CN115327007A

CN115327007A - Method for detecting fingerprint spectrum of ephedra decoction

Info

Publication number: CN115327007A
Application number: CN202210986926.7A
Authority: CN
Inventors: 蔡伟; 龚佳; 杨丽萍; 尚祥伟
Original assignee: Zhejiang Shuangshi Pharmaceutical Co ltd
Current assignee: Zhejiang Shuangshi Pharmaceutical Co ltd
Priority date: 2022-08-17
Filing date: 2022-08-17
Publication date: 2022-11-11

Abstract

The invention relates to a method for detecting a fingerprint of ephedra decoction, which comprises the following steps of S1, preparing an ephedra decoction, and concentrating the decoction to an extract with the density of 1.2-1.3 g/mL; s2, sampling the extract and carrying out fingerprint detection, wherein the chromatographic conditions are as follows: ALLTECH C18, 4.6X 250mm,5 μm; the wavelength is 210nm; solvent: 10% methanol; mobile phase system: acetonitrile-0.09% phosphoric acid. The invention determines the chromatographic condition of the ephedra decoction fingerprint detection, and tests prove the accuracy and the effectiveness of the chromatographic condition, and the detection can be directly carried out according to the chromatographic condition in the process of batch production of the ephedra decoction, thereby ensuring the high efficiency and the accuracy of the detection.

Description

Method for detecting ephedra decoction fingerprint spectrum

Technical Field

The invention belongs to the technical field of ephedra decoction detection, and particularly relates to an ephedra decoction fingerprint spectrum detection method.

Background

The ephedra decoction is an exterior syndrome relieving agent and has the efficacies of sweating, relieving exterior syndrome, ventilating lung and relieving asthma. It is mainly indicated for exterior excess syndrome due to wind-cold of exogenous pathogenic factor. Aversion to cold, fever, pain of the head and body, no sweat, dyspnea, thin and white tongue coating, and superficial and tense pulse. It is clinically used to treat exterior syndrome of wind-cold type, such as common cold, influenza, acute bronchitis, bronchial asthma, etc.

Fingerprint detection is a common detection means of traditional Chinese medicine medicaments, can ensure the safety and effectiveness of the traditional Chinese medicine medicaments, ensures the stable quality of the medicaments, is beneficial to deeply researching the action mechanism of the traditional Chinese medicine, and specifically comprises thin-layer chromatography, high-performance liquid chromatography, gas chromatography, spectrometry and the like.

Although fingerprint detection is a mature detection technology for traditional Chinese medicines, different traditional Chinese medicines need different detection parameters, and how to determine the detection parameters (such as wavelength, flow rate, column temperature and solvent) is a difficult point of detection. At present, some scholars do related research on the detection of the ephedra decoction fingerprint, but detection parameters are not standardized, and strict and effective detection standards are required when the ephedra decoction is produced in large quantities. For example, CN202110600880 and CN202110761770 disclose methods for quantitative chromatographic testing, but the solvents, mobile phase systems, and mobile phase system gradients used therein are not optimal.

Disclosure of Invention

The invention aims to solve the technical problem of providing a method for detecting the fingerprint of the ephedra decoction, which can realize the standardization of detection and is used for batch production.

In order to solve the problems, the technical scheme adopted by the invention is as follows: the fingerprint spectrum detection method of the ephedra decoction comprises the following steps

S1, preparing a ephedra decoction, and concentrating the decoction to obtain an extract with the density of 1.2-1.3 g/mL;

s2, sampling the extract and carrying out fingerprint detection, wherein the chromatographic conditions are as follows:

