WO2024007532A1 - Method for determining dissolution rates of eight representative components in daochi pills - Google Patents

Abstract

The present invention relates to the field of drug analysis and detection, and provides a method for determining dissolution rates of eight representative components in Daochi pills, which comprises the following steps: preparing a mixed reference solution: taking 200 mL of degassed hydrochloric acid solution having a pH value of 1.0 as a dissolution medium, keeping the temperature at 37±0.5°C and the rotating speed at 80 r/min, lowering the solution into a container by means of a paddle, performing sampling after 180 min to obtain 2 mL of a product, and filtering same by using a 0.22 μm microporous filter membrane, so as to obtain the mixed reference solution; preparing a test solution; and precisely suctioning 1 μl of the mixed reference solution and 1 μl of the test solution, respectively, injecting same into an ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) instrument, performing determination, and calculating content by means of an external standard method. According to the present invention, the dissolution rates of baicalin, berberine hydrochloride, aloe-emodin, calceolarioside B, harpagoside, geniposide, paeoniflorin, and forsythiaside A are determined by using a UPLC-MS/MS method, so that the medicine quality of Daochi pills is comprehensively evaluated, and a reference can be provided for quality control and efficacy verification of Daochi pills.

Description

A method for determining the dissolution of 8 representative ingredients in Daochi Pills Technical field

The invention belongs to the field of drug analysis and detection, and is specifically a method for measuring the dissolution of eight representative components in Daochi Pills.

Background technique

Daochi Pills are composed of ten medicinal herbs, including Forsythia suspensa, Coptis chinensis, Gardenia jasminoides (fried with ginger), Akebia bark, Scrophulariaceae, Trichosanthes trichosanthes, red peony root, rhubarb, skullcap, and talc. Volume 6 of "Minheji Bureau Prescriptions" is now included in the 2020 edition of "Chinese Pharmacopoeia". It is commonly used clinically for sores on the mouth and tongue, sore throat, irritability in the chest and chest, short red urine, and constipation caused by internal heat accumulation. Traditional Chinese medicine preparations are mostly produced using traditional techniques. Differences in raw materials, excipients, production conditions and processes among various manufacturers can cause uneven product quality, thus affecting the clinical efficacy of finished medicines. The dissolution rate of a drug refers to the speed and degree of drug dissolution in a specified solvent. The dissolution rate has a direct impact on the absorption and utilization of the drug in the body. Drug dissolution testing is an effective means to evaluate the quality and process level of preparations. It can reflect differences in prescription composition, excipients, and production processes to a certain extent. Therefore, it is necessary to conduct dissolution testing on Daochi Pills. Currently, the UPLC-MS/MS method is used to determine 8 representative ingredients in Daochi Pills: baicalin, berberine hydrochloride, aloe-emodin, Akebia phenylethanoid B, harpagoside, gardeniposide, paeoniflorin, and forsythiarin. The dissolution rate of glycoside A has not been reported.

Contents of the invention

The purpose of the present invention is to address the above problems and provide a method for measuring the dissolution of 8 representative ingredients in Daochi Pills, which includes the following steps:

A. Prepare mixed reference solution;

B. Prepare the test solution: Take 6 pills of Daochiwan Shuimiwan, use 200mL of degassed pH=1.0 hydrochloric acid solution as the dissolution medium, keep the temperature at 37±0.5℃, rotate at 80r/min, and lower the paddle into the container. , start timing immediately, take a sample of 2 mL at 5, 15, 40, 80, 120, 150, and 180 minutes respectively, add the same amount of dissolution medium at the same temperature, and filter with a 0.22 μm microporous membrane to obtain;

C. Precisely draw 1 μl each of the mixed reference solution and the test solution, inject into the ultra-high performance liquid chromatography mass spectrometer, measure, and calculate the content with the external standard method, and you get;

Chromatographic conditions: Chromatographic column: phenomenon C18 column 2.6μm, 2.1×100mm; acetonitrile as mobile phase A, 0.1% formic acid as mobile phase B, gradient elution; column temperature: 40°C, flow rate: 0.3ml/min; injection Volume: 1μl;

Mass spectrometry conditions: electrospray ion source ESI, scanning mode: simultaneous scanning of positive and negative ions, monitoring mode: multiple reaction monitoring mode MRM, positive ionization voltage: 5500V, desolvation gas temperature: 500°C, negative ionization voltage: -4500V, desolvation Air temperature: 500℃.

Preferably, the mixed reference solution is prepared by the following steps: accurately weighing baicalin, berberine hydrochloride, aloe-emodin, Akebia phenylethanoid B, harpagoside, gardeniposide, paeoniflorin, Forsythiaside A reference substance is an appropriate amount, and methanol is added to prepare it. Each 1ml contains about 70 μg of baicalin, 22 μg of berberine hydrochloride, 1 μg of aloe emodin, 1 μg of Akebia phenylethanoid glycoside B, 2 μg of harpagoside, and 60 μg of gardeniposide. A mixed solution of 14 μg of paeoniflorin and 70 μg of forsythiaside A is obtained.

