WO2023024322A1 - 一种中药组合物的指纹图谱的测定方法 - Google Patents

一种中药组合物的指纹图谱的测定方法 Download PDF

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WO2023024322A1
WO2023024322A1 PCT/CN2021/136486 CN2021136486W WO2023024322A1 WO 2023024322 A1 WO2023024322 A1 WO 2023024322A1 CN 2021136486 W CN2021136486 W CN 2021136486W WO 2023024322 A1 WO2023024322 A1 WO 2023024322A1
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emodin
weigh
solution
reference substance
acid
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PCT/CN2021/136486
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French (fr)
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贾振华
蔡艳
王慧娜
杨文�
田恺玥
孙健
高贤
何凤军
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石家庄以岭药业股份有限公司
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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
    • G01N30/50—Conditioning of the sorbent material or stationary liquid
    • G01N30/52—Physical parameters
    • G01N30/54—Temperature
    • 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
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/74—Optical detectors

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  • the invention relates to a method for measuring the fingerprint of a traditional Chinese medicine composition.
  • the invention belongs to the technical field of traditional Chinese medicine detection.
  • Fingerprint of traditional Chinese medicine refers to the chromatogram or spectrum that can mark the chemical characteristics of some traditional Chinese medicinal materials or preparations after proper treatment and certain analysis methods.
  • Traditional Chinese medicine fingerprint is a comprehensive and quantifiable means of identification. It is based on the systematic research on the chemical components of traditional Chinese medicine and is mainly used to evaluate the authenticity, goodness and stability of the quality of Chinese medicinal materials and semi-finished products of traditional Chinese medicine preparations. "Integrity” and “ambiguity” are its salient features.
  • chromatography methods are the mainstream methods, especially HPLC, TLCS and GC have become three recognized routine analysis methods.
  • HPLC has the characteristics of high separation efficiency, high selectivity, high detection sensitivity, fast analysis speed, and wide application range; most of the components of traditional Chinese medicine can be analyzed and detected on high-performance liquid chromatography, and rich application experience has been accumulated. Therefore, high performance liquid chromatography has become the preferred method for fingerprinting of traditional Chinese medicine. With the application of combined techniques such as HPLC-MS and GC-MS, the fingerprint technology of traditional Chinese medicine is becoming more and more perfect.
  • the patent application number is 200810089447.5, and the name of the invention is: a medicine containing ephedra for treating bronchitis and its preparation method, which discloses a Chinese medicine composition containing ephedra, forsythia, bitter almond, pinellia and other ingredients and its preparation method , as the quality control method of the traditional Chinese medicine composition, this application investigates the determination of the fingerprint of the traditional Chinese medicine composition, and the determination method has not been disclosed. The fingerprints are used to control the quality of the listed varieties, effectively ensuring the drug safety of the people.
  • the invention provides a fingerprint determination method of a traditional Chinese medicine composition.
  • the fingerprint assay method is as follows:
  • test solution take 0.5-1.5 g of the traditional Chinese medicine composition, accurately weigh it, put it in a stoppered Erlenmeyer flask, add 40-100% methanol 10-50 mL accurately, weigh it, and ultrasonically treat it for 20-40 minutes , let it cool, weigh again, make up the lost weight with 40-100% methanol, shake well, filter, and take the filtrate to obtain final product;
  • Preparation of reference solution weigh neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isoforsythiaside A, forsythiaside A, hesperidin, baicalin, arctiin, aloe-emodin, licorice Ammonium acid, chrysophanol, emodin, rhein, emodin methyl ether reference substance, add methanol to make a mixed reference substance solution;
  • Preparation of reference substance solution Take an appropriate amount of baicalin reference substance, weigh it accurately, add methanol to make a solution containing 60 ⁇ g per 1 ml, and obtain it;
  • the mobile phase is acetonitrile-0.1% phosphoric acid aqueous solution, gradient elution, and the elution ratio is as follows:
  • the chromatographic column is a C 18 chromatographic column; the detection wavelength is 245-254nm, and the flow rate is 0.1-0.5ml/min;
  • Determination method Precisely draw 1-10 ⁇ L each of the reference substance solution and the test solution, inject it into an ultra-high liquid chromatograph, measure, record the chromatogram, and obtain it.
  • the Chinese medicine composition preparation detected is made of the following raw materials by weight:
  • the Chinese medicine composition preparation detected is preferably made of the following crude drugs in parts by weight:
  • the detected Chinese medicine composition preparation can also preferably be made from the following crude drugs in parts by weight:
  • Ephedra 69 Gypsum 259; Forsythia 259; Scutellaria baicalensis 104; Morus alba 259; Licorice 52.
  • the Chinese medicine composition preparation that detects also can preferably be made of the crude drug of following weight part:
  • the preparation method of the detected Chinese medicine composition active ingredient is:
  • the detected Chinese medicine composition can be made into capsules, tablets, granules or oral liquid, wherein the preparation method of the tablet is:
  • step D spray-dry the combined clear paste obtained in step C, and collect the spray powder for subsequent use;
  • step E The spray powder obtained in step D and the fine powder obtained in step A, use ethanol as a binder to make a soft material, sieve, granulate and dry, then granulate, add sodium carboxymethyl starch, microcrystalline cellulose, magnesium stearate, and mix well , ready to be pressed into tablets.
  • Fingerprint spectrum determination method of the present invention preferably comprises the following steps:
  • test solution take 1-1.2 g of the traditional Chinese medicine composition, accurately weigh it, place it in a stoppered Erlenmeyer flask, add 50 mL of methanol precisely, weigh it, ultrasonically treat it for 30 minutes, let it cool, and weigh it again , use methanol to make up for the lost weight, shake well, filter, and take the filtrate to obtain the final product;
  • Preparation of reference solution weigh neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isoforsythiaside A, forsythiaside A, hesperidin, baicalin, arctiin, aloe-emodin, licorice Ammonium acid, chrysophanol, emodin, rhein, and emodin methyl ether reference substances were added to methanol to prepare 0.07-0.09mg/ml neochlorogenic acid, 0.07-0.09mg/ml chlorogenic acid, and 0.07-0.09mg /ml cryptochlorogenic acid, 0.07-0.09mg/ml isoforsythiaside A, 0.1-0.3mg/ml forsythiaside A, 0.1-0.3mg/ml hesperidin, 0.1-0.3mg/ml baicalin , 0.1-0.3mg/ml arctiin, 0.04-0.06mg/m
  • Preparation of reference substance solution Take an appropriate amount of baicalin reference substance, weigh it accurately, add methanol to make a solution containing 60 ⁇ g per 1 ml, and obtain it;
  • the mobile phase is acetonitrile-0.1% phosphoric acid aqueous solution, gradient elution, and the elution ratio is as follows:
  • the chromatographic column is a C 18 chromatographic column; the detection wavelength is 254nm, and the flow rate is 0.3ml/min;
  • Determination method Accurately draw 1 ⁇ L each of the reference substance solution and the test solution, inject it into an ultra-high liquid chromatograph, measure, record the chromatogram, and obtain it.
