WO2023116476A1 - 一种四季三黄片溶出度的检测方法 - Google Patents

一种四季三黄片溶出度的检测方法 Download PDF

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WO2023116476A1
WO2023116476A1 PCT/CN2022/138245 CN2022138245W WO2023116476A1 WO 2023116476 A1 WO2023116476 A1 WO 2023116476A1 CN 2022138245 W CN2022138245 W CN 2022138245W WO 2023116476 A1 WO2023116476 A1 WO 2023116476A1
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dissolution
sijisanhuang
solution
tablets
baicalin
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PCT/CN2022/138245
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English (en)
French (fr)
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刘永利
周亚楠
白洁
张元元
苏建
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河北省药品医疗器械检验研究院(河北省化妆品检验研究中心)
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Publication of WO2023116476A1 publication Critical patent/WO2023116476A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
    • G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N2030/042—Standards
    • G01N2030/047—Standards external

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  • the invention belongs to the technical field of detection of traditional Chinese medicines, in particular to a detection method for the dissolution rate of Sijisanhuang Tablets.
  • Drug dissolution refers to the dissolution rate and degree of drug components from solid preparations such as tablets, capsules or granules under specified conditions, and is an effective means to evaluate the quality and process level of solid preparations.
  • solid preparations such as tablets, capsules or granules under specified conditions
  • the object of the present invention is to address the above problems, providing a detection method for the dissolution rate of Sijisanhuang Tablets, using high performance liquid chromatography to measure the contents of geniposide, berberine hydrochloride, baicalin, and wogonin. Simple and accurate, it is suitable for the dissolution test of Sijisanhuang Tablets.
  • the present invention provides the following technical solutions: a method for detecting the dissolution of Sijisanhuang Tablets, comprising the following steps:
  • test solution adopt the paddle method, use the degassed aqueous solution as the dissolution medium, control the temperature of the dissolution apparatus at 37 ⁇ 0.5°C, and the speed of rotation is 120-150r/min, in 5min, 15min, 40min, Take 2mL of samples at 80min, 120min, 150min, 180min, and 240min, filter through the filter membrane immediately, and take the subsequent filtrate as the test solution;
  • Dissolution measurement accurately draw respectively 6 ⁇ l of mixed reference substance solution and need testing solution, inject into ultra-high performance liquid chromatograph, sample injection determination, external standard method to calculate content, to obtain final product;
  • Chromatographic conditions Chromatographic column: Waters HSS C18 (1.7 ⁇ m, 2.1 ⁇ 100mm), with acetonitrile as mobile phase A, 0.1% trifluoroacetic acid aqueous solution as mobile phase B, gradient elution; detection wavelength 254nm, column temperature: 30-40 °C, flow rate: 0.2-0.3ml ⁇ min -1 .
  • the filter membrane in the step (1) adopts a 0.22 ⁇ m microporous filter membrane.
  • the rotating speed in the step (1) is 120r/min.
  • the gradient elution program is 0-10min, 10%-15%A; 10-20min, 15%-30%A; 20-30min, 30%-42%A; 30-45min, 42%-75% A; 45-50 min, 10% A.
  • the column temperature is 30°C.
  • the flow rate is 0.3ml ⁇ min -1 .
  • the invention adopts a high-performance liquid chromatography method to measure the contents of geniposide, berberine hydrochloride, baicalin and wogonoside.
  • the method is easy to operate and has accurate results, and is suitable for detecting the dissolution rate of Sijisanhuang tablets.
  • Figures 1-6 are the dissolution HPLC graphs of geniposide reference substance, baicalin reference substance, berberine hydrochloride reference substance, wogonoside reference substance, Sijisanhuang Tablet sample and negative sample respectively;
  • Figures 7-10 are respectively the cumulative dissolution curves of geniposide from manufacturers A, B, C, and D;
  • Figures 11-14 are respectively the cumulative dissolution curves of manufacturers A, B, C, and D baicalin;
  • Figures 15-18 are respectively the cumulative dissolution curves of manufacturers A, B, C, and D berberine hydrochloride
  • Figures 19-22 are respectively the cumulative dissolution curves of wogonoside from manufacturers A, B, C, and D;
  • Figure 23 is a comparison chart of cumulative dissolution curves of geniposide from manufacturers A, B, C, and D;
  • Figure 24 is a comparison chart of cumulative dissolution curves of baicalin from manufacturers A, B, C, and D;
  • Figure 25 is a comparison chart of cumulative dissolution curves of berberine hydrochloride from manufacturers A, B, C, and D;
  • Figure 26 is a comparison chart of the cumulative dissolution curves of wogonoside from manufacturers A, B, C, and D.