Preferably, the pH=1.0 hydrochloric acid solution in step B is prepared by precisely measuring 9.00 mL of hydrochloric acid, diluting it with water to 1000 mL, and shaking evenly.

Preferably, the gradient elution procedure is as follows:

Mobile phase gradient elution procedure

Compared with the prior art, the beneficial effects of the present invention are as follows:

The present invention has established a method for testing the dissolution of Daochi Pills and Water Honey Pills using the UPLC-MS/MS method. The active ingredient baicalin of Scutellaria baicalensis, Coptis chinensis, rhubarb, Akebia, Scrophulariaceae, Gardenia, red peony root and Forsythia suspensa in the prescription is , berberine hydrochloride, aloe-emodin, phytoside B, harpagoside, gardeniposide, paeoniflorin, and forsythiaside A dissolution were measured and analyzed, thereby conducting a comprehensive evaluation of the drug quality of Daochi Pills. The evaluation can provide reference for its quality control and drug efficacy verification.

Description of the drawings

Figures 1 to 24 respectively show baicalin, berberine hydrochloride, aloe-emodin, Akebia phenylethanoid glycoside B, harpagoside, gardeniposide, paeoniflorin, and forsythiaside in different dissolution cups of the same batch of Daochi pills. The dissolution curve of A (with time as the abscissa and dissolution as the ordinate);

Figures 25 to 32 respectively show the average values of baicalin, berberine hydrochloride, aloe-emodin, Akebia phenylethanoid B, harpagoside, gardeniposide, paeoniflorin, and forsythiaside A in different batches of Daochi Pills. Dissolution curve graph (with time as the abscissa and average dissolution as the ordinate).

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

The purpose of the present invention is to provide a method for measuring the dissolution of eight representative components of Daochi Pills. The present invention uses the UPLC-MS/MS method to determine baicalin, berberine hydrochloride, aloe-emodin, and Akebia phenylethanoid glycoside B. , dissolution of harpagoside, gardeniposide, paeoniflorin, and forsythiaside A. The content determination method of the present invention adopts the paddle method for dissolution, using hydrochloric acid solution with pH=1.0 as the dissolution medium, the rotation speed is 80r/min, and the sampling time is 5, 15, 40, 80, 120, 150, and 180min. Chromatographic conditions: Chromatographic column: phenomenon C18 column (2.6μm, 2.1×100mm); use acetonitrile as mobile phase A and 0.1% formic acid as mobile phase B. The gradient elution procedure is shown in Table 1; column temperature: 40°C, flow rate: 0.3 ml/min; injection volume: 1μl.

Table 1 Mobile phase gradient elution procedure

Detected by triple quadrupole tandem mass spectrometer; electrospray ion source (ESI), scanning mode: simultaneous scanning of positive and negative ions, monitoring mode: multiple reaction monitoring mode (MRM), positive ionization voltage: 5500V, desolvation gas temperature: 500 ℃, negative ionization voltage: -4500V, desolvation gas temperature: 500℃; mass spectrometry parameters are detailed in Table 2.

Table 2 Qualitative and quantitative ions and mass spectrometry conditions

Preparation of the mixed reference solution: Precisely weigh appropriate amounts of baicalin, berberine hydrochloride, aloe-emodin, aphylloside B, hapagoside, gardeniposide, paeoniflorin, and forsythiaside A reference substances. Add methanol to make each 1ml containing about 70μg of baicalin, 22μg of berberine hydrochloride, 1μg of aloe-emodin, 1μg of Akebia phenylethanoid glycoside B, 2μg of hapagoside, 60μg of gardeniposide, 14μg of paeoniflorin, and forsythiaside. A 70μg mixed solution is obtained.

Preparation of dissolution medium: Prepare the dissolution medium solution according to the method of dissolution medium in "Technical Guidelines for Dissolution Test of General Oral Solid Preparations", pH=1.0 hydrochloric acid solution: Precisely measure 9.00mL of hydrochloric acid, dilute it with water to 1000mL, shake well, that is have to.

