CN112014508A

CN112014508A - Quality detection method of liver-protecting tablets

Info

Publication number: CN112014508A
Application number: CN202010916306.7A
Authority: CN
Inventors: 关彦斌
Original assignee: Sunflower Medicine Ltd
Current assignee: Sunflower Medicine Ltd
Priority date: 2020-09-03
Filing date: 2020-09-03
Publication date: 2020-12-01
Anticipated expiration: 2040-09-03
Also published as: CN112014508B

Abstract

The invention discloses a quality detection method of liver-protecting tablets, and relates to the technical field of pharmacy. The quality detection method of the liver protection tablet adopts liquid chromatography to detect the content of eight components of chlorogenic acid, vitexin, glycohyodeoxycholic acid, saikosaponin b2, schisandrin, saikosaponin b1, deoxyschizandrin and schisandrin B in a sample to be detected, which is prepared from the liver protection tablet, and comprises the following steps: carrying out gradient elution by using the mobile phase A and the mobile phase B, determining a detection map and calculating the content of the eight components; by controlling the composition of the mobile phase A and the mobile phase B, each component can form a detection spectrogram, the content of the eight components can be effectively detected, and the method has the characteristics of high efficiency, systematicness, characteristics, stability and the like.

Description

Quality detection method of liver-protecting tablets

Technical Field

The invention relates to the technical field of pharmacy, and in particular relates to a quality detection method of a liver protection tablet.

Background

The liver protection tablet takes six medicinal materials of radix bupleuri, oriental wormwood, isatis root, Chinese magnoliavine fruit, pig gall powder and mung bean powder as main raw materials, has the functions of soothing liver, regulating qi, strengthening spleen, promoting digestion and reducing transaminase, and is used for treating chronic hepatitis and early cirrhosis. The liver-protecting tablets have been clinically applied for many years and are also accepted by patients, and the liver-protecting tablets on the market have various types but have different product qualities. The quality control of the liver protection tablets by a quality detection method is not comprehensive, and at present, a plurality of researches on the liver protection tablets are basically single fingerprint spectrum researches or single-component content measurement and multi-component content measurement.

Wherein, the single fingerprint spectrum can integrally evaluate the quality of the medicine but is difficult to control the quality of a single medicinal material, and the component content measurement can well control the main components but is difficult to control the quality of other medicinal materials. In addition, the existing method has a long detection period, and the two quality detection methods are not practical and are difficult to apply to actual production.

In view of this, the present application is presented.

Disclosure of Invention

The invention aims to provide a quality detection method of a liver protection tablet, aiming at detecting the quality of the liver protection tablet more systematically and efficiently, and the detection method has better characteristics and stability.

The technical problem to be solved by the invention is realized by adopting the following technical scheme.

The invention provides a quality detection method of liver protection tablets, which adopts liquid chromatography to detect chlorogenic acid, vitexin, glycohyodeoxycholic acid and saikosaponin b in a sample to be detected, which is prepared from the liver protection tablets₂Schisandrin, bupleurum saponin b₁The contents of the schizandrin A and the schizandrin B comprise the following eight components:

carrying out gradient elution by using the mobile phase A and the mobile phase B, determining a detection map and calculating the content of the eight components;

wherein the mobile phase A is a phosphoric acid solution with the volume fraction of 0.03-0.1%, and the mobile phase B is at least one selected from acetonitrile and methanol.

The embodiment of the invention provides a quality detection method of liver-protecting tablets, which has the beneficial effects that: the liver protection tablet is prepared into a sample to be detected, the content of eight components including chlorogenic acid, vitexin, glycohyodeoxycholic acid, saikosaponin B2, schisandrin A, saikosaponin B1, deoxyschizandrin and schisandrin B in the liver protection tablet is detected by liquid chromatography, and the components can form a detection spectrogram by controlling the composition of a mobile phase A and the mobile phase B, so that the content of the eight components can be effectively detected, and the liver protection tablet has the characteristics of high efficiency, systematicness, characteristics, stability and the like.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a reference positioning chart for the specificity test of the detection method in the embodiment of the present invention;

FIG. 2 is a diagram of attribution of a specific characteristic peak of the detection method according to the embodiment of the present invention;

FIG. 3 is a characteristic peak spectral absorbance plot;

FIG. 4 is a characteristic peak spectral absorbance plot;

FIG. 5 is a control map of eight components;

FIG. 6 is an overlay of fingerprint spectra from batches;

FIG. 7 is a comparison of chromatograms of different mobile phases;

FIG. 8 is a chromatogram of a sample obtained by different extraction methods;

FIG. 9 is a chromatogram of the sample obtained at different detection wavelengths;

FIG. 10 is a graph comparing the results of different flow rate chromatograms;

FIG. 11 is a comparison of chromatographic results at different column temperatures;

FIG. 12 is a comparative graph of testing of different chromatography columns.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The method for detecting the quality of the liver protection tablet provided by the embodiment of the invention is specifically explained below.

