阅读说明：本技术 同时测定四黄止痢颗粒中4种有效成分的方法 (Method for simultaneously determining 4 effective components in Sihuang dysentery stopping granules ) 是由 毕海林 肖裕章 张超 袁启峰 于 2020-05-25 设计创作，主要内容包括：本发明提供了一种同时检验四黄止痢颗粒中4种有效成分的方法,属于兽药检测技术领域。该方法以四黄止痢颗粒原标准及其有效成分为依据,对四黄止痢颗粒中的4种有效成分进行准确的分析检测,能进一步有效控制四黄止痢颗粒的质量,实现对制剂质量进行全面地评价,从而最大限度地保证产品质量的稳定性及兽用药的安全性、有效性,且该方法的专属性强、精密度高、稳定性好、准确度高,操作简便,试剂与耗材价廉易得,能够有效地控制产品生产质量及其稳定性,提升产品的品质及市场竞争力,在医药行业中具有推广实用价值。(The invention provides a method for simultaneously detecting 4 effective components in Sihuang dysentery stopping granules, belonging to the technical field of veterinary drug detection. The method is based on the original standard of the four-yellow dysentery stopping particles and the effective components thereof, and carries out accurate analysis and detection on the 4 effective components in the four-yellow dysentery stopping particles, so that the quality of the four-yellow dysentery stopping particles can be further effectively controlled, the quality of a preparation can be comprehensively evaluated, the stability of the product quality and the safety and effectiveness of veterinary drugs can be furthest guaranteed, the method is strong in specificity, high in precision, good in stability, high in accuracy, simple and convenient to operate, cheap and easily available in reagents and consumables, the production quality and the stability of the product can be effectively controlled, the quality and the market competitiveness of the product are improved, and the method has popularization and practical values in the pharmaceutical industry.)

1. The method for simultaneously measuring 4 effective components in the Sihuang dysentery stopping granules is characterized in that the method adopts a high performance liquid chromatography-diode array detector (HP L C-DAD) method and comprises the following steps:

1) chromatographic conditions

Chromatographic column packing agent: octadecylsilane chemically bonded silica;

mobile phase and elution mode: using phosphoric acid water solution as mobile phase A and acetonitrile as mobile phase B, carrying out gradient elution according to the following procedures,

0-5 min: the mobile phase is a mixed liquid of a mobile phase A and a mobile phase B in a volume ratio of 92: 8;

5-10 min: the mobile phase is a mobile phase A-mobile phase B mixed solution with the volume ratio of 85: 15;

10-20 min: the mobile phase is a mobile phase A-mobile phase B mixed solution with the volume ratio of 80: 20;

20-30 min: the mobile phase is a mobile phase A-mobile phase B mixed solution with the volume ratio of 75: 25;

30-45 min: the mobile phase is a mixed liquid of a mobile phase A and a mobile phase B in a volume ratio of 65: 35;

45-65 min: the mobile phase is a mobile phase A-mobile phase B mixed solution with the volume ratio of 35: 65;

65-70 min: the mobile phase is a mixed liquid of a mobile phase A and a mobile phase B in a volume ratio of 92: 8;

70-75 min: the mobile phase is a mixed liquid of a mobile phase A and a mobile phase B in a volume ratio of 92: 8;

the flow rate is 0.8m L/min;

a detector: a DAD detector;

2) preparation of test solution

Taking a test sample and preparing a test sample solution;

3) preparation of reference solution and Mixed reference solution

Precisely weighing emodin, (R, S) -goitrin, baicalin and liquiritin, respectively dissolving with methanol solution, sequentially making into 4 kinds of reference solutions, respectively precisely weighing 4 kinds of reference solutions, mixing, and diluting with methanol solution to obtain mixed reference solution;

4) measurement of

Accurately measuring emodin, (R, S) -goitrin, baicalin and liquiritin reference substance solutions and mixed reference substance solutions in sequence, respectively, injecting into a liquid chromatograph, recording chromatogram, accurately measuring test substance solution, injecting into the liquid chromatograph, measuring by the same method, and calculating the content of each effective component in the test substance by peak area according to an external standard method.

2. The method for simultaneously determining 4 effective components in Sihuang Zhili granules as claimed in claim 1, wherein the phosphoric acid aqueous solution is 0.1% by weight.

3. The method for simultaneously determining 4 effective components in Sihuang Zhili granules as claimed in claim 1, wherein the methanol solution is 75% by weight.

4. The method for simultaneously determining 4 effective components in Sihuang Zhili granule as claimed in claim 3, wherein the sample solution is prepared by precisely weighing the sample, grinding, placing in a 100m L volumetric flask, adding 75% methanol solution, performing ultrasonic treatment for 20min, cooling to room temperature, dissolving in 100m L with 75% methanol solution, shaking, filtering, and collecting the filtrate.

