A method for constructing HPLC characteristic spectrum of Qinglin granules

Abstract

The present invention provides a method for constructing an HPLC characteristic spectrum of Qinglin granules, comprising: A) dissolving Qinglin granules with a solvent to obtain a test liquid; B) determining the test liquid with high performance liquid chromatography to obtain an HPLC characteristic spectrum of Qinglin granules; the chromatographic conditions of the high performance liquid chromatography are: the chromatographic column is a C18 column; the mobile phase A is acetonitrile, the mobile phase B is 0.2% phosphoric acid, and the gradient elution. The present invention adopts high performance liquid chromatography, selects acetonitrile-0.2% phosphoric acid as the mobile phase for gradient elution, and uses gardenia glycoside and geniposide as reference substances to establish an HPLC characteristic spectrum of Qinglin granules. 10 batches of HPLC characteristic spectrum of Qinglin granules were established, 9 characteristic peaks were determined, and a standard characteristic spectrum of Qinglin granules was established. The present invention has the characteristics of convenience, speed, stability, high precision, good reproducibility, etc., and can effectively control the quality of Qinglin granules.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection, and in particular to a method for constructing a HPLC characteristic spectrum of Qinglin particles. Background Art

[0002] Qinglin Granules are composed of eight medicinal materials: Dianthus superbus, Polygonum multiflorum, Akebia, Plantago asiatica, Talc, Gardenia jasminoides, Rhubarb, and Glycyrrhiza uralensis. The standard is included in the 2020 edition of the Chinese Pharmacopoeia. The quality standard of this product includes the properties of the granules; TLC identification of Rhubarb and Gardenia jasminoides; and the determination of the content of Gardenia jasminoides. Its quality standard only controls the qualitative and quantitative detection methods of one medicine, making it difficult to comprehensively control the product quality.

[0003] Therefore, it is necessary to establish a method for constructing HPLC characteristic spectrum of Qinglin Granules to provide a basis for effectively controlling and comprehensively evaluating the quality of Qinglin Granules. Summary of the invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a method for constructing an HPLC characteristic spectrum of Qinglin granules. The HPLC characteristic spectrum method of Qinglin granules constructed by the present invention is stable and reliable, and can control the quality of Qinglin granules.

[0005] The present invention provides a method for constructing a HPLC characteristic spectrum of Qinglin granules, comprising:

[0006] A) dissolving the Qinglin granules with a solvent to obtain a test solution;

[0007] B) using high performance liquid chromatography to measure the test solution to obtain a HPLC characteristic spectrum of Qinglin granules;

[0008] The chromatographic conditions of the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; the mobile phase A is acetonitrile, the mobile phase B is 0.2% phosphoric acid, and the elution is gradient.

[0009] Preferably, the method further comprises preparing a reference solution: taking gardenia glycoside and geniposide respectively, dissolving them in methanol to obtain a reference solution;

[0010] The reference solution is measured by high performance liquid chromatography to obtain a chromatogram of the reference; and the components of the HPLC characteristic spectrum of the Qinglin granules are qualitatively measured according to the chromatogram of the reference.

[0011] Preferably, the gradient elution is specifically:

[0012]

[0013] Preferably, the chromatographic column is Agilent ZORBAX SB-C18, with specifications of 5 μm, 4.6×250 mm; and a column temperature of 30° C. to 40° C.

[0014] Preferably, the mobile phase flow rate is 0.9-1.1 mL / min; the detection wavelength is 235-245 nm; and the injection volume is 10-20 μL.

[0015] Preferably, the mobile phase flow rate is 1.0 mL / min; the detection wavelength is 240 nm; the injection volume is 10 μL;

[0016] In the reference solution, the concentration of geniposide is 100 μg / mL; the concentration of geniposide is 100 μg / mL.

[0017] Preferably, the similarity of the HPLC characteristic spectrum of Qinglin Granules is evaluated by using a Chinese medicine chromatographic fingerprint similarity evaluation system to obtain a Qinglin Granules HPLC standard characteristic spectrum consisting of 9 characteristic peaks, wherein peak No. 3 is the gardenia glycoside peak and peak No. 8 is the geniposide peak; the 9 characteristic peaks are attributed: characteristic peaks No. 1, 2, and 4 are attributed to the medicinal material of roasted licorice, characteristic peaks No. 3 and 5 are attributed to the medicinal material of gardenia, characteristic peaks No. 6 and 7 are attributed to the medicinal material of rhubarb, and characteristic peaks No. 8 and 9 are attributed to the medicinal material of salt plantain.

[0018] Preferably, in the standard characteristic spectrum, the gardenoside peak is used as the reference peak S peak, and the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.70-peak 1, 0.91-peak 2, 1.00-peak 3, 1.60-peak 4, 1.67-peak 5, 1.83-peak 6, 2.27-peak 7, 2.76-peak 8, and 2.89-peak 9.

[0019] Preferably, in step A), the solvent is methanol; the dissolving is ultrasonic extraction; the ultrasonic power is 400W, 40kHz; and the ultrasonic time is 20-40min.

[0020] Preferably, in step A), the ratio of the mass g of the clearing particles to the volume mL of the solvent is (1-2): (20-50).

[0021] Compared with the prior art, the present invention provides a method for constructing an HPLC characteristic spectrum of Qinglin granules, comprising: A) dissolving Qinglin granules with a solvent to obtain a test solution; B) determining the test solution with a high performance liquid chromatography method to obtain an HPLC characteristic spectrum of Qinglin granules; the chromatographic conditions of the high performance liquid chromatography method are: a chromatographic column is a C18 column; mobile phase A is acetonitrile, mobile phase B is 0.2% phosphoric acid, and gradient elution. The present invention adopts high performance liquid chromatography, selects acetonitrile-0.2% phosphoric acid as the mobile phase for gradient elution, and uses gardenia glycoside and geniposide as reference substances to establish an HPLC characteristic spectrum of Qinglin granules. HPLC characteristic spectrums of 10 batches of Qinglin granules were established, 9 characteristic peaks were determined, and a standard characteristic spectrum of Qinglin granules was established. The present invention has the characteristics of convenience, speed, stability, high precision, good reproducibility, etc., and can effectively control the quality of Qinglin granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1-Figure 5 It is the HPLC characteristic spectrum of Qinglin granules measured by the present invention;

[0023] Figure 6 These are the characteristic spectra and common patterns of 10 batches of Qinglin granules;

[0024] Figure 7 It is the standard characteristic spectrum of Qinglin granules;

[0025] Figure 8 HPLC characteristic spectrum of Qinglin particles with different mobile phases in Comparative Example 1;

[0026] Fig. 9 This is the HPLC characteristic spectrum of Qinglin granules in different acid aqueous solutions in Comparative Example 2. DETAILED DESCRIPTION

[0027] The present invention provides a method for constructing a HPLC characteristic spectrum of Qinglin granules. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they all fall within the scope of protection of the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0028] The present invention provides a method for constructing a HPLC characteristic spectrum of Qinglin granules, comprising:

[0029] A) dissolving the Qinglin granules with a solvent to obtain a test solution;

[0030] B) using high performance liquid chromatography to measure the test solution to obtain a HPLC characteristic spectrum of Qinglin granules;

[0031] The chromatographic conditions of the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; the mobile phase A is acetonitrile, the mobile phase B is 0.2% phosphoric acid, and the elution is gradient.

[0032] The method for constructing a HPLC characteristic spectrum of Qinglin granules provided by the present invention firstly takes Qinglin granules and dissolves them in a solvent to obtain a test solution. The solvent is preferably methanol.

[0033] The present invention adopts the above extraction solvent, the chromatographic peak information volume is large and the effect is good.

