CN109239250B - Method for measuring fingerprint of brain-benefiting heart tablet and standard fingerprint thereof - Google Patents
Method for measuring fingerprint of brain-benefiting heart tablet and standard fingerprint thereof Download PDFInfo
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Abstract
The invention discloses a method for measuring HPLC (high performance liquid chromatography) fingerprint of a brain-benefiting heart tablet and a standard fingerprint thereof, wherein the method comprises the following steps of: (1) preparation of control solutions: preparing sodium danshensu, protocatechualdehyde, puerarin, penoniflorin, liquiritin, polygonin, and salvianolic acid B reference solutions respectively; (2) preparation of a test solution: extracting the brain heart benefiting tablet, and filtering the extract with microporous membrane to obtain a test solution; (3) the determination method comprises the following steps: the determination is carried out by adopting a high performance liquid chromatography, wherein the chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filler for the chromatographic column; gradient elution is adopted, wherein a mobile phase A is 0.01-3% phosphoric acid water solution, and a mobile phase B is acetonitrile; the flow rate is 0.3-1.5 ml/min; the column temperature is 20-45 ℃; the detection wavelength is 230 nm.
Description
Technical Field
The invention relates to a traditional Chinese medicine fingerprint spectrum analysis method, in particular to an HPLC fingerprint spectrum determination method of a brain-benefiting heart tablet and a fingerprint spectrum of the brain-benefiting heart tablet preparation obtained by the method.
Technical Field
Cardiovascular and cerebrovascular diseases are common diseases seriously endangering human health in recent years. As the quality of life of human beings is improved, cardiovascular and cerebrovascular diseases are on an increasing trend. Although the single-target action strength of the traditional Chinese medicine is weaker than that of western medicine, the traditional Chinese medicine is far better than the western medicine in the advantages of multiple ways, multiple targets, dynamic overall treatment and small toxic and side effects, and particularly the comprehensive treatment effect of the Chinese patent medicine with definite curative effect is better than that of the western medicine.
The brain-benefiting heart tablet is prepared from 15 traditional Chinese medicines of salvia miltiorrhiza, ligusticum wallichii, radix puerariae, earthworm, red paeony root, safflower, radix curcumae, prepared polygonum multiflorum, rhizoma alismatis, medlar, spina date seed (fried), polygala tenuifolia, irkutsk anemone rhizome, achyranthes bidentata and liquorice, and has the effects of promoting blood circulation, removing blood stasis, promoting qi circulation, reducing phlegm, dredging collaterals, relieving pain and the like. Wherein, the salvia miltiorrhiza is the monarch drug of the preparation, the main active ingredients of the salvia miltiorrhiza comprise tanshinol, salvianolic acid B and the like, and the salvia miltiorrhiza has the effects of treating chest and rib pain, rheumatic arthralgia, lump in the abdomen, sore and ulcer gall, traumatic injury pain, irregular menstruation, amenorrhea dysmenorrhea, postpartum stasis pain and the like; paeoniflorin contained in radix Paeoniae Rubra can be used for treating coronary atherosclerotic heart disease, improving body constitution and immunity, relieving inflammation and cough, eliminating phlegm, and relieving asthma, especially can be used as adjuvant medicine in the treatment of senile chronic respiratory diseases; ferulic acid (sodium ferulate) contained in rhizoma Ligustici Chuanxiong has effects of resisting platelet aggregation, enhancing prostaglandin activity, relieving pain, and relieving vasospasm; puerarin in radix Puerariae has effects of improving immunity, enhancing myocardial contraction force, protecting myocardial cell, lowering blood pressure, and resisting platelet aggregation.
The brain-benefiting heart tablet contains 15 traditional Chinese medicinal materials, and only one component of danshensu is detected by content determination of the existing quality standard. In addition, according to the literature reports, the current content measurement method can simultaneously measure 6 effective components in the preparation at most, including danshensu, protocatechuic aldehyde, salvianolic acid B, paeoniflorin, ferulic acid and puerarin.
