CN111912911A

CN111912911A - Method for determining content of effective components in honeysuckle antipyretic mixture by HPLC method

Info

Publication number: CN111912911A
Application number: CN201910373742.1A
Authority: CN
Inventors: 孙国栋; 杜世霞; 廉英杰
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-05-07
Filing date: 2019-05-07
Publication date: 2020-11-10

Abstract

The invention relates to a method for determining the content of active ingredients in a honeysuckle antipyretic mixture by an HPLC method, namely the content of chlorogenic acid in the honeysuckle antipyretic mixture is measured. Extracting the effective components of the medicinal materials in the prescription by water decoction and distillation concentration to obtain mixture, and measuring by HPLC, and purifying by Diamonsil G8ODS chromatographic column (4.6nm × 200mm, 5 μm); 50 percent of methanol-0.4 percent of phosphoric acid water is used as a mobile phase, the flow rate is 1.0ml/min, the column temperature is 30 ℃, the detection wavelength is 327nm, and the sample volume is 10 mul. The retention time of chlorogenic acid is 18.126min, the linear range is 0.991-9.93 (r = 0.9999), the average sample recovery rate is 99.16%, and the detection limit is 0.1 mug/ml. The honeysuckle antipyretic mixture has stable preparation process, high efficiency, accuracy and stability in determining the content of chlorogenic acid by an HPLC method, and feasible quality standard.

Description

Method for determining content of effective components in honeysuckle antipyretic mixture by HPLC method

Technical Field

The invention relates to a method for determining the content of active ingredients in a honeysuckle antipyretic mixture by an HPLC method, namely the content of chlorogenic acid in the honeysuckle antipyretic mixture is measured.

Background

The honeysuckle antipyretic mixture (original honeysuckle mixture) is prepared by a hospital for years, is applied in the hospital for years, particularly for the first years of epidemic outbreak of hand-foot-and-mouth disease, plays an active role in treatment when western medicines are clinically used and do not have good curative effect, and is praised by doctors and patients. The preparation contains 14 Chinese medicines of honeysuckle, forsythia, patchouli, talcum powder, oriental wormwood, divaricate saposhnikovia root, angelica dahurica, reed rhizome, kudzuvine root, isatis root, bistort, astragalus, largehead atractylodes rhizome and liquorice, and has certain mutual interference because the types of the Chinese medicines of the preparation are as many as 14, and no good method exists in quality control, particularly in the determination of the content of main components.

At present, no method for measuring the content of the effective components of the honeysuckle antipyretic mixture is found.

Disclosure of Invention

In order to make up for the defects of the prior art, the invention provides the method for determining the content of the effective components in the honeysuckle antipyretic mixture by the HPLC method, which is simple, anti-interference and stable and accurate in measurement result.

The invention is realized by the following technical scheme: the method for determining the content of the effective components in the honeysuckle antipyretic mixture by the HPLC method is characterized by comprising the following steps:

(1) preparation of the solution

Control solution: precisely weighing appropriate amount of chlorogenic acid control dried to constant weight, adding 50% methanol to dissolve, and making into control solution containing chlorogenic acid 0.1 mg/ml;

test solution: precisely measuring 10ml of honeysuckle antipyretic mixture, placing in a 25ml volumetric flask, adding 50% methanol to scale, shaking up, filtering with a filter membrane, discarding the primary filtrate, and taking the subsequent filtrate as the test solution;

(2) detection of control solutions

Precisely measuring a certain amount of reference substance solution, placing in a volumetric flask, adding 50% methanol to constant volume, then taking 10 μ l of sample, measuring by high performance liquid chromatography to obtain peak area, and recording; taking the peak area as a vertical coordinate Y and the concentration of the sample as a horizontal coordinate X, and performing linear analysis to obtain a regression equation;

(3) detection of test solution

And (3) detecting the sample solution by using a high performance liquid chromatography to obtain the peak area of the sample solution, substituting the peak area serving as a Y value into the regression equation in the step (2), and calculating X according to the Y value to obtain the content of chlorogenic acid in the honeysuckle antipyretic mixture.

Preferably, the regression equation is Y =1563200.216X-1638.212 (r = 0.9999), and the chlorogenic acid has good linearity in the concentration range of 0.991-9.93 mg/ml.

Preferably, the filter membrane is a microporous filter membrane with the pore size of 0.5 mu m.

Preferably, the reference solution is measured in the test of the reference solution in 1ml, 2ml, 4ml, 6ml and 8ml, and the volumetric flask is 10 ml.

As a preferred embodiment, the chromatographic conditions are: a Diamonsil G8ODS column (4.6 nm. times.200 mm, 5 μm) was used; the mobile phase is 50% methanol-0.4% phosphoric acid water solution, the flow rate is 1.0ml/min, the detection wavelength is 327nm, the column temperature is 30 ℃, and the sample injection amount is 10 mul.

