1. A method for measuring the content of stilbene glucoside in brain-benefiting heart tablets comprises the following steps:

step 1, preparation of a reference solution: taking stilbene glucoside reference substance, adding diluted ethanol to obtain reference substance solution;

step 2, preparation of a test solution: taking the brain heart benefiting tablet, adding a proper amount of dilute ethanol solution, performing ultrasound for 30min, and filtering to obtain filtrate as a test solution;

and 3, injecting the reference solution and the test solution into a high performance liquid chromatograph, recording a chromatogram, and calculating the content of the stilbene glucoside in the test solution according to the peak area of the chromatogram and an external standard method.

2. The method according to claim 1, characterized by the steps of:

step 1, preparation of a reference solution: taking a proper amount of stilbene glucoside reference substance, precisely weighing, and adding diluted ethanol to prepare a solution containing 20-60 μ g of stilbene glucoside per 1 ml;

step 2, preparation of a test solution: grinding the brain-benefiting heart-mind tablets, weighing 1-3g, adding appropriate amount of diluted ethanol, performing ultrasonic treatment, fixing volume, shaking up after quantification, and filtering to obtain;

and 3, injecting 5-20 mu l of the reference solution and the test solution into a high performance liquid chromatograph, recording a chromatogram, and calculating the content of the stilbene glucoside in the test solution according to the peak area of the chromatogram and an external standard method.

3. The method according to claim 1, characterized by the steps of:

step 1, preparation of a reference solution: taking a proper amount of stilbene glucoside reference substance, precisely weighing, and adding diluted ethanol to prepare a solution containing 40 μ g of stilbene glucoside in each 1 ml;

step 2, preparation of a test solution: taking 10 tablets of the product, removing the coating, grinding, weighing 1g, precisely weighing, placing in a conical flask with a plug, precisely adding 30-50ml of dilute ethanol, sealing the plug, weighing, carrying out ultrasonic treatment (power 250W, frequency 40kHz) for 30 minutes, cooling, weighing again, complementing the weight loss by dilute ethanol, shaking up, and filtering (through a 0.22 mu m filter membrane), thus obtaining the product;

and 3, injecting 10 mu l of the reference solution and the test solution into a high performance liquid chromatograph, recording a chromatogram, and calculating the content of the stilbene glucoside in the test solution according to the peak area of the chromatogram and an external standard method.

4. The method of claim 1, wherein the chromatographic conditions are as follows:

the instrument comprises the following steps: agilent 1200

A chromatographic column: agilent Eclipse XDB-C18 (4.6X 250mm,5 μm)

A detector: DAD detector

Mobile phase: acetonitrile-water 15:85

Detection wavelength: 320 nm.

Flow rate: 1.0ml/min

Sample introduction amount: 10 μ l

The number of theoretical plates should not be less than 2000 calculated by stilbene glycoside peak.

Background

The traditional Chinese medicine is named as brain-heart benefiting tablet. Is prepared from Saviae Miltiorrhizae radix, rhizoma Ligustici Chuanxiong, radix Puerariae, Lumbricus, radix Paeoniae Rubra, Carthami flos, radix Curcumae, radix Polygoni Multiflori Preparata, Alismatis rhizoma, fructus Lycii, parched semen Ziziphi Spinosae, cortex et radix Polygalae, rhizoma anemones Altaicae, Achyranthis radix, and Glycyrrhrizae radix. Has effects of promoting blood circulation, dispelling blood stasis, promoting qi circulation, eliminating phlegm, dredging collaterals, and relieving pain. Can be used for treating stagnation of qi and blood stasis, stagnation of phlegm and obstruction of collaterals, thoracic obstruction, stabbing pain, angina pectoris, dizziness, headache, coronary heart disease, myocardial infarction, cerebral arteriosclerosis, and cerebral thrombosis.