The existing detection method is as follows:
in the national drug Standard of the State food and drug administration (YBZ04282005-2011Z), the content of danshensu is determined by an HPLC method. Methanol is used as a mobile phase B, 0.5% glacial acetic acid solution is used as a mobile phase A, and the gradient elution procedure is as follows: 0min → 15min → 20min → 25min → 30min, mobile phase A95% → 95% → 70% → 70% → 70% → 95%, the flow rate is 1ml/min, and the detection wavelength is 280 nm. The preparation method of the test solution comprises the following steps: taking 10 tablets of the product, removing a coating, grinding, weighing 2g, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of hydrochloric acid solution (1ml → 100ml), sealing the plug, weighing, carrying out ultrasonic treatment (power 100W, frequency 50KHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with hydrochloric acid solution (1ml → 100ml), shaking up, filtering, precisely weighing 10ml of subsequent filtrate, adding 1g of sodium chloride for dissolving, shaking up and extracting with ethyl acetate for 4 times, 15ml each time, combining ethyl acetate solutions, evaporating to dryness, dissolving residues with mobile phase, transferring to a 5ml measuring flask, adding the mobile phase to a scale, shaking up, filtering, and taking the subsequent filtrate to obtain the product.
Jianghui and Wangshouzhi, etc. adopt HPLC method to simultaneously determine the contents of 6 components in the brain-benefiting and heart-nourishing capsule, including danshensu, protocatechuic aldehyde, salvianolic acid B, paeoniflorin, ferulic acid and puerarin. The mobile phase is acetonitrile-methanol-0.5% phosphoric acid water solution (gradient elution), and the gradient elution procedure is as follows: 0min → 7min → 10min → 35min → 45min, mobile phase A95% → 95% → 70% → 70% → 70% → 95%, the flow rate is 1.0ml/min, the detection wavelengths are 280nm, 230nm, 250nm, 320nm, and the column temperature is 30 ℃. The preparation method of the test solution comprises the following steps: precisely weighing about 0.5g of LINAOXINJIAO Capsule, placing in a 10ml measuring flask, adding ultrapure water for dissolving, performing ultrasonic treatment (power: 80W, frequency: 20kHz) for 30min, cooling to room temperature, adding ultrapure water to supplement volume, centrifuging at centrifugation radius of 5cm and 1500 r/min for 30min, filtering with 0.45 μm microporous membrane, and collecting filtrate.
It has been found that it is difficult to effectively control the overall quality of the composition by measuring the above components, and the safety and effectiveness of the administration cannot be ensured. Further research is needed to control the quality of the Naoxinpian tablet as a whole.
Disclosure of Invention
The invention aims to: provides a fingerprint spectrum measuring method of brain-benefiting heart tablets and a characteristic fingerprint spectrum thereof. The invention provides a new method for the quality control of the brain-benefiting heart tablet by researching the liquid-phase fingerprint of the brain-benefiting heart tablet, overcomes the defect of the prior quality control technology, and ensures that the quality control technology of the brain-benefiting heart tablet is more perfect and scientific.
Therefore, the invention provides a method for establishing a Linaoxing tablet liquid chromatogram standard fingerprint and measuring the Linaoxing tablet in production and sale by using the fingerprint.
An HPLC detection method of a brain-benefiting heart tablet, which comprises the following steps:
(1) preparation of a test solution: extracting the brain heart benefiting tablet, and filtering the extract with microporous membrane to obtain a test solution;
(2) the determination method comprises the following steps: injecting the test solution into a high performance liquid chromatograph to obtain a chromatogram, wherein the chromatogram conditions are as follows:
octadecylsilane chemically bonded silica is used as a filler for the chromatographic column;
gradient elution is carried out, wherein a mobile phase A is 0.01-3% phosphoric acid water solution, and a mobile phase B is acetonitrile; the flow rate is 0.3-1.5 ml/min; the column temperature is 20-45 ℃; elution procedure: 0min → 7min → 10min → 30min → 50min → 65min, acetonitrile 1% → 10% → 15% → 38% → 60% → 60%; the detection wavelength is 230 nm;
(3) comparing the obtained chromatogram with a standard fingerprint, judging the product with similarity of more than 0.75, and evaluating the similarity by adopting '2012 version of a traditional Chinese medicine chromatogram fingerprint similarity evaluation system'.
Wherein the concentration of the reference substance solution in the step (1) is 20-500 mug/mL.
Wherein the weight of the brain-benefiting mind tablets in the step (2) is 0.1-10 g.
In the step (2), the methanol aqueous solution is adopted for ultrasonic extraction or distilled water is adopted for ultrasonic extraction, and the extraction time is 10-80 min.
The aperture of the microporous filter membrane in the step (2) is 0.2-0.8 μm.
