A UPLC identification method for Poria cocos, Poria cocos peel, and formulated granules.

Abstract

This invention discloses a UPLC method for identifying Poria cocos (including its sclerotium), Poria cocos (including its bark), and its formulated granules. The method uses high-performance liquid chromatography (HPLC) to determine the specified relative retention times of these medicinal materials and granules. The relative retention times of the corresponding test solutions fall within ±10% of the specified values ​​for each of the three medicinal materials. This invention can effectively identify Poria cocos (including its sclerotium), Poria cocos (including its bark), and its formulated granules.

Description

Technical Field

[0001] This invention relates to the field of identification of formula granules, and in particular to a method for identifying Poria cocos, Poria cocos, Poria cocos peel medicinal materials and formula granules. Background Technology

[0002] Poria cocos is the dried sclerotium of the fungus Poria cocos (Polyporaceae family); Poria cocos peel is the dried outer skin of the sclerotium of the fungus Poria cocos (Polyporaceae family); Poria cocos sclerotium (Polyporaceae family) contains the pine root-like part in the middle of the dried sclerotium. All three medicinal materials contain various components including pachymic acid A reference standard, pachymic acid B reference standard, dehydrotemocarboxylic acid reference standard, 3-epidehydrotemocarboxylic acid reference standard, pachymic acid C reference standard, and pachymic acid AM.

[0003] Traditional Chinese medicine (TCM) formula granules are produced by extracting single-herb medicinal slices using water as a solvent, and then processing them into granules through physical methods such as solid-liquid separation, concentration, drying, and granulation. TCM formula granules retain a variety of effective components from the medicinal materials.

[0004] Currently, research on Poria cocos, Poria cocos peel, and Poria cocos sclerotium mainly focuses on pachymic acid A and pachymic acid B. The research is limited to a single component and cannot effectively distinguish between Poria cocos, Poria cocos peel, Poria cocos sclerotium medicinal materials and formula granules. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for identifying Poria cocos, Poria cocos, Poria cocos peel and its formulation granules. The method effectively identifies Poria cocos, Poria cocos, Poria cocos peel and its formulation granules using high performance liquid chromatography.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A UPLC identification method for Poria cocos sclerotium, Poria cocos, Poria cocos peel and its formulation granules, comprising the following steps:

[0008] (1) Preparation of reference solution: Take the reference standards of pachymica acid A, pachymica acid B, dehydrotomonic acid, epidehydrotomonic acid, pachymica acid C, and pachymica acid AM, dissolve them to obtain the reference solution;

[0009] (2) Inject the reference solution and the test solution into the high-performance liquid chromatograph, respectively, and use gradient elution to pass the mobile phase through the column. The chromatographic conditions are as follows:

[0010] The mobile phase consists of mobile phase A and mobile phase B; mobile phase A is acetonitrile, and mobile phase B is 0.1% phosphoric acid.

[0011] The gradient elution method is as follows:

[0012] During the period of 0 to 5 minutes, the mass fraction of mobile phase A in the mobile phase is 63-65%, and the mass fraction of mobile phase B in the mobile phase is 37-35%.

[0013] During the period of 5 to 16 minutes, the mass fraction of mobile phase A in the mobile phase is 65-90%, and the mass fraction of mobile phase B in the mobile phase is 35-10%.

[0014] During the period of 16 to 20 minutes, the mass fraction of mobile phase A in the mobile phase is 90-100%, and the mass fraction of mobile phase B in the mobile phase is 10-0%.

[0015] During the period of 20.1–25 min, the mass fraction of mobile phase A in the mobile phase was 63%, and the mass fraction of mobile phase B in the mobile phase was 37%.

[0016] (3) Discrimination is made by counting the number of characteristic peaks and relative retention time of the characteristic spectrum of the sample to be identified, and by comparing the characteristic spectrum of the sample to be identified with the characteristic spectrum of the reference standard.

[0017] Furthermore, the mobile phase flows through the chromatographic column at a rate of 0.2 ml per minute.

