Method for determining contents of forsythin and amygdalin in mulberry leaf and chrysanthemum flower cold granules

Abstract

The invention discloses a method for determining the content of forsythin and amygdalin in Sangju cold granules through high performance liquid chromatography. The method comprises the following steps: preparing a methanol contrast solution of phillyrin (30 [mu] g / ml) and amygdalin (0.1 mg / ml), taking about 2g of Sangju cold granules, adding 25ml of methanol, carrying out ultrasonic treatment for 15-60 minutes, filtering, and taking the subsequent filtrate as a sample solution; high performance liquid chromatography is adopted, phillyrin uses a C18 column, amygdalin uses a Phenyl column, the flow rate is 1 ml / min, and the column temperature is 30 DEG C. Injecting 10 microliters of solutions into a chromatographic instrument, recording peak areas, and calculating the content by using an external standard method. The method has the advantages of high sensitivity, good separation effect, strong repeatability and the like, and can provide technical support for improving the quality standard of the mulberry leaf and chrysanthemum cold granules.

Description

Technical Field

[0001] The invention relates to a detection technology for effective ingredients in a traditional Chinese medicine preparation, and in particular to a method for determining the contents of forsythiaside and amygdalin in Sangju Ganmao granules by high performance liquid chromatography. Background Art

[0002] Sangju Ganmao Granule is a classic Chinese patent medicine preparation, and its prescription is included in the "Chinese Herbal Medicine Formula Preparation" (Volume 2), and the implementation standard is WS3-B-0397-90. The medicine is composed of 8 Chinese medicinal materials, including chrysanthemum, mulberry leaf, forsythia, reed root, licorice, bitter almond, mint, and platycodon. The various medicinal materials in the prescription are reasonably matched to play the role of dispelling wind and heat, promoting lung function and relieving cough. It is an important medicine for treating wind-heat cold, sore throat, cough and phlegm.

[0003] Specifically, the chrysanthemum in the prescription has the effect of calming liver fire and dispersing wind-heat, which can not only relieve cold headaches, but also clear the liver and improve eyesight, and assist in treating fever, sore throat and other symptoms caused by wind-heat colds; mulberry leaves are good at nourishing yin and blood, clearing lungs and moistening dryness, clearing heat and dispersing wind, and are often used to treat symptoms of exogenous wind-heat, coughs, etc. Chrysanthemum and mulberry leaves complement each other. The combination of the two can significantly enhance the effect of clearing the lungs and relieving coughs, and dispersing wind-heat in the upper part of the body, which is one of the core ingredients of this prescription.

[0004] In addition, the role of Forsythia suspensa in this prescription cannot be ignored. It has the effects of reducing swelling and dispersing nodules, clearing away heat and detoxifying, and evacuating wind-heat. It is especially suitable for treating symptoms such as sore throat and headache caused by exogenous wind-heat, and it also has a good auxiliary therapeutic effect on infectious diseases caused by heat toxins. Phragmites australis is known for clearing heat and promoting body fluid, quenching thirst and vomiting, and removing phlegm and relieving cough. It can be used to relieve symptoms such as dry mouth and tongue, cough and phlegm associated with colds. It also has a good therapeutic effect on cough caused by lung heat and spitting pus caused by lung abscesses.

[0005] Licorice is a commonly used blending ingredient in traditional Chinese medicine prescriptions. It not only plays a role in blending various medicines, but also has the effects of clearing away heat and detoxifying, moistening the lungs and relieving coughs. It is often used in clinical practice to treat symptoms such as sore throat, asthma and coughs, and can also alleviate the toxicity and potency of other drugs. Bitter almonds are good at moistening the intestines and relieving constipation, relieving asthma and relieving coughs. The amygdalin it contains can produce trace amounts of hydrocyanic acid after metabolism in the body, which has a significant antitussive and antiasthmatic effect. It is particularly suitable for treating symptoms such as cough, sputum, and asthma caused by lung qi failure.

[0006] Peppermint is a pungent and cool antipyretic medicine that has the effects of sweating, relieving fever, dispersing wind-heat, and clearing the throat. It can effectively relieve symptoms such as sore throat, headache, and fever caused by colds when taken internally, and can also relieve skin itching, eczema, and other symptoms when used externally. Platycodon grandiflorum is an important lung-clearing and expectorant medicine that has the effects of clearing lungs and draining pus, relieving cough and expectoration, and reducing inflammation. Its active ingredients can promote the discharge of respiratory secretions and have a significant effect on relieving symptoms such as cough and sore throat.

