甲醚在不同的色谱柱上出峰时间不同，因此在此时间范围内，设定乙腈-0.1vol％磷酸(60:40)等度洗脱10min，以确保测试方法对不同色谱柱耐用性，也提升测试准确度。
以下对本发明中总蒽醌和游离蒽醌的测定方法进行方法学验证：
1专属性考察
取大黄阴性样品按总蒽醌/游离蒽醌供试品溶液制备方法分别制备总蒽醌/游离蒽醌阴性样品溶液。将游离蒽醌供试品溶液、总蒽醌供试品溶液、游离蒽醌阴性样品溶液、总蒽醌阴性样品溶液和对照品溶液各10μL分别注入液相色谱仪，进行测试，其结果参见图1～图5。其中，各个对照品的出峰位置如下表所示：
表1 化湿败毒组合物总蒽醌/游离蒽醌含量测定专属性考察出峰位置表
Figure PCTCN2020114894-appb-000003
结果显示，供试品色谱在与对照品色谱相应的保留时间处有相同的色谱峰，游离蒽醌阴性样品无干扰，总蒽醌阴性样品无干扰，该方法专属性良好。
2线性关系考察
2.1游离蒽醌含量测定线性关系考察
精密称定芦荟大黄素对照品3.570mg、大黄素对照品2.751mg、大黄酚对照品1.631mg、大黄素甲醚对照品3.051mg，各置50mL量瓶中，加甲醇分别制成每1mL含芦荟大黄素71.405μg、大黄素55.015μg、大黄酚32.629μg、大黄素甲醚61.024μg的溶液；精密测定大黄酸对照品2.057mg，再精密吸取上述芦荟大黄素、大黄素、大黄酚对照品溶液各5mL，大黄素甲醚对照品1mL，共置100mL量瓶中，加甲醇制成每1mL含芦荟大黄素3.570μg、大黄酸20.565μg、大黄素2.751μg、大黄酚1.631μg、大黄素甲醚0.610μg的混合溶液，作为游离蒽醌对照品母液。精密吸取游离蒽醌对照品母液5mL、3mL、1mL、0.5mL、0.2mL，各置10mL量瓶中，分别制成每1mL含0.071μg、0.179μg、0.357μg、1.071μg、1.785μg的芦荟大黄素，每1mL含0.411μg、1.028μg、2.057μg、6.170μg、10.283μg的大黄酸，每1mL含0.055μg、0.138μg、0.275μg、0.825μg、1.375μg的大黄素，每1mL含0.033μg、0.082μg、0.163μg、0.489μg、0.816μg的大黄酚，每1mL含0.012μg、0.030μg、0.061μg、0.183μg、0.305μg的大黄素甲醚对照品溶液。
分别精密吸取上述6个不同浓度的对照品溶液10μL，测试分析，记录色谱峰面积。以峰面积为纵坐标(y)，对照品浓度为横坐标(x)，并绘制标准曲线，其结果如图6～图10所示。
由图6可以看出：芦荟大黄素的回归方程为：y＝66,512.6130x+128.8558，其相关系数R 2＝0.9994，表明芦荟大黄素在进样浓度0.071μg/mL～3.570μg/mL的范围内进样质量与峰面积线性关系良好。
由图7可以看出，大黄酸的回归方程为：y＝49,694.7092x-1,493.7898，其相关系数R 2＝0.9998，表明大黄酸在进样浓度0.411μg/mL～20.565μg/mL的范围内 进样质量与峰面积线性关系良好。
由图8可以看出，大黄素的回归方程为：y＝50,043.3431x+269.0534，其相关系数R 2＝0.9995，表明大黄素在进样浓度0.055μg/mL～2.751μg/mL的范围内进样质量与峰面积线性关系良好。
由图9可以看出，大黄酚的回归方程为y＝68,915.3676x+180.1324，其相关系数R 2＝0.9995，表明大黄酚在进样浓度0.033μg/mL～1.631μg/mL的范围内进样质量与峰面积线性关系良好。
由图10可以看出，大黄素甲醚的回归方程为：y＝51,006.3529x+707.5671，其相关系数R 2＝0.995，表明大黄素甲醚在进样浓度0.012μg/mL～0.610μg/mL的范围内进样质量与峰面积线性关系良好。
2.2总蒽醌含量测定线性关系考察