1.Instruments and reagents

1.1 Instruments

AB SCIEX Qtrap 6500 ultra-high performance liquid chromatography-ion trap mass spectrometer; analytical balance: Mettler XPE26 (one part per million); Mettler XS105 (one hundred thousandth) (Shanghai Mettler Instrument Co., Ltd.); KQ -400KDE ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.);

1.2 Test medicine

Baicalin (CCI, batch number: 110715-201117, content 91.7%)

Berberine hydrochloride (CCI, batch number: 110713-201814, content 86.7%)

Aloe emodin (CCI, batch number: 110795-201308, content 97.8%)

Akebia phenylethanol glycoside B (CCI, batch number: 111910-201604, content 98.2%)

Harpagoside (CCI, batch number: 111730-201709, content 95.9%)

Paeoniflorin (CCI, batch number: 110736-201942, content 95.1%)

Forsythiaside A (CCI, batch number: 111810-201606, content 93.4%)

Geniposide (CCI, batch number: 110749-201919, content 97.1%)

Reagents: methanol, acetonitrile, and formic acid are all chromatographically pure, and water is ultrapure water. 10 batches of samples of Daochi Pills and Water Honey Pills.

2. Methods and results

2.1 Chromatographic conditions

Use acetonitrile as mobile phase A and 0.1% formic acid as mobile phase B. The gradient elution program is shown in Table 1. The flow rate is 0.3mL·min-1, the column temperature is 40°C, and the injection volume is 1 μL.

Table 1 Mobile phase gradient elution procedure

2.2 Preparation of reference solution: Accurately weigh appropriate amounts of baicalin, berberine hydrochloride, aloe-emodin, aphylloside B, hapagoside, gardeniposide, paeoniflorin, and forsythiaside A reference substances, and add Each 1ml made of methanol contains about 70 μg of baicalin, 22 μg of berberine hydrochloride, 1 μg of aloe-emodin, 1 μg of Ayatoside phenylethanoid glycoside B, 2 μg of hapagoside, 60 μg of gardeniposide, 14 μg of paeoniflorin, and 70 μg of forsythiaside A. The mixed solution is obtained.

2.3 Preparation of dissolution medium: Prepare the dissolution medium solution according to the method of dissolution medium in "Technical Guidelines for Dissolution Test of General Oral Solid Preparations", pH=1.0 hydrochloric acid solution: Precisely measure 9.00mL of hydrochloric acid, dilute to 1000mL with water, shake well, That’s it.

2.4 Screening of dissolution conditions

2.4.1 Investigation of the dissolution medium: Taking Daochi pills (batch number: 20201001) produced by manufacturer A as the research object, take 6 pills and place them in 200mL of the medium under "2.3" and 200mL of water dissolution medium. According to the 2020 version of "China The paddle method was operated under the general chapter "Dissolution and Release Measurement Methods" of the Pharmacopoeia (Part IV), and the above two dissolution media were placed respectively. The results showed that in the two media, baicalin and berberine hydrochloride in Daochi Pills , aloe-emodin, harpagoside, gardeniposide, paeoniflorin, and forsythiaside A are best dissolved in hydrochloric acid solution at pH=1.0, but poorly dissolved in water. Considering all things considered, the dissolution medium of this sample is pH=1.0 Hydrochloric acid solution is preferred.

2.4.2 Selection of rotation speed: Through preliminary tests, it was found that the rotation speed has a greater impact on the dissolution of this product. The dissolution conditions of the rotation speeds of 40, 80 and 120 r/min were investigated respectively. The results showed that the dissolution of Daochi Pills was better at 80 r/min. It has more discriminating power, so 80r/min was chosen to conduct the dissolution study of Daochi Pills.

2.4.3 Investigation of sampling time: Taking the 20201001 batch number Daochi Pills as the research object, the dissolution rates of 5, 15, 40, 80, 120, 150, 180, and 240 minutes were investigated respectively. The results showed that the dissolution of components such as baicalin had reached equilibrium within 180 minutes, so the sampling dissolution time was determined to be 180 minutes.

2.5 Evaluation of intra-batch homogeneity of samples

According to the second paddle method under the "Dissolution and Release Measurement Method" of the Four General Chapters of the 2020 Edition of the "Chinese Pharmacopoeia", 3 different batch numbers were selected, 6 pills were tested at one time, and 200 mL of degassed pH=1.0 hydrochloric acid was used. The solution is the dissolution medium, the temperature is maintained at (37±0.5)℃, and the rotation speed is 80r/min. Lower the paddle into the container and start timing immediately. Take 2 mL samples at 5, 15, 40, 80, 120, 150, and 180 minutes (while adding an equal amount of dissolution medium at the same temperature), and filter with a 0.22 μm microporous membrane. , take 1 μL of the continued filtrate, inject a sample, and determine the content of baicalin, berberine hydrochloride and other components. Divide the dissolution volume of baicalin, berberine hydrochloride, etc. in different dissolution cups by the maximum dissolution volume of each component in the dissolution sample to calculate different dissolutions The dissolution rate of baicalin, berberine hydrochloride, etc. in the cup, and the dissolution curve is drawn with time as the abscissa and dissolution rate as the ordinate. The results are shown in Figure 1-Figure 24. The results showed that different batches had good intra-batch homogeneity and good repeatability, and the determination method had high accuracy.

