A method for constructing a characteristic chromatogram of danqi qing'e tablets and quality control

Abstract

The application discloses a kind of Danzhi Qing'e tablet characteristic atlas construction and quality control method, wherein, a kind of Danzhi Qing'e tablet characteristic atlas construction method, the following chromatographic conditions are used to carry out gradient elution to test product solution;Chromatographic column: with octadecylsilane bonding silica gel as filler, column temperature is 40 DEG C ~ 45 DEG C;Mobile phase A is 0.2% formic acid aqueous solution, mobile phase B is methanol;The preparation method of test product solution is as follows: taking the mass M1 of test product, accurately weighed, ultrasonic treatment is added with solvent, and cold, solvent constant volume to volume V1, shake, centrifugation is taken supernatant or filter is taken filter liquor, and test product solution is obtained.The application is optimized by each chromatographic condition and test product solution cooperation, and only needs to carry out gradient elution at least effective simultaneously realizes the detection of the index component containing salt bux, salvia miltiorrhiza, salt eucommia ulmoid oliver, jiumu, with the advantages of short detection cycle, fast, high efficiency, and the method is simple, and has strong specificity.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of traditional Chinese medicine detection, and particularly relates to a method for constructing a characteristic chromatogram of Danzhiqing'e tablets and quality control. BACKGROUND

[0002] Danzhiqing'e tablets are based on Qing'e pills (hereinafter referred to as "Qing'e recipe") in "Taiping Hui Ming He Ji Lu Fang", and are prepared from scientific extraction of salt Duzhong, Danshen and Zhimu, etc. by starting from "Bu Yang method" and retaining salt Duzhong, etc. in the Qing'e recipe for tonifying kidney and assisting yang. The weight parts of the components are as follows: salt Buganqi 1 part, Danshen 2 parts, salt Duzhong 2 parts, and Zhimu 1 part.

[0003] Danzhiqing'e tablets are a compound preparation containing multiple effective components, and the main index components are psoralen, isopsoralen, salvianolic acid B, mangiferin and chlorogenic acid, etc. Danzhiqing'e tablets have the functions of tonifying liver and kidney and clearing heat to relieve restlessness, and are used for perimenopausal period and menopausal syndrome caused by liver and kidney deficiency, with symptoms of hot flashes, sweating, restlessness, palpitation, fatigue, soreness of the waist and knees, aversion to cold and cold limbs, pale or reddish tongue, thin white fur, and sunken and fine pulse.

[0004] At present, the quality control method of Danzhiqing'e tablets mainly includes thin layer identification of each medicinal material, and content determination of index components in Buganqi, Danshen and Zhimu. Although this method can control the quality of Danzhiqing'e tablets to a certain extent, it still cannot comprehensively reflect the quality status of Danzhiqing'e tablets. For example, salt Duzhong is the monarch drug, but there is no suitable index to evaluate and control its quality. In the quality standard research of Danzhiqing'e tablets disclosed in the prior art, the index components of all raw materials cannot be detected simultaneously, and each index component is detected by using different chromatographic conditions, for example: the index component salvianolic acid B in Danshen is detected by using acetonitrile-0.1% formic acid aqueous solution (24:76) as the mobile phase and detecting at a wavelength of 286 nm; the index components psoralen and isopsoralen in Buganqi are detected by using methanol-water (51:49) as the mobile phase and detecting at a wavelength of 246 nm; and the index component mangiferin in Zhimu is detected by using acetonitrile-0.2% glacial acetic acid aqueous solution (12:88) as the mobile phase and detecting at a wavelength of 258 nm. The detection is very inconvenient. In order to make up for the defects of the existing quality control method of Danzhiqing'e tablets, it is necessary to construct a standard characteristic chromatogram of the product, so as to better control the quality of the product. SUMMARY

[0005] Therefore, the technical problem to be solved by the present application is to overcome the defect that there is no method capable of effectively realizing the overall quality control of Danzhiqing'e tablets in the prior art, so as to provide a Danzhiqing'e tablet characteristic chromatogram construction and quality control method capable of simultaneously realizing the detection of index components containing salt-bu Zhuli, Danshen, salt-Duzhong and Zhimu under only one chromatographic condition.

