WO2024007538A1

WO2024007538A1 - Content measurement method for six alkaloid components in small meridian-activating pill

Info

Publication number: WO2024007538A1
Application number: PCT/CN2022/139273
Authority: WO
Inventors: 赵振霞; 刘永利; 段吉平; 雷蓉; 王璐; 朱靖
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: 2022-07-06
Filing date: 2022-12-15
Publication date: 2024-01-11
Also published as: CN115372497B; CN115372497A

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

A method for determining the content of six alkaloids in Xiaohuoluo Pills

Technical field

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.

Background technique

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.

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.

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.

Contents of the invention

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. Prepare mixed reference solution;

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. 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;

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.

Preferably, the ultrasonic treatment in step B: power 400W, frequency 40kHz, water temperature below 40°C.

Preferably, the centrifugal speed in step B is 6000 rpm.

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.

Preferably, the chromatographic conditions are:

Chromatographic column: PICKERING C18 column 4.6×250mm, 5μm

Mobile phase A: methanol, mobile phase B: acetonitrile, mobile phase C: 0.1% phosphoric acid

gradient elution

Column temperature: 30℃

Flow rate: 1ml/min

Detection wavelength: 232nm

Injection volume: 15μl.

Preferably, the gradient elution procedure is as follows:

Compared with the prior art, the beneficial effects of the present invention are 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.

Description of the drawings

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.

Detailed ways

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.

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.

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, 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.

Mobile phase gradient elution procedure

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.

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.

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.Instruments and reagents

1.1 Instruments

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 Test medicine

Reference substances (all purchased from China Institute of Food and Drug Control):

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%)

Benzoyl aconitine (batch number: 111795-201604, content based on 94.0%)

Benzoylaconitine (batch number: 111794-201705, content based on 99.1%)

Benzoyl aconitine (batch number: 111796-201906, content based on 97.2%)

5 batches of Xiaohuoluo pill samples: all purchased from pharmacies, detailed information is shown in Table 1.

Table 1 5 batches of Xiaohuoluo pill sample information

Reagents: methanol, acetonitrile (chromatographically pure, Merck, Germany), water is ultrapure water; other reagents are of analytical grade.

2. Methods and results

2.1 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 program is shown in Table 2; column temperature: 30°C , flow rate: 1ml/min; detection wavelength: 232nm; injection volume: 15μl.

Table 2 Mobile phase gradient elution procedure

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 Preparation of solution

2.2.1 Mix the reference solution

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 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 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 Preparation of negative sample solution

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 Specificity test

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 Investigation of linear relationship

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.

Table 3 Linear relationship, detection limit and quantitation limit of each component

2.5 Precision experiment

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 Stability experiment

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 Repeatability test

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.

Table 4 Repeatability test results

2.8 Recovery rate test

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.

Table 5 Sample recovery test results (n=9)

2.9 Sample determination

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.

Table 6 Content determination results of Xiaohuoluo Pills

(μg/g)

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

A method for determining the content of six alkaloid components in Xiaohuoluo Pills, which is characterized by including the following steps:

A. Prepare mixed reference solution;

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. 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.
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.
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.
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.
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.
A method for determining the content of 6 alkaloid components in Xiaohuoluo Pills according to claim 1, characterized in that: the chromatographic conditions are:

Chromatographic column: PICKERING C18 column 4.6×250mm, 5μm

Mobile phase A: methanol, mobile phase B: acetonitrile, mobile phase C: 0.1% phosphoric acid

gradient elution

Column temperature: 30℃

Flow rate: 1ml/min

Detection wavelength: 232nm

Injection volume: 15μl.
A method for determining the content of 6 alkaloid components in Xiaohuoluo pills according to claim 6, characterized in that: the gradient elution procedure is as follows: