A method for determining the content of 10 triterpenoid components in Poria cocos

Abstract

This invention relates to the field of analytical detection technology and proposes a method for determining the content of 10 triterpenoid components in Poria cocos, comprising the following steps: S1, preparing a reference solution containing dehydropachyric acid and pachyric acid; S2, preparing a test solution containing Poria cocos; S3, determining the content of the 10 triterpenoid components in the reference solution and the test solution respectively using high performance liquid chromatography; the triterpenoid components are pachyric acid B, dehydropachyric acid, pachyric acid A, pachyric acid C, 3-epi-dehydropachyric acid, dehydropachyric acid, pinocembrinic acid, pachyric acid, 3-O-acetyl-16α-hydroxy-hydropachyric acid, and pachyric acid. This technical solution solves the problem in existing technologies where the quality control of Poria cocos relies on a single legal quality standard and lacks content determination quality control items.

Description

Technical Field

[0001] This invention relates to the field of analytical detection technology, specifically to a method for determining the content of 10 triterpenoid components in Poria cocos. Background Technology

[0002] Poria cocos was first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica) and listed as a superior-grade herb. It is a major traditional Chinese medicine used for both medicinal and culinary purposes for thousands of years. Poria cocos is a fungus belonging to the Polyporaceae family. Poria cocos (Schw.) Wolf The dried sclerotium of Poria cocos has a sweet, bland, and neutral taste. It enters the heart, lung, spleen, and kidney meridians and has the effects of promoting diuresis and eliminating dampness, strengthening the spleen and calming the mind. It can be used to regulate and treat edema with scanty urine, phlegm retention with dizziness and palpitations, spleen deficiency with poor appetite, and restlessness. Poria cocos is known as the "divine medicine of all four seasons" and is said to contain "nine out of ten medicines". The 2020 edition of the Chinese Pharmacopoeia contains more than 250 preparations containing Poria cocos. Among the 100 prescriptions included in the "Catalogue of Famous Ancient Prescriptions (First Batch)", 25 contain Poria cocos. It is a major Chinese medicine with a production volume of tens of thousands of tons and is known as the "holy product" for removing dampness and the "essential medicine" for strengthening the spleen. It is also an important raw material for a large number of foods, health products, cosmetics, and traditional Chinese veterinary medicines, with extremely broad application prospects.

[0003] Poria cocos contains various chemical components, including polysaccharides, triterpenoids, sterols, volatile oils, and proteins. The two main active components are triterpenoids and polysaccharides. Modern pharmacological studies have shown that Poria cocos has diuretic, hepatoprotective, sedative, and immunomodulatory effects, as well as antibacterial, anti-ulcer, and hypoglycemic effects. It also has good protective effects on the central nervous system, immune system, and digestive system. The legal quality standard for Poria cocos is the 2020 edition of the Chinese Pharmacopoeia, which has relatively simple testing items and lacks content determination, failing to comprehensively and accurately reflect the quality of the medicinal material. Market research and literature review have revealed that the quality control of Poria cocos suffers from a single legal quality standard and the absence of content determination quality control items. Therefore, it is essential to establish a method for determining the content of 10 triterpenoid components in Poria cocos using a single test and multiple evaluation methods, providing a simple and efficient basis for the quality control of Poria cocos. Summary of the Invention

[0004] This invention proposes a method for determining the content of 10 triterpenoid components in Poria cocos, which solves the problem in related technologies that the quality control of Poria cocos has a single statutory quality standard and no quality control items for content determination.

[0005] The technical solution of the present invention is as follows: This invention proposes a method for determining the content of 10 triterpenoid components in Poria cocos, comprising the following steps: S1. Prepare a reference solution containing dehydropachymic acid and pachymic acid; S2. Prepare a test solution containing Poria cocos; S3. The contents of 10 triterpenoid components in the reference solution and the test solution were determined by high performance liquid chromatography. The triterpenoid components are pachymic acid B, dehydro-termoic acid, pachymic acid A, porphyric acid C, 3-epidehydro-termoic acid, dehydropachymic acid, pinocytic acid, termoic acid, 3-O-acetyl-16α-hydroxy-hydro-pinocytic acid, and pachymic acid.

[0006] As a further technical solution, the solvents of the reference solution and the test solution are each independently methanol.

[0007] The methanol solvent used in this invention yields the highest content of triterpenoid components, with good peak shape, fewer impurity peaks, and easier filtration.