[0018] Furthermore, during the high-performance liquid chromatography (HPLC) detection process, the column temperature of the chromatographic column is 30°C.

[0019] Furthermore, in the high-performance liquid chromatography detection process, the packing agent is octadecylsilane-bonded silica gel.

[0020] Furthermore, during the high-performance liquid chromatography (HPLC) detection process, the detection wavelength is 242 nm.

[0021] Furthermore, in step (3), the specified values ​​of the relative retention times of the medicinal materials and formula granules of Poria cocos, Poria cocos, Poria cocos peel, and the corresponding test solution are determined, and the relative retention times of the test solution fall within ±10% of the specified values ​​of the medicinal materials and formula granules of Poria cocos, Poria cocos, Poria cocos peel, and the corresponding formula granules.

[0022] The beneficial effects of this invention are:

[0023] High-performance liquid chromatography (HPLC) can effectively identify Poria cocos, Poria cocos peel, and its formulation granules. Attached Figure Description

[0024] Figure 1 Characteristic chromatograms of reference standards for pachymic acid A, pachymic acid B, dehydrotemocarboxylic acid, 3-epidehydrotemocarboxylic acid, pachymic acid C, and pachymic acid AM.

[0025] Figure 2The characteristic chromatogram of the Poria cocos formula granules in Example 1;

[0026] Figure 3 The chromatogram of the Poria cocos formula granules in Example 2 is shown.

[0027] Figure 4 The chromatogram of the Poria cocos peel formula granules in Example 3 is shown.

[0028] Figure 5 This is a characteristic chromatogram of Poria cocos in Example 4;

[0029] Figure 6 The characteristic chromatogram of Poria cocos in Example 5;

[0030] Figure 7 This is a characteristic chromatogram of the Poria cocos peel medicinal material in Example 6. Detailed Implementation

[0031] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features described therein can be combined with each other.

[0032] It should be noted that the illustrations provided in the following embodiments are only schematic representations of the basic concept of the present invention. Therefore, the drawings only show the components related to the present invention and are not drawn according to the actual number, shape and size of the components in the actual implementation. In the actual implementation, the form, quantity and proportion of each component can be arbitrarily changed, and the layout of the components may also be more complex.

[0033] The following specific examples all use high-performance liquid chromatography (HPLC) to detect the test samples and obtain the corresponding characteristic spectra:

[0034] The mobile phases used in the high-performance liquid chromatography detection process are mobile phase A and mobile phase B; mobile phase A is acetonitrile, and mobile phase B is 0.1% phosphoric acid.

[0035] The high-performance liquid chromatography (HPLC) detection process employs gradient elution to pass the mobile phase through the chromatographic column. The gradient elution method is as follows:

[0036] During the period from 0 to 5 minutes, the mass fraction of mobile phase A in the mobile phase is 63-65, and the mass fraction of mobile phase B in the mobile phase is 37-35.

[0037] Between 5 and 16 minutes, the mass fraction of mobile phase A in the mobile phase increases from 63-65 to 65-90, and the mass fraction of mobile phase B in the mobile phase decreases from 37-35 to 35-10.

[0038] During 16–20 min, the mass fraction of mobile phase A in the mobile phase increased from 65–90 to 90–100, and the mass fraction of mobile phase B in the mobile phase decreased from 35–10 to 10–0.

[0039] During the period of 20.1 to 25 minutes, the mass fraction of mobile phase A in the mobile phase was 63, and the mass fraction of mobile phase B in the mobile phase was 37.

[0040] During the high-performance liquid chromatography (HPLC) detection process, the flow rate of the mobile phase through the chromatographic column is 0.2 ml per minute;

[0041] The column temperature is 30℃; the packing material is octadecylsilane-bonded silica gel.

[0042] During the high-performance liquid chromatography (HPLC) detection process, the detection wavelength is 242 nm; the theoretical plate number, calculated based on the pamoate A peak, should be no less than 10,000.

