CN113791152B

CN113791152B - Method for determining contents of various effective components in Xianyu capsule by HPLC (high performance liquid chromatography)

Info

Publication number: CN113791152B
Application number: CN202111064701.8A
Authority: CN
Inventors: 胡小艳; 张琼; 吕慧锋
Original assignee: Xi' An Chiho Pharmaceutical Co ltd
Current assignee: Xi' An Chiho Pharmaceutical Co ltd
Priority date: 2021-09-10
Filing date: 2021-09-10
Publication date: 2023-02-28
Anticipated expiration: 2041-09-10
Also published as: CN113791152A

Abstract

The invention discloses a method for determining the content of various active ingredients in Xianyu capsule by HPLC, which simultaneously determines gastrodine and p-hydroxybenzyl alcohol in gastrodia elata and tenuifolin and polygala tenuifolia in polygala tenuifolia by adopting a liquid chromatography method

Description

Method for determining contents of various effective components in Xianyu capsule by HPLC (high performance liquid chromatography)

Technical Field

The invention belongs to the technical field of medicine detection and analysis, and particularly relates to a method for determining contents of various effective components in Xianyu capsules by an HPLC (high performance liquid chromatography) method.

Background

Epilepsy includes primary and secondary epilepsy, which can be seen in all age groups. The children have higher epilepsy incidence rate than adults, and the epilepsy incidence rate is reduced with the increase of the age. In the elderly (after 65 years), the incidence of epilepsy is increasing due to cerebrovascular disease, senile dementia and degenerative changes of nervous system. Epilepsy is a chronic disease, and although the epilepsy has little influence on patients in a short period, the long-term frequent seizures can cause the physical, mental and intelligence of the patients to have serious influence. Epileptics often suddenly attack at any time, place and environment and without self control, and are easy to fall injury, scald, drowning, traffic accidents and the like; the patients are often distinguished by the society, have difficulties in employment, marriage, family life and the like, and are greatly influenced by mental depression and physical and mental health of the patients. The main manifestations are dysmnesia, mental retardation, character change, and finally gradually losing the working ability and even the living ability.

Epilepsy is considered to belong to the category of epilepsy by traditional Chinese medicine, and is a paroxysmal disease characterized by phlegm, fire, stasis, congenital factors and the like, qi and blood disorder, resuscitation and masking, sudden coma, tetany, moving time and self-waking, and waking after waking. The traditional Chinese medicine considers that the occurrence of epilepsy is mainly due to congenital factors, acquired seven-emotion imbalance, improper diet, overstrain and ease, or other diseases, which cause viscera imbalance and turbid phlegm obstruction; the treatise on the three causes of the extreme one disease, the treatise on epilepsy and pain: "Fu epilepsy" is caused by convulsions, or by wind-cold, summer-heat and dampness, or improper diet, which is adverse to the zang-organs. "Danxi Xin Fa-Dian" (epilepsy) also indicates the occurrence of this disease, rather than without phlegm and congestion of saliva, and stuffy orifices. Medical compendium. Epilepsy: for epilepsy, phlegm pathogen also goes upwards. That is, the "phlegm returning to heart orifice" is the pathogenesis of this disease. Phlegm is the main cause of epilepsy, and the important disease is in the brain and heart, because phlegm turbidity obstructs the heart orifice, and inevitably causes dysfunction of heart governing mind and abnormal spirit. Phlegm is a pathological product transformed by the body fluids of the zang-fu organs, and the body fluids are the nutritive tissues and organs of the human body derived from the nutrient substances of diet. Because of emotional disorder, improper diet, damage to the spleen and stomach, weak constitution and insufficiency of middle qi, the spleen fails to transport its fluids and stagnates and accumulates to form phlegm, so called "spleen is also the source of phlegm". Clinically, the severity of epilepsy is related to the depth of turbid phlegm and the abundance or insufficiency of healthy qi. At the beginning, healthy qi is not weakened and phlegm is not heavy, so the attack duration is short, the intermittence period is long, if the attack is repeated, the healthy qi is gradually weakened, the phlegm is not changed, the disease is more frequent and more frequent, the healthy qi is further weakened, and the disease is caused mutually, so the disease is also serious, and the disease is often mixed with deficiency and excess, and the origin is deficient and marked with excess. Deficiency syndrome is usually manifested as qi deficiency and blood deficiency; excessive syndrome is usually caused by upward disturbance of phlegm-fire and stagnation of phlegm-qi. Generally speaking, epilepsy is more deficient than excessive, and because it has a slow onset and a long course of disease, healthy qi is gradually weakened and qi and blood are weak. The treatment aims at benefiting qi, nourishing blood, strengthening body resistance, consolidating constitution, tranquilizing mind, arresting convulsion, calming endogenous wind, relieving spasm, restoring healthy qi, eliminating phlegm, dredging heart orifice, and recovering spirit.

