Method for establishing a compound medicine fingerprint

Abstract

The application belongs to the technical field of pharmaceutical technology analysis, and particularly relates to a method for establishing a fingerprint spectrum of a compound medicine. The method comprises preparation of a test sample solution, preparation of a control sample solution, and adoption of a specific gradient elution procedure. The method is simple, stable, reproducible, and highly precise, and can improve the quality control method of the compound medicine containing psoralea, walnut kernel, radix morindae officinalis, cistanche deserticola, eucommia ulmoides carbon, vinegar frankincense and vinegar myrrh, guarantee the reliable and stable quality of the compound medicine, and effectively control the quality of the compound medicine containing psoralea, walnut kernel, radix morindae officinalis, cistanche deserticola, eucommia ulmoides carbon, vinegar frankincense and vinegar myrrh, such as the quality of the modified Qingmo pill, so as to guarantee the stability, reliability and safety of the product.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical technology, and particularly relates to a method for establishing a fingerprint spectrum of a compound medicine. BACKGROUND

[0002] Traditional Chinese medicine compound is an important part of the traditional Chinese medicine treasure, and has a long history of use and clear efficacy. At present, the quality control of traditional Chinese medicine preparation mainly includes appearance, identification and quantitative analysis of single or several components. However, traditional Chinese medicine compound contains multiple medicinal materials, and the effective substance basis for playing curative effect is relatively complex. The mechanism is often multi-target coordination. It is difficult to comprehensively reflect the quality of traditional Chinese medicine preparation by using single or several components as quality control indicators.

[0003] As a comprehensive and quantifiable quality control method, traditional Chinese medicine fingerprint spectrum can comprehensively and effectively reflect the quality of traditional Chinese medicine and its preparation, which is consistent with the multi-target and holistic characteristics of traditional Chinese medicine. In recent years, the modern traditional Chinese medicine fingerprint spectrum analysis technology established by combining computer software analysis is widely used in various fields of traditional Chinese medicine research, and is currently internationally recognized as a quality control mode of traditional Chinese medicine. The method for establishing the fingerprint spectrum includes various chromatographic methods, spectroscopic methods, spectral methods, etc., and chromatography is the most commonly used method.

[0004] Jiawei Qingye Pill is a traditional Chinese medicine compound pill prepared from salted psoralea, walnut kernel, fried baijiatian, wine cistanche, eucommia charcoal, vinegar frankincense and vinegar myrrh. Its main effects are tonifying kidney, dispelling cold and relieving pain. It is used for treating lumbago and leg pain caused by kidney deficiency and cold, impotence and spermatorrhea, frequent urination and cold pain in the lower abdomen, and has significant clinical curative effect. The existing quality control method of Jiawei Qingye Pill mainly performs qualitative identification of psoralea and walnut kernel through microscopic identification and thin layer identification, which is difficult to comprehensively reflect the quality of the medicine. There is no report on using traditional Chinese medicine chromatographic fingerprint spectrum as the quality control method of Jiawei Qingye Pill. Therefore, it is of great significance to establish the fingerprint spectrum of Jiawei Qingye Pill for comprehensive control of the quality of the product. SUMMARY

[0005] Therefore, the technical problem to be solved by the present application is to overcome the problems of insufficient quality control of Jiawei Qingye Pill in the prior art and difficulty in comprehensively reflecting the quality of the medicine, so as to provide a method for establishing a fingerprint spectrum of a compound medicine.

[0006] Therefore, the present application provides the following technical solutions.

[0007] The method for establishing a fingerprint spectrum of a compound medicine provided by the present application comprises the following steps:

[0008] (1) Preparation of a test sample solution;

[0009] (2) Preparation of a control sample solution;

[0010] (3) separately draw the test sample solution and the control sample solution, and determine by high performance liquid chromatography; wherein, the chromatographic conditions: octadecylsilane bonded silica gel as the filler, acetonitrile as the mobile phase A, aqueous phosphoric acid as the mobile phase B, gradient elution, the program of gradient elution: 0-5min, 3% A, 97% B; 5-15min, 3%→9% A, 97%→91% B; 15-25min, 9%→13% A, 91%→87% B; 25-45min, 13%→15% A, 87%→85% B; 45-65min, 15%→35% A, 85%→65% B; 65-75min, 35% A, 65% B; 75-80min, 35%→95% A, 65%→5% B;

[0011] The raw materials of the compound medicine include Fructus Psoraleae, walnut kernel, Radix Morindae Officinalis, Cistanche, Eucommia ulmoides, Olibanum and Myrrha.