按照蒽醌对照品溶液制备方法制备每1mL含芦荟大黄素15.807μg、大黄酸30.167μg、大黄素16.039μg、大黄酚26.563μg、大黄素甲醚5.221μg的溶液，作为总蒽醌对照品母液。精密吸取总蒽醌对照品母液5mL、3mL、1mL、0.5mL、0.2mL，各置10mL量瓶中，分别制成每1mL含0.316μg、0.790μg、1.581μg、4.742μg、7.903μg的芦荟大黄素，每1mL含0.603μg、1.508μg、3.017μg、9.050μg、15.084μg的大黄酸，每1mL含0.321μg、0.802μg、1.604μg、4.812μg、8.109μg的大黄素，每1mL含0.531μg、1.328μg、2.656μg、7.969μg、13.282μg的大黄酚，每1mL含0.104μg、0.261μg、0.522μg、1.566μg、2.611μg的大黄素甲醚对照品溶液。
分别精密吸取上述6个不同浓度的对照品溶液10μL，测试分析，记录色谱峰面积。以峰面积为纵坐标(y)，对照品浓度为横坐标(x)，并绘制标准曲线，其结果如图11～图15所示。
由图11可以看出，芦荟大黄素的回归方程为：y＝58108.7967x-793.9060，其相关系数R 2＝0.9999，表明芦荟大黄素在进样浓度0.316μg/mL～15.807μg/mL的范围内进样质量与峰面积线性关系良好。
由图12可以看出，大黄酸的回归方程为：y＝50,539.6466x-3,498.3368，其相关系数R 2＝1.0000，表明大黄酸在进样浓度0.603μg/mL～30.167μg/mL的范围内进样质量与峰面积线性关系良好。
由图13可以看出，大黄素的回归方程为：y＝43,070.1473x-501.3586，其相 关系数R 2＝0.9999，表明大黄素在进样浓度0.321μg/mL～16.039μg/mL的范围内进样质量与峰面积线性关系良好。
由图14可以看出，大黄酚的回归方程为：y＝61,967.3466x-2,584.2033，其相关系数R 2＝0.9999，表明大黄酚在进样浓度0.531μg/mL～26.563μg/mL的范围内进样质量与峰面积线性关系良好。
由图15可以看出，大黄素甲醚的回归方程为：y＝30,079.1466x+505.0118，其相关系数R 2＝1.000，表明大黄素甲醚在进样浓度0.104μg/mL～5.221μg/mL的范围内进样质量与峰面积线性关系良好。
3精密度考察
3.1仪器精密度
精密吸取对照品溶液10μL，测试分析，以芦荟大黄素、大黄酸、大黄素、大黄酚和大黄素甲醚峰面积计算，测定结果见表2。
表2 化湿败毒组合物游离蒽醌含量测定仪器精密度考察结果表
Figure PCTCN2020114894-appb-000004
结果显示，同一份对照品溶液连续进样5针，各指标峰面积RSD值均小于3.0％，说明仪器精密度良好。
3.2中间精密度考察
选择不同实验人员在不同的时间、不同的高效液相色谱仪测定，取化湿败毒组合物适量，研细，取约0.5g，精密称定，平行6份，分别制备游离蒽醌供试品溶液、总蒽醌供试品溶液，精密吸取游离蒽醌供试品溶液或总蒽醌供试品溶液、对照品溶液各10μL，测试分析，计算游离蒽醌、总蒽醌含量及RSD值，与重复性考察试验结果相比较，结果见表3～4。
表3 化湿败毒组合物游离蒽醌含量测定中间精密度考察结果表
Figure PCTCN2020114894-appb-000005
表4 化湿败毒组合物总蒽醌含量测定中间精密度考察结果表
Figure PCTCN2020114894-appb-000006
结果显示，同一批样品由不同的人员于不同的时间在不同的仪器上操作，重复测定6次，游离蒽醌含量RSD值为0.49％，与重复性试验6个数据的RSD 值为0.45％；总蒽醌含量RSD值为0.75％，与重复性试验6个数据的RSD值为0.92％，根据中国药典2015年版“药品质量标准分析方法验证指导原则”规定样品中待测成分含量在0.01％～0.1％时，中间精密度RSD限度为＜6％，因此，不同分析人员在不同日期和不同色谱仪下操作，该方法中间精密度良好。
4稳定性考察
精密吸取游离蒽醌供试品溶液和总蒽醌供试品溶液10μL，分别在0、2.5、6、9.5、13、17、20.5、25.5小时进样，测定供试品溶液中芦荟大黄素、大黄酸、大黄素、大黄酚和大黄素甲醚的峰面积，计算峰面积RSD值，测定结果见表5和表6。
表5 化湿败毒组合物游离蒽醌含量测定稳定性考察结果表
Figure PCTCN2020114894-appb-000007
