CN115356420A - Pudilan anti-inflammatory tablet quality evaluation method based on one-test-multiple evaluation - Google Patents

Abstract

The invention discloses a Pudilan anti-inflammatory tablet quality evaluation method based on one test and multiple evaluations, which comprises the steps of preparing a single reference substance solution, preparing a mixed reference substance solution, preparing a test sample solution, feeding the mixed reference substance solution into a high performance liquid chromatograph, carrying out sample injection measurement to obtain a liquid chromatogram, recording the peak area of each component, and calculating relative correction factors of other 6 components to be tested by adopting a multipoint correction method and taking baicalin as an internal reference substance; analyzing and determining the positioning basis of the chromatographic peak of the component to be detected. The invention creates a quick, accurate and low-cost method for controlling and evaluating the quality of the Pudilan anti-inflammatory tablet, and the contents of 7 index components are simultaneously measured by one-time evaluation, namely, only one reference substance of baicalin is needed, and the contents of other 6 components can be obtained by calculating relative correction factors.

Description

Pudilan anti-inflammatory tablet quality evaluation method based on one-test-multiple evaluation

Technical Field

The invention belongs to the technical field of traditional Chinese medicine component detection, and relates to a Pudilan anti-inflammatory tablet quality evaluation method based on one-test-multiple evaluation.

Background

The Pudilan anti-inflammatory tablet is a common Chinese medicinal compound preparation, consists of 4 Chinese medicaments of dandelion, scutellaria baicalensis, corydalis bungeana and isatis root, has the functions of clearing heat and removing toxicity, resisting inflammation and diminishing swelling, is mainly used for treating furuncle, parotitis, pharyngitis, lymphadenitis and the like clinically, and is a common non-prescription medicament. As the variety is not loaded in Chinese pharmacopoeia all the time, the quality standard is basically the enterprise registration standard, and no unified standard is used for quality analysis and evaluation. The Pudilan antiphlogistic tablet has complex components, the existing quality standard quantitative analysis only determines a single component of baicalin, and the quality control method only depends on the single component determination, so that the real quality of the Pudilan antiphlogistic tablet is difficult to objectively and effectively evaluate. At present, basic research on the pharmacodynamic substances of traditional Chinese medicines reveals the activity or index components of the traditional Chinese medicines as much as possible, and quantitative analysis is carried out, so that the method becomes a new trend of traditional Chinese medicine quality evaluation. However, the problem of large use amount of each component reference substance is faced in the actual measurement process of multi-index components, the separation difficulty and the price of the chemical component reference substances of the traditional Chinese medicine are high, and the problem of reference substance supply becomes a bottleneck restricting the quantitative analysis of the multi-index components of the traditional Chinese medicine.

In view of this, it is urgent to establish a quality control and evaluation method which has the characteristics of convenience, accuracy and low cost and can objectively reflect the internal quality of the traditional Chinese medicine compound preparation.

A QAMS (quantitative assessment method) is a multi-index quality evaluation mode suitable for Chinese medicinal characteristics, and is characterized in that only one component (a reference substance is easy to obtain) is measured by utilizing the inherent functional relationship and proportional relationship of the effective components of the Chinese medicinal herbs, so that the synchronous measurement of a plurality of components (the reference substance is difficult to obtain or supply) is realized. Under the condition of insufficient reference substances, the quality control of the traditional Chinese medicine by using the relative correction factor is convenient, rapid and cheap, and the method is a development direction of multi-component synchronous quantification of the traditional Chinese medicine. At present, the research on the content determination of multiple components of the Pudilan antiphlogistic tablet has been reported in documents, but the detection methods adopted in the documents are all external standard methods, the methods are complicated, and the detection cost is high, but a one-test multiple-evaluation method for synchronously determining the multiple index components of the Pudilan antiphlogistic tablet has not been reported.

Disclosure of Invention

The invention aims to provide a Pudilan anti-inflammatory tablet quality evaluation method based on one-test-multiple evaluation, and establishes a quality control and evaluation method which has the characteristics of convenience, accuracy and low cost and can objectively reflect the internal quality of a traditional Chinese medicine compound preparation. The invention creates a quality control and evaluation method for simultaneously measuring multiple index components of the Pudilan anti-inflammatory tablet based on one-measurement multiple evaluation on the basis of analysis of the effective components (index components) of the Pudilan anti-inflammatory tablet, namely, the content of one component (easily obtained reference substance) in a preparation is measured through the functional relation and the proportional relation existing between the effective components (index components) in the preparation, so that the synchronous measurement of the multiple components (difficultly obtained reference substance) is realized. The invention adopts a multi-wavelength switching HPLC method, uses baicalin as a reference substance to determine relative correction factors of other 6 index components (monocaffeoyltartaric acid, chicoric acid, oroxylin A-7-0-beta-D-glucuronide, wogonoside, baicalein and wogonin) in the Pudilan anti-inflammatory tablet, can realize synchronous determination of 7 components by only using a baicalin reference substance, can greatly reduce the detection cost by one-test and multi-evaluation determination of the multiple index components of the Pudilan anti-inflammatory tablet, and provides a convenient and reliable new method for quality evaluation of the Pudilan anti-inflammatory tablet.