[0012] The raw materials of the compound medicine include Fructus Psoraleae, walnut kernel, Radix Morindae Officinalis, Cistanche, Eucommia ulmoides, Olibanum and Myrrha. The raw materials can be processed products, for example, Fructus Psoraleae can be salted Fructus Psoraleae, Cistanche can be wine Cistanche, Eucommia ulmoides can be Eucommia ulmoides charred, Olibanum can be vinegar Olibanum, and Myrrha can be vinegar Myrrha. Further, the compound medicine can be flavored Qinggao compound medicine in different dosage forms, for example, flavored Qinggao pills.

[0013] In the chromatographic conditions: the column temperature is 25-40℃; and / or,

[0014] the flow rate is 0.8-1.2ml / min; and / or,

[0015] the concentration of phosphoric acid in the aqueous phosphoric acid is 0.05-0.2%; and / or,

[0016] the detection wavelength is 230-250nm; and / or,

[0017] the injection volume is 5-20μL.

[0018] In the chromatographic conditions: the column temperature is 30℃; the flow rate is 1.0ml / min; the concentration of phosphoric acid in the aqueous phosphoric acid is 0.1%; the detection wavelength is 240nm; and the injection volume is 10μL.

[0019] In the establishment method, a solvent is added to the test sample, ultrasonic treatment is performed, filtration is performed, and the filtrate is taken to obtain the test sample solution.

[0020] The solvent is at least one of methanol, ethanol and water;

[0021] Preferably, the solvent is 50-100% methanol and / or 50-100% ethanol.

[0022] Preferably, the mass of the test sample to the volume of the solvent is (2.5-15):(25-100); wherein the unit of the mass of the test sample is g, and the unit of the volume of the solvent is ml;

[0023] Preferably, the power of the ultrasonic is 200-500 W, and the time is 20-90 min.

[0024] The method for preparing the test sample solution further comprises the step of adding diatomite.

[0025] Preferably, when preparing the test sample solution, the amount of diatomite added is 0.2-2 times the mass of the test sample. When the test sample is honeyed pills, the extraction solvent cannot easily penetrate into the honeyed pills due to the high viscosity of the honeyed pills, and therefore diatomite is added as a dispersant to improve the mixing uniformity of the solvent and the test sample.

[0026] In the method for establishing, at least one of the following is used as a control: ericoidin, geniposidic acid, echinacoside, verbascoside, psoralen and isopsoralen.

[0027] Preferably, 1 ml of the control solution contains 60-180 μg of ericoidin; and / or,

[0028] 1 ml of the control solution contains 20-60 μg of geniposidic acid; and / or,

[0029] 1 ml of the control solution contains 120-360 μg of echinacoside; and / or,

[0030] 1 ml of the control solution contains 40-120 μg of verbascoside; and / or,

[0031] 1 ml of the control solution contains 55-165 μg of psoralen; and / or,

[0032] 1 ml of the control solution contains 50-150 μg of isopsoralen.

[0033] The fingerprint spectrum of the compound medicine obtained by the method for establishing comprises 11 characteristic peaks, including peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, peak 9, peak 10 and peak 11.

[0034] Taking peak 1 as the reference peak 1, the specified values of the relative retention times of peak 2, peak 3, peak 4, peak 5 and peak 6 are 1.19, 3.19, 3.74, 6.96 and 7.44, respectively.

[0035] The relative retention time of the peak 2, the peak 8, the peak 9, the peak 10 and the peak 11 is 1.39, 1.45, 1.80 and 1.85 respectively, with the peak 7 as a reference peak.

[0036] The relative retention time of the characteristic peak is ±10% of the specified value.

[0037] The peak 1 is verticinone, the peak 3 is geniposidic acid, the peak 7 is echinacoside, the peak 8 is verbascoside, the peak 10 is psoralen and the peak 11 is isopsoralen.