[0008] As a further technical solution, in step S2, the preparation process employs one of ultrasonic mixing, reflux mixing, or shaking mixing.

[0009] As a further technical solution, when the preparation is carried out by ultrasonic mixing, the ultrasonic mixing time is 15~45min, the power is 400~500W, and the frequency is 40~50kHz.

[0010] As a further technical solution, in step S2, the mass-to-volume ratio of Poria cocos to solvent in the test solution is 1g:10~15mL.

[0011] As a further technical solution, in step S3, the detection conditions of the high-performance liquid chromatography include: the mobile phase consists of phase A and phase B, phase A is acetonitrile, and phase B is a 0.1% (v / v) phosphoric acid solution, and gradient elution is performed; the gradient elution conditions are as follows: From 0 to 5 minutes, the volume fraction of phase A increased from 53% to 55%, while the volume fraction of phase B decreased from 47% to 45%. Within 5–10 minutes, the volume fraction of phase A increased from 55% to 65%, while the volume fraction of phase B decreased from 45% to 35%. Within 10-12 minutes, the volume fraction of phase A increased from 65% to 85%, while the volume fraction of phase B decreased from 35% to 15%. Within 12-15 minutes, the volume fraction of phase A increased from 85% to 90%, while the volume fraction of phase B decreased from 15% to 10%.

[0012] The gradient elution conditions set in this invention have the advantages of short elution time and high efficiency, which further improves the accuracy of detecting the content of 10 triterpenoid components in Poria cocos.

[0013] As a further technical solution, the detection conditions of the high performance liquid chromatography method also include using a C18 column with a size of 2.1×100mm, a packing particle size of 1.7µm, a flow rate of 0.4mL / min, and a column temperature of 30℃.

[0014] In this invention, a C18 chromatographic column with a packing particle size of 1.7µm and a size of 2.1×100mm is used, which can better separate the chromatographic peaks and impurity peaks of Poria cocos samples, and the peak shape does not tail, and the analysis time of Poria cocos samples is shorter.

[0015] As a further technical solution, the detection wavelengths of high performance liquid chromatography are as follows: 242 nm for pamoic acid B, dehydrotomonic acid, pamoic acid A, pamoic acid C, 3-epi-dehydrotomonic acid, dehydropamoic acid and pinocembrinic acid; and 210 nm for tomonic acid, 3-O-acetyl-16α-hydroxy-hydropamoic acid and pamoic acid.

[0016] The present invention employs a dual-wavelength method to establish a one-to-many evaluation-dual-wavelength coupled ultra-high performance liquid chromatography method that can determine the content of 10 triterpenoid components in Poria cocos, providing a simple and efficient basis for the quality control of Poria cocos.

[0017] As a further technical solution, the method for determining the content of 10 triterpenoid components in Poria cocos using high performance liquid chromatography is as follows: A1. Using the peak corresponding to dehydropamoic acid as peak S1, calculate the relative retention times of pamoic acid B, dehydropamoic acid, pamoic acid A, pamoic acid C, 3-epi-dehydropamoic acid, and pinocembrinic acid, and then multiply them by correction factors to calculate the contents of pamoic acid B, dehydropamoic acid, pamoic acid A, pamoic acid C, 3-epi-dehydropamoic acid, dehydropamoic acid, and pinocembrinic acid. A2. Using the corresponding peak of pamoic acid as the S2 peak, calculate the relative retention times of tomonic acid and 3-O-acetyl-16α-hydroxy-hydrogenated pamoic acid, and then multiply them by correction factors to calculate the contents of tomonic acid, 3-O-acetyl-16α-hydroxy-hydrogenated pamoic acid, and pamoic acid.

[0018] As a further technical solution, the relative retention time of pachymic acid B is 0.36, with a correction factor of 1.04; the relative retention time of dehydrotemoic acid is 0.38, with a correction factor of 1.02; the relative retention time of pachymic acid A is 0.48, with a correction factor of 1.09; the relative retention time of pachymic acid C is 0.57, with a correction factor of 0.94; and the relative retention time of 3-epi-dehydrotemoic acid is 0.65, with a correction factor of 1.00. The relative retention time of the dehydropachymic acid is 1.00, with a correction factor of 1.00; the relative retention time of the pinacol is 1.15, with a correction factor of 0.93; the relative retention time of the tumanoic acid is 0.39, with a correction factor of 0.98; the relative retention time of the 3-O-acetyl-16α-hydroxy-hydropachymic acid is 0.90, with a correction factor of 0.82; and the relative retention time of the poriacoic acid is 1.00, with a correction factor of 1.00.