ALLTECH C18，4.6×250mm，5μm；

the wavelength is 210nm;

solvent: 10% methanol;

mobile phase system: acetonitrile-0.09% phosphoric acid;

the gradient of the mobile phase system is as follows:

at time 0, acetonitrile percentage 4%,0.09% phosphoric acid percentage 96%;

when the time is 25min, the percentage of acetonitrile is 8 percent, and the percentage of 0.09 percent phosphoric acid is 92 percent;

when the time is 45min, the percentage of acetonitrile is 9 percent, and the percentage of 0.09 percent phosphoric acid is 91 percent;

when the time is 55min, the percentage of acetonitrile is 15 percent, and the percentage of 0.09 percent phosphoric acid is 85 percent;

when the time is 68min, the percentage of acetonitrile is 20 percent, and the percentage of 0.09 percent phosphoric acid is 80 percent;

at the time of 78min, the percentage of acetonitrile is 30 percent, and the percentage of 0.09 percent phosphoric acid is 70 percent;

when the time is 85min, the percentage of acetonitrile is 35 percent, and the percentage of 0.09 percent phosphoric acid is 65 percent;

when the time is 90min, the percentage of acetonitrile is 50 percent, and the percentage of 0.09 percent phosphoric acid is 50 percent;

when the time is 95min, the percentage of acetonitrile is 4 percent, and the percentage of 0.09 percent phosphoric acid is 96 percent;

at 105min, the percentage of acetonitrile was 4% and the percentage of 0.09% phosphoric acid was 96%.

Further, the chromatographic conditions further comprise: the flow rate was 1.0ml/min.

Further, the chromatographic conditions further comprise: the column temperature was 35 ℃.

Further, the chromatographic conditions further comprise: the sample size is 10ul.

Further, in step S1, the preparation process of the decoction of ephedra decoction is as follows:

weighing herba Ephedrae, soaking in water for at least 30min;

weighing ramulus Cinnamomi, mel processed Glycyrrhrizae radix and semen Armeniacae amarum, mixing, and soaking in water for at least 30min;

filtering ramulus Cinnamomi, mel radix Glycyrrhizae Preparata and semen Armeniacae amarum, adding the filtrate into a container for soaking herba Ephedrae, boiling with strong fire, and decocting with slow fire for 10-15 min;

adding soaked ramulus Cinnamomi, honey-processed Glycyrrhrizae radix and semen Armeniacae amarum into a container for decocting herba Ephedrae, decocting for 10min, and filtering to obtain primary medicinal residue and primary medicinal liquid;

adding water into the primary decoction dregs, decocting for 20-25 min, filtering to obtain secondary decoction dregs and a secondary decoction, and mixing the primary decoction and the secondary decoction to obtain a decoction of ephedra decoction.

Further, the ephedra herb is soaked and decocted by a stainless steel pot.

The beneficial effects of the invention are: the invention further optimizes the chromatographic condition of the ephedra decoction fingerprint detection, and proves the accuracy and the effectiveness of the chromatographic condition through test verification, and can directly carry out detection according to the chromatographic condition in the process of batch production of the ephedra decoction, thereby ensuring the high efficiency and the accuracy of the detection.

Drawings

FIG. 1 is a schematic diagram showing the detection results of the decoction, the extract aqueous solution and the-20% methanol decoction;

FIG. 2 is a graph showing the detection results of the extract at various wavelengths;

FIG. 3 is a graph of the results of the corresponding wavelength tests for the Ma Huang, ku xing ren and Gui Zhi controls;

FIG. 4 is a graph showing the results of the test on the control licorice mixture at the corresponding wavelength;

FIG. 5 is a graph of the wavelength response of the control of Ephedra, bitter apricot seed and cassia twig;

FIG. 6 is a comparison of decoction and extract of herba Ephedrae decoction at a wavelength of 262 nm;

FIG. 7 is a graph comparing the wavelength of decoction and extract of herba Ephedrae at 258 nm;

FIG. 8 is a comparative plot of various solvent screens;

FIG. 9 is a graph of methanol/acetonitrile-0.09% phosphoric acid comparison;

FIG. 10 is a comparison of acetonitrile-various acids;

FIG. 11 is a fingerprint at each wavelength;

FIG. 12 is a fingerprint map gradient comparison graph;

figure 13 is a map of the location of fingerprint samples and controls.

Detailed Description

The invention is further illustrated by the following examples in conjunction with the drawings.

The fingerprint spectrum detection method of the ephedra decoction comprises the following steps

S1, preparing a decoction of the ephedra decoction, and concentrating the decoction to obtain an extract with the density of 1.2-1.3 g/mL.