[0038] The technical scheme of the present application has the following advantages:

[0039] 1. The method for establishing the fingerprint spectrum of the compound medicine provided by the present application, which comprises the preparation of a test sample solution, the preparation of a control sample solution and the use of a specific gradient elution procedure. The method is simple, stable, reproducible and precise, and can improve the quality control method of the compound medicine containing psoralen, walnut kernel, radix morindae officinalis, cistanche deserticola, eucommia ulmoides carbon, frankincense and myrrh, thereby ensuring the reliable and stable quality of the compound medicine, and effectively controlling the quality of the compound medicine containing psoralen, walnut kernel, radix morindae officinalis, cistanche deserticola, eucommia ulmoides carbon, frankincense and myrrh, such as the flavored Qingmo pill, so as to ensure the stability, reliability and safety of the product. BRIEF DESCRIPTION OF DRAWINGS

[0040] In order to more clearly illustrate the specific embodiments of the present application or the technical scheme in the prior art, the following will briefly introduce the drawings needed to be used in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creating any inventive labor.

[0041] Figure 1 is the fingerprint spectrum of the flavored Qingmo pill in seven batches in Example 1 of the present application;

[0042] Figure 2 is the control fingerprint spectrum in Example 1 of the present application;

[0043] Figure 3 is the spectrum of the control sample solution 1 in Example 1 of the present application;

[0044] Figure 4 is the spectrum of each control sample in the control sample solution 1 in Example 1 of the present application;

[0045] Figure 5 is the spectrum of the control sample solution, the test sample solution and each reference sample solution when the characteristic peak fingerprint is obtained in Example 1 of the present application;

[0046] Figure 6 is the chromatogram obtained from the different wavelengths of the experimental example 1 of the present application;

[0047] Figure 7 is the chromatogram obtained from the different gradient elution procedures of the experimental example 1 of the present application;

[0048] Figure 8 is the chromatogram obtained from the different column temperatures of the experimental example 1 of the present application;

[0049] Figure 9 is the chromatogram obtained from the different column temperatures of the experimental example 1 of the present application;

[0050] Figure 10 is the chromatogram obtained from the different column temperatures of the experimental example 1 of the present application;

[0051] Figure 11 is the chromatogram obtained from the different column temperatures of the experimental example 1 of the present application;

[0052] Figure 12 is the chromatogram obtained from the different column temperatures of the experimental example 1 of the present application. DETAILED DESCRIPTION

[0053] The following examples are provided to better further understand the present application, and are not limited to the best mode, and do not constitute a limitation to the content and protection scope of the present application, and any person under the inspiration of the present application or the combination of the present application with other prior art features, any product same or similar to the present application falls within the protection scope of the present application.

[0054] The specific experimental steps or conditions are not indicated in the examples, and can be performed according to the conventional experimental steps or conditions described in the literature in the art. The reagents or instruments used are not indicated by the manufacturer, and are conventional reagent products that can be obtained by purchase.

[0055] The instruments and reagents used in the following examples are as follows:

[0056] Instruments:

[0057] Waters E2695 high performance liquid chromatograph; chromatographic column 1: Kromasil KR100-5-C18 (4.6 mm x 250 mm, 5 μm); chromatographic column 2: Agilent Zorbax SB-C18 (4.6 mm x 250 mm, 5 μm).

[0058] Reagents:

[0059] The batch numbers of the Jiawei Qingmo Pills are 18015267, 17015978, 18015062, 20010731, 21010064, 2022031630, and 2022031660; the raw materials of the Jiawei Qingmo Pills include salted Psoralea, walnut kernel, fried Bajitian, wine Cistanche, Duzhong charcoals, vinegar frankincense, and vinegar myrrh.

[0060] The salted Psoralea, walnut kernel, fried Bajitian, wine Cistanche, Duzhong charcoals, vinegar frankincense, and vinegar myrrh were provided by the Tongrentang Pharmaceutical Factory of Beijing Tongrentang Co., Ltd.; the water crystal lan glycoside, geniposidic acid, trilobatin, verbascoside, psoralen, and isopsoralen control samples were purchased from the China Institute for Control of Biological and Pharmaceutical Products;

[0061] The batch number of the gallic acid is 110831-201906, China Institute for Control of Biological and Pharmaceutical Products;

[0062] The batch number of the pinoresinol diglucoside is 111537-202107, China Institute for Control of Biological and Pharmaceutical Products;

[0063] Acetonitrile is chromatographically pure; phosphoric acid is analytically pure.