[0019] The working principle and beneficial effects of this invention are as follows: 1. In this invention, high performance liquid chromatography (HPLC) is used to determine the content of triterpenoids in Poria cocos. Ten triterpenoids can be detected simultaneously. When preparing the test solution, ultrasonic extraction is used on Poria cocos. Compared with reflux extraction and shaking extraction, the operation is simpler and more convenient. The method of this invention has the advantages of good repeatability, short detection time, simplicity and efficiency in detecting ten triterpenoids in Poria cocos, which provides a basis for the quality control of Poria cocos.

[0020] 2. This invention constructs a method for the detection of triterpenoid components in Poria cocos using a single-method-multiple-evaluation approach. Using dehydropachyric acid and pachyric acid standards as references, the relative correction factors between dehydropachyric acid and pachyric acid B, dehydrotomolic acid, pachyric acid A, pachyric acid C, 3-epi-dehydrotomolic acid, and pinocembrinic acid, as well as the correction factors between pachyric acid and tomolic acid, 3-O-acetyl-16α-hydroxy-hydropachyric acid, and pachyric acid, are determined. This method can effectively detect the content of 10 triterpenoid components in Poria cocos, not only reducing detection costs but also improving detection efficiency. Furthermore, the results obtained using the single-method-multiple-evaluation approach are basically consistent with those obtained using the external standard method, indicating accurate results, good repeatability, and good robustness. Attached Figure Description

[0021] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0022] Figure 1 This is the liquid chromatogram of Poria cocos in Example 1 of the present invention; Figure 2 This is the liquid chromatogram of Poria cocos in Comparative Example 1 of the present invention; Figure 3 This is the liquid chromatogram of Poria cocos in Comparative Example 2 of the present invention; Figure 4This is the liquid chromatogram of Poria cocos in Comparative Example 3 of the present invention; Figure 5 A comparison chart of the content determination by external standard method and the content determination by one-to-many method with correction factor for samples of batch numbers 1-20; Figure 6 A comparison chart of the content determination by external standard method and the content determination by one-to-many method with correction factor for sample 21-40; Figure 7 This is a comparison chart of the content determination by external standard method and the content determination by one-to-many method with correction factor for sample No. 41-60. Detailed Implementation

[0023] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0024] Instruments and reagents Instruments: Waters ACQuity ultra-high performance liquid chromatograph (quaternary pump, diode array detector), Mettler XPE26 analytical balance (0.001mg), Mettler XS105DU analytical balance (0.01mg), Millipore ultrapure water system, Waters ACQUITY UPLC BEH, C18 (1.7µm, 2.1×100mm).

[0025] Reagents: Methanol (Sinopharm Chemical Reagent Co., Ltd., batch number: 10014118, analytical grade); Acetonitrile (Thermo Fisher Scientific (China) Co., Ltd., batch number: F22M2A201, chromatographic grade); Water was ultrapure water; Reference standards: pachymic acid A (batch number 12682, 98.6% purity), pachymic acid B (batch number 15639, 97.7% purity), pachymic acid C (batch number 14725, 99.9% purity), 3-O-acetyl-16α-hydroxy-hydrogenated pachymic acid (batch number 9102, 99.0% purity), dehydropachymic acid (batch number 15265, 99.0% purity). The following substances were purchased from Shanghai Shidander Standard Technical Service Co., Ltd.: 9% (presumably referring to a specific concentration of 9%), pine resin reference standard (batch number 13609, presumably 98.6%), pachymic acid (batch number 15736, presumably 98.0%), dehydrotumoic acid (batch number 15734, presumably 98.0%), tumoic acid (batch number 15764, presumably 98.0%), and 3-epi-dehydrotumoic acid (batch number 15759, presumably 98.0%). Example 1 Preparation of reference solution: Weigh the reference standard accurately, add methanol to prepare a mixed solution containing 100 μg / mL each of dehydropachymic acid and pachymic acid, and the reference solution is obtained. Preparation of the test solution: Take 1g of Poria cocos powder, accurately weigh it, place it in a stoppered conical flask, add 10mL of methanol, weigh it, sonicate it for 30 minutes at a power of 400W and a frequency of 40kHz, cool it, weigh it again, replenish the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain the test solution. Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B. Gradient elution was performed according to the specifications in Table 1, with a flow rate of 0.4 mL / min; the column temperature was 30℃; the detection wavelengths were: 242 nm for pamoic acid B, dehydro-termoic acid, pamoic acid A, pamoic acid C, 3-epi-dehydro-termoic acid, dehydropamoic acid, and pinocembrinic acid; and 210 nm for termoic acid, 3-O-acetyl-16α-hydroxy-hydro-pamoic acid, and pamoic acid; the theoretical plate number calculated based on the dehydropamoic acid peak should not be less than 10,000.