2.6 Determination of dissolution curves of different batches of samples

Take 5 different batches of Daochi pills, 6 pills in each batch, and measure according to the second method of the slurry method under the "Dissolution and Release Determination Method" of the Four General Chapters of the 2015 edition of the "Chinese Pharmacopoeia", using 200 mL of degassed The aqueous solution is the dissolution medium, the temperature is maintained at (37±0.5)℃, and the rotation speed is 80r/min. Lower the paddle into the container and start timing immediately. Take 2 mL samples at 5, 15, 40, 80, 120, 150, and 180 minutes (while adding an equal amount of dissolution medium at the same temperature), and filter with a 0.22 μm microporous membrane. , take 1 μL of the continued filtrate, inject a sample, and determine the content of gardeniposide, baicalin, etc., and calculate the difference by dividing the average dissolution amount of gardeniposide, baicalin, etc. at different times by the maximum dissolution amount of gardeniposide, baicalin, etc. in the dissolution sample The average dissolution of gardeniposide, baicalin, etc. in Shidaochi Pills was calculated, and the dissolution curve was drawn with time as the abscissa and the average dissolution as the ordinate. The results are shown in Figure 25-Figure 32.

In summary, the method of the present invention can treat the active ingredients of baicalin, berberine hydrochloride, aloe-emodin, baicalin, berberine hydrochloride, and aloe-emodin in the prescription of Daochi Pills. The dissolution rates of Akebia acutaphylla glycoside B, harpagoside, gardeniposide, paeoniflorin, and forsythiaside A were measured and analyzed, thereby comprehensively evaluating the quality of Daochi Pills, which can be used for quality control and pharmaceutical preparation. Provide reference for validation.

It should be noted that in this application, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply that these entities or operations There is no such actual relationship or sequence between them. Furthermore, the terms "comprises," "comprises," or any other variations thereof are intended to cover a non-exclusive inclusion such that a process, method, article, or apparatus that includes a list of elements includes not only those elements, but also those not expressly listed other elements, or elements inherent to the process, method, article or equipment.

Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art will understand that various changes, modifications, and substitutions can be made to these embodiments without departing from the principles and spirit of the invention. and modifications, the scope of the invention is defined by the appended claims and their equivalents.

Claims (4)

1. A method for measuring the dissolution of 8 representative ingredients in Daochi Pills, which is characterized by including the following steps:

   A. Prepare mixed reference solution;

   B. Prepare the test solution: Take 6 pills of Daochiwan Shuimiwan, use 200mL of degassed pH=1.0 hydrochloric acid solution as the dissolution medium, keep the temperature at 37±0.5℃, rotate at 80r/min, and lower the paddle into the container. , start timing immediately, take a sample of 2 mL at 5, 15, 40, 80, 120, 150, and 180 minutes respectively, add the same amount of dissolution medium at the same temperature, and filter with a 0.22 μm microporous filter membrane to obtain;

   C. Precisely draw 1 μl each of the mixed reference solution and the test solution, inject into the ultra-high performance liquid chromatography mass spectrometer, measure, and calculate the content with the external standard method, and you get;

   Chromatographic conditions: Chromatographic column: phenomenon C18 column 2.6μm, 2.1×100mm; acetonitrile as mobile phase A, 0.1% formic acid as mobile phase B, gradient elution; column temperature: 40°C, flow rate: 0.3ml/min; injection Volume: 1μl;

   Mass spectrometry conditions: electrospray ion source ESI, scanning mode: simultaneous scanning of positive and negative ions, monitoring mode: multiple reaction monitoring mode MRM, positive ionization voltage: 5500V, desolvation gas temperature: 500°C, negative ionization voltage: -4500V, desolvation Air temperature: 500℃.
2. A method for measuring the dissolution of 8 representative components in Daochi pills according to claim 1, characterized in that the mixed reference solution is configured by the following steps: weigh baicalin and berberine hydrochloride respectively , aloe-emodin, phytoside B, harpagoside, gardeniposide, paeoniflorin, and forsythiaside A reference substances were prepared by adding methanol and containing 70 μg of baicalin, 22 μg of berberine hydrochloride, and aloe vera per 1 ml. A mixed solution of 1 μg of emodin, 1 μg of Ayatoside B, 2 μg of Harpagoside, 60 μg of Geniposide, 14 μg of Paeoniflorin, and 70 μg of Forsythiaside A is obtained.
3. A dissolution method for measuring 8 representative ingredients in Daochi pills according to claim 1, characterized in that the preparation of the pH=1.0 hydrochloric acid solution in step B: measure 9.00 mL of hydrochloric acid and dilute it with water to 1000mL, shake well and get it.
4. [Amended in accordance with Rule 26 06.01.2023]
   A method for measuring the dissolution of 8 representative components in Daochi pills according to claim 1, characterized in that the gradient elution procedure is as follows:
   Mobile phase gradient elution procedure
   

   

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