  • the assay method of fingerprint spectrum of the present invention more preferred steps are:
  • test solution take 1 g of the traditional Chinese medicine composition, accurately weigh it, place it in a stoppered Erlenmeyer flask, add 50 mL of methanol precisely, weigh it, ultrasonically treat it for 30 minutes, let it cool, weigh it again, and use methanol Make up for the lost weight, shake well, filter, and take the filtrate to get it;
  • Preparation of reference solution weigh neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isoforsythiaside A, forsythiaside A, hesperidin, baicalin, arctiin, aloe-emodin, licorice Ammonium acid, chrysophanol, emodin, rhein, and emodin methyl ether reference substances were added to methanol to prepare 0.07-0.09mg/ml neochlorogenic acid, 0.07-0.09mg/ml chlorogenic acid, and 0.07-0.09mg /ml cryptochlorogenic acid, 0.07-0.09mg/ml isoforsythiaside A, 0.1-0.3mg/ml forsythiaside A, 0.1-0.3mg/ml hesperidin, 0.1-0.3mg/ml baicalin , 0.1-0.3mg/ml arctiin, 0.04-0.05mg/ml
  • Preparation of reference substance solution Take an appropriate amount of baicalin reference substance, weigh it accurately, add methanol to make a solution containing 60 ⁇ g per 1 ml, and obtain it;
  • the mobile phase is acetonitrile-0.1% phosphoric acid aqueous solution, gradient elution, and the elution ratio is as follows:
  • the chromatographic column is a C 18 chromatographic column; the column length is 100mm, the inner diameter is 2.1mm, and the particle size is 1.8 ⁇ m; the detection wavelength is 254nm, and the flow rate is 0.3ml/min; column temperature: 30°C; injection volume: 1 ⁇ L;
  • Determination method Accurately draw 1 ⁇ L each of the reference substance solution and the test solution, inject it into an ultra-high liquid chromatograph, measure, record the chromatogram, and obtain it.
  • evaluation method is as follows:
  • the gradient elution table is shown in Table 2. Determine the chromatographic column ACQUITY UPLC HSS T3C 18 1.8 ⁇ m, 2.1*100mm; set the following parameters as the initial conditions, wavelength 254nm; column temperature: 30°C; injection volume: 1 ⁇ L.
  • the chromatographic column was inspected at different column temperatures.
  • the chromatographic peak tailing at the column temperature of 25°C was excellent, and the chromatographic peak was excellent at 30°C.
  • the symmetry of the chromatographic peak with a larger peak area was poor at the column temperature of 35°C.
  • Comprehensive comparison Select the column temperature as 30°C.
  • Neochlorogenic Acid Reference Substance Solution Accurately weigh 21.95mg of Neochlorogenic Acid Reference Substance, put it in a 10mL volumetric flask, add methanol to dissolve, dilute to the mark, shake well, and set aside.
  • Preparation of cryptochlorogenic acid reference substance solution Accurately weigh 19.07mg of cryptochlorogenic acid reference substance, put it in a 10mL volumetric flask, add methanol to dissolve, dilute to the mark, shake well, and set aside.
  • isoforsythiaside A reference substance solution Accurately weigh 10.19 mg of isoforsythiaside A reference substance, put it in a 5mL volumetric flask, add methanol to dissolve, set the volume to the mark, shake well, and set aside.
  • Preparation of forsythiaside A reference substance solution Accurately weigh 11.89 mg of forsythiaside A reference substance, put it in a 5mL volumetric flask, add methanol to dissolve, dilute to the mark, shake well, and set aside.
  • baicalin reference substance solution Accurately weigh 15.42mg of baicalin reference substance, put it in a 10mL volumetric flask, add methanol to dissolve, dilute to the mark, shake well, and set aside.
  • hesperidin standard solution Accurately weigh 13.63mg of hesperidin reference substance, put it in a 10mL volumetric flask, add methanol to dissolve, dilute to the mark, shake well, and set aside.
  • arctiin reference substance solution Accurately weigh 12.80 mg of arctiin reference substance, put it in a 5mL volumetric flask, add methanol to dissolve, dilute to the mark, shake well, and set aside.
  • aloe-emodin reference substance solution Accurately weigh 8.29mg of aloe-emodin reference substance, put it in a 10mL volumetric flask, add methanol to dissolve, dilute to the mark, shake well, and set aside.
  • rhein reference substance solution Accurately weigh 10.58mg of rhein reference substance, put it in a 10mL volumetric flask, add methanol to dissolve, dilute to the mark, shake well, and set aside.
  • Chrysophanol Reference Substance Solution Accurately weigh 10.56mg of Chrysophanol Reference Substance, put in a 10mL volumetric flask, add methanol to dissolve, dilute to the mark, shake well, and set aside.
  • emodin methyl ether reference substance solution Preparation of emodin methyl ether reference substance solution: Accurately weigh about 9.58mg of emodin methyl ether reference substance, put it in a 25mL volumetric flask, add methanol to dissolve, dilute to the mark, shake well, and set aside.
  • the traditional Chinese medicine composition described in the present invention is a listed Chinese patent medicine.
  • the quality control of this variety adopts the separate identification of some medicinal materials.
  • This variety is a large compound Chinese medicine product, and a small amount of identification is not enough to reflect the quality of the product.
  • To ensure the quality of the product and ensure the safety of medication for the people we studied the fingerprint of the Chinese medicine composition, and conducted fingerprint research on 7 batches of medicines, and screened out 22 representative common peaks in the fingerprint analysis. , and identified 14 chromatographic peaks, and the resolution of each chromatographic peak is good.
  • the use of fingerprints for quality control can fully reflect the overall quality of Chinese patent medicines, effectively avoiding the disadvantages of traditional Chinese medicines that only add individual ingredients that need to be tested in the process of listing inspections and can pass the legal procedures.
  • the detection method of fingerprint chromatogram described in the present invention is a complete technical scheme, wherein has included several technical characteristics, for any one technical characteristic wherein, the contriver has all carried out the test method investigation for many times, although each technical characteristic sees It seems simple, but the overall technical solution composed of numerous technical features is the most preferred technical solution for the quality control of the preparation.
  • the similarity of fingerprints between 7 batches carried out by this method is all greater than 95%, indicating that this product is stable and controllable.
  • the method is simple in operation, strong in implementability and short in time, and solves the difficult problem of quality control of large compound preparations of traditional Chinese medicines. From the above experimental results, it can be seen that the method has good precision, stability, and repeatability, and provides a new method for improving the quality control of the traditional Chinese medicine composition.
  • Fig. 1 reference substance collection of graphs
  • Fig. 2 fingerprint spectrum of test product
  • Peak 1 Neochlorogenic acid Peak 2: Chlorogenic acid Peak 3: Cryptochlorogenic acid Peak 5: Isorsythiaside A Peak 7: Forsythiaside A Peak 10: Hesperidin Peak 12: Baicalin Peak 13: Arctiin Peak 17: Aloe-Emodin Peak 18: Ammonium Glycyrrhizinate Peak 19: Rhein Peak 20: Emodin Peak 21: Chrysophanol Peak 22: Emodin Methyl Ether
  • Fig. 7 reflux extracts fingerprint spectrum
  • Fig. 12 need testing solution chromatogram
  • step D spray-dry the combined clear paste obtained in step C, and collect the spray powder for subsequent use;
  • test solution take 1 g of the traditional Chinese medicine composition, accurately weigh it, place it in a stoppered Erlenmeyer flask, add 50 mL of methanol precisely, weigh it, ultrasonically treat it for 30 minutes, let it cool, weigh it again, and use methanol Make up for the lost weight, shake well, filter, and take the filtrate to get it;
  • Preparation of reference solution weigh neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isoforsythiaside A, forsythiaside A, hesperidin, baicalin, arctiin, aloe-emodin, licorice Ammonium acid, chrysophanol, emodin, rhein, and emodin methyl ether reference substances were added to methanol to prepare 0.088mg/ml neochlorogenic acid, 0.078mg/ml chlorogenic acid, and 0.076mg/ml cryptochlorogenic acid.
  • Preparation of reference substance solution Take an appropriate amount of baicalin reference substance, weigh it accurately, add methanol to make a solution containing 60 ⁇ g per 1 ml, and obtain it;
  • the mobile phase is acetonitrile-0.1% phosphoric acid aqueous solution, gradient elution, and the elution ratio is as follows:
  • Determination method Accurately draw 1 ⁇ L each of the reference substance solution and the test solution, inject it into an ultra-high liquid chromatograph, measure, record the chromatogram, and obtain it.
  • step D Spray-dry the combined clear paste obtained in step C, collect the spray powder, and make tablets according to conventional methods.