At present, the medicines for treating the epileptic disease are various, and the conventional treatment methods comprise medicine treatment, diet treatment, operation treatment and the like, but each method has respective advantages and disadvantages, and the pathogenetic factors and the conditions of the epileptic patient are different, so that the most effective treatment method is only selected. The Chinese patent medicine for treating epilepsy has the advantages of mild medicine property, small side effect, no damage to human bodies and the like, and the commonly used anti-epilepsy medicine epilepsy curing capsule is proved to have good treatment effect on epilepsy through a large number of repeated clinical tests for a long time and is sold on the market for many years.

The Xianqianghe capsule is an exclusive variety produced by Xian Qian standing grain pharmaceutical industry Limited company, has been sold on the market for many years, has good effects on treating epileptic convulsion, infantile convulsion and facial spasm caused by wind-phlegm obstruction, and comprises the following main components: radix astragali, radix Codonopsis, saviae Miltiorrhizae radix, bupleuri radix, semen Ziziphi Spinosae, cortex et radix Polygalae, rhizoma Gastrodiae, ramulus Uncariae cum uncis, rhizoma Acori Graminei, rhizoma arisaematis cum bile, radix Angelicae sinensis, bombyx Batryticatus, massa Medicata Fermentata, radix Curcumae, glycyrrhrizae radix, and rhizoma Typhonii preparata. Has effects in eliminating phlegm, inducing resuscitation, tranquilizing mind, arresting convulsion, calming endogenous wind, and relieving spasm. In the existing detection method of the Xianyu capsule, only the content of the monarch drug rhizoma gastrodiae and the ministerial drug liquorice is measured, the ministerial drug radix astragali, the salvia miltiorrhiza and the codonopsis pilosula are identified in a thin layer, and the adjuvant drug stiff silkworm is identified in a microscopic way, so the quality control indexes cannot comprehensively reflect the quality of the medicine and cannot control the clinical curative effect index, therefore, the detection method of the medicine is necessary to be further perfected, and the control index is associated with the clinical curative effect, so the quality and the curative effect of the product are further ensured.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a method for determining the contents of various active ingredients in the Xianyu capsule by an HPLC method

Ketone III and 3,6' -two mustard seed acyl sucrose, bupleurum root effective constituent Bupleurum saponin a and Bupleurum root saponin d total 7 effective constituents were tested at the same time. The detection method can make up the defects of the existing method, provides a more comprehensive and effective drug control quality standard, and further effectively ensures the product quality.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for determining the content of various active ingredients in a Xianyu capsule by an HPLC method is disclosed, wherein the Xianyu capsule is prepared from the following 16 medicinal materials: radix astragali, radix Codonopsis, saviae Miltiorrhizae radix, bupleuri radix, semen Ziziphi Spinosae, cortex et radix Polygalae, rhizoma Gastrodiae, ramulus Uncariae cum uncis, rhizoma Acori Graminei, rhizoma arisaematis cum bile, radix Angelicae sinensis, bombyx Batryticatus, massa Medicata Fermentata, radix Curcumae, glycyrrhrizae radix, and rhizoma Typhonii preparata; performing high performance liquid chromatography on gastrodine and p-hydroxybenzyl alcohol in rhizoma Gastrodiae, and tenuifolin and cortex et radix Polygalae saponin in cortex et radix Polygalae

Ketone III, 3,6' -mustard seed acyl sucrose, saikosaponin a and saikosaponin d in bupleurum are subjected to content detection, and the detection method comprises the following steps:

(1) Chromatographic conditions and System suitability test

Octadecylsilane chemically bonded silica is used as a filling agent; gradient elution is carried out by taking an organic solvent as an A phase and a dilute acid aqueous solution with a certain concentration as a B phase, wherein the organic solvent is selected from methanol, acetonitrile or acetonitrile-methanol mixed solution, and the dilute acid aqueous solution is selected from a phosphoric acid aqueous solution with a volume percentage concentration of 0.01-1.0% or an acetic acid aqueous solution with a volume percentage concentration of 0.01-0.5%; the column temperature is 20-40 ℃; an ultraviolet detector or a diode array detector is adopted to detect the wavelength of 200-400nm; the flow rate is 0.8ml/min-1.2ml/min; the theoretical plate number of tenuifolin peak is not less than 3000;

preferably, the phase A is acetonitrile, and the phase B is a phosphoric acid aqueous solution with the volume percentage concentration of 0.05 percent; the flow rate is 1.0ml/min; the column temperature is 35 ℃; the detection wavelength is 215nm.

Preferably, when the gradient elution is performed, the gradient elution procedure is as follows: 0 to 1695in, 5% by weight of A; 16-19min, 5% -20% of A;19 to 40min,20% by weight A; 40-50min, 20% -5% A; 50-60min, 5 percent A; the specific gradient elution procedure was:

time (minutes)	Mobile phase A (%)	Mobile phase B (%)
			0～16	5	95
16～19	5→20	95→80
			19～40	20	80
40～50	20→5	80→95
			50～60	5	95

(2) Preparation of control solutions

Taking reference substances including gastrodine, p-hydroxybenzyl alcohol, tenuifolin and cortex et radix Polygalae

Ketone III, 3,6' -di-sinapoyl sucrose, saikosaponin a, saikosaponin d, precisely weighing, and adding methanol to obtain a solution containing 50 μ g gastrodine, 25 μ g p-hydroxybenzyl alcohol, 1.0mg tenuifolin, and 0.15mg senega

Ketone III, 0.2mg 3,6' -di sinapoyl sucrose, 0.4mg saikosaponin a, 0.5mg saikosaponin d mixed solution;

(3) Preparation of test solution

Taking the content of the Xianyu capsule, grinding, weighing 1.0-2.0g, precisely weighing, placing in a conical flask with a plug, precisely adding 50-100ml of 70% methanol, weighing, ultrasonically treating (200W, 40kHz) for 30-90min, cooling, supplementing the lost weight with 70% methanol, shaking uniformly, centrifuging, filtering, and taking the subsequent filtrate.

Preferably, the epilepsy curing capsule content is taken, ground, weighed to be 1.5g, precisely weighed, placed in a conical flask with a plug, precisely added with 50ml of 70% methanol, weighed, ultrasonically treated (200W, 40kHz) for 60min, cooled, complemented with 70% methanol to the loss weight, shaken up, centrifuged, filtered, and the subsequent filtrate is taken, thus obtaining the epilepsy curing capsule.

(4) Measurement of

Precisely sucking 10-20 μ l of each of the reference solution and the sample solution, respectively, injecting into high performance liquid chromatograph, and measuring to obtain measurement result.

The epilepsy healing capsule provided by the invention has the specific formula as follows: radix astragali, radix Codonopsis, saviae Miltiorrhizae radix, bupleuri radix, semen Ziziphi Spinosae, cortex et radix Polygalae, rhizoma Gastrodiae, ramulus Uncariae cum uncis, rhizoma Acori Graminei, rhizoma arisaematis cum bile, radix Angelicae sinensis, bombyx Batryticatus, massa Medicata Fermentata, radix Curcumae, glycyrrhrizae radix, and rhizoma Typhonii preparata. Has effects in eliminating phlegm, inducing resuscitation, tranquilizing mind, arresting convulsion, calming endogenous wind, and relieving spasm.

Compared with the prior art, the invention has the following beneficial technical effects:

the invention provides a detection method for simultaneously detecting a plurality of active ingredients in Xianyu capsules, which adopts the same detection wavelength to detect the active ingredients including gastrodin and parahydroxybenzene methanol in the monarch drug rhizoma gastrodiae and the adjuvant drug radix polygalaeThe effective components of tenuifolin and polygala tenuifolia

The detection method can comprehensively and effectively make up the defects of the existing detection method, can simultaneously detect 7 effective components in the Xianyu capsule, such as monarch drug, adjuvant drug and conductant drug under the same chromatographic condition, effectively controls the quality of the drugs, improves the detection efficiency, reduces the detection cost, associates the quality of the drugs with the effective components of clinical treatment, achieves the stable, controllable, efficient and safe standards, further effectively ensures the quality of the product, can better meet the requirements of the clinical treatment, and provides more bases for the quality standard improvement of the product.