In order to achieve the purpose, the invention adopts the following technical scheme:

a Pudilan anti-inflammatory tablet quality evaluation method based on one-test-multiple evaluation comprises the following steps:

1) Preparation of a single control solution: respectively dissolving single caffeoyltartaric acid, chicoric acid, baicalin, oroxylin A-7-0-beta-D-glucuronide, wogonoside, baicalein and wogonin with methanol, and diluting to obtain single reference substance stock solution;

2) Preparation of mixed control solutions: respectively taking the single reference substance stock solutions obtained in the step 1), placing the single reference substance stock solutions into a measuring flask, and adding a methanol solution for dilution to obtain a mixed reference substance solution;

3) Preparing a test solution: taking a sample, placing the sample in a conical flask, adding a methanol solution, sealing, weighing, heating and refluxing, taking out, cooling, weighing again, complementing the lost weight with the methanol solution, shaking up, filtering, and taking a filtrate to obtain a sample solution;

4) Feeding the mixed reference substance solution obtained in the step 2) into a high performance liquid chromatograph, carrying out sample injection measurement to obtain a liquid chromatogram, recording peak areas of all components, and calculating relative correction factors of other 6 components to be measured by adopting a multipoint correction method and taking baicalin as an internal reference substance;

5) Analyzing and determining the positioning basis of the chromatographic peak of the component to be detected.

As a preferred embodiment of the present invention, in step 1), monocaffeoyltartaric acid 271.46. Mu.g/mL, chicoric acid 443.49. Mu.g/mL, baicalin 915.08. Mu.g/mL, oroxylin A-7-0- β -D-glucuronide 580.55. Mu.g/mL, wogonin 376.27. Mu.g/mL, baicalin 291.55. Mu.g/mL and wogonin 159.54. Mu.g/mL are measured by mass concentration.

As a preferred embodiment of the present invention, in step 2), the amount of monocaffeoyltartaric acid 21.716. Mu.g/mL, chicoric acid 17.740. Mu.g/mL, baicalin 292.82. Mu.g/mL, oroxylin A-7-0- β -D-glucuronide 46.444. Mu.g/mL, wogonin 75.254. Mu.g/mL, baicalin 11.662. Mu.g/mL and wogonin 6.382. Mu.g/mL are measured by mass concentration.

As a preferable embodiment of the present invention, in the step 3), the ratio of the amount of the sample added to the volume of methanol is 0.4g:50mL.

In a preferred embodiment of the present invention, in step 3), the volume percentage concentration of the methanol as the extraction solvent is 70%.

As a preferable scheme of the invention, in the step 3), the heating reflux time is 2h, and the temperature is reduced to the room temperature after the heating reflux time is taken out.

As a preferred embodiment of the present invention, in step 4), the chromatographic conditions are: agilent Poroshell 120SB-C ₁₈ 2.1X 50mm,2.7 μm; mobile phase: the mobile phase A is methanol, the mobile phase B is acetonitrile, and the mobile phase C is 0.1 percent phosphoric acid solution;

the gradient elution was: 0-5 min, mobile phase A,10% → 20%, mobile phase B,5%, mobile phase C,85% → 75%;

5-20 min, mobile phase A,20% → 25%, mobile phase B,5% → 10%, mobile phase C,75% → 65%;

20-25 min, mobile phase A,25% → 40%, mobile phase B,10% → 15%, and mobile phase C,65% → 45%;

30-35 min, mobile phase A,40%, mobile phase B,15% and mobile phase C,45%;

flow rate: 0.3mL/min; detection wavelength: 0-13min, 326nm; 14-35min, 274nm; the column temperature is 30 ℃; sample injection amount: 2 μ L.

As a preferable scheme of the invention, in the step 4), baicalin is taken as an internal reference, and the relative correction factor f of other 6 components to be measured is calculated, wherein the calculation formula is f = f _s /f _k ＝(C _k ×A _s )/(C _s ×A _k )；

Wherein C is _k To determine the content of the component A _k Is the peak area of the component to be measured, C _s Is the content of an internal reference substance, A _s The peak area of the internal reference substance is shown.

As a preferred embodiment of the present invention, in step 5), a relative retention value method is adopted as a chromatographic peak positioning basis, and the deviation is limited to (+/-5%).

Compared with the prior art, the invention has the following beneficial effects:

1) The invention researches the drug effect substance basis of the Pudilan anti-inflammatory tablet, and obtains 2 phenolic acid components capable of representing monarch drug-dandelion and 5 flavonoid components capable of representing ministerial drug-scutellaria baicalensis through spectral effect analysis.

2) The invention creates a quick, accurate and low-cost method for controlling and evaluating the quality of the Pudilan anti-inflammatory tablet, and the contents of 7 index components are simultaneously measured by a one-measurement and multi-evaluation method, namely the contents of other 6 components can be calculated by relative correction factors only by using baicalin as a reference substance.