  • test solution take 1 g of the traditional Chinese medicine composition, accurately weigh it, place it in a stoppered Erlenmeyer flask, add 50 mL of methanol precisely, weigh it, ultrasonically treat it for 30 minutes, let it cool, weigh it again, and use methanol Make up for the lost weight, shake well, filter, and take the filtrate to get it;
  • Preparation of reference solution weigh neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isoforsythiaside A, forsythiaside A, hesperidin, baicalin, arctiin, aloe-emodin, licorice Ammonium acid, chrysophanol, emodin, rhein, and emodin methyl ether reference substances were added to methanol to prepare 0.079mg/ml neochlorogenic acid, 0.088mg/ml chlorogenic acid, and 0.085mg/ml cryptochlorogenic acid.
  • Preparation of reference substance solution Take an appropriate amount of baicalin reference substance, weigh it accurately, add methanol to make a solution containing 60 ⁇ g per 1 ml, and obtain it;
  • the mobile phase is acetonitrile-0.1% phosphoric acid aqueous solution, gradient elution, and the elution ratio is as follows:
  • Determination method Accurately draw 1 ⁇ L each of the reference substance solution and the test solution, inject it into an ultra-high liquid chromatograph, measure, record the chromatogram, and obtain it.
  • the formula of raw materials is: Ephedra 69g; Gypsum 259g; Forsythia 259g; Scutellaria baicalensis 104g; Morus alba 259g; 52g; bellflower 61g; licorice 52g.
  • step D Spray-dry the combined clear paste obtained in step C, collect the spray powder, and make granules according to conventional methods.
  • test solution take 0.5 g of the traditional Chinese medicine composition, accurately weigh it, place it in a stoppered Erlenmeyer flask, add 20 mL of 50% methanol accurately, weigh it, ultrasonically treat it for 20 minutes, let it cool, and weigh it again , make up the lost weight with 50% methanol, shake well, filter, and take the filtrate to get final product;
  • Preparation of reference solution weigh neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isoforsythiaside A, forsythiaside A, hesperidin, baicalin, arctiin, aloe-emodin, licorice Ammonium acid, chrysophanol, emodin, rhein, and emodin methyl ether reference substances were added to methanol to prepare 0.080mg/ml neochlorogenic acid, 0.082mg/ml chlorogenic acid, and 0.085mg/ml cryptochlorogenic acid.
  • Preparation of reference substance solution Take an appropriate amount of baicalin reference substance, weigh it accurately, add methanol to make a solution containing 60 ⁇ g per 1 ml, and obtain it;
  • the mobile phase is acetonitrile-0.1% phosphoric acid aqueous solution, gradient elution, and the elution ratio is as follows:
  • the chromatographic column is ACQUITY UPLC BEH C 18 1.7 ⁇ m, 2.1*100mm chromatographic column; the detection wavelength is 245nm, and the flow rate is 0.1ml/min;
  • Determination method Accurately draw 1 ⁇ L each of the reference substance solution and the test solution, inject it into an ultra-high liquid chromatograph, measure, record the chromatogram, and obtain it.
  • the raw material drug formula is: take by weighing:
  • step D Spray-dry the combined clear paste obtained in step C, collect the spray powder, and make pills according to conventional methods.
  • test solution take 1.5 g of the traditional Chinese medicine composition, accurately weigh it, place it in a stoppered conical flask, accurately add 50 mL of 80% methanol, weigh it, ultrasonically treat it for 40 minutes, let it cool, and weigh it again , make up the lost weight with 80% methanol, shake well, filter, and take the filtrate to get final product;