[0006] A Danzhiqing'e tablet characteristic chromatogram construction method, wherein the following chromatographic conditions are used for gradient elution of the test sample solution:

[0007] The chromatographic column is filled with octadecylsilane-bonded silica gel, and the column temperature is 40-45 DEG C; the mobile phase A is 0.2% formic acid aqueous solution, and the mobile phase B is methanol;

[0008] The gradient elution procedure is as follows:

[0009]

[0010]

[0011] The preparation method of the test sample solution is as follows: a test sample with a mass of M1 is precisely weighed, ultrasonically treated with a solvent, cooled, and then diluted with a solvent to a volume of V1, uniformly shaken, and then centrifuged or filtered to obtain the test sample solution.

[0012] The centrifugation condition is 13000-20000 rpm for 5-15 min;

[0013] The filtration condition is 0.22-0.80 um microporous membrane filtration;

[0014] And / or, the ultrasonic condition is 220 V, 600 W, and the ultrasonic treatment is performed for 10-30 min;

[0015] And / or, the solvent is methanol or methanol aqueous solution with a volume concentration of greater than 50%;

[0016] The solid-liquid ratio of M1 to V1 is 1 g:(80-500) mL.

[0017] The solid-liquid ratio of M1 to V1 is 1 g:(200-300) mL, and preferably 1 g:250 mL.

[0018] The specification of the chromatographic column is 4.6 mm x 150 mm, 5 um;

[0019] And / or, the detection wavelength is 190-400 nm;

[0020] And / or, the column temperature is 35-45 DEG C;

[0021] And / or, the flow rate is 1.0 mL·min -1 ;

[0022] And / or, the sample injection amount is 5 μL-10 μL.

[0023] The detection wavelength is 254 nm.

[0024] A quality control method of Danzhiqing'e tablets, comprising:

[0025] The characteristic spectrum of the test sample is obtained by using the above-mentioned method for constructing a characteristic spectrum of Danzhiqing'e tablets, and the characteristic spectrum of the test sample at least includes four characteristic peaks corresponding to chlorogenic acid, mangiferin, salvianolic acid B, and psoralen.

[0026] The characteristic spectrum of the test sample at least includes 10 characteristic peaks corresponding to the standard characteristic spectrum.

[0027] The characteristic spectrum of the test sample at least includes characteristic peaks corresponding to chlorogenic acid, mangiferin, salvianolic acid B, psoralen, isopsoralen, neopsoralen isoflavone, psoralinnin, psoralen dihydroflavone methyl ether, tanshinone IIa, and psoralen phenol in the standard characteristic spectrum.

[0028] In the standard characteristic spectrum, the peak corresponding to the reference peak is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated; in the characteristic spectrum of the test Danzhiqing'e tablet, the relative standard deviation between the relative retention time of the characteristic peak corresponding to the standard characteristic spectrum and the relative retention time of the corresponding characteristic peak in the standard characteristic spectrum is within ± 5%.

[0029] The technical scheme of the present application has the following advantages:

[0030] 1. In the method for constructing a characteristic spectrum of Danzhiqing'e tablets provided by the present application, by optimizing the chromatographic conditions and the test sample solution, at least the detection of the index components of salt psoralen, salvia, salt eucommia, and anemarrhena can be effectively realized at the same time by performing gradient elution only once, specifically, the content determination of psoralen, chlorogenic acid, salvianolic acid B, and mangiferin can be simultaneously realized, the detection cycle is short, fast, and efficient, the method is simple to operate and has strong specificity, and it provides a good premise for the overall quality control of Danzhiqing'e tablets.

[0031] 2. In the quality control method of Danzhiqing'e tablets provided by the present application, the control of psoralen, chlorogenic acid, salvianolic acid B, and mangiferin can be simultaneously realized, the index components of salt psoralen, salvia, salt eucommia, and anemarrhena can be identified at the same time, the overall characteristics of the quality of Danzhiqing'e tablets can be more comprehensively reflected, and the requirements of the overall quality control of Danzhiqing'e tablets can be met.