The invention has the beneficial effects that: 1. the invention establishes a method for measuring the content of chlorogenic acid which is the main effective component of the monarch drug honeysuckle in the honeysuckle antipyretic mixture by an HPLC method for the first time according to the general rules of the four departments of the 2015 edition of pharmacopoeia of the people's republic of China. 2. The measuring method is simple, efficient and stable, and has accurate measuring result and certain anti-interference performance. 3. The invention is also beneficial to the further research and development of the honeysuckle antipyretic mixture.

Drawings

FIG. 1 is a chromatogram of a control solution of the present invention;

FIG. 2 is a chromatogram of a test solution according to the present invention;

FIG. 3 is a negative control chromatogram of the present invention.

Detailed Description

The invention is further described with reference to the accompanying drawings and specific embodiments.

Instrument and reagent

1. Instrument for measuring the position of a moving object

Waters-HPLC (Waters, USA); 2847 ultraviolet detector (Qingdao Vast chromatography, Inc.); model TC0-250 ultrasonic cleaner (guangzhou south china ultrasonic equipment ltd); EYELAN-1100 model rotary evaporator (Tokyo, Japan, chemical and physical instruments Co., Ltd.); AUW120D electronic analytical balance (shimadzu, japan).

2. Reagent and reagent

Chlorogenic acid as a reference (batch No. 110753-201615), China institute for food and drug testing; chromatographically pure methanol, (shanghai zhenxing chemical industry one factory); phosphoric acid (analytically pure, Guiyang Linshan chemical Co., Ltd.).

Second, honeysuckle antipyretic mixture prescription composition and preparation method

Honeysuckle antipyretic mixture, a perennial self-made mixture in traditional Chinese medicine institute of Neze city (approval document: Shandong medicine preparation Z20150039, specification: 100 m/bottle).

1. Prescription composition

Honeysuckle, fructus forsythiae, cablin potchouli herb, talcum powder, oriental wormwood, divaricate saposhnikovia root, radix angelicae, rhizoma phragmitis, radix puerariae, radix isatidis, bistort rhizome, astragalus, bighead atractylodes rhizome and liquorice.

2. Preparation process

Distilling fructus forsythiae, herba Agastaches, radix Saposhnikoviae, herba Artemisiae Scopariae, radix Angelicae Dahuricae, and parched Atractylodis rhizoma with water, and collecting distillate 200 ml; decocting the residues and the rest eight medicines such as honeysuckle in water twice, decocting the residues and the rest eight medicines in 10 times of amount of the medicines for 2.5 hours for the first time, decocting the residues and the rest eight medicines in 8 times of amount of the medicines for 2 hours for the second time, mixing the decoctions, standing for 12 hours, naturally precipitating, filtering supernatant, concentrating to a relative density of 1.20-1.30 (80 ℃), adding 3g of the distillate and sodium benzoate, mixing uniformly, standing, filtering, adding water to 1000ml, stirring uniformly, and filling to obtain the traditional Chinese medicine.

Third, method and results

1. Chromatographic conditions

A Diamonsil G8ODS column (4.6 nm. times.200 mm, 5 μm) was used; the mobile phase is 50% methanol-0.4% phosphoric acid water solution, the flow rate is 1.0ml/min, the detection wavelength is 327nm, the column temperature is 30 ℃, and the sample injection amount is 10 mul.

2. Preparation of the solution

Control solution: precisely weighing appropriate amount of chlorogenic acid control dried to constant weight, adding appropriate amount of 50% methanol to dissolve, and making into control solution containing chlorogenic acid 0.1 mg/ml.

Test solution: precisely measuring 10ml of honeysuckle antipyretic mixture, placing in a 25ml volumetric flask, adding 50% methanol to scale, shaking, filtering with 0.5 μm microporous membrane, discarding the primary filtrate, and collecting the subsequent filtrate as the sample solution.

Negative control solution: taking the honeysuckle-free antipyretic mixture with the same formula and preparation method as the honeysuckle-free antipyretic mixture, precisely measuring 10ml, putting the mixture into a 25ml volumetric flask, adding 50% methanol to scale, shaking up, filtering with a 0.5 mu m microporous membrane, discarding the primary filtrate, and taking the subsequent filtrate as a negative control solution.

3. Methodology investigation

The linear relationship is: precisely measuring 1ml, 2ml, 4ml, 6ml and 8ml of reference substance solution, placing the reference substance solution in a 10ml volumetric flask, adding 50% methanol to a constant volume, sampling 10 μ l of each sample, measuring according to the chromatographic conditions, recording peak area, performing linear regression by taking the peak area as ordinate Y and the sample concentration as abscissa X (mg/ml), and obtaining equation Y =1563200.216X-1638.212 (r = 0.9999), wherein the chlorogenic acid is in a good linear range of 0.991-9.93 (r = 0.9999).