  • Preparation of reference solution weigh neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isoforsythiaside A, forsythiaside A, hesperidin, baicalin, arctiin, aloe-emodin, licorice Ammonium acid, chrysophanol, emodin, rhein, and emodin methyl ether reference substances were added to methanol to prepare 0.080mg/ml neochlorogenic acid, 0.082mg/ml chlorogenic acid, and 0.085mg/ml cryptochlorogenic acid.
  • Preparation of reference substance solution Take an appropriate amount of baicalin reference substance, weigh it accurately, add methanol to make a solution containing 60 ⁇ g per 1 ml, and obtain it;
  • the mobile phase is acetonitrile-0.1% phosphoric acid aqueous solution, gradient elution, and the elution ratio is as follows:
  • the chromatographic column is CORTECS UPLC C 18 1.6 ⁇ m, 2.1*100mm chromatographic column; the detection wavelength is 245nm, and the flow rate is 0.1ml/min;
  • Determination method Accurately draw 1 ⁇ L each of the reference substance solution and the test solution, inject it into an ultra-high liquid chromatograph, measure, record the chromatogram, and obtain it.

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Abstract

一种中药组合物的指纹图谱测定方法，该中药组合物由以下原料药组成：麻黄、石膏、连翘、黄芩、桑白皮、苦杏仁、前胡、半夏、陈皮、贝母、牛蒡子、金银花、大黄、桔梗、甘草。指纹图谱测定方法，标定了22个主要色谱峰，指认了其中14个，分别为新绿原酸、绿原酸、隐绿原酸、异连翘酯苷A、连翘酯苷A、橙皮苷、黄芩苷、牛蒡苷、芦荟大黄素、甘草酸铵、大黄酚、大黄素、大黄酸、大黄素甲醚。

Description

一种中药组合物的指纹图谱的测定方法 技术领域
本发明涉及到一种中药组合物的指纹图谱的测定方法。属于中药检测技术领域。
背景技术
中药指纹图谱是指某些中药材或中药制剂经适当处理后，采用一定的分析手段，得到的能够标示其化学特征的色谱图或光谱图。中药指纹图谱是一种综合的，可量化的鉴定手段，它是建立在中药化学成分系统研究的基础上，主要用于评价中药材以及中药制剂半成品质量的真实性、优良性和稳定性。“整体性”和“模糊性”为其显著特点。
中药及其制剂均为多组分复杂体系，因此评价其质量应采用与之相适应的，能提供丰富鉴别信息的检测方法，但现行的显微鉴别、理化鉴别和含量测定等方法都不足以解决这一问题，建立中药指纹图谱将能较为全面地反映中药及其制剂中所含化学成分的种类与数量，进而对药品质量进行整体描述和评价。这也正好符合中医药整体学说。在此基础上，如果进一步开展谱效学研究，可使中药质量与其药效真正结合起来，有助于阐明中药作用机理。总之，中药指纹图谱的研究和建立，对于提高中药质量，促进中药现代化具有重要意义。
目前，中药指纹图谱技术已涉及众多方法，包括薄层扫描(TLCS)、高效液相色谱法(HPLC)、气相色谱法(GC)和高效毛细管电泳法(HPCE)等色谱法以及紫外光谱法(UV)、红外光谱法(IR)、质谱法(MS)、核磁共振法(NMR)和X-射线衍射法等光谱法。其中色谱方法为主流方法，尤其是HPLC、TLCS和GC已成为公认的三种常规分析手段。由于HPLC具有分离效能高、选择性高、检测灵敏度高、分析速度快、应用范围广等特点；中药成分绝大多数可在高效液相色谱仪上进行分析检测，且积累较丰富的应用经验。因此高效液相色谱法已成为中药指纹图谱技术的首选方法。随着HPLC-MS和GC-MS等联用技术的应用，中药指纹图谱技术更趋完善。
专利申请号为200810089447.5，发明名称为：一种含有麻黄的治疗支气管炎的药物及其制备方法，公开了一种含有麻黄、连翘、苦杏仁、半夏等成分的中药组合物及其制备方法，本申请作为该中药组合物的质控方法，考察了该中药组合物的指纹图谱测定，该测定方法并未被公开过。采用该指纹图谱控制该上市品种的质量，有力的保证人民群众的用药安全。
发明内容
本发明提供一种中药组合物的指纹图谱测定方法。
所述指纹图谱测定方法如下：
供试品溶液制备：取所述中药组合物0.5-1.5g，精密称定，置具塞锥形瓶中，精密加入40-100％甲醇10-50mL，称定重量，超声处理20-40分钟，放冷，再称定重量，用40-100％的甲醇补足减失的重量，摇匀，滤过，取续滤液，即得；
对照品溶液的制备：分别称取新绿原酸、绿原酸、隐绿原酸、异连翘酯苷A、连翘酯苷A、橙皮苷、黄芩苷、牛蒡苷、芦荟大黄素、甘草酸铵、大黄酚、大黄素、大黄酸、大黄素甲醚对照品，加入甲醇，制成混合对照品溶液即得；
参照物溶液的制备：取黄芩苷对照品适量，精密称定，加甲醇制成每1ml含60μg的溶液，即得；
流动相为乙腈-0.1％磷酸水溶液，梯度洗脱，洗脱比例如下表：
Figure PCTCN2021136486-appb-000001
色谱柱为C 18色谱柱；检测波长245-254nm，流速0.1-0.5ml/min；
测定法：分别精密吸取对照品溶液与供试品溶液各1-10μL，注入超高液相色谱仪，测定，记录色谱图，即得。
本发明所述的指纹图谱测定方法，检测的中药组合物制剂是由如下重量份的原料药制成：
麻黄52；石膏324；连翘194；黄芩78；桑白皮194；苦杏仁130；前胡78；半夏130；陈皮78；贝母78；牛蒡子130；金银花130；大黄39；桔梗76；甘草65。
本发明所述的指纹图谱测定方法，检测的中药组合物制剂优选由下列重量份的原料药制成：
麻黄86；石膏194；连翘324；黄芩130；桑白皮324；苦杏仁78；前胡130；半夏78；陈皮130；贝母130；牛蒡子78；金银花78；大黄65；桔梗46；甘草39。
本发明所述的指纹图谱测定方法，检测的中药组合物制剂还可优选由下列重量份的原料药制成：
麻黄69；石膏259；连翘259；黄芩104；桑白皮259；苦杏仁104；前胡104；半夏104；陈皮104；贝母104；牛蒡子104；金银花104；大黄52；桔梗61；甘草52。
本发明所述的指纹图谱测定方法，检测的中药组合物制剂也可优选由下列重量份的原料药制成：
麻黄55；石膏254；连翘318；黄芩107；桑白皮203；苦杏仁107；前胡82；半夏105；陈皮84；贝母125；牛蒡子122；金银花113；大黄42；桔梗60；甘草50。
本发明所述的指纹图谱测定方法，所检测的中药组合物活性成分的制备方法为：
A、按组方比例称取贝母，粉碎成细粉，备用；
B、按组方比例称取麻黄、连翘、苦杏仁、半夏、牛蒡子、大黄加40-70％乙醇回流提取2次，每次1-4小时，第一次加8-10倍量，第二次加6-9倍量，提取液合并，滤过，滤液减压回收乙醇，浓缩至60℃热测相对密度为1.04-1.16的清膏，备用；