The brain-benefiting heart tablet is prepared by the following process: decocting Saviae Miltiorrhizae radix, Polygoni Multiflori radix (preparata), fructus Lycii, radix Paeoniae Rubra, radix Puerariae and Lumbricus with appropriate amount of water for three times, 3 hr for the first time, 2 hr for the second time, and 1 hr for the third time, filtering, mixing filtrates, and concentrating under reduced pressure to obtain fluid extract with relative density of 1.2-1.5; and crushing a certain amount of nine ingredients of ligusticum wallichii, safflower, curcuma aromatica, rhizoma alismatis, spina date seed, polygala tenuifolia, irkutsk anemone rhizome, achyranthes bidentata, liquorice and the like into fine powder, sieving the fine powder by a 100-mesh sieve, adding the fine powder into the clear paste, uniformly mixing, drying, crushing the dry paste into fine powder, adding 5g of carboxymethyl starch sodium and 40g of microcrystalline cellulose, wetting the fine powder by 60 percent ethanol, granulating, drying, adding 2g of magnesium stearate and adjusting the total amount of starch to 400g, pressing the mixture into 1000 tablets, coating a film coat and packaging to obtain the tablet. "

The existing detection method is as follows:

taking 15 tablets of the product, removing a film coat, grinding, taking 4g, adding 50ml of methanol, carrying out ultrasonic extraction for 30 minutes, filtering, evaporating filtrate to dryness, adding 15ml of water into residues for dissolving, shaking and extracting for 3 times by using diethyl ether, 20ml each time, combining ethyl ether solutions, shaking and extracting for 3 times by using ethyl acetate, 20ml each time, combining ethyl acetate solutions, adding 20ml of 5% sodium bicarbonate solution for washing for 1 time, drying the ethyl acetate solutions to dryness, and adding 5ml of methanol into residues for dissolving to obtain a sample solution. Taking 2,3,5, 4' -tetrahydroxystilbene-2-O-beta-D-glucoside reference substance, adding methanol to obtain solution containing 0.2mg per 1ml as reference substance solution. Performing thin layer chromatography (SOP-QC-6060) test, sucking 5 μ l of the above two solutions, respectively dropping on the same silica gel G thin layer plate, developing with toluene-acetone-methanol (6:1:2) as developing agent, taking out, air drying, inspecting under ultraviolet lamp (365nm), and displaying fluorescent spots of the same color on the corresponding positions of the sample chromatogram and the control chromatogram.

The defects of the prior art are as follows:

the stilbene glucoside in the original quality standard is identified by thin-layer chromatography, which can only be qualitative and cannot be quantitative. The method has the advantage that the content of the stilbene glucoside in the brain benefiting heart tablet is accurately measured by adopting an HPLC-UV external standard method. The new detection method is advanced, has good precision and reproducibility, can better control the quality of the heart and brain tablets, and ensures the stability and uniformity of the product quality and the medication safety.

Disclosure of Invention

The invention provides a method for measuring the content of stilbene glucoside in a brain-benefiting heart tablet, which comprises the following steps:

step 1, preparation of a reference solution: taking stilbene glucoside reference substance, adding diluted ethanol to obtain reference substance solution;

step 2, preparation of a test solution: taking the brain heart benefiting tablet, adding a proper amount of dilute ethanol solution, performing ultrasound for 30min, and filtering to obtain filtrate as a test solution;

step 3, preparation of negative control solution: weighing other medicinal materials except the radix polygoni multiflori preparata according to the prescription, weighing other medicinal materials and auxiliary materials which do not contain the radix polygoni multiflori preparata according to the prescription, the extraction process and the preparation process under the same prescription proportion to prepare final granules before tabletting of the radix polygoni multiflori preparata, and then preparing a radix polygoni multiflori preparata negative sample solution according to the preparation method of the test solution in the step 2;

and 4, injecting the reference substance solution, the test solution and the negative reference solution into a high performance liquid chromatograph, recording a chromatogram, and calculating the content of the stilbene glucoside in the test solution according to the peak area of the chromatogram and an external standard method.

In the above method, the negative control solution is optional, i.e., the negative control solution may or may not be used during the assay.