表6 化湿败毒组合物总蒽醌含量测定稳定性考察结果表
Figure PCTCN2020114894-appb-000008
Figure PCTCN2020114894-appb-000009
结果显示，同一份游离蒽醌供试品溶液，分别在0.0、2.5、6.0、9.5、13.0、17.0、20.5、25.5小时进样测定，各指标成分峰面积RSD值均小于3.0％，说明供试品溶液在24小时内稳定性良好。
同一份总蒽醌供试品溶液，分别在0.0、2.5、6.0、9.5、13.0、17.0、20.5、25.5小时进样测定，各指标成分除了大黄素甲醚峰面积RSD值小于3.0％，其余成分均大于3％，建议总蒽醌供试品溶液在13小时完成测定。
5重复性考察
精密吸取游离蒽醌供试品溶液和总蒽醌供试品溶液10μL，进行测试，计算游离蒽醌、总蒽醌含量及RSD值，测定结果见表7和表8。
表7 化湿败毒组合物游离蒽醌含量测定重复性考察结果表
Figure PCTCN2020114894-appb-000010
表8 化湿败毒组合物总蒽醌含量测定重复性考察结果表
Figure PCTCN2020114894-appb-000011
Figure PCTCN2020114894-appb-000012
结果显示，同一批样品重复测定6次，游离蒽醌及总蒽醌各指标含量RSD值均小于3％，说明该分析方法的重复性良好。
6准确度考察
6.1游离蒽醌含量测定准确度考察
精密称定芦荟大黄素对照品2.455mg、大黄酸对照品2.485mg、大黄素对照品1.625mg、大黄酚对照品1.950mg、大黄素甲醚对照品0.845mg，分别置250、50、250、500、500mL量瓶中，加甲醇分别制成每1mL含芦荟大黄素0.00982mg、大黄酸0.04969mg、大黄素0.00650mg、大黄酚0.00390mg、大黄素甲醚0.00169mg的溶液，作为加样回收母液。精密吸取上述母液各1mL于锥形瓶中，平行6份，挥干溶剂。再取化湿败毒组合物，制备游离蒽醌供试品溶液，测定供试品中游离蒽醌含量，计算加样回收率，结果见表9～13。
表9 化湿败毒组合物游离蒽醌芦荟大黄素含量测定加样回收率考察结果表
Figure PCTCN2020114894-appb-000013
表10 化湿败毒组合物游离蒽醌大黄酸含量测定加样回收率考察结果表
Figure PCTCN2020114894-appb-000014
Figure PCTCN2020114894-appb-000015
表11 化湿败毒组合物游离蒽醌大黄素含量测定加样回收率考察结果表
Figure PCTCN2020114894-appb-000016
表12 化湿败毒组合物游离蒽醌大黄酚含量测定加样回收率考察结果表
Figure PCTCN2020114894-appb-000017
表13 化湿败毒组合物游离蒽醌大黄素甲醚含量测定加样回收率考察结果表
Figure PCTCN2020114894-appb-000018
从表中可以看出，芦荟大黄素回收率为112.08％，大黄酸回收率为86.17％，大黄素回收率为107.31％，大黄酚回收率为97.79％，大黄素甲醚回收率为105.93％，根据中国药典2015年版“药品质量标准分析方法验证指导原则”规定 样品中待测成分含量在0.001％～0.01％时，回收率限度为80％～115％，本发明测定方法准确度良好
6.2总蒽醌含量测定准确度考察
精密称定芦荟大黄素对照品2.497mg、大黄酸对照品3.960mg、大黄素对照品2.707mg、大黄酚对照品2.313mg、大黄素甲醚对照品1.995mg，分别置50mL量瓶中，加甲醇分别制成每1mL含芦荟大黄素0.04994mg、大黄酸0.07920mg、大黄素0.05414mg、大黄酚0.04625mg、大黄素甲醚0.03990mg的溶液，作为加样回收母液。精密吸取上述芦荟大黄素、大黄素、大黄素甲醚母液各1mL，大黄酸、大黄酚各2mL于锥形瓶中，平行6份，挥干溶剂；再取化湿败毒组合物适量，研细，分别取0.25g于上述6个锥形瓶中，制备总蒽醌供试品溶液，并测定供试品中总蒽醌含量，计算加样回收率，结果见表14～18。
表14 化湿败毒组合物总蒽醌芦荟大黄素含量测定加样回收率考察结果表