[0026] Table 1 Gradient elution program

[0027] Accurately pipette 2 μL each of the reference solution and the test solution, and inject them into the ultra-high performance liquid chromatograph for analysis. Obtain the liquid chromatogram of Poria cocos, as shown below. Figure 1 As shown in the figure, peaks 1 to 10 correspond to 1: pachymic acid B, 2: dehydrotomolic acid, 3: pachymic acid A, 4: pachymic acid C, 5: 3-epi-dehydrotomolic acid, 6: dehydropachymic acid, 7: pinoresinic acid, 8: tomolic acid, 9: 3-O-acetyl-16α-hydroxy-hydro-pinoresinic acid, and 10: pachymic acid.

[0028] Using dehydropachyric acid and pachyric acid reference standards as references, and with the corresponding peak of dehydropachyric acid as peak S1, calculate the relative retention times of pachyric acid B, dehydrotomolic acid, pachyric acid A, porphyric acid C, 3-epi-dehydrotomolic acid, and pinocembrinic acid. Using the corresponding peak of pachyric acid as peak S2, calculate the relative retention times of tomolic acid and 3-O-acetyl-16α-hydroxy-hydropiperazic acid. The relative retention times should be within ±10% of the specified values. Using dehydropachyric acid reference standard as a control, multiply by correction factors to calculate the contents of pachyric acid B, dehydrotomolic acid, pachyric acid A, porphyric acid C, 3-epi-dehydrotomolic acid, dehydropachyric acid, and pinocembrinic acid. Using pachyric acid reference standard as a control, multiply by correction factors to calculate the contents of tomolic acid, 3-O-acetyl-16α-hydroxy-hydropiperazic acid, and pachyric acid. The relative retention times and correction factors are shown in Table 2. Table 2 Relative Retention Time and Correction Factor

[0029] Methodology validation - System suitability testing 1. Examination of linear relationships 5.285 mg of pachymic acid B, 5.243 mg of dehydrotomonic acid, 5.314 mg of tomonic acid, 5.007 mg of pachymic acid A, 5.183 mg of pachymic acid C, 5.178 mg of 3-epi-dehydrotomonic acid, 5.210 mg of 3-O-acetyl-16α-hydroxy-hydropachymic acid, 5.000 mg of dehydropachymic acid, 5.539 mg of pachymic acid, and 5.408 mg of pineacinic acid were weighed and dissolved in methanol to prepare a series of concentration solutions. These solutions were then injected into the solutions at a volume of 2 μL, and the peak areas were recorded. A standard curve was plotted with the peak area integral as the ordinate and the standard injection volume as the abscissa, as shown in Tables 3-12. Tables 3-12 show that the 10 triterpenoid components in Poria cocos exhibit good linearity.

[0030] Table 3. Results of linearity study of pamoate B

[0031] Table 4. Results of linearity study of dehydrotumomolybic acid

[0032] Table 5. Results of linearity study of pamoate A

[0033] Table 6. Results of linearity study of porphyrin C

[0034] Table 73 - Results of linearity study of dehydrotumoric acid

[0035] Table 8. Results of linearity study of dehydropachymic acid

[0036] Table 9. Results of linearity study of Songling Xin Acid

[0037] Table 10 Results of linearity study of tumolybdenum

[0038] Table 1. Results of linearity study of 113-O-acetyl-16α-hydroxy-hydropiperazine

[0039] Table 12 Results of linearity study of pamoate.

[0040] 2. Repeatability experiment 1 g of Poria cocos powder (batch number 20221204) was accurately weighed and prepared into a test solution according to the method described above for "Preparation of Test Solution". The content (mg / g) of 10 triterpenoid components in Poria cocos was determined. The results are shown in Table 15. Table 13 shows that the repeatability of this method is good.