[0032] 3、The application further limits the characteristic peak components, adopts 10 chromatographic peaks of chlorogenic acid, mangiferin, salvianolic acid B, psoralen, isopsoralen, neopsoralen isoflavone, psoralenning, psoralen dihydroflavone methyl ether, tanshinone IIa and psoralen phenol with larger peak area, better peak shape and better separation degree as characteristic fingerprint peaks, more comprehensively realizes the overall quality control of Danzhiqing'e tablets, and improves the precision of overall quality control. BRIEF DESCRIPTION OF DRAWINGS

[0033] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed to be used in the specific embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0034] Figure 1 is the characteristic spectrum of the test solution in the present application;

[0035] Figure 2 is the characteristic spectrum of the reference solution of the control in the present application;

[0036] Figure 3 is the fingerprint spectrum of the test solution in the present application detected under the condition of 45℃;

[0037] Figure 4 is the fingerprint spectrum of the test solution in the present application detected under the condition of 35℃;

[0038] Figure 5 is the fingerprint spectrum of the test solution in the present application detected under the condition of 50℃. DETAILED DESCRIPTION

[0039] Example 1

[0040] A method for constructing a characteristic spectrum of Danzhiqing'e tablets, comprising:

[0041] (1) Preparation of the test sample: 0.2g of Danzhiqing'e tablet powder (passed through a No. 3 sieve) was accurately weighed and placed in a 25ml volumetric flask, and an appropriate amount of methanol was added. Ultrasonic treatment (voltage 220V, power 600W) was carried out for 20 minutes, and then the sample was taken out and placed at room temperature. The methanol was diluted to the mark, shaken well, and centrifuged at 20000rpm for 5min. The supernatant was obtained;

[0042] (2) Preparation of the reference solution of the control:

[0043] Take chlorogenic acid, mangiferin, salvianolic acid B, psoralen, isopsoralen, new psoralen isoflavone, psoralen, psoralen dihydroflavone methyl ether, tanshinone IIa, and psoralen phenol reference substance, accurately weigh, add methanol to dissolve and prepare a mixed solution containing 50.2 μg of chlorogenic acid, 51.3 μg of mangiferin, 49.8 μg of salvianolic acid B, 50.2 μg of psoralen, 49.6 μg of isopsoralen, 52.5 μg of new psoralen isoflavone, 48.9 μg of psoralen, 52.3 μg of psoralen dihydroflavone methyl ether, 50.4 μg of tanshinone IIa, and 51.7 μg of psoralen phenol per 1 ml, as a reference solution of the reference substance.

[0044] (3) Determination method: 5ul of the reference solution and the test solution are respectively accurately taken, injected into the liquid chromatograph, and the chromatographic conditions of the liquid chromatograph are as follows:

[0045] The mobile phase A is a 0.2% formic acid aqueous solution, the mobile phase B is methanol, gradient elution is carried out according to the gradient elution program shown in Table 1 below, the detection wavelength is 254 nm, the column temperature is 40 DEG C, the flow rate is 1.0 ml / min, the chromatographic column is Agilent Eclipse XDB-C18, the specification of the chromatographic column is 4.6mm*150mm, and the filler particle size is 5um.

[0046] Table 1

[0047]

[0048] The characteristic chromatograms of the reference solution and the test solution obtained by the above detection are shown in Figure 1 and Figure 2 .

[0049] It can be known from Figure 1 and Figure 2 that the present application obtains a chromatogram by one gradient elution, at least effectively realizes the detection of the index components in salt psoralen, salvia, salt eucommia, and anemarrhena asphodeloides, for example: the detection of the effective components such as psoralen and isopsoralen in salt psoralen, salvianolic acid B in salvia, mangiferin in anemarrhena asphodeloides, and chlorogenic acid in salt eucommia can be simultaneously realized. The detection period of the method is short, fast, efficient, the operation of the method is simple, the detection of the index components of all raw materials can be realized, the specificity is strong, and therefore the method is used for the overall quality control of Danzhiqing'e tablets.