Preferably, the method of the invention comprises the following steps:

step 1, preparation of a reference solution: taking a proper amount of stilbene glucoside reference substance, precisely weighing, and adding diluted ethanol to prepare a solution containing 20-60 μ g of stilbene glucoside per 1 ml;

step 2, preparation of a test solution: grinding the brain-benefiting heart-mind tablets, weighing 1-3g, adding appropriate amount of diluted ethanol, performing ultrasonic treatment, fixing volume, shaking up after quantification, and filtering to obtain;

and 3, injecting 5-20 mu l of the reference solution and the test solution into a high performance liquid chromatograph, recording a chromatogram, and calculating the content of the stilbene glucoside in the test solution according to the peak area of the chromatogram and an external standard method.

Most preferably, the method of the present invention comprises the following steps:

step 1, preparation of a reference solution: taking a proper amount of stilbene glucoside reference substance, precisely weighing, and adding diluted ethanol to prepare a solution containing 40 μ g of stilbene glucoside in each 1 ml;

step 2, preparation of a test solution: taking 10 tablets of the product, removing the coating, grinding, weighing 1g, precisely weighing, placing in a conical flask with a plug, precisely adding 30-50ml of dilute ethanol, sealing the plug, weighing, carrying out ultrasonic treatment (power 250W, frequency 40kHz) for 30 minutes, cooling, weighing again, complementing the weight loss by dilute ethanol, shaking up, and filtering (through a 0.22 mu m filter membrane), thus obtaining the product;

step 3, injecting 10 mu l of reference solution and test solution into a high performance liquid chromatograph, recording a chromatogram, and calculating the content of stilbene glucoside in the test solution according to the peak area of the chromatogram and an external standard method;

1. the chromatographic conditions were as follows:

the instrument comprises the following steps: agilent 1200

A chromatographic column: agilent Eclipse XDB-C18 (4.6X 250mm,5 μm)

A detector: DAD detector

Mobile phase: acetonitrile-water 15:85

Detection wavelength: 320 nm.

Flow rate: 1.0ml/min

Sample introduction amount: 10 μ l

The number of theoretical plates should not be less than 2000 calculated by stilbene glycoside peak.

The chromatographic conditions (proportion and flow rate of mobile phase) of the invention are obtained by screening, and the screening process is as follows:

1.1 selection of the proportions of the mobile phases

The influence of different mobile phase ratios (13% acetonitrile, 15% acetonitrile, 17% acetonitrile) on the chromatographic peak of stilbene glucoside was examined. As can be seen from FIG. 2, the separation from impurities is poor when the peak is formed by using the mobile phase 17% acetonitrile, and the peak is formed by using the mobile phase 13% acetonitrile after the peak is formed; the 15% acetonitrile has better peak shape and better separation from impurities, so the 15% acetonitrile can be selected.

1.2 selection of different flow rates

The influence of different flow rates (0.8mL/min, 1.0mL/min, 1.2mL/min) on the chromatographic peak of stilbene glucoside was examined. As can be seen from FIG. 3, the flow rate had a greater effect on the time to peak of stilbene glucoside, with the 0.8mL/min flow rate having a later time to peak. Meanwhile, the magazine has a large influence on the target peak. And the peak change is smaller within the range of 1.0mL/min to 1.2 mL/min. Thus, a flow rate of 1.0mL/min can be selected.

1.3 selection of different sample sizes

The effect of different sample volumes (10. mu.L, 20. mu.L) on the chromatographic peak of stilbene glucoside was examined. As seen from FIG. 4, the impurity peak area increased with the increase of the amount of the sample to 20. mu.L, but the degree of separation of stilbene glucoside was not greatly affected, and 10. mu.L having a small amount of the sample was selected as the condition.

The method of the invention is proved to have good effect by verification.

2 methodology examination

2.1 specificity

Respectively and precisely absorbing 10ul of each of the test solution, the negative sample solution, the reference solution and the blank solution, injecting the solution into a chromatograph, recording a spectrum, and showing a result in a figure 1: the stilbene glucoside has better peak shape, the separation condition of the target component peak and the impurity peak is good, and no negative interference exists.