Wherein, the detection procedure of the procedure HPLC detection in the step (3) is as follows: the detection wavelength was 230 nm.
And (3) in the determination, injecting 0.5-20 mu l of sample by using an automatic sample injector of a high performance liquid chromatograph, and determining according to the high performance liquid chromatography to obtain the fingerprint.
A method for establishing a standard fingerprint spectrum is characterized by comprising the following steps: the method comprises the following steps:
(1) preparation of reference control solutions:
A. preparation of sodium danshensu reference solution: precisely weighing a proper amount of danshensu sodium reference substance, and preparing a solution containing 20-500 mu g of danshensu per 1ml by using 50% methanol;
B. preparation of protocatechuic aldehyde control solution: precisely weighing a proper amount of protocatechuic aldehyde reference substances, and preparing a solution containing 20-500 mu g of protocatechuic aldehyde per 1ml by using 50% methanol;
C. preparation of puerarin reference solution: precisely weighing a proper amount of puerarin reference substances, and preparing a solution containing 20-500 mu g of puerarin per 1ml by using 50% methanol;
D. preparation of paeoniflorin control solution: precisely weighing a proper amount of paeoniflorin reference substance, and preparing a solution containing 20-500 mu g of paeoniflorin per 1ml by using 50% methanol;
E. preparation of liquiritin reference solution: precisely weighing a proper amount of liquiritin reference substance, and preparing a solution containing 20-500 mu g of liquiritin per 1ml by using 50% methanol;
F. preparation of polygonin reference solution: precisely weighing a proper amount of the polygonin reference substance, and preparing a solution containing 20-500 mu g of the polygonin per 1ml by using 50% methanol;
G. preparation of salvianolic acid B reference solution: precisely weighing a proper amount of salvianolic acid B reference substance, and preparing a solution containing 20-500 mu g of salvianolic acid B per 1ml by using 50% methanol;
(2) preparation of a test solution: weighing multiple batches of qualified brain-benefiting heart tablets, removing coatings, grinding, weighing 0.1-10 g, adding 0-100% methanol water solution, carrying out ultrasonic extraction for 10-80 min, preferably for 30min, and filtering the extract by using a microporous filter membrane (the pore diameter is 0.2-0.8 μm, preferably 0.45 μm) to obtain a test solution;
(3) injecting the reference substance solution and the test substance solution into a high performance liquid chromatograph, wherein the sample volume is 0.5-20 mu l, and obtaining a plurality of batches of qualified chromatogram maps of the heart of the mind;
wherein the chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filler for the chromatographic column; gradient elution is adopted, wherein a mobile phase A is 0.01-3% phosphoric acid water solution, and a mobile phase B is acetonitrile; the gradient elution time is 20-180 min, and the gradient elution procedure is as follows: 0min → 7min → 10min → 30min → 50min → 65min, acetonitrile 1% → 10% → 15% → 38% → 60% → 60%, and a flow rate of 0.3-1.5 ml/min; the column temperature is 20-45 ℃; the detection wavelength is 230 nm.
(4) Fitting the obtained multiple batches of qualified chromatogram maps of the brain-benefiting heart tablets into a standard comparison fingerprint by a computer.
Wherein (1) the preparation method of the reference substance solution is as follows: taking each standard substance, placing into a conical flask with a plug, adding 20ml of 50% methanol precisely, sealing the plug, weighing, ultrasonically treating for 30min, cooling, weighing again, supplementing the lost weight with extraction solvent, filtering with 0.45 μm microporous membrane, and collecting the filtrate as reference control solution.
Wherein (2) the preparation method of the test solution comprises the following steps: taking the brain-benefiting heart tablets, removing the coating, grinding, weighing 1g, placing in a conical flask with a plug, precisely adding 20ml of 50% methanol, sealing the plug, weighing, carrying out ultrasonic treatment for 30min, cooling, weighing again, supplementing the loss weight by using an extraction solvent, filtering by using a 0.45 mu m microporous filter membrane, and obtaining the subsequent filtrate which is the test solution.
Preferably, the liquid phase capillary column used is of the type Eclipse XDB-C18 (4.6X 15cm,3.5 um). The number of batches is at least 10, preferably 20, 30 and 50.
