A method for the joint detection of chlorogenic acid and baicalin in Yindan Detoxification Granules

Abstract

This invention relates to the field of component detection technology for Yindan Detoxification Granules, and discloses a method for the joint detection of chlorogenic acid and baicalin in Yindan Detoxification Granules, comprising the following steps: S1, preparation of the test solution: accurately weigh 0.3g of the powder, place it in a stoppered conical flask, accurately add 25ml of 10% methanol, seal tightly, weigh, sonicate, cool, weigh again, replenish the lost weight with 10% methanol, shake well, filter, and collect the filtrate to obtain the sample solution; S2, preparation of the reference solution; S3, determination method: accurately pipette 1μl each of the reference solution and the test solution, inject them into the liquid chromatograph, and perform the determination. This scheme first dissolves the sample in 10% methanol, sonicates, and then performs liquid chromatography determination according to a gradient. This scheme is simple to operate and can efficiently and accurately quantitatively control chlorogenic acid and baicalin simultaneously, greatly reducing the interference of the other 9 components in Yindan.

Description

Technical Field

[0001] This invention relates to the field of component detection technology for Yindan Detoxification Granules, specifically a method for the joint detection of chlorogenic acid and baicalin in Yindan Detoxification Granules. Background Technology

[0002] Yindan Detoxification Granules are based on the classic formulas Qingwen Baidu Decoction and Ma Xing Shi Gan Decoction, and have the effects of clearing the lungs and expelling pathogens, detoxifying and cooling the blood.

[0003] Honeysuckle, the principal herb, has anti-inflammatory and antipyretic properties, enhances immunity, promotes phagocytosis of leukocytes and inflammatory cells, and has significant antioxidant effects. Scutellaria baicalensis, the assistant herb, can inhibit the excessive release of pro-inflammatory cytokines and promote the expression of anti-inflammatory cytokines, thereby inhibiting the body's systemic inflammatory response syndrome, reducing the immune inflammatory pathological damage to the lungs, the target organ caused by influenza virus FM1 infection, and promoting the repair of inflammatory lesions in lung tissue.

[0004] Therefore, quantitative control of representative components of honeysuckle and scutellaria (chlorogenic acid in honeysuckle and baicalin in scutellaria) is of great significance. To quantitatively control chlorogenic acid and baicalin, it is necessary to detect them separately. Existing methods involve establishing separate liquid chromatography methods for chlorogenic acid and baicalin, requiring two separate detection methods, which increases detection time and the consumption of experimental consumables. Furthermore, since Yindan Detoxification Granules contain 11 herbs, the components of the other 9 herbs also pose challenges to the control of these two components. Currently, no suitable method for simultaneously detecting these two components has been found. Therefore, this invention proposes a method for the joint detection of chlorogenic acid and baicalin in Yindan Detoxification Granules. Summary of the Invention

[0005] The purpose of this invention is to provide a method for the joint detection of chlorogenic acid and baicalin in Yindan Detoxification Granules, so as to solve the problems in the background art mentioned above.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for the joint detection of chlorogenic acid and baicalin in Yindan Detoxification Granules, comprising the following steps:

[0007] S1, Preparation of the test solution:

[0008] Take 0.3g of the powder, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 10% methanol, stopper tightly, weigh it, sonicate it, cool it, weigh it again, make up the lost weight with 10% methanol, shake well, filter it, and take the filtrate to obtain the sample solution.

[0009] S2, Preparation of the reference solution:

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

[0011] S3, Determination method:

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

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

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

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

[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] Over 30–31 minutes, 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] Between 31 and 49 minutes, 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] Over 49–54 minutes, 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] Over 54–55 minutes, 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] This invention has at least the following beneficial effects:

[0023] This invention provides a method for the joint detection of chlorogenic acid and baicalin in Yindan Detoxification Granules. The method involves first dissolving the sample in 10% methanol, then sonicating it, and finally performing liquid chromatography determination according to a gradient. This method is simple to operate and can efficiently and accurately quantify chlorogenic acid and baicalin simultaneously, greatly reducing the interference of the other nine components in Yindan. Attached Figure Description

[0024] Figure 1 The linear curve of chlorogenic acid in this invention is shown.

[0025] Figure 2 The linear curve of baicalin of the present invention is shown.

[0026] Figure 3 The chromatogram of baicalin is shown.

[0027] Figure 4 This is the chromatogram of chlorogenic acid. Detailed Implementation

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] A method for the joint detection of chlorogenic acid and baicalin in Yindan Detoxification Granules includes the following steps:

[0030] S1, Preparation of the test solution:

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

[0032] S2, Preparation of the reference solution:

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

[0034] S3, Determination method:

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

[0036] Chromatographic conditions and system suitability test:

[0037] A Waters CORTECS T3 (2.1 × 100 mm) column was used; methanol was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, with gradient elution performed according to the specifications in the table below; 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 pretreatment conditions, as detailed below:

[0045] Table 3. Exploration of Ultrasound and Reflux Conditions

[0046]

[0047]

[0048] The data in the table show that the content results of ultrasound pretreatment and reflux pretreatment are not significantly different in the range of 15 to 45 minutes. Considering the time cost and tediousness of the pretreatment process, the ultrasound time is selected as 30 minutes.

[0049] Table 4. Exploration of Sample Dissolution Reagents

[0050]

[0051]

[0052] This invention uses content determination values ​​as indicators to screen out the optimal extraction solvent, extraction method, and extraction time, namely, precisely adding 25 ml of 10% methanol and ultrasonic treatment for 30 minutes.