2.2 sample tightness

Taking a proper amount of stilbene glucoside reference substance, adding a proper amount of diluted ethanol to prepare 450.8 mu g/ml reference substance mother liquor. Precisely measuring 1.0mL of reference product mother solution, placing in a 10mL volumetric flask, respectively injecting 6 needles with 10 muL of each, and recording the retention time and peak area of the component to be measured. The retention time of stilbene glucoside in the reference solution and the RSD value of peak area are respectively 0.46% and 0.32%, see Table 1, the result shows that the precision of sample introduction of the instrument is good.

TABLE 1 sample introduction precision results

2.2 linearity

Taking a proper amount of stilbene glucoside reference substance, adding a proper amount of diluted ethanol to prepare 450.8 mu g/ml reference substance mother liquor. Precisely measuring the reference product mother liquor by 0.1 mL, 0.2 mL, 0.5 mL, 1.0mL, 1.2mL and 1.5mL, respectively placing in 10mL volumetric flasks, respectively adding ethanol to dilute to scale marks, shaking up, precisely injecting 10 μ L of sample, and recording the peak area of the component to be measured.

Drawing a standard curve graph through statistical experimental data to obtain a linear regression equation as follows: 0.5904x +0.0425, R²0.9990, which shows that stilbene glucoside has a good linear relationship with the peak area in the range of 4.058-60.88. mu.g/mL.

2.3 repeatability

Taking the brain heart benefiting tablets, removing the film coating, grinding, and sieving with a 100-mesh sieve. Weighing 1.0g, precisely weighing, weighing 6 parts in parallel to obtain test solution, injecting sample respectively, each 10 μ L, recording peak area of component to be measured, and calculating content and RSD value.

The average stilbene glucoside content value is 0.6600mg/g after 6 repeated determinations, the RSD is 0.34% (n is 6) (the RSD is less than 2%), and the content accords with the specification of 2015 version Chinese pharmacopoeia. The result shows that the method has good repeatability. See table below.

TABLE 2 repeatability results

2.4 stability

Taking the test solution under the repeatability item, respectively sampling and analyzing for 0, 2, 4, 6, 8, 10, 12, 16, 20 and 24h, respectively, 10 mu L, and recording the peak area of the component to be measured.

The RSD value of the peak area of the stilbene glucoside within 24 hours is 1.30 percent, and is shown in the following table. The results show that the test solution is stable within 24h at room temperature.

TABLE 3 stability results

Time (h)	Peak area
		0	14.588
2	14.724
		4	14.731
6	14.934
		8	14.779
10	14.849
		12	14.797
16	14.445
		20	14.487
24	14.351
		Mean value	14.669
RSD(％)	1.30

2.5 degree of accuracy

Accurately weighing a proper amount of stilbene glucoside control, and preparing a stilbene glucoside control solution with the concentration of 0.01342 mg/mL.

Taking the known content of the brain benefiting heart tablets, removing the film coating, grinding and sieving by a 100-mesh sieve. Precisely weighing about 0.5g, weighing 6 parts in parallel, precisely transferring 25mL of the prepared reference substance adding solution, preparing a sample solution according to the method under item 2.2.1 in this section, respectively injecting 10 μ L of sample, recording peak areas of components to be detected, and calculating sample injection recovery rate.

The results show (see the table below) that the average sample recovery rate of the method is 103.6%, and the RSD% value is 1.10% (RSD < 2%), which meets the requirements. The method is proved to be good in accuracy.

TABLE 4 accuracy results

2.6 sample determination

Taking 3 batches of samples of batch No. 1, batch No. 2 and batch No. 3, respectively preparing sample solutions, precisely injecting 10 mu L of the sample solutions, recording peak areas, and calculating the content of the stilbene glucoside, wherein the results are shown in the following table.