  • RCZ-8M dissolution tester ultra-high performance liquid chromatography (PDA detector, American Thermo Company); XS105DU electronic analytical balance (Switzerland METTLER TOLEDO company); KQ-500DE numerical control ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.) , ultrapure water instrument (Millipore, USA).
  • Reagents acetonitrile (chromatographically pure, Merck, Germany), methanol (analytical pure, Tianjin Guangfu Technology Development Co., Ltd.), trifluoroacetic acid (chromatographically pure, Tianjin Chemical Co., Ltd.), and water was ultrapure water.
  • Sample information Four Seasons Three Yellow Tablets (manufacturer A batch number: 181145, 190908, 191240, 200302, specification: 0.29g/tablet, film-coated tablet; manufacturer B batch number: 190601, 190901, 191201, 200301, specification: 0.45g/tablet, film coated tablet; manufacturer C batch number: 20190501, 20190802, 20180801, 20190801, specification: 0.3g/tablet, sugar-coated tablet; manufacturer D batch number: 1000011, 1000009, 1000010, 1000013, specification: 0.25g/tablet, sugar-coated tablet)
  • Chromatographic column Shimadzu Shim-pack gist C18 (2.1 ⁇ 100mm, 2 ⁇ m); Acetonitrile is used as mobile phase A, 0.1% trifluoroacetic acid aqueous solution is mobile phase B, gradient elution program is shown in Table 1; detection wavelength is 254nm; column Temperature: 30°C, flow rate: 0.3ml ⁇ min -1 .
  • Reference Substance Solution Accurately weigh appropriate amount of reference substances of geniposide, berberine hydrochloride, baicalin and wogonoside respectively, and add methanol to make each 1ml contains about 20 ⁇ g of geniposide, 35 ⁇ g of baicalin, and 80 ⁇ g of berberine hydrochloride , a mixed solution of 35 ⁇ g of wogonoside, ready to be obtained.
  • Preparation of the test solution adopt the paddle method, use the degassed aqueous solution as the dissolution medium, control the temperature of the dissolution apparatus at (37 ⁇ 0.5)°C, and the rotation speed is 120r/min, and sample 2mL at 150min (after each sampling, at the same time Add the same amount of dissolution medium at the same temperature), immediately filter through a 0.22 ⁇ m microporous membrane, take the subsequent filtrate as the test solution, and determine the contents of geniposide, baicalin, berberine hydrochloride, and wogonin according to the law.
  • the selection of rotating speed adopts the preparation method of need testing solution, takes Sijisanhuang tablet (lot number: 181145) of manufacturer A as research object, has investigated the dissolution situation that rotating speed is 75, 100 and 120r/min respectively, and the result shows that when 120r/min The dissolution rate of the four ingredients in Sijisanhuang Tablets is good, as shown in Table 1, so 120r/min was chosen for the dissolution study of Sijisanhuang Tablets.
  • Repeatability test takes manufacturer A Sijisanhuang Tablets (batch number: 181145) as the research object, takes 6 tablets in parallel, adopts the preparation method of the test sample, and measures them.
  • the average contents were 0.926mg, 3.761mg, 0.898mg, 0.686mg respectively, and the RSDs were 1.5%, 1.7%, 2.5%, 2.0%. It shows that the method repeatability of each component to be tested is good.
