WO2024007538A1 - Content measurement method for six alkaloid components in small meridian-activating pill - Google Patents
Content measurement method for six alkaloid components in small meridian-activating pill Download PDFInfo
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- WO2024007538A1 WO2024007538A1 PCT/CN2022/139273 CN2022139273W WO2024007538A1 WO 2024007538 A1 WO2024007538 A1 WO 2024007538A1 CN 2022139273 W CN2022139273 W CN 2022139273W WO 2024007538 A1 WO2024007538 A1 WO 2024007538A1
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- 238000000691 measurement method Methods 0.000 title abstract 2
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the invention belongs to the technical field of traditional Chinese medicine detection, and is specifically a method for determining the content of six alkaloid components in Xiaohuoluo Pills.
- Xiaohuoluo Pill is a traditional Chinese patent medicine, formerly known as Huoluo Dan, also known as Xiaohuoluo Dan.
- Huoluo Dan also known as Xiaohuoluo Dan.
- the 1977 edition of "Chinese Pharmacopoeia” was revised to "Xiaohuoluo Pills", and subsequent editions of the Pharmacopoeia were all recorded as "Xiaohuoluo Pills”.
- Xiaohuoluo Pill is composed of six traditional Chinese medicines: Dannanxing, Zhichuanwu, Zhicaowu, Dilong, Frankincense (made), and Myrrh (made).
- symptoms include pain in limb joints, or cold pain, or tingling, or pain that is severe at night, difficulty in joint flexion and extension, numbness and spasm, etc.
- Zhichuanwu and Zhicaowu warm the meridians, dispel cold, expel wind and dampness, relieve numbness and relieve pain, and are the king medicines;
- Dannanxing removes dampness and resolves phlegm, removes wind, phlegm and dampness in the meridians, and can relieve pain, and is the prescription.
- the Aconite Sichuan and Aconite Radix in the prescription are traditional Chinese medicines from the Ranunculaceae family.
- the diterpenoid diester alkaloids (MA, HA, AC) contained in them are highly toxic, mainly causing damage to the nervous and cardiovascular systems. Serious injuries. When Aconitum plants are used as clinical medicines, poisoning or even death often occurs due to improper processing or medication. After processing, these medicinal materials can be hydrolyzed into the corresponding diterpene monoester alkaloids (BMA, BAC, BHA), which have active activity, reduced toxicity, and improved therapeutic index. They are often used in clinical applications as the main medicinal ingredients. In order to reduce raw material quality risks, ensure efficacy and prevent low-limit feeding, it is necessary to control the content of finished products.
- the 2020 version of the "Chinese Pharmacopoeia" quality standard for Xiaohuoluo Pills only contains the TLC limit inspection item for aconitine. It is difficult to fully control the quality of the toxic medicinal flavor in the prescription, and the control limit for the limit of aconitine is relatively high. May cause risk of poisoning.
- the present invention provides a method for determining the content of 6 alkaloid components in Xiaohuoluo Pills, which includes the following steps:
- test solution Take an appropriate amount of small Huoluo pills, cut into pieces, take 3g, weigh it, place it in a stoppered Erlenmeyer flask, add 25ml of 0.2mol/L hydrochloric acid solution, stopper it tightly, weigh it, and conduct ultrasonic treatment for 30 minutes, let cool, weigh again, use 0.2mol/L hydrochloric acid solution to make up for the lost weight, shake well, centrifuge for 20 minutes, measure 15ml of the additional filtrate, place it on the processed solid phase extraction column, and in turn use 0.05 Elute with 10 ml each of mol/L hydrochloric acid solution and acetonitrile. Discard the eluent and leave it for 5 minutes.
- the preparation method of the mixed reference solution is: taking benzoyl aconitine, benzoyl aconitine, benzoyl aconitine reference substance and aconitine diester alkaloids Weigh the control extract accurately, add acetonitrile to make a mixed solution containing 300 ⁇ g, 100 ⁇ g, 100 ⁇ g, and 300ug per 1 ml respectively.
- As the reference substance stock solution measure 5 ml of the reference substance stock solution and place it in a 50 ml measuring bottle. Add the volume ratio Dilute 90:10 acetonitrile-concentrated ammonia solution to the mark, shake well, and it is ready.
- the ultrasonic treatment in step B power 400W, frequency 40kHz, water temperature below 40°C.
- the centrifugal speed in step B is 6000 rpm.
- the solid-phase extraction column in step B is a solid-phase extraction column filled with a mixed cation exchange reversed-phase adsorbent, 500 mg, 6 ml, and is eluted with 6 ml of acetonitrile and 6 ml of water in sequence before use.
- a mixed cation exchange reversed-phase adsorbent 500 mg, 6 ml, and is eluted with 6 ml of acetonitrile and 6 ml of water in sequence before use.
- the chromatographic conditions are:
- Mobile phase A methanol
- mobile phase B acetonitrile
- mobile phase C 0.1% phosphoric acid
- the gradient elution procedure is as follows:
- the HPLC method of the present invention has the advantages of high efficiency, sensitivity, easy operation, and high accuracy.
- the present invention establishes an HPLC method for simultaneous determination of the contents of six alkaloids in Xiaohuoluo pills.
- the method is simple, highly specific, and can simultaneously measure single
- the control of ester and diester alkaloids is more conducive to the quality control of Aconitum medicinal flavor, ensuring the safety and effectiveness of clinical medication, and providing a reference for product quality control and quality evaluation.
- Figure 1 is the chromatogram of the mixed reference solution, including: 1-benzoylaconitine; 2-benzoylaconitine; 3-benzoylaconitine; 4-benzoylaconitine Aconitine; 5-aconitine; 6-aconitine;
- Figure 2 is the chromatogram of the test solution, including: 1-benzoyl aconitine; 2-benzoyl aconitine; 3-benzoyl aconitine; 4-benzoyl aconitine Alkali; 5-aconitine; 6-aconitine;
- Figure 3 is the chromatogram of the negative sample solution.
- any reference to “one embodiment” means that the specific features, structures or parameters, steps, etc. described in the embodiment are included in at least one embodiment according to the present invention. Therefore, in the description of the present invention, if terms such as “according to one embodiment of the present invention” and “in an embodiment” are used, they are not used to specifically refer to the same embodiment. If terms such as “in an embodiment” are used, Terms such as “in other embodiments”, “according to different embodiments of the present invention”, “according to other embodiments of the present invention” are not used to specifically indicate that the mentioned features can only be included in specific different embodiments. . Those skilled in the art should understand that each specific feature, structure or parameter, step, etc. disclosed in one or more embodiments of the present invention may be combined in any suitable manner.