[0034] Specifically, the Qinglin granules are dissolved in a solvent, ultrasonicated, cooled, shaken, and filtered to obtain the product.

[0035] The ultrasonic power of the present invention is preferably 400W, 40kHz; the extraction time is preferably 20-40min; more preferably 30min.

[0036] The ratio of the mass g of the Qinglin particles to the volume mL of the solvent is preferably (1-2): (20-50); more preferably (1-1.5): (20-40); and most preferably 1:25.

[0037] The present invention does not limit the specific manufacturer of the Qinglin granules, and all of them can be subjected to quality control and qualitative testing by the method of the present invention.

[0038] The present invention also includes preparing a reference solution: taking gardenia glycoside and geniposide respectively, dissolving them in methanol to obtain a reference solution;

[0039] In the reference solution of the present invention, the concentration of geniposide is 100 μg / mL; the concentration of geniposide is 100 μg / mL.

[0040] The reference solution is measured by high performance liquid chromatography to obtain a chromatogram of the reference; and the components of the HPLC characteristic spectrum of the Qinglin granules are qualitatively measured according to the chromatogram of the reference.

[0041] The mobile phase A of the present invention is acetonitrile, the mobile phase B is 0.2% phosphoric acid solution, and the elution is performed by gradient elution.

[0042] The gradient elution of the present invention is preferably specifically:

[0043]

[0044] The present invention has good baseline separation under the above elution gradient, good separation of each peak and stable baseline.

[0045] C18 column, preferably Agilent ZORBAX SB-C18, specification is 5μm, 4.6×250mm; column temperature is 30℃~40℃; preferably 35℃.

[0046] The chromatographic column of the present invention has symmetrical chromatographic peaks and good separation under the above 35°C condition.

[0047] The flow rate of the mobile phase is 0.9~1.1 ml / min; preferably 1 ml / min.

[0048] The present invention found that the chromatographic peaks were better separated and the peak shapes were more symmetrical at a flow rate of 1 ml / min, which was the most preferred solution.

[0049] The detection wavelength of the present invention is 235-245 nm, preferably 240 nm.

[0050] The inventors found that at 240 nm, the chromatographic information is rich, each component has good absorption, the response value is moderate, the peak separation is good, and the baseline is stable.

[0051] The injection volume of the present invention is 10-20 μL, preferably 10 μL.

[0052] The beneficial effect of the present invention is that under a liquid chromatography condition, the substance group of Qinglin granules is controlled by fingerprint spectrum, and the fingerprint spectrum is positioned by gardenia glycoside and geniposide; the cost of detection can be greatly reduced, and qualitative detection can be achieved.

[0053] The similarity of the HPLC characteristic spectrum of Qinglin Granules was evaluated by the similarity evaluation system of Chinese medicine chromatographic fingerprint, and the standard HPLC characteristic spectrum of Qinglin Granules consisting of 9 characteristic peaks was obtained, among which peak 3 was gardenia glycoside peak and peak 8 was geniposide peak. The 9 characteristic peaks were attributed as follows: characteristic peaks 1, 2, and 4 were attributed to Radix Glycyrrhizae Preparata, characteristic peaks 3 and 5 were attributed to Gardenia, characteristic peaks 6 and 7 were attributed to Rhei, and characteristic peaks 8 and 9 were attributed to Plantago asiatica.

[0054] Taking the Gardenia glycoside peak as the reference peak S peak, the relative retention time of each characteristic peak and the S peak was calculated, and the relative retention time was within ±10% of the specified value, and the specified values ​​were: 0.70-peak 1, 0.91-peak 2, 1.00-peak 3, 1.60-peak 4, 1.67-peak 5, 1.83-peak 6, 2.27-peak 7, 2.76-peak 8, and 2.89-peak 9.