The technical scheme of the invention is obtained by screening, and the screening process is as follows:
1. instruments and reagents
1.1 instruments
A one-tenth-ten-thousandth electronic balance (METTLER TOLEDO), a Waters H-Class liquid chromatograph, and an Agilent Eclipse XDB-C18 (4.6X 15cm,3.5um) chromatographic column.
1.2 reagents
Sodium danshensu reference substance, protocatechualdehyde reference substance, puerarin reference substance, penoniflorin reference substance, polygonin reference substance, liquiritin reference substance, salvianolic acid B reference substance, methanol, acetonitrile
2. Conditions of the liquid chromatography system:
gradient elution is adopted, wherein a mobile phase A is 0.5% phosphoric acid water solution, a mobile phase B is acetonitrile, the elution time is 65min, and the flow rate is 1.0 ml/min; the column temperature is 35 ℃; ultraviolet detection wavelength: 230 nm;
gradient elution procedure: 0min → 7min → 10min → 30min → 50min → 65min, acetonitrile 1% → 10% → 15% → 38% → 60% (volume percentage).
3. Preparation of test solution
Taking the brain-benefiting heart tablets, removing the coating, grinding, weighing 1g, placing in a conical flask with a plug, precisely adding 20ml of 50% methanol, sealing the plug, weighing, carrying out ultrasonic treatment for 30min, cooling, weighing again, supplementing the loss weight by using an extraction solvent, filtering by using a 0.45 mu m microporous filter membrane, and obtaining the subsequent filtrate which is the test solution.
4. Preparation of control solutions
A. Preparation of sodium danshensu reference solution: precisely weighing appropriate amount of sodium danshensu reference substance, and preparing into solution containing 55.8 μ g danshensu per 1ml with 50% methanol;
B. preparation of protocatechuic aldehyde control solution: precisely weighing appropriate amount of protocatechuic aldehyde reference substance, and preparing into solution containing protocatechuic aldehyde 52.6 μ g per 1ml with 50% methanol;
C. preparation of puerarin reference solution: precisely weighing appropriate amount of puerarin reference substance, and preparing into solution containing puerarin 55.3 μ g per 1ml with 50% methanol;
D. preparation of paeoniflorin control solution: accurately weighing appropriate amount of penoniflorin reference substance, and preparing into solution containing penoniflorin 27.6 μ g per 1ml with 50% methanol;
E. preparation of polygonin reference solution: precisely weighing appropriate amount of polygonin reference substance, and preparing into solution containing 25.5 μ g of polygonin per 1ml with 50% methanol;
F. preparation of liquiritin reference solution: precisely weighing appropriate amount of liquiritin reference substance, and preparing into solution containing liquiritin 51.8 μ g per 1ml with 50% methanol;
G. preparation of salvianolic acid B reference solution: precisely weighing appropriate amount of liquiritin reference substance, and preparing into solution containing 86.3 μ g of salvianolic acid B per 1ml with 50% methanol;
5. methodology validation
5.1 extraction time study
Weighing 4 parts of the product in parallel, each 1g, precisely weighing, placing in a conical flask with a plug, precisely adding 20ml of 50% methanol, sealing the plug, weighing, ultrasonically treating for 10min, 20min, 30min and 40min, cooling, weighing again, supplementing the loss by using an extraction solvent, filtering by using a 0.45 mu m microporous filter membrane, and filtering to obtain the final product.
The results of comparative experiments show that the ultrasonic treatment effect is better for 30min and 40 min. Therefore, 30 minutes of sonication was chosen as the extraction time.
5.2 precision test
The same sample solution was sampled and the sample injection was repeated for 6 needles continuously to examine the precision. The results show that the relative retention time RSD of the common peak is less than 0.08 percent, the relative peak area RSD is not more than 2.00 percent, the similarity between 6 repeated sample injection needles is 1.000, and the precision is good. Tables 1 and 2 show the results of the precision evaluation.
TABLE 1 precision test results (relative Retention time)
TABLE 2 precision test results (relative peak area)
5.3 repeatability
6 parts of the same batch of test sample are weighed, and the repeatability is inspected according to the preparation method of the test sample solution and parallel operation. The results show that the relative retention time RSD of the common peak is not more than 0.10 percent, the relative peak area RSD is not more than 3.65 percent, the similarity among 6 samples is 1.000, and the repeatability is good. Tables 3 and 4 show the results of the reproducibility evaluation.