- the object of the present invention is to provide a method for simultaneously determining the content of six alkaloid components in Xiaohuoluo Pills.
- the present invention uses HPLC method to determine benzoyl aconitine (BMA), benzoyl aconitine aconitine (BAC), benzoyl aconitine (BHA), aconitine (MA), aconitine (HA) and aconitine (AC) content.
- the content determination method of the present invention adopts HPLC method, and the method is as follows:
- Chromatographic conditions Chromatographic column: PICKERING C18 column (4.6 ⁇ 250mm, 5 ⁇ m); use: methanol as mobile phase A, acetonitrile as mobile phase B, 0.1% phosphoric acid as mobile phase C, the gradient elution procedure is shown in the table below; column temperature :30°C, flow rate: 1ml/min; detection wavelength: 232nm; injection volume: 15 ⁇ l.
- Preparation of mixed reference solution Take appropriate amounts of benzoyl aconitine, benzoyl aconitine, benzoyl aconitine reference substance and aconitine diester alkaloid control extract, and weigh them accurately. Determine, add acetonitrile to make a mixed solution containing 300 ⁇ g, 100 ⁇ g, 100 ⁇ g, and 300ug per 1 ml respectively.
- As the reference substance stock solution accurately measure 5 ml of the reference substance stock solution and place it in a 50 ml measuring bottle.
- test solution Take an appropriate amount of this product, cut into pieces, take about 3g, weigh it accurately, place it in a stoppered conical flask, add 25ml of 0.2mol/L hydrochloric acid solution accurately, stopper it tightly, weigh it, and ultrasonicate it.
- Assay method Precisely draw 15 ⁇ l each of the mixed reference solution and the test solution, inject them into the liquid chromatograph, measure, and calculate the content using the external standard method.
- High performance liquid chromatograph equipped with quaternary pump, DAD detector, Waters e2695; analytical balance: Mettler XPE26 (parts per million) (Shanghai Mettler Instrument Co., Ltd.); Mettler XS105 (parts per hundred thousand) (Shanghai Mettler Instrument Co., Ltd.); KQ-400KDE ultrasonic cleaning instrument (Kunshan Ultrasonic Instrument Co., Ltd.); ultrapure water instrument (American Millipore Company).
- Aconitine diester alkaloid control extract (batch number: 112029-201601, for content determination, content is based on neo-aconitine 31.7%, hypo-aconitine 30.0%, aconitine 31.8%)
- Benzoylaconitine (batch number: 111794-201705, content based on 99.1%)
- Reagents methanol, acetonitrile (chromatographically pure, Merck, Germany), water is ultrapure water; other reagents are of analytical grade.
- Chromatographic column PICKERING C18 column (4.6 ⁇ 250mm, 5 ⁇ m); use: methanol as mobile phase A, acetonitrile as mobile phase B, 0.1% phosphoric acid as mobile phase C, the gradient elution program is shown in Table 2; column temperature: 30°C , flow rate: 1ml/min; detection wavelength: 232nm; injection volume: 15 ⁇ l.
- the method of the present invention is simple, highly specific, and simultaneously controls monoester and diester alkaloids, which is more conducive to the quality control of Aconitum medicinal flavor and ensures the safety and effectiveness of clinical medication. , which can provide reference for product quality control and quality evaluation.
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Abstract
The present invention relates to the technical field of traditional Chinese medicine detection, and specifically relates to a content measurement method for six alkaloid components in a small meridian-activating pill, comprising: preparing a mixed reference solution; preparing a test solution; respectively and precisely suctioning 15 μl of the mixed reference solution and 15 μl of the test solution, injecting the mixed reference solution and the test solution into a liquid chromatograph, measuring, and calculating the content by means of an external standard method. In the present invention, an HPLC method is established for simultaneously measuring the content of six alkaloid components in the small meridian-activating pill, the method is simple and convenient, and has high specificity, and monoester and diester alkaloids are controlled at the same time, such that quality control of herbal medicine Aconitum is facilitated, the safety and effectiveness of clinical medication are ensured, and reference is provided for quality control and quality evaluation of products.
Description
本发明属于中药检测技术领域,具体是一种小活络丸中6种生物碱成分的含量测定方法。The invention belongs to the technical field of traditional Chinese medicine detection, and is specifically a method for determining the content of six alkaloid components in Xiaohuoluo Pills.
小活络丸为传统中成药,原名活络丹,又称小活络丹。1977年版《中国药典》修改为“小活络丸”,以后历版药典均以“小活络丸”收载。小活络丸由胆南星、制川乌、制草乌、地龙、乳香(制)、没药(制)六味中药组成,临床常用于风寒湿邪闭阻、痰瘀阻络所致的痹病,症见肢体关节疼痛,或冷痛,或刺痛,或疼痛夜甚、关节屈伸不利、麻木拘挛等。方中制川乌、制草乌温经散寒、祛风除湿,通痹止痛,为君药;胆南星燥湿化痰,祛经络中之风痰及湿邪,并能止痛,为本方臣药;乳香、没药行气活血,以化络中之淤血,并能止痛,为佐药;地龙通经活络,引导诸药直达病所,为使药;诸药合用,共奏祛风散寒,化痰除湿,活血止痛之功效。Xiaohuoluo Pill is a traditional Chinese patent medicine, formerly known as Huoluo Dan, also known as Xiaohuoluo Dan. The 1977 edition of "Chinese Pharmacopoeia" was revised to "Xiaohuoluo Pills", and subsequent editions of the Pharmacopoeia were all recorded as "Xiaohuoluo Pills". Xiaohuoluo Pill is composed of six traditional Chinese medicines: Dannanxing, Zhichuanwu, Zhicaowu, Dilong, Frankincense (made), and Myrrh (made). It is commonly used clinically for paralysis caused by obstruction of wind-cold dampness and phlegm and blood stasis. , symptoms include pain in limb joints, or cold pain, or tingling, or pain that is severe at night, difficulty in joint flexion and extension, numbness and spasm, etc. In the prescription, Zhichuanwu and Zhicaowu warm the meridians, dispel cold, expel wind and dampness, relieve numbness and relieve pain, and are the king medicines; Dannanxing removes dampness and resolves phlegm, removes wind, phlegm and dampness in the meridians, and can relieve pain, and is the prescription. Ministerial drugs: frankincense and myrrh promote qi and activate blood circulation, dissolve congestion in the collaterals, and relieve pain, and are used as adjuvant drugs; Dilong stimulates the meridians and activates the collaterals, guiding all the drugs directly to the diseased location, and is used as an adjuvant drug; all the drugs are used together to eliminate the phlegm. It has the effects of dispersing wind and cold, resolving phlegm and removing dampness, promoting blood circulation and relieving pain.
