WO2024007531A1 - 一种同时测定导赤丸中24种成分含量的方法 - Google Patents

一种同时测定导赤丸中24种成分含量的方法 Download PDF

Info

Publication number
WO2024007531A1
WO2024007531A1 PCT/CN2022/138252 CN2022138252W WO2024007531A1 WO 2024007531 A1 WO2024007531 A1 WO 2024007531A1 CN 2022138252 W CN2022138252 W CN 2022138252W WO 2024007531 A1 WO2024007531 A1 WO 2024007531A1
Authority
WO
WIPO (PCT)
Prior art keywords
content
daochi
ingredients
hydrochloride
mass spectrum
Prior art date
Application number
PCT/CN2022/138252
Other languages
English (en)
French (fr)
Inventor
曹春琪
刘永利
孙慧珠
赵振霞
段琼
苏建
Original Assignee
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)
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 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) filed Critical 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)
Publication of WO2024007531A1 publication Critical patent/WO2024007531A1/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
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • 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
    • 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 field of drug analysis and detection, and is specifically a method for simultaneously measuring the contents of 24 ingredients in Daochi pills.
  • Daochiwan comes from Volume 6 of "Taiping Huimin Heji Bureau Prescriptions” compiled by Taiping Huimin Heji Bureau in the Song Dynasty.
  • Daochi Pills are composed of ten herbs, including Forsythia suspensa, Coptis chinensis, Gardenia (fried with ginger), Akebia, Scrophulariaceae, Trichosanthin, red peony root, rhubarb, skullcap, and talc.
  • Coptis chinensis clears away heat and dampness, purges fire and detoxifies; Scutellaria baicalensis clears away heat and detoxifies, clears away heat and dampness; Gardenia jasminoides purges fire and relieves troubles, cools blood and detoxifies.
  • Forsythia suspensa dispels wind-heat, Akebia diuresis and relieves stranguria; Rhubarb purges and relieves attacks.
  • Ji Scrophulariaceae clearing away heat and cooling blood, red peony root dispersing blood stasis and relieving pain are the ministerial drugs; talc is a diuretic and relieves stranguria, and trichosanthin is an adjuvant drug for promoting body fluid and quenching thirst. All the medicines are combined to clear away heat, purge fire, diuresis and relieve constipation. Clinically, it is often used for sores on the mouth and tongue, sore throat, irritability in the chest and chest, short and red urine, and constipation caused by excessive internal heat.
  • the purpose of this invention is to address the above problems and provide a method for simultaneously determining the content of 24 ingredients in Daochi Pills, which includes the following steps:
  • Chromatographic conditions Chromatographic column: phenomenon C18 column 2.6 ⁇ m, 2.1 ⁇ 100mm; acetonitrile as mobile phase A, 0.1% formic acid as mobile phase B, gradient elution; column temperature: 40°C, flow rate: 0.3ml/min; injection Volume: 1 ⁇ l;
  • Mass spectrometry conditions electrospray ion source ESI, scanning mode: simultaneous scanning of positive and negative ions, monitoring mode: multiple reaction monitoring mode MRM, positive ionization voltage: 5500V, desolvation gas temperature: 500°C, negative ionization voltage: -4500V, desolvation Air temperature: 500°C.
  • the mixed reference solution is configured by the following steps: Take each component reference solution to be measured, weigh it accurately, add 70% methanol to prepare a solution containing 60 ⁇ g of gardeniposide, 70 ⁇ g of forsythiaside A, and Crocin I 4 ⁇ g, Crocin II 1 ⁇ g, palmatine hydrochloride 7 ⁇ g, epiberberine 3 ⁇ g, coptisine hydrochloride 9 ⁇ g, chrysophanol 2 ⁇ g, baicalein 3 ⁇ g, paeoniflorin 14 ⁇ g, harpaside 4 ⁇ g, jatrorrhizine hydrochloride 6 ⁇ g , Wogonin 1 ⁇ g, Harpagoside 2 ⁇ g, Melaleucain A 1 ⁇ g, Trichophyton phenylethanoid glycoside B 1 ⁇ g, Forsythiarin 4 ⁇ g, Baicalin 70 ⁇ g, Wogonin 25 ⁇ g, Berberine hydrochloride 22 ⁇ g