[0050] In summary, when the characteristic spectrum of the present application is used for overall quality control, the chromatogram of the to-be-tested Danzhiqing'e tablets can be compared with the standard characteristic spectrum. If the same four characteristic peaks corresponding to chlorogenic acid, mangiferin, salvianolic acid B, and psoralen appear in the chromatogram of the to-be-tested Danzhiqing'e tablets, preferably, the chromatogram of the to-be-tested Danzhiqing'e tablets includes at least 10 characteristic peaks compared with the standard characteristic spectrum, the peaks corresponding to the reference peaks are S peaks, the relative retention time of each characteristic peak and the relative retention time of each characteristic peak in the standard characteristic spectrum are calculated, and the relative standard deviation is within ± 5%, then the quality of the Danzhiqing'e tablets is qualified, otherwise it is not qualified, so that the quality of the Danzhiqing'e tablets can be quickly detected and judged. In this embodiment, the 10 characteristic peaks are chlorogenic acid (peak 1), mangiferin (peak 2), salvianolic acid B (peak 3), psoralen (peak 4), isopsoralen (peak 5), neopsoralen isoflavone (peak 6), psoralinnin (peak 7), psoralen dihydroflavone methyl ether (peak 8), tanshinone IIa (peak 9), and psoralenol (peak 10).

[0051] In this embodiment, the fingerprint spectrum of multiple batches of Danzhiqing'e tablets is obtained by using the above detection conditions, and a standard characteristic spectrum is obtained. The reference peak of each characteristic peak in the standard characteristic spectrum is the isopsoralen peak, and the relative retention time of each peak and the isopsoralen chromatographic peak is 0.31, 0.49, 0.80, 0.94, 1.00, 1.45, 1.55, 1.79, 2.02, and 2.11, respectively.

[0052] Example 2

[0053] The difference between this embodiment and Example 1 is that in the preparation of the sample solution, the solvent is replaced by 50% or 75% methanol aqueous solution instead of methanol, and the other conditions are the same as in Example 1. The peak area of each index component is determined, and the ratio of peak area to sample weight (A / g) is calculated. The detection results are shown in Table 2.

[0054] Table 2

[0055]

[0056] As shown in Table 2, when the extraction solvent is pure methanol, the measured values of the other index components in Danzhiqing'e tablets are relatively high except for chlorogenic acid, and the detection results of chlorogenic acid obtained by different extraction solvents have little difference. Considering comprehensively, pure methanol is selected as the extraction solvent for Danzhiqing'e tablets.

[0057] Example 3

[0058] The difference between this embodiment and embodiment 1 is that the ratio of the sample solution preparation is different, specifically, the sample solution is prepared by taking Danzhiqing'e tablet powder (0.3g, 0.2g, 0.1g), and the sample is precisely weighed and placed in different volume flasks (25mL, 50mL), and the detection is carried out according to the method of embodiment 1, the peak area of each index component is determined, and the ratio of the peak area of each index component to the sample weight (A / g) is calculated, and the detection results are shown in Table 3.

[0059] Table 3

[0060]

[0061] As shown in Table 3, compared with 3:250, 1:125 and 1:500, the detection results of the index components under different conditions are relatively small, and the determination value of the index components in Danzhiqing'e tablet is relatively high when the ratio of the sample solution (m:v) is 1:250, therefore, the optimal extraction solvent volume is determined to be 1:250.

[0062] Example 4

[0063] In the preparation of the sample solution, the extraction time is different, specifically, the sample solution is prepared by ultrasonic treatment (voltage 220V, power 600W) for different times (10min, 20min, 30min), and other operations are carried out according to the method of embodiment 1, the peak area of each index component is determined, and the ratio of the peak area of each index component to the sample weight (A / g) is calculated, and the detection results are shown in Table 4.

[0064] Table 4

[0065]

[0066] As shown in Table 4, compared with 10min and 30min, the detection results of the index components under different conditions are relatively small, and the determination value of the index components in Danzhiqing'e tablet is relatively high when the ultrasonic treatment time is 20min, therefore, the optimal extraction time is selected to be 20min.