方中制川乌、制草乌为毛茛科乌头类中药材,其中所含的二萜类双酯型生物碱(MA、HA、AC)有大毒,主要是对神经和心血管系统造成严重伤害,乌头属植物作为临床用药时,常因炮制或用药不当引起中毒甚至死亡。此类药材经过炮制加工后,可水解为相应的二萜类单酯型生物碱(BMA、BAC、BHA),活性存在,毒性降低,治疗指数提高,常作为主要的药效成分应用于临床。要想降低原料质量风险、保证疗效以及防止低限投料等都需要通过成品的含量控制才能实现。 目前尚未有采用HPLC法同时测定小活络丸中苯甲酰新乌头原碱(BMA)、苯甲酰乌头原碱(BAC)、苯甲酰次乌头原碱(BHA)、新乌头碱(MA)、次乌头碱(HA)和乌头碱(AC)含量的方法,因此,建立小活络丸中二萜类生物碱含量的测定方法,并控制其范围,对保证临床疗效和用药安全具有重要意义。The Aconite Sichuan and Aconite Radix in the prescription are traditional Chinese medicines from the Ranunculaceae family. The diterpenoid diester alkaloids (MA, HA, AC) contained in them are highly toxic, mainly causing damage to the nervous and cardiovascular systems. Serious injuries. When Aconitum plants are used as clinical medicines, poisoning or even death often occurs due to improper processing or medication. After processing, these medicinal materials can be hydrolyzed into the corresponding diterpene monoester alkaloids (BMA, BAC, BHA), which have active activity, reduced toxicity, and improved therapeutic index. They are often used in clinical applications as the main medicinal ingredients. In order to reduce raw material quality risks, ensure efficacy and prevent low-limit feeding, it is necessary to control the content of finished products. At present, there is no HPLC method for simultaneous determination of benzoyl aconitine (BMA), benzoyl aconitine (BAC), benzoyl aconitine (BHA), and aconitine in Xiaohuoluo Pills. aconitine (HA) and aconitine (AC) content. Therefore, establishing a method for determining the content of diterpenoid alkaloids in Xiaohuoluo Pills and controlling its range is important for ensuring clinical efficacy and Medication safety is of great significance.
《中国药典》2020年版一部小活络丸质量标准中仅收载有乌头碱的TLC限量检查项,难以全面控制方中毒性药味的质量,且对乌头碱限量的控制限度较高,有可能引起中毒的风险。The 2020 version of the "Chinese Pharmacopoeia" quality standard for Xiaohuoluo Pills only contains the TLC limit inspection item for aconitine. It is difficult to fully control the quality of the toxic medicinal flavor in the prescription, and the control limit for the limit of aconitine is relatively high. May cause risk of poisoning.
发明内容Contents of the invention
本发明针对以上问题,提供了一种小活络丸中6种生物碱成分的含量测定方法,包括以下步骤:In view of the above problems, the present invention provides a method for determining the content of 6 alkaloid components in Xiaohuoluo Pills, which includes the following steps:
A.制备混合对照品溶液;A. Prepare mixed reference solution;
B.制备供试品溶液:取小活络丸适量,剪碎,取3g,称定,置具塞锥形瓶中,加入0.2mol/L盐酸溶液25ml,密塞,称定重量,超声处理30分钟,放冷,再称定重量,用0.2mol/L盐酸溶液补足减失的重量,摇匀,离心20分钟,量取续滤液15ml,置于处理好的固相萃取柱上,依次以0.05mol/L盐酸溶液、乙腈各10ml洗脱,弃去洗脱液,放置5分钟,继续用体积比为90:10的乙腈-浓氨溶液5ml洗脱,洗脱液收集于5ml量瓶中,并定容至刻度,摇匀,滤过,取续滤液,即得。B. Prepare the test solution: Take an appropriate amount of small Huoluo pills, cut into pieces, take 3g, weigh it, place it in a stoppered Erlenmeyer flask, add 25ml of 0.2mol/L hydrochloric acid solution, stopper it tightly, weigh it, and conduct ultrasonic treatment for 30 minutes, let cool, weigh again, use 0.2mol/L hydrochloric acid solution to make up for the lost weight, shake well, centrifuge for 20 minutes, measure 15ml of the additional filtrate, place it on the processed solid phase extraction column, and in turn use 0.05 Elute with 10 ml each of mol/L hydrochloric acid solution and acetonitrile. Discard the eluent and leave it for 5 minutes. Continue to elute with 5 ml of acetonitrile-concentrated ammonia solution with a volume ratio of 90:10. The eluent is collected in a 5 ml measuring bottle. Dilute the volume to the mark, shake well, filter, and take the remaining filtrate to get it.
C.分别吸取混合对照品溶液与供试品溶液各15μl,注入液相色谱仪,测定,外标法计算含量,即得;C. Take 15 μl each of the mixed reference solution and the test solution, inject them into the liquid chromatograph, measure, and calculate the content with the external standard method, and you get;
优选的,所述混合对照品溶液的制备方法为:取苯甲酰新乌头原碱、苯甲酰乌头原碱、苯甲酰次乌头原碱对照品与乌头双酯型生物碱对照提取物,精密称定,加乙腈制成每1ml分别含300μg、100μg、100μg、300ug的混合溶液,作为对照品贮备液,量取对照品贮备液5ml,置50ml量瓶中,加体积比为90:10的乙腈-浓氨溶液稀释至刻度,摇匀,即得。Preferably, the preparation method of the mixed reference solution is: taking benzoyl aconitine, benzoyl aconitine, benzoyl aconitine reference substance and aconitine diester alkaloids Weigh the control extract accurately, add acetonitrile to make a mixed solution containing 300 μg, 100 μg, 100 μg, and 300ug per 1 ml respectively. As the reference substance stock solution, measure 5 ml of the reference substance stock solution and place it in a 50 ml measuring bottle. Add the volume ratio Dilute 90:10 acetonitrile-concentrated ammonia solution to the mark, shake well, and it is ready.