Figure PCTCN2020114894-appb-000019
表15 化湿败毒组合物总蒽醌大黄酸含量测定加样回收率考察结果表
Figure PCTCN2020114894-appb-000020
表16 化湿败毒组合物总蒽醌大黄素含量测定加样回收率考察结果表
Figure PCTCN2020114894-appb-000021
Figure PCTCN2020114894-appb-000022
表17 化湿败毒组合物总蒽醌大黄酚含量测定加样回收率考察结果表
Figure PCTCN2020114894-appb-000023
表18 化湿败毒组合物总蒽醌大黄素甲醚含量测定加样回收率考察结果表
Figure PCTCN2020114894-appb-000024
结果显示，芦荟大黄素回收率为95.05％，大黄酸回收率为88.68％，大黄素回收率为101.25％，大黄酚回收率为98.83％，大黄素甲醚回收率为101.53％，根据中国药典2015年版“药品质量标准分析方法验证指导原则”规定样品中待测成分含量在0.001％～0.01％时，回收率限度为80％～115％，该方法准确度良好。
7耐用性考察
7.1不同色谱柱考察
选用三种色谱柱，分别是菲罗门Kinetex-EVO C18色谱柱(4.6mm×150mm， 5μm；编号：JS-091)，Waters HSS T3色谱柱(4.6mm×150mm，5μm；编号：JS-136)，Waters X-bridge C18色谱柱(4.6mm×150mm，5μm；编号：JS-154)。
取游离蒽醌供试品溶液及总蒽醌供试品溶液各10μL，分别采用以上三种色谱柱进行测定，计算游离蒽醌及总蒽醌含量及RSD值，实验结果见表19。
表19 化湿败毒组合物总/游离蒽醌含量测定不同色谱柱考察结果表
Figure PCTCN2020114894-appb-000025
结果显示，各色谱柱分离效果良好，含量RSD值≤5％，表明该分析方法在不同色谱柱下分析耐用性良好。
7.2不同柱温考察
分别设定柱温为28℃、30℃和32℃，考察柱温对对化湿败毒组合物游离蒽醌及总蒽醌含量测定的影响。
取游离蒽醌供试品溶液及总蒽醌供试品溶液各10μL，分别采用以上柱温进行测定，计算游离蒽醌及总蒽醌含量及RSD值，实验结果见表20。
表20 化湿败毒组合物总/游离蒽醌含量测定不同柱温考察结果表
Figure PCTCN2020114894-appb-000026
结果显示，不同柱温的化湿败毒组合物游离蒽醌及总蒽醌含量值，其RSD值均大于5％，表明该分析方法对柱温较为敏感，因此，严格控制柱温在30℃。
7.3不同流速考察
分别设定流速为0.7mL/min、0.8mL/min和0.9mL/min，考察流速对对化湿败毒组合物游离蒽醌及总蒽醌含量测定的影响。
取游离蒽醌供试品溶液及总蒽醌供试品溶液各10μL，分别采用以上流速进 行测定，计算游离蒽醌及总蒽醌含量及RSD值，实验结果见表21。
表21 化湿败毒组合物总/游离蒽醌含量测定不同流速考察结果表
Figure PCTCN2020114894-appb-000027
结果显示，三种不同流速的化湿败毒组合物游离蒽醌及总蒽醌含量值，其RSD值均小于5％。但当流速为0.7mL/min时，总蒽醌中的大黄素甲醚出现裂峰，建议分析时控制流速应不低于0.8mL/min。
8不同批次化湿败毒组合物供试品的含量测定
取10批化湿败毒组合物，分别制备游离蒽醌供试品溶液及总蒽醌供试品溶液，并测定10批化湿败毒组合物中游离蒽醌含量及总蒽醌含量，并计算结合蒽醌含量；实验结果见表22。
表22 化湿败毒组合物结合蒽醌含量测定结果表
Figure PCTCN2020114894-appb-000028
综上所述，本发明建立了化湿败毒组合物中结合蒽醌的测定方法，该检测方法的专属性良好，准确可靠，稳定性好。通过上述测定方法可对化湿败毒组合物中总蒽醌和游离蒽醌的含量进行测定，进而计算出结合蒽醌含量，从而保证化湿败毒组合物产品质量稳定性和可控性，为大生产提供良好的基础。
以上所述是发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也视为本发明的保护范围。