[0067] Example 5

[0068] The difference between this embodiment and embodiment 1 is that the column temperature is adjusted to 45℃ during chromatographic elution, and the detection is carried out according to the method of embodiment 1, and the detection chromatogram is shown in Figure 3 .

[0069] As shown in Figure 3 , the sample solution is detected according to the method of embodiment 1, and the column temperature is 45℃, and the separation effect of each component chromatographic peak is good.

[0070] Example 6

[0071] The difference between this example and Example 1 is that the centrifugal condition for preparing the sample solution is 13000 rpm for 15 min, and the other conditions are the same as those in Example 1.

[0072] Example 7

[0073] The difference between this example and Example 1 is that the sample solution is obtained by filtering the filtrate, and the other conditions are the same as those in Example 1. Specifically, the sample solution is filtered by using a 0.22 μm microporous filter membrane in this example.

[0074] Example 8

[0075] The difference between this example and Example 1 is that the sample solution is obtained by filtering the filtrate, and the other conditions are the same as those in Example 1. Specifically, the sample solution is filtered by using a 0.80 μm microporous filter membrane in this example.

[0076] The detection is performed according to the method of Example 1, the peak area of each index component is determined, and the ratio of the peak area of each index component to the sample weight (A / g) is calculated. The detection results of Examples 6-8 are shown in Table 5.

[0077] Table 5

[0078]

[0079] It can be seen from Table 5 that the detection results of the index components under different conditions have little difference, indicating that the process conditions of the sample solution have little effect on the detection results.

[0080] Comparative Example 1

[0081] The difference between this comparative example and Example 1 is that the column temperature is adjusted to 35°C during the chromatographic elution, and the detection is performed according to the method of Example 1. The detection chromatogram is shown in Figure 4 .

[0082] It can be seen from Figure 4 that when the sample solution is detected according to the method of Example 1, and the column temperature is adjusted to 35°C or below, the separation effect of No. 7 chromatographic peak (Boschniakine) and No. 8 chromatographic peak (Boschniakine dihydroflavone methyl ether) is poor.

[0083] Comparative Example 2

[0084] The difference between this comparative example and Example 1 is that the column temperature is adjusted to 50°C during the chromatographic elution, and the detection is performed according to the method of Example 1. The detection chromatogram is shown in Figure 5 .

[0085] It can be seen from Figure 5It can be seen that when the column temperature is adjusted to 50°C or above, the separation effect of No. 3 chromatographic peak (salvianolic acid B) and No. 7 chromatographic peak (Buguqinning) is poor.

[0086] Experimental Example - Precision Test

[0087] 1. Precision Test

[0088] 1.1. Intra-day Precision Test

[0089] The test sample solution was prepared by the same preparation method as in Example 1, and the same portion of the test sample solution was precisely taken and injected repeatedly for 6 times to determine the peak area and retention time of each common peak. Taking No. 5 peak as the reference peak, the relative retention time and relative peak area of each common peak were calculated, and the results are shown in Table 6 and Table 7.

[0090] Table 6

[0091]

[0092] Table 7

[0093]

[0094] It can be seen from Table 6 and Table 7 that the RSD values of the relative retention time and relative peak area of each common peak are less than 2.47%, indicating that the precision of the method is good.

[0095] 1.2. Inter-day Precision

[0096] A test sample solution was prepared according to the method of Example 1, and the same portion of the test sample solution was precisely taken and injected repeatedly for 6 times every day, continuously for three days, to determine the peak area and retention time of each common peak. Taking No. 5 peak as the reference peak, the relative retention time and relative peak area of each common peak were calculated, and the results are shown in Table 8 and Table 9.

[0097] Table 8

[0098]

[0099] Table 9

[0100]

[0101] It can be seen from the above data in Table 8 and Table 9 that the RSD values of the relative retention time and relative peak area of each common peak are less than 2.17%, indicating that the precision of the method is good.

[0102] 2. Reproducibility

[0103] Six sample solutions were prepared in parallel by the same tester according to the method of Example 1, the peak area and retention time of each common peak were determined, and the relative retention time and relative peak area of each common peak were calculated with No. 5 peak as the reference peak. The results are shown in Table 10 and Table 11.