  • the test solution is prepared by the following steps: Grind or cut Daochi pills into pieces, mix well, take about 0.3g, add 25ml of 70% methanol, weigh, plug tightly, and ultrasonicate for 30 minutes , take it out, let it cool, make up for the lost weight with 70% methanol, shake well, filter, take the remaining filtrate, and filter it through a 0.22 ⁇ m microporous filter membrane to get it.
  • the ultrasonic treatment power 250W, frequency 40kHz.
  • the gradient elution procedure is as follows:
  • the present invention has established a UPLC-MS/MS method for simultaneous determination of 24 ingredients in Daochi Pills. It is more specific and can control more ingredients. It also conducts all-round measurement and analysis of different medicinal materials in the prescription, and conducts a more comprehensive assessment of drug quality. , and also embodies the concept of convenient, efficient and resource-saving inspection and testing work.
  • Figure 1 shows the mass spectrum of baicalin reference substance
  • Figure 2 is the mass spectrum of the baicalin test product
  • Figure 3 is the mass spectrum of wogonin reference substance
  • Figure 4 is the mass spectrum of the wogonin test sample
  • Figure 5 is the mass spectrum of baicalein reference substance
  • Figure 6 is the mass spectrum of the baicalein test sample
  • Figure 7 is the mass spectrum of wogonin reference substance
  • Figure 8 is the mass spectrum of the wogonin test sample
  • Figure 9 is the mass spectrum of Melaleuca A reference substance
  • Figure 10 is the mass spectrum of the Melaleuca A test sample
  • Figure 11 is the mass spectrum of berberine hydrochloride reference substance
  • Figure 12 is the mass spectrum of the berberine hydrochloride test product
  • Figure 13 is the mass spectrum of epiberberine reference substance
  • Figure 14 is the mass spectrum of the berberine test sample
  • Figure 15 is the mass spectrum of coptisine hydrochloride reference substance
  • Figure 16 is the mass spectrum of coptisine hydrochloride test product
  • Figure 17 is the mass spectrum of jatrorrhizine hydrochloride reference substance
  • Figure 18 is the mass spectrum of the test sample of jatrorrhizine hydrochloride
  • Figure 19 is the mass spectrum of palmatine hydrochloride reference substance
  • Figure 20 is the mass spectrum of the palmatine hydrochloride test product
  • Figure 21 is the mass spectrum of emodin reference substance
  • Figure 22 is the mass spectrum of the emodin test sample
  • Figure 23 is the mass spectrum of rhein reference substance
  • Figure 24 is the mass spectrum of the rhein test sample
  • Figure 25 is the mass spectrum of emodin methyl ether reference substance
  • Figure 26 is the mass spectrum of the test sample of emodin methyl ether
  • Figure 27 is the mass spectrum of the aloe-emodin reference substance
  • Figure 28 is the mass spectrum of the aloe-emodin test sample
  • Figure 29 is the mass spectrum of chrysophanol reference substance
  • Figure 30 is the mass spectrum of chrysophanol test sample
  • Figure 31 is the mass spectrum of the gardeniposide reference substance
  • Figure 32 is the mass spectrum of the gardeniposide test sample
  • Figure 33 is the mass spectrum of crocin I reference substance
  • Figure 34 is the mass spectrum of the crocin I test sample
  • Figure 35 is the mass spectrum of crocin II reference substance
  • Figure 36 is the mass spectrum of the crocin II test sample
  • Figure 37 is the mass spectrum of harpaside reference substance
  • Figure 38 is the mass spectrum of the harpaside test sample
  • Figure 39 is the mass spectrum of harpagoside reference substance
  • Figure 40 is the mass spectrum of the harpagoside test sample