[0055] Quality judgment standard: Take a sample of Qinglin Granules and operate according to the same method as above to obtain the characteristic spectrum of Qinglin Granules. Use the "Similarity Evaluation System of Chromatographic Fingerprints of Traditional Chinese Medicines" (2012 edition) of the National Pharmacopoeia Committee to analyze the standard characteristic spectrum of Qinglin Granules and the sample characteristic spectrum, and the similarity is greater than 0.90.

[0056] The method provided by the present invention can effectively monitor the quality of different batches of Qinglin granules to ensure stable quality. The method has the characteristics of high precision and good reproducibility, and is conducive to comprehensive monitoring of product quality.

[0057] The characteristic spectrum of Qinglin granules established in the present invention uses gardenia glycoside and geniposide as references, focuses on the order of each characteristic peak and the correlation with medicinal materials and intermediate products, can comprehensively evaluate the overall quality features of the product, and the method is scientific and reliable.

[0058] The newly developed characteristic spectrum method of the present invention can detect the components in Qinglin granules. The sample preparation method is simple and easy to operate, and relatively more characteristic peaks can be identified. The characteristic spectrum of Qinglin granules can be accurately and reliably detected. The authenticity, quality consistency and stability of Qinglin granules can be effectively detected and controlled.

[0059] The present invention provides a method for constructing an HPLC characteristic spectrum of Qinglin granules, comprising: A) dissolving Qinglin granules with a solvent to obtain a test solution; B) measuring the test solution with a high performance liquid chromatography method to obtain an HPLC characteristic spectrum of Qinglin granules; the chromatographic conditions of the high performance liquid chromatography method are: a chromatographic column is a C18 column; mobile phase A is acetonitrile, mobile phase B is 0.2% phosphoric acid, and gradient elution. The present invention adopts high performance liquid chromatography, selects acetonitrile-0.2% phosphoric acid as the mobile phase for gradient elution, and uses gardenia glycoside and geniposide as reference substances to establish an HPLC characteristic spectrum of Qinglin granules. HPLC characteristic spectrums of 10 batches of Qinglin granules are established, 9 characteristic peaks are determined, and a standard characteristic spectrum of Qinglin granules is established. The present invention has the characteristics of convenience, speed, stability, high precision, good reproducibility, etc., and can effectively control the quality of Qinglin granules.

[0060] The present invention establishes a common mode of HPLC characteristic spectrum of the Chinese patent medicine "Qinglin Granules", calibrates 9 characteristic peaks, and the established characteristic spectrum can comprehensively reflect the types and quantities of the chemical components contained, avoids the singleness and one-sidedness of the quality control of Qinglin Granules, and is conducive to comprehensive control of product quality.

[0061] The present invention selects to carry out the measurement at a detection wavelength of 240 nm, which has many peaks, good peak shape, easy identification, high similarity, good stability, accuracy and reliability.

[0062] The invention has the characteristics of convenience, speed, stability, high precision, good reproducibility, etc., and can effectively control the quality of the qinglin particles.

[0063] In order to further illustrate the present invention, a method for constructing a HPLC characteristic spectrum of Qinglin granules provided by the present invention is described in detail below in conjunction with examples.

[0064] Example 1: A method for constructing a HPLC characteristic spectrum of Qinglin granules

[0065] Instrument: Agilent 1200 high performance liquid chromatograph, MS205DU analytical balance

[0066] Reagents: Gardenia glycoside reference substance, geniposide reference substance, acetonitrile for liquid chromatography analysis was of chromatographic grade, other reagents were of analytical grade, water was ultrapure water, and Qinglin granules were provided by Xiuzhen Pharmaceutical Group Co., Ltd.

[0067] Preparation of test solution: Take 1.0g of Qinglin granular powder, weigh it accurately, put it in a stoppered conical flask, add 20ml of methanol accurately, weigh it, treat it with ultrasound for 20 minutes, cool it, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain the solution.

[0068] Preparation of reference solution: Take appropriate amount of gardenia glycoside and geniposide reference substances, weigh accurately, add methanol to make solutions containing 100 μg of gardenia glycoside and 100 μg of geniposide per 1 ml respectively.