The preparation process of the decoction of the ephedra decoction comprises the following steps:

weighing herba Ephedrae, soaking in water for at least 30min;

The ephedra herb can be soaked and decocted by adopting an earthen pot and the like, and preferably, the ephedra herb is soaked and decocted by utilizing a stainless steel pot. The stainless steel pot is corrosion-resistant, does not react with the medicinal material to most large-scale boiling pan on the market is the stainless steel pot, directly purchases and can produce.

ALLTECH C18，4.6×250mm，5μm；

the wavelength is 210nm;

solvent: 10% methanol;

the flow rate is 1.0ml/min; the column temperature is 35 ℃;

the sample amount is 10ul;

mobile phase system: acetonitrile-0.09% phosphoric acid;

the gradient of the mobile phase system is as follows:

at time 0, acetonitrile percentage 4%,0.09% phosphoric acid percentage 96%;

when the time is 78min, the percentage of acetonitrile is 30 percent, and the percentage of 0.09 percent phosphoric acid is 70 percent;

In the invention, the concentration of the solution is volume concentration, for example, 10% methanol, 0.09% phosphoric acid and the like are volume concentration, and the acetonitrile percentage of 4% means volume concentration of 4%.

In fingerprint detection, any detection condition may affect the detection result, and therefore, a plurality of detection conditions need to be tested to determine a better detection condition.

Example one

Weighing 45g of herba Ephedrae in a stainless steel pot, adding about 300ml of water, soaking for 45min, weighing 30g of ramulus Cinnamomi, 15g of radix Glycyrrhizae Preparata with honey and 30g of semen Armeniacae amarum, soaking in 500ml of water in another container for 45min, filtering, adding the filtrate into the stainless steel pot in which herba Ephedrae is soaked, boiling with strong fire, decocting with slow fire for 15min, and removing foam. Then adding the other 3 soaked medicinal materials into the pan, decocting for 10min, and filtering with 300 mesh gauze to obtain decoction and residue.

Adding 800ml of water into the residue, boiling with strong fire, boiling with small fire (1000W) for 25min, and filtering with 300-mesh gauze to obtain a second decoction.

Mixing the decoctions obtained in 2 times to obtain decoction, heating and concentrating the decoction until the concentration density is 1.3g/ml, and stopping concentrating to obtain batch 1 extract.

Example two

Weighing 180g herba Ephedrae in a stainless steel pot, adding appropriate amount of water, soaking for 30min, weighing 120g ramulus Cinnamomi, 60g radix Glycyrrhizae Preparata, and 120g semen Armeniacae amarum, soaking in appropriate amount of water for 30min in another container, filtering, adding the filtrate into the stainless steel pot with herba Ephedrae soaked therein, boiling with strong fire, decocting with slow fire (1000W) for 15min, and removing foam. Then adding the other 3 soaked medicinal materials into the pan, decocting for 10min, and filtering with 300 mesh gauze to obtain decoction and residue.

Adding 3200ml of water into the residue, boiling with strong fire, boiling with slow fire for 25min, and filtering with 300-mesh gauze to obtain second decoction.

Mixing the decoctions obtained in 2 times to obtain 6000ml (50 ml, calculating dry extract rate), heating and concentrating the decoction until the concentration density is 1.3g/ml, and stopping concentrating to obtain batch 2 of extract.

EXAMPLE III

Weighing 45g of herba Ephedrae in a stainless steel pot, adding about 300ml of water, soaking for 45min, weighing 30g of ramulus Cinnamomi, 15g of radix Glycyrrhizae Preparata with honey and 30g of semen Armeniacae amarum, soaking in 500ml of water in another container for 45min, filtering, adding the filtrate into the stainless steel pot in which herba Ephedrae is soaked, boiling with strong fire, decocting with slow fire for 11min, and removing foam. Then adding the other 3 soaked medicinal materials into the pan, decocting for 10min, and filtering with 300 mesh gauze to obtain decoction and residue.

Adding 800ml water into the residue, boiling with strong fire, boiling with slow fire for 21min, and filtering with 300 mesh gauze to obtain the second decoction.