  • Figure 41 is the mass spectrum of the Akebia phenylethanol glycoside B reference substance
  • Figure 42 is the mass spectrum of the test sample of Akebia phenylethanol glycoside B
  • Figure 43 is the mass spectrum of paeoniflorin reference substance
  • Figure 44 is the mass spectrum of the paeoniflorin test product
  • Figure 45 is the mass spectrum of the forsythin reference substance
  • Figure 46 is the mass spectrum of the forsythin test sample
  • Figure 47 is the mass spectrum of the forsythiaside A reference substance
  • Figure 48 is the mass spectrum of the test sample of forsythiaside A.
  • the present invention provides a method for simultaneously determining the contents of 24 components in Daochi Pills.
  • the present invention adopts the UPLC-MS/MS method to determine baicalin, wogonin, baicalein, wogonin, melaleuca A, and hydrochloric acid.
  • the content determination method of the present invention adopts the UPLC-MS/MS method, which is as follows:
  • Chromatographic conditions Chromatographic column: phenomenon C18 column (2.6 ⁇ m, 2.1 ⁇ 100mm); use acetonitrile as mobile phase A and 0.1% formic acid as mobile phase B.
  • the gradient elution procedure is shown in Table 1; column temperature: 40°C, flow rate: 0.3 ml/min; injection volume: 1 ⁇ l.
  • baicalin (Inspection Institute of China, batch number: 110715-201117, content 91.7%)
  • Palmatine hydrochloride (CCI, batch number: 110732-201913, content 85.7%)
  • Aloe emodin (CCI, batch number: 110795-201308, content 97.8%)
  • Crocin I (Inspection Institute of China, batch number: 111588-201303, content 92.6%)
  • Crocin II Crocin II (Chinese Institute of Technology, batch number: 111589-201705, content 92.2%)
  • Reagents methanol, acetonitrile, formic acid, and ammonium acetate are all chromatographically pure, and water is ultrapure water.
  • Chromatographic conditions Chromatographic column: phenomenon C18 column (2.6 ⁇ m, 2.1 ⁇ 100mm); use acetonitrile as mobile phase A and 0.1% formic acid as mobile phase B.
  • the gradient elution procedure is shown in Table 1; column temperature: 40°C, flow rate: 0.3 ml/min; injection volume: 1 ⁇ l.
  • Determination method Precisely draw 1 ⁇ l each of the mixed reference solution and test solution, inject into the ultra-high performance liquid chromatography mass spectrometer, measure, and calculate the content with the external standard method.
  • statin 7 ⁇ g of statin, 3 ⁇ g of epiberberine, 9 ⁇ g of coptisine hydrochloride, 2 ⁇ g of chrysophanol, 3 ⁇ g of baicalein, 14 ⁇ g of paeoniflorin, 4 ⁇ g of harpagoside, 6 ⁇ g of jatrorrhizine hydrochloride, 1 ⁇ g of wogonin, 2 ⁇ g of harpagoside, and melaleuca A 1 ⁇ g, Akebia phenylethanoid glycoside B 1 ⁇ g, Forsythiarin 4 ⁇ g, Baicalin 70 ⁇ g, Wogonin 25 ⁇ g, Berberine hydrochloride 22 ⁇ g, Emodin methyl ether 1 ⁇ g, Aloe emodin 1 ⁇ g, Rhein 1 ⁇ g, Emodin 1 ⁇ g Mix the solution and get it.
  • test solution Take this product, grind water honey pills or large honey pills into pieces, mix well, take about 0.3g, add 25ml of 70% methanol, weigh, seal tightly, and ultrasonic treatment (power 250W, frequency 40kHz ) for 30 minutes, take it out, let it cool, make up for the lost weight with 70% methanol, shake well, filter, take the remaining filtrate, and filter it through a 0.22 ⁇ m microporous filter membrane to get it.
  • ultrasonic treatment power 250W, frequency 40kHz
  • the UPLC/MS/MS method established by the present invention for the simultaneous determination of 24 ingredients in Daochi Pills is more specific, controls more ingredients, and performs comprehensive measurement and analysis of different medicinal materials in the prescription, and improves drug quality. It provides a more comprehensive assessment and also embodies the concept of convenient, efficient and resource-saving inspection and testing work.