  • the dissolution equilibrium of geniposide, baicalin, berberine hydrochloride, and wogonoside all reached the dissolution equilibrium in about 40 minutes;
  • the cumulative dissolution rate of baicalin, berberine hydrochloride, and wogonin was greater than 80%, reaching the dissolution equilibrium; while the cumulative dissolution rate of manufacturer D was still less than 50% at 80 minutes, and the dissolution was slow.
  • the cumulative dissolution rates of glycosides, berberine hydrochloride, and wogonoside were about 90%, and there was a tendency to continue to increase.
  • a method for measuring the dissolution rate of Sijisanhuang Tablets adopts the paddle method, uses water as the dissolution medium, and controls the speed of dissolution, so that the active ingredients in Sijisanhuang Tablets can be better Dissolution.
  • the method is accurate, reliable, and reproducible, and is suitable for the dissolution testing of Sijisanhuang Tablets.
  • Chromatographic conditions Chromatographic column: Waters HSS C18 (1.7 ⁇ m, 2.1 ⁇ 100 mm), with acetonitrile as mobile phase A and 0.1% trifluoroacetic acid aqueous solution as mobile phase B, gradient elution.
  • the gradient elution program is 0 ⁇ 10min, 10% ⁇ 15%A; 10 ⁇ 20min, 15% ⁇ 30%A; 20 ⁇ 30min, 30% ⁇ 42%A; 30 ⁇ 45min, 42% ⁇ 75%A; 45 ⁇ 50min, 10%A.
  • the detection wavelength is 254nm, the column temperature: 30°C, and the flow rate: 0.3ml ⁇ min -1 .
  • Preparation of reference solution Accurately weigh appropriate amount of reference substances of geniposide, baicalin, berberine hydrochloride, and wogonoside respectively, and add methanol to make each 1ml contains about 20 ⁇ g of geniposide, 35 ⁇ g of baicalin, and berberine hydrochloride. A mixed solution of 80 ⁇ g and 35 ⁇ g of wogonoside was obtained.
  • Preparation of the test solution adopt the paddle method, use the degassed aqueous solution as the dissolution medium, control the temperature of the dissolution apparatus at (37 ⁇ 0.5)°C, and the rotation speed is 120r/min, take a sample of 2mL at 120min, and immediately pass through a 0.22 ⁇ m micrometer The pore filter membrane was filtered, and the subsequent filtrate was taken as the test solution, and the contents of geniposide, baicalin, berberine hydrochloride, and wogonoside in Sijisanhuang Tablets were determined according to the law.
  • Determination method Precisely draw and mix 6 ⁇ l of the reference substance solution and the test solution respectively, inject into the ultra-high performance liquid chromatograph, inject the sample for determination, calculate the content by the external standard method, and obtain it.
  • Chromatographic conditions Chromatographic column: Waters HSS C18 (1.7 ⁇ m, 2.1 ⁇ 100 mm), with acetonitrile as mobile phase A and 0.1% trifluoroacetic acid aqueous solution as mobile phase B, gradient elution.
  • the gradient elution program is 0 ⁇ 10min, 10% ⁇ 15%A; 10 ⁇ 20min, 15% ⁇ 30%A; 20 ⁇ 30min, 30% ⁇ 42%A; 30 ⁇ 45min, 42% ⁇ 75%A; 45 ⁇ 50min, 10%A.
  • the detection wavelength is 254nm, the column temperature: 30°C, and the flow rate: 0.3ml ⁇ min -1 .
  • Preparation of reference solution Accurately weigh appropriate amount of reference substances of geniposide, baicalin, berberine hydrochloride, and wogonoside respectively, and add methanol to make each 1ml contains about 20 ⁇ g of geniposide, 35 ⁇ g of baicalin, and berberine hydrochloride. A mixed solution of 80 ⁇ g and 35 ⁇ g of wogonoside was obtained.
  • Preparation of the test solution adopt the paddle method, use the degassed aqueous solution as the dissolution medium, control the temperature of the dissolution apparatus at (37 ⁇ 0.5)°C, and the rotation speed is 120r/min, take a sample of 2mL at 120min, and immediately pass through a 0.22 ⁇ m micrometer The pore filter membrane was filtered, and the subsequent filtrate was taken as the test solution, and the contents of geniposide, baicalin, berberine hydrochloride, and wogonoside in Sijisanhuang Tablets were determined according to the law.