The embodiment of the invention provides a quality detection method of liver protection tablets, which adopts liquid chromatography to detect chlorogenic acid, vitexin, glycohyodeoxycholic acid and saikosaponin b in a sample to be detected, which is prepared from the liver protection tablets₂Schisandrin, bupleurum saponin b₁The contents of the schizandrin A and the schizandrin B comprise the following eight components: carrying out gradient elution by using the mobile phase A and the mobile phase B, determining a detection map and calculating the content of the eight components; wherein the mobile phase A is a phosphoric acid solution with the volume fraction of 0.03-0.1%, and the mobile phase B is at least one selected from acetonitrile and methanol.

It should be noted that, the inventor can precisely detect the content of each component by optimizing the composition of the mobile phase to form an effective detection map for all eight components. If the mobile phase A is replaced by other components, an effective detection map cannot be formed, and if formic acid is adopted, the baseline can be collapsed.

In order to further increase the detection precision, the inventor further optimizes the composition of the mobile phase, wherein the mobile phase A is a phosphoric acid solution with the volume fraction of 0.04-0.06%, and the mobile phase B is acetonitrile.

Controlling the flow rate to be 0.7-0.9mL/min and 0.75-0.85mL/min in the gradient elution process; the column temperature is 28-32 ℃; the column is a C18 column, such as a Cortecs T3 (4.6X 150mm, 2.7 μm) column. The components are effectively separated by controlling the flow rate and the column temperature, and the detection accuracy is improved.

During the gradient elution, the elution conditions were controlled to be in table 1, in terms of volume ratio:

TABLE 1 gradient elution conditions

Time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	95	5
70	32	68
			75	95	5
85	95	5

Further, within 0-25min, the detection wavelength is 320-340 nm; within 25-42min, the detection wavelength is 200-210 nm; at 42-85min, the detection wavelength is 240-260 nm; preferably, the detection wavelength is 330nm within 0-25 min; within 25-42min, the detection wavelength is 205 nm; the detection wavelength is 250nm in 42-85 min. By further controlling the detection wavelength, each component can detect the peak, and if the technical scheme of the wavelength is not adopted, the situation that some components cannot be detected is likely to occur.

In some preferred embodiments, the sample to be tested is prepared by ethanol extraction and methanol reconstitution. Specifically, the preparation process of the sample to be tested comprises the following steps: removing coatings of the liver protection tablets, crushing, performing ultrasonic extraction by using an ethanol extracting solution, filtering, evaporating filtrate to dryness to obtain residues, re-dissolving the residues by using a methanol solution, and filtering to obtain filtrate as a sample to be detected; the volume fraction of the methanol solution is 40-60%, and the volume fraction of the ethanol extract is 90-98% in the ultrasonic extraction process by adopting the ethanol extract; the ultrasonic power is 300-400W, the ultrasonic frequency is 30-45kHz, and the ultrasonic time is 20-30 min. By adopting the extraction method, the effective components in the sample can be effectively extracted, and the detection accuracy is improved.

In some preferred embodiments, eight components are adopted to prepare a reference substance solution, a reference spectrum is formed, and the similarity between the fingerprint of the sample to be detected and the reference spectrum is greater than or equal to 0.90 according to the calculation of the traditional Chinese medicine fingerprint similarity evaluation system. The preparation of the control solution included: mixing the eight components with solvent, and controlling chlorogenic acid, vitexin, glycohyodeoxycholic acid, and saikosaponin b₂Schisandrin, bupleurum saponin b₁The concentrations of the deoxyschizandrin and the schisandrin B are 28-32 mug/mL, 3-5 mug/mL, 0.5-0.7mg/mL, 13-17 mug/mL, 90-110 mug/mL, 4-6 mug/mL, 18-22 mug/mL and 38-42 mug/mL in sequence; the solvent used was methanol.

Further, calculating the content of chlorogenic acid, vitexin, glycohyodeoxycholic acid, and saikosaponin b according to detection chromatogram₂Schisandrin, bupleurum saponin b₁The minimum contents of the deoxyschizandrin and the schisandrin B are as follows in sequence: 0.26mg/g, 0.03mg/g, 10.0mg/g, 0.11mg/g, 0.80mg/g, 0.13mg/g, 0.07mg/g, 0.03mg/g, and is acceptable if the content of the eight components is greater than or equal to the corresponding minimum content. By further controlling the minimum content of the eight components, a quality evaluation criterion is formed to evaluate whether the test sample is qualified.