Claims (12)

  1. 一种化湿败毒组合物的检测方法，其特征在于，所述化湿败毒组合物主要包括以下组分：麻黄，炒苦杏仁，生石膏，甘草，广藿香，厚朴，麸炒苍术，炒草果仁，法半夏，茯苓，大黄，黄芪，葶苈子，赤芍；
    所述化湿败毒组合物的检测方法包括采用高效液相色谱法测定化湿败毒组合物中总蒽醌含量和游离蒽醌含量，并计算结合蒽醌含量；
    其中，结合蒽醌含量＝总蒽醌含量-游离蒽醌含量。
  2. 如权利要求1所述的化湿败毒组合物的检测方法，其特征在于，所述总蒽醌含量的测定方法包括：
    (1)取芦荟大黄素、大黄酸、大黄素、大黄酚、大黄素甲醚适量，加甲醇制成混合溶液，制得对照品溶液；
    (2)取化湿败毒组合物，利用甲醇提取，制得总蒽醌供试品溶液；
    (3)吸取对照品溶液和总蒽醌供试品溶液，注入液相色谱仪，所述液相色谱仪以十八烷基硅烷键合硅胶为填充剂，以乙腈为流动相A，磷酸水溶液为流动相B进行梯度洗脱，测定得到化湿败毒组合物中总蒽醌含量。
  3. 如权利要求1所述的化湿败毒组合物的检测方法，其特征在于，所述游离蒽醌含量的测定方法包括：
    (一)取芦荟大黄素、大黄酸、大黄素、大黄酚、大黄素甲醚适量，加甲醇制成混合溶液，制得对照品溶液；
    (二)取化湿败毒组合物，利用甲醇提取，制得游离蒽醌供试品溶液；
    (三)吸取对照品溶液和游离蒽醌供试品溶液，注入液相色谱仪，所述液相色谱仪以十八烷基硅烷键合硅胶为填充剂，以乙腈为流动相A，磷酸水溶液为流动相B进行梯度洗脱，测定得到化湿败毒组合物中游离蒽醌含量。
  4. 如权利要求2或3所述的化湿败毒组合物的检测方法，其特征在于，所述总蒽醌含量的测定方法和游离蒽醌含量的测定方法均采用以下洗脱程序进行：
    0～10min，流动相A从35％→40％，流动相B从65％→60％；
    10～38min，流动相A从40％→60％，流动相B从60％→40％；
    38～48min，流动相A为60％，流动相B为40％。
  5. 如权利要求2所述的化湿败毒组合物的检测方法，其特征在于，所述总蒽醌供试品溶液由下述方法制得：
    取化湿败毒组合物0.2～0.5g，置具塞锥形瓶中，加入甲醇20～30mL，加热回流提取20～30分 钟，取出，放冷，再称定重量，用甲醇补足减失的重量，摇匀，滤过，取续滤液10～15mL，减压回收溶剂至干，加8％盐酸溶液10～15mL，超声处理2～5分钟，再加三氯甲烷10～20mL，加热回流1～3小时，放冷，置分液漏斗中，用少量三氯甲烷洗涤容器，并入分液漏斗中，分取三氯甲烷层，酸液再用三氯甲烷提取2～5次，每次10～15mL，合并三氯甲烷液，减压回收溶剂至干，残渣加甲醇使溶解，转移至10mL量瓶中，加甲醇至刻度，摇匀，滤过，取续滤液，即得。
  6. 如权利要求3所述的化湿败毒组合物的检测方法，其特征在于，所述游离蒽醌供试品溶液由下述方法制得：
    取化湿败毒组合物0.2～0.5g，置具塞锥形瓶中，加入甲醇25～30mL，称定重量，加热回流20～60分钟，取出，放冷，再称定重量，用甲醇补足减失的重量，摇匀，滤过，取续滤液，即得。