Mixing the decoctions obtained in 2 times, and concentrating under heating until the concentration density is 1.2g/ml to obtain batch 3 of extract.

Example four

Weighing 180g of herba Ephedrae in a stainless steel pot, adding 1400ml of water, soaking for 30min, weighing 120g of ramulus Cinnamomi, 60 honey processed Glycyrrhrizae radix and 120g of semen Armeniacae amarum, soaking in 600ml of water in another container for 30min, filtering, adding the filtrate into the stainless steel pot in which herba Ephedrae is soaked, boiling with strong fire, decocting with slow fire for 11min, and removing foams. Then adding the other 3 soaked medicinal materials into the pan, decocting for 10min, and filtering with 300 mesh gauze to obtain decoction and residue.

Adding 3200ml water into the residue, boiling with strong fire, boiling with slow fire for 21min, and filtering with 300 mesh gauze to obtain the second decoction.

Mixing the decoctions for 2 times to obtain 4500ml total (50 ml is taken, and dry extract rate is calculated) to obtain decoction, heating and concentrating the decoction until the concentration density is 1.2g/ml, and stopping concentrating to obtain batch 4 of extract.

EXAMPLE five

267.9g of herba Ephedrae is weighed in a stainless steel pot, 2600ml of herba Ephedrae is added and soaked for 30min, 180.4g of ramulus Cinnamomi, 90.4g of radix Glycyrrhizae Preparata and 180.8g of semen Armeniacae amarum are weighed and soaked in a proper amount of water in another container for 30min, filtering is carried out, the filtrate is added into the stainless steel pot soaked with herba Ephedrae, the total volume of water is 3600ml, the mixture is boiled with big fire (270 ℃), and then the mixture is decocted with small fire (160 ℃) for 15min, and foam is removed. Then adding the other 3 soaked medicinal materials into the pan, decocting for 10min, and filtering with 300 mesh gauze to obtain decoction and residue.

Adding 9300ml of water into the residue, boiling with strong fire (270 deg.C), boiling with slow fire (160 deg.C) for 25min, and filtering with 300 mesh gauze to obtain 8600ml of the second decoction.

Mixing the decoction obtained in 2 times to obtain 9800ml (about 50ml, calculating dry extract rate) to obtain decoction, heating the decoction and concentrating until the concentration density is 1.2g/ml, and stopping concentrating to obtain batch 5 of extract.

Determining fingerprint detection conditions:

1. investigation of chromatographic columns

Preparation of a test solution: accurately weighing 0.1149g of the decoction of the ephedra decoction in the preparation process of the first embodiment in a 25ml volumetric flask, adding a 10% methanol solvent for dilution, and fixing the volume to the scale to obtain the ephedra decoction.

Chromatographic conditions are as follows:

sepax Polar-Phenyl (4.6X 250mm,5 μm) and ALLTECH C18 (4.6X 250mm,5 μm),

flow rate: 1.0ml/min of the mixture is added,

column temperature: at a temperature of 35 c,

sample introduction amount: 10 mu L of the mixed solution is prepared,

detection wavelength: 210. 285, 290, 207, 237, 254nm,

mobile phase: acetonitrile-0.15% of phosphoric acid,

gradient elution was performed as gradient 2:

gradient 2 mobile phase gradiometer

Through tests, compared with the operation of Sepax Polar-Phenyl and ALLTECH C18, the peak number and the peak pattern of the sample obtained at different wavelengths are more and better when the ALLTECH C18 chromatographic column is used. In addition, the characteristics of each medicinal material in the prescription need to be characterized in consideration of the whole prescription and the purpose of fingerprint spectrum, so that an ALLTECH C18 chromatographic column is selected for subsequent experiments.

From the chromatogram results of the decoction sample, the overall peak height of the chromatogram peak under each wavelength is low and should be related to the low concentration of the decoction sample; therefore, the decoction is directly filtered and injected to compare the chromatographic peak height and the peak number of the decoction of-20 percent methanol solvent and the extract water solution for subsequent research.