[0007] The eight herbs in this prescription work together to dispel wind and heat, clear the lungs and relieve cough through scientific compatibility and synergy, and are especially suitable for patients with the initial onset of wind-heat cold or with cough and sore throat, and have a wide range of clinical application value. However, the current drug implementation standard (WS3-B-0397-90) does not yet include requirements for the content determination of key active ingredients, which to some extent limits the comprehensive evaluation and control of the drug's quality.

[0008] Since forsythiaside and amygdalin are two important active ingredients in Sangju Cold Granules, their contents directly affect the efficacy and quality stability of the drug, so it is of great significance to accurately determine their contents. Forsythiaside is the main chemical component extracted from Forsythia suspensa, which has anti-inflammatory, antibacterial, antiviral and other pharmacological effects, and is of great significance for relieving cold symptoms and inhibiting pathogenic microorganisms. Amygdalin is an active ingredient present in almonds. It can be decomposed by enzymes in the body to produce benzaldehyde and hydrocyanic acid with antitussive and antiasthmatic effects, significantly improving the patient's cough symptoms. The contents of these two ingredients not only directly affect the efficacy of Sangju Cold Granules, but are also closely related to its quality stability. However, due to the complex ingredients of traditional Chinese medicine preparations, differences in factors such as the source of medicinal materials, processing technology and storage conditions, the contents of forsythiaside and amygdalin may fluctuate greatly, thus affecting product consistency and clinical efficacy.

[0009] At present, there is still a lack of unified and accurate analytical methods for the determination of forsythiaside and amygdalin in Sangju Ganmao Granules. Traditional detection methods, such as thin layer chromatography or ultraviolet spectrophotometry, have certain limitations in sensitivity, accuracy and repeatability, and are difficult to meet the requirements of modern Chinese medicine quality control. In addition, the complex composition of Chinese medicine and the large number of interferences also pose higher challenges to detection technology. Therefore, the development of an efficient, rapid and accurate content determination method will not only help to achieve effective control of the quality of Sangju Ganmao Granules, but also provide technical support for optimization and adjustment in the production process. It is of great significance in improving the quality of drugs and ensuring the safety of patients' medication. It also lays the foundation for the modernization and standardization of Chinese medicine. Summary of the invention

[0010] The technical problem to be solved by the present invention is to provide a detection method based on high performance liquid chromatography (HPLC) that can accurately determine the content of forsythiaside and amygdalin in Sangju Ganmao Granules, thereby effectively controlling the intrinsic quality of the drug. The method has the advantages of high sensitivity, good separation effect, strong repeatability, etc., and can provide technical support for the improvement of the quality standard of Sangju Ganmao Granules.

[0011] To achieve the above object, the present invention adopts the following technical solutions:

[0012] To achieve the above object, the present invention provides a method for detecting the content of forsythiaside and amygdalin in Sangju Ganmao granules, the method specifically comprising the following steps:

[0013] A method for detecting the content of forsythiaside and amygdalin in Sangju Ganmao granules, the method comprising the following steps:

[0014] Step (1) Preparation of reference solution:

[0015] Take an appropriate amount of forsythin reference substance, weigh it accurately, and add methanol to make a solution containing 30 μg forsythin per 1 ml;

[0016] Take an appropriate amount of amygdalin reference substance, weigh it accurately, and add methanol to make a solution containing 0.1 mg of amygdalin per 1 ml;

[0017] Step (2) Preparation of the sample solution to be tested:

[0018] Take about 2g of Sangju Ganmao Granules, weigh accurately, add 25ml of methanol, and ultrasonically treat for 15 to 60 minutes. After filtering, take the filtrate as the sample solution to be tested;

[0019] Step (3) Chromatographic conditions:

[0020] The chromatographic conditions of forsythin are as follows: the filler is octadecylsilane bonded silica gel (C18 column), the mobile phase is acetonitrile-water = 18.6-19.4:81.4-80.6 (volume ratio), the detection wavelength is 230nm, the flow rate is 0.8-1.2ml / min, and the column temperature is 25-35°C;

[0021] The chromatographic conditions of amygdalin are as follows: the filler is phenylsilane bonded silica gel (Phenyl column), the mobile phase is methanol-water = 10.7-11.3:89.3-88.7 (volume ratio), the detection wavelength is 207nm, the flow rate is 0.8-1.2ml / min, and the column temperature is 30-35°C;

[0022] Step (4) Content determination: 10 μl of the reference solution and the sample solution to be tested were accurately aspirated and injected into a high performance liquid chromatograph, the peak areas were recorded, and the contents of forsythiaside and amygdalin in the sample to be tested were calculated according to the external standard method.