In some preferred embodiments, multiple batches of liver protecting tablets are tested according to the quality detection method, the experimental data of the multiple batches of liver protecting tablets are introduced into the traditional Chinese medicine chromatogram fingerprint similarity evaluation system software, a median method is utilized, the chromatograms of random batches of liver protecting tablets are taken as reference spectra, automatic matching is carried out after multipoint correction, and a comparison fingerprint is generated.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

The embodiment provides a quality detection method of a liver-protecting tablet, namely, the fingerprint spectrum and the multi-component content of the liver-protecting tablet are detected, and the specific steps of the liver-protecting tablet fingerprint spectrum determination are as follows:

the instrument comprises the following steps: waters e2695(PDA detector); thermo U3000(PDA detector); agilent 1260(VWD detector); thermo U3000-QE high resolution mass spectrometer; electronic balance (one hundred thousand), XSE105DU, mettler-toledo; electronic balance (parts per million), MS204TS/02, mettler-toledo; sonicator, P120H, Elma Schmidbauer GmbH, germany; ultrapure water preparation apparatus, Elix advance 15, merck millipore.

Materials: liver-protecting tablet (batch: 201705024, 201903092, 201903034, 201902044, 201902037, 201902021, 201902020, 201812170, 201812052, 201710026 and 201707026), negative preparation lacking fructus Schisandrae chinensis, lacking bupleuri radix, lacking herba Artemisiae Scopariae, lacking radix Isatidis, lacking semen Phaseoli Radiati and lacking pulvis Fellis suis, Heilongjiang sunflower pharmaceutical Co.

Comparison products: schisandrin with purity of 99.7% is detected in the middle school; chlorogenic acid with purity of 96.8% and middle school; vitexin with purity of 94.9% and is collected in the inspection center; 98.7 percent of glycohyodeoxycholic acid and source leaf organisms; saikosaponin b2, 98.2%, source leaf organism; schizandrin A, 99.5%, middle school; schisandrin B, 99.1%, Zhongzhong hospital.

Acetonitrile (chromato graphic, honeyy-well), formic acid (mass chromato graphic, honeyy-well), phosphoric acid (chromato graphic, Fisher), ultrapure water (laboratory preparation).

Chromatographic conditions and System suitability test A Cortecs T3 (4.6X 150mm, 2.7 μm) column; gradient elution was performed with 0.05% phosphoric acid as mobile phase a and acetonitrile as mobile phase B as specified in table 1; the flow rate was 0.8mL per minute; the column temperature is 30 ℃; the detection wavelength is as follows: 0-25min, 330nm, 25-42min, 205 nm; 42-85min, 250nm, and the theoretical plate number is not less than 3000 according to the schizandrol A peak.

Preparation of control solutions: collecting chlorogenic acid, vitexin, and saikosaponin b₂Glycohyoxy, cholic acid, schizandrol A, and saikosaponin b₁The right amount of deoxyschizandrin, schizandrin A and schizandrin B reference substances are precisely weighed and added with methanol to prepare the medicine containing 30 mug chlorogenic acid, 4 mug vitexin and 0 glycohyodeoxycholic acid per 1 mL.6mg, 215 mug of saikosaponin B, 100 mug of schizandrol A, 15 mug of saikosaponin B, 20 mug of deoxyschizandrin and 40 mug of schisandrin B.

Preparation of a test solution: taking 10 tablets of the product, removing the coating, grinding, taking about 1.4g, precisely weighing, placing in a conical flask with a plug, precisely adding 20mL of 95% ethanol, sealing the plug, weighing, ultrasonically treating (with the power of 330W and the frequency of 37kHz) for 25 minutes, cooling, weighing again, supplementing the lost weight with 95% ethanol, shaking up, filtering, precisely weighing 10mL of subsequent filtrate, evaporating to dryness, dissolving the residue with 50% methanol, transferring to a 10mL measuring flask, adding 50% methanol to the scale, shaking up, filtering, and taking the subsequent filtrate as a liver protection tablet sample solution.

The determination method comprises the following steps: precisely sucking 10 μ L of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring. Taking 10 batches of liver protecting tablets of different manufacturers to test according to the method, introducing the experimental data of the 10 different manufacturers into traditional Chinese medicine chromatogram fingerprint similarity evaluation system software, using a median method, taking the chromatogram of a random batch of liver protecting tablets as a reference chromatogram, adopting multi-point correction and then automatically matching to generate a reference fingerprint, and adopting an external standard method to calculate the contents of chlorogenic acid, vitexin, glycohyodeoxycholic acid, saikosaponin b2, schizandrol A, saikosaponin b1, deoxyschizandrin and schizandrin B.