TABLE 3 repeatability test results (relative retention time)
TABLE 4 repeatability test results (relative peak area)
5.4 stability Studies
And (3) injecting the same sample solution into a liquid chromatograph for 0h,2h,4h,6h,8h and 10h respectively. The result shows that the relative retention time RSD of the common peak is not more than 0.06 percent, the relative peak area RSD is not more than 3.05 percent, the similarity is 1.000, and the test solution is stable within 10 hours. Tables 5 and 6 show the results of the reproducibility evaluation.
TABLE 5 stability test results (relative Retention time)
TABLE 6 stability test results (relative peak area)
5.5 sample determination
Extracting 10 batches of brain benefiting heart tablets according to a test article extraction method, measuring according to the fingerprint spectrum measuring method, and recording various spectrograms.
6. Data analysis
And (3) calculating according to 2012 edition of Chinese pharmacopoeia committee 'traditional Chinese medicine chromatogram fingerprint similarity evaluation software' to obtain similarity values of each batch. The sample similarity is 0.75-0.99.
Compared with the prior art, the invention has the improvement points that:
1. the fingerprint detection method of the invention can simultaneously identify at least 7 effective components in the brain-benefiting heart tablet.
2. The standard fingerprint spectrum of the invention has 19 fingerprint characteristic peaks, can reflect the whole quality of the brain-benefiting heart tablet, and effectively represents the content of the main components of the brain-benefiting heart tablet.
The method of the invention has the following remarkable advantages and uses:
1. the invention discloses the establishment of the fingerprint spectrum of the brain-benefiting heart tablet by HPLC detection for the first time, 19 peaks are summarized in total, and the quality of the main components of the brain-benefiting heart tablet can be effectively represented;
2. the fingerprint maps pay attention to the mutual relation of all the formed fingerprint characteristic peaks and the overall facial features, so that the integral quality one-sidedness caused by measuring individual chemical components is avoided, and the possibility of manual treatment for reaching the quality standard is reduced;
3. the test sample is simple and convenient to prepare, and chromatographic conditions are easy to realize;
4. the brain-benefiting heart tablet fingerprint spectrum obtained by the method has multiple peaks, good peak shape, easy identification, high similarity, accuracy and reliability;
5. the quality of the brain-benefiting heart tablet is effectively represented, and the quality monitoring of products is facilitated; has the advantages of simple and stable method, high precision and good reproducibility; the authenticity of the product can be quickly and accurately identified.
The present invention will be further described with reference to the following examples and the accompanying drawings.
Drawings
FIG. 1 Standard fingerprint of Linaoxinpian
FIG. 2 chromatogram of brain-benefiting heart tablet
FIG. 3 is a comparative atlas of the brain-benefiting heart tablet
Figure 4. Linaoxinpian 10 batches of fingerprint
Detailed Description
The present invention will be further described with reference to the following examples in order to understand the present invention in more detail.
Example 1
The preparation method of the test solution in the step (1) comprises the following steps: taking the brain-benefiting heart tablets, removing the coating, grinding, weighing 1g, placing in a conical flask with a plug, precisely adding 20ml of 50% methanol, sealing the plug, weighing, carrying out ultrasonic treatment for 30min, cooling, weighing again, supplementing the loss weight by using an extraction solvent, filtering by using a 0.45 mu m microporous filter membrane, and obtaining the subsequent filtrate which is the test solution.
The preparation concentrations of the reference substance solution in the step (2) are respectively as follows: the concentration of the danshensu reference substance is 55.8 mug/ml, the concentration of the protocatechuic aldehyde reference substance is 52.6 mug/ml, the concentration of the puerarin reference substance is 55.3 mug/ml, the concentration of the paeoniflorin reference substance is 27.6 mug/ml, the concentration of the polygonin reference substance is 25.5 mug/ml, the concentration of the liquiritin reference substance is 51.8 mug/ml, and the concentration of the salvianolic acid B reference substance is 86.3 mug/ml.
And (3) injecting the test solution and the reference solution into a chromatograph to obtain a chromatogram.
Conditions of the liquid chromatography system:
gradient elution is adopted, wherein the mobile phase A is 0.5 percent phosphoric acid water solution, the mobile phase B is acetonitrile, the elution time is 65min, and the flow rate is 1.0 ml/min; the column temperature is 35 ℃; ultraviolet detection wavelength: 230 nm; the chromatographic column was Agilent eclipse XDB-C18 (4.6X 15cm,3.5um), and the sample volume was 10. mu.l.