Drawings

The invention is further described with reference to the accompanying drawings in which:

FIG. 1 is a chromatogram of a mixed control of an example of the invention;

FIG. 2 is a chromatogram of a test sample according to an embodiment of the present invention;

FIG. 3 is a chromatogram of a negative sample of Gastrodia elata Blume of an embodiment of the present invention;

FIG. 4 is a chromatogram of a polygala negative sample of an embodiment of the present invention;

FIG. 5 is a chromatogram of a bupleuri radix negative sample according to an embodiment of the present invention;

wherein, in the figure, 1 is gastrodin peak, 2 is p-hydroxybenzyl alcohol peak, 3 is tenuifolin senega saponin peak, 4 is saikosaponin a peak, 5 is 3,6' -erucyl sucrose peak, 6 is saikosaponin d peak, 7 is tenuifolin senega saponin d peak

Ketoiii peak.

FIG. 6 is a linear relationship chart of 7 effective components; in the figure, from top to bottom and from left to right, gastrodine, p-hydroxybenzyl alcohol, tenuifolin and polygala tenuifolia are sequentially

A linear relationship diagram of ketone III, 3,6' -disinapoyl sucrose, saikosaponin a and saikosaponin d.

Detailed Description

The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way. The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials, reagents and materials used in the following examples are all commercially available products unless otherwise specified.

Example 1: prescription of 'Xianyu' capsule and its preparing process

Prescription:

140g of astragalus, 140g of codonopsis pilosula, 140g of salvia miltiorrhiza, 70g of radix bupleuri, 105g of spina date seed, 70g of polygala tenuifolia, 105g of gastrodia elata, 105g of uncaria, 70g of rhizoma acori graminei, 70g of arisaema cum bile, 140g of angelica, 105g of stiff silkworm, 70g of medicated leaven, 70g of radix curcumae, 70g of liquorice and 35g of rhizoma typhonii preparata;

the preparation method comprises the following steps: pulverizing Bombyx Batryticatus and Massa Medicata Fermentata into fine powder, and sieving; decocting the rest fourteen medicines such as astragalus root and the like with water for three times, adding 10 times of water for 2 hours for the first time, adding 8 times of water for 1.5 hours for the second time, adding 6 times of water for 1 hour for the third time, mixing the decoctions, filtering, concentrating the filtrate under reduced pressure to obtain thick paste with the relative density of 1.20-1.25 (60 ℃), adding the fine powder, uniformly mixing, drying in vacuum (60-80 ℃), crushing into fine powder, adding 22g of calcium carbonate and 22g of calcium hydrophosphate, uniformly mixing, granulating by using a proper amount of 80-85% ethanol, drying (75-80 ℃), and filling capsules to prepare 1000 granules.

Example 2: content determination research in Xianyu capsule

2.1 instruments and materials

Shimadzu LC-2030C Plus type high performance liquid chromatograph; AS5150A ultrasonic cleaner, kunshan ultrasonic instruments ltd; GB204 electronic balance, AT201 electronic balance, mettler-toledo; LD4-2 electric centrifuge, jintan City Kongwang laboratory Instrument plant.

Methanol/acetonitrile (chromatographically pure) was purchased from semer fisher technologies; the other reagents are purchased from analytical purifiers and purchased from Tianjin Dagmang chemical reagent factories;

the Xianyu capsule used in the experiment is produced by Qian He pharmaceutical industry Co., ltd. The reference samples were purchased from Shaanxi general medicine science and technology, inc., and produced by the institute of pharmaceutical and biological products in China.

2.2 selection of wavelength

Scanning the gastrodin and p-hydroxybenzene methanol reference substance solution at the wavelength of 200-400nm, wherein the result shows that the reference substance solution has maximum absorption at both 215nm and 220nm, and 215nm with less interference and better separation effect is selected as the detection wavelength considering that the absorption of the effective components at 220nm is also higher but the separation effect is not good.

Scanning the tenuifolin reference substance solution at 200-400nm, wherein the result shows that the absorption is maximum at 210nm and 215nm, and 215nm with less interference and better peak shape is selected as the detection wavelength considering that the absorption of the effective component at 210nm is also larger, but the peak shape is trailing and the interference is more.