The chromatogram of the test sample should show the following 8 characteristic peaks, and the relative retention time of each characteristic peak is calculated by using schizandrol A as reference peak, and the result should be within + -5% of the specified value. And (3) regulating value: 0.25 (peak 1), 0.42 (peak 2), 0.88 (peak 3), 0.97 (peak 4), 1.00 (peak 5/S peak), 1.02 (peak 6), 1.51 (peak 7), 1.61 (peak 8). According to the similarity evaluation system of the traditional Chinese medicine chromatogram fingerprints, the similarity of the sample fingerprint and the comparison fingerprint is calculated to be not lower than 0.90.

And (3) testing results: corresponding chromatographic peaks with same retention time of reference substance chromatographic peaks are respectively presented in the fingerprint of the test sample; calculated according to the similarity evaluation system of the traditional Chinese medicine chromatogram fingerprint, the similarity of the fingerprint of the test sample and the comparison fingerprint should be not lower than 0.90, and the contents of chlorogenic acid, vitexin, glycohyodesoxycholic acid, saikosaponin b2, schisandrin, saikosaponin b1, deoxyschizandrin and schisandrin B should be not lower than 0.09 mg/tablet, 0.012 mg/tablet, 3.50 mg/tablet, 0.04 mg/tablet, 0.28 mg/tablet, 0.045 mg/tablet, 0.024 mg/tablet, 0.012 mg/tablet and 0.024 mg/tablet.

Test example 1

The specificity of the method of example 1 was tested and the results are shown in FIGS. 1 and 2, in which FIG. 1 shows the control location (from top to bottom: blank solution, schizandrol A, chlorogenic acid, vitexin, glycohyodeoxycholic acid, saikosaponin b₂And saikosaponin b₁Schizandrin a, schizandrin b and liver protection tablets 201705024), fig. 2 shows characteristic peak attribution (from top to bottom: herba Artemisiae Scopariae-deficient negative preparation, semen Phaseoli Radiati-deficient negative preparation, pulvis Fellis suis-deficient negative preparation, bupleuri radix-deficient negative preparation, fructus Schisandrae chinensis-deficient negative preparation, radix Isatidis-deficient negative preparation, and liver-protecting tablet 201705024).

As can be seen from FIGS. 1-2, the blank solution has no interference to fingerprint determination of hepatoprotective tablet, and of the 8 characteristic peaks, chlorogenic acid is attributed to herba Artemisiae Scopariae, vitexin is attributed to semen Phaseoli Radiati, glycohyodeoxycholic acid is attributed to pulvis Fellis suis, saikosaponin B2 and saikosaponin B1 are attributed to radix bupleuri, and schisandrin, deoxyschizandrin and schisandrin B are attributed to fructus Schisandrae chinensis. As can be seen from FIGS. 3-4, the spectral absorptions (190 nm-400 nm) of 8 characteristic peaks in the liver-protecting tablet sample are consistent with those of the corresponding reference substance, indicating that the specificity of the method is good.

Test example 2

The precision of the method in example 1 was tested, 6 parts of liver protection tablet test solution were prepared in parallel according to the test solution preparation method, the relative retention time and the relative peak area of each characteristic peak were calculated using the schizandrol a peak as a reference peak, and the similarity was calculated using the traditional Chinese medicine chromatogram fingerprint similarity evaluation software, and the results are shown in tables 2-4.

TABLE 2 precision results (relative Retention time)

TABLE 3 precision results (Peak area)

TABLE 4 precision results (similarity)

As can be seen from tables 2-4, in 6 test sample chromatographs, the relative retention time RSD% of each characteristic peak is between 0.01 and 0.11, and meets the requirement (the RSD% value is less than or equal to 1%); the RSD% value of the relative peak area of each characteristic peak is between 1.57 and 12.59, and meets the requirement (the RSD% value is less than or equal to 15%); the similarity of 6 test samples is greater than 0.997, which meets the requirement (not less than 0.95), thus the method has good precision.

Test example 3

The method of example 1 was tested for accuracy, and samples of the standard recovery rates of Hugan tablets were prepared at three concentrations, high, medium and low (the ratio of the amount of the added control to the amount of the component to be tested in the sample was about 1.5:1, 1:1, 0.5: 1), each concentration was prepared in triplicate, and liquid phase analysis was performed and the recovery rates calculated, the results are shown in Table 5.