  • Determination method Accurately draw and mix 5 ⁇ l of the reference substance solution and the test solution respectively, inject into the ultra-high performance liquid chromatograph, inject the sample for determination, calculate the content by the external standard method, and obtain it.
  • Chromatographic conditions Chromatographic column: Shimadzu Shim-pack gist C 18 (2.1 ⁇ 100mm, 2 ⁇ m), with acetonitrile as mobile phase A and 0.2% trifluoroacetic acid aqueous solution as mobile phase B, gradient elution.
  • the gradient elution program is 0 ⁇ 10min, 10% ⁇ 15%A; 10 ⁇ 20min, 15% ⁇ 30%A; 20 ⁇ 30min, 30% ⁇ 42%A; 30 ⁇ 45min, 42% ⁇ 75%A; 45 ⁇ 50min, 10%A.
  • the detection wavelength is 254nm, the column temperature: 30°C, and the flow rate: 0.3ml ⁇ min -1 .
  • Preparation of reference solution Accurately weigh appropriate amount of reference substances of geniposide, baicalin, berberine hydrochloride, and wogonoside respectively, and add methanol to make each 1ml contains about 20 ⁇ g of geniposide, 35 ⁇ g of baicalin, and berberine hydrochloride. A mixed solution of 80 ⁇ g and 35 ⁇ g of wogonoside was obtained.
  • Preparation of the test solution adopt the paddle method, use the degassed aqueous solution as the dissolution medium, control the temperature of the dissolution apparatus at (37 ⁇ 0.5)°C, and the rotation speed is 120r/min, take a sample of 2mL at 120min, and immediately pass through a 0.22 ⁇ m micrometer The pore filter membrane was filtered, and the subsequent filtrate was taken as the test solution, and the contents of geniposide, baicalin, berberine hydrochloride, and wogonoside in Sijisanhuang Tablets were determined according to the law.