[0023] The ultrasonic treatment time in step (2) is 30 to 50 minutes.

[0024] The filler specification in the forsythiaside chromatography conditions described in step (3) is an octadecylsilane bonded silica gel column of 5 μm and 4.6×250 mm.

[0025] The mobile phase ratio in the forsythiaside chromatography conditions described in step (3) is acetonitrile-water = 19:81 (volume ratio).

[0026] The filler specification in the amygdalin chromatography conditions described in step (3) is a 5 μm, 4.6×250 mm phenylsilane bonded silica gel column.

[0027] The mobile phase ratio in the amygdalin chromatography conditions described in step (3) is methanol-water = 11:89 (volume ratio).

[0028] The preparation of the sample solution to be tested in step (2) comprises the following steps: taking 2 g of Sangju Ganmao Granules, grinding them into powder, accurately weighing them, placing them in a stoppered conical flask, accurately adding 25 ml of methanol, weighing them, ultrasonically treating them for 45 minutes, cooling them, weighing them again, making up the lost weight with methanol, shaking them well, filtering them, and taking the filtrate to obtain the sample solution to be tested.

[0029] In step (4), the contents of forsythiaside and amygdalin in the sample to be tested are calculated by the external standard method, and the calculation formula is:

[0030]

[0031] Where: C R is the concentration of the reference solution, in μg / ml;

[0032] A X is the peak area of ​​the test product;

[0033] A R is the peak area of ​​the reference substance;

[0034] V is the dilution volume, in ml;

[0035] W is the sample weight, unit is g.

[0036] The present invention uses high performance liquid chromatography (HPLC) to accurately measure the content of two key active ingredients in Sangju Ganmao Granules, namely, forsythin and amygdalin. Compared with the detection method that does not include the content determination item in the existing quality standards, the present invention provides a more scientific and rigorous quality control method. This method can directly evaluate and control the intrinsic quality of the drug by detecting the content of forsythin and amygdalin, making up for the deficiency of the traditional method that only relies on sensory indicators, physical and chemical indicators or single component qualitative analysis.

[0037] In the specific determination process, the present invention particularly optimizes the separation conditions of amygdalin, and adopts high performance liquid chromatography to achieve a good separation effect, thereby ensuring the sensitivity and accuracy of the detection. At the same time, the experimental results show that the stability and repeatability of the present method in different batches of samples are excellent, and the determination results of forsythin and amygdalin have a low relative standard deviation (RSD), which further verifies the reliability and operability of the method.

[0038] Through the above technical improvements, the present invention can not only accurately reflect the quality level of the key active ingredients in Sangju Ganmao Granules, but also significantly improve the scientificity and repeatability of product quality control, thereby providing reliable guarantees for the consistent production of Sangju Ganmao Granules and the stable performance of the efficacy. In addition, with the high sensitivity and high precision of this method, deviations in the production process can be discovered and corrected in a timely manner, achieving full process control of product quality, and providing technical support for the modern quality management of Chinese medicine preparations.

[0039] The advantages of the present invention are as follows:

[0040] 1. Enhanced quality control: The present invention can more accurately reflect the intrinsic quality of the drug by determining the contents of forsythiaside and amygdalin in Sangju Ganmao Granules, thus making up for the deficiency of no content determination item in the existing standards.

[0041] 2. The method is highly scientific: The present invention adopts high performance liquid chromatography, which has the characteristics of high sensitivity, good separation effect, stability and strong repeatability, and is particularly effective in the separation and detection of amygdalin.

[0042] 3. Simple operation: The sample pretreatment of this method is simple and efficient, and is suitable for large-scale application in production and quality control.