As can be seen from Table 5, the recovery rate of chlorogenic acid in the liver-protecting tablet is between 98.9% and 107.4%, the average value is 103.5%, and the RSD value is 3.09, which meets the regulation (the recovery rate is more than or equal to 85% and less than or equal to 110%, and the RSD is more than or equal to 4%); the recovery rate of vitexin in the liver-protecting tablet is between 98.8 and 109.7 percent, the average value is 103.4 percent, the RSD value is 3.4, and the regulation is met (the recovery rate is more than or equal to 80 percent and less than or equal to 115 percent, and the RSD value is less than or equal to 6 percent); the recovery rate of glycohyodeoxycholic acid in the liver protection tablet is between 97.9% and 103.6%, the average value is 101.8%, the RSD% value is 1.4, the recovery rate of saikosaponin b2 in the liver protection tablet is between 98.0% and 106.6% according to the specification (the recovery rate is more than or equal to 90% and less than or equal to 108%, and the RSD% value is more than or equal to 3), the average value is 107.7%, the RSD% value is 1.3, the recovery rate is more than or equal to 110%, and the recovery rate of RSD% is less than or equal to 4); the recovery rate of schisandrin in the liver-protecting tablet is between 97.1% and 101.7%, the average value is 99.2%, the RSD value is 1.3, the recovery rate meets the requirements (the recovery rate is more than or equal to 90% and less than or equal to 108%, the RSD% is more than or equal to 3), the recovery rate of saikosaponin b1 in the liver-protecting tablet is between 98.2% and 109.3%, the average value is 105.2%, the RSD value is 3.1, the recovery rate meets the requirements (the recovery rate is more than or equal to 85% and less than or equal to 110%, and the RSD% is less than or equal.

The recovery rate of the schizandrin in the liver protection tablet is between 98.5 and 106.7 percent, the average value is 101.8 percent, the RSD value is 1.5, the recovery rate is up to 108 percent (90 percent to 3 percent), the recovery rate of the schizandrol in the liver protection tablet is between 101.6 and 105.4 percent, the average value is 103.0 percent, the RSD value is 1.3, and the recovery rate is up to 108 percent (90 percent to 3 percent).

TABLE 5 recovery test results

Test example 4

The stability of the method of example 1 was tested by taking one sample solution and examining the stability at 0h, 4h, 8h, 12h, 24h and 36h, respectively, and the results are shown in tables 6-7.

TABLE 6 stability test results (relative Retention time)

TABLE 7 stability test results (Peak area)

The result shows that the relative retention time RSD% value of each characteristic peak is between 0.008 and 0.73 and is less than 1.0 percent; the RSD% value of the relative peak area of each characteristic peak is between 0.2 and 1.7 and is less than 2.0 percent; the chromatogram obtained at each time point had a similarity of 1.000. In conclusion, the stability of the liver protection tablet test solution is good within 36 hours.

Test example 5

The method of example 1 was tested for column durability and the blank solution, the mixed control solution and the liver protection test solution were analyzed using different lot numbers of columns (LC04701 and LC04702), respectively, and the results are shown in tables 8 to 9.

TABLE 8 durability-chromatographic column test results (relative retention time)

TABLE 9 durability-results of column experiments (Peak area)

The results show that: the blank solution has no interference to the measurement of the fingerprint of the liver protecting tablet by using different chromatographic columns; by using different chromatographic columns, the relative retention time RSD% value of each characteristic peak is between 0.002 and 1.04 and is less than 2.0 percent; the RSD% value of the relative peak area of each characteristic peak is between 0.04 and 1.04 and is less than 2.0 percent; the two chromatographic columns have a similarity of 1.000, which meets the specification. In conclusion, the method has good durability when operated on chromatographic columns of the same model and different batches.

Test example 6

The method of example 1 was tested for durability against the amount of phosphoric acid, and the blank solution, the mixed control solution, and the liver protection test solution were analyzed with mobile phases (aqueous phases) of different amounts of phosphoric acid added, respectively, and the results are shown in tables 10 to 11.

TABLE 10 durability-phosphoric acid dosage test results (relative retention time)

TABLE 11 durability-phosphoric acid dosage test results (peak area)

The results show that: the blank solution has no interference to the measurement of the fingerprint of the liver protecting tablet by using different phosphoric acid addition amounts; by using different phosphoric acid addition amounts, the relative retention time RSD% value of each characteristic peak is between 0.02 and 0.52 and is less than 1.0 percent; the RSD% value of the relative peak area of each characteristic peak is between 0.17 and 1.43 and is less than 2.0 percent; the chromatographic results obtained with different amounts of phosphoric acid added were 1.000 in similarity, which met the specifications. In conclusion, the method has good durability when the addition amount of the phosphoric acid in the mobile phase (water phase) is between 0.04% and 0.06%.