  • Determination method Precisely draw and mix 6 ⁇ l of the reference substance solution and the test solution respectively, inject into the ultra-high performance liquid chromatograph, inject the sample for determination, calculate the content by the external standard method, and obtain.

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Abstract

一种四季三黄片溶出度的检测方法，涉及中药检测技术领域。包括以下步骤：(1)供试品溶液的制备；(2)对照品溶液的制备；(3)溶出度测定；色谱条件：色谱柱：Waters HSS C18(1.7μm，2.1×100mm)，以乙腈为流动相A，0.1％三氟乙酸水溶液为流动相B，梯度洗脱；检测波长254nm，柱温：30-40℃，流速：0.2-0.3ml·min -1。采用高效液相色谱方法测定了栀子苷、盐酸小檗碱、黄芩苷、汉黄芩苷的含量，方法操作简便、结果准确，适用于四季三黄片溶出度的检测。

Description

一种四季三黄片溶出度的检测方法 技术领域
本发明属于中药检测技术领域，具体是一种四季三黄片溶出度的检测方法。
背景技术
四季三黄片由大黄、黄芩、黄柏、栀子四中味药组成，这四味中药均为临床常用的清热解毒药物。方中大黄泻下攻积、清热泻火；黄芩清热燥湿、泻火解毒；黄柏清热解毒、清热燥湿，三者共为君药。栀子泻火除烦、凉血解毒，为佐药。诸药相合，共奏消炎退热、通便利水之功效。目前该药的质量标准收录于《卫生部药品标准》中药成方制剂第二十册。
药物溶出度是指药物中成分从片剂、胶囊剂或颗粒剂等固体制剂在规定条件下溶出的速率和程度，是评价固体制剂品质和工艺水平的一种有效手段。鉴于中药的复杂因素如流浸膏或有效部位或生药投料，复杂基质下多成分共同作用难以用某一种成分作为溶出指标，溶出和测定方法也难以建立，因此，对中药固体制剂进行溶出度研究较少。四季三黄片自1998年批准上市以来，一直没有人做过溶出度研究。
本研究以四季三黄片中主要有效成分栀子苷、盐酸小檗碱、黄芩苷、汉黄芩苷为指标，建立了超高效液相色谱含量分析方法，对市场上4个厂家的产品进行体外溶出研究，绘制了累积溶出曲线，考察不同厂家四季三黄片的溶出度情况，为本品药效的评价提供参考。
发明内容
本发明的目的是针对以上问题，提供了一种四季三黄片溶出度的检测方法，采用高效液相色谱方法测定了栀子苷、盐酸小檗碱、黄芩苷、汉黄芩苷的含量，方法操作简便、结果准确，适用于四季三黄片溶出度的检测。
为实现上述目的，本发明提供如下技术方案：一种四季三黄片溶出度的检测方法，包括以下步骤：
(1)供试品溶液的制备：采用桨法，以经脱气处理的水溶液为溶出介质，控制溶出仪温度在37±0.5℃，转速为120-150r/min，于5min、15min、40min、80min、120min、150min、180min、240min取样2mL，立即经滤膜过滤，取续滤液作为供试品溶液；
(2)对照品溶液的制备：分别精密称取栀子苷、黄芩苷、盐酸小檗碱、汉黄芩苷对照品适量，加甲醇制成每1ml含栀子苷约20μg、黄芩苷35μg、盐酸小檗碱80μg、汉黄芩苷35μg的混合溶液，即得；
(3)溶出度测定：分别精密吸取混合对照品溶液与供试品溶液各6μl，注入超高效液相色谱仪，进样测定，外标法计算含量，即得；
色谱条件：色谱柱：Waters HSS C18(1.7μm，2.1×100mm)，以乙腈为流动相A，0.1％三氟乙酸水溶液为流动相B，梯度洗脱；检测波长254nm，柱温：30-40℃，流速：0.2-0.3ml·min -1。
优选的，所述步骤(1)中的滤膜采用0.22μm的微孔滤膜。
优选的，所述步骤(1)中的转速为120r/min。
优选的，梯度洗脱程序为0～10min，10％～15％A；10～20min，15％～30％A；20～30min，30％～42％A；30～45min，42％～75％A；45～50min，10％A。
优选的，所述柱温为30℃。
优选的，所述流速为0.3ml·min -1。
与现有技术相比，本发明的有益效果如下：
本发明采用高效液相色谱方法测定了栀子苷、盐酸小檗碱、黄芩苷、汉黄芩苷的含量，方法操作简便、结果准确，适用于四季三黄片溶出度的检测。
附图说明