[0043] 4. Ensure medication safety: Accurately measure the content of active ingredients to improve the consistency and stability of drug production, providing reliable protection for patients' medication safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0045] Figure 1 Schematic diagram of the forsythiaside reference solution described in Example 2 of the present invention

[0046] Figure 2 Schematic diagram of the sample solution (forsythiaside) to be tested according to Example 2 of the present invention

[0047] Figure 3 Schematic diagram of the negative sample solution of Forsythia suspensa described in Example 2 of the present invention

[0048] Figure 4 Schematic diagram of the amygdalin solution described in Example 2 of the present invention

[0049] Figure 5 Schematic diagram of the sample solution (amygdalin) to be tested according to Example 2 of the present invention

[0050] Figure 6 Schematic diagram of the bitter almond negative sample solution described in Example 2 of the present invention

[0051] Figure 7 Schematic diagram of the standard curve of forsythiaside described in Example 2 of the present invention

[0052] Figure 8 Schematic diagram of the standard curve of amygdalin described in Example 2 of the present invention DETAILED DESCRIPTION

[0053] The following content is described in detail with reference to the embodiments and accompanying drawings to explain the technical solution of the present invention, so that those skilled in the art can more clearly understand the technical contents of the present invention. However, these embodiments are only used to illustrate the specific technical solution of the present invention and do not constitute a limitation to the scope of protection of the present invention. In the following examples, the technical terms used have the meanings generally understood by those skilled in the art, and the test reagents used are all conventional biochemical reagents, and the experimental methods are all conventional technical methods.

[0054] The instruments and materials used in the following examples are as follows. Other materials are commercially available unless otherwise specified.

[0055] Instruments: electronic balance (AL204-IC, METTLER TOLEDO), electronic balance (Ms303s, METTLER TOLEDO), ultrasonic cleaner (TH400-BQE, Jining Tianhua Ultrasonic Electronic Instrument Co., Ltd.), water purifier (Milli-Q, Merck Millipore), high performance liquid chromatograph (e2695, Waters, USA), high performance liquid chromatograph (1260, Agilent, USA);

[0056] Materials: Forsythiaside (batch number: 110821-202318, content 95.8%) and amygdalin (batch number: 110820-202410, content 94.9%) were purchased from the China Food and Drug Inspection Institute; Sangju Cold Granules (batch numbers: 0900002; 0900004; 0900006) were produced by Lerentang Pharmaceutical Factory of Tianjin Medicine Darentang Group.

[0057] Reagents: methanol (chromatographic grade, batch number: I1338907416, Merck), acetonitrile (chromatographic grade, batch number: I1336830415, Merck);

[0058] Chromatographic columns: Waters Xbridge C18 (specifications 5 μm, 4.6×250 mm), Siphila Megassil PolarPhenyl (specifications 5 μm, 4.6×250 mm).

[0059] Example 1

[0060] The method for determining the contents of forsythiaside and amygdalin in Sangju Ganmao granules comprises the following steps:

[0061] Step (1) Preparation of reference solution

[0062] Take an appropriate amount of forsythiaside reference substance, weigh it accurately, and add methanol to prepare a solution containing 30 μg forsythiaside per 1 ml.

[0063] Take an appropriate amount of amygdalin reference substance, weigh it accurately, and add methanol to make a solution containing 0.1 mg of amygdalin per 1 ml.

[0064] Step (2) Preparation of sample solution to be tested

[0065] Take an appropriate amount of this product, grind it into powder, take about 2g, weigh it accurately, put it in a stoppered conical flask, accurately add 25ml of methanol, weigh it, treat it ultrasonically (power 500W, frequency 40kHz) for 45 minutes, let it cool, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0066] Step (3) Chromatographic conditions

[0067] Chromatographic conditions for forsythiaside: octadecylsilane bonded silica gel as filler; acetonitrile-water (19:81) as mobile phase; detection wavelength at 230nm; flow rate at 1ml / min; column temperature at 30℃.

[0068] Chromatographic conditions for amygdalin: phenylsilane bonded silica gel as filler; methanol-water (11:89) as mobile phase; detection wavelength at 207nm; flow rate at 1ml / min; column temperature at 30℃.

[0069] Step (4) Determination of the content of the sample to be tested

[0070] Accurately pipette 10 μl of the forsythiaside reference substance of step (1) and the sample solution prepared in step (2), respectively, and inject them into a high performance liquid chromatograph. Determine and analyze according to the forsythiaside chromatographic conditions of step (3) to obtain the peak areas of the forsythiaside reference substance and the sample to be tested. Calculate the forsythiaside content in the sample to be tested by the external standard method, and further calculate that the forsythiaside content in the Sangju Ganmao Granules is 0.2722 mg / g.