TABLE 5 results of content measurement of samples

Batch number	Stilbene glucoside content (mg/g)	RSD(％)
			1	0.6553±0.0006	0.09
2	0.6955±0.0075	1.07
			3	0.7387±0.0012	0.16

The result shows that the content of the stilbene glucoside in the three batches of samples measured by the liquid phase method has good result. The invention does not adopt the existing method for detecting the stilbene glucoside in the Xinnaoxing tablet, but develops a new high performance liquid chromatography, and the improved method can accurately determine the stilbene glucoside content in the Xinnaoxing tablet, can effectively control the quality of the Xinnaoxing tablet and is more beneficial to the effectiveness and safety of medication.

Drawings

FIG. 1 a specificity graph

FIG. 2 comparison of different flow phase ratios

FIG. 3 comparison of different flow chromatograms

FIG. 4 comparison of chromatograms for different sample volumes

Detailed Description

Example 1

Preparation of a test solution: the brain-benefiting heart tablets (batch No. 1) are taken, the film coating is removed, the tablets are ground and sieved by a 100-mesh sieve. Weighing about 1.0g, precisely weighing, precisely transferring 25mL of dilute ethanol, adding into a conical flask with a plug, and weighing. Ultrasonic treatment for 30min (power 250W, frequency 40 kHz). Taking out, cooling to room temperature, weighing, complementing weight loss, shaking, filtering, and filtering with 0.45 μm filter membrane.

Preparation of control solutions: precisely weighing about 10mg of stilbene glucoside reference substance, placing in a 25mL volumetric flask, adding diluted ethanol for dissolving, fixing the volume to a scale mark, and shaking up to obtain the reference substance mother liquor with the concentration of 0.4059 mg/mL.

And precisely injecting 10 mu L of sample, recording peak areas, and calculating the content of the stilbene glucoside, wherein the results are shown in the following table.

Results of sample content measurement

Batch number	Stilbene glucoside content (mg/g)	RSD(％)
			Batch number 1	0.6553±0.0006	0.09

Example 2

Preparation of a test solution: the brain-benefiting heart tablets (batch No. 2) are taken, the film coating is removed, the tablets are ground and sieved by a 100-mesh sieve. Weighing about 1.0g, precisely weighing, precisely transferring 25mL of dilute ethanol, adding into a conical flask with a plug, and weighing. Ultrasonic treatment for 30min (power 250W, frequency 40 kHz). Taking out, cooling to room temperature, weighing, complementing weight loss, shaking, filtering, and filtering with 0.45 μm filter membrane.

Preparation of control solutions: precisely weighing about 10mg of stilbene glucoside reference substance, placing in a 25mL volumetric flask, adding diluted ethanol for dissolving, fixing the volume to a scale mark, and shaking up to obtain the reference substance mother liquor with the concentration of 0.4059 mg/mL.

And precisely injecting 10 mu L of sample, recording peak areas, and calculating the content of the stilbene glucoside, wherein the results are shown in the following table.

Results of sample content measurement

Batch number	Stilbene glucoside content (mg/g)	RSD(％)
			Batch number 2	0.6955±0.0075	1.07

Example 3

Preparation of a test solution: the brain heart benefiting tablets (batch 3) are taken, the film coating is removed, the tablets are ground and sieved by a 100-mesh sieve. Weighing about 1.0g, precisely weighing, precisely transferring 25mL of dilute ethanol, adding into a conical flask with a plug, and weighing. Ultrasonic treatment for 30min (power 250W, frequency 40 kHz). Taking out, cooling to room temperature, weighing, complementing weight loss, shaking, filtering, and filtering with 0.45 μm filter membrane.

Preparation of control solutions: precisely weighing about 10mg of stilbene glucoside reference substance, placing in a 25mL volumetric flask, adding diluted ethanol for dissolving, fixing the volume to a scale mark, and shaking up to obtain the reference substance mother liquor with the concentration of 0.4059 mg/mL.

And precisely injecting 10 mu L of sample, recording peak areas, and calculating the content of the stilbene glucoside, wherein the results are shown in the following table.

Results of sample content measurement

Batch number	Stilbene glucoside content (mg/g)	RSD(％)
			Batch No. 3	0.7387±0.0012	0.16