优选的,所述步骤B中的超声处理:功率400W,频率40kHz,水温40℃以下。Preferably, the ultrasonic treatment in step B: power 400W, frequency 40kHz, water temperature below 40°C.
优选的,所述步骤B中离心转速为每分钟6000转。Preferably, the centrifugal speed in step B is 6000 rpm.
优选的,所述步骤B中固相萃取柱为以混合型阳离子交换反相吸附剂为填充剂的固相萃取柱,500mg,6ml,用前依次用乙腈、水各6ml洗脱。Preferably, the solid-phase extraction column in step B is a solid-phase extraction column filled with a mixed cation exchange reversed-phase adsorbent, 500 mg, 6 ml, and is eluted with 6 ml of acetonitrile and 6 ml of water in sequence before use.
优选的,色谱条件为:Preferably, the chromatographic conditions are:
色谱柱:PICKERING C18柱4.6×250mm,5μmChromatographic column: PICKERING C18 column 4.6×250mm, 5μm
流动相A:甲醇,为流动相B:乙腈,流动相C:0.1%磷酸Mobile phase A: methanol, mobile phase B: acetonitrile, mobile phase C: 0.1% phosphoric acid
梯度洗脱gradient elution
柱温:30℃Column temperature: 30℃
流速:1ml/minFlow rate: 1ml/min
检测波长:232nmDetection wavelength: 232nm
进样量:15μl。Injection volume: 15μl.
优选的,所述梯度洗脱程序如下:Preferably, the gradient elution procedure is as follows:
与现有技术相比,本发明的有益效果如下:Compared with the prior art, the beneficial effects of the present invention are as follows:
本发明的HPLC法具有高效、灵敏、操作简便、准确性高等优点,本发明建立了HPLC法同时测定小活络丸中6种生物碱类成分的含量,方法简便、专属性强,且同时对单酯型和双酯型生物碱进行控制,更有利于对乌头属药味的质量控制,保证了临床用药的安全性与有效性,为产品的质量控制和质量评价提供参考。The HPLC method of the present invention has the advantages of high efficiency, sensitivity, easy operation, and high accuracy. The present invention establishes an HPLC method for simultaneous determination of the contents of six alkaloids in Xiaohuoluo pills. The method is simple, highly specific, and can simultaneously measure single The control of ester and diester alkaloids is more conducive to the quality control of Aconitum medicinal flavor, ensuring the safety and effectiveness of clinical medication, and providing a reference for product quality control and quality evaluation.
图1为混合而对照品溶液的色谱图,其中:1-苯甲酰新乌头原碱;2-苯甲酰乌头原碱;3-苯甲酰次乌头原碱;4-新乌头碱;5-次乌头碱;6-乌头碱;Figure 1 is the chromatogram of the mixed reference solution, including: 1-benzoylaconitine; 2-benzoylaconitine; 3-benzoylaconitine; 4-benzoylaconitine Aconitine; 5-aconitine; 6-aconitine;
图2为供试品溶液的色谱图,其中:1-苯甲酰新乌头原碱;2-苯甲酰乌头原碱;3-苯甲酰次乌头原碱;4-新乌头碱;5-次乌头碱;6-乌头碱;Figure 2 is the chromatogram of the test solution, including: 1-benzoyl aconitine; 2-benzoyl aconitine; 3-benzoyl aconitine; 4-benzoyl aconitine Alkali; 5-aconitine; 6-aconitine;
图3为阴性样品溶液的色谱图。Figure 3 is the chromatogram of the negative sample solution.
为让本发明的上述特征和优点能更明显易懂,下文特举实施例,并配合附图,作详细说明如下,但本发明并不限于此。In order to make the above-mentioned features and advantages of the present invention more obvious and easy to understand, embodiments are given below and described in detail along with the accompanying drawings, but the present invention is not limited thereto.
在本发明的说明书中,提及“一个实施例”时均意指在该实施例中描述的具体特征、结构或者参数、步骤等至少包含在根据本发明的 一个实施例中。因而,在本发明的说明书中,若采用了诸如“根据本发明的一个实施例”、“在一个实施例中”等用语并不用于特指在同一个实施例中,若采用了诸如“在另外的实施例中”、“根据本发明的不同实施例”、“根据本发明另外的实施例”等用语,也并不用于特指提及的特征只能包含在特定的不同的实施例中。本领域的技术人员应该理解,在本发明说明书的一个或者多个实施例中公开的各具体特征、结构或者参数、步骤等可以以任何合适的方式组合。In the description of the present invention, any reference to "one embodiment" means that the specific features, structures or parameters, steps, etc. described in the embodiment are included in at least one embodiment according to the present invention. Therefore, in the description of the present invention, if terms such as "according to one embodiment of the present invention" and "in an embodiment" are used, they are not used to specifically refer to the same embodiment. If terms such as "in an embodiment" are used, Terms such as "in other embodiments", "according to different embodiments of the present invention", "according to other embodiments of the present invention" are not used to specifically indicate that the mentioned features can only be included in specific different embodiments. . Those skilled in the art should understand that each specific feature, structure or parameter, step, etc. disclosed in one or more embodiments of the present invention may be combined in any suitable manner.
本发明的目的在于提供了一种同时测定小活络丸中6种生物碱类成分的含量测定方法,本发明采用HPLC法测定苯甲酰新乌头原碱(BMA)、苯甲酰乌头原碱(BAC)、苯甲酰次乌头原碱(BHA)、新乌头碱(MA)、次乌头碱(HA)和乌头碱(AC)含量。The object of the present invention is to provide a method for simultaneously determining the content of six alkaloid components in Xiaohuoluo Pills. The present invention uses HPLC method to determine benzoyl aconitine (BMA), benzoyl aconitine aconitine (BAC), benzoyl aconitine (BHA), aconitine (MA), aconitine (HA) and aconitine (AC) content.
本发明含量测定方法采用HPLC法,该方法如下:The content determination method of the present invention adopts HPLC method, and the method is as follows:
色谱条件:色谱柱:PICKERING C18柱(4.6×250mm,5μm);以:以甲醇为流动相A,乙腈为流动相B,0.1%磷酸为流动相C,梯度洗脱程序见下表;柱温:30℃,流速:1ml/min;检测波长:232nm;进样量:15μl。Chromatographic conditions: Chromatographic column: PICKERING C18 column (4.6×250mm, 5μm); use: methanol as mobile phase A, acetonitrile as mobile phase B, 0.1% phosphoric acid as mobile phase C, the gradient elution procedure is shown in the table below; column temperature :30℃, flow rate: 1ml/min; detection wavelength: 232nm; injection volume: 15μl.