[0064] Example 1

[0065] The present embodiment provides a method for establishing the fingerprint spectrum of Jiawei Qingmo Pills, comprising the following steps:

[0066] Preparation of the test sample solution: take Jiawei Qingmo Pills, cut and crush, accurately weigh, add about 0.6 times of diatomite, crush and mix, take about 9 g, accurately weigh, place in a conical bottle with a plug, accurately add 50 ml of methanol, tightly plug, weigh, soak for 30 min, ultrasonically treat (power 500 W, frequency 40 kHz) for 60 min, cool, weigh again, make up the weight loss with methanol, shake well, filter, and take the filtrate to obtain the test sample solution.

[0067] Preparation of the control sample solution: take water crystal lan glycoside, geniposidic acid, trilobatin, verbascoside, psoralen, and isopsoralen control samples, accurately weigh, add methanol to prepare a mixed solution containing 114.54 μg of water crystal lan glycoside, 41.24 μg of geniposidic acid, 236.85 μg of trilobatin, 75.68 μg of verbascoside, 106.2 μg of psoralen, and 93.72 μg of isopsoralen per 1 ml, shake well, pass through a 0.45 μm microporous filter membrane to obtain the control sample solution.

[0068] The test sample solution and the control sample solution are respectively taken and injected into a high performance liquid chromatograph for determination and recording of a chromatogram; wherein, the chromatographic conditions are as follows: a chromatographic column: Kromasil KR100-5-C18 (specification: 4.6 mm x 250 mm, 5 μm); a detection wavelength: 240 nm; a column temperature: 30 DEG C; a flow rate: 1.0 ml / min; an injection amount: 10 μL; a mobile phase A: acetonitrile; a mobile phase B: 0.1% phosphoric acid aqueous solution, in a gradient elution manner; a gradient elution procedure: 0-5 min, 3% A, 97% B; 5-15 min, 3%→9% A, 97%→91% B; 15-25 min, 9%→13% A, 91%→87% B; 25-45 min, 13%→15% A, 87%→85% B; 45-65 min, 15%→35% A, 85%→65% B; 65-75 min, 35% A, 65% B; 75-80 min, 35%→95% A, 65%→5% B.

[0069] Confirmation of characteristic peaks

[0070] Seven batches of Jiawei Qinggao Pills were taken, and test sample solutions were prepared according to the above method, and were determined to obtain the fingerprint chromatograms of each batch of Jiawei Qinggao Pills, and the results are shown in Figure 1 , Figure 1 The batch numbers of Jiawei Qinggao Pills from top to bottom correspond to 2022031660, 2022031630, 21010064, 20010731, 18015062, 17015978, and 18015267. The fingerprint chromatograms of different batches of Jiawei Qinggao Pills were introduced into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2.0 version) compiled by the Pharmacopoeia Commission, 11 chromatographic peaks common to all batches of samples were selected as common peaks, and a control fingerprint chromatogram of Jiawei Qinggao Pills was generated, the relative peak area and relative retention time of each common peak were calculated, and the results are shown in Table 1 and Table 2, and the control fingerprint chromatogram is shown in Figure 2 .

[0071] The fingerprint of Jiawei Qinggumacihang Pill comprises 11 characteristic peaks, including Peak 1, Peak 2, Peak 3, Peak 4, Peak 5, Peak 6, Peak 7, Peak 8, Peak 9, Peak 10 and Peak 11, Peak 1 is verticinone, Peak 3 is geniposidic acid, Peak 7 is trilobatin, Peak 8 is verbascoside, Peak 10 is psoralen, and Peak 11 is isopsoralen, the specified values of the relative retention time of each characteristic peak are as follows: taking Peak 1 as the reference, the specified values of the relative retention time of Peak 2, Peak 3, Peak 4, Peak 5 and Peak 6 are 1.19, 3.19, 3.74, 6.96 and 7.44, respectively; taking Peak 7 as the reference, the specified values of the relative retention time of Peak 8, Peak 9, Peak 10 and Peak 11 are 1.39, 1.45, 1.80 and 1.85, respectively; wherein the relative retention time of the characteristic peak is within ±10% of the specified value.

[0072] Table 1 Relative peak area of common peaks in the fingerprint of 7 batches of Jiawei Qinggumacihang Pill

[0073]

[0074] Table 2 Relative retention time of common peaks in the fingerprint of 7 batches of Jiawei Qinggumacihang Pill

[0075]

[0076]

[0077] The similarity of the fingerprint of 7 batches of Jiawei Qinggumacihang Pill was evaluated using the Similarity Evaluation System of Traditional Chinese Medicine Chromatographic Fingerprint (2.0 edition) prepared by the Pharmacopoeia Commission, and the similarity was greater than 0.99, and the results are shown in Table 3.