Radix Polygalae

The peak of ketone III and the 3,6' -dibapinyl sucrose control solution are scanned at the wavelength of 200-400nm, the result shows that the maximum absorption is generated at 215nm and 320nm, and 215nm with less interference and better separation effect is selected as the detection wavelength considering that the absorption of the effective components at 320nm is also higher but the separation effect is not good.

The saikosaponin a and saikosaponin d reference substance solutions are subjected to wavelength scanning within the range of 200-400nm, the results show that the maximum absorption exists at 210nm and 215nm, and 215nm with less interference and better peak shape is selected as the detection wavelength considering that the absorbance of other components in the prescription is also higher at about 210nm, but the peak shape is trailing and the interference is more.

2.3 selection of the Mobile phase

In order to effectively separate 7 active ingredients in a sample and simultaneously meet the requirements of peak shape, theoretical plate number and the like, the flow properties of acetonitrile-0.5% glacial acetic acid aqueous solution, methanol-0.5% phosphoric acid aqueous solution, methanol-0.1% formic acid aqueous solution, methanol-acetonitrile-0.05% phosphoric acid aqueous solution, acetonitrile-0.05% phosphoric acid aqueous solution and the like in different proportions are respectively adopted for preliminary tests. The isocratic elution separation effect is the best by using acetonitrile-0.05 percent phosphoric acid aqueous solution, the base line is the most stable, but the retention time is long, and partial peaks are overlapped; therefore, the mobile phase A is acetonitrile, and the mobile phase B is phosphoric acid water solution with the volume percentage concentration of 0.05 percent to carry out gradient elution optimization test.

Table 1: results of mobile phase selection test

After repeated experiments, the conventional isocratic system is difficult to effectively separate the 7 effective components, the retention time is too long, or partial components are overlapped, in order to consider the content determination of the 7 effective components in the medicine, a gradient system is carried out, and the results show that the mobile phase is adjusted in different proportions and time for multiple times: the mobile phase system consisting of acetonitrile-0.05 percent of phosphoric acid aqueous solution is adopted, the separation effect is best under the condition of gradient elution, the baseline is stable, chromatographic peaks of 7 components can be completely separated, and the peak time, the peak type and the separation degree of each component meet the requirements, so the condition can meet the related requirements of analysis and determination.

3. Research on specialization

3.1 preparation of each negative sample:

according to the prescription and preparation method of the Xianyu capsule (example 1), negative samples without gastrodia elata, polygala tenuifolia and radix bupleuri are respectively prepared; and (3) preparing a gastrodia elata negative sample: pulverizing Bombyx Batryticatus and Massa Medicata Fermentata into fine powder, and sieving; decocting the rest thirteen medicines such as astragalus root and the like with water for three times, adding 10 times of water for 2 hours for the first time, adding 8 times of water for 1.5 hours for the second time, adding 6 times of water for 1 hour for the third time, mixing the decoction, filtering, concentrating the filtrate under reduced pressure to obtain thick paste with the relative density of 1.20-1.25 (60 ℃), adding the fine powder, uniformly mixing, drying in vacuum (60-80 ℃), crushing into fine powder, adding 22g of calcium carbonate and 22g of calcium hydrophosphate, uniformly mixing, granulating by using a proper amount of 80-85% ethanol, drying (75-80 ℃), and filling capsules to prepare 1000 capsules.

3.2 preparation of negative sample solution:

weighing 1.5g of each negative sample respectively, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 70% methanol, weighing, ultrasonically treating (200W, 40kHz) for 60min, cooling, supplementing the lost weight with 70% methanol, shaking uniformly, centrifuging, filtering, and taking the subsequent filtrate.

3.3 preparation of test solution:

the test method comprises the following steps: taking the contents of the Xianyu capsule, grinding, weighing 1.5g, accurately weighing, placing in a conical flask with a plug, accurately adding 50% methanol, 70% methanol or 100% methanol 50ml, weighing, performing ultrasonic treatment (200W, 40kHz) for 30-90min, cooling, complementing the weight loss, shaking up, centrifuging, filtering, and taking the subsequent filtrate to obtain the Xianyu capsule.