[0104] Table 10

[0105]

[0106] Table 11

[0107]

[0108] From the data in Table 10 and Table 11, it can be seen that the RSD values of the relative retention time and relative peak area of each common peak are less than 1.72%, indicating that the method has good repeatability.

[0109] 3. Stability

[0110] The sample solution was placed at room temperature, and samples were taken at different time points for detection to investigate the stability of the sample solution under room temperature conditions. The peak area and retention time of each common peak were determined, and the relative retention time and relative peak area of each common peak were calculated with No. 5 peak as the reference peak. The detection results are shown in Table 12-Table 13.

[0111] Table 12

[0112]

[0113] Table 13

[0114]

[0115] From Table 12 and Table 13, it can be seen that the RSD values of the relative retention time and relative peak area of each common peak are less than 2.11%, indicating that the sample is basically stable under room temperature conditions for 12h.

[0116] From the data of the experimental examples, through systematic methodological investigation, the RSD values of the relative retention time and relative peak area of precision, stability, and repeatability are less than 2.47%, indicating that the constructed fingerprint method of Danzhiqing'e tablets meets the requirements of fingerprint research.

[0117] Obviously, the above examples are only examples for clarity and do not limit 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 impossible to exhaust all embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present invention.

Claims

1. A method for constructing a characteristic spectrum of Danzhiqing'e slices, characterized in that: The test solution was gradient eluted using the following chromatographic conditions: Chromatographic column: Octadecylsilane bonded silica gel as filler, column temperature 40°C~45°C; mobile phase A is 0.2% formic acid in water, mobile phase B is methanol; The gradient elution procedure is: The preparation method of the test solution is as follows: take the test sample with a mass of M1, accurately weigh it, add solvent to perform ultrasonic treatment, cool it, add solvent to make the volume V1, shake it well, centrifuge it to obtain the supernatant or filter it to obtain the filtrate to obtain the test solution; The characteristic spectrum of the test solution includes at least 10 characteristic peaks, and the 10 characteristic peaks are the characteristic peaks corresponding to chlorogenic acid, mangiferin, salvianolic acid B, psoralen, isopsoralen, neopsoralen isoflavones, psoralen, psoralea corylifolia methyl ether, tanshinone IIa and bakuchiol.

2. The construction method according to claim 1, characterized in that The centrifugation condition is 13000 rpm to 20000 rpm for 5 min to 15 min; The filtration condition is 0.22μm~0.80μm microporous membrane filtration; And / or, the ultrasonic condition is 220V, 600W, ultrasonication for 10 min to 30 min; And / or, the solvent is methanol or a methanol aqueous solution with a volume concentration greater than 50%; And / or, the material-liquid ratio of M1 to V1 is 1 g: (80-500) mL.

3. The construction method according to claim 2, characterized in that The material-liquid ratio of M1 to V1 is 1 g: (200-300) mL.

4. The construction method according to claim 3, characterized in that The material-liquid ratio of M1 to V1 is 1 g:250 mL.

5. The construction method according to any one of claims 1 to 3, characterized in that: The specifications of the chromatographic column are 4.6 mm × 150 mm, 5 μm; and / or, the detection wavelength is 190 nm to 400 nm; and / or, flow rate of 1.0 mL min -1 ; And / or, the injection volume is 5μL~10μL.

6. The construction method according to claim 5, characterized in that: The detection wavelength is 254 nm.

7. The construction method according to claim 1, characterized in that The peak corresponding to isopsoralen was used as the reference peak, and the reference peak was the S peak. The relative retention times of the other characteristic peaks and the S peak were calculated. The relative retention times of the corresponding characteristic peaks in the standard characteristic spectrum were 0.31, 0.49, 0.80, 0.94, 1.00, 1.45, 1.55, 1.79, 2.02, and 2.11, respectively. In the characteristic spectrum of Danzhiqing'e tablets to be tested, the relative standard deviations between the relative retention times of the corresponding characteristic peaks and the relative retention times of the corresponding characteristic peaks in the standard characteristic spectrum were within ±5%.

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