[0043] For high performance liquid chromatography, please refer to General Chapter 0512 of the 2020 edition of the Chinese Pharmacopoeia.

[0044] Prepare a test solution from the medicinal material or granule formulation to be identified:

[0045] Take an appropriate amount of the medicinal material or formula granules to be identified, grind them into a fine powder, take 1g, weigh it accurately, place it in a stoppered conical flask, add 10ml of methanol accurately, stopper tightly, weigh it, sonicate for 10 minutes (power 250W, frequency 35kHz), cool it, weigh it again, make up the lost weight with methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0046] Take appropriate amounts of pachymic acid A reference standard, pachymic acid B reference standard, dehydrotemocarboxylic acid reference standard, 3-epi-dehydrotemocarboxylic acid reference standard, pachymic acid C reference standard, and pachymic acid AM reference standard, accurately weigh them, and add methanol to prepare a mixed solution containing 10 μg of pachymic acid A, 10 μg of pachymic acid B, 10 μg of dehydrotemocarboxylic acid, 10 μg of 3-epi-dehydrotemocarboxylic acid, 10 μg of pachymic acid C, and 10 μg of pachymic acid AM per 1 ml, as the reference solution.

[0047] Accurately pipette 1 μl of the reference solution and inject it into the liquid chromatograph. Analyze the reference solution using the high-performance liquid chromatography method described in Example 3 to obtain the characteristic chromatogram of the reference solution. Figure 1 As shown.

[0048] Accurately pipette 1 μl each of the test solution and the reference solution, inject them into the liquid chromatograph, and perform the determination.

[0049] Example 1:

[0050] This embodiment provides a method for identifying Poria cocos formula granules. For high-performance liquid chromatography (HPLC) detection, please refer to the aforementioned steps. The characteristic chromatogram of Poria cocos formula granules is as follows: Figure 2 As shown, the characteristic chromatogram of the Poria cocos formula granules exhibits 10 characteristic peaks, which correspond to the retention times of the 10 characteristic peaks in the characteristic chromatogram of the Poria cocos reference material. Six of the 10 characteristic peaks in the characteristic chromatogram of the Poria cocos formula granules correspond to the retention times of the corresponding reference peaks in the characteristic chromatograms of pachymic acid A, pachymic acid B, dehydrotemocarboxylic acid, 3-epi-dehydrotemocarboxylic acid, pachymic acid C, and pachymic acid AM reference materials. The peak corresponding to the 3-epi-dehydrotemocarboxylic acid reference peak is peak S. The relative retention times of peaks 4, 6, 9, and 10 with peak S were calculated, and the specified values ​​for the relative retention times were determined to be 0.86 (peak 4), 0.92 (peak 6), 1.44 (peak 9), and 1.75 (peak 10), respectively.

[0051] If the relative retention time of the test sample solution falls within ±10% of the above-mentioned specified value, the test sample is determined to be a Poria cocos formula granule.

[0052] Example 2:

[0053] This embodiment provides a method for identifying Poria cocos granules. The high-performance liquid chromatography (HPLC) method is the same as in Example 1. The characteristic chromatogram of the Poria cocos granules is as follows: Figure 3 As shown, the characteristic spectrum of the Fu Shen formula granules exhibits 14 characteristic peaks, which correspond to the retention times of the 14 characteristic peaks in the characteristic spectrum of the Fu Shen reference medicinal material. Six of the 14 characteristic peaks in the characteristic spectrum of the Fu Shen formula granules correspond to the retention times of the corresponding reference peaks in the characteristic spectra of pachymic acid A, pachymic acid B, dehydrotemoic acid, 3-epi-dehydrotemoic acid, pachymic acid C, and pachymic acid AM. The peak corresponding to the reference peak of 3-table dehydrotumoic acid is the S peak. The relative retention times of peaks 4, 6, 9, 10, 11, 12, 13, and 14 with peak S are calculated, and the specified values ​​of the relative retention times are determined to be: 0.86 (peak 4), 0.92 (peak 6), 1.27 (peak 9), 1.28 (peak 10), 1.34 (peak 11), 1.44 (peak 12), 1.75 (peak 13), and 1.90 (peak 14).