3) In HPLC determination, the invention adopts multi-wavelength switching to ensure that different types of index components in the Pudilan anti-inflammatory tablet obtain the best chromatographic response, thereby greatly improving the detection sensitivity of each component.

4) The flavonoid components in the scutellaria baicalensis are similar in structure and large in quantity, and the chromatographic separation difficulty is high, a fluidity system of 3 solvent combinations is established, and through gradient elution, various flavonoid components in the scutellaria baicalensis are well separated from a base line, so that the accuracy and reliability of the positioning of each chromatographic peak in the determination of one-test multi-evaluation method are ensured.

5) According to the invention, the pretreatment extraction mode of the Pudilan anti-inflammatory tablet sample is investigated, and the stability of the ultrasonic extraction method loaded by the national drug standard of part of the Pudilan anti-inflammatory tablet is proved to be poor through the stability investigation and reason analysis of the sample solution, so that the ultrasonic extraction method does not meet the formulated requirement of the drug standard.

Drawings

FIG. 1 is an HPLC chart of the mixed control (A) and sample (B) of the present invention.

Figure 2 is a full wavelength scan of DAD detection of monocaffeyltartaric acid.

FIG. 3 is a full wavelength scan of the DAD detection of chicoric acid.

FIG. 4 is a full wavelength scan of the DAD detection of baicalin.

FIG. 5 is a full wavelength scan of the DAD detection of oroxylin A-7-0- β -D-glucuronide.

FIG. 6 is a full wavelength scan of the DAD detection of wogonoside.

FIG. 7 is a full wavelength scan of the DAD detection of baicalein.

FIG. 8 is a full wavelength scan of the DAD detection of wogonin.

In the figure, 1, monocaffeyltartaric acid, 2, chicoric acid, 3, baicalin, 4, oroxylin A-7-0-beta-D-glucuronide, 5, wogonoside, 6, baicalein, 7 and wogonin.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. 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.

The instruments and materials used in the present invention are as follows:

instrument

Agilent 1260 high performance liquid chromatograph (Agilent, USA), waters 2695 high performance liquid chromatograph (Waters, USA), XSE205DU electronic balance (Mettler-Tooliduo instruments (Shanghai) Co., ltd.), HHS-11-8 electric heating constant temperature water bath (Shanghai Bocheng Shi Co., ltd.).

Material

Comparison products: chicoric acid (batch No. 111752-201703, content: 97.6%), baicalin (batch No. 111752-201703, content: 93.5%), baicalein (batch No. 111595-201808, content: 97.8%), wogonin (batch No. 112002-201702, content: 98.5%), and wogonin (batch No. 111514-201304) were purchased from the institute of food and drug testing; monocaffeoyltartaric acid (batch: G29J12L139066, content ≥ 98%) was purchased from Shanghai-derived leaf Biotech Co., ltd; oroxylin A-7-O-beta-D-glucuronide (batch number: CFS201801, content ≥ 98%) was purchased from Wuhantian Biotechnology GmbH.

Reagent: phosphoric acid is super pure (national drug group chemical reagent limited); both methanol and acetonitrile are chromatographically pure (Merck, germany); the water is ultrapure water.

Sample preparation: the 10 purplish blue anti-inflammatory tablet samples come from 3 manufacturers: guangdong Xinbao pharmaceutical science and technology Co., ltd (batch No.: 20210502, 20210702, 20210117, 20211204).

Foshan Yibaosheng pharmaceutical Co., ltd (batch Nos.: 120041, 121015, 121020).

Tianchangyi Sai pharmaceuticals Ltd (batch Nos.: 210203, 210601, 201202).

The invention provides a Pudilan anti-inflammatory tablet quality evaluation method based on one-test-multiple evaluation, which comprises the following steps:

1) Preparation of single control solutions: respectively dissolving single caffeoyl tartaric acid, chicoric acid, baicalin, oroxylin A-7-0-beta-D-glucuronide, wogonoside, baicalein and wogonin with methanol, and diluting to obtain single reference stock solution;

2) Preparation of mixed control solutions: respectively taking the single reference substance stock solutions obtained in the step 1), placing the single reference substance stock solutions into a measuring flask, and adding a methanol solution for dilution to obtain a mixed reference substance solution;

3) Preparing a test solution: taking a sample, placing the sample in a conical flask, adding a methanol solution, sealing, weighing, heating and refluxing, taking out, cooling, weighing again, complementing the lost weight with the methanol solution, shaking up, filtering, and taking a filtrate to obtain a sample solution;

4) Feeding the mixed reference substance solution obtained in the step 2) into a high performance liquid chromatograph, carrying out sample injection measurement to obtain a liquid chromatogram, recording peak areas of all components, and calculating relative correction factors of other 6 components to be measured by adopting a multipoint correction method and taking baicalin as an internal reference substance;

5) Analyzing and determining the positioning basis of the chromatographic peak of the component to be detected.