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)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

一种同时测定导赤丸中24种成分含量的方法，包括如下步骤：制备混合对照品溶液；制备供试品溶液：分别精密吸取混合对照品溶液与供试品溶液各1μL，注入超高效液相色谱质谱联用仪，测定，外标法计算含量。UPLC-MS/MS同时测定导赤丸中24种成分的方法，专属性强，控制成分多，且对处方中不同药材进行全方位测定分析，对药品质量进行了更全面的评估，同时也体现了便捷、高效、节约资源的检验检测工作理念。

Description

一种同时测定导赤丸中24种成分含量的方法 技术领域
本发明属于药物分析检测领域，具体是一种同时测定导赤丸中24种成分含量的方法。
背景技术
导赤丸，出自宋代太平惠民和剂局编写的《太平惠民和剂局方》卷六。《中国药典》1963年版一部收录了导赤丸，以后历版药典均予以收载。现收载于《中国药典》2020年版。导赤丸由连翘、黄连、栀子(姜炒)、木通、玄参、天花粉、赤芍、大黄、黄芩、滑石共十味药组成。处方中黄连清热燥湿、泻火解毒，黄芩清热解毒、清热燥湿，栀子泻火除烦、凉血解毒共为君药；连翘疏散风热、木通利尿通淋、大黄泻下攻积、玄参清热凉血、赤芍散瘀止痛为臣药；滑石利尿通淋、天花粉生津止渴为佐药。诸药相合，共奏清热泻火，利尿通便之功效。临床常用于火热内盛所致的口舌生疮、咽喉疼痛、心胸烦热、小便短赤、大便秘结。
导赤丸现行药典标准，虽有薄层色谱鉴别项及含量测定项，但除显微鉴别外，仅控制了其中2味药，仅仅为处方量的五分之一，质量标准覆盖药味少，且测定成分单一，并不能全面反映成药的质量。在药品检验的过程中，难以发现以次充好、偷工减料等问题。
发明内容
本发明的目的是针对以上问题，提供了一种同时测定导赤丸中24种成分含量的方法，包括以下步骤：
A.制备混合对照品溶液；
B.制备供试品溶液：
C.分别精密吸取混合对照品溶液与供试品溶液各1μl，注入超高效液相色 谱质谱联用仪，测定，外标法计算含量，即得；
色谱条件：色谱柱：phenomenon C18柱2.6μm，2.1×100mm；以乙腈为流动相A，0.1％甲酸为流动相B，梯度洗脱；柱温：40℃，流速：0.3ml/min；进样量：1μl；
质谱条件：电喷雾离子源ESI，扫描方式：正负离子同时扫描，监测模式：多反应监测模式MRM，正离子化电压：5500V，脱溶剂气温度：500℃，负离子化电压：-4500V，脱溶剂气温度：500℃。
优选的，所述混合对照品溶液由如下步骤配置而成：取各待测成分对照品，精密称定，加70％甲醇分别制成每1ml含栀子苷60μg、连翘酯苷A 70μg、西红花苷Ⅰ4μg、西红花苷Ⅱ1μg、盐酸巴马汀7μg、表小檗碱3μg、盐酸黄连碱9μg、大黄酚2μg、黄芩素3μg、芍药苷14μg、哈巴苷4μg、盐酸药根碱6μg、汉黄芩素1μg、哈巴俄苷2μg、千层纸素A 1μg、木通苯乙醇苷B 1μg、连翘苷4μg、黄芩苷70μg、汉黄芩苷25μg、盐酸小檗碱22μg、大黄素甲醚1μg、芦荟大黄素1μg、大黄酸1μg、大黄素1μg的混合溶液，即得。
优选的，所述供试品溶液由如下步骤配置而成：取导赤丸研细或剪碎，混匀，取约0.3g，加70％甲醇25ml，称定重量，密塞，超声处理30分钟，取出，放冷，用70％甲醇补足减失的重量，摇匀，滤过，取续滤液，经0.22μm微孔滤膜滤过，即得。
优选的，所述超声处理：功率250W，频率40kHz。
优选的，梯度洗脱程序如下：
流动相梯度洗脱程序
Figure PCTCN2022138252-appb-000001
Figure PCTCN2022138252-appb-000002
与现有技术相比，本发明的有益效果如下：
本发明建立了UPLC-MS/MS同时测定导赤丸中24种成分的方法，专属性更强，控制成分更多，且对处方中不同药材进行全方位测定分析，药品质量进行了更全面的评估，同时也体现了便捷、高效、节约资源的检验检测工作理念。
附图说明
图1为黄芩苷对照品的质谱图；
图2为黄芩苷供试品的质谱图；
图3为汉黄芩苷对照品的质谱图；
图4为汉黄芩苷供试品的质谱图；
图5为黄芩素对照品的质谱图；
图6为黄芩素供试品的质谱图；
图7为汉黄芩素对照品的质谱图；
图8为汉黄芩素供试品的质谱图；
图9为千层纸素A对照品的质谱图；
图10为千层纸素A供试品的质谱图；
图11为盐酸小檗碱对照品的质谱图；
图12为盐酸小檗碱供试品的质谱图；
图13为表小檗碱对照品的质谱图；
图14为表小檗碱供试品的质谱图；
图15为盐酸黄连碱对照品的质谱图；
图16为盐酸黄连碱供试品的质谱图；
图17为盐酸药根碱对照品的质谱图；
图18为盐酸药根碱供试品的质谱图；
图19为盐酸巴马汀对照品的质谱图；
图20为盐酸巴马汀供试品的质谱图；
图21为大黄素对照品的质谱图；
图22为大黄素供试品的质谱图；
图23为大黄酸对照品的质谱图；
图24为大黄酸供试品的质谱图；
图25为大黄素甲醚对照品的质谱图；
图26为大黄素甲醚供试品的质谱图；
图27为芦荟大黄素对照品的质谱图；
图28为芦荟大黄素供试品的质谱图；
图29为大黄酚对照品的质谱图；
图30为大黄酚供试品的质谱图；
图31为栀子苷对照品的质谱图；
图32为栀子苷供试品的质谱图；