5. The method for simultaneously measuring 4 effective components in Sihuang Zhili Keli according to claim 3, wherein the control solution and the mixed control solution are prepared by precisely weighing emodin, (R, S) -goitrin, baicalin and liquiritin, respectively, adding 75% methanol to dissolve and fix the volume to obtain 4 control solutions with mass concentrations of 0.4mg/m L, 0.03mg/m L, 0.1mg/m L and 0.5mg/m L, precisely weighing 4 control solutions with mass concentrations of 2.0m L, respectively, placing in a 25m L container, adding 75% methanol to dissolve and fix the volume to obtain the mixed control solution.

Technical Field

The invention relates to the technical field of veterinary drug detection, and particularly relates to a method for simultaneously determining 4 effective components in Sihuang dysentery stopping granules.

Background

The Sihuang Zhili granule is prepared by decocting six traditional Chinese medicines of coptis chinensis, phellodendron, rhubarb, scutellaria baicalensis, isatis root and liquorice with water twice, concentrating, granulating with auxiliary materials and drying, has the functions of clearing heat and purging fire and stopping dysentery, has good treatment effect on chicken colibacillosis, and is recorded in the second part of 2015 edition Chinese veterinary pharmacopoeia.

More patents are available in China about the four-yellow dysentery stopping granules, for example, the Chinese patent 200910066083.3 discloses a preparation method of the four-yellow dysentery stopping effervescent granules, and the four-yellow dysentery stopping effervescent granules are prepared from the following raw materials and auxiliary materials in parts by weight: raw materials: 200 parts of coptis chinensis, 200 parts of golden cypress, 100 parts of rheum officinale, 200 parts of scutellaria baicalensis, 200 parts of isatis root and 100 parts of liquorice; auxiliary materials: 108-132 parts of sodium bicarbonate, 90-110 parts of fumaric acid, 549-671 parts of diluent and 18-22 parts of flavoring agent. Meanwhile, the effervescent granules are prepared by adopting a method of separately granulating by acid and alkali. The invention adopts sodium bicarbonate as an alkaline agent, fumaric acid as an acidic agent and other auxiliary materials to prepare the Sihuang dysentery stopping effervescent granule, can reduce the consumption of the auxiliary materials, and has the advantages of simple preparation process, easy forming, difficult moisture absorption, high effervescence speed, good effervescence effect, good dissolvability, good clarity of liquid medicine after effervescence, no coke foreign matter and the like.

The Chinese medicinal compound preparation can exert better curative effect because of the synergistic effect of various Chinese medicinal components, wherein the main component of the scutellaria baicalensis in the four-yellow dysentery stopping granules is baicalin, the main components of the coptis chinensis and the phellodendron are berberine, the main component of the liquorice is liquiritin, the main component of the rhubarb is emodin, and the main component (R, S) -goivy of the isatis root. The 2015 version of Chinese veterinary pharmacopoeia requires only measuring the content of baicalin, thin-layer identification of berberine hydrochloride and rhubarb reference medicinal materials, and other components do not require detection, so that the degradation of effective substances at high temperature is obvious, and the quality of the Sihuang dysentery stopping granules is difficult to control comprehensively.

The method for measuring the content of berberine, emodin, (R, S) -goivol, baicalin and liquiritin has related literature reports, for example, application No. 201310739482.8 discloses a method for testing the quality of Sihuang dysentery stopping granules, which is obtained by revising the product quality standard by combining the actual situation on the basis of the original standard of the Sihuang dysentery stopping granules, increases the identification of scutellaria baicalensis, and provides a method for measuring berberine hydrochloride by high performance liquid chromatography (HP L C).

However, the simultaneous determination of several components has been rarely reported. In order to effectively control the quality of the Sihuang Zhili granules and ensure the safety, effectiveness and stability of products, it is very important to establish a method for simultaneously measuring the contents of 4 main components, namely emodin, (R, S) -goitrin, baicalin and liquiritin, in the Sihuang Zhili granules.

Disclosure of Invention

In view of the above, the present invention aims to provide a method for simultaneously determining 4 effective ingredients in a Sihuang Zhili granule with simple operation and low cost.

Through research, the invention provides the following technical scheme:

a method for simultaneously measuring 4 effective components in Sihuang Zhili particles adopts a high performance liquid chromatography-diode array detector (HP L C-DAD) method, and comprises the following steps:

1) chromatographic conditions

Chromatographic column packing agent: octadecylsilane chemically bonded silica;

mobile phase and elution mode: using phosphoric acid water solution as mobile phase A and acetonitrile as mobile phase B, carrying out gradient elution according to the following procedures,

0-5 min: the mobile phase is a mixed liquid of a mobile phase A and a mobile phase B in a volume ratio of 92: 8;

5-10 min: the mobile phase is a mobile phase A-mobile phase B mixed solution with the volume ratio of 85: 15;