[0078] Table 3 Similarity calculation results of 7 batches of Jiawei Qinggumacihang Pill

[0079]

[0080] Identification of characteristic peaks

[0081] (1) Control solution 1: Take verticinone, gallic acid, geniposidic acid, pinoresinol diglucoside, trilobatin, verbascoside, psoralen and isopsoralen control substances, accurately weigh, add methanol to prepare a mixed solution containing 190.9 μg of verticinone, 136.6 μg of gallic acid, 103.1 μg of geniposidic acid, 75.2 μg of pinoresinol diglucoside, 94.74 μg of trilobatin, 126.1 μg of verbascoside, 106.2 μg of psoralen and 78.1 μg of isopsoralen per 1 ml, shake well, and it is obtained.

[0082] The control solution 1 was determined according to Example 1, and the results are shown in Figure 3The spectra of monardocin, gallic acid, geniposidic acid, pinoresinol diglucoside, trillin, verbascoside, psoralen and isopsoralen are shown in Figure 4 Figure 3 As can be seen from the comparison results, the chromatographic peaks of monardocin, geniposidic acid, trillin, verbascoside, psoralen and isopsoralen appear in the chromatogram, and no peak of gallic acid appears; the retention time of peak No. 5 is close to that of pinoresinol diglucoside, but the spectra of peak No. 5 and pinoresinol diglucoside are different, and no peak of pinoresinol diglucoside appears in the reference chromatogram. Therefore, it is determined that the fingerprint chromatogram contains monardocin, geniposidic acid, trillin, verbascoside, psoralen and isopsoralen.

[0083] (2) The total amount of the test sample and diatomite in Example 1 is about 9 g, and the mixture formed by the test sample and diatomite contains psoralae (salt-fried) 0.96 g, walnuts 0.64 g, radix cistanche (processed) 0.48 g, cistanche (wine-fried) 0.48 g, eucommia ulmoides oliver (charred) 0.96 g, olibanum (vinegar-fried) 0.143 g, and myrrh (vinegar-fried) 0.143 g.

[0084] Salt psoralae reference solution: salt psoralae was crushed and uniformly mixed, about 0.96 g was accurately weighed and placed in a conical flask with a stopper, 50 ml of methanol was accurately added, the weight was determined, and the flask was tightly sealed. After immersion for 30 min, ultrasonic treatment (power 500 W, frequency 40 kHz) was performed for 60 min, and the weight was determined again. The lost weight was made up with methanol, and the mixture was shaken and filtered. The filtrate was collected to obtain the salt psoralae reference solution.

[0085] Radix cistanche reference solution: the processed radix cistanche was crushed and uniformly mixed, about 0.48 g was accurately weighed and placed in a conical flask with a stopper, 50 ml of methanol was accurately added, the weight was determined, and the flask was tightly sealed. After immersion for 30 min, ultrasonic treatment (power 500 W, frequency 40 kHz) was performed for 60 min, and the weight was determined again. The lost weight was made up with methanol, and the mixture was shaken and filtered. The filtrate was collected to obtain the radix cistanche reference solution.

[0086] Cistanche deserticola Y.C.Ma reference solution: cistanche deserticola Y.C.Ma was crushed and uniformly mixed, about 0.48 g was accurately weighed and placed in a conical flask with a stopper, 50 ml of methanol was accurately added, the weight was determined, and the flask was tightly sealed. After immersion for 30 min, ultrasonic treatment (power 500 W, frequency 40 kHz) was performed for 60 min, and the weight was determined again. The lost weight was made up with methanol, and the mixture was shaken and filtered. The filtrate was collected to obtain the cistanche deserticola Y.C.Ma reference solution.

[0087] ​Eucommia ulmoides Oliv. charcoal reference solution: Eucommia ulmoides Oliv. charcoal was crushed and mixed, about 0.96 g was accurately weighed, put into a conical flask with a stopper, sealed, the weight was determined, soaked for 30 min, treated by ultrasonic (power 500 W, frequency 40 kHz) for 60 min, cooled, the weight was determined again, the lost weight was made up with methanol, shaken well, filtered, and the filtrate was collected to obtain the Eucommia ulmoides Oliv. charcoal reference solution.