[0069] Determination: Chromatographic conditions: The chromatographic column is Agilent ZORBAX SB-C18 (5μm, 4.6×250mm) column, the mobile phase A is acetonitrile, the mobile phase B is 0.2% phosphoric acid solution, the gradient elution, the flow rate is 0.8ml / min, the column temperature is 30℃, and the detection wavelength is 235nm. The volume ratio concentration configuration of the gradient elution program is as follows:

[0070]

[0071] Accurately pipette 20 μl of the reference solution and the test solution respectively, inject them into the high performance liquid chromatograph, and determine them according to the high performance liquid chromatography method to obtain the HPLC characteristic spectrum of "Qinglin Granules". Figure 1 shown.

[0072] The HPLC characteristic spectrum of Qinglin Granules was obtained, and the HPLC standard characteristic spectrum of Qinglin Granules consisting of 9 characteristic peaks was obtained. Among them, the No. 3 peak was the Gardenia glycoside peak, the No. 8 peak was the Geniposide peak, and the peak corresponding to the Gardenia glycoside reference peak was the S peak. The relative retention time of Peak 1-Peak 9 and the S peak was calculated, and the relative retention time was within ±10% of the specified value, and the specified values ​​were: 0.70-Peak 1, 0.91-Peak 2, 1.00-Peak 3, 1.60-Peak 4, 1.67-Peak 5, 1.83-Peak 6, 2.27-Peak 7, 2.76-Peak 8, and 2.89-Peak 9.

[0073] Example 2: A method for constructing a HPLC characteristic spectrum of Qinglin granules

[0074] Instrument: Agilent 1200 high performance liquid chromatograph, MS205DU analytical balance

[0075] Reagents: Gardenia glycoside reference substance, geniposide reference substance, acetonitrile for liquid chromatography analysis was of chromatographic grade, other reagents were of analytical grade, water was ultrapure water, and Qinglin granules were provided by Xiuzhen Pharmaceutical Group Co., Ltd.

[0076] Preparation of test solution: Take 1.5g of Qinglin granular powder, weigh it accurately, put it in a stoppered conical flask, add 25ml of methanol accurately, weigh it, treat it with ultrasound for 40 minutes, cool it, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0077] Preparation of reference solution: Take appropriate amount of gardenia glycoside and geniposide reference substances, weigh accurately, add methanol to make solutions containing 100 μg of gardenia glycoside and 100 μg of geniposide per 1 ml respectively.

[0078] Determination: Chromatographic conditions: The chromatographic column is Agilent ZORBAX SB-C18 (5μm, 4.6×250mm), the mobile phase A is acetonitrile, the mobile phase B is 0.2% phosphoric acid solution, the gradient elution, the flow rate is 1.1ml / min, the column temperature is 40℃, and the detection wavelength is 245nm. The volume ratio concentration configuration of the gradient elution program is as follows:

[0079]

[0080] Accurately pipette 15 μl of the reference solution and the test solution respectively, inject them into the high performance liquid chromatograph, and determine them according to the high performance liquid chromatography method to obtain the HPLC characteristic spectrum of "Qinglin Granules". Figure 2 shown.

[0081] The HPLC characteristic spectrum of Qinglin Granules was obtained, and the HPLC standard characteristic spectrum of Qinglin Granules consisting of 9 characteristic peaks was obtained. Among them, the No. 3 peak was the Gardenia glycoside peak, the No. 8 peak was the Geniposide peak, and the peak corresponding to the Gardenia glycoside reference peak was the S peak. The relative retention time of Peak 1-Peak 9 and the S peak was calculated, and the relative retention time was within ±10% of the specified value, and the specified values ​​were: 0.70-Peak 1, 0.91-Peak 2, 1.00-Peak 3, 1.60-Peak 4, 1.67-Peak 5, 1.83-Peak 6, 2.27-Peak 7, 2.76-Peak 8, and 2.89-Peak 9.