The comparative test of the decoction solvent, the decoction and the extract water solution comprises the following steps:

chromatographic conditions are as follows:

ALLTECH C18(4.6×250mm，5μm)，

flow rate: 1.0ml/min of the mixture is added,

column temperature: at a temperature of 35 c,

sample introduction amount: 10 mu L of the mixture is added into the solution,

detection wavelength: 210. 285, 290, 207, 237, 254nm,

mobile phase: acetonitrile-0.15% of phosphoric acid,

gradient elution was performed as gradient 9.

Gradient 9 mobile phase gradiometer

Preparing a test extract aqueous solution: accurately weighing 0.2026g of batch 1 extract in a 10ml volumetric flask, adding water for dilution, fixing the volume to scale, and mixing uniformly to obtain the traditional Chinese medicine.

The decoction prepared in the first example was used as the sample for the decoction.

Preparing a test decoction solution: precisely weighing 0.5536g of the decoction prepared in the first embodiment in a 25ml volumetric flask, adding 20% methanol solvent for dilution, and fixing the volume to the scale to obtain the finished product.

The results of the tests on the three test samples are shown in FIG. 1. According to the comparison result of the chromatographic peaks in fig. 1, the peak height and peak area of the extract chromatographic peak of the ephedra decoction are both obviously greater than those of the extract chromatographic peak of the ephedra decoction, so that the subsequent experiment uses the extract to carry out the fingerprint spectrum research.

2. Selection of wavelength

Preparing a test solution: accurately weighing 0.2026g of batch 1 extract in a 10ml volumetric flask, adding water for dilution, metering volume to scale, and mixing uniformly to obtain the traditional Chinese medicine composition. The decoction is directly filtered and injected.

The decoction of example one was taken as a sample of the decoction.

Preparation of a reference solution:

preparing a herba ephedrae mixed alkaloid reference substance solution: precisely weighing 11.37mg of ephedrine hydrochloride and 10.47mg of pseudoephedrine hydrochloride in a 25ml volumetric flask, adding methanol for dilution and fixing volume to scale, weighing 1ml again in a 10ml volumetric flask, adding methanol for dilution and fixing volume to scale, and mixing. The concentration of ephedrine hydrochloride is 45.48 μ g/ml, and the concentration of pseudoephedrine hydrochloride is 41.80 μ g/ml.

Preparation of amygdalin reference solution: weighing amygdalin 12.50mg in 25ml measuring flask, adding methanol to dilute and fixing volume to scale. Mixing to obtain the final product. And precisely measuring 1ml in a 10ml volumetric flask, adding methanol for dilution and fixing the volume to the scale. Mixing to obtain the final product.

Preparation of cinnamic acid control solution: 9.94mg of cinnamic acid is precisely weighed into a 10ml volumetric flask, diluted by adding methanol and fixed to the volume to the scale. Mixing to obtain the final product. And precisely measuring 1ml in a 10ml volumetric flask, adding methanol for dilution and fixing the volume to the scale. Mixing to obtain the final product.

Preparing a mixed reference substance solution of ephedra mixed alkaloid, amygdalin and cinnamic acid: precisely measuring 1ml of herba Ephedrae mixed alkaloid reference solution, amygdalin reference solution, and cinnamic acid reference solution in 10ml volumetric flasks, adding methanol to desired volume, and mixing.

Preparing a liquorice mixed reference substance solution: precisely weighing 5.09mg of liquiritin and 5.95mg of ammonium glycyrrhizinate in a 5ml volumetric flask, adding methanol for dilution and dissolution, fixing the volume to a scale, and mixing uniformly to obtain the liquiritin-ammonium glycyrrhizinate-water dispersible tablet.

Chromatographic conditions are as follows:

ALLTECH C18 column (4.6X 250mm,5 μm),

flow rate: 1.0ml/min of the mixture is added,

column temperature: at a temperature of 35 c,

sample introduction amount: 10 mu L of the mixture is added into the solution,

mobile phase: acetonitrile-0.15% of phosphoric acid,

the detection wavelength is selected according to the wavelength extracted by the wavelength scanning of 190nm-400 nm: 204. 206, 214, 216, 222, 237, 244, 256, 258, 262, 272, 274, 276, 282, 284nm.