C、按组方比例称取石膏、桑白皮、前胡、陈皮、金银花、桔梗、甘草，加水煎煮两次，每次1-4小时，第一次加9-11倍量，第二次加7-9倍量，煎液合并，滤过，浓缩至60℃热测相对密度为1.04-1.16的清膏，与步骤B所得的清膏合并，备用；
步骤A所得细粉和步骤C所得合并后的清膏共同构成该药物组合物的活性成分。
本发明所述的指纹图谱测定方法，检测的中药组合物可制成胶囊剂、片剂、颗粒剂或口服液，其中片剂的制备方法是：
A、按组方比例称取贝母，粉碎成细粉，备用；
B、按组方比例称取麻黄、连翘、苦杏仁、半夏、牛蒡子、大黄加50％乙醇回流提取2次，每次3小时，第一次加10倍量，第二次加6倍量，提取液合并，滤过，滤液减压回收乙醇，浓缩至60℃热测相对密度为1.15的清膏，备用；
C、按组方比例称取石膏、桑白皮、前胡、陈皮、金银花、桔梗、甘草，加水煎煮两次，每次2小时，第一次加10倍量，第二次加7倍量，煎液合并，滤过，浓缩至60℃热测相对密度为1.15的清膏，与步骤B所得的清膏合并，备用；
D、将步骤C所得合并的清膏，喷雾干燥，收集喷雾粉备用；
E、步骤D所得喷雾粉与步骤A所得细粉，以乙醇为黏合剂制软材，过筛制粒干燥后整粒，加入羧甲淀粉钠、微晶纤维素、硬脂酸镁，混匀，压片即得。
本发明所述的指纹图谱测定方法，优选的包括以下步骤：
供试品溶液制备：取所述中药组合物1-1.2g，精密称定，置具塞锥形瓶中，精密加入甲醇50mL，称定重量，超声处理30分钟，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；
对照品溶液的制备：分别称取新绿原酸、绿原酸、隐绿原酸、异连翘酯苷A、连翘酯 苷A、橙皮苷、黄芩苷、牛蒡苷、芦荟大黄素、甘草酸铵、大黄酚、大黄素、大黄酸、大黄素甲醚对照品，加入甲醇，制成分别含0.07-0.09mg/ml新绿原酸、0.07-0.09mg/ml绿原酸、0.07-0.09mg/ml隐绿原酸、0.07-0.09mg/ml异连翘酯苷A、0.1-0.3mg/ml连翘酯苷A、0.1-0.3mg/ml橙皮苷、0.1-0.3mg/ml黄芩苷、0.1-0.3mg/ml牛蒡苷、0.04-0.06mg/ml甘草酸铵、0.01-0.02mg/ml芦荟大黄素、0.01-0.03mg/ml大黄酸、0.01-0.03mg/ml大黄素、0.01-0.03mg/ml大黄酚、0.005-0.01mg/ml大黄素甲醚混合对照品溶液即得；
参照物溶液的制备：取黄芩苷对照品适量，精密称定，加甲醇制成每1ml含60μg的溶液，即得；
流动相为乙腈-0.1％磷酸水溶液，梯度洗脱，洗脱比例如下表：
Figure PCTCN2021136486-appb-000002
色谱柱为C 18色谱柱；检测波长254nm，流速0.3ml/min；
测定法：分别精密吸取对照品溶液与供试品溶液各1μL，注入超高液相色谱仪，测定，记录色谱图，即得。
本发明指纹图谱的测定方法，更优选的步骤为：
供试品溶液制备：取所述中药组合物1g，精密称定，置具塞锥形瓶中，精密加入甲醇50mL，称定重量，超声处理30分钟，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；
对照品溶液的制备：分别称取新绿原酸、绿原酸、隐绿原酸、异连翘酯苷A、连翘酯苷A、橙皮苷、黄芩苷、牛蒡苷、芦荟大黄素、甘草酸铵、大黄酚、大黄素、大黄酸、大黄素甲醚对照品，加入甲醇，制成分别含0.07-0.09mg/ml新绿原酸、0.07-0.09mg/ml绿原酸、0.07-0.09mg/ml隐绿原酸、0.07-0.09mg/ml异连翘酯苷A、0.1-0.3mg/ml连翘酯苷A、0.1-0.3mg/ml橙皮苷、0.1-0.3mg/ml黄芩苷、0.1-0.3mg/ml牛蒡苷、0.04-0.05mg/ml甘草酸铵、0.01-0.02mg/ml芦荟大黄素、0.01-0.03mg/ml大黄酸、0.01-0.02mg/ml大黄素、0.01-0.02mg/ml大黄酚、0.005-0.009mg/ml大黄素甲醚混合对照品溶液即得；
参照物溶液的制备：取黄芩苷对照品适量，精密称定，加甲醇制成每1ml含60μg的溶液，即得；
流动相为乙腈-0.1％磷酸水溶液，梯度洗脱，洗脱比例如下表：
Figure PCTCN2021136486-appb-000003
色谱柱为C 18色谱柱；柱长为100mm，内径为2.1mm，粒径为1.8μm；检测波长254nm，流速0.3ml/min；柱温：30℃；进样体积：1μL；
测定法：分别精密吸取对照品溶液与供试品溶液各1μL，注入超高液相色谱仪，测定，记录色谱图，即得。
用实施例1制备的样品，对本发明中药组合物的指纹图谱测定方法，从多个方面评价其可行性，评价方法如下：
1.仪器与材料
1.1仪器
Waters ACQUITY UPLC H-CLASS超高效液相色谱(PDA检测器，Empower 3.0色谱工作站)，循环式多用真空泵(郑州长城科工贸有限公司)，KQ250DB型超声波清洗器(昆山市超声仪器有限公司)，瑞士METTLER TOLEDOAL型电子分析天平。
1.2材料
1.2.1对照品信息
表1 对照品来源信息表
Figure PCTCN2021136486-appb-000004
Figure PCTCN2021136486-appb-000005
1.2.2样品信息
本发明中药组合物(石家庄以岭药业股份有限公司)
1.2.3试剂
乙腈(色谱纯，Merck，批号JB091230)，甲醇(色谱纯，Merck，批号l1061607948)。磷酸(分析纯，天津市科密欧化学试剂有限公司，批号20190518)，
2.色谱条件选择
2.1检测波长的选择
对所述中药组合物样品进行全波长测定，对样品进行全波长测定，在不同波长(220nm、254nm、280nm、300nm、330nm)下分别提取色谱图，经过仔细比较，在254nm波长处，提取的色谱峰最多，能较全面反应样品的图谱信息，故选择254nm作为检测波长。
2.2流动相系统的选择
本研究考察了甲醇-水、甲醇-0.1％甲酸水溶液、甲醇-0.1％磷酸水溶液、乙腈-水、乙腈-0.1％甲酸水溶液、结果各色谱峰的峰形不对称，有的色谱峰不能达到基线分离，考察了乙腈-0.1％磷酸水溶液，在此条件下各色谱峰峰形较好，且能达到基线分离。结果显示乙腈-0.1％磷酸系统色谱峰的分离度最好，因此流动相系统为流动相：乙腈—酸水(0.1％磷酸水溶液)，流速：0.3mL/min。梯度洗脱表见表2。确定色谱柱ACQUITY UPLC HSS T3C 18 1.8μm，2.1*100mm；设置如下参数为初始条件，波长254nm；柱温：30℃；进样体积：1μL。
表2 流动相梯度洗脱表
Figure PCTCN2021136486-appb-000006
2.3流速的选择
考察了不同流速0.2ml/min、0.3ml/min、0.4ml/min对色谱图的影响，流速为0.2ml/min的色谱图，出峰时间较长、峰面积大的化合物色谱峰对称性不好；流速为0.4ml/min的色谱图，多数色谱峰没有达到基线分离，综合考虑，流速为0.3ml/min的色谱图峰形较好且大多数色谱峰基本达到基线分离，所以流速选择0.3ml/min。
2.4柱温选择
在不同柱温下对色谱柱进行了考察，柱温为25℃色谱峰峰型拖尾，30℃时色谱峰优秀，柱温为35℃峰面积较大的色谱峰对称性较差，综合比较选择柱温为30℃。
2.5色谱柱的选择
考察了ACQUITY UPLC HSS T3 C 18 1.8μm，2.1*100mm；CORTECS UPLC C 18 1.6μm，2.1*100mm；ACQUITY UPLC BEH C 18 1.7μm，2.1*100mm不同色谱柱，从色谱峰的数量及峰形综合比较，选择ACQUITY UPLC HSS T3 C 18 1.8μm，2.1*100mm。
2.6对照品、供试品指纹图谱
在已确定的色谱条件下，分别对对照品溶液和供试品溶液进样，供试品溶液的色谱图中确定14个主要色谱峰。根据色谱峰的保留时间和光谱特征，确定指纹图谱中的色谱峰峰1：新绿原酸，峰2：绿原酸，峰3：隐绿原酸，峰5：异连翘酯苷A，峰7：连翘酯苷A，峰10：橙皮苷，峰12：黄芩苷，峰13：牛蒡苷，峰17：芦荟大黄素，峰18：甘草酸铵，峰19：大黄酸，峰20：大黄素，峰21：大黄酚，峰22：大黄素甲醚。以上对照品与供试品色谱图及光谱图见附图1-2。