  7. 如权利要求2或3所述的化湿败毒组合物的检测方法，其特征在于，所述对照品溶液由下述方法制得：
    称定芦荟大黄素对照品1.581mg、大黄酸对照品3.017mg、大黄素对照品1.604mg、大黄酚对照品2.656mg、大黄素甲醚对照品5.221mg，置100mL量瓶中，加甲醇分别制成每1mL含芦荟大黄素15.807μg、大黄酸30.167μg、大黄素16.039μg、大黄酚26.563μg、大黄素甲醚52.213μg的母液；分别精密吸取上述芦荟大黄素、大黄酸、大黄素、大黄酚母液各1mL，大黄素甲醚母液0.1mL，置10mL量瓶中，加甲醇制成每1mL含芦荟大黄素1.581μg、大黄酸3.017μg、大黄素1.604μg、大黄酚2.656μg、大黄素甲醚0.522μg的混合溶液，即得。
  8. 如权利要求2或3所述的化湿败毒组合物的检测方法，其特征在于，所述总蒽醌含量测定方法和游离蒽醌含量的测定方法中，以十八烷基硅烷键合硅胶为填充剂，以乙腈为流动相A，0.1vol％磷酸水溶液为流动相B进行梯度洗脱，流速为0.6～1mL/min，检测波长为253～256nm，柱温为25～35℃。
  9. 如权利要求8所述的的化湿败毒组合物的检测方法，其特征在于，所述总蒽醌含量的测定方法和游离蒽醌含量的测定方法中，以十八烷基硅烷键合硅胶为填充剂，以乙腈为流动相A，0.1vol％磷酸水溶液为流动相B进行梯度洗脱，流速为0.8mL/min，检测波长为254nm，柱温为30℃。
  10. 如权利要求2所述的化湿败毒组合物的检测方法，其特征在于，所述总蒽醌含量的测定方法中，所述对照品溶液、总蒽醌供试品溶液的进样量为10μL。
  11. 如权利要求3所述的化湿败毒组合物的检测方法，其特征在于，所述游离蒽醌含量的测定方法中，所述对照品溶液、游离蒽醌供试品溶液的进样量为10μL。
  12. 如权利要求1所述的化湿败毒组合物的检测方法，其特征在于，所述化湿败毒组合物主要包括下述组分：麻黄3-60份，炒苦杏仁4.5-90份，生石膏7.5-150份，甘草1.5-30份，广藿香5-100份，厚朴5-100份，麸炒苍术7.5-150份，炒草果仁5-100份，法半夏4.5-90份，茯苓7.5-150份，大黄2.5-50份，黄芪5-100份，葶苈子5-100份，赤芍5-100份，辅料适量；
    所述化湿败毒组合物被制成中药制剂，所述中药制剂为颗粒剂、汤剂、散剂、胶囊剂、口服液、片剂或丸剂。
PCT/CN2020/114894 2020-08-19 2020-09-11 化湿败毒组合物的检测方法 WO2022036780A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010834342.9A CN112180000B (zh) 2020-08-19 2020-08-19 化湿败毒组合物的检测方法
CN202010834342.9 2020-08-19