[0054] If the relative retention time of the test sample solution falls within ±10% of the above-mentioned specified value, then the test sample is determined to be a Poria cocos formula granule.

[0055] Example 3:

[0056] This embodiment provides a method for identifying Poria cocos peel granules. The high-performance liquid chromatography (HPLC) method is the same as in Example 1. The characteristic chromatogram of the Poria cocos peel granules is as follows: Figure 4 As shown, the characteristic chromatogram of the Poria cocos peel formula granules shows nine characteristic peaks, which correspond to the retention times of the nine characteristic peaks in the characteristic chromatogram of the Poria cocos peel reference material. Five of the nine characteristic peaks in the characteristic chromatogram of the Poria cocos peel formula granules correspond to the retention times of the corresponding reference peaks in the characteristic chromatograms of pachymic acid A, pachymic acid B, dehydrotemocarboxylic acid, 3-epi-dehydrotemocarboxylic acid, and pachymic acid C. The peak corresponding to the 3-epi-dehydrotemocarboxylic acid reference peak is peak S. The relative retention times of peaks 5, 7, 8, and 9 with peak S were calculated, and the specified values ​​for the relative retention times were determined to be 0.92 (peak 5), 1.44 (peak 7), 1.75 (peak 8), and 1.90 (peak 9), respectively.

[0057] If the relative retention time of the test sample solution falls within ±10% of the above-mentioned specified value, the test sample is determined to be a Poria cocos peel formula granule.

[0058] Example 4:

[0059] This embodiment provides a method for identifying Poria cocos medicinal material. The high-performance liquid chromatography (HPLC) method is the same as in Example 1. The characteristic chromatogram of Poria cocos medicinal material is as follows: Figure 5 As shown, the characteristic chromatogram of Poria cocos presents 11 characteristic peaks. Six of these peaks correspond to the retention times of the reference peaks in the characteristic chromatograms of pachymic acid A, pachymic acid B, dehydrotemocarboxylic acid, 3-epi-dehydrotemocarboxylic acid, pachymic acid C, and pachymic acid AM, respectively. The peak corresponding to the 3-epi-dehydrotemocarboxylic acid reference peak is designated as peak S. The relative retention times of peaks 7, 8, 9, 10, and 11 with peak S were calculated, and the specified values ​​for these relative retention times were determined to be: 1.23 (peak 7), 1.34 (peak 8), 1.43 (peak 9), 1.75 (peak 10), and 1.90 (peak 11), respectively.

[0060] If the relative retention time of the test sample solution falls within ±10% of the above-mentioned specified value, the test sample is determined to be Poria cocos.

[0061] Example 5:

[0062] This embodiment provides a method for identifying Poria cocos (Fu Shen) medicinal material. The high-performance liquid chromatography (HPLC) method is the same as in Example 1. The characteristic chromatogram of Poria cocos (Fu Shen) medicinal material is as follows: Figure 6As shown in the diagram, the characteristic chromatogram of Poria cocos (Fu Shen) shows 10 characteristic peaks. Six of these peaks correspond to the retention times of the reference peaks in the characteristic chromatograms of pachymic acid A, pachymic acid B, dehydrotemocarboxylic acid, 3-epi-dehydrotemocarboxylic acid, pachymic acid C, and pachymic acid AM, respectively. The peak corresponding to the 3-epi-dehydrotemocarboxylic acid reference peak is peak S. The relative retention times of peaks 7, 8, 9, and 10 with peak S were calculated, and the specified values ​​for these relative retention times were determined to be 1.34 (peak 7), 1.44 (peak 8), 1.75 (peak 9), and 1.90 (peak 10), respectively.

[0063] If the relative retention time of the test sample solution falls within ±10% of the above-mentioned specified value, the test sample is determined to be Poria cocos.