[0088] Walnut kernel, acetic Commiphora molmol, and acetic Commiphora myrrha mixed reference solution: Walnut kernel was crushed and mixed, about 0.64 g was accurately weighed; acetic Commiphora molmol was crushed and mixed, about 0.143 g was accurately weighed; acetic Commiphora myrrha was crushed and mixed, about 0.143 g was accurately weighed. The above three medicines were put into the same conical flask with a stopper, 50 ml of methanol was accurately added, sealed, the weight was determined, soaked for 30 min, treated by ultrasonic (power 500 W, frequency 40 kHz) for 60 min, cooled, the weight was determined again, the lost weight was made up with methanol, shaken well, filtered, and the filtrate was collected to obtain the mixed reference solution.

[0089] The test sample solution, the reference solution, and the above-mentioned reference solutions were injected into the high performance liquid chromatograph respectively, and the chromatograms were obtained by determination. The results are shown in Figure 5 , Figure 5 From top to bottom correspond to the reference solution, the test sample solution, the mixed reference solution, the Eucommia ulmoides Oliv. charcoal reference solution, the Cistanche deserticola reference solution, the Morinda officinalis reference solution, and the Psoralea corylifolia reference solution. As can be seen from the figure, peaks 1 and 2 come from Morinda officinalis; peaks 3, 4, 7, 8, and 9 come from Cistanche deserticola; and peaks 5, 6, 10, and 11 come from Psoralea corylifolia.

[0090] Determination of chromatographic conditions

[0091] 1.1 Detection wavelength

[0092] The same batch of Jiawei Qinggao Pills was used to prepare the test sample solution according to Example 1, and the fingerprint chromatograms under different wavelengths were obtained by determination, wherein the detection wavelengths were 220 nm, 240 nm, 280 nm, and 330 nm. The results are shown in Figure 6 .From bottom to top correspond to the wavelengths of 220 nm, 240 nm, 280 nm, and 330 nm. Figure 6 As can be seen from the results, when the detection wavelength is 240 nm, the fingerprint chromatogram has the most data of chromatographic peaks, and the separation degree between the peaks is good, so 240 nm is preferably selected as the detection wavelength.

[0093] 1.2 Gradient elution program

[0094]

[0095] Using the gradient elution program as a variable, tests were conducted according to the gradient elution programs in the table below. The same batch of Jiawei Qing'e Pills was used to prepare test solutions according to Example 1, and the fingerprint spectra of different gradient elution programs were obtained. The results are shown in the table below. Figure 7 , Figure 7 The gradient elution procedures 1-3 correspond to the order from bottom to top.

[0096] Gradient elution program 1

[0097]

[0098]

[0099] Gradient elution program 2

[0100] Time (min) Mobile phase A (%) Mobile phase B (%) 0～5 3 97 5～15 3→9 97→91 15～40 9→17 91→83 40～55 17→37 83→63 55～75 37→45 63→55 75～80 45→55 55→45

[0101] Gradient elution program 3

[0102] Time (min) Mobile phase A (%) Mobile phase B (%) 0～5 3 97 5～15 3→9 97→91 15～25 9→13 91→87 25～45 13→15 87→85 45～65 15→35 85→65 65～75 35 65 75～80 35→95 65→5

[0103] The results show that the fingerprint spectrum obtained by gradient elution program 3 has the highest separation degree and the best peak shape. Therefore, gradient elution program 3 is selected as the gradient elution program for Jiawei Qing'e Pill.

[0104] 1.3 Column Temperature

[0105] Using column temperature as a variable, the same batch of flavored Qing'e pills was used to prepare test solutions according to Example 1, and the fingerprint spectra at different column temperatures were obtained. The results are shown in [Figure 1]. Figure 8 ;in, Figure 8 The corresponding column temperatures from bottom to top are 30℃ and 40℃. The results show that the peak resolution and peak shape are not significantly different between column temperatures of 30℃ and 40℃. Taking all factors into consideration, 30℃ was chosen as the column temperature for the fingerprint chromatogram of Jiawei Qing'e Pill.