Gradient elution procedure: 0min → 7min → 10min → 30min → 50min → 65min, acetonitrile 1% → 10% → 15% → 38% → 60% → 60% (volume percentage).
And (3) calculating according to 2012 edition of Chinese pharmacopoeia committee 'traditional Chinese medicine chromatogram fingerprint similarity evaluation software', comparing the similarity of the standard fingerprint with the measured fingerprint, wherein the similarity is qualified when the similarity is 0.75-0.99, and is unqualified when the similarity is less than 0.75.
Example 2
The fingerprint spectrum measurement of the cardiotonic tablets 171202 in batches comprises the following specific steps:
the preparation method of the test solution in the step (1) comprises the following steps: taking the brain-benefiting heart tablets 171202, removing the coating, grinding, weighing 1g, placing in a conical flask with a plug, precisely adding 20ml of double distilled water, sealing the plug, weighing, carrying out ultrasonic treatment for 30min, cooling, weighing again, supplementing the loss weight by using an extraction solvent, filtering by using a 0.45 mu m microporous filter membrane, and obtaining the subsequent filtrate which is the test solution.
The preparation concentrations of the reference substance solution in the step (2) are respectively as follows: the concentration of the danshensu reference substance is 55.8 mug/ml, the concentration of the protocatechuic aldehyde reference substance is 52.6 mug/ml, the concentration of the puerarin reference substance is 55.3 mug/ml, the concentration of the paeoniflorin reference substance is 27.6 mug/ml, the concentration of the polygonin reference substance is 25.5 mug/ml, the concentration of the liquiritin reference substance is 51.8 mug/ml, and the concentration of the salvianolic acid B reference substance is 86.3 mug/ml.
And (3) injecting the test solution and the reference solution into a chromatograph to obtain a chromatogram.
Conditions of the liquid chromatography system:
gradient elution is adopted, wherein the mobile phase A is 0.5 percent phosphoric acid water solution, the mobile phase B is acetonitrile, the elution time is 65min, and the flow rate is 1.0 ml/min; the column temperature is 35 ℃; ultraviolet detection wavelength: 230 nm; the chromatographic column was Agilent eclipse XDB-C18 (4.6X 15cm,3.5um), and the sample volume was 10. mu.l.
Gradient elution procedure: 0min → 7min → 10min → 30min → 50min → 65min, acetonitrile 1% → 10% → 15% → 38% → 60% → 60% (volume percentage).
And (3) calculating according to the 2012 edition of the Chinese pharmacopoeia committee 'traditional Chinese medicine chromatogram fingerprint similarity evaluation software', and comparing the similarity of the standard fingerprint and the measured fingerprint.
And (3) test results: comparing 171202 batches of brain benefiting heart tablets with the standard map, the similarity is 0.88, and the requirement of not less than 0.75 is met.
Example 3
The fingerprint spectrum measurement of the cardiotonic tablets 180303 in batches comprises the following specific steps:
the preparation method of the test solution in the step (1) comprises the following steps: taking the brain-benefiting heart tablets 180303, removing the coating, grinding, weighing 1g, placing in a conical flask with a plug, precisely adding 20ml of double distilled water, sealing the plug, weighing, carrying out ultrasonic treatment for 30min, cooling, weighing again, supplementing the loss weight by using an extraction solvent, filtering by using a 0.45 mu m microporous filter membrane, and obtaining the subsequent filtrate which is the test solution.
The preparation concentrations of the reference substance solution in the step (2) are respectively as follows: the concentration of the danshensu reference substance is 55.8 mug/ml, the concentration of the protocatechuic aldehyde reference substance is 52.6 mug/ml, and the concentration of the puerarin reference substance is 55.3 mug/ml; the concentration of paeoniflorin control is 27.6 μ g/ml; the concentration of the polygonin reference substance is 25.5 mug/ml; the concentration of liquiritin reference substance is 51.8 μ g/ml, and the concentration of salvianolic acid B reference substance is 86.3 μ g/ml.
And (3) injecting the test solution and the reference solution into a chromatograph to obtain a chromatogram.