The method comprises the following steps: precisely adding 50ml of methanol, precisely weighing, ultrasonically extracting for 30min, cooling to complement weight, and filtering;

the method 2 comprises the following steps: adding 50ml of 50% methanol, weighing, ultrasonic extracting for 30min, cooling, and filtering;

the method 3 comprises the following steps: precisely adding 70% methanol 50ml, precisely weighing, ultrasonically extracting for 90min, cooling to make up the weight, and filtering;

the method 4 comprises the following steps: precisely adding 70% methanol 50ml, precisely weighing, ultrasonically extracting for 60min, cooling to make up the weight, and filtering;

precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into high performance liquid chromatograph, and measuring to obtain measurement result. The results are detailed in table 2.

Table 2: results of selection test of test article preparation method

The results show that: by adopting the method 1, gastrodin is insufficiently extracted, and peak area is highSignificantly lower than other methods; by adopting the method 2, the effective components of gastrodine, p-hydroxybenzyl alcohol, tenuifolin and polygala tenuifolia

The peak areas for Ketone III and 3,6' -Disinapoylsucrose are significantly lower than for method 4; when the method 3 is adopted, the transfer rate of the other effective components is equivalent to that of the method 4 except that the peak areas of the effective components of the saikosaponin a and the saikosaponin d are slightly lower than that of the method 4, and the saikosaponin a and the saikosaponin d are broken or lost or converted possibly due to overlong ultrasonic time.

Therefore, the preparation method for determining the optimal test solution comprises the following steps: taking the content of the Xianyu capsule, grinding, weighing 1.5g, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 70% methanol, weighing, carrying out ultrasonic treatment (200W, 40kHz) for 60min, cooling, supplementing the loss weight with 70% methanol, shaking uniformly, centrifuging, filtering, and taking the subsequent filtrate.

3.4 chromatographic conditions and System suitability test

Octadecylsilane chemically bonded silica is used as a filling agent; the mobile phase was as specified in Table 1 at a flow rate of 1.0ml/min; the column temperature was 35 ℃; the detection wavelength is 215nm; the sample injection volume is 10 mul; the theoretical plates are not lower than 3000;

preparation of control solutions:

precisely weighing appropriate amount of each reference, and adding methanol to obtain solution containing gastrodin 0.0512mg/ml, p-hydroxybenzyl alcohol 0.0264mg/ml, tenuifolin 1.0251mg/ml, and cortex et radix Polygalae

ketoIII peak 0.1632mg/ml, 3,6' -di-sinapoyl sucrose 0.2215mg/ml, saikosaponin a 0.5215mg/ml, saikosaponin d 0.6253 mg/ml; 1ml of each of the 7 stock solutions was sequentially and precisely aspirated and placed in the same 10ml volumetric flask, dissolved in methanol to a constant volume, and shaken up to obtain mixed control solutions with mass concentrations of 5.12,2.64, 102.51, 16.32, 22.15, 52.15 and 62.53. Mu.g/ml.

Taking the content of the Xianyu capsule, grinding, weighing 1.5g, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of 70% methanol, weighing, carrying out ultrasonic treatment (200W, 40kHz) for 60min, cooling, supplementing the loss weight with 70% methanol, shaking uniformly, centrifuging, filtering, and taking the subsequent filtrate.

Precisely sucking each negative sample solution, mixed reference solution and sample solution 10 μ l each, injecting into high performance liquid chromatograph, and measuring.

The results show that: in the chromatogram of the test solution, the same chromatographic peaks are arranged at the corresponding positions of the chromatographic peaks of the 7 reference substances; the negative is not interfered, and the separation degree and the number of theoretical plates both meet the requirements; indicating that the chromatographic conditions are well specified. See in particular figures 1-5 below.

4. Repeatability test

4.1 chromatographic conditions and System suitability test are as in 3.4.

4.2 precisely absorbing 10 mul of the same sample solution, injecting into a liquid chromatograph, repeatedly injecting for 6 times, and calculating the peak area RSD. The specific results are shown in Table 3.

Table 3: repeatability experiment 7 effective component peak area data table

4.3 results show that the RSD values of the peak areas of the 7 effective components are respectively 0.12%, 1.01%, 0.76%, 0.50%, 0.34%, 0.18% and 1.07%, and are all less than 1.5%; indicating good precision.

5. Linear range of

5.1 chromatographic conditions and System suitability test are the same as 3.4.