[0106] 1.4 Chromatographic Column

[0107] Using the chromatographic column as a variable, the same batch of Jiawei Qing'e Pills was used to prepare test solutions according to Example 1, and the fingerprints of different chromatographic columns were obtained. The results are shown in [Figure 1]. Figure 9 and Table 4; where, Figure 9 From bottom to top, these correspond to column 1 and column 2; column 1 is a Kromasil 100-5-C18 (4.6×250mm, 5μm); column 2 is an Agilent Zorbax SB-C18 (4.6mm×250mm, 5μm).

[0108] Table 4. Relative retention times of fingerprint peaks on different chromatographic columns.

[0109]

[0110] The above results show that the retention time of the characteristic peaks obtained by different chromatographic columns is different, but the relative retention time is basically stable, so both of the two chromatographic columns are suitable.

[0111] 1.5 Flow rate

[0112] Taking the same batch of Jiawei Qinggao Pills as an example, the test sample solution was prepared according to the preparation method of the test sample solution in Example 1, and the fingerprint spectrum of different flow rates was determined, and the results are shown in Table 2. Figure 10 ; wherein, Figure 10 The flow rates from bottom to top are 0.8 ml / min and 1.0 ml / min, respectively. It can be seen from the results that when the flow rate is 1.0 ml / min, the separation degree of the characteristic peaks in the obtained fingerprint spectrum is better.

[0113] 1.6 Injection amount

[0114] Taking the same batch of Jiawei Qinggao Pills as an example, the test sample solution was prepared according to the preparation method of the test sample solution in Example 1, and the fingerprint spectrum of different injection amounts was determined, and the results are shown in Table 3. Figure 11 ; wherein, Figure 11 The injection amounts from bottom to top are 5 μL, 10 μL and 20 μL, respectively.

[0115] It can be seen from the results that when the injection amount is 10 μL, there are more peaks in the fingerprint spectrum, and the separation degree of each chromatographic peak is optimal, so the injection amount is preferably 10 μL.

[0116] 1.7 Extraction solvent

[0117] In the preparation of the test sample solution, the extraction solvent was used as a variable, and the test sample solution was prepared according to the preparation method of the test sample solution in Example 1, and was determined; wherein the extraction solvent was methanol and 50% methanol, respectively, and the results are shown in Table 4. Figure 12 , Figure 12 The methanol and 50% methanol from bottom to top. It can be seen from the results that different concentrations of methanol have little effect on the fingerprint spectrum, and 50-100% methanol is suitable for the present application.

[0118] In summary, the chromatographic conditions of the present application are preferably: gradient elution program 3, column temperature is 30℃; the flow rate is 1.0 ml / min; the detection wavelength is 240 nm; the injection amount is 10 μL.

[0119] Experimental Example 2 Methodology investigation

[0120] 2.1 Precision

[0121] Take the same batch of Jia Wei Qinggao Pills, prepare the test sample solution according to Example 1, and continuously inject 6 times according to the chromatographic conditions of Example 1, respectively obtain the peak area and retention time of the characteristic peaks, calculate the relative peak area and relative retention time, and the results are shown in the table below. The experimental results show that the relative retention time of each characteristic peak has an RSD of less than 1%, and the relative peak area has an RSD of less than 5%, indicating good precision.

[0122] Table 5 Precision relative retention time calculation results

[0123]

[0124] Table 6 Precision relative peak area calculation results

[0125]

[0126]

[0127] 2.2 Stability

[0128] Take the same batch of Jia Wei Qinggao Pills, prepare the test sample solution according to Example 1, and inject at 0, 3, 6, 9, 12, 18, and 24 h after the preparation of the test sample solution, respectively, according to Example 1, calculate the relative retention time and relative peak area of each characteristic peak, and the results are shown in the table below. The experimental results show that the relative retention time of each common peak has an RSD of less than 1%, and the relative peak area has an RSD of less than 5%, indicating that the test sample solution has good stability within 24 h.

[0129] Table 7 Stability relative retention time calculation results

[0130]

[0131] Table 8 Stability relative peak area calculation results

[0132]

[0133] 2.3 Reproducibility

[0134] Take the same batch of Jia Wei Qinggao Pills, prepare 6 test sample solutions according to Example 1, and measure and calculate the relative retention time and relative peak area of each characteristic peak, and the results are shown in the table below. The experimental results show that the relative retention time of each common peak has an RSD of less than 1%, and the relative peak area has an RSD of less than 5%, indicating that the established Jia Wei Ganmao Pills fingerprint detection method has good reproducibility.