图33为西红花苷Ⅰ对照品的质谱图；
图34为西红花苷Ⅰ供试品的质谱图；
图35为西红花苷Ⅱ对照品的质谱图；
图36为西红花苷Ⅱ供试品的质谱图；
图37为哈巴苷对照品的质谱图；
图38为哈巴苷供试品的质谱图；
图39为哈巴俄苷对照品的质谱图；
图40为哈巴俄苷供试品的质谱图；
图41为木通苯乙醇苷B对照品的质谱图；
图42为木通苯乙醇苷B供试品的质谱图；
图43为芍药苷对照品的质谱图；
图44为芍药苷供试品的质谱图；
图45为连翘苷对照品的质谱图；
图46为连翘苷供试品的质谱图；
图47为连翘酯苷A对照品的质谱图；
图48为连翘酯苷A供试品的质谱图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明提供了一种同时测定导赤丸中24种成分的含量测定方法，本发明采用UPLC-MS/MS法测定黄芩苷、汉黄芩苷、黄芩素、汉黄芩素、千层纸素A、盐酸小檗碱、盐酸黄连碱、盐酸巴马汀、表小檗碱、盐酸药根碱、连翘苷、连翘酯苷A、栀子苷、西红花苷Ⅰ、西红花苷Ⅱ、木通苯乙醇苷B、哈巴苷、哈巴俄苷、芍药苷、大黄酚、大黄素甲醚、芦荟大黄素、大黄酸、大黄素含量。
本发明含量测定方法采用UPLC-MS/MS法，该方法如下：
色谱条件：色谱柱：phenomenon C18柱(2.6μm，2.1×100mm)；以乙腈为流动相A，0.1％甲酸为流动相B，梯度洗脱程序见表1；柱温：40℃，流速：0.3ml/min；进样量：1μl。
表1流动相梯度洗脱程序
Figure PCTCN2022138252-appb-000003
以三重四极杆串联质谱仪检测；电喷雾离子源(ESI)，扫描方式：正负离子同时扫描，监测模式：多反应监测模式(MRM)，正离子化电压：5500V，脱溶剂气温度：500℃，负离子化电压：-4500V，脱溶剂气温度：500℃；质谱参数详见表2。
表2定性和定量离子及质谱条件表
Figure PCTCN2022138252-appb-000004
Figure PCTCN2022138252-appb-000005
混合对照品溶液的制备：
取各待测成分对照品适量，精密称定，加70％甲醇分别制成每1ml含栀子苷60μg、连翘酯苷A 70μg、西红花苷Ⅰ 4μg、西红花苷Ⅱ 1μg、盐酸巴马汀7μg、表小檗碱3μg、盐酸黄连碱9μg、大黄酚2μg、黄芩素3μg、芍药苷14μg、哈巴苷4μg、盐酸药根碱6μg、汉黄芩素1μg、哈巴俄苷2μg、千层纸素A 1μg、木通苯乙醇苷B 1μg、连翘苷4μg、黄芩苷70μg、汉黄芩苷25μg、盐酸小檗碱22μg、大黄素甲醚1μg、芦荟大黄素1μg、大黄酸1μg、大黄素1μg的混合溶液，即得。
供试品溶液的制备：
取本品，水蜜丸研细或大蜜丸剪碎，混匀，取约0.3g，加70％甲醇25ml，称定重量，密塞，超声处理(功率250W，频率40kHz)30分钟，取出，放冷，用70％甲醇补足减失的重量，摇匀，滤过，取续滤液，经0.22μm微孔滤膜滤过，即得。
测定法：
分别精密吸取混合对照品溶液与供试品溶液各1μl，注入超高效液相色谱质谱联用仪，测定，外标法计算含量，即得。
1.仪器与试药
1.1仪器
AB SCIEX Qtrap 6500超高效液相色谱-离子阱质谱联用仪；分析天平：Mettler XPE26(百万分之一)；MettlerXS105(十万分之一)(上海梅特勒仪器有限公司)；KQ-400KDE超声波清洗仪(昆山市超声仪器有限公司)；
1.2试药
对照品：黄芩苷(中检院，批号：110715-201117，含量91.7％)
汉黄芩苷(中检院，批号：112002-201702，含量98.5％)
黄芩素(中检院，批号：111595-201808，含量97.9％)
汉黄芩素(中检院，批号：111514-201706，含量100％)
千层纸素A(上海诗丹德，批号：ST23660120，含量98.0％)
盐酸小檗碱(中检院，批号：110713-201814，含量86.7％)
盐酸巴马汀(中检院，批号：110732-201913，含量85.7％)
盐酸黄连碱(中检院，批号：112026-201601，含量95.1％)
盐酸药根碱(中检院，批号：110733-201108，含量90.3％)
表小檗碱(上海诗丹德，批号：ST23680105，含量98.0％)
大黄酚(中检院，批号：110796-201922，含量99.4％)
大黄素甲醚(中检院，批号：110758-201817，含量99.2％)
芦荟大黄素(中检院，批号：110795-201308，含量97.8％)
大黄素甲醚(中检院，批号：110758-201817，含量99.2％)
大黄酸(中检院，批号：110757-201607，含量99.3％)
木通苯乙醇苷B(中检院，批号：111910-201604，含量98.2％)
哈巴苷(中检院，批号：111729-201707，含量96.8％)
哈巴俄苷(中检院，批号：111730-201709，含量95.9％)
芍药苷(中检院，批号：110736-201942，含量95.1％)
连翘苷(中检院，批号：110821-201816，含量95.1％)
连翘酯苷A(中检院，批号：111810-201606，含量93.4％)
栀子苷(中检院，批号：110749-201919，含量97.1％)
西红花苷Ⅰ(中检院，批号：111588-201303，含量92.6％)