3.对照品及样品的配制
3.1对照品溶液的配制
新绿原酸对照品溶液的配制：精密称取新绿原酸对照品21.95mg，置10mL容量瓶，加甲醇溶解，稀释至刻度，摇匀，备用。
绿原酸对照品溶液的配制：精密称取绿原酸对照品20.23mg，置5mL容量瓶，加甲醇溶解，稀释至刻度，摇匀，备用。
隐绿原酸对照品溶液的配制：精密称取隐绿原酸对照品19.07mg，置10mL容量瓶，加甲醇溶解，稀释至刻度，摇匀，备用。
异连翘酯苷A对照品溶液的配制：精密称取异连翘酯苷A对照品10.19mg，置5mL容量瓶，加甲醇溶解，定容至刻度，摇匀，备用。
连翘酯苷A对照品溶液的配制：精密称取连翘酯苷A对照品11.89mg，置5mL容量瓶，加甲醇溶解，稀释至刻度，摇匀，备用。
黄芩苷对照品溶液的配制：精密称取黄芩苷对照品15.42mg，置10mL容量瓶，加甲醇溶解，稀释至刻度，摇匀，备用。
橙皮苷照品溶液的配制：精密称取橙皮苷对照品13.63mg，置10mL容量瓶，加甲醇溶解，定容至刻度，摇匀，备用。
牛蒡苷对照品溶液的配制：精密称取牛蒡苷对照品12.80mg，置5mL容量瓶，加甲醇溶解，稀释至刻度，摇匀，备用。
甘草酸铵对照品溶液的配制：精密称取甘草酸铵对照品11.36mg，置10mL容量瓶，加 甲醇溶解，稀释至刻度，摇匀，备用。
芦荟大黄素、大黄酸、大黄素、大黄酚、大黄素甲醚对照品母液的配制：
芦荟大黄素对照品溶液的配制：精密称取芦荟大黄素对照品8.29mg，置10mL容量瓶，加甲醇溶解，稀释至刻度，摇匀，备用。
大黄酸对照品溶液的配制：精密称取大黄酸对照品10.58mg，置10mL容量瓶，加甲醇溶解，稀释至刻度，摇匀，备用。
大黄素对照品溶液的配制：精密称取对照品12.84mg，置25mL容量瓶，加甲醇溶解，稀释至刻度，摇匀，备用。
大黄酚对照品溶液的配制：精密称取大黄酚对照品10.56mg，置10mL容量瓶，加甲醇溶解，稀释至刻度，摇匀，备用。
大黄素甲醚对照品溶液的配制：精密称取大黄素甲醚对照品约9.58mg，置25mL容量瓶，加甲醇溶解，稀释至刻度，摇匀，备用。
芦荟大黄素、大黄酸、大黄素、大黄酚、大黄素甲醚混合对照品溶液的配制：
精密量取1mL芦荟大黄素对照品溶液，1mL大黄酸对照品溶液，1mL大黄素对照品溶液，1mL大黄酚对照品溶液，1mL大黄素甲醚对照品溶液，至50ml容量瓶中，用甲醇定容至刻度，配成芦荟大黄素、大黄酸、大黄素、大黄酚、大黄素甲醚混合对照品溶液。
精密量取1mL新绿原酸对照品溶液，0.5mL绿原酸对照品溶液，1mL隐绿原酸对照品溶液，1mL异连翘酯苷A对照品溶液，2mL连翘酯苷A对照品溶液，2mL黄芩苷对照品溶液，3mL橙皮苷对照品溶液，2mL牛蒡苷对照品溶液，1mL甘草酸铵对照品溶液，芦荟大黄素、大黄酸、大黄素、大黄酚、大黄素甲醚混合对照品溶液10ml,至25mL容量瓶中,用甲醇定容至刻度，配成混合对照品溶液，备用。
3.2供试品溶液的配制
取所述中药组合物适量，研细，取1g，精密称定，置具塞锥形瓶中，精密加入甲醇50mL，称定重量，超声处理(功率500W，频率40kHz)30分钟，放冷，再称定重量，用甲醇补足减失的重量，摇匀，离心(1400rpm离心10min)，取上清液，0.22μm滤过，取续滤液，即得。
4供试品溶液和参照物溶液的制备
4.1提取溶剂的考察
分别比较了50％甲醇，80％甲醇，100％甲醇的提取效果，见附图3、附图4、附图5，结果表明100％甲醇提取所得指纹图谱中色谱峰最多，因此选择甲醇作为提取溶剂。
4.2提取方法的考察
分别比较了超声和回流两种提取方法见附图6、附图7，超声和回流提取两种方法所得 的指纹图谱色谱峰的数目和强度没有明显区别，由于超声提取方法简便，因此选择超声提取。
4.3超声时间的考察
分别比较了超声20分钟、30分钟、40分钟，，结果表明超声30分钟与40分钟所得指纹图谱中色谱峰信息量大，无明显区别，因此最终确定超声时间为30分钟，具体信息见附图8、附图9、附图10。
4.4参照物溶液的制备
取黄芩苷对照品适量，精密称定，加甲醇制成每1ml含60μg的溶液，即得，参照物的色谱信息见附图11。
4.5测定法：精密吸取供试品溶液1μL，注入超高液相色谱仪，测定，记录色谱图，即得。
供试品的色谱信息见附图12。
5.方法学考察
5.1精密度试验：取所述中药组合物样品，按3.2的“供试品溶液制备方法”制备，按照色谱条件连续进样6次，各主要色谱峰的相对保留时间和相对峰面积的RSD均小于2％，表明进样精密度良好，结果见表3和表4。
表3 共有峰相对保留时间考察结果
Figure PCTCN2021136486-appb-000007
Figure PCTCN2021136486-appb-000008
表4 共有峰相对峰面积考察结果
Figure PCTCN2021136486-appb-000009
5.2重复性试验：取所述中药组合物内容物6份，按3.2的“供试品溶液制备方法”制备，注入液相色谱仪检测，测定各主要色谱峰保留时间的RSD均小于2％，峰面积的RSD均小于3％，表明方法重复性较好，结果见表5和表6。
表5 相对保留时间试验结果
Figure PCTCN2021136486-appb-000010
Figure PCTCN2021136486-appb-000011
表6 相对峰面积试验结果
Figure PCTCN2021136486-appb-000012
5.3稳定性试验：取供试品溶液，分别在0h，2h，4h，8h，12h，24h进样，测定各主要色谱峰保留时间的RSD均小于2％，峰面积的RSD均小于3％，表明供试品溶液在24h内稳定，结果见表7和表8。
表7 共有峰相对保留时间试验结果
Figure PCTCN2021136486-appb-000013
Figure PCTCN2021136486-appb-000014
表8 共有峰相对峰面积试验结果
Figure PCTCN2021136486-appb-000015
5.4指纹图谱的建立及相似度分析
称取7批所述中药组合物样品，按3.2项下要求制备供试品溶液，在上述色谱条件下进样分析，得到7批所述中药组合物样品的指纹图谱，具体色谱信息见附图13，并采用国家 药典委员会开发的中药色谱指纹图谱相似度评价系统2012.0版软件对7批样品的指纹图谱进行分析，采用中位数法对各指纹图谱色谱峰进行了多点校正和自动匹配，计算相似度并生成对照指纹图谱，相似度结果见表9，对照指纹图谱见附图14，其中共有色谱峰22个，其中以出峰稳定，峰面积较大的12号色谱峰黄芩苷为参照峰。
表9 7批所述中药组合物指纹图谱相似度
Figure PCTCN2021136486-appb-000016
5.5 7批所述中药组合物样品含量测定，将5.4的供试品溶液和3.1的对照品混标溶液注入液相色谱仪检测，进行含量测定，结果如下见表10。
表10 7批所述中药组合物样品含量测定结果
Figure PCTCN2021136486-appb-000017
本发明所述中药组合物为上市品种中成药，目前该品种的质控采用了对部分药材进行单独鉴别，该品种为大复方中药产品，少量的鉴别不足以反映该产品的质量，为了更好保证产品的质量，保障人民群众的用药安全，我们研究了该中药组合物的指纹图谱，对不同7批次的药品进行了指纹图谱研究，指纹图谱分析中筛选出有代表性的22个共有峰，并指认了其中的14个色谱峰，各色谱峰分离度较好。采用指纹图谱进行质量控制，能完整的反映中成药的整体质量，有效的避免了中药品种在上市检验过程中，只添加需要检测的个别成 分就可以通过法定程序检测的弊端。
本发明所述的指纹图谱的检测方法是一个完整的技术方案，其中包括了若干技术特征，针对于其中的任何一个技术特征，发明人都进行过多次的试验方法考察，虽然各个技术特征看似简单，但是由无数个技术特征组成的整体的技术方案是为了该制剂的质量控制的最优选的技术方案。特别是通过本方法进行的7批次之间的指纹图谱相似度均大于95％，说明本产品是稳定可控的。
本方法操作简单，可实施性强，用时较短，解决了中药大复方制剂质量控制的难题。由以上实验结果可知，本方法具有良好的精密度和稳定性，以及重复性，为提高该中药组合物的质量控制提供一种新方法。
附图说明
图1对照品图谱；
图2供试品指纹图谱；