Conditions of the liquid chromatography system:
gradient elution is adopted, wherein the mobile phase A is 0.5 percent phosphoric acid water solution, the mobile phase B is acetonitrile, the elution time is 65min, and the flow rate is 1.0 ml/min; the column temperature is 35 ℃; ultraviolet detection wavelength: 230 nm; the chromatographic column was Agilent eclipse XDB-C18 (4.6X 15cm,3.5um), and the sample volume was 10. mu.l.
Gradient elution procedure: 0min → 7min → 10min → 30min → 50min → 65min, acetonitrile 1% → 10% → 15% → 38% → 60% → 60% (volume percentage).
And (3) calculating according to the 2012 edition of the Chinese pharmacopoeia committee 'traditional Chinese medicine chromatogram fingerprint similarity evaluation software', and comparing the similarity of the standard fingerprint and the measured fingerprint.
And (3) test results: comparing 180303 batches of brain benefiting heart tablets with the standard map, the similarity is 0.85, and the requirement of not less than 0.75 is met.
Example 4
The fingerprint spectrum measurement of the cardiotonic tablets 180701 in batches comprises the following specific steps:
the preparation method of the test solution in the step (1) comprises the following steps: taking the brain-benefiting heart tablets 180701, removing the coating, grinding, weighing 1g, placing in a conical flask with a plug, precisely adding 20ml of double distilled water, sealing the plug, weighing, carrying out ultrasonic treatment for 30min, cooling, weighing again, supplementing the loss weight by using an extraction solvent, filtering by using a 0.45 mu m microporous filter membrane, and obtaining the subsequent filtrate which is the test solution.
The preparation concentrations of the reference substance solution in the step (2) are respectively as follows: the concentration of the danshensu reference substance is 55.8 mug/ml, the concentration of the protocatechuic aldehyde reference substance is 52.6 mug/ml, the concentration of the puerarin reference substance is 55.3 mug/ml, the concentration of the paeoniflorin reference substance is 27.6 mug/ml, the concentration of the polygonin reference substance is 25.5 mug/ml, the concentration of the liquiritin reference substance is 51.8 mug/ml, and the concentration of the salvianolic acid B reference substance is 86.3 mug/ml.
And (3) injecting the test solution and the reference solution into a chromatograph to obtain a chromatogram.
Conditions of the liquid chromatography system:
gradient elution is adopted, wherein the mobile phase A is 0.5 percent phosphoric acid water solution, the mobile phase B is acetonitrile, the elution time is 65min, and the flow rate is 1.0 ml/min; the column temperature is 35 ℃; ultraviolet detection wavelength: 230 nm; the chromatographic column was Agilent eclipse XDB-C18 (4.6X 15cm,3.5um), and the sample volume was 10. mu.l.
Gradient elution procedure: 0min → 7min → 10min → 30min → 50min → 65min, acetonitrile 1% → 10% → 15% → 38% → 60% → 60% (volume percentage).
And (3) calculating according to the 2012 edition of the Chinese pharmacopoeia committee 'traditional Chinese medicine chromatogram fingerprint similarity evaluation software', and comparing the similarity of the standard fingerprint and the measured fingerprint.
And (3) test results: comparing 180701 batches of brain benefiting heart tablets with the standard map, the similarity is 0.90, and the requirement of not less than 0.75 is met.
Claims (7)
1. An HPLC detection method of a brain-benefiting heart tablet, which comprises the following steps:
(1) preparation of a test solution: taking the brain benefiting heart tablet, ultrasonically extracting the brain benefiting heart tablet by adopting a methanol water solution or ultrasonically extracting the brain benefiting heart tablet by adopting distilled water, wherein the extraction time is 10-80 min, and filtering an extracting solution by using a microporous filter membrane to obtain a test solution;
(2) the determination method comprises the following steps: injecting the test solution into a high performance liquid chromatograph to obtain a chromatogram, wherein the chromatogram condition is as follows:
octadecylsilane chemically bonded silica is used as a filler for the chromatographic column;
gradient elution is carried out, wherein a mobile phase A is 0.01-3% phosphoric acid water solution, and a mobile phase B is acetonitrile; the flow rate is 0.3-1.5 ml/min; the column temperature is 20-45 ℃; elution procedure: 0min → 7min → 10min → 30min → 50min → 65min, acetonitrile 1% → 10% → 15% → 38% → 60% → 60%; the detection wavelength is 230 nm;
(3) comparing the obtained chromatogram with standard fingerprint, determining the product with similarity greater than 0.75, and evaluating the similarity by using Chinese medicinal chromatogram fingerprint similarity evaluation system 2004A edition.