10-20 min: the mobile phase is a mobile phase A-mobile phase B mixed solution with the volume ratio of 80: 20;

20-30 min: the mobile phase is a mobile phase A-mobile phase B mixed solution with the volume ratio of 75: 25;

30-45 min: the mobile phase is a mixed liquid of a mobile phase A and a mobile phase B in a volume ratio of 65: 35;

45-65 min: the mobile phase is a mobile phase A-mobile phase B mixed solution with the volume ratio of 35: 65;

65-70 min: the mobile phase is a mixed liquid of a mobile phase A and a mobile phase B in a volume ratio of 92: 8;

70-75 min: the mobile phase is a mixed liquid of a mobile phase A and a mobile phase B in a volume ratio of 92: 8;

the flow rate is 0.8m L/min;

a detector: a DAD detector;

2) preparation of test solution

Taking a test sample and preparing a test sample solution;

3) preparation of reference solution and Mixed reference solution

Precisely weighing emodin, (R, S) -goitrin, baicalin and liquiritin, respectively dissolving with methanol solution, sequentially making into 4 kinds of reference solutions, respectively precisely weighing 4 kinds of reference solutions, mixing, and diluting with methanol solution to obtain mixed reference solution;

4) measurement of

Accurately measuring emodin, (R, S) -goitrin, baicalin and liquiritin reference substance solutions and mixed reference substance solutions in sequence, respectively, injecting into a liquid chromatograph, recording chromatogram, accurately measuring test substance solution, injecting into the liquid chromatograph, measuring by the same method, and calculating the content of each effective component in the test substance by peak area according to an external standard method.

Preferably, the phosphoric acid aqueous solution is 0.1% by mass.

Preferably, the methanol solution is 75% by mass.

More preferably, the sample solution is prepared by precisely weighing the sample, grinding, placing in a 100m L volumetric flask, adding 75% methanol solution, performing ultrasonic treatment for 20min, cooling to room temperature, dissolving in 100m L with 75% methanol solution, shaking, filtering, and collecting the filtrate.

More preferably, the control solution and the mixed control solution are prepared by precisely weighing emodin, (R, S) -goitrin, baicalin and liquiritin respectively, adding 75% methanol to dissolve and fix the volume to 4 control solutions with mass concentrations of 0.4mg/m L, 0.03mg/m L, 0.1mg/m L and 0.5mg/m L respectively, precisely weighing 4 control solutions of 2.0m L respectively, placing in a 25m L container, adding 75% methanol to dissolve and fix the volume to obtain the mixed control solution.

The method for simultaneously determining the 4 effective components in the four-yellow dysentery stopping granules provided by the invention is based on the original standard of the four-yellow dysentery stopping granules and the effective components thereof, and can be used for accurately analyzing and detecting the 4 effective components in the four-yellow dysentery stopping granules, so that the quality of the four-yellow dysentery stopping granules can be further effectively controlled, the quality of a preparation can be comprehensively evaluated, and the stability of the product quality and the safety and effectiveness of veterinary drugs can be furthest guaranteed.

The invention has the beneficial effects that:

1) the method for simultaneously determining 4 effective components in the four-yellow dysentery stopping particles provided by the invention adopts an HP L C-DAD combined method, and a DAD detector can simultaneously detect a plurality of wavelengths, so that the accurate analysis and detection of 4 effective components of emodin, (R, S) -goitrin, baicalin and liquiritin in the four-yellow dysentery stopping particles can be realized, the quality of the four-yellow dysentery stopping particles can be further effectively controlled, the comprehensive evaluation of the preparation quality can be realized, and the stability of the product quality and the safety and effectiveness of veterinary drugs can be furthest guaranteed;

2) the method for simultaneously measuring 4 effective components in the four-yellow dysentery stopping particles provided by the invention has the advantages of strong specificity, high precision, good stability, high accuracy, simplicity and convenience in operation, cheap and easily-obtained reagents and consumables, capability of effectively controlling the production quality and stability of products, promotion of the quality and market competitiveness of the products and popularization and practical value in the pharmaceutical industry.

Drawings

FIG. 1 is a chromatogram of an emodin control solution;

FIG. 2 is a chromatogram of a control solution of (R, S) -goitrin;

FIG. 3 is a chromatogram of a baicalin control solution;

FIG. 4 is a chromatogram of a liquiritin control solution;

FIG. 5 is a chromatogram of a mixed control solution;

FIG. 6 is a chromatogram of a test solution;

FIG. 7 is a chromatogram of a Scutellaria baicalensis-deficient negative mixed sample solution;

FIG. 8 is a chromatogram of a licorice-deficient negative mixed sample solution;

FIG. 9 is a chromatogram of a mixed sample solution negative for rheum officinale deficiency;

FIG. 10 is a chromatogram of a mixed sample solution negative for lack of radix Isatidis.

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.