Publications (1)

Publication Number Publication Date
WO2022036780A1 true WO2022036780A1 (zh) 2022-02-24

Family

ID=73919440

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/114894 WO2022036780A1 (zh) 2020-08-19 2020-09-11 化湿败毒组合物的检测方法

Country Status (2)

Country Link
CN (1) CN112180000B (zh)
WO (1) WO2022036780A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116223656A (zh) * 2022-12-30 2023-06-06 广东一方制药有限公司 化湿败毒组合物的检测方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104483399A (zh) * 2014-11-07 2015-04-01 无锡济民可信山禾药业股份有限公司 黄氏响声丸制剂的质量控制方法
CN109541049A (zh) * 2018-11-02 2019-03-29 漳州片仔癀药业股份有限公司 一种清火片的质量控制方法
CN111735889A (zh) * 2020-08-19 2020-10-02 广东一方制药有限公司 化湿败毒组合物的质量检测方法
CN111983106A (zh) * 2020-08-19 2020-11-24 广东一方制药有限公司 化湿败毒组合物的质量控制方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103235082B (zh) * 2013-04-27 2015-01-21 贵州盛世龙方制药股份有限公司 精乌胶囊的检测方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104483399A (zh) * 2014-11-07 2015-04-01 无锡济民可信山禾药业股份有限公司 黄氏响声丸制剂的质量控制方法
CN109541049A (zh) * 2018-11-02 2019-03-29 漳州片仔癀药业股份有限公司 一种清火片的质量控制方法
CN111735889A (zh) * 2020-08-19 2020-10-02 广东一方制药有限公司 化湿败毒组合物的质量检测方法
CN111983106A (zh) * 2020-08-19 2020-11-24 广东一方制药有限公司 化湿败毒组合物的质量控制方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GUO QI, HUI ZHANG, GUI-BEN LI, LI HAI-YAN: "Determination of Free Anthraquinones, Total Anthraquinones and Conjugated Anthraquinones in Lidan Paishi Tablets", SHIPIN YU YAOPIN = FOOD AND DRUG, SHANDONG SHENG SHENGWU YAOWU YANJIUYUAN, CHINA, vol. 17, no. 6, 20 November 2015 (2015-11-20), China , pages 426 - 430, XP055902179, ISSN: 1672-979X *
YANG XIUWEI: "Material Basis Ｒesearch of Anti-COVID-19 Huashibaidu Granule Gormula", MODERN CHINESE MEDICINE, ZHONG GUO XIAN DAI ZHONG YAO BIAN JI BU, CN, vol. 22, no. 5, 16 May 2020 (2020-05-16), CN , pages 672 - 689, XP055901093, ISSN: 1673-4890, DOI: 10.13313/j.issn.1673-4890.20200331002 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116223656A (zh) * 2022-12-30 2023-06-06 广东一方制药有限公司 化湿败毒组合物的检测方法
CN116223656B (zh) * 2022-12-30 2024-04-23 广东一方制药有限公司 化湿败毒组合物的检测方法

Also Published As

Publication number Publication date
CN112180000B (zh) 2021-09-10
CN112180000A (zh) 2021-01-05

Similar Documents

Publication Publication Date Title
WO2022036781A1 (zh) 化湿败毒组合物的质量控制方法
CN111735889B (zh) 化湿败毒组合物的质量检测方法
CN111735896B (zh) 化湿败毒组合物特征图谱的构建方法
WO2022036795A1 (zh) 化湿败毒组合物的鉴别方法
WO2022036779A1 (zh) 化湿败毒颗粒及其制备方法和抗病毒药物
WO2021168855A1 (zh) 一种物质含量测定方法及其应用
CN110780004A (zh) 一种具有调节抑郁情绪作用的中药组合物或其制剂指纹图谱的建立方法
CN104758515A (zh) 一种治疗肾病的中药组合物及其制备方法和检测方法
CN107402260B (zh) 一种药物组合物的检测方法
CN104614450B (zh) 一种消渴清制剂的指纹图谱检测方法
CN103344737A (zh) 一种治疗鼻窦炎的中药片剂的质量控制方法
WO2022036780A1 (zh) 化湿败毒组合物的检测方法
CN110954645A (zh) 一种高质量四黄止痢颗粒的检测方法
CN113769023B (zh) 一种治疗高血压的中药复方组合物及其质量检测方法
CN106706766A (zh) 一种治疗艾滋病的中药唐草片新增检测成分的测定方法
CN102049012B (zh) 一种治疗儿童性早熟的中药组合物及其制备和检测方法
CN108226325A (zh) 葶苈生脉口服液组合物指纹图谱的建立方法
CN107389827A (zh) 益母草或其复方制剂中盐酸水苏碱含量的测定方法
CN116223656B (zh) 化湿败毒组合物的检测方法
CN113499382B (zh) 一种清肺解毒的中药组合物和药物制剂及其制备方法和应用
WO2024060536A1 (zh) 一种中药提取物、药物及其检测方法
CN114791472B (zh) 祛风活络喷雾剂成分检测方法
CN113484427B (zh) 化湿败毒组合物中苦杏仁苷的测定方法及其应用
CN114689776B (zh) 一种乌药汤高效液相特征图谱的构建方法
CN114720620B (zh) 一种伤风停胶囊的质量检测方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20949984

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20949984

Country of ref document: EP

Kind code of ref document: A1