Chromatographic conditions

A chromatographic column: agilent Poroshell 120SB-C ₁₈ (2.1X 50mm,2.7 μm); mobile phase: methanol (a) -acetonitrile (B) -0.1% phosphoric acid solution (C) was subjected to gradient elution according to table 1; flow rate: 0.3mL/min; detection wavelength: 326nm (0-13 min) and 274nm (14-35 min); the column temperature is 30 ℃; sample introduction amount: 2 μ L. Analyzing according to the above chromatographic conditions, separating each peak in sample chromatogram well, counting with baicalin with theoretical plate number more than 30000, and showing reference and sample chromatogram in figure 1.

Table 1 hplc mobile phase gradient chart

Time	Mobile phase A/%)	Mobile phase B/%)	Mobile phase C/%)
				0～5	10→20	5	85→75
5～20	20→25	5→10	75→65
				20～30	25→40	10→15	65→45
30～35	40	15	45

Example 1

Preparation of the solution

Step 1) preparation of single control solution:

taking appropriate amount of monocaffeoyltartaric acid, chicoric acid, baicalin, oroxylin A-7-0-beta-D-glucuronide, wogonoside, baicalein and wogonin as reference substances, precisely weighing, and respectively adding methanol to dissolve and dilute into single reference substance storage liquid with mass concentration of 271.46, 443.49, 915.08, 580.55, 376.27, 291.55 and 159.54 mu g/mL.

Step 2) preparation of mixed reference substance solution: taking appropriate amount of each single control stock solution in the step 1), placing the single control stock solution in a 25mL measuring flask, adding methanol to dilute the single control stock solution into a mixed control solution I (for linear measurement) with the mass concentration of 21.716, 17.740, 292.82, 46.444, 75.254, 11.662 and 6.382 mu g/mL respectively; then taking each appropriate amount of the single control stock solution in the step 1), placing the single control stock solution into a 500mL measuring flask, adding 20% methanol to dilute the single control stock solution into a mixed control solution II (used for determining the recovery rate) with the mass concentration of 13.571, 17.740, 183.02, 29.034, 37.635, 11.662 and 3.1925 mu g/mL respectively.

Step 3) preparation of a test solution:

taking 10 tablets of the product, removing the coating, grinding, taking about 0.4g, precisely weighing, placing in a conical flask with a plug, precisely adding 50mL of 70% methanol, sealing the plug, weighing, heating and refluxing for 2h, taking out, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking up, filtering, and taking the subsequent filtrate.

Example 2

Investigation of linear relationships

Precisely sucking 0.5, 1, 2, 4 and 8 μ L of the mixed reference substance solution I obtained in the step 2), injecting into a liquid chromatograph, performing sample injection measurement according to the chromatographic conditions, and drawing a standard curve by taking the mass of the reference substance as a horizontal coordinate (X) and the peak area as a vertical coordinate (Y), wherein the results are shown in Table 2. The correlation coefficient (r) of 7 components is greater than 0.999, which indicates that the components are in good linear relation in the corresponding mass range.

TABLE 2.7 component regression equation, correlation coefficient, linear Range

Precision test

Precisely sucking 2 mu L of the mixed reference substance solution I in the step 2), continuously injecting samples for 6 times according to the chromatographic conditions, and determining that the RSD of the peak areas of the monocaffeyltartaric acid, the chicoric acid, the baicalin, the oroxylin A-7-0-beta-D-glucuronide, the wogonoside, the baicalein and the wogonoside is 0.38%, 0.45%, 0.29%, 0.39%, 0.37%, 0.40% and 0.61% respectively, which indicates that the precision of the instrument is good.

Repeatability test

Taking about 0.4g of fine powder of the same batch of samples, paralleling 6 parts, precisely weighing, preparing a test solution according to the method of the step 3), and measuring according to the chromatographic conditions by sample injection, wherein the contents of the monocaffeoyltartaric acid, the chicoric acid, the baicalin, the oroxylin A-7-0-beta-D-glucuronide, the wogonoside, the baicalein and the wogonin are 3.611, 4.480, 48.38, 6.403, 9.462, 2.707 and 0.729mg/g respectively, and the contents of the RSD are 0.22%, 0.70%, 0.30%, 0.42%, 0.39%, 0.41% and 0.85% respectively, which indicates that the method has good repeatability.

Stability test

A sample solution with the same repeatability test is taken, and is subjected to sample injection measurement at room temperature for 0, 4, 8, 12, 18 and 24 hours respectively, according to the chromatographic conditions, the peak areas RSD of the monocaffeyltartaric acid, the chicoric acid, the baicalin, the oroxylin A-7-0-beta-D-glucuronide, the wogonin, the baicalin and the wogonin are respectively 0.61%, 0.83%, 1.0%, 0.38%, 0.41%, 1.1% and 0.69%, and the sample solution has good stability within 24 hours.

Sample application recovery test

Taking 6 parts of sample fine powder with known content, wherein each part is about 0.2g, precisely weighing, respectively placing in a conical flask with a plug, precisely adding 50mL of mixed reference substance solution II obtained in the step 2), parallelly preparing a 1:1 ratio sample standard solution according to the method of the step 3), measuring according to the chromatographic conditions, and calculating the recovery rate of each component. The average sample recovery rates of monocaffeoyltartaric acid, chicoric acid, baicalin, oroxylin A-7-0-beta-D-glucuronide, wogonoside, baicalein and wogonoside were 99.13%, 100.6%, 98.68%, 101.8%, 98.91%, 98.14% and 101.1%, respectively, and the RSDs were 0.37%, 0.77%, 0.68%, 0.84%, 0.89%, 0.56% and 1.06%, respectively, as shown in Table 3.

TABLE 3 sample application recovery test results (n = 6)

6 component relative correction factor calculation

Precisely sucking the mixed reference substance solution I in the step 2), injecting samples of 0.5, 1, 2, 4 and 8 mu L respectively according to the chromatographic conditions, recording peak areas of the components, calculating relative correction factors f of other 6 components to be detected by using baicalin as an internal reference by adopting a multipoint correction method, wherein the calculation formula is f = f _s /f _k ＝(C _k ×A _s )/(C _s ×A _k ) In which C is _k To determine the content of the component A _k Is the peak area of the component to be measured, C _s Is the content of an internal reference substance, A _s The peak area of the internal reference substance shows that the RSD of 4,6 components f is less than 3 percent, which indicates that the method is stable and has good repeatability.

TABLE 4.6 relative correction factors for the components

One-test and multi-evaluation system durability investigation

During the establishment of a one-test-multiple-evaluation method, all factors that cause changes in the response values of the detectors of the chromatography system may cause fluctuations in the relative correction factors, thereby introducing errors between the calculated and measured values. Therefore, the established relative correction factor is ensured to have good reproducibility, the method is the key for the accurate analysis result of the one-test-multiple-evaluation method, and the durability of the system is investigated from 4 factors of different instruments, chromatographic columns, column temperature and flow rate.

Investigation of different instruments and chromatographic columns

Selecting 2 different chromatographic systems (Agilent 1260 high performance liquid chromatograph and Waters 2695 high performance liquid chromatograph)Liquid chromatograph) and 3 different types of columns (column 1 ₁₈ (2.1X 50mm,2.7 μm); column 2. Yuekxu Botimate EXT C ₁₈ (2.1X 50mm,2.7 μm); column 3 ₁₈ XP (2.1 mm. Times.50mm, 2.5 μm)) examined the relative correction factors for each component, see Table 5. The results show that different chromatographs and different chromatographic columns have no significant difference in the relative correction factor effects of each component (RSD is less than 3% in each case).

TABLE 5 Effect of different chromatographs and columns on relative correction factors

Investigation of different column temperatures

Other chromatographic conditions were unchanged, and the relative correction factors for each component were determined with different column temperatures (25, 30, 35, 40 ℃) set, as shown in Table 6. The results show that different column temperatures have no significant difference in the relative correction factor effect of each component (RSD is less than 3%).

TABLE 6 influence of different column temperatures on the relative correction factor

Investigation of different flow rates

Other chromatographic conditions were unchanged and different flow rates (0.28, 0.30, 0.32, 0.34 mL/min) were set to determine the relative correction factors for each component, see Table 7. The results show that different flow rates have no significant difference in the relative correction factor effect for each component (RSD less than 3% for each).

TABLE 7 Effect of different flow rates on relative correction factor

Example 3

Localization of 6 component chromatographic peaks

In practical application, the one-test-multiple-evaluation method utilizes the relative correction factor to calculate the content, and does not need to use a reference substance of the components to be tested except for a reference substance, so that the problem of how to position chromatographic peaks of the components is bound to be encountered. The traditional Chinese medicine components are complex, and besides the chromatographic peak of the component to be detected, a plurality of other chromatographic peaks exist in the chromatogram, so that the correct positioning of the chromatographic peak of the component to be detected is the premise of accurate determination by the one-test-multiple-evaluation method. At present, the adopted chromatographic peak positioning method mainly comprises 2 methods, namely, the relative retention time ratio of the component to be detected and an internal reference material is adopted for positioning, namely, a relative retention value method t _i/s ＝t _i /t _s Wherein t is _i Retention time of the test object, t _s The retention time of the internal reference substance is shown. Secondly, the difference between the retention time of the component to be measured and the retention time of the internal reference material is utilized for positioning, namely the retention time difference method delta t _is ＝t _i -t _s . The invention adopts a relative retention value method for positioning, and examines t under different chromatographic systems and different chromatographic column conditions _i/s The results are shown in Table 8. The results show that under different chromatographic systems and different chromatographic column conditions, the average relative retention values of monocaffeoyltartaric acid, chicoric acid, oroxylin A-7-0-beta-D-glucuronide, wogonoside, baicalein and wogonin are 0.0932, 0.5311, 1.3115, 1.3542, 1.5219 and 1.7892 respectively, the relative retention value RSD of each component is less than 3 percent, and the chromatographic peak positioning requirement of the one-test-multiple-evaluation method is met.

TABLE 8 relative Retention values determined by different instruments and columns

Comparison of sample one-test-multiple evaluation method and external standard method

Preparing a test solution for 10 batches of Pudilan anti-inflammatory tablet samples according to the method in the step 3), carrying out sample injection determination according to the chromatographic conditions, and recording a chromatogram map. The contents of the components are respectively calculated by adopting a one-test-multiple-evaluation (QAMS) method and an External Standard Method (ESM), the calculation results of the contents of the two methods are compared by using Relative Deviation (RD), the calculation formula is RD = (QAMS value-ESM value)/ESM value multiplied by 100%, and the calculation results are shown in tables 9 and 10. The result shows that the content determination result RD obtained by the two methods is less than 5 percent, and the paired sample T test shows that the two methods have no significant difference. The relative correction factor established by the invention is accurate and reliable, and can be used for determination of monocaffeoyltartaric acid, chicoric acid, baicalin, oroxylin A-7-0-beta-D-glucuronide, wogonoside, baicalein and wogonin and evaluation of preparation quality.

TABLE 9 comparison of results of content determination for samples by external standard method and one-test-multiple evaluation method (mg/g, n = 2)

TABLE 10 comparison of results of content determination for samples by external standard method and one-test-multiple evaluation method (mg/g, n = 2)

When the content of the multi-index components of the traditional Chinese medicine is determined by a multi-evaluation method, one of the effective components or index components with definite pharmacological action is generally used as an internal reference, and the commercial component which is cheap and easy to obtain, has stable chromatographic information and has high content is used as the internal reference. Among the index components of the 7 Pudilan anti-inflammatory tablets, baicalin meets the above conditions, and the chromatographic peak is centered, so that the invention takes the baicalin as an internal reference substance.

The dandelion in the dandelion anti-inflammatory tablet formula is a monarch drug, phenolic acid which is one of main components of the dandelion water extraction process is the basis of important drug effect substances, wherein the caftaric acid and the chicoric acid are 2 phenolic acid components with the highest dandelion content, the contents of other phenolic acids (protocatechuic acid, chlorogenic acid, caffeic acid, etc.) are all low, so the above 2 phenolic acids are selected as index components of dandelion. In the formula, although scutellaria baicalensis is used as a ministerial drug, the dosage is larger, and the dosage of more than 1/2 of the formula in the preparation method is directly added as raw powder, so that the content of 4' -deoxy-flavonoid components represented by baicalin is higher in actual detection, wherein 5 components of baicalin, oroxylin A-7-0-beta-D-glucuronide, wogonin, baicalin and wogonin have the highest content and are also the basis of main drug effect substances of scutellaria baicalensis, and the 5 flavonoid components are selected as the index components of the scutellaria baicalensis.

Optimization of pretreatment conditions of test sample

Selecting an extraction mode:

the influence of different extraction modes (ultrasonic and heating reflux) on the detection result is inspected, the content of 7 index components is selected as an inspection object, the content of phenolic acid substances in different extraction modes is basically the same, the content of flavonoid substances is slightly different, the content of baicalin and wogonoside in the ultrasonic-extracted sample is lower than that of the reflux-extracted sample, but the content of baicalein and wogonoside in the ultrasonic-extracted sample is higher than that of the reflux-extracted sample. And (4) carrying out stability investigation after the test solution extracted by different extraction modes is placed for 12 h. The results show that the test solution extracted by ultrasonic wave has poor stability within 12h, the contents of baicalin and wogonin are reduced in different degrees, and the contents of baicalein and wogonin are increased in different degrees, which shows that a certain amount of baicalinase existing in the sample can be effectively inactivated under the condition of heating reflux extraction, but the enzyme can not be inactivated by ultrasonic wave, so the extraction mode of the invention selects heating reflux.

Selection of extraction time:

and (3) investigating the influence of different reflux times (0.5, 1, 1.5, 2, 2.5 and 3 h) on the detection result, comparing the content of each component under different reflux times, wherein the content change trends of each component are basically consistent along with the change of the reflux time, and the content measurement result of each component is the largest at 2h, so that the extraction time is selected to be 2h.

Selection of extraction solvent concentration:

and (3) examining the influence of methanol solutions with different volume percentage concentrations (10%, 30%, 50%, 70% and 90%) on the detection result, and comparing the content of each component in the extraction solvents with different concentrations, wherein the content change trends of the components are basically consistent along with the change of the volume percentage concentration of the methanol. The volume percentage concentration of the methanol is between 10 percent and 50 percent, the content of each component is in a relatively obvious rising trend, basically tends to be gentle between 50 percent and 70 percent, and is in a slightly descending trend of 90 percent, so that the 70 percent methanol is finally selected as the extraction solvent.

Optimization of chromatographic conditions

Selection of mobile phase:

the influence of different mobile phase systems (methanol-0.1% phosphoric acid, acetonitrile-0.1% phosphoric acid, methanol-acetonitrile-0.1% phosphoric acid) on the separation efficiency of the components of the Pudilan anti-inflammatory tablet is examined. As a result, when methanol-0.1% phosphoric acid is used as a mobile phase, the chromatographic peak has a wide peak shape, and chromatographic peaks after the peaks are relatively large are difficult to separate from a base line; when acetonitrile-0.1% phosphoric acid is used as a mobile phase, the peak emergence time is too fast, the component peak with the earliest peak emergence is difficult to separate from a solvent peak, and the separation degree between several chromatographs with intermediate peak emergence time is also poor; when methanol-acetonitrile-0.1% phosphoric acid is used as a mobile phase, the peak shape of each chromatographic peak is good, and the baseline separation can be achieved. Therefore, a methanol-acetonitrile-0.1% phosphoric acid system is finally selected as a mobile phase, and gradient elution is adopted.

Selection of detection wavelength:

the invention adopts DAD detector to carry out full-wavelength scanning on 7 index components, and ultraviolet spectrograms of each component of the sample are shown in figures 2-8 and are completely consistent with ultraviolet spectrograms of a reference substance by comparison. 2-8 show that the maximum absorption wavelength of 5 flavonoids is 272-277 nm, 2 phenolic acid components have maximum absorption at 326nm, and the absorption at 274nm is very low. Therefore, in order to take account of the detection sensitivity of each component, the invention adopts an online wavelength switching method, the detection wavelength of the monocaffeoyltartaric acid and the chicoric acid is set to be 326nm, and the detection wavelength of the baicalin, the oroxylin A-7-0-beta-D-glucuronide, the wogonoside, the baicalein and the wogonin is set to be 274nm.

Selection of relative correction factor calculation method:

there are 2 methods for calculating relative correction factors of components to be measured in the one-test-multiple-evaluation method, namely a slope method and a multipoint correction method. The invention compares relative correction factors of each component obtained by 2 methods, takes the mixed reference substance solution I in the step 2), samples 0.5, 1, 2, 4 and 8 mu L respectively according to chromatographic conditions, records peak areas of each component and obtains a standard curve of each component. Using baicalin as an internal reference, and calculating F value of each component by adopting a slope method, wherein F = Fs/F _k In which F is _S Calibrating the slope of the curve for the internal reference, F _k The slope of the standard curve of the component to be measured is shown. The f values of the monocaffeoyltartaric acid, the chicoric acid, the wogonoside, the baicalein and the wogonin are 1.0334, 0.9444, 1.3863, 0.8451, 0.6552 and 0.5917 in sequence. The f values of the components calculated by adopting a multipoint correction method are 1.0081, 0.9209, 1.3693, 0.8572, 0.6423 and 0.6068 in sequence. The relative deviations of the f values of the components obtained by the slope method compared with the f values obtained by the multipoint correction method are respectively 2.48%, 2.52%, 1.23%, -1.42%, 1.99% and-2.52%, and are less than +/-3.0%, which indicates that the 2 calculation methods have no significant difference. However, the invention selects a more reasonable multipoint correction method considering that the intercept caused by system errors and instrument operation errors in the standard curve on which the slope method is based influences the accuracy of the result.

Selecting a locating method of a chromatographic peak of a component to be detected:

accurate positioning of chromatographic peaks is a premise for ensuring application of a one-test-multiple-evaluation method, and a relative retention value method and a retention time difference method are two most commonly used positioning methods at present. The invention compares the stability of the two positioning methods under different chromatographic systems and different chromatographic column conditions, and the result shows that the t is calculated by the two positioning methods in two chromatographs of different brands and three chromatographic columns of different brands _i/s And Δ t _is The RSD of (A) is obviously different. 6 components t in the Pudilan antiphlogistic tablet _i/s RSD of the calculated results is less than 3%, and delta t _is The RSD of the calculation result is between 2% and 5%. Currently, one-test and multiple-evaluation methodThe RSD requirement for chromatographic peak location is less than 5%, although the RSD of both location methods meet the requirement, the RSD of the relative retention value method is significantly less than the retention time difference method. Therefore, the invention adopts a relative retention value method as a chromatographic peak positioning basis and uses t _i/s The deviation is limited to a specified range (+ -5%).

The invention establishes a method for simultaneously determining the content of 7 index components in Pudilan anti-inflammatory tablets by a one-test-multiple evaluation method, and calculates relative correction factors of monocaffeoyltartaric acid, chicoric acid, oroxylin A-7-0-beta-D-glucuronide, wogonin, baicalein and wogonin relative to baicalin by taking baicalin as an internal reference. And the 6 component relative correction factors are examined from the aspects of different chromatographic systems, chromatographic columns, column temperature, flow rate and the like, and the result shows that the method has good durability. 10 batches of Pudilan anti-inflammatory tablets are subjected to one-test multi-evaluation method to calculate the content of each component and are compared with the actual measurement result of the external standard method so as to verify the accuracy and feasibility of the one-test multi-evaluation method, and no significant difference exists between the calculated value of the one-test multi-evaluation method and the actual measurement value of the external standard method. The relative correction factor established by the invention is accurate and reliable, the difficulty that part of component reference substances are in short supply, expensive and high in detection cost when the baicalin is used as an internal reference substance is effectively solved by a one-test-multiple-evaluation method, the detection cost is greatly reduced, and a new method is provided for quality control and evaluation of the Pudilan anti-inflammatory tablet.