西红花苷Ⅱ(中检院，批号：111589-201705，含量92.2％)
试剂：甲醇、乙腈、甲酸、乙酸铵均为色谱纯，水为超纯水。
导赤丸样品26批，水蜜丸10批，大蜜丸16批，分别为4家企业生产。
甲醇、乙腈、甲酸(色谱纯，Merck公司)；水为去离子水；其它试剂均为分析纯。
2.方法与结果
2.1色谱及质谱条件
色谱条件：色谱柱：phenomenon C18柱(2.6μm，2.1×100mm)；以乙腈为流动相A，0.1％甲酸为流动相B，梯度洗脱程序见表1；柱温：40℃，流速：0.3ml/min；进样量：1μl。
以三重四极杆串联质谱仪检测；电喷雾离子源(ESI)，扫描方式：正负离子同时扫描，监测模式：多反应监测模式(MRM)，正离子化电压：5500V，脱溶剂气温度：500℃，负离子化电压：-4500V，脱溶剂气温度：500℃；质谱参数详见表2。
测定法：别精密吸取混合对照品溶液与供试品溶液各1μl，注入超高效液相色谱质谱联用仪，测定，外标法计算含量，即得。
2.2溶液的制备
2.2.1混合对照品溶液
取各待测成分对照品适量，精密称定，加70％甲醇分别制成每1ml含栀子苷60μg、连翘酯苷A 70μg、西红花苷Ⅰ4μg、西红花苷Ⅱ1μg、盐酸巴马汀7μg、表小檗碱3μg、盐酸黄连碱9μg、大黄酚2μg、黄芩素3μg、芍 药苷14μg、哈巴苷4μg、盐酸药根碱6μg、汉黄芩素1μg、哈巴俄苷2μg、千层纸素A 1μg、木通苯乙醇苷B 1μg、连翘苷4μg、黄芩苷70μg、汉黄芩苷25μg、盐酸小檗碱22μg、大黄素甲醚1μg、芦荟大黄素1μg、大黄酸1μg、大黄素1μg的混合溶液，即得。
2.2.2供试品溶液的制备
供试品溶液的制备取本品，水蜜丸研细或大蜜丸剪碎，混匀，取约0.3g，加70％甲醇25ml，称定重量，密塞，超声处理(功率250W，频率40kHz)30分钟，取出，放冷，用70％甲醇补足减失的重量，摇匀，滤过，取续滤液，经0.22μm微孔滤膜滤过，即得。
2.3线性关系考察
分别精密量取各对照品储备液配制系列浓度混合对照品溶液，分别注入超高效液相色谱-质谱联用仪，按正文条件进行测定，以进样浓度(μg/ml)为横坐标，峰面积积分值为纵坐标，进行线性回归，建立标准曲线。结果见表3，各待测成分线性关系良好。
表3各待测成分线性关系考察结果
Figure PCTCN2022138252-appb-000006
Figure PCTCN2022138252-appb-000007
2.4精密度实验
取混合对照品溶液连续进样6次，分别计算各待测成分的精密度。结果各待测成分的RSD范围为2.1％～8.7％，仪器精密度良好。
2.5重复性试验
取导赤丸样品(批号：20200701)适量，研细，分别取0.3g，共六份，精密称定，照正文方法制备供试品溶液，按拟定的色谱条件测定，计算重复性。结果表明本方法各成分重复性良好。见表4。
表4重复性试验结果
Figure PCTCN2022138252-appb-000008
Figure PCTCN2022138252-appb-000009
2.6回收率试验
取导赤丸样品(批号：20200701)适量，研细，分别取0.15g，共六份，精密称定，精密加入用70％甲醇溶液配制的混合对照品溶液25ml，按正文方法制备供试品溶液，按拟定的色谱条件测定，计算回收率。结果表明本方法各成分回收率良好，见表5。
表5回收率试验结果
Figure PCTCN2022138252-appb-000010
Figure PCTCN2022138252-appb-000011
Figure PCTCN2022138252-appb-000012
Figure PCTCN2022138252-appb-000013
Figure PCTCN2022138252-appb-000014
Figure PCTCN2022138252-appb-000015
Figure PCTCN2022138252-appb-000016
2.7样品测定
取全部26批样品，分别进行测定，各成分含量测定结果见表6。
表6各批次样品各成分含量测定结果列表
Figure PCTCN2022138252-appb-000017
Figure PCTCN2022138252-appb-000018
Figure PCTCN2022138252-appb-000019
Figure PCTCN2022138252-appb-000020
Figure PCTCN2022138252-appb-000021
Figure PCTCN2022138252-appb-000022
综上所述，本发明建立的UPLC/MS/MS同时测定导赤丸中24种成分的方法，专属性更强，控制成分更多，且对处方中不同药材进行全方位测定分析，药品质量进行了更全面的评估，同时也体现了便捷、高效、节约资源的检验检测工作理念。
需要说明的是，在本文中，诸如第一和第二等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来，而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者设备所固有的要素。
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。