流动相梯度洗脱程序Mobile phase gradient elution procedure
混合对照品溶液的制备:取苯甲酰新乌头原碱、苯甲酰乌头原碱、 苯甲酰次乌头原碱对照品与乌头双酯型生物碱对照提取物适量,精密称定,加乙腈制成每1ml分别含300μg、100μg、100μg、300ug的混合溶液,作为对照品贮备液,精密量取对照品贮备液5ml,置50ml量瓶中,加乙腈-浓氨(90:10)溶液稀释至刻度,摇匀,即得。Preparation of mixed reference solution: Take appropriate amounts of benzoyl aconitine, benzoyl aconitine, benzoyl aconitine reference substance and aconitine diester alkaloid control extract, and weigh them accurately. Determine, add acetonitrile to make a mixed solution containing 300 μg, 100 μg, 100 μg, and 300ug per 1 ml respectively. As the reference substance stock solution, accurately measure 5 ml of the reference substance stock solution and place it in a 50 ml measuring bottle. Add acetonitrile-concentrated ammonia (90: 10) Dilute the solution to the mark, shake well, and it is ready.
供试品溶液的制备:取本品适量,剪碎,取约3g,精密称定,置具塞锥形瓶中,精密加入0.2mol/L盐酸溶液25ml,密塞,称定重量,超声处理(功率400W,频率40kHz)30分钟(水温40℃以下),放冷,再称定重量,用0.2mol/L盐酸溶液补足减失的重量,摇匀,离心(转速为每分钟6000转)20分钟,精密量取续滤液15ml,置于处理好的固相萃取柱(以混合型阳离子交换反相吸附剂为填充剂的固相萃取柱,500mg,6ml,用前依次用乙腈、水各6ml洗脱)上,依次以0.05mol/L盐酸溶液、乙腈各10ml洗脱,弃去洗脱液,放置5分钟,继用乙腈-浓氨试液(90:10)5ml洗脱,洗脱液收集于5ml量瓶中,并定容至刻度,摇匀,滤过,取续滤液,即得。Preparation of test solution: Take an appropriate amount of this product, cut into pieces, take about 3g, weigh it accurately, place it in a stoppered conical flask, add 25ml of 0.2mol/L hydrochloric acid solution accurately, stopper it tightly, weigh it, and ultrasonicate it. (power 400W, frequency 40kHz) for 30 minutes (water temperature below 40℃), let cool, weigh again, use 0.2mol/L hydrochloric acid solution to make up for the lost weight, shake well, and centrifuge (speed: 6000 rpm) for 20 Minutes, accurately measure 15 ml of the additional filtrate, and place it on a prepared solid-phase extraction column (a solid-phase extraction column filled with mixed cation exchange reversed-phase adsorbent, 500 mg, 6 ml). Before use, use 6 ml of acetonitrile and 6 ml of water. Elution), elute with 10 ml each of 0.05 mol/L hydrochloric acid solution and acetonitrile, discard the eluent, leave it for 5 minutes, and then elute with 5 ml of acetonitrile-concentrated ammonia test solution (90:10). Collect in a 5ml measuring flask, adjust the volume to the mark, shake well, filter, and take the remaining filtrate to get it.
测定法分别精密吸取混合对照品溶液与供试品溶液各15μl,注入液相色谱仪,测定,外标法计算含量,即得。Assay method: Precisely draw 15 μl each of the mixed reference solution and the test solution, inject them into the liquid chromatograph, measure, and calculate the content using the external standard method.
1.仪器与试药1.Instruments and reagents
1.1仪器1.1 Instruments
高效液相色谱仪(配备四元泵,DAD检测器,Waters e2695);分析天平:Mettler XPE26(百万分之一)(上海梅特勒仪器有限公司);Mettler XS105(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);超纯水仪(美国Millipore 公司)。High performance liquid chromatograph (equipped with quaternary pump, DAD detector, Waters e2695); analytical balance: Mettler XPE26 (parts per million) (Shanghai Mettler Instrument Co., Ltd.); Mettler XS105 (parts per hundred thousand) (Shanghai Mettler Instrument Co., Ltd.); KQ-400KDE ultrasonic cleaning instrument (Kunshan Ultrasonic Instrument Co., Ltd.); ultrapure water instrument (American Millipore Company).
1.2试药1.2 Test medicine
对照品(均购自中国食品药品检定研究院):Reference substances (all purchased from China Institute of Food and Drug Control):
乌头双酯型生物碱对照提取物(批号:112029-201601,供含量测定用,含量以新乌头碱31.7%,次乌头碱30.0%,乌头碱31.8%计)Aconitine diester alkaloid control extract (batch number: 112029-201601, for content determination, content is based on neo-aconitine 31.7%, hypo-aconitine 30.0%, aconitine 31.8%)
苯甲酰新乌头原碱(批号:111795-201604,含量以94.0%计)Benzoyl aconitine (batch number: 111795-201604, content based on 94.0%)
苯甲酰乌头原碱(批号:111794-201705,含量以99.1%计)Benzoylaconitine (batch number: 111794-201705, content based on 99.1%)
苯甲酰次乌头原碱(批号:111796-201906,含量以97.2%计)Benzoyl aconitine (batch number: 111796-201906, content based on 97.2%)
5批次小活络丸样品:均购自药店,详细信息见表1。5 batches of Xiaohuoluo pill samples: all purchased from pharmacies, detailed information is shown in Table 1.
表1 5批次小活络丸样品信息Table 1 5 batches of Xiaohuoluo pill sample information
试剂:甲醇、乙腈(色谱纯,Merck,Germany),水为超纯水;其它试剂均为分析纯。Reagents: methanol, acetonitrile (chromatographically pure, Merck, Germany), water is ultrapure water; other reagents are of analytical grade.