Gradient elution was performed in gradients 9, 13, 14, 15. The gradient methods are consistent, and the wavelengths of the gradient 13, 14 and 15 methods are inconsistent; gradient 13 wavelengths are 204, 206, 214, 216, 222, 228, 237, 244nm, gradient 14 wavelengths are 256, 258, 262, 272, 274, 276, 282, 284nm, and gradient 15 wavelengths are 258, 262nm.

Gradient 13 mobile phase gradiometer

Gradient 14 mobile phase gradiometer

Gradient 15 mobile phase gradiometer

Through tests, the results of the extract at various wavelengths are shown in fig. 2, the test results of the ephedra herb, bitter apricot seed and cassia twig control products at corresponding wavelengths are shown in fig. 3, the test results of the licorice mixed control product at corresponding wavelengths are shown in fig. 4, the test results of the ephedra herb, bitter apricot seed and cassia twig control products at corresponding wavelengths are shown in fig. 5, the ratio of the ephedra decoction to the extract at the wavelength of 262nm is shown in fig. 6, and the ratio of the ephedra decoction to the extract at the wavelength of 258nm is shown in fig. 7.

According to the chromatogram, when the wavelength is 262nm, the number of chromatographic peaks, the peak height, the prescription integrity and the like of each reference solution are in a relatively ideal state, so that the wavelength of 262nm is temporarily selected for subsequent fingerprint spectrum research by combining the ephedra decoction extract, the decoction, the number of chromatographic peaks, the peak height, the prescription integrity, the fingerprint spectrum characterization and the like of each reference solution.

3. Selection of mobile phase system

Selecting a mobile phase system and a phosphoric acid concentration according to the angle of mixing the ephedra and the alkaloid.

3.1 testing of different concentrations of phosphoric acid in the acetonitrile-phosphoric acid System

Preparing a ephedra mixed alkaloid reference substance solution:

accurately weighing 10.47mg and 11.37mg of ephedrine hydrochloride and pseudoephedrine hydrochloride respectively in a 25ml volumetric flask, adding methanol for dilution and fixing the volume to the scale; and then taking 1ml to be put into a 10ml volumetric flask, adding methanol to dilute and fix the volume to the scale.

0.04% phosphoric acid solution: precisely measuring 0.2ml of phosphoric acid in a 500ml measuring cylinder, adding water to the scale, transferring to a beaker, uniformly mixing and carrying out ultrasonic filtration to obtain the product.

0.07% phosphoric acid solution: precisely measuring 0.35ml of phosphoric acid in a 500ml measuring cylinder, adding water to scale, transferring to a beaker, uniformly mixing, and performing ultrasonic filtration.

0.09% phosphoric acid solution: precisely measuring 0.9ml of phosphoric acid in a 1000ml measuring cylinder, adding water to the scale, transferring to a beaker, uniformly mixing and carrying out ultrasonic filtration to obtain the product.

0.15% phosphoric acid solution: precisely measuring 0.75ml of phosphoric acid in a 500ml measuring cylinder, adding water to the scale, transferring to a beaker, uniformly mixing and carrying out ultrasonic filtration to obtain the product.

Water: weighing 500ml of water and filtering to obtain the product.

Chromatographic conditions are as follows:

ALLTECH C18 column (4.6X 250mm,5 μm),

flow rate: 1.0ml/min of the mixture is added,

column temperature: at a temperature of 35 c,

sample introduction amount: 10 mu L of the mixed solution is prepared,

detection wavelength: the wavelength of the light source is 262nm,

mobile phase: acetonitrile-phosphoric acid (0.04%, 0.07%, 0.09%, 0.15%), acetonitrile-water.

Gradient elution was performed as gradient 4.

Gradient 4 mobile phase gradiometer

From the chromatogram results of the mixed alkaloid components of the ephedra, the peak shape is better when the concentration of the phosphoric acid solution is 0.09 percent compared with other concentrations; the concentration of phosphoric acid was therefore determined by taking the concentration of 0.09% in the aqueous phase.