[0071] Accurately pipette 10 μl of the amygdalin reference solution of step (1) and the sample solution prepared in step (2), respectively, and inject them into a high performance liquid chromatograph. Determine and analyze according to the amygdalin chromatographic conditions of step (3) to obtain the peak areas of the amygdalin reference solution and the sample solution. Calculate the amygdalin content in the sample solution according to the external standard method, and further calculate that the amygdalin content in the Sangju Ganmao Granules is 1.024 mg / g.

[0072] The calculation formula is:

[0073]

[0074] Where: C R is the concentration of the reference solution, in μg / ml;

[0075] A X is the peak area of ​​the test product;

[0076] A R is the peak area of ​​the reference substance;

[0077] V is the dilution volume, in ml;

[0078] W is the sample weight, unit is g.

[0079] Example 2

[0080] In order to ensure the effect of the present invention, the present invention has carried out corresponding method verification on the content determination method of forsythiaside and amygdalin in Sangju Ganmao granules, as follows:

[0081] 1. Specificity experiment

[0082] Take Sangju Ganmao Granules and prepare the test solution in the same manner as in step (2) of Example 1. Take an appropriate amount of the negative sample of Forsythia suspensa, grind it into powder, take about 2 g, weigh it accurately, put it in a stoppered conical flask, accurately add 25 ml of methanol, weigh it, and ultrasonically treat it (power 500 W, frequency 40 kHz) for 45 minutes. Let it cool, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain the negative sample solution of Forsythia suspensa. Accurately pipette 10 μl of the Forsythia suspensa reference solution, the test solution, and the Forsythia suspensa negative sample solution respectively, inject them into a high performance liquid chromatograph, and determine and analyze the Forsythia suspensa chromatographic conditions according to step (3) of Example 1. The chromatogram is shown in the figure. Figure 1 , Figure 2 and Figure 3 As shown, the separation degree between forsythiaside and impurity peaks in the test solution is 6.121, which is well separated, and the forsythia negative sample has no interference at the forsythiaside chromatographic peak.

[0083] Take Sangju Ganmao Granules and prepare the test solution in the same manner as in step (2) of Example 1. Take an appropriate amount of bitter almond negative sample, grind it into powder, take about 2 g, accurately weigh it, put it in a stoppered conical flask, accurately add 25 ml of methanol, weigh it, and ultrasonically treat it (power 500 W, frequency 40 kHz) for 45 minutes. Let it cool, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain the bitter almond negative sample solution. Accurately pipette 10 μl of the amygdalin reference solution, the test solution, and the bitter almond negative sample solution, respectively, inject them into a high performance liquid chromatograph, and determine and analyze the amygdalin chromatographic conditions according to step (3) of Example 1. The chromatogram is as shown below. Figure 4 Figure 5 and Figure 6 As shown, the separation degree between amygdalin and impurity peaks in the test solution is 2.036, which is well separated, and the bitter almond negative sample has no interference at the amygdalin chromatographic peak.

[0084] 2. Accuracy Experiment

[0085] Take an appropriate amount of Sangju Ganmao Granules (Batch No.: 0900006), grind it into powder, take about 1g, weigh it accurately, put it in a stoppered conical flask, a total of 9 portions, accurately add 25ml of three reference solutions of high, medium and low concentrations (the high concentration reference solution contains 22.20165μg forsythiaside and 96.1.11149μg amygdalin per 1ml, the medium concentration reference solution contains 14.8011μg forsythiaside and 62.00766μg amygdalin per 1ml, and the low concentration reference solution contains 7.40055μg forsythiaside and 31.00383μg amygdalin per 1ml), each concentration of 3 portions, weigh the weight, ultrasonically treat (power 500W, frequency 40kHz) for 45 minutes, cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the filtrate.

[0086] According to the chromatographic conditions of step (3) of Example 1, forsythin and amygdalin were respectively measured and analyzed, and the recovery rates were calculated. The results showed that the average recovery rate of forsythin was 104.5%, with an RSD of 1.89%; the average recovery rate of amygdalin was 103.4%, with an RSD of 1.65%. The results are shown in Tables 1 and 2.