峰1：新绿原酸 峰2：绿原酸 峰3：隐绿原酸 峰5：异连翘酯苷A 峰7：连翘酯苷A 峰10：橙皮苷 峰12：黄芩苷 峰13：牛蒡苷 峰17：芦荟大黄素 峰18：甘草酸铵 峰19：大黄酸 峰20：大黄素 峰21：大黄酚 峰22：大黄素甲醚
图3 50％甲醇的提取效果；
图4 80％甲醇的提取效果；
图5 100％甲醇的提取效果；
图6超声提取指纹图谱；
图7回流提取指纹图谱；
图8超声提取20分钟指纹图谱；
图9超声提取30分钟指纹图谱；
图10超声提取40分钟指纹图谱；
图11参照物溶液色谱图；
图12供试品溶液色谱图；
图13 7批所述中药组合物色谱指纹图谱；
图14所述中药组合物对照指纹图谱。
具体实施方式
以下结合附图及实施例详细说明本发明的技术方案，但本发明的保护范围包括但是不限于此。
实施例1
按比例称取：麻黄52g；石膏324g；连翘194g；黄芩78g；桑白皮194g；苦杏仁130g；前胡78g；半夏130g；陈皮78g；贝母78g；牛蒡子130g；金银花130g；大黄39g；桔梗76g；甘草65g。
按照以下工艺提取：
A、按组方比例称取贝母，粉碎成细粉，备用；
B、按组方比例称取麻黄、连翘、苦杏仁、半夏、牛蒡子、大黄加50％乙醇回流提取2次，每次3小时，第一次加10倍量，第二次加6倍量，提取液合并，滤过，滤液减压回收乙醇，浓缩至60℃热测相对密度为1.15的清膏，备用；
C、按组方比例称取石膏、桑白皮、前胡、陈皮、金银花、桔梗、甘草，加水煎煮两次，每次2小时，第一次加10倍量，第二次加7倍量，煎液合并，滤过，浓缩至60℃热测相对密度为1.15的清膏，与步骤B所得的清膏合并，备用；
D、将步骤C所得合并的清膏，喷雾干燥，收集喷雾粉备用；
E、按常规方法制成胶囊剂。
指纹图谱测定方法：
供试品溶液制备：取所述中药组合物1g，精密称定，置具塞锥形瓶中，精密加入甲醇50mL，称定重量，超声处理30分钟，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；
对照品溶液的制备：分别称取新绿原酸、绿原酸、隐绿原酸、异连翘酯苷A、连翘酯苷A、橙皮苷、黄芩苷、牛蒡苷、芦荟大黄素、甘草酸铵、大黄酚、大黄素、大黄酸、大黄素甲醚对照品，加入甲醇，制成分别含0.088mg/ml新绿原酸、0.078mg/ml绿原酸、0.076mg/ml隐绿原酸、0.080mg/ml异连翘酯苷A、0.177mg/ml连翘酯苷A、0.157mg/ml橙皮苷、0.115mg/ml黄芩苷、0.202mg/ml牛蒡苷、0.045mg/ml甘草酸铵、0.016mg/ml芦荟大黄素、0.021mg/ml大黄酸、0.010mg/ml大黄素、0.015mg/ml大黄酚、0.008mg/ml大黄素甲醚混合对照品溶液即得；
参照物溶液的制备：取黄芩苷对照品适量，精密称定，加甲醇制成每1ml含60μg的溶液，即得；
流动相为乙腈-0.1％磷酸水溶液，梯度洗脱，洗脱比例如下表：
Figure PCTCN2021136486-appb-000018
Figure PCTCN2021136486-appb-000019
确定色谱柱ACQUITY UPLC HSS T3 C18 1.8μm，2.1*100mm；设置如下参数为初始条件，流速：0.3mL/min；波长254nm；柱温：30℃；进样体积：1μL。
测定法：分别精密吸取对照品溶液与供试品溶液各1μL，注入超高液相色谱仪，测定，记录色谱图，即得。
实施例2
按比例称取：麻黄86g；石膏194g；连翘324g；黄芩130g；桑白皮324g；苦杏仁78g；前胡130g；半夏78g；陈皮130g；贝母130g；牛蒡子78g；金银花78g；大黄65g；桔梗46g；甘草39g
按照以下工艺提取：
A、按组方比例称取贝母，粉碎成细粉，备用；
B、按组方比例称取麻黄、连翘、苦杏仁、半夏、牛蒡子、大黄加50％乙醇回流提取2次，每次1小时，第一次加8倍量，第二次加9倍量，提取液合并，滤过，滤液减压回收乙醇，浓缩至60℃热测相对密度为1.04-1.16的清膏，备用；
C、按组方比例称取石膏、桑白皮、前胡、陈皮、金银花、桔梗、甘草，加水煎煮两次，每次2小时，煎液合并，滤过，浓缩至60℃热测相对密度为1.04-1.16的清膏，与步骤B所得的清膏合并，备用；
D、将步骤C所得合并的清膏，喷雾干燥，收集喷雾粉，按常规方法制成片剂。
指纹图谱测定方法：
供试品溶液制备：取所述中药组合物1g，精密称定，置具塞锥形瓶中，精密加入甲醇50mL，称定重量，超声处理30分钟，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；
对照品溶液的制备：分别称取新绿原酸、绿原酸、隐绿原酸、异连翘酯苷A、连翘酯苷A、橙皮苷、黄芩苷、牛蒡苷、芦荟大黄素、甘草酸铵、大黄酚、大黄素、大黄酸、大黄素甲醚对照品，加入甲醇，制成分别含0.079mg/ml新绿原酸、0.088mg/ml绿原酸、0.085mg/ml隐绿原酸、0.081mg/ml异连翘酯苷A、0.179mg/ml连翘酯苷A、0.148mg/ml橙皮苷、0.109mg/ml黄芩苷、0.226mg/ml牛蒡苷、0.049mg/ml甘草酸铵、0.018mg/ml芦荟大黄素、0.025mg/ml大黄酸、0.015mg/ml大黄素、0.018mg/ml大黄酚、0.009mg/ml大黄素甲醚混合对照品溶液即得；
参照物溶液的制备：取黄芩苷对照品适量，精密称定，加甲醇制成每1ml含60μg的溶液，即得；
流动相为乙腈-0.1％磷酸水溶液，梯度洗脱，洗脱比例如下表：
Figure PCTCN2021136486-appb-000020
确定色谱柱ACQUITY UPLC HSS T3 C 18 1.8μm，2.1*100mm；设置如下参数为初始条件，流速：0.3mL/min；波长254nm；柱温：30℃；进样体积：1μL；
测定法：分别精密吸取对照品溶液与供试品溶液各1μL，注入超高液相色谱仪，测定，记录色谱图，即得。
实施例3
原料药配方为：麻黄69g；石膏259g；连翘259g；黄芩104g；桑白皮259g；苦杏仁104g；前胡104g；半夏104g；陈皮104g；贝母104g；牛蒡子104g；金银花104g；大黄52g；桔梗61g；甘草52g。
按照以下工艺提取：
A、按组方比例称取贝母，粉碎成细粉，备用；
B、按组方比例称取麻黄、连翘、苦杏仁、半夏、牛蒡子、大黄加70％乙醇回流提取2次，每次2小时，第一次加10倍量，第二次加9倍量，提取液合并，滤过，滤液减压回收乙醇，浓缩至60℃热测相对密度为1.04-1.16的清膏，备用；
C、按组方比例称取石膏、桑白皮、前胡、陈皮、金银花、桔梗、甘草，加水煎煮两次，每次3小时，第一次加10倍量，第二次加10倍量，煎液合并，滤过，浓缩至60℃热测相对密度为1.04-1.16的清膏，与步骤B所得的清膏合并，备用；
D、将步骤C所得合并的清膏，喷雾干燥，收集喷雾粉，按常规方法制成颗粒剂。
指纹图谱测定方法：
供试品溶液制备：取所述中药组合物0.5g，精密称定，置具塞锥形瓶中，精密加入50％甲醇20mL，称定重量，超声处理20分钟，放冷，再称定重量，用50％的甲醇补足减失的重量，摇匀，滤过，取续滤液，即得；
对照品溶液的制备：分别称取新绿原酸、绿原酸、隐绿原酸、异连翘酯苷A、连翘酯苷A、橙皮苷、黄芩苷、牛蒡苷、芦荟大黄素、甘草酸铵、大黄酚、大黄素、大黄酸、大黄素甲醚对照品，加入甲醇，制成分别含0.080mg/ml新绿原酸、0.082mg/ml绿原酸、0.085mg/ml隐绿原酸、0.085mg/ml异连翘酯苷A、0.180mg/ml连翘酯苷A、0.166mg/ml橙皮苷、0.116mg/ml黄芩苷、0.232mg/ml牛蒡苷、0.046mg/ml甘草酸铵、0.019mg/ml芦荟大黄素、0.020mg/ml大黄酸、0.011mg/ml大黄素、0.016mg/ml大黄酚、0.009mg/ml大黄素甲醚混合对照品溶液即得；
参照物溶液的制备：取黄芩苷对照品适量，精密称定，加甲醇制成每1ml含60μg的溶液，即得；
流动相为乙腈-0.1％磷酸水溶液，梯度洗脱，洗脱比例如下表：
Figure PCTCN2021136486-appb-000021
色谱柱为ACQUITY UPLC BEH C 18 1.7μm，2.1*100mm色谱柱；检测波长245nm，流速0.1ml/min；
测定法：分别精密吸取对照品溶液与供试品溶液各1μL，注入超高液相色谱仪，测定，记录色谱图，即得。
实施例4