3.2 selection of the types of acids in the organic and aqueous phases of the mobile phase System

Preparing a herba ephedrae mixed alkaloid reference substance solution: precisely weighing 13.94mg and 10.45mg of ephedrine hydrochloride and pseudoephedrine hydrochloride respectively in a 10ml volumetric flask, adding methanol for dilution and fixing the volume to the scale.

Chromatographic conditions are as follows:

DIKMA C18 (4.6X 250mm,5 μm) column,

flow rate: 1.0ml/min of the mixture is added,

column temperature: at a temperature of 35 c,

sample injection amount: 10 mu L of the mixed solution is prepared,

detection wavelength: 262nm.

Mobile phase system 1: methanol/acetonitrile-0.09% phosphoric acid is used as a mobile phase, and the mobile phase has the same gradient as the gradient 4.

Mobile phase system 2: acetonitrile-0.09% formic acid is used as a mobile phase, and the mobile phase gradient is the same as the gradient 4.

Mobile phase system 3: acetonitrile-0.09% glacial acetic acid is used as a mobile phase, and the gradient of the mobile phase is the same as the gradient 4.

Through experiments, methanol/acetonitrile-0.09% phosphoric acid contrast is shown in figure 9, acetonitrile-various acids contrast is shown in figure 10, and from the comparison chart, methanol and acetonitrile contrast, the elution of acetonitrile is stronger than methanol; thus acetonitrile was chosen as the organic phase; in comparison graphs of acetonitrile and various acids, formic acid and glacial acetic acid are inferior to phosphoric acid in baseline and peak patterns, so that the phosphoric acid is selected to be determined as the acid type; in combination with the above determination of the phosphoric acid concentration, the acetonitrile-0.09% phosphoric acid system was thus determined as a defined mobile phase system.

4. Selection of solvents

Preparing a test sample: precisely weighing 1.5226g, 1.2938g, 1.1312g, 1.1766g and 1.1271g of batch 3 extract samples, dissolving in 10% methanol, 20% methanol, 30% methanol, 40% methanol and 50% methanol solutions respectively, fixing the volume to a volume corresponding to 25ml volumetric flask, adding a corresponding solvent for dilution and dissolution, fixing the volume to a scale, and uniformly mixing to obtain a sample solution.

Chromatographic conditions are as follows:

DIKMA C18 (4.6X 250mm,5 μm) column,

flow rate: 1.0ml/min of the mixture is added,

column temperature: at a temperature of 35 c,

sample introduction amount: 10 mu L of the mixture is added into the solution,

wavelength: the wavelength of the light source is 262nm,

mobile phase system: acetonitrile-0.09% phosphoric acid.

The mobile phase gradient is the same as gradient 4.

The solvent screening pairs are shown in fig. 8, and it can be seen that the integrity of each peak is highest when 10% methanol is used, so 10% methanol is selected as the extraction solvent for the fingerprint sample.

5. Gradient optimization

Preparing a test sample: precisely weighing 1.5982g of batch 4 extract sample in a 25ml volumetric flask, adding 10% methanol solvent for dilution and dissolution, fixing the volume to the scale, and uniformly mixing to obtain a test solution.

Preparing a ephedra decoction fingerprint mixed reference solution: accurately weighing 11.10mg of ephedrine hydrochloride, 13.13mg of pseudoephedrine hydrochloride, 9.34mg of amygdalin, 9.94mg of cinnamic acid, 7.41mg of liquiritin and 8.35 mg of ammonium glycyrrhizinate in 10ml volumetric flasks respectively, adding methanol for dilution to constant volume to scale, and mixing uniformly to obtain the final product; precisely weighing 1ml of each reference solution in a 25ml volumetric flask, adding methanol for dilution, fixing the volume to the scale, and mixing uniformly to obtain the product.

Chromatographic conditions are as follows:

DIKMA C18 (4.6X 250mm,5 μm) column,

flow rate: 1.0ml/min of the mixture is added,

column temperature: at a temperature of 35 c,

sample injection amount: 10 mu L of the mixture is added into the solution,

detection wavelength: 262. the wavelength of the light source is 210nm, 220 nm and 230nm,

optimization of the

gradients

13, 15, 30, 31, 34 was performed.