2.方法与结果2. Methods and results
2.1色谱条件2.1 Chromatographic conditions
色谱柱:PICKERING C18柱(4.6×250mm,5μm);以:以甲醇为流动相A,乙腈为流动相B,0.1%磷酸为流动相C,梯度洗脱程序 见表2;柱温:30℃,流速:1ml/min;检测波长:232nm;进样量:15μl。Chromatographic column: PICKERING C18 column (4.6×250mm, 5μm); use: methanol as mobile phase A, acetonitrile as mobile phase B, 0.1% phosphoric acid as mobile phase C, the gradient elution program is shown in Table 2; column temperature: 30°C , flow rate: 1ml/min; detection wavelength: 232nm; injection volume: 15μl.
表2流动相梯度洗脱程序Table 2 Mobile phase gradient elution procedure
分别精密吸取混合对照品溶液与供试品溶液各15μl,注入液相色谱仪,测定,外标法计算含量,即得。Precisely draw 15 μl each of the mixed reference solution and the test solution, inject them into the liquid chromatograph, measure, and calculate the content using the external standard method.
2.2溶液的制备2.2 Preparation of solution
2.2.1混合对照品溶液2.2.1 Mix the reference solution
取苯甲酰新乌头原碱、苯甲酰乌头原碱、苯甲酰次乌头原碱对照品与乌头双酯型生物碱对照提取物适量,精密称定,加乙腈制成每1ml分别含300μg、100μg、100μg、300ug的混合溶液,作为对照品贮备液,精密量取对照品贮备液5ml,置50ml量瓶中,加乙腈-浓氨(90:10)溶液稀释至刻度,摇匀,即得。Take an appropriate amount of benzoyl aconitine, benzoyl aconitine, benzoyl aconitine reference substance and aconitine diester alkaloid control extract, weigh it accurately, and add acetonitrile to prepare each solution. 1 ml of mixed solutions containing 300 μg, 100 μg, 100 μg, and 300ug respectively is used as the reference substance stock solution. Precisely measure 5 ml of the reference substance stock solution and place it in a 50 ml volumetric flask. Add acetonitrile-concentrated ammonia (90:10) solution to dilute to the mark. Shake well and you have it.
2.2.2供试品溶液的制备2.2.2 Preparation of test solution
取本品适量,剪碎,取约3g,精密称定,置具塞锥形瓶中,精密加入0.2mol/L盐酸溶液25ml,密塞,称定重量,超声处理(功率400W,频率40kHz)30分钟(水温40℃以下),放冷,再称定重量,用0.2mol/L盐酸溶液补足减失的重量,摇匀,离心(转速为每分钟6000转)20分钟,精密量取续滤液15ml,置于处理好的固相萃取柱(以混合型阳离子交换反相吸附剂为填充剂的固相萃取柱,500mg, 6ml,用前依次用乙腈、水各6ml洗脱)上,依次以0.05mol/L盐酸溶液、乙腈各10ml洗脱,弃去洗脱液,放置5分钟,继用乙腈-浓氨试液(90:10)5ml洗脱,洗脱液收集于5ml量瓶中,并定容至刻度,摇匀,滤过,取续滤液,即得。Take an appropriate amount of this product, cut into pieces, take about 3g, weigh it accurately, place it in a stoppered Erlenmeyer flask, add 25ml of 0.2mol/L hydrochloric acid solution accurately, stopper it tightly, weigh it, and treat it ultrasonically (power 400W, frequency 40kHz) 30 minutes (water temperature below 40°C), let cool, weigh again, use 0.2mol/L hydrochloric acid solution to make up for the lost weight, shake well, centrifuge (speed: 6000 rpm) for 20 minutes, and accurately measure the remaining filtrate 15ml, placed on the treated solid-phase extraction column (solid-phase extraction column filled with mixed cation exchange reversed-phase adsorbent, 500mg, 6ml, eluted with 6ml of acetonitrile and 6ml of water before use), and sequentially Elute with 10 ml each of 0.05 mol/L hydrochloric acid solution and acetonitrile. Discard the eluent and leave it for 5 minutes. Then elute with 5 ml of acetonitrile-concentrated ammonia test solution (90:10). The eluent is collected in a 5 ml measuring bottle. Dilute the volume to the mark, shake well, filter, and take the remaining filtrate to get it.
2.2.3阴性样品溶液的制备2.2.3 Preparation of negative sample solution
按小活络丸处方比例与制备工艺,制备缺制川乌与制草乌的阴性样品,按“供试品溶液”项下方法制备阴性样品溶液。According to the prescription ratio and preparation process of Xiaohuoluo Pills, prepare negative samples lacking Zhichuanwu and Zhicaowu, and prepare negative sample solutions according to the method under "Test solution".
2.3专属性试验2.3 Specificity test
分别取混合对照品溶液、供试品溶液及阴性样品溶液,按“2.1”项下色谱条件,分别进样15μl进行分析,记录色谱图,见附图。在样品色谱图中,分别显示与对照品苯甲酰新乌头原碱、苯甲酰乌头原碱、苯甲酰次乌头原碱、新乌头碱、次乌头碱与乌头碱保留时间一致的色谱峰,阴性样品色谱图中无相应的色谱峰,说明除制川乌、制草乌外的其它药味不干扰待测成分的测定,方法专属性良好。Take the mixed reference solution, test solution and negative sample solution respectively, according to the chromatographic conditions under "2.1", inject 15 μl of each sample for analysis, record the chromatogram, see the attached figure. In the sample chromatogram, the comparison with the reference substances benzoyl aconitine, benzoyl aconitine, benzoyl aconitine, neoaconitine, hypoaconitine and aconitine are shown respectively. There are chromatographic peaks with consistent retention times, but there are no corresponding chromatographic peaks in the chromatogram of the negative sample, indicating that other medicinal flavors except Chuanwu and Caowu do not interfere with the determination of the components to be tested, and the method has good specificity.