[0087] Table 1 Forsythiaside accuracy test results

[0088]

[0089] Table 2 Amygdalin accuracy test results

[0090]

[0091]

[0092] 3. Repeatability Experiment

[0093] Take Sangju Ganmao Granules, prepare the test solution in the same manner as in step (2) of Example 1, prepare 6 portions in parallel, accurately aspirate 10 μl of the reference solution of forsythiaside or amygdalin and the test solution, inject into the high performance liquid chromatograph, and measure and analyze the forsythiaside and amygdalin chromatographic conditions in step (3) of Example 1, respectively, and calculate the content of forsythiaside and amygdalin. The result shows that the content of forsythiaside is 0.2722 mg / g, and the RSD is 1.52%; the content of amygdalin is 1.024 mg / g, and the RSD is 1.06%. The results are shown in Tables 3 and 4.

[0094] Table 3 Results of repeatability test of forsythiaside

[0095]

[0096] Table 4 Amygdalin repeatability test results

[0097]

[0098] 4. Intermediate precision experiment

[0099] Take Sangju Ganmao Granules, prepare the test solution in the same way as in step (2) of Example 1, prepare 6 portions in parallel, change the date, personnel and instruments, accurately aspirate 10 μl of the reference solution of forsythiaside or amygdalin and the test solution, inject into the high performance liquid chromatograph, and measure and analyze the forsythiaside and amygdalin chromatographic conditions in step (3) of Example 1, respectively, and calculate the contents of forsythiaside and amygdalin based on the comprehensive reproducibility results. The results show that the content of forsythiaside is 0.2727 mg / g, with an RSD of 1.23%; the content of amygdalin is 1.046 mg / g, with an RSD of 2.34%. The results are shown in Tables 5 and 6.

[0100] Table 5 Results of intermediate precision test of forsythiaside

[0101]

[0102] Table 6 Results of intermediate precision test of amygdalin

[0103]

[0104]

[0105] 5. Linear Relationship

[0106] Take an appropriate amount of forsythiaside reference substance, accurately weigh it, add methanol to prepare reference substance solutions containing 1.48011, 2.96022, 5.92044, 14.8011, 29.6022, 74.0055, and 148.011 μg of forsythiaside per 1 ml, accurately pipette 10 μl of each, inject into high performance liquid chromatograph, and measure and analyze forsythiaside according to the chromatographic conditions of step (3) of Example 1, prepare a standard curve with forsythiaside concentration as the horizontal axis (X) and peak area as the vertical axis (Y), and calculate the regression equation and correlation coefficient. Results are shown in Figure 7 As shown, Y=20364.29X-3703.61 (r=1.0), the concentration of forsythiaside has a good linear relationship in the range of 1.48011 to 148.011 μg / ml.

[0107] Take an appropriate amount of amygdalin reference substance, accurately weigh it, add methanol to prepare reference substance solutions containing 6.21595, 12.4319, 24.8638, 62.1595, 93.2393, 186.479, and 310.798 μg of amygdalin per 1 ml, accurately pipette 10 μl of each, inject into a high performance liquid chromatograph, and measure and analyze the amygdalin chromatographic conditions according to step (3) of Example 1, prepare a standard curve with amygdalin concentration as the horizontal axis (X) and peak area as the vertical axis (Y), and calculate the regression equation and correlation coefficient. Results are shown in Figure 8 As shown, Y = 9.8504X + 2.1332 (r = 0.9999), and the concentration of amygdalin has a good linear relationship in the range of 6.21595 to 310.798 μg / ml.

[0108] 6. Durability-Solution Stability Test

[0109] Take Sangju Ganmao Granules, prepare a test solution in the same manner as in step (2) of Example 1, and measure and analyze the forsythin and amygdalin chromatographic conditions in step (3) of Example 1 at regular intervals, and calculate the relative standard deviation of the peak area. The results show that the forsythin test solution has good stability within 172 hours; the amygdalin test solution has good stability within 122 hours. The results are shown in Tables 7 and 8.

[0110] Table 7 Stability test results of forsythiaside test solution

[0111]

[0112]

[0113] Table 8 Amygdalin test solution stability test results

[0114]

[0115] 7. Durability - Chromatographic parameters

[0116] Take Sangju Ganmao Granules and prepare the test solution in the same manner as in Example 1 (2). Accurately pipette 10 μl of the forsythiaside or amygdalin reference solution and the test solution, respectively, and inject them into a high performance liquid chromatograph. Follow the chromatographic conditions of forsythiaside and amygdalin in step (3) of Example 1, respectively, and slightly change the detection wavelength, injection volume, column temperature, flow rate, mobile phase and chromatographic column. Measure and analyze them respectively, and calculate the relative standard deviation of the measured contents when the chromatographic parameters are different. As a result, the RSD of the measured forsythiaside and amygdalin contents when the chromatographic conditions are slightly changed is less than 3%, indicating that the method has good durability.