5.2 precisely sucking 1.5ml of each mass concentration reference substance respectively, putting the reference substance into a 10ml measuring flask, fixing the volume by using methanol to obtain a mixed reference substance solution, precisely sucking 2 mul, 5 mul, 8 mul, 10 mul, 15 mul and 20 mul of the reference substance solution respectively, detecting according to the chromatographic conditions described in the embodiment 3, recording a chromatogram, performing linear regression analysis on the concentration (X) by using a peak area (Y), drawing a linear relation graph and reporting a linear equation. The results are shown in Table 4, FIG. 6.

Table 4: standard curve equation of 7 components and correlation coefficient and linear range

6. Accuracy (recovery) test

6.1 chromatographic conditions and System suitability test are the same as 3.4.

6.2 preparation of control solutions:

accurately weighing gastrodin reference substance, and adding methanol to obtain reference substance solution with concentration of 52.36 μ g/ml;

6.3 preparation of test solution:

taking and grinding the content of the Xianyu capsule, respectively weighing 9 parts of 0.5g of sample, taking and grinding the content of the Xueping capsule, respectively weighing 0.5g and 9 parts, precisely weighing, placing in a conical flask with a plug, respectively and precisely sucking and weighing 9 parts of gastrodin reference substances of 0.5ml, 1.0ml and 1.5ml, sequentially adding into the conical flask after sample adding, precisely adding 50ml of 70% methanol, weighing, ultrasonically treating (200W, 40kHz) for 60min, cooling, complementing the loss weight with 70% methanol, shaking uniformly, centrifuging, filtering, and taking the subsequent filtrate to obtain the product.

Percent recovery = (C-A)/B × 100%

Wherein A is the amount of the component to be tested contained in the test sample; b is the amount of the added reference substance; c is an observed value.

Table 5: recovery test data sheet

The standard requires that: the recovery rate is 85-110%, and the RSD is less than 4.0%; the experimental result shows that the method has good accuracy.

7. Intermediate precision test

7.1 chromatographic conditions and System suitability test are the same as 3.4.

7.2 in the same laboratory, different experimenters A and B perform experiments on different dates by using different chromatographs, and the content of each effective component is calculated.

Table 6: intermediate precision test data table

The results are shown in Table 6, and show that the inventive method has good precision.

8. Durability test

8.1 chromatographic conditions and System suitability test

Table 7: stability test data sheet

8.2 taking the same sample solution, standing at room temperature for 0, 2, 4, 8, 12 and 24 hours respectively, and precisely sucking 10 mul to inject into a high performance liquid chromatograph; the result is shown in Table 6, and the result shows that the RSD maximum value of the peak retention area of the test solution within 24 hours is only 1.48 percent and is less than the standard 2.0 percent; the test solution was stable for 24h at room temperature.

In summary, the following steps: the content determination method of various active ingredients in the Xianyu capsule verifies the scientificity of the method in the aspects of specificity, repeatability, accuracy (recovery rate), intermediate precision, durability and the like. By measuring the content of the effective components in 7 of the 3 medicinal materials in the prescription, the product quality assurance is further improved, the safety and the effectiveness of the Xianyu capsule are ensured, and the defects of the prior art are overcome.

9. Stability test

9.1 chromatographic conditions and System suitability test are the same as 3.4.

9.2 taking commercially available samples of Xiandu capsules (batch numbers: 20190601, 20190602, 20190701, produced by Xian Qian standing grain pharmaceutical industry Co., ltd.) to carry out an accelerated stability experiment and a room temperature long-term stability experiment. Accelerated test conditions: placing the sample in a stability test box with the relative humidity of 75% +/-5% and the temperature of 40 +/-2 ℃ to carry out 6-month accelerated test investigation, and sampling in 0 th, 1 st, 2 th, 3 th and 6 th months respectively to carry out detection; room temperature experimental conditions: the samples were placed in a stability testing cabinet with a relative humidity of 60% + -5% and a temperature of 25 deg.C + -2 deg.C for a long period of 24 months for experimental investigation, and examined at 0, 3,6, 12, 18, and 24 months, respectively. The test results are shown in Table 8.