2.4线性关系考察2.4 Investigation of linear relationship
用乙腈-浓氨试液(90:10)分别配制苯甲酰新乌头原碱、苯甲酰乌头原碱、苯甲酰次乌头原碱、新乌头碱、次乌头碱与乌头碱浓度依次为:272.5675、108.3225、105.183、91.8127、86.889、92.1023μg/ml的对照品储备液溶液,依次稀释为系列浓度对照品溶液,分别注入高效液相色谱仪,按“2.1”项下的色谱条件测定,以对照品进样量(ng)为横坐标,峰面积积分值为纵坐标,绘制标准曲线,结果见表3,表明各成分在各自线性范围内线性关系良好。另取混合对照 品溶液逐级稀释,按“2.1”项下的色谱条件进样测定,当信噪比(S/N)为3:1时得检出限,当信噪比(S/N)为10:1时得定量限,结果见表3。Use acetonitrile-concentrated ammonia test solution (90:10) to prepare benzoyl aconitine, benzoyl aconitine, benzoyl aconitine, aconitine, aconitine and The reference substance stock solutions with aconitine concentrations of 272.5675, 108.3225, 105.183, 91.8127, 86.889, and 92.1023 μg/ml are diluted into serial concentration reference substance solutions in sequence, and injected into the high-performance liquid chromatograph respectively. Press item "2.1" The chromatographic conditions were measured under the conditions. Taking the injection volume of the reference substance (ng) as the abscissa and the peak area integrated value as the ordinate, a standard curve was drawn. The results are shown in Table 3, which shows that each component has a good linear relationship within its respective linear range. Another mixed reference solution is diluted step by step, and the sample is injected and measured according to the chromatographic conditions under "2.1". When the signal-to-noise ratio (S/N) is 3:1, the detection limit is obtained. When the signal-to-noise ratio (S/N) ) is 10:1, the limit of quantification is obtained, and the results are shown in Table 3.
表3各成分线性关系、检出限和定量限Table 3 Linear relationship, detection limit and quantitation limit of each component
2.5精密度实验2.5 Precision experiment
吸取混合对照品溶液15μl按“2.1”项下色谱条件进样,连续进样6次,记录各成分峰面积积分值,并计算RSD,结果苯甲酰新乌头原碱、苯甲酰乌头原碱、苯甲酰次乌头原碱、新乌头碱、次乌头碱和乌头碱的RSD值分别为0.8%、0.5%、0.3%、0.7%、0.6%和0.5%,结果表明仪器精密度良好。Aspirate 15 μl of the mixed reference solution and inject according to the chromatographic conditions under "2.1". Inject the sample 6 times continuously. Record the integrated value of the peak area of each component and calculate the RSD. The results show that benzoyl neoconitine, benzoyl aconitine The RSD values of protopine, benzoylhypoconitine, neoconitine, hypoconitine and aconitine are 0.8%, 0.5%, 0.3%, 0.7%, 0.6% and 0.5% respectively. The results show that The instrument precision is good.
2.6稳定性实验2.6 Stability experiment
精密吸取正文方法制备的批号为210603供试品溶液各15μl,分别在配制后0、2、4、8、12、18、24h按“2.1”项下色谱条件进样测定,记录各成分峰面积积分值,计算RSD,结果苯甲酰新乌头原碱、苯甲酰乌头原碱、苯甲酰次乌头原碱、新乌头碱、次乌头碱和乌头碱 的RSD值分别为0.5%、0.4%、1.2%、0.8%、0.5%、0.7%,结果表明实验制得的供试品溶液在24h内稳定。Precisely absorb 15 μl of each test solution of the batch number 210603 prepared by the method in the text. Inject samples according to the chromatographic conditions under "2.1" at 0, 2, 4, 8, 12, 18, and 24 hours after preparation. Record the peak area of each component. Integrate the value and calculate the RSD. The results are the RSD values of benzoyl aconitine, benzoyl aconitine, benzoyl aconitine, aconitine, aconitine and aconitine respectively. They are 0.5%, 0.4%, 1.2%, 0.8%, 0.5%, and 0.7%. The results show that the test solution prepared in the experiment is stable within 24 hours.
2.7重复性试验2.7 Repeatability test
取小活络丸样品(批号:210603)适量,剪碎,分别取1.5、3.0、4.5g,各取3份,精密称定,按正文方法制备供试品溶液,按“2.1”项的色谱条件进样测定,记录各成分峰面积积分值,计算含量及其RSD,结果见表4,表明该方法重复性良好。Take an appropriate amount of Xiaohuoluo Pill sample (batch number: 210603), cut it into pieces, take 1.5, 3.0, and 4.5g respectively, and take 3 parts each, weigh accurately, prepare the test solution according to the method in the text, and follow the chromatographic conditions of item "2.1" Inject samples for measurement, record the integrated peak area value of each component, and calculate the content and its RSD. The results are shown in Table 4, which shows that the method has good repeatability.
表4重复性试验结果Table 4 Repeatability test results
2.8回收率试验2.8 Recovery rate test
取小活络丸样品(批号:210603)适量,剪碎,精密称取9份,每份约1.5g,每三份为一组,分别加入用0.2mol/L盐酸溶液配制的低、中、高三个浓度的对照品溶液25ml,按“2.2.2”项下方法分别 制备供试溶液。由于本方法中双酯型生物碱(新乌头碱、次乌头碱、乌头碱)为限量成分,制剂中一般含量较低,所以加入对照品的量以限度为依据进行折算。按“2.1”项下的色谱条件进样测定,计算回收率,结果见表5,表明本方法回收率较好。Take an appropriate amount of Xiaohuoluo Pill sample (batch number: 210603), cut into pieces, and accurately weigh 9 portions, each portion is about 1.5g, and each three portions are a group. Add low, medium and high three portions prepared with 0.2mol/L hydrochloric acid solution. 25ml of the reference solution of each concentration, prepare the test solutions respectively according to the method under "2.2.2". Since the diester alkaloids (neoaconitine, hypoconitine, and aconitine) are limited ingredients in this method, and the content in the preparation is generally low, the amount of the reference substance added is calculated based on the limit. Inject the sample according to the chromatographic conditions under "2.1" and calculate the recovery rate. The results are shown in Table 5, which shows that the recovery rate of this method is good.
表5加样回收率试验结果(n=9)Table 5 Sample recovery test results (n=9)
2.9样品测定2.9 Sample determination
采用所建立的方法对收集到的5批次小活络丸样品进行6种生物碱成分的含量测定,结果见表6。The established method was used to determine the content of six alkaloid components in five batches of Xiaohuoluo pill samples collected. The results are shown in Table 6.
表6小活络丸含量测定结果Table 6 Content determination results of Xiaohuoluo Pills
(μg/g)(μg/g)
综上所述,本发明方法简便、专属性强,且同时对单酯型和双酯型生物碱进行控制,更有利于对乌头属药味的质量控制,保证临床用药的安全性与有效性,可为产品的质量控制和质量评价提供参考。In summary, the method of the present invention is simple, highly specific, and simultaneously controls monoester and diester alkaloids, which is more conducive to the quality control of Aconitum medicinal flavor and ensures the safety and effectiveness of clinical medication. , which can provide reference for product quality control and quality evaluation.