图1-6分别为栀子苷对照品、黄芩苷对照品、盐酸小檗碱对照品、汉黄芩苷对照品、四季三黄片样品、阴性样品的溶出度HPLC图；
图7-10分别为厂家A、B、C、D栀子苷的累积溶出度曲线图；
图11-14分别为厂家A、B、C、D黄岑苷的累积溶出度曲线图；
图15-18分别为厂家A、B、C、D盐酸小檗碱的累积溶出度曲线图；
图19-22分别为厂家A、B、C、D汉黄芩苷的累积溶出度曲线图；
图23为厂家A、B、C、D栀子苷的累积溶出度曲线对比图；
图24为厂家A、B、C、D黄岑苷的累积溶出度曲线对比图；
图25为厂家A、B、C、D盐酸小檗碱的累积溶出度曲线对比图；
图26为厂家A、B、C、D汉黄芩苷的累积溶出度曲线对比图。
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。
1仪器与试药
1.1仪器
RCZ-8M溶出试验仪、超高效液相色谱仪(PDA检测器，美国Thermo公司)；XS105DU电子分析天平(瑞士METTLER TOLEDO公司)；KQ-500DE型数控超声波清洗器(昆山市超声仪器有限公司)，超纯水仪(美国Millipore公司)。
1.2试药
对照品：栀子苷(批号：110749-201919，含量以97.1％计)、黄芩苷(批号：110715-201821，95.4％)、盐酸小檗碱(批号：110713-201212，86.8％)、 汉黄芩苷(批号：112002-201702，98.5％)，均购自中国食品药品检定研究院。
试剂：乙腈(色谱纯，Merck，Germany)，甲醇(分析纯，天津市光复科技发展有限公司)三氟乙酸(色谱纯，天津化学有限公司)，水为超纯水。
样品信息：四季三黄片(厂家A批号：181145、190908、191240、200302，规格：0.29g/片，薄膜衣片；厂家B批号：190601、190901、191201、200301，规格：0.45g/片，薄膜衣片；厂家C批号：20190501、20190802、20180801、20190801，规格：0.3g/片，糖衣片；厂家D批号：1000011、1000009、1000010、1000013，规格：0.25g/片，糖衣片)
2.方法与结果
2.1色谱条件
色谱柱：Shimadzu Shim-pack gist C18(2.1×100mm，2μm)；以乙腈为流动相A，0.1％的三氟乙酸水溶液为流动相B，梯度洗脱程序见表1；检测波长为254nm；柱温：30℃，流速：0.3ml·min -1。
表1流动相梯度洗脱程序
Figure PCTCN2022138245-appb-000001
2.2溶液的制备
对照品溶液的制备分别精密称取栀子苷、盐酸小檗碱、黄芩苷、汉黄芩苷对照品适量，加甲醇制成每1ml含栀子苷约20μg、黄芩苷35μg、盐酸小檗碱80μg、汉黄芩苷35μg的混合溶液，即得。
供试品溶液的制备：采用桨法，以经脱气处理的水溶液为溶出介质，控 制溶出仪温度在(37±0.5)℃，转速为120r/min，于150min取样2mL(每次取样后同时补加同温等量的溶出介质)，立即经0.22μm微孔滤膜过滤，取续滤液作为供试品溶液，依法测定栀子苷、黄芩苷、盐酸小檗碱、汉黄芩苷含量。
2.3专属性考察
按处方比例和制法制备不含大黄的阴性样品，按供试品溶液的制备方法制备阴性样品溶液，测定，并在二极管阵列检测器上分析各色谱峰纯度。结果表明，阴性样品不干扰栀子苷、黄芩苷、盐酸小檗碱、汉黄芩苷的测定，色谱图见图1-6。
2.4转速的选择及取样时间的考察
转速的选择采用供试品溶液的制备方法，以厂家A的四季三黄片(批号：181145)为研究对象，分别考察了转速为75、100和120r/min的溶出情况，结果表明120r/min时四季三黄片中4种成分的溶出较好，见表1，因此选择120r/min来进行四季三黄片的溶出度研究。
表1 不同转速的溶出度结果(mg)
Figure PCTCN2022138245-appb-000002
取样时间的考察分别以4批次四季三黄片(厂家A批号：181145；厂家B批号：190601；厂家C批号：20190501；厂家D批号：1000011)为研究对象，考察了5、15、40、80、120、150、180、240min的溶出度结果，结果样品基本在120分钟时达到溶出平衡，故选择120分钟为溶出时间。