Claims (4)

  1. 一种同时测定导赤丸中24种成分含量的方法，其特征在于，包括以下步骤：
    A.制备混合对照品溶液；
    B.制备供试品溶液：取导赤丸研细或剪碎，混匀，取约0.3g，加70％甲醇25ml，称定重量，密塞，超声处理30分钟，取出，放冷，用70％甲醇补足减失的重量，摇匀，滤过，取续滤液，经0.22μm微孔滤膜滤过，即得；
    C.分别精密吸取混合对照品溶液与供试品溶液各1μl，注入超高效液相色谱质谱联用仪，测定，外标法计算含量，即得；
    色谱条件：色谱柱：phenomenon C18柱2.6μm，2.1×100mm；以乙腈为流动相A，0.1％甲酸为流动相B，梯度洗脱；柱温：40℃，流速：0.3ml/min；进样量：1μl；
    质谱条件：电喷雾离子源ESI，扫描方式：正负离子同时扫描，监测模式：多反应监测模式MRM，正离子化电压：5500V，脱溶剂气温度：500℃，负离子化电压：-4500V，脱溶剂气温度：500℃。
  2. 根据权利要求1所述的一种同时测定导赤丸中24种成分含量的方法，其特征在于，所述混合对照品溶液由如下步骤配置而成：取各待测成分对照品，精称定，加70％甲醇分别制成每1ml含栀子苷60μg、连翘酯苷A 70μg、西红花苷Ⅰ4μg、西红花苷Ⅱ1μg、盐酸巴马汀7μg、表小檗碱3μg、盐酸黄连碱9μg、大黄酚2μg、黄芩素3μg、芍药苷14μg、哈巴苷4μg、盐酸药根碱6μg、汉黄芩素1μg、哈巴俄苷2μg、千层纸素A 1μg、木通苯乙醇苷B 1μg、连翘苷4μg、黄芩苷70μg、汉黄芩苷25μg、盐酸小檗碱22μg、大黄素甲醚1μg、芦荟大黄素1μg、大黄酸1μg、大黄素1μg的混合溶液，即得。
  3. 根据权利要求1所述的一种同时测定导赤丸中24种成分含量的方法，其特征在于，所述步骤B中超声处理：功率250W，频率40kHz。
  4. 根据权利要求1所述的一种同时测定导赤丸中24种成分含量的方法，其特征在于，梯度洗脱程序如下：
    流动相梯度洗脱程序
    Figure PCTCN2022138252-appb-100001
PCT/CN2022/138252 2022-07-04 2022-12-10 一种同时测定导赤丸中24种成分含量的方法 WO2024007531A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210779412.4 2022-07-04
CN202210779412.4A CN115876908A (zh) 2022-07-04 2022-07-04 一种同时测定导赤丸中24种成分含量的方法

Publications (1)

Publication Number Publication Date
WO2024007531A1 true WO2024007531A1 (zh) 2024-01-11

Family

ID=85769480

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/138252 WO2024007531A1 (zh) 2022-07-04 2022-12-10 一种同时测定导赤丸中24种成分含量的方法

Country Status (2)