[0041] Table 13 Repeatability Tests (Content of each component is in mg / g)

[0042] 3. Accuracy Experiment Take 1g of Poria cocos powder with known content (batch number 20221204), take 9 portions of the powder, weigh them accurately, add high, medium and low concentration reference solutions, and prepare the test solution according to the method of "Preparation of Test Solution" above. Calculate the recovery rate. The results are shown in Tables 14 to 23. The results show that the accuracy of this method is high.

[0043] Table 14 Results of the Poria cocos acid B recovery test

[0044] Table 15 Results of Dehydrotumoic Acid Recovery Test

[0045] Table 16 Results of the recovery rate test of pamoate A

[0046] Table 17 Results of the recovery rate test of porphyrin C

[0047] Table 183 - Results of Dehydrothomoleic Acid Recovery Test

[0048] Table 19 Results of Dehydropachymic Acid Recovery Test

[0049] Table 20 Results of the Songling New Acid Recovery Rate Test

[0050] Table 21 Results of the Tumoic Acid Recovery Rate Test

[0051] Table 223 Results of Recovery Test of 3-O-acetyl-16α-hydroxy-hydropinene

[0052] Table 23 Results of the Poria cocos acid recovery test

[0053] 4. Stability test The same test solution was sampled and measured at 0, 2, 4, 8, 12, 18 and 24 hours, and the peak area fraction was recorded as shown in Table 24. The results show that the test solution is stable within 24 hours.

[0054] Table 24 Results of the stability test of the test sample solution

[0055] Example 2 The only difference between this embodiment and Example 1 is that the test solution is not subjected to ultrasonic treatment during preparation, but instead is subjected to shaking treatment for 30 minutes.

[0056] Example 3 The only difference between this embodiment and Example 1 is that the test solution is not subjected to ultrasonic treatment during preparation, but instead is subjected to reflux treatment for 30 minutes.

[0057] The effects of Examples 1 and 2-3 on the content of Poria cocos are summarized in Table 25. Table 25 shows that there was no significant difference in the content of triterpenoids obtained by different extraction methods. Considering simplicity and ease of operation, the ultrasonic extraction method was chosen.

[0058] Table 25. Effects of different extraction methods on the content (mg / g) of Poria cocos.

[0059] Comparative Example 1 The only difference between this comparative example and Example 1 is the chromatographic column used: Waters ACQUITY UPLC, BEHC18, 1.7 μm, 2.1 × 50 mm, with the gradient elution program shown in Table 26 below. Table 26

[0060] The chromatogram obtained from the test is as follows Figure 2 As shown in the figure, the Poria cocos sample has relatively few chromatographic peaks and the peaks are tailed.

[0061] Comparative Example 2 The only difference between this comparative example and Example 1 is the chromatographic column used: Waters ACQUITY UPLC, BEHC18, 1.7 μm, 2.1 × 50 mm, with the gradient elution program shown in Table 27 below. Table 27

[0062] The chromatogram obtained from the test is as follows Figure 3 As shown in the figure, the chromatographic peaks of the Poria cocos sample have poor shapes, and some chromatographic peaks and impurity peaks are not separated.

[0063] Comparative Example 3 The only difference between this comparative example and Example 1 is the chromatographic column used: Waters CORTECS, Phenyl, 2.7 μm, 4.6 × 150 mm, with the gradient elution program shown in Table 28 below. Table 28

[0064] The chromatogram obtained from the test is as follows Figure 4 As shown in the figure, it can be seen that using chromatographic columns with different particle sizes and packing materials results in longer analysis times and fewer chromatographic peaks for Poria cocos samples.

[0065] Comparative Example 4 The only difference between this comparative example and Example 1 is that the methanol is replaced with methanol with a volume fraction of 70%.

[0066] Comparative Example 5 The only difference between this comparative example and Example 1 is that the methanol is replaced with methanol with a volume fraction of 50%.

[0067] The effects of Example 1 and Comparative Examples 4-5 on the content of Poria cocos are summarized in Table 28. Table 29 shows that the content of triterpenoids extracted with methanol solvent was the highest, with better peak shape, fewer impurity peaks, and easier filtration. Therefore, methanol was chosen as the extraction solvent.

[0068] Table 29. Effects of different extraction solvents on the content (mg / g) of Poria cocos.

[0069] One test with multiple assessments Triterpenoids are the main components and active ingredients in Poria cocos. To simplify the experiment, a multi-evaluation study was conducted using dehydropachymic acid as a reference. The names, structural formulas, and molecular formulas of 10 triterpenoids in Poria cocos and