[0135] Table 9 Reproducibility relative retention time calculation results

[0136]

[0137] Table 10 Reproducibility relative peak area calculation results

[0138]

[0139] It is apparent that the above-described embodiments are merely example for the purpose of clear illustration, but are not limitation to the embodiments. Based on the above description, other different forms of changes or variations can be made by those of ordinary skill in the art. Here, it is not necessary and also impossible to exhaust all the embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A method for establishing the fingerprint spectrum of a compound drug, characterized in that, Includes the following steps: (1) Preparation of test solution: Take an appropriate amount of Jiawei Qing'e pills, soak them in methanol and then sonicate them to obtain the test solution; (2) Preparation of reference solution: Take appropriate amounts of crystallizing glycoside, genipin, echinacoside, verbascoside, psoralen and isopsoralen reference standards, add methanol to prepare reference solution; (3) Take the test solution and reference solution separately and determine them by high performance liquid chromatography; wherein, the chromatographic conditions are as follows: the chromatographic column is C18, 4.6 mm × 250 mm, 5 μm, with acetonitrile as mobile phase A and phosphoric acid aqueous solution as mobile phase B, gradient elution, detection wavelength is 240 nm; column temperature is 30 ℃; flow rate is 1.0 ml / min, gradient elution program is: 0~5 min, 3% A, 97% B; 5~ 15min, 3%→9%A, 97%→91%B; 15~25min, 9%→13%A, 91%→87%B; 25~45min, 13%→15%A, 87%→85% B; 45~65min, 15%→35%A, 85%→65%B; 65~75min, 35%A, 65%B; 75~80min, 35%→95%A, 65%→5%B; The raw materials of the compound drug include psoralea corylifolia, walnut kernel, morinda officinalis, cistanche deserticola, eucommia ulmoides, frankincense, and myrrh.

2. The method for establishing according to claim 1, characterized in that, In the chromatographic conditions: The concentration of phosphoric acid in the aqueous solution is 0.05-0.2%; and / or, The injection volume is 5-20 μL.

3. The method for establishing according to claim 1, characterized in that, The chromatographic conditions are as follows: the concentration of phosphoric acid in the phosphoric acid aqueous solution is 0.1%; the injection volume is 10 μL.

4. The method for establishing according to claim 1, characterized in that, Add solvent to the test sample, sonicate, filter, and collect the filtrate to obtain the test sample solution.

5. The method for establishing according to claim 4, characterized in that, The mass ratio of the test sample to the volume of the solvent is (2.5-15):(25-100); wherein the mass of the test sample is in g and the volume of the solvent is in ml.

6. The method for establishing according to claim 4, characterized in that, The ultrasound power is 200-500W, and the duration is 20-90min.

7. The method for establishing according to claim 4, characterized in that, The method for preparing the test solution also includes the step of adding diatomaceous earth.

8. The method for establishing according to claim 7, characterized in that, The amount of diatomaceous earth added is 0.2-2 times the mass of the test sample.

9. The method for establishing according to claim 1, characterized in that, Each 1 ml of the reference solution contains 60-180 μg of crystalline lecithin; and / or, Each 1 ml of the reference solution contains 20-60 μg of genipinic acid; and / or, Each 1 ml of the reference solution contains 120-360 μg of echinacoside; and / or, Each 1 ml of the reference solution contains 40-120 μg of verbascoside; and / or, Each 1 ml of the reference solution contains 55-165 μg of psoralen; And / or, Each 1 ml of the reference solution contains 50-150 μg of isopsoralen.

10. The method for establishing according to any one of claims 1-9, characterized in that, The fingerprint spectrum of the compound drug obtained by the establishment method includes 11 characteristic peaks, including peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, peak 9, peak 10 and peak 11. With peak 1 as the reference, the specified values ​​for the relative retention times of peaks 1, 2, 3, 4, 5, and 6 are 1.19, 3.19, 3.74, 6.96, and 7.44, respectively. With peak 7 as the reference, the specified values ​​for the relative retention times of peaks 2, 8, 9, 10 and 11 are 1.39, 1.45, 1.80 and 1.85, respectively. The relative retention time of the characteristic peak is within ±10% of the specified value.

11. The method for establishing according to claim 10, characterized in that, Peak 1 is crystalloside, peak 3 is genipin, peak 7 is echinacoside, peak 8 is verbascoside, peak 10 is psoralen, and peak 11 is isopsoralen.

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