Test example 7

Liquid phase analysis is carried out on 17 batches of liver protection tablet samples by using a liver protection tablet fingerprint spectrum method, schizandrol A is taken as a reference peak, and the relative retention time and the relative peak area of each peak are calculated (tables 12-15). The software of the traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012.13723 version) is used for processing chromatographic data of 17 batches of liver protection tablet samples, a median method is adopted, the time window width is 0.10, the characteristic chromatographic peaks are subjected to multi-point correction, Marck peak matching is carried out on the fingerprint chromatographic peaks of the 17 batches of liver protection tablet samples to generate a comparison spectrum (shown in figure 5) which is used as the fingerprint of the liver protection tablets, a fingerprint superposition graph is shown in figure 6, and the similarity results of the 17 batches of liver protection tablet samples are shown in figure 13.

In FIG. 5, 1-chlorogenic acid, 2-vitexin, 3-glycohyodeoxycholic acid, 4-saikosaponin B2, 5-schizandrol A, 6-saikosaponin B1, 7-deoxyschizandrin, and 8-schisandrin B. In FIG. 6, S1-201705024, S2-201706019, S3-201707026, S4-201710026, S5-201711006, S6-201801009, S7-201812052, S8-201812170, S9-201812179, S10-20191003, S11-201902020, S12-201902021, S13-201902037, S14-201902044, S15-201903034, S16-201903092, S17-201903099.

TABLE 12 sample determination test results (relative Retention time)

TABLE 13 determination of samples the results of the experiments (peak area)

TABLE 14 sample determination test results (similarity)

(S1-201705024、S2-201706019、S3-201707026、S4-201710026、S5-201711006、S6-201801009、S7-201812052、 S8-201812170、S9-201812179、S10-20191003、S11-201902020、S12-201902021、S13-201902037、S14-201902044、 S15-201903034、S16-201903092、S17-201903099)

TABLE 15 content determination of samples Experimental results (unit: mg/tablet)

The results show that the RSD% value of the characteristic peak relative retention time (taking the schizandrol A as a reference peak) of the 17 batches of liver protection tablets is between 0.01 and 0.36 and is less than 1 percent; the chromatographic similarities of the 17 batches were all greater than 0.97.

Comparative test

To demonstrate the superiority of the chromatographic condition control in this application, the following comparative example was added.

Comparative example 1

The comparative example provides a quality detection method of liver-protecting tablets, which is different from the example 1 only in that: mobile phase a was replaced with 0.2% formic acid (volume fraction) for 0.05% phosphoric acid (volume fraction). The spectrum of the test patterns of example 1 and comparative example 1 vs. figure 7, the baseline "collapse" when formic acid was added to the mobile phase. The results show that the base line of the chromatogram for adjusting the wavelength can be more stable when acetonitrile-0.05% phosphoric acid aqueous solution is used as a mobile phase for gradient elution, and the 0.05% phosphoric acid is selected as a water phase, so that the absorption of the pig gall powder and the isatis root is not influenced, and the characteristic peak is not interfered.

Comparative example 2

The comparative example provides a quality detection method of liver-protecting tablets, which is different from the example 1 only in that: the test results are shown in fig. 8, in which the method for preparing the test solution in example 1 is replaced by the method of water decoction, water ultrasound, ethanol ultrasound, methanol ultrasound, 50% methanol ultrasound, or ethyl acetate ultrasound.

The method comprises the following specific steps: taking 10 tablets of the product, removing the coating, grinding, taking about 1.4g, precisely weighing, placing in a conical flask with a plug, precisely adding 20mL of 95% ethanol, sealing the plug, weighing, ultrasonically treating (with the power of 330W and the frequency of 37kHz) for 25 minutes, cooling, weighing again, supplementing the lost weight with 95% ethanol, shaking up, filtering, precisely weighing 10mL of subsequent filtrate, evaporating to dryness, dissolving the residue with 50% methanol, transferring to a 10mL measuring flask, adding 50% methanol to the scale, shaking up, filtering, and taking the subsequent filtrate as a liver protection tablet sample solution. Wherein, ultrasonic water treatment, ultrasonic ethanol treatment, ultrasonic methanol treatment, ultrasonic 50% methanol treatment and ultrasonic ethyl acetate treatment are all performed by replacing 95% ethanol with the above solvents.

The preparation method of the water decoction comprises the following steps: taking 10 tablets of the product, removing the coating, grinding, taking about 1.4g, precisely weighing, placing in a conical flask with a plug, precisely adding 20mL of water, weighing, heating and refluxing for 25 minutes, cooling, weighing again, supplementing the lost weight with water, shaking up, filtering, precisely weighing 10mL of subsequent filtrate, evaporating to dryness, dissolving the residue with 50% methanol, transferring to a 10mL measuring flask, adding 50% methanol to scale, shaking up, filtering, and taking the subsequent filtrate as a liver protection tablet sample solution.

The results show that: the chromatographic information of the liver protection tablet test sample extracted by ethanol, methanol and 50% methanol in an ultrasonic mode is similar and most comprehensive, wherein the baseline of the test sample prepared from the ethanol is lifted to the minimum when the wavelength is switched, but the peak shape before 25min is affected by the solvent effect and is poorer.

It is to be noted that the extraction modes of water decoction, water ultrasound, ethanol ultrasound, methanol ultrasound, 50% methanol ultrasound and ethyl acetate ultrasound are contrastively investigated, and the method of ethanol extraction-50% methanol redissolution is adopted as the preparation method of the test sample, so that the operation is simple, the time and the efficiency are saved, and the chlorogenic acid, the vitexin and the saikosaponin b in the liver-protecting tablets can be effectively and comprehensively extracted₂Glycohyoxy, cholic acid, schizandrol A, and saikosaponin b₁The schizandrin A and the schizandrin B active ingredients eliminate the interference of other ingredients and can reduce the problem of poor peak shape caused by the preparation effect 25min before.

Comparative example 3

The comparative example provides a quality detection method of liver-protecting tablets, which is different from the example 1 only in that: the wavelength control in example 1 was adjusted to maintain 250nm at all times and the test results are shown in FIG. 9.

The results show that: the method can detect characteristic peaks of all medicinal materials by adopting a wavelength switching detection method, can better and comprehensively control the quality of the liver-protecting tablet, can detect only a few characteristic peaks by adopting a single wavelength (250nm), and cannot detect chlorogenic acid, vitexin and glycohyodeoxycholic acid.

It is to be supplemented that the selection of wavelength in chromatographic conditions of the invention, according to the comparison analysis of each negative preparation and finished product chromatogram, determine characteristic peak information of each unit of medicinal material, select the wavelength switching detection method, more comprehensively control the quality of the hepatoprotective tablet, make each chromatographic peak sensitivity and response value detected better, meanwhile exclude the interference of other components, and obtain the commonly used evaporation light detector of the present method for detecting glycohyodeoxycholic acid content according to literature search, but the method can utilize the ultraviolet detector to detect the glycohyodeoxycholic acid content in the hepatoprotective tablet through wavelength switching detection, reduce the trouble of detecting glycohyodeoxycholic acid by utilizing evaporation light again, save the detection cost.

Comparative example 4

The comparative example provides a quality detection method of liver-protecting tablets, which is different from the example 1 only in that: the flow rate in example 1 was adjusted to 1mL per minute and the results are shown in FIG. 10.

The results show that: when the flow rate is 1.0mL/min, the saikosaponin b2 and the schizandrin A peak are not completely separated.

Comparative example 5

The comparative example provides a quality detection method of liver-protecting tablets, which is different from the example 1 only in that: the column temperature was adjusted from 30 ℃ in example 1 to 35 ℃.

As shown in fig. 11, the experimental results show that: at the same flow rate, the temperature rises, and the No. 6 peak can separate; while peak 14 (saikosaponin b2) and peak 15 (schizandrol A) showed complete overlap. The column temperature is 30 ℃, so that the stability of each characteristic peak in the chromatogram is good, the chromatographic peaks with strong absorption have good separation degree, and the peak shape of each chromatographic peak is good.

Comparative example 6

The comparative example provides a quality detection method of liver-protecting tablets, which is different from the example 1 only in that: the column was replaced by Agilent Poroshell 120 SB-C18 and Waters X-bridge C18 from Waters Cortecs T3 in example 1, and the results are shown in FIG. 12.

As is clear from FIG. 12, under the same chromatographic conditions, the column Waters Cortecs T3(2.7 μm, 4.6X 150 mm) exhibited the best performance, and therefore, this column was selected as the column for liver protection fingerprint optimization.

In summary, the quality detection method of the liver protection tablet provided by the invention is characterized in that the liver protection tablet is prepared into a sample to be detected, the liquid chromatography is utilized to detect the content of eight components including chlorogenic acid, vitexin, glycohyodeoxycholic acid, saikosaponin B2, schizandrol A, saikosaponin B1, schizandrin A and schizandrin B, the composition of the mobile phase A and the mobile phase B is controlled to enable each component to form a detection spectrogram, the content of the eight components is effectively detected, and the liver protection tablet has the characteristics of high efficiency, systematicness, characteristics, stability and the like.

The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Claims

1. A quality detection method of Hugan tablet is characterized in that liquid chromatography is adopted to detect chlorogenic acid, vitexin, glycohyodeoxycholic acid and saikosaponin b in a sample to be detected, which is prepared from Hugan tablet₂Schisandrin, bupleurum saponin b₁The contents of the schizandrin A and the schizandrin B comprise the following eight components:

2. The quality detection method of the liver protection tablet as claimed in claim 1, wherein the mobile phase A is a phosphoric acid solution with a volume fraction of 0.04-0.06%, and the mobile phase B is acetonitrile.

3. The quality detection method of liver protection tablets according to claim 2, characterized in that in the gradient elution process, the flow rate is controlled to be 0.7-0.9mL/min, preferably 0.75-0.85 mL/min;

preferably, the column temperature is 28-32 ℃; preferably, the column is a C18 column.

4. The method for detecting the quality of a liver protection tablet as claimed in claim 2, wherein the detection wavelength is 320-340nm within 0-25 min; within 25-42min, the detection wavelength is 200-210 nm; at 42-85min, the detection wavelength is 240-260 nm;

preferably, the detection wavelength is 330nm within 0-25 min; within 25-42min, the detection wavelength is 205 nm; the detection wavelength is 250nm in 42-85 min.

5. The quality detection method of liver protection tablets according to claim 2, wherein in the gradient elution process, the elution conditions are controlled as follows in terms of volume ratio:

6. The quality detection method of the liver protection tablet according to claim 1, wherein the sample to be detected is prepared by a method of ethanol extraction and methanol redissolution;

preferably, the preparation process of the sample to be tested comprises the following steps: removing the coating of the liver protection tablet, crushing, performing ultrasonic extraction by using an ethanol extracting solution, filtering, evaporating filtrate to dryness to obtain residues, re-dissolving the residues by using a methanol solution, and filtering to obtain filtrate as the sample to be detected;

preferably, the volume fraction of the methanol solution is 40-60%.

7. The quality detection method of the liver protection tablet as claimed in claim 6, wherein in the ultrasonic extraction process by using the ethanol extract, the volume fraction of the ethanol extract is 90-98%;

preferably, the ultrasonic power is 300-400W, the ultrasonic frequency is 30-45kHz, and the ultrasonic time is 20-30 min.

8. The quality detection method of liver-protecting tablets according to claim 1, characterized in that the eight components are adopted to prepare a reference solution, a reference spectrum is formed, and the similarity between the fingerprint spectrum of the sample to be detected and the reference spectrum is required to be greater than or equal to 0.90 according to the calculation of a traditional Chinese medicine fingerprint spectrum similarity evaluation system.

9. The method for detecting the quality of a liver-protecting tablet according to claim 8, wherein the control isThe preparation of the product solution comprises: mixing the eight components with solvent, and controlling chlorogenic acid, vitexin, glycohyodeoxycholic acid, and saikosaponin b₂Schisandrin, bupleurum saponin b₁The concentrations of the deoxyschizandrin and the schisandrin B are 28-32 mug/mL, 3-5 mug/mL, 0.5-0.7mg/mL, 13-17 mug/mL, 90-110 mug/mL, 4-6 mug/mL, 18-22 mug/mL and 38-42 mug/mL in sequence;

preferably, the solvent used in the preparation of the control solution is methanol.

10. The quality detection method of the liver protection tablet of any one of claims 1 to 9, wherein the content of each component is calculated according to a detection map, and the liver protection tablet is qualified if the content is greater than or equal to the respective minimum content of the eight components, and the minimum content of chlorogenic acid, vitexin, glycohyodeoxycholic acid, saikosaponin b2, schizandrol A, saikosaponin b1, deoxyschizandrin and schisandrin B are sequentially: 0.26mg/g, 0.03mg/g, 10.0mg/g, 0.11mg/g, 0.80mg/g, 0.13mg/g, 0.07mg/g, 0.03 mg/g;

preferably, a plurality of batches of liver protecting tablets are tested according to the quality detection method of any one of claims 1 to 9, the experimental data of the plurality of batches of liver protecting tablets are introduced into the traditional Chinese medicine chromatogram fingerprint similarity evaluation system software, the chromatogram of a random batch of liver protecting tablets is used as a reference spectrum by a median method, and the reference fingerprint is generated by automatic matching after multipoint correction.