And (3) specific investigation: the prepared test solution, control solution and negative control solution are used for detecting chlorogenic acid retention time of 18.126min under chromatographic condition, and good separation is obtained, as shown in FIGS. 1-3.

4. And (3) precision experiment: precisely sucking 10 μ l of control solution, detecting according to the above chromatographic conditions, and continuously injecting sample for 8 times with Relative Standard Deviation (RSD) of 0.86%, which indicates good precision.

5. And (3) repeatability experiment: 9 parts of honeysuckle antipyretic mixture of the same batch are respectively taken and prepared according to the preparation method of the test solution, and the detection is carried out according to the chromatographic conditions, so that the Relative Standard Deviation (RSD) of the result is 1.1 percent, which indicates that the method has good repeatability.

6. And (3) stability test: sampling the same batch of sample solution, respectively performing sample injection measurement within 0, 2, 4, 6, 8 and 12h, recording peak areas, and determining that the Relative Standard Deviation (RSD) of the result is 1.6%, which indicates that the sample solution is stable within 12 h.

7. Sample adding and recovering experiment: precisely measuring 9 parts of test solution with known content and 3 parts of 1 group, respectively, adding precisely weighed low, medium and high chlorogenic acid reference substances into each group, respectively measuring according to the chromatographic conditions, and calculating the recovery rate as shown in Table 1:

。

8. detection limit and quantitation limit: and (3) adding 50% methanol into the control solution, respectively diluting the control solution into solutions with different concentrations, and determining, wherein the detection limit is 0.1 mu m/ml, and the signal-to-noise ratio is more than or equal to 3: 1, limit of quantitation is 1.0. mu.g/ml.

9. Content determination: taking 3 parts of test solution, the method is used for measuring the content of chlorogenic acid in the honeysuckle antipyretic mixture, and the result is shown in table 2:

。

honeysuckle flower, a monarch drug in the honeysuckle flower antipyretic mixture, has the functions of clearing away heat and toxic materials, cooling and dispersing wind heat, resisting pathogenic microorganisms, resisting inflammation, relieving fever, regulating immunity and the like, and an organic acid compound represented by chlorogenic acid is a main component of the honeysuckle flower antipyretic mixture, so that the honeysuckle flower is selected as a measured object. According to the verification guiding principle of the four-part general rule drug quality standard analysis method of pharmacopoeia of the people's republic of China 2015 edition, the quality standard for determining the content of chlorogenic acid as the active ingredient of the honeysuckle monarch drug in the honeysuckle antipyretic mixture by an HPLC method is established. The components in the formula are relatively complex, and considering that the interference of other components is reduced in order to effectively separate the active ingredient chlorogenic acid, a series of experiments are carried out to investigate the separation condition of the chlorogenic acid when the mobile phase acetonitrile-water, the methanol-0.2% of phosphoric acid water and the 50% of methanol-0.4% of phosphoric acid water are taken as the mobile phase, so that the separation condition of the chlorogenic acid is good, the peak shape is ideal and the interference is small. The invention adopts HPLC to measure the main effective components in the honeysuckle antipyretic mixture, has simple method and accurate and stable detection result, and is beneficial to the development of new medicines of the honeysuckle antipyretic mixture and the research of quality control.

Claims

1. A method for determining the content of active ingredients in a honeysuckle antipyretic mixture by an HPLC method is characterized by comprising the following steps:

(1) preparation of the solution

(2) detection of control solutions

(3) detection of test solution

2. The method for determining the content of the active ingredients in the honeysuckle antipyretic mixture by the HPLC method according to claim 1, wherein the regression equation is Y =1563200.216X-1638.212 (r = 0.9999), and the chlorogenic acid has good linearity in the concentration range of 0.991-9.93 mg/ml.

3. The method for determining the content of the active ingredients in the honeysuckle antipyretic mixture by the HPLC method as claimed in claim 1, wherein the filter membrane is a microporous filter membrane with a pore size of 0.5 μm.

4. The method for determining the content of the active ingredients in the honeysuckle antipyretic mixture by the HPLC method according to claim 1, wherein the reference solutions measured in the detection of the reference solution are 1ml, 2ml, 4ml, 6ml and 8ml respectively, and the volumetric flask is 10 ml.

5. The method for determining the content of the active ingredients in the honeysuckle antipyretic mixture by the HPLC method according to claim 1, wherein the chromatographic conditions are as follows: a Diamonsil G8ODS column (4.6 nm. times.200 mm, 5 μm) was used; the mobile phase is 50% methanol-0.4% phosphoric acid water solution, the flow rate is 1.0ml/min, the detection wavelength is 327nm, the column temperature is 30 ℃, and the sample injection amount is 10 mul.