表2 不同企业不同取样时间的溶出度结果(mg)
Figure PCTCN2022138245-appb-000003
Figure PCTCN2022138245-appb-000004
2.5方法的重复性考察
重复性试验以厂家A四季三黄片(批号：181145)为研究对象，平行取 6片，采用供试品制备方法，进行测定，样品中栀子苷、黄芩苷、盐酸小檗碱、汉黄芩苷平均含量分别为0.926mg、3.761mg、0.898mg、0.686mg，RSD分别为1.5％、1.7％、2.5％、2.0％。表明各待测成分方法重复性良好。
2.6不同企业溶出度考察
按《中国药典》2020年版四部通则“溶出度与释放度测定法”项下第二法桨法测定，选取4家生产企业各1个批次，每批次试验6片，以200mL经脱气处理的水溶液为溶出介质，温度保持在(37±0.5)℃，转速120r/min。桨降入容器中，立即开始计时，分别于5、15、40、80、120、150min(厂家D考察到180min)时取样2mL(同时补加同温等量的溶出介质)，用0.45μm微孔滤膜滤过，取续滤液，进样测定栀子苷、黄芩苷、盐酸小檗碱、汉黄芩苷的含量，以不同时间栀子苷、黄芩苷、盐酸小檗碱、汉黄芩苷溶出量除以溶出样品中的含量计算不同时刻上述4种成份的累积溶出度，并以时间为横坐标，累积溶出度为纵坐标绘制累积溶出曲线。结果见图7-22。结果表明四个厂家产品批内均一性良好，重复性良好。
2.7不同企业溶出曲线测定
取不同企业(厂家A、B、C、D)四季三黄片各3个批次，每个批次1片，采用上述供试品制备方法，测定栀子苷、黄芩苷、盐酸小檗碱、汉黄芩苷的含量，并计算平均含量。以不同时间4种成份的平均溶出量除以溶出样品中4种成份含量计算不同时刻四季三黄片中栀子苷、黄芩苷等的累积溶出度，并以时间为横坐标，累积溶出度为纵坐标绘制不同企业同一成分累积溶出曲线，结果见图23-26。表明不同厂家同一成份的溶出行为具有明显的差异性。厂家A与厂家B的的溶出曲线相似，溶出速率较快，栀子苷、黄芩苷、盐酸小檗碱、汉黄芩苷均在40min左右已达到溶出平衡；厂家C在80min时，栀子苷、黄芩苷、盐酸小檗碱、汉黄芩苷累积溶出度已大于80％，达到溶出平衡；而厂家D在80min时，累积溶出度仍小于50％，溶出较慢，180min 时，栀子苷、黄芩苷、盐酸小檗碱、汉黄芩苷的累积溶出度在90％左右，且有继续增加的趋势。
综上所述，本发明所提供的一种四季三黄片溶出度的测定方法，采用桨法，以水为溶出介质，通过控制溶出时的转速，可以将四季三黄片中的有效成分较好的溶出。该方法准确、可靠，重现性好，适合用于四季三黄片溶出度的检测。
实施例1
色谱条件：色谱柱：Waters HSS C18(1.7μm，2.1×100mm)，以乙腈为流动相A，0.1％三氟乙酸水溶液为流动相B，梯度洗脱。梯度洗脱程序为0～10min，10％～15％A；10～20min，15％～30％A；20～30min，30％～42％A；30～45min，42％～75％A；45～50min，10％A。检测波长254nm，柱温：30℃，流速：0.3ml·min -1。
对照品溶液的制备：分别精密称取栀子苷、黄芩苷、盐酸小檗碱、汉黄芩苷对照品适量，加甲醇制成每1ml含栀子苷约20μg、黄芩苷35μg、盐酸小檗碱80μg、汉黄芩苷35μg的混合溶液，即得。
供试品溶液的制备：采用桨法，以经脱气处理的水溶液为溶出介质，控制溶出仪温度在(37±0.5)℃，转速为120r/min，于120min取样2mL，立即经0.22μm微孔滤膜过滤，取续滤液作为供试品溶液，依法测定四季三黄片中栀子苷、黄芩苷、盐酸小檗碱、汉黄芩苷含量。
测定法：分别精密吸取混合对照品溶液与供试品溶液各6μl，注入超高效液相色谱仪，进样测定，外标法计算含量，即得。
实施例2
色谱条件：色谱柱：Waters HSS C18(1.7μm，2.1×100mm)，以乙腈为流动相A，0.1％三氟乙酸水溶液为流动相B，梯度洗脱。梯度洗脱程序为0～10min，10％～15％A；10～20min，15％～30％A；20～30min，30％～42％ A；30～45min，42％～75％A；45～50min，10％A。检测波长254nm，柱温：30℃，流速：0.3ml·min -1。