[0082] Example 3: A method for constructing a HPLC characteristic spectrum of Qinglin granules

[0083] Instrument: Dionex U-3000 high performance liquid chromatograph, MS205DU analytical balance

[0084] Reagents: Gardenia glycoside reference substance, geniposide reference substance, acetonitrile for liquid chromatography analysis was of chromatographic grade, other reagents were of analytical grade, water was ultrapure water, and Qinglin granules were provided by Xiuzhen Pharmaceutical Group Co., Ltd.

[0085] Preparation of test solution: Take 2.0g of Qinglin granular powder, weigh it accurately, put it in a stoppered conical flask, add 25ml of methanol accurately, weigh it, treat it with ultrasound for 30 minutes, cool it, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain the solution.

[0086] Preparation of reference solution: Take appropriate amount of gardenia glycoside and geniposide reference substances, weigh accurately, add methanol to make solutions containing 100 μg of gardenia glycoside and 100 μg of geniposide per 1 ml respectively.

[0087] Determination: Chromatographic conditions: The chromatographic column is Agilent ZORBAX SB-C18 (5μm, 4.6×250mm), the mobile phase A is acetonitrile, the mobile phase B is 0.2% phosphoric acid solution, the gradient elution, the flow rate is 1.0ml / min, the column temperature is 35℃, and the detection wavelength is 240nm. The volume ratio concentration configuration of the gradient elution program is as follows:

[0088]

[0089] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the high performance liquid chromatograph, and determine them according to the high performance liquid chromatography method to obtain the HPLC characteristic spectrum of "Qinglin Granules". Figure 3 shown.

[0090] The HPLC characteristic spectrum of Qinglin Granules was obtained, and the HPLC standard characteristic spectrum of Qinglin Granules consisting of 9 characteristic peaks was obtained. Among them, the No. 3 peak was the Gardenia glycoside peak, the No. 8 peak was the Geniposide peak, and the peak corresponding to the Gardenia glycoside reference peak was the S peak. The relative retention time of Peak 1-Peak 9 and the S peak was calculated, and the relative retention time was within ±10% of the specified value, and the specified values ​​were: 0.70-Peak 1, 0.91-Peak 2, 1.00-Peak 3, 1.60-Peak 4, 1.67-Peak 5, 1.83-Peak 6, 2.27-Peak 7, 2.76-Peak 8, and 2.89-Peak 9.

[0091] Example 4: Establishment of a HPLC standard characteristic spectrum of Qinglin granules

[0092] Instrument: Dionex U-3000 high performance liquid chromatograph, MS205DU analytical balance

[0093] Reagents: Gardenia glycoside reference substance, geniposide reference substance, chromatographically pure acetonitrile for liquid chromatography analysis, other reagents of analytical grade, ultrapure water, Qinglin Granules (batch numbers: 200418, 200419, 200420, 200421, 200422, 200518, 200519, 200520, 200521, 200522) were provided by Xiuzhen Pharmaceutical Group Co., Ltd.

[0094] Preparation of test solution: Take 1.0g of Qinglin granular powder, weigh it accurately, put it in a stoppered conical flask, add 25ml of methanol accurately, weigh it, treat it with ultrasound for 30 minutes, cool it, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain the solution.

[0095] Preparation of reference solution: Take appropriate amount of gardenia glycoside and geniposide reference substances, weigh accurately, add methanol to make solutions containing 100 μg of gardenia glycoside and 100 μg of geniposide per 1 ml respectively.

[0096] Chromatographic conditions: The chromatographic column was Agilent ZORBAX SB-C18 (5 μm, 4.6 × 250 mm), mobile phase A was acetonitrile, mobile phase B was 0.2% phosphoric acid solution, gradient elution, flow rate 1.0 ml / min, column temperature 35 ° C, detection wavelength 240 nm. The volume ratio concentration configuration of the gradient elution program is as follows:

[0097]

[0098] Determination: Accurately pipette 10 μl of reference solution and test solution respectively, inject into HPLC, and determine according to HPLC method. Figure 4 shown.