[0117] Table 9 Forsythiaside durability-chromatographic parameter test results

[0118]

[0119]

[0120] Table 10 Amygdalin durability-chromatographic parameter test results

[0121]

[0122] Example 3

[0123] Three batches of Sangju Ganmao Granules were taken and the test solution was prepared in the same manner as in step (2) of Example 1. 10 μl of the forsythiaside or amygdalin reference solution and the test solution were accurately aspirated and injected into a high performance liquid chromatograph. The forsythiaside and amygdalin chromatographic conditions of step (3) of Example 1 were used for determination and analysis. The forsythiaside and amygdalin contents were calculated by the external standard method. The results are shown in the following table.

[0124] Table 11 Test results of multiple batches of samples

[0125]

[0126] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting the content of forsythiaside and amygdalin in Sangju Ganmao granules, characterized in that: The method comprises the following steps: Step (1) Preparation of reference solution: Take an appropriate amount of forsythin reference substance, weigh it accurately, and add methanol to make a solution containing 30 μg forsythin per 1 ml; Take an appropriate amount of amygdalin reference substance, weigh it accurately, and add methanol to make a solution containing 0.1 mg of amygdalin per 1 ml; Step (2) Preparation of the sample solution to be tested: Take about 2g of Sangju Ganmao Granules, weigh accurately, add 25ml of methanol, and ultrasonically treat for 15 to 60 minutes. After filtering, take the filtrate as the sample solution to be tested; Step (3) Chromatographic conditions: The chromatographic conditions of forsythin are as follows: the filler is octadecylsilane bonded silica gel (C18 column), the mobile phase is acetonitrile-water = 18.6-19.4:81.4-80.6 (volume ratio), the detection wavelength is 230nm, the flow rate is 0.8-1.2ml / min, and the column temperature is 25-35°C; The chromatographic conditions of amygdalin are as follows: the filler is phenylsilane bonded silica gel (Phenyl column), the mobile phase is methanol-water = 10.7-11.3:89.3-88.7 (volume ratio), the detection wavelength is 207nm, the flow rate is 0.8-1.2ml / min, and the column temperature is 30-35°C; Step (4) Content determination: 10 μl of the reference solution and the sample solution to be tested were accurately aspirated and injected into a high performance liquid chromatograph, the peak areas were recorded, and the contents of forsythiaside and amygdalin in the sample to be tested were calculated according to the external standard method.

2. The detection method according to claim 1, characterized in that: The ultrasonic treatment time in step (2) is 30 to 50 minutes.

3. The detection method according to claim 1, characterized in that: The filler specification in the forsythiaside chromatography conditions described in step (3) is an octadecylsilane bonded silica gel column of 5 μm and 4.6×250 mm.

4. The detection method according to claim 1, characterized in that: The mobile phase ratio in the forsythiaside chromatography conditions described in step (3) is acetonitrile-water = 19:81 (volume ratio).

5. The detection method according to claim 1, characterized in that: The filler specification in the amygdalin chromatography conditions described in step (3) is a 5 μm, 4.6×250 mm phenylsilane bonded silica gel column.

6. The detection method according to claim 1, characterized in that: The mobile phase ratio in the amygdalin chromatography conditions described in step (3) is methanol-water = 11:89 (volume ratio).

7. The detection method according to claim 1, characterized in that: The preparation of the sample solution to be tested in step (2) comprises the following steps: Take 2g of Sangju Ganmao Granules, grind them into powder, weigh them accurately, put them in a stoppered conical flask, add 25ml of methanol accurately, weigh them, treat them with ultrasound for 45 minutes, let them cool, weigh them again, make up the lost weight with methanol, shake them well, filter them, and take the filtrate to obtain the sample solution to be tested.

8. The detection method according to claim 1, characterized in that: In step (4), the contents of forsythiaside and amygdalin in the sample to be tested are calculated by the external standard method, and the calculation formula is: Where: C R is the concentration of the reference solution, in μg / ml; A X is the peak area of ​​the test product; A R is the peak area of ​​the reference substance; V is the dilution volume, in ml; W is the sample weight, unit is g.

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