The fingerprint spectrum under each wavelength is shown in fig. 11, the peak heights of the ephedra mixed alkaloid and amygdalin related to the previous wavelengths are compared before gradient optimization, and the peak heights are found to be too low because the absorption is different under different wavelengths, which can be reflected but is not obvious; the ephedra and bitter apricot seed medicinal materials are in monarch and minister relationship in the prescription, and are still determined as main components to be reflected on the fingerprint according to the efficacy and the fingerprint principle; therefore, when the wavelengths 210, 220 and 230nm are re-operated and compared with the wavelength 262nm, the components of the ephedra herb and the bitter apricot seed are concentrated in the low wavelength, so that the wavelength 210nm with higher absorption is selected for subsequent gradient optimization.

Gradients 13, 15 see gradient 13 table and gradient 15 table above, with

gradients

30, 31, 34 as follows:

gradient 30 mobile phase gradiometer

Gradient 31 mobile phase gradiometer

Gradient 34 mobile phase gradiometer

The test shows that the fingerprint gradient pair is shown in figure 12, and the positions of the fingerprint sample and the reference product are shown in figure 13. From the figure, gradient 34 is determined as fingerprint gradient, each reference component position is corresponding, the baseline is stable, the separation degree of ephedra mixed alkaloid is 1.7, the separation degree of amygdalin is 1.3, the separation degree of liquiritin is 2.1, the separation degree of cinnamic acid is 6.6, and the separation degree of glycyrrhizic acid is 5.6.

In conclusion, the chromatographic conditions of the ephedra decoction extract are as follows:

ALLTECH C18，4.6×250mm，5μm；

the wavelength is 210nm;

solvent: 10% methanol;

the flow rate is 1.0ml/min; the column temperature is 35 ℃;

the sample amount is 10ul;

mobile phase system: acetonitrile-0.09% phosphoric acid;

the gradient of the mobile phase system is as follows:

the invention determines reasonable wavelength, solvent, mobile phase system and mobile phase system gradient through a plurality of tests, can sample and detect the ephedra decoction extract according to the chromatographic conditions of the invention in the batch production process, and can accurately detect whether the drug effect and the like of the product meet the requirements, thereby judging whether the product is qualified and ensuring the drug effect of the product.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The method for detecting the fingerprint spectrum of the ephedra decoction is characterized by comprising the following steps

ALLTECH C18，4.6×250mm，5μm；

the wavelength is 210nm;

solvent: 10% methanol;

mobile phase system: acetonitrile-0.09% phosphoric acid;

the gradient of the mobile phase system is as follows:

at time 0, acetonitrile percentage 4%,0.09% phosphoric acid percentage 96%;

2. The method for detecting the fingerprint of the ephedra decoction according to claim 1, wherein the chromatographic conditions further comprise: the flow rate was 1.0ml/min.

3. The method for detecting the fingerprint of the ephedra decoction according to claim 1, wherein the chromatographic conditions further comprise: the column temperature was 35 ℃.

4. The method for detecting the fingerprint of the ephedra decoction according to claim 1, wherein the chromatographic conditions further comprise: the sample size is 10ul.

5. The method for detecting the fingerprint of the ephedra decoction according to claim 1, wherein in the step S1, the preparation process of the ephedra decoction is as follows:

weighing herba Ephedrae, and soaking in water for at least 30min;

weighing ramulus Cinnamomi, mel preparata Glycyrrhrizae radix and semen Armeniacae amarum, mixing ramulus Cinnamomi, mel preparata Glycyrrhrizae radix and semen Armeniacae amarum, adding water, and soaking for at least 30min;

filtering ramulus Cinnamomi, mel-processed Glycyrrhrizae radix and semen Armeniacae amarum, adding the filtrate into a container for soaking herba Ephedrae, boiling with strong fire, and decocting with slow fire for 10-15 min;

adding water into the primary decoction dregs, decocting for 20-25 min again, filtering to obtain secondary decoction dregs and a secondary decoction, and mixing the primary decoction and the secondary decoction to obtain the decoction of the ephedra decoction.

6. The method of claim 5, wherein the ephedra herb is soaked and decocted in a stainless steel pot.