[0099] Verification example:

[0100] Methodological investigation

[0101] Precision test: Take the same sample solution and inject it 6 times continuously, record the chromatogram, and compare the relative retention time of each characteristic peak. The RSD value of the relative retention time of all characteristic peaks is less than 3.0%, indicating that the instrument has good precision. The detailed results are as follows:

[0102]

[0103] Repeatability test: Take 6 portions of Qinglin granules from the same batch (batch number: 160101), prepare the test solution according to the test solution preparation method, inject the sample, and record the chromatogram. The RSD value of the relative retention time of all characteristic peaks is less than 3.0%, indicating that the method has good repeatability. The detailed results are as follows:

[0104]

[0105] Stability test: Take the same sample solution of Qinglin granules and inject them at 0, 2, 6, 8, 12, 18, and 24 hours, and record the chromatogram. The RSD value of the relative retention time of all characteristic peaks is less than 3.0%, indicating that the solution has good stability for 24 hours. The detailed results are as follows:

[0106]

[0107] Example 5

[0108] Characteristic spectrum determination

[0109] The above method was used to measure 10 batches of Qinglin granules, record the characteristic spectra, and compare the relative retention times. The results showed that the RSD values ​​of the relative retention times of the 10 batches of Qinglin granules were less than 3.0%. Figure 5-6 The detailed results are as follows:

[0110]

[0111] The characteristic spectra of 10 batches of Qinglin granules were tested against the standard characteristic spectra. The detailed results are as follows:

[0112]

[0113] The standard characteristic spectrum of Qinglin granules established by the above method is as follows: Figure 7 shown.

[0114] Comparative Example 1:

[0115] (1) Preparation of test solution: Grind 1.0 g of Qinglin granules into fine powder, weigh accurately, place in a stoppered conical flask, add 25 ml of methanol accurately, weigh, ultrasonicate for 30 min, cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the filtrate.

[0116] (2) Preparation of reference solution: Take appropriate amount of gardenia glycoside and geniposide reference substances, weigh accurately, add methanol to prepare solutions containing 100 μg gardenia glycoside and 100 μg geniposide per 1 ml.

[0117] (3) Determination: Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into a high performance liquid chromatograph, and determine them according to the high performance liquid chromatography method to obtain the HPLC characteristic spectrum of "Qinglin Granules".

[0118] The chromatographic column was an Agilent ZORBAX SB-C18 (5 μm, 4.6×250 mm) column, the mobile phase A was methanol, the mobile phase B was 0.1% phosphoric acid solution, the gradient elution was at a flow rate of 1.0 ml / min, the column temperature was 35°C, and the detection wavelength was 240 nm.

[0119] During the gradient elution process, the ratio of mobile phase A and B changes as follows: 0-10 min, 5% phase A, 95% phase B; 10-15 min, 5%-10% phase A, 95%-90% phase B; 15-45 min, 10%-20% phase A, 90%-80% phase B; 45-60 min, 20%-30% phase A, 80%-70% phase B; 60-75 min, 30%-5% phase A, 70%-95% phase B; that is, the gradient elution program table:

[0120]

[0121] Results: The HPLC standard characteristic spectrum of Qinglin Granules consisting of 6 characteristic peaks was obtained. Peak No. 2 was the peak of Gardenia glycoside, and Peak No. 5 was the peak of Geniposide. Compared with acetonitrile as mobile phase A, the number of chromatographic peaks was less and could not fully characterize the intrinsic quality of the Qinglin Granules sample. Therefore, acetonitrile was determined as mobile phase A. Figure 8 shown.

[0122] Comparative Example 2:

[0123] (1) Preparation of test solution: Grind 1.0 g of Qinglin granules into fine powder, weigh accurately, place in a stoppered conical flask, add 25 ml of methanol accurately, weigh, ultrasonicate for 30 min, cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the filtrate.