[0064] Example 6:

[0065] This embodiment provides a method for identifying Poria cocos peel, using high-performance liquid chromatography (HPLC) as described in Example 1. The characteristic chromatogram of Poria cocos peel is shown below. Figure 7 As shown in the diagram, the characteristic chromatogram of Poria cocos peel shows 12 characteristic peaks. Six of these peaks correspond to the retention times of the reference peaks in the characteristic chromatograms of pachymic acid A, pachymic acid B, dehydrotemocarboxylic acid, 3-epi-dehydrotemocarboxylic acid, pachymic acid C, and pachymic acid AM, respectively. The peak corresponding to the 3-epi-dehydrotemocarboxylic acid reference peak is designated as peak S. The relative retention times of peaks 7, 8, 9, 10, 11, and 12 with peak S were calculated, and the specified values ​​for the relative retention times were determined to be: 1.44 (peak 7), 1.49 (peak 8), 1.64 (peak 9), 1.75 (peak 10), 1.91 (peak 11), and 2.05 (peak 12), respectively.

[0066] If the relative retention time of the test sample solution falls within ±10% of the above-mentioned specified value, the test sample is determined to be Poria cocos peel.

[0067] In summary, the above embodiments are merely illustrative examples of preferred embodiments of the present invention and do not encompass all aspects of the invention. Those skilled in the art can make various modifications and refinements without departing from the spirit and scope of the present invention; therefore, the scope of protection of the present invention is defined by the claims.

Claims

1. A UPLC method for identifying Poria cocos sclerotium, Poria cocos, Poria cocos peel, and formulated granules, characterized in that, Includes the following steps: (1) Preparation of reference solution: Take pachymica acid A, pachymica acid B, dehydrotomonic acid, epidehydrotomonic acid, pachymica acid C and pachymica acid AM reference standards, dissolve them to obtain the reference solution; (2) Inject the reference solution and the test solution into the high performance liquid chromatograph, respectively, and use gradient elution to pass the mobile phase through the column. The chromatographic conditions are as follows: The mobile phase consists of mobile phase A and mobile phase B; mobile phase A is acetonitrile, and mobile phase B is 0.1% phosphoric acid. The gradient elution method is as follows: During the period from 0 to 5 minutes, the mass fraction of mobile phase A in the mobile phase is 63-65%, and the mass fraction of mobile phase B in the mobile phase is 37-35%. During the period of 5–16 min, the mass fraction of mobile phase A in the mobile phase is 65–90%, and the mass fraction of mobile phase B in the mobile phase is 35–10%. During the 16–20 min period, the mass fraction of mobile phase A in the mobile phase is 90–100%, and the mass fraction of mobile phase B in the mobile phase is 10–0%. During the period of 20.1–25 min, the mass fraction of mobile phase A in the mobile phase was 63%, and the mass fraction of mobile phase B in the mobile phase was 37%. (3) Discrimination is made by counting the number of characteristic peaks and relative retention time of the characteristic spectrum of the sample to be identified, and by comparing the characteristic spectrum of the sample to be identified with that of the reference standard. In the high-performance liquid chromatography (HPLC) detection process, the packing material is octadecylsilane-bonded silica gel; During the high-performance liquid chromatography (HPLC) detection process, the detection wavelength is 242 nm. The samples to be identified are Poria cocos (shen), Poria cocos (pi), Poria cocos (pi), Poria cocos (formula granules), Poria cocos (formula granules), and Poria cocos (pi) (formula granules).

2. The method according to claim 1, characterized in that, The mobile phase flows through the chromatographic column at a rate of 0.2 ml per minute.

3. The method according to claim 1, characterized in that, During the high-performance liquid chromatography (HPLC) detection process, the column temperature of the chromatographic column is 30℃.

4. The method according to claim 1, characterized in that, In step (3), the specified values ​​of the relative retention time of Poria cocos, Poria cocos, Poria cocos peel and formula granules are determined, and the relative retention time of the corresponding test solution falls within ±10% of the specified values ​​of Poria cocos, Poria cocos, Poria cocos peel and formula granules.

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