2. The method of claim 1, wherein: the weight of the brain-benefiting heart tablets in the step (1) is 0.1-10 g.
3. The method of claim 1, wherein: the aperture of the microporous filter membrane in the step (1) is 0.2-0.8 μm.
4. The method of claim 1, wherein: and (3) in the determination of the step (2), injecting 0.5-20 mu l of sample by an automatic sample injector of a high performance liquid chromatograph, and determining according to the high performance liquid chromatography to obtain a chromatogram.
5. The method for establishing a standard fingerprint spectrum according to claim 1, wherein the standard fingerprint spectrum comprises the following steps: the method comprises the following steps:
(1) preparation of reference control solutions:
A. preparation of sodium danshensu reference solution: precisely weighing a proper amount of a danshensu sodium reference substance, and preparing a solution containing 20-500 mu g of danshensu sodium per 1ml by using 50% methanol;
B. preparation of protocatechuic aldehyde control solution: precisely weighing a proper amount of protocatechuic aldehyde reference substances, and preparing a solution containing 20-500 mu g of protocatechuic aldehyde per 1ml by using 50% methanol;
C. preparation of puerarin reference solution: precisely weighing a proper amount of puerarin reference substances, and preparing a solution containing 20-500 mu g of puerarin per 1ml by using 50% methanol;
D. preparation of paeoniflorin control solution: precisely weighing a proper amount of paeoniflorin reference substance, and preparing a solution containing 20-500 mu g of paeoniflorin per 1ml by using 50% methanol;
E. preparation of liquiritin reference solution: precisely weighing a proper amount of liquiritin reference substance, and preparing a solution containing 20-500 mu g of liquiritin per 1ml by using 50% methanol;
F. preparation of polygonin reference solution: precisely weighing a proper amount of the polygonin reference substance, and preparing a solution containing 20-500 mu g of the polygonin per 1ml by using 50% methanol;
G. preparation of salvianolic acid B reference solution: precisely weighing a proper amount of salvianolic acid B reference substance, and preparing a solution containing 20-500 mu g of salvianolic acid B per 1ml by using 50% methanol;
(2) preparation of a test solution: weighing multiple batches of qualified brain-benefiting heart tablets, removing coatings, grinding, weighing 0.1-10 g, adding 0-100% methanol water solution, carrying out ultrasonic extraction for 10-80 min, and filtering an extracting solution by using a microporous filter membrane with the pore diameter of 0.2-0.8 mu m to obtain a test solution;
(3) injecting the reference substance solution and the test substance solution into a high performance liquid chromatograph, wherein the sample volume is 0.5-20 mu l, and obtaining a plurality of batches of qualified chromatogram maps of the heart of the mind;
wherein the chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filler for the chromatographic column; gradient elution is adopted, wherein a mobile phase A is 0.01-3% phosphoric acid water solution, and a mobile phase B is acetonitrile; the gradient elution procedure was as follows: 0min → 7min → 10min → 30min → 50min → 65min, acetonitrile 1% → 10% → 15% → 38% → 60% → 60%, and a flow rate of 0.3-1.5 ml/min; the column temperature is 20-45 ℃; the detection wavelength is 230 nm;
(4) fitting the obtained multiple batches of qualified chromatogram maps of the brain-benefiting heart tablets into a standard comparison fingerprint by a computer.
6. The method of establishing according to claim 5, wherein: wherein (1) the preparation method of the reference substance solution is as follows: taking each standard substance, placing into a conical flask with a plug, adding 20ml of 50% methanol precisely, sealing the plug, weighing, ultrasonically treating for 30min, cooling, weighing again, supplementing the lost weight with 50% methanol, filtering with a 0.45 μm microporous membrane, and collecting the filtrate as reference control solution.
7. The method of establishing according to claim 5, wherein: wherein (2) the preparation method of the test solution comprises the following steps: taking the brain-benefiting heart tablets, removing the coating, grinding, weighing 1g, placing in a conical flask with a plug, precisely adding 20ml of 50% methanol, sealing the plug, weighing, carrying out ultrasonic treatment for 30min, cooling, weighing again, supplementing the loss weight by using an extraction solvent, filtering by using a 0.45 mu m microporous filter membrane, and obtaining the subsequent filtrate which is the test solution.
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