[0053] The method provided by this invention was subjected to experimental analyses including spiking recovery, precision, stability, linearity, repeatability, and robustness, as detailed below:

[0054] 1. Recovery rate:

[0055] The recovery rate refers to the degree to which the result determined by the established method is close to the true value or reference value, and is generally expressed as recovery rate (%).

[0056] The recovery rate of sample loading not only reflects the operator's skill level, but more importantly, it reflects whether the analytical method is suitable for the matrix being tested. It helps the analyst to identify problems in the analysis in a timely manner and ensure that the analytical data are accurate and reliable.

[0057] This invention sets up 6 experimental groups and 2 control groups for comparative analysis:

[0058] Table 5. Experimental groups for sample recovery

[0059]

[0060]

[0061] Table 6. Control group for sample recovery

[0062]

[0063] Table 7. Chlorogenic acid recovery rate of each experimental group after sample addition.

[0064]

[0065] Table 8. Recovery rates of baicalin in each experimental group after sample addition.

[0066]

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

[0068] 2. Precision:

[0069] Precision refers to the degree of similarity between the results obtained from multiple sampling and determination of the same homogeneous sample under specified test conditions.

[0070] In this experimental analysis, six 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 this invention is accurate for the detection of baicalin and chlorogenic acid.

[0074] 3. Stability:

[0075] Stability studies examine how the physical, chemical, and biological properties of traditional Chinese medicine (TCM) preparations change over time under different environmental conditions (such as temperature, humidity, and light) to understand and predict the quality change trends of TCM preparations, providing a scientific basis for determining the production, packaging, storage, and transportation conditions of TCM preparations and for establishing their shelf life.

[0076] In this experimental analysis, the stability was analyzed by comparing the results at different time points from 0 to 26 hours, as detailed below:

[0077] Table 10 Stability

[0078]

[0079] In summary, the data meet the pharmacopoeia requirements, and the method provided by this invention results in samples with high 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 Linearity of chlorogenic acid

[0083]

[0084]

[0085] Fitting the values ​​in the table above, the final linear curve for chlorogenic acid is Y = 0.1709X - 0.162, R0. 2 =0.9998, its linear graph is as follows Figure 1 As shown.

[0086] Table 12 Linearity of baicalin

[0087]

[0088] Fitting the values ​​in the table above, the final linear curve for baicalin is Y = 0.2639X + 4.0932, R0. 2 =0.9997, its linear graph is as follows Figure 2 As shown.

[0089] In summary, the data meet the requirements of the pharmacopoeia, and chlorogenic acid and baicalin in the method of this invention both have good linear relationships.

[0090] 5. Repeatability:

[0091] Within the specified range, test samples of the same concentration (the sample determination concentration specified by the analytical method, equivalent to 100% concentration level) are used to evaluate the results using at least 6 test results.

[0092] Table 13 Repeatability Test Groups

[0093]

[0094]

[0095] Table 14 Repeat Control Groups

[0096]

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

[0098] 6. Durability:

[0099] Table 15 Experimental Groups

[0100]

[0101] Table 16 Control Group

[0102]

[0103]

[0104] In summary, the data meet the requirements of the pharmacopoeia, and the detection method for chlorogenic acid and baicalin in this invention has universality.

[0105] 7. Intermediate precision

[0106] To examine the impact of random variations, 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 for chlorogenic acid and baicalin in this invention is accurate.

[0111] 8. Exclusivity

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

[0113] Chlorogenic acid was derived solely from honeysuckle, and baicalin was derived solely from scutellaria baicalensis. Neither the solvent nor the excipients affected the content determination.

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

[0115] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or basic characteristics. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0116] While 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 these embodiments without departing from the principles and spirit of the invention, and that the scope of the 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 Detoxification Granules, characterized in that, The following steps are involved: S1, Preparation of the test solution: Take 0.3g of the powder, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 10% methanol, stopper tightly, weigh it, sonicate it, cool it, weigh it again, make up the lost weight with 10% methanol, shake well, filter it, and take the filtrate to obtain the sample solution. S2, Preparation of the reference solution: Take an appropriate amount of chlorogenic acid reference standard, accurately weigh it, and add 75% methanol to prepare a solution containing 0.28 mg of chlorogenic acid per ml to obtain the chlorogenic acid reference solution; take an appropriate amount of baicalin reference standard, accurately weigh it, and add methanol to prepare a solution containing 60 μg of baicalin per ml to obtain the baicalin reference solution. S3, Determination method: Accurately pipette 1 μl each of the reference solution and the test solution and inject them into the liquid chromatograph for determination; A Waters CORTEC ST3 column was used; methanol was used as mobile phase A, and 0.05–0.15% phosphoric acid solution was used as mobile phase B. The detection wavelength was 327 nm, and the mobile phase gradient program was 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%. Over 30–31 minutes, 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%. Between 31 and 49 minutes, 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%. Over 49–54 minutes, 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%. Over 54–55 minutes, 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%.

2. The method for the joint detection of chlorogenic acid and baicalin in Yindan Detoxification Granules according to claim 1, characterized in that, The ultrasonic treatment in S1 has a power of 500W, a frequency of 40kHz, and a treatment time of 30min.

3. The method for the joint detection of chlorogenic acid and baicalin in Yindan Detoxification Granules according to claim 1, characterized in that, The S3 detection wavelength is 327nm, the flow rate is 0.3ml / min, and the column temperature is 35℃.

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