Claims (7)
- 一种小活络丸中6种生物碱成分的含量测定方法,其特征在于,包括以下步骤:A method for determining the content of six alkaloid components in Xiaohuoluo Pills, which is characterized by including the following steps:A.制备混合对照品溶液;A. Prepare mixed reference solution;B.制备供试品溶液:取小活络丸适量,剪碎,取3g,称定,置具塞锥形瓶中,加入0.2mol/L盐酸溶液25ml,密塞,称定重量,超声处理30分钟,放冷,再称定重量,用0.2mol/L盐酸溶液补足减失的重量,摇匀,离心20分钟,量取续滤液15ml,置于处理好的固相萃取柱上,依次以0.05mol/L盐酸溶液、乙腈各10ml洗脱,弃去洗脱液,放置5分钟,继续用体积比为90:10的乙腈-浓氨溶液5ml洗脱,洗脱液收集于5ml量瓶中,并定容至刻度,摇匀,滤过,取续滤液,即得。B. Prepare the test solution: Take an appropriate amount of small Huoluo pills, cut into pieces, take 3g, weigh it, place it in a stoppered Erlenmeyer flask, add 25ml of 0.2mol/L hydrochloric acid solution, stopper it tightly, weigh it, and conduct ultrasonic treatment for 30 minutes, let cool, weigh again, use 0.2mol/L hydrochloric acid solution to make up for the lost weight, shake well, centrifuge for 20 minutes, measure 15ml of the additional filtrate, place it on the processed solid phase extraction column, and in turn use 0.05 Elute with 10 ml each of mol/L hydrochloric acid solution and acetonitrile. Discard the eluent and leave it for 5 minutes. Continue to elute with 5 ml of acetonitrile-concentrated ammonia solution with a volume ratio of 90:10. The eluent is collected in a 5 ml measuring bottle. Dilute the volume to the mark, shake well, filter, and take the remaining filtrate to get it.C.分别吸取混合对照品溶液与供试品溶液各15μl,注入液相色谱仪,测定,外标法计算含量,即得。C. Take 15 μl each of the mixed reference solution and the test solution, inject them into the liquid chromatograph, measure, and calculate the content using the external standard method.
- 根据权利要求1所述的一种小活络丸中6种生物碱成分的含量测定方法,其特征在于:所述混合对照品溶液的制备方法为:取苯甲酰新乌头原碱、苯甲酰乌头原碱、苯甲酰次乌头原碱对照品与乌头双酯型生物碱对照提取物,称定,加乙腈制成每1ml分别含300μg、100μg、100μg、300ug的混合溶液,作为对照品贮备液,量取对照品贮备液5ml,置50ml量瓶中,加体积比为90:10的乙腈-浓氨溶液稀释至刻度,摇匀,即得。A method for determining the content of 6 alkaloid components in Xiaohuoluo pills according to claim 1, characterized in that: the preparation method of the mixed reference solution is: taking benzoyl neoaconitine, benzoyl Weigh the aconitine, benzoylhypoconitine reference substance and aconitine diester alkaloid control extract, add acetonitrile to make a mixed solution containing 300μg, 100μg, 100μg and 300ug per 1ml respectively. As the reference substance stock solution, measure 5 ml of the reference substance stock solution, place it in a 50 ml measuring bottle, add acetonitrile-concentrated ammonia solution with a volume ratio of 90:10 and dilute it to the mark, shake well, and it is ready.
- 根据权利要求1所述的一种小活络丸中6种生物碱成分的含量测定方法,其特征在于:所述步骤B中的超声处理:功率400W,频 率40kHz,水温40℃以下。The content determination method of 6 alkaloid components in Xiaohuoluo Pills according to claim 1, characterized in that: the ultrasonic treatment in step B: power 400W, frequency 40kHz, water temperature below 40°C.
- 根据权利要求1所述的一种小活络丸中6种生物碱成分的含量测定方法,其特征在于:所述步骤B中离心转速为每分钟6000转。A method for determining the content of 6 alkaloid components in Xiaohuoluo Pills according to claim 1, characterized in that: the centrifugal speed in step B is 6000 revolutions per minute.
- 根据权利要求1所述的一种小活络丸中6种生物碱成分的含量测定方法,其特征在于:所述步骤B中固相萃取柱为以混合型阳离子交换反相吸附剂为填充剂的固相萃取柱,500mg,6ml,用前依次用乙腈、水各6ml洗脱。A method for determining the content of 6 alkaloid components in Xiaohuoluo pills according to claim 1, characterized in that: in step B, the solid phase extraction column is filled with a mixed cation exchange reversed phase adsorbent. Solid phase extraction column, 500 mg, 6 ml, elute with 6 ml each of acetonitrile and water in sequence before use.
- 根据权利要求1所述的一种小活络丸中6种生物碱成分的含量测定方法,其特征在于:色谱条件为:A method for determining the content of 6 alkaloid components in Xiaohuoluo Pills according to claim 1, characterized in that: the chromatographic conditions are:色谱柱:PICKERING C18柱4.6×250mm,5μmChromatographic column: PICKERING C18 column 4.6×250mm, 5μm流动相A:甲醇,为流动相B:乙腈,流动相C:0.1%磷酸Mobile phase A: methanol, mobile phase B: acetonitrile, mobile phase C: 0.1% phosphoric acid梯度洗脱gradient elution柱温:30℃Column temperature: 30℃流速:1ml/minFlow rate: 1ml/min检测波长:232nmDetection wavelength: 232nm进样量:15μl。Injection volume: 15μl.
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HONGYAN CHEN, SUN XIAO; ZHANG XIAOLONG; YAO CHENXU; JIANG YAN: "Determination of Nine Aconitum Alkaloids in Xiaohuoluo Pills with Quantitative Analysis of Multi-components", CHINA PHARMACIST, vol. 20, no. 9, 5 September 2017 (2017-09-05), pages 1545 - 1549, XP093124729 * |
YUHUA SHI, HUANG WENKANG, HUANG QINWEI, ZHANG WENTING, ZHAO WEILIANG: "Content Determination of Six Alkaloids in Fengshigutong Tablet by SPE-HPLC", CHINESE JOURNAL OF MODERN APPLIED PHARMACY, vol. 35, no. 12, 24 December 2018 (2018-12-24), pages 1797 - 1800, XP093124723, DOI: 10.13748/j.cnki.issn1007-7693.2018.12.009 * |
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