Country Link
CN (1) CN115876908A (zh)
WO (1) WO2024007531A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117686622A (zh) * 2024-02-02 2024-03-12 江西中医药大学 八味齐薷散中多成分的分析方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102139008A (zh) * 2010-02-03 2011-08-03 天津中新药业集团股份有限公司隆顺榕制药厂 小儿导赤片的质量控制方法
CN103405664A (zh) * 2013-07-12 2013-11-27 康美药业股份有限公司 导赤散配方颗粒及其制备方法、用途和检测方法
CN110646537A (zh) * 2019-09-29 2020-01-03 陕西起源农业科技有限责任公司 一种基于hplc波长切换技术同时测定三黄片中多种成分含量的方法
CN115290767A (zh) * 2022-07-04 2022-11-04 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) 一种导赤丸中8种代表性成分的溶出度测定方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102139008A (zh) * 2010-02-03 2011-08-03 天津中新药业集团股份有限公司隆顺榕制药厂 小儿导赤片的质量控制方法
CN103405664A (zh) * 2013-07-12 2013-11-27 康美药业股份有限公司 导赤散配方颗粒及其制备方法、用途和检测方法
CN110646537A (zh) * 2019-09-29 2020-01-03 陕西起源农业科技有限责任公司 一种基于hplc波长切换技术同时测定三黄片中多种成分含量的方法
CN115290767A (zh) * 2022-07-04 2022-11-04 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) 一种导赤丸中8种代表性成分的溶出度测定方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LI LING-YUN; WANG YUN; LIU MENG-JIAO; ZHANG XUE; LI XIAO-QING; DAI YE-JIA; MA YIN-LIAN; ZHANG CUN: "Analysis of Chemical Components in Zhizi Jinhua Wan by UPLC-Q-TOF-MS/MS", CHINESE JOURNAL OF EXPERIMENTAL TRADITIONAL MEDICAL FORMULAE, ZHONGGUO ZHONGYI KEXUEYUAN ZHONGYAO YANJIUSUO, CN, vol. 23, no. 14, 20 July 2017 (2017-07-20), CN , pages 1 - 11, XP009551700, ISSN: 1005-9903 *
LU BINAN, LIU YUETAO, YIN LIANHONG, WANG XIAONA, PENG JINYONG: "Simple and reliable methods for the determination of sixteen marker components for quality control of Daochi pill by HPLC coupled with diode array detection", PHYTOCHEMICAL ANALYSIS, J. WILEY, HOBOKEN, USA, vol. 20, no. 5, 1 September 2009 (2009-09-01), Hoboken, USA, pages 385 - 394, XP093125676, ISSN: 0958-0344, DOI: 10.1002/pca.1138 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117686622A (zh) * 2024-02-02 2024-03-12 江西中医药大学 八味齐薷散中多成分的分析方法
CN117686622B (zh) * 2024-02-02 2024-05-14 江西中医药大学 八味齐薷散中多成分的分析方法

Also Published As

Publication number Publication date
CN115876908A (zh) 2023-03-31

Similar Documents

Publication Publication Date Title
Feng et al. Simultaneous determination of baicalin, baicalein, wogonin, berberine, palmatine and jatrorrhizine in rat plasma by liquid chromatography–tandem mass spectrometry and application in pharmacokinetic studies after oral administration of traditional Chinese medicinal preparations containing scutellaria–coptis herb couple
CN109307721B (zh) 一种hplc-qqq/ms法测定龙生蛭胶囊中有效成分的含量检测方法
WO2024007532A1 (zh) 一种导赤丸中8种代表性成分的溶出度测定方法
CN107703244B (zh) 一种中药组合物中14种化学成分含量的测定方法
WO2024007531A1 (zh) 一种同时测定导赤丸中24种成分含量的方法
CN110849983A (zh) 一种大鼠血浆中黄芪建中丸十二种成分的定量分析方法
CN109828062A (zh) 一种黄芪建中丸中十八种成分含量的测定方法
CN105181855A (zh) Uhplc-ms/ms同时测定防己黄芪汤煎剂中10种化学成分含量的方法
CN108152399B (zh) 一种柏子仁药材uplc特征图谱的构建及其检测方法
CN112505221A (zh) 基于uhplc-q-tof/ms识别消痰通腑方化学成分的分析方法
CN103424498B (zh) 一种清毒安肾胶囊指纹图谱的建立方法及其应用
CN111624295B (zh) 一种首荟通便胶囊质量检测方法
CN108776183B (zh) 大黄甘草汤的质量控制方法
CN111007190B (zh) 一种土贝母药材uplc特征图谱的构建方法及其成分含量测定方法
CN112946108A (zh) 一种大黄粉指纹图谱的检测方法及其应用
CN114113403B (zh) 荷丹片液质联用测定方法
CN105158397B (zh) 一种可同时评价补血类中药复方制剂四种原药材质量的方法
CN114994220B (zh) 一种七清败毒颗粒的指纹图谱的构建方法和其成分含量的测定方法及应用
CN110988198A (zh) 一种痹痛宁胶囊的含量测定方法
CN116298001A (zh) 一种荆银颗粒及其指纹图谱的检测方法与应用
Yang et al. Rapid analysis of differential chemical compositions of Poria cocos using thin-layer chromatography spray ionization-mass spectrometry
Guo et al. Quantitative determination of multi-class bioactive constituents for quality control of Yiqi Jiangzhi Granules
Yuan et al. Chemical profile Analysis of ling-gui-zhu-Gan decoction by LC–QTOF MS and simultaneous determination of nine major components using QAMS method
Zheng et al. Simultaneous determination of 14 components in Shuanghua Baihe tablets by ultrasonic-assisted extraction and HPLC-MS/MS
CN114563497A (zh) 一种小柴胡胶囊成分的定量指纹图谱检测方法

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: 22950088

Country of ref document: EP

Kind code of ref document: A1