Table 8: accelerated and long-term stability test data of Xianyu capsule

The experimental results show that the effective components detected by the test of 6 months of accelerated test and 24 months of long-term test are gastrodine, p-hydroxybenzyl alcohol, tenuifolin and polygala tenuifolia

The contents of ketone III, 3,6' -erucyl sucrose, saikosaponin a and saikosaponin d all meet the requirements, which shows that the detection method can effectively control the quality of the medicine and can be considered to be included in the quality standard. In addition, from the results of detection of 24 months after the end of three consecutive batches of products (batch numbers: 20190601, 20190602, 20190701): the content determination method of 7 effective components in the Xianyu capsule determines the scientificity of the method from the aspects of a repetitive chromatographic expression chart, a test sample solution stability chart, instrument precision, recovery rate, linearity, specificity and the like. The content determination of monarch drug and precious drug of the preparation is increased, the detection cost is reduced, the detection efficiency is improved, the safety and effectiveness of the preparation are ensured, the defects of the prior art are overcome, and meanwhile, the detection accuracy is further improved.

The embodiments given above are preferable examples for implementing the present invention, and the present invention is not limited to the above-described embodiments. Any non-essential addition or replacement made by a person skilled in the art according to the technical features of the technical solution of the present invention is within the scope of the present invention.

Claims

1. A method for determining contents of multiple effective components in XIANYU Capsule by HPLC method is characterized by simultaneously determining gastrodine and p-hydroxybenzyl alcohol in rhizoma Gastrodiae, and tenuifolin and cortex et radix Polygalae saponin in cortex et radix Polygalae by HPLC method

Ketone III, 3,6' -mustard seed acyl sucrose, bupleurum root in saikosaponin a, saikosaponin d content, comprising the following steps:

(1) Chromatographic conditions and System suitability test

Octadecylsilane chemically bonded silica is used as a filling agent; taking acetonitrile as a mobile phase A phase and phosphoric acid aqueous solution with the volume percentage concentration of 0.05 percent as a mobile phase B phase, and performing gradient elution, wherein the gradient elution procedure is as follows: 0 to 1695in, 5% by weight of A;16 to 19min,5 to 20% of A;19 to 40min,20% by weight A; 40-50min, 20% -5% A; 50-60min, 5 percent A; detecting with ultraviolet detector or diode array detector with wavelength of 200-400nm; the flow rate is 0.8ml/min-1.2ml/min; the theoretical plate number of tenuifolin peak is not less than 3000;

(2) Preparation of control solutions

Ketone III, 3,6' -di-sinapoylsucrose, saikosaponin a and saikosaponin d, precisely weighing, and adding methanol to obtain a solution containing 50 μ g gastrodine, 25 μ g p-hydroxybenzyl alcohol, 1.0mg tenuifolin and 0.15mg senega

(3) Preparation of test solution

Taking the content of the Xianyu capsule, grinding, weighing 1.0-2.0g, precisely weighing, placing in a conical flask with a plug, precisely adding 50-100ml of 70% methanol, weighing, ultrasonically treating for 30-90min under the conditions of ultrasonic power of 200W and ultrasonic frequency of 40kHz, cooling, complementing the loss weight with 70% methanol, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the Xianyu capsule;

(4) Measurement of

Respectively sucking 10-20 μ l of each of the reference solution and the sample solution, injecting into high performance liquid chromatograph, and measuring.

2. The HPLC method for determining the content of the active ingredients in Xiyu capsules according to claim 1, wherein the HPLC method comprises the following steps: the epilepsy curing capsule is prepared from the following medicinal materials: radix astragali, radix Codonopsis, saviae Miltiorrhizae radix, bupleuri radix, semen Ziziphi Spinosae, cortex et radix Polygalae, rhizoma Gastrodiae, ramulus Uncariae cum uncis, rhizoma Acori Graminei, rhizoma arisaematis cum bile, radix Angelicae sinensis, bombyx Batryticatus, massa Medicata Fermentata, radix Curcumae, glycyrrhrizae radix, and rhizoma Typhonii preparata.

3. The HPLC method for determining the content of various effective components in Xiyu capsules according to claim 1, wherein: the flow rate is 1.0ml/min; the column temperature was 35 ℃; the detection wavelength is 215nm.

4. The HPLC method for determining the content of various effective components in Xiyu capsules according to claim 1, wherein: grinding the content of XIANYU Capsule, weighing 1.5g, precisely weighing, placing in conical flask with plug, and precisely adding 70%

Weighing 50ml of methanol, carrying out ultrasonic treatment for 60min under the conditions of 200W of ultrasonic power and 40kHz of ultrasonic frequency,

cooling, adding 70% methanol to make up the lost weight, shaking, centrifuging, filtering, and collecting the filtrate.