原料药配方为：按比例称取：
麻黄55g；石膏254g；连翘318g；黄芩107g；桑白皮203g；苦杏仁107g；前胡82g；半夏105g；陈皮84g；贝母125g；牛蒡子122g；金银花113g；大黄42g；桔梗60g；甘草50g。
按照以下工艺提取：
A、按组方比例称取贝母，粉碎成细粉，备用；
B、按组方比例称取麻黄、连翘、苦杏仁、半夏、牛蒡子、大黄加40-70％乙醇回流提取2次，每次3小时，第一次加8倍量，第二次加7倍量，提取液合并，滤过，滤液减压回收乙醇，浓缩至60℃热测相对密度为1.04-1.16的清膏，备用；
C、按组方比例称取石膏、桑白皮、前胡、陈皮、金银花、桔梗、甘草，加水煎煮两次，每次2小时，第一次加10倍量，第二次加8倍量，煎液合并，滤过，浓缩至60℃热测相对密度为1.04-1.16的清膏，与步骤B所得的清膏合并，备用；
D、将步骤C所得合并的清膏，喷雾干燥，收集喷雾粉，按常规方法制成丸剂。
指纹图谱测定方法：
供试品溶液制备：取所述中药组合物1.5g，精密称定，置具塞锥形瓶中，精密加入80％甲醇50mL，称定重量，超声处理40分钟，放冷，再称定重量，用80％的甲醇补足减失的重量，摇匀，滤过，取续滤液，即得；
对照品溶液的制备：分别称取新绿原酸、绿原酸、隐绿原酸、异连翘酯苷A、连翘酯苷A、橙皮苷、黄芩苷、牛蒡苷、芦荟大黄素、甘草酸铵、大黄酚、大黄素、大黄酸、大黄素甲醚对照品，加入甲醇，制成分别含0.080mg/ml新绿原酸、0.082mg/ml绿原酸、0.085mg/ml隐绿原酸、0.085mg/ml异连翘酯苷A、0.180mg/ml连翘酯苷A、0.166mg/ml橙皮苷、0.116mg/ml黄芩苷、0.232mg/ml牛蒡苷、0.046mg/ml甘草酸铵、0.019mg/ml芦荟大黄素、0.020mg/ml大黄酸、0.011mg/ml大黄素、0.016mg/ml大黄酚、0.009mg/ml大黄素甲醚混合对照品溶液即得；
参照物溶液的制备：取黄芩苷对照品适量，精密称定，加甲醇制成每1ml含60μg的溶液，即得；
流动相为乙腈-0.1％磷酸水溶液，梯度洗脱，洗脱比例如下表：
Figure PCTCN2021136486-appb-000022
色谱柱为CORTECS UPLC C 18 1.6μm，2.1*100mm色谱柱；检测波长245nm，流速0.1ml/min；
测定法：分别精密吸取对照品溶液与供试品溶液各1μL，注入超高液相色谱仪，测定，记录色谱图，即得。

Claims (5)

  1. 一种中药组合物的指纹图谱测定方法，该中药组合物由如下重量份的原料药制成：麻黄52-86；石膏194-324；连翘194-324；黄芩78-130；桑白皮194-324；苦杏仁78-130；前胡78-130；半夏78-130；陈皮78-130；贝母78-130；牛蒡子78-130；金银花78-130；大黄39-65；桔梗46-76；甘草39-65，其特征在于该中药组合物制剂指纹图谱的测定方法如下：
    供试品溶液制备：取所述中药组合物0.5-1.5g，精密称定，置具塞锥形瓶中，精密加入40-100％甲醇10-50mL，称定重量，超声处理20-40分钟，放冷，再称定重量，用40-100％的甲醇补足减失的重量，摇匀，滤过，取续滤液，即得；
    对照品溶液的制备：分别称取新绿原酸、绿原酸、隐绿原酸、异连翘酯苷A、连翘酯苷A、橙皮苷、黄芩苷、牛蒡苷、芦荟大黄素、甘草酸铵、大黄酚、大黄素、大黄酸、大黄素甲醚对照品，加入甲醇，制成混合对照品溶液即得；
    参照物溶液的制备：取黄芩苷对照品适量，精密称定，加甲醇制成每1ml含60μg的溶液，即得；
    流动相为乙腈-0.1％磷酸水溶液，梯度洗脱，洗脱比例如下表：
    Figure PCTCN2021136486-appb-100001
    色谱柱为C 18色谱柱；检测波长245-254nm，流速0.1-0.5ml/min；
    测定法：分别精密吸取对照品溶液与供试品溶液各1-10μL，注入超高液相色谱仪，测定，记录色谱图，即得。
  2. 如权利要求1所述的指纹图谱测定方法，其特征在于所述指纹图谱测定方法如下：
    供试品溶液制备：取所述中药组合物1-1.2g，精密称定，置具塞锥形瓶中，精密加入甲醇50mL，称定重量，超声处理30分钟，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；
    对照品溶液的制备：分别称取新绿原酸、绿原酸、隐绿原酸、异连翘酯苷A、连翘酯苷A、橙皮苷、黄芩苷、牛蒡苷、芦荟大黄素、甘草酸铵、大黄酚、大黄素、大黄酸、大 黄素甲醚对照品，加入甲醇，制成分别含0.07-0.09mg/ml新绿原酸、0.07-0.09mg/ml绿原酸、0.07-0.09mg/ml隐绿原酸、0.07-0.09mg/ml异连翘酯苷A、0.1-0.3mg/ml连翘酯苷A、0.1-0.3mg/ml橙皮苷、0.1-0.3mg/ml黄芩苷、0.1-0.3mg/ml牛蒡苷、0.04-0.06mg/ml甘草酸铵、0.01-0.02mg/ml芦荟大黄素、0.01-0.03mg/ml大黄酸、0.01-0.03mg/ml大黄素、0.01-0.03mg/ml大黄酚、0.005-0.01mg/ml大黄素甲醚混合对照品溶液即得；
    参照物溶液的制备：取黄芩苷对照品适量，精密称定，加甲醇制成每1ml含60μg的溶液，即得；
    流动相为乙腈-0.1％磷酸水溶液，梯度洗脱，洗脱比例如下表：
    Figure PCTCN2021136486-appb-100002
    色谱柱为C 18色谱柱；检测波长254nm，流速0.3ml/min；柱温为30℃；
    测定法：分别精密吸取对照品溶液与供试品溶液各1μL，注入超高液相色谱仪，测定，记录色谱图，即得。
  3. 如权利要求2所述的指纹图谱测定方法，其特征在于所述指纹图谱测定方法如下：
    供试品溶液制备：取所述中药组合物1g，精密称定，置具塞锥形瓶中，精密加入甲醇50mL，称定重量，超声处理30分钟，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；
    对照品溶液的制备：分别称取新绿原酸、绿原酸、隐绿原酸、异连翘酯苷A、连翘酯苷A、橙皮苷、黄芩苷、牛蒡苷、芦荟大黄素、甘草酸铵、大黄酚、大黄素、大黄酸、大黄素甲醚对照品，加入甲醇，制成分别含0.07-0.09mg/ml新绿原酸、0.07-0.09mg/ml绿原酸、0.07-0.09mg/ml隐绿原酸、0.07-0.09mg/ml异连翘酯苷A、0.1-0.3mg/ml连翘酯苷A、0.1-0.3mg/ml橙皮苷、0.1-0.3mg/ml黄芩苷、0.1-0.3mg/ml牛蒡苷、0.04-0.05mg/ml甘草酸铵、0.01-0.02mg/ml芦荟大黄素、0.01-0.03mg/ml大黄酸、0.01-0.02mg/ml大黄素、0.01-0.02mg/ml大黄酚、0.005-0.009mg/ml大黄素甲醚混合对照品溶液即得；
    参照物溶液的制备：取黄芩苷对照品适量，精密称定，加甲醇制成每1ml含60μg的溶液，即得；
    流动相为乙腈-0.1％磷酸水溶液，梯度洗脱，洗脱比例如下表：
    Figure PCTCN2021136486-appb-100003
    色谱柱为C 18色谱柱；柱长为100mm，内径为2.1mm，粒径为1.8μm；检测波长254nm，流速0.3ml/min；柱温为30℃；
    测定法：分别精密吸取对照品溶液与供试品溶液各1μL，注入超高液相色谱仪，测定，记录色谱图，即得。
  4. 根据权利要求1-3任一所述的指纹图谱的测定方法，其特征在于所用色谱柱型号为ACQUITY UPLC HSS T3 C 181.8μm，2.1*100mm。
  5. 根据权利要求1-3任一所述的指纹图谱的测定方法，其特征在于还包括以下步骤：a获得对照指纹图谱：将现行标准评价合格的样品的供试品图谱导入《中药色谱指纹图谱相似度评价系统》合成对照指纹图谱；
    b相似度评价：将待评价样品的供试品指纹图谱导入《中药色谱指纹图谱相似度评价系统》，计算相似度，供试品图谱与对照指纹图谱的相似度不得低于0.9。
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