对照品溶液的制备：分别精密称取栀子苷、黄芩苷、盐酸小檗碱、汉黄芩苷对照品适量，加甲醇制成每1ml含栀子苷约20μg、黄芩苷35μg、盐酸小檗碱80μg、汉黄芩苷35μg的混合溶液，即得。
供试品溶液的制备：采用桨法，以经脱气处理的水溶液为溶出介质，控制溶出仪温度在(37±0.5)℃，转速为120r/min，于120min取样2mL，立即经0.22μm微孔滤膜过滤，取续滤液作为供试品溶液，依法测定四季三黄片中栀子苷、黄芩苷、盐酸小檗碱、汉黄芩苷含量。
测定法：分别精密吸取混合对照品溶液与供试品溶液各5μl，注入超高效液相色谱仪，进样测定，外标法计算含量，即得。
实施例3
色谱条件：色谱柱：Shimadzu Shim-pack gist C 18(2.1×100mm，2μm)，以乙腈为流动相A，0.2％三氟乙酸水溶液为流动相B，梯度洗脱。梯度洗脱程序为0～10min，10％～15％A；10～20min，15％～30％A；20～30min，30％～42％A；30～45min，42％～75％A；45～50min，10％A。检测波长254nm，柱温：30℃，流速：0.3ml·min -1。
对照品溶液的制备：分别精密称取栀子苷、黄芩苷、盐酸小檗碱、汉黄芩苷对照品适量，加甲醇制成每1ml含栀子苷约20μg、黄芩苷35μg、盐酸小檗碱80μg、汉黄芩苷35μg的混合溶液，即得。
供试品溶液的制备：采用桨法，以经脱气处理的水溶液为溶出介质，控制溶出仪温度在(37±0.5)℃，转速为120r/min，于120min取样2mL，立即经0.22μm微孔滤膜过滤，取续滤液作为供试品溶液，依法测定四季三黄片中栀子苷、黄芩苷、盐酸小檗碱、汉黄芩苷含量。
测定法：分别精密吸取混合对照品溶液与供试品溶液各6μl，注入超高效 液相色谱仪，进样测定，外标法计算含量，即得。
3个实施例栀子苷、黄芩苷、盐酸小檗碱、汉黄芩苷含量结果(mg)如下表：
  栀子苷 黄芩苷 盐酸小檗碱 汉黄芩苷
实施例1 0.925 3.754 0.898 0.688
实施例2 0.922 3.766 0.889 0.681
实施例3 0.931 3.747 0.901 0.690
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 (6)

  1. 一种四季三黄片溶出度的检测方法，其特征在于，包括以下步骤：
    (1)供试品溶液的制备：采用桨法，以经脱气处理的水溶液为溶出介质，控制溶出仪温度在37±0.5℃，转速为120-150r/min，于5min、15min、40min、80min、120min、150min、180min、240min取样2mL，立即经滤膜过滤，取续滤液作为供试品溶液；
    (2)对照品溶液的制备：分别精密称取栀子苷、黄芩苷、盐酸小檗碱、汉黄芩苷对照品适量，加甲醇制成每1ml含栀子苷约20μg、黄芩苷35μg、盐酸小檗碱80μg、汉黄芩苷35μg的混合溶液，即得；
    (3)溶出度测定：分别精密吸取混合对照品溶液与供试品溶液各6μl，注入超高效液相色谱仪，进样测定，外标法计算含量，即得；
    色谱条件：色谱柱：Waters HSS C18(1.7μm，2.1×100mm)，以乙腈为流动相A，0.1％三氟乙酸水溶液为流动相B，梯度洗脱；检测波长254nm，柱温：30-40℃，流速：0.2-0.3ml·min -1。
  2. 根据权利要求1所述的一种四季三黄片溶出度的检测方法，其特征在于，所述步骤(1)中的滤膜采用0.22μm的微孔滤膜。
  3. 根据权利要求1所述的一种四季三黄片溶出度的检测方法，其特征在于，所述步骤(1)中的转速为120r/min。
  4. 根据权利要求1所述的一种四季三黄片溶出度的检测方法，其特征在于，梯度洗脱程序为0～10min，10％～15％A；10～20min，15％～30％A；20～30min，30％～42％A；30～45min，42％～75％A；45～50min，10％A。
  5. 根据权利要求1所述的一种四季三黄片溶出度的检测方法，其特征在于，所述柱温为30℃。
  6. 根据权利要求1所述的一种四季三黄片溶出度的检测方法，其特征在于，所述流速为0.3ml·min -1。
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