Method for jointly detecting chlorogenic acid and baicalin in Yindan detoxification granules

Abstract

The invention relates to the technical field of component detection of Yindan detoxification granules, and discloses a joint detection method of chlorogenic acid and baicalin in Yindan detoxification granules, which comprises the following steps: S1, preparation of a test solution: taking 0.3 g of the product powder, precisely weighing, placing in a conical flask with a plug, precisely adding 25 ml of 10% methanol, sealing the plug, weighing, carrying out ultrasonic treatment, cooling, re-weighing, and taking out the test solution; supplementing the lost weight with 10% methanol, uniformly shaking, filtering, and taking a subsequent filtrate to obtain a sample solution; s2, preparing a reference substance solution; and S3, determining: precisely sucking 1 microliter of the reference solution and 1 microliter of the test solution respectively, injecting the solutions into a liquid chromatograph, and determining. According to the scheme, firstly, a sample is dissolved in 10% methanol, ultrasonic treatment is conducted, then liquid chromatography determination is conducted according to gradient, the scheme is easy to operate, chlorogenic acid and baicalin can be efficiently and accurately subjected to quantitative control at the same time, and interference of the other nine components in the silver plumbum rubrum is greatly reduced.

Description

Technical Field

[0001] The invention relates to the technical field of Yindan Jiedu granule component detection, and specifically to a method for jointly detecting chlorogenic acid and baicalin in Yindan Jiedu granules. Background Art

[0002] Yindan Detoxification Granules are modified based on the classic prescriptions Qingwen Baidu Yin and Ma Xing Shi Gan Tang, and have the effects of clearing the lungs, expelling evil, detoxifying and cooling the blood.

[0003] Among them, the main drug honeysuckle has the functions of anti-inflammatory and antipyretic, enhancing immunity, promoting phagocytosis of leukocytes and inflammatory cells, and has significant antioxidant effects. The auxiliary drug scutellaria baicalensis can inhibit the excessive release of proinflammatory cytokines and promote the expression of anti-inflammatory cytokines, thereby inhibiting the body's systemic inflammatory response syndrome, reducing the immune inflammatory pathological damage of the target organ lung caused by influenza virus FM1 infection, and promoting the repair of inflammatory lesions in lung tissue.

[0004] Therefore, it is of great significance to quantitatively control the representative components of honeysuckle and scutellaria (chlorogenic acid in honeysuckle and baicalin in scutellaria). In order to quantitatively control chlorogenic acid and baicalin, it is necessary to detect chlorogenic acid and baicalin. The existing detection method is to establish liquid chromatography content determination for chlorogenic acid and baicalin separately. Two detection methods need to be established, which prolongs the detection time and consumes more experimental consumables. In addition, since Yindan Jiedu Granules have a total of 11 medicinal flavors, the ingredients in the other 9 medicinal flavors will also bring difficulties to the control of the above two ingredients. At present, no method with reference significance for simultaneously detecting the above two ingredients has been found. In this regard, the present invention proposes a method for jointly detecting chlorogenic acid and baicalin in Yindan Jiedu Granules. Summary of the invention

[0005] The object of the present invention is to provide a method for co-detecting chlorogenic acid and baicalin in Yindan Jiedu Granules to solve the problems in the above-mentioned background technology.

[0006] To achieve the above object, the present invention provides the following technical solution: a method for co-detecting chlorogenic acid and baicalin in Yindan Jiedu granules, comprising the following steps:

[0007] S1, Preparation of test solution:

[0008] Take 0.3g of the powder of this product, weigh it accurately, put it in a stoppered conical flask, add 25ml of 10% methanol accurately, stopper it tightly, weigh it, treat it with ultrasound, let it cool, weigh it again, make up the lost weight with 10% methanol, shake it well, filter it, and take the filtrate to get the sample solution;

[0009] S2, Preparation of reference solution:

[0010] Take an appropriate amount of chlorogenic acid reference substance, accurately weigh it, add 75% methanol to prepare a solution containing 0.28 mg of chlorogenic acid per 1 ml, and obtain a chlorogenic acid reference solution; take an appropriate amount of baicalin reference substance, accurately weigh it, add methanol to prepare a solution containing 60 μg of baicalin per 1 ml, and obtain a baicalin reference solution;

[0011] S3, determination method:

[0012] Accurately pipette 1 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph for determination.

[0013] Preferably, the power of the ultrasonic treatment in S1 is 500 W, the frequency is 40 kHz, and the ultrasonic treatment time is 30 min.

[0014] Preferably, the S3 uses methanol as mobile phase A and 0.1% phosphoric acid solution as mobile phase B.

[0015] Preferably, the S3 detection wavelength is 327 nm, the flow rate is 0.3 ml / min, and the column temperature is 35°C.

[0016] Preferably, the mobile phase gradient program is as follows:

[0017] From 0 to 30 min, the volume fraction of mobile phase A changed from 5% to 11%, and the volume fraction of mobile phase B changed from 95% to 89%;

[0018] From 30 to 31 min, the volume fraction of mobile phase A changed from 11% to 30%, and the volume fraction of mobile phase B changed from 89% to 70%;

[0019] From 31 to 49 min, the volume fraction of mobile phase A changed from 30% to 35%, and the volume fraction of mobile phase B changed from 70% to 65%;

[0020] From 49 to 54 min, the volume fraction of mobile phase A changed from 35% to 90%, and the volume fraction of mobile phase B changed from 65% to 10%;

[0021] From 54 to 55 min, the volume fraction of mobile phase A changed from 90% to 5%, and the volume fraction of mobile phase B changed from 10% to 95%.

[0022] The present invention has at least the following beneficial effects:

[0023] The present invention provides a method for jointly detecting chlorogenic acid and baicalin in Yindan Jiedu granules. The sample is first dissolved in 10% methanol, ultrasonically treated, and then liquid chromatography is performed according to a gradient. The scheme is simple to operate, and can efficiently and accurately perform quantitative control of chlorogenic acid and baicalin at the same time, greatly reducing the interference of the other 9 components in Yindan. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is a linear curve graph of chlorogenic acid of the present invention;

[0025] Figure 2 is a linear curve diagram of baicalin of the present invention;

[0026] Figure 3 is the chromatogram of baicalin;

[0027] Figure 4 This is the chromatogram of chlorogenic acid. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] A method for co-detecting chlorogenic acid and baicalin in Yindan Jiedu granules comprises the following steps:

[0030] S1, Preparation of test solution:

[0031] Take 0.3g of the powder of this product, weigh it accurately, put it in a stoppered conical flask, add 25ml of 10% methanol accurately, stopper it tightly, weigh it, and treat it ultrasonically (power 500W, frequency 40kHz) (or reflux) for 15, 30, 45, 60min, let it cool, weigh it again, make up the lost weight with 10% methanol, shake it well, filter it, and take the filtrate to obtain the sample solution;

[0032] S2, Preparation of reference solution:

[0033] Take an appropriate amount of chlorogenic acid reference substance, accurately weigh it, add 75% methanol to prepare a solution containing 0.28 mg of chlorogenic acid per 1 ml, and obtain a chlorogenic acid reference solution; take an appropriate amount of baicalin reference substance, accurately weigh it, add methanol to prepare a solution containing 60 μg of baicalin per 1 ml, and obtain a baicalin reference solution;

[0034] S3, determination method:

[0035] Accurately pipette 1 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph for determination.

[0036] Chromatographic conditions and system suitability test:

[0037] Waters CORTECS T3 (2.1×100 mm) was used as the chromatographic column; methanol was used as the mobile phase A, 0.1% phosphoric acid solution was used as the mobile phase B, and gradient elution was performed according to the provisions in the following table; the detection wavelength was 327 nm, the flow rate was 0.3 ml / min, and the column temperature was 35° C. The mobile phase gradient program is shown in Table 1 below:

[0038] Table 1 Mobile phase gradient

[0039]

[0040] The chromatographic conditions are shown in Table 2 below:

[0041] Table 2 Chromatographic conditions

[0042]

[0043]

[0044] This patent explores the pre-treatment conditions, as follows:

[0045] Table 3 Exploration of ultrasound and reflow conditions

[0046]

[0047]

[0048] From the data in the table, it can be seen that the overall difference in content results between ultrasonic pretreatment and reflux pretreatment in the range of 15 to 45 minutes is not large. Considering the time cost and tediousness of the pretreatment process, the ultrasonic time is selected to be 30 minutes.

[0049] Table 4 Exploration of sample dissolution reagents

[0050]

[0051]

[0052] The present invention selects the optimal extraction solvent, extraction method and extraction time by taking the content determination value as an index, that is, 25 ml of 10% methanol is accurately added and ultrasonic treatment is performed for 30 minutes.

[0053] The method provided by the present invention was tested and analyzed in terms of sample recovery, precision, stability, linearity, repeatability, durability, etc., as follows:

[0054] 1. Sample recovery rate:

[0055] The sample recovery rate refers to the degree of closeness between the result measured by the established method and the true value or reference value, and is generally expressed as recovery rate (%).

[0056] The size of the sample recovery rate not only reflects the analyst's operational technical level, but more importantly, it reflects whether the analysis method is suitable for the matrix being tested, helping analysts to promptly discover problems in the analysis and ensure that the analysis data is accurate and reliable.

[0057] The present invention sets up 6 test groups and 2 control groups for comparative analysis:

[0058] Table 5 Test groups of sample recovery

[0059]

[0060]

[0061] Table 6 Control group of sample recovery

[0062]

[0063] Table 7 Recovery rate of chlorogenic acid in each test group after adding sample

[0064]

[0065] Table 8 Recovery rate of baicalin in each test group

[0066]

[0067] In summary, the data meet the requirements of the pharmacopoeia, and the method provided by the present invention is feasible for the detection of baicalin and chlorogenic acid.

[0068] 2. Precision:

[0069] Precision refers to the degree of closeness between the results obtained from multiple sampling and measurement of the same uniform test sample under specified measurement conditions.

[0070] In this experimental analysis, 6 experimental groups were set up, as shown in Table 9 below:

[0071] Table 9 Precision

[0072]

[0073] In summary, the data meet the requirements of the pharmacopoeia, and the method provided by the present invention is accurate for the detection of baicalin and chlorogenic acid.

[0074] 3. Stability:

[0075] Stability research is to understand and predict the quality change trend of Chinese medicine preparations by examining the changes in the physical, chemical and biological characteristics of Chinese medicine preparations under different environmental conditions (such as temperature, humidity, light, etc.) over time, and to provide a scientific basis for determining the production, packaging, storage, transportation conditions and the establishment of shelf life of Chinese medicine preparations.

[0076] In this test analysis, the stability was analyzed by comparing the results of different time periods from 0 to 26 hours, as follows:

[0077] Table 10 Stability

[0078]

[0079] In summary, the data meet the requirements of the pharmacopoeia, and the method provided by the present invention has high sample stability.

[0080] 4. Linearity:

[0081] Linearity refers to the ability of a linear test result to be directly proportional to the concentration of the analyte in the sample within the designed range.

[0082] Table 11 Chlorogenic acid linearity

[0083]

[0084]

[0085] The values ​​in the above table were fitted, and the linear curve of chlorogenic acid was finally obtained as Y=0.1709X-0.162, R 2 =0.9998, its linear graph is as follows Figure 1 shown.

[0086] Table 12 Baicalin linearity

[0087]

[0088] The values ​​in the above table were fitted, and the linear curve of baicalin was finally obtained as Y=0.2639X+4.0932, R 2 =0.9997, its linear graph is as follows Figure 2 shown.

[0089] In summary, the data meet the requirements of the pharmacopoeia, and chlorogenic acid and baicalin in the method of the present invention have a good linear relationship.

[0090] 5. Repeatability:

[0091] Within the specified range, take the same concentration (the sample measurement concentration proposed by the analytical method, equivalent to 100% concentration level) of the test sample and evaluate it with at least 6 measurement results.

[0092] Table 13 Repeatability test group

[0093]

[0094]

[0095] Table 14 Repeatability control group

[0096]

[0097] In summary, the data meet the requirements of the pharmacopoeia, and both chlorogenic acid and baicalin in the method of the present invention have good accuracy.

[0098] 6. Durability:

[0099] Table 15 Test Group

[0100]

[0101] Table 16 Control group

[0102]

[0103]

[0104] In summary, the data meet the requirements of the pharmacopoeia, and the detection method of chlorogenic acid and baicalin in the method of the present invention is universal.

[0105] 7. Intermediate precision

[0106] To examine the effects of random variation factors, such as different dates, different analysts, and different instruments on precision, intermediate precision tests should be conducted.

[0107]

[0108]

[0109]

[0110] In summary, the data meet the requirements of the pharmacopoeia, and the detection method of chlorogenic acid and baicalin in the method of the present invention is accurate.

[0111] 8. Exclusivity

[0112] Specificity refers to the ability of the analytical method to correctly determine the analyte in the presence of other components (such as impurities, degradation products, excipients, etc.).

[0113] Chlorogenic acid only comes from honeysuckle, baicalin only comes from scutellaria baicalensis, and solvents and excipients have no effect on the content determination.

[0114] Figure 3 is the chromatogram of baicalin. Figure 4 This is the chromatogram of chlorogenic acid.

[0115] The above shows and describes the basic principles, main features and advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention.

[0116] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for the joint detection of chlorogenic acid and baicalin in Yindan Jiedu granules, characterized in that: The following steps are involved: S1, Preparation of test solution: Take 0.3g of the powder of this product, weigh it accurately, put it in a stoppered conical flask, add 25ml of 10% methanol accurately, stopper it tightly, weigh it, treat it with ultrasound, let it cool, weigh it again, make up the lost weight with 10% methanol, shake it well, filter it, and take the filtrate to get the sample solution; S2, Preparation of reference solution: Take an appropriate amount of chlorogenic acid reference substance, accurately weigh it, add 75% methanol to prepare a solution containing 0.28 mg of chlorogenic acid per 1 ml, and obtain a chlorogenic acid reference solution; take an appropriate amount of baicalin reference substance, accurately weigh it, add methanol to prepare a solution containing 60 μg of baicalin per 1 ml, and obtain a baicalin reference solution; S3, determination method: Accurately pipette 1 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph for determination.

2. The method for co-detecting chlorogenic acid and baicalin in Yindan Jiedu Granules according to claim 1, characterized in that: The power of the ultrasonic treatment in S1 is 500 W, the frequency is 40 kHz, and the ultrasonic treatment time is 30 min.

3. The method for co-detecting chlorogenic acid and baicalin in Yindan Jiedu Granules according to claim 1, characterized in that: The S3 uses methanol as mobile phase A and 0.1% phosphoric acid solution as mobile phase B.

4. The method for co-detecting chlorogenic acid and baicalin in Yindan Jiedu Granules according to claim 3, characterized in that: The S3 detection wavelength was 327 nm, the flow rate was 0.3 ml / min, and the column temperature was 35°C.

5. The method for co-detecting chlorogenic acid and baicalin in Yindan Jiedu Granules according to claim 4, characterized in that: The mobile phase gradient program is as follows: From 0 to 30 min, the volume fraction of mobile phase A changed from 5% to 11%, and the volume fraction of mobile phase B changed from 95% to 89%; From 30 to 31 min, the volume fraction of mobile phase A changed from 11% to 30%, and the volume fraction of mobile phase B changed from 89% to 70%; From 31 to 49 min, the volume fraction of mobile phase A changed from 30% to 35%, and the volume fraction of mobile phase B changed from 70% to 65%; From 49 to 54 min, the volume fraction of mobile phase A changed from 35% to 90%, and the volume fraction of mobile phase B changed from 65% to 10%; From 54 to 55 min, the volume fraction of mobile phase A changed from 90% to 5%, and the volume fraction of mobile phase B changed from 10% to 95%.

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