[0124] (2) Preparation of reference solution: Take appropriate amount of gardenia glycoside and geniposide reference substances, weigh accurately, add methanol to prepare solutions containing 100 μg gardenia glycoside and 100 μg geniposide per 1 ml.

[0125] (3) Determination: Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into a high performance liquid chromatograph, and determine them according to the high performance liquid chromatography method to obtain the HPLC characteristic spectrum of "Qinglin Granules".

[0126] The chromatographic column was Agilent ZORBAX SB-C18 (5 μm, 4.6×250 mm) column, mobile phase A was acetonitrile, mobile phase B was 0.3% glacial acetic acid solution, gradient elution, flow rate was 1.0 ml / min, column temperature was 35°C, and detection wavelength was 240 nm.

[0127] During the gradient elution process, the ratio of mobile phase A and B changes as follows: 0-5min, phase A 0%, phase B 100%; 5-10min, phase A 5%-15%, phase B 95%-85%; 10-35min, phase A 15%-20%, phase B 85%-80%; 35-65min, phase A 20%-50%, phase B 80%-50%; 65-75min, phase A 30%-0%, phase B 70%-100%; that is, the gradient elution program table:

[0128]

[0129] Results: A standard characteristic spectrum of Qinglin Granules HPLC consisting of 9 characteristic peaks was obtained. Peak No. 3 was the peak of Gardenia glycoside, and Peak No. 8 was the peak of Geniposide. Compared with the elution using the mobile phase of the embodiment, the chromatographic separation was lower and the baseline was unstable, which could not fully characterize the intrinsic quality of the Qinglin Granules sample. Fig. 9 shown.

[0130] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for constructing a HPLC characteristic spectrum of Qinglin granules, comprising: Preparation of test solution: Take 1.0g of Qinglin granule powder, weigh accurately, put it in a stoppered conical flask, add 20ml of methanol accurately, weigh it, treat it with ultrasound for 20 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; Preparation of reference solution: Take appropriate amount of gardenia glycoside and geniposide reference substances, weigh accurately, add methanol to prepare solutions containing 100 μg gardenia glycoside and 100 μg geniposide per 1 ml respectively; Determination: Chromatographic conditions: The chromatographic column is Agilent ZORBAX SB-C18 column, 5μm, 4.6×250mm, mobile phase A is acetonitrile, mobile phase B is 0.2% phosphoric acid solution, gradient elution, flow rate is 0.8ml / min, column temperature is 30℃, detection wavelength is 235nm; the volume ratio concentration configuration of the gradient elution program is as follows: Accurately pipette 20 μl of reference solution and test solution respectively, inject into HPLC, and determine according to HPLC method; The similarity of the HPLC characteristic spectrum of Qinglin Granules was evaluated by the similarity evaluation system of Chinese medicine chromatographic fingerprint, and the standard HPLC characteristic spectrum of Qinglin Granules consisting of 9 characteristic peaks was obtained, among which the peak No. 3 was the peak of gardenia glycoside and the peak No. 8 was the peak of geniposide. The 9 characteristic peaks were attributed as follows: the characteristic peaks No. 1, 2, and 4 were attributed to the medicinal material of Zhi Gancao, the characteristic peaks No. 3 and 5 were attributed to the medicinal material of Gardenia, the characteristic peaks No. 6 and 7 were attributed to the medicinal material of Rhei, and the characteristic peaks No. 8 and 9 were attributed to the medicinal material of Plantago. In the standard characteristic spectrum, the gardenoside peak is used as the reference peak S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time is within ±10% of the specified value, and the specified values ​​are: 0.70-peak 1, 0.91-peak 2, 1.00-peak 3, 1.60-peak 4, 1.67-peak 5, 1.83-peak 6, 2.27-peak 7, 2.76-peak 8, and 2.89-peak 9.

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