While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that the foregoing and other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any equivalent changes, modifications and evolutions of the above embodiments according to the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims (9)

1. A Pudilan anti-inflammatory tablet quality evaluation method based on one-test-multiple evaluation is characterized by comprising the following steps:

1) Preparation of a single control solution: respectively dissolving single caffeoyl tartaric acid, chicoric acid, baicalin, oroxylin A-7-0-beta-D-glucuronide, wogonoside, baicalein and wogonin with methanol, and diluting to obtain single reference stock solution;

2) Preparation of mixed control solutions: respectively taking the single reference substance stock solutions obtained in the step 1), placing the single reference substance stock solutions into a measuring flask, and adding a methanol solution for dilution to obtain a mixed reference substance solution;

3) Preparing a test solution: taking a sample, placing the sample in a conical flask, adding a methanol solution, sealing, weighing, heating and refluxing, taking out, cooling, weighing again, complementing the lost weight with the methanol solution, shaking up, filtering, and taking a filtrate to obtain a sample solution;

4) Feeding the mixed reference substance solution obtained in the step 2) into a high performance liquid chromatograph, carrying out sample injection measurement to obtain a liquid chromatogram, recording peak areas of all components, and calculating relative correction factors of other 6 components to be measured by adopting a multipoint correction method and taking baicalin as an internal reference substance;

5) Analyzing and determining the positioning basis of the chromatographic peak of the component to be detected.

2. The method for evaluating the quality of the Pudilan anti-inflammatory tablet based on one test and multiple tests as claimed in claim 1, which is characterized in that: in step 1), the single caffeoyltartaric acid 271.46 ug/mL, chicoric acid 443.49 ug/mL, baicalin 915.08 ug/mL, oroxylin A-7-0-beta-D-glucuronide 580.55 ug/mL, wogonoside 376.27 ug/mL, baicalein 291.55 ug/mL and wogonon 159.54 ug/mL are measured by mass concentration.

3. The method for evaluating the quality of the Pudilan anti-inflammatory tablet based on one test and multiple tests as claimed in claim 1, wherein the method comprises the following steps: in step 2), the monocaffeoyltartaric acid 21.716. Mu.g/mL, chicoric acid 17.740. Mu.g/mL, baicalin 292.82. Mu.g/mL, oroxylin A-7-0-beta-D-glucuronide 46.444. Mu.g/mL, wogonoside 75.254. Mu.g/mL, baicalein 11.662. Mu.g/mL and wogonoside 6.382. Mu.g/mL are measured by mass concentration.

4. The method for evaluating the quality of the Pudilan anti-inflammatory tablet based on one test and multiple tests as claimed in claim 1, wherein the method comprises the following steps: in step 3), the ratio of the amount of the sample added to the volume of methanol was 0.4g:50mL.

5. The method for evaluating the quality of the Pudilan anti-inflammatory tablet based on one test and multiple tests as claimed in claim 1, wherein the method comprises the following steps: in the step 3), the volume percentage concentration of the extraction solvent methanol is 70%.

6. The method for evaluating the quality of the Pudilan anti-inflammatory tablet based on one test and multiple tests as claimed in claim 1, wherein the method comprises the following steps: in the step 3), the heating reflux time is 2h, and the temperature is reduced to the room temperature after the heating reflux time is taken out.

7. The method for evaluating the quality of the Pudilan anti-inflammatory tablet based on one test and multiple tests as claimed in claim 1, wherein the method comprises the following steps: in the step 4), the chromatographic conditions are as follows: agilent Poroshell 120SB-C ₁₈ 2.1X 50mm,2.7 μm; mobile phase: the mobile phase A is methanol, the mobile phase B is acetonitrile, and the mobile phase C is 0.1 percent phosphoric acid solution;

the gradient elution was: 0-5 min, mobile phase A,10% → 20%, mobile phase B,5%, mobile phase C,85% → 75%;

5-20 min, mobile phase A,20% → 25%, mobile phase B,5% → 10%, mobile phase C,75% → 65%;

20-25 min, mobile phase A,25% → 40%, mobile phase B,10% → 15%, and mobile phase C,65% → 45%;

30-35 min, mobile phase A,40%, mobile phase B,15% and mobile phase C,45%;

flow rate: 0.3mL/min; detection wavelength: 0-13min, 326nm; 14-35min, 274nm; the column temperature is 30 ℃; sample introduction amount: 2 μ L.

8. The method for evaluating the quality of the Pudilan anti-inflammatory tablet based on one test and multiple tests as claimed in claim 1, wherein the method comprises the following steps: in the step 4), taking baicalin as an internal reference, calculating relative correction factors f of other 6 components to be detected, wherein the calculation formula is f = f _s /f _k ＝(C _k ×A _s )/(C _s ×A _k )；

Wherein C is _k To determine the content of the component A _k Is the peak area of the component to be measured, C _s Is the content of an internal reference substance, A _s The peak area of the internal reference substance is shown.

9. The method as claimed in claim 1, wherein in step 5), the relative retention method is used as the basis for locating chromatographic peaks, and the deviation is limited to (+/-5%), and the calculated value of the one-test-multiple-evaluation method and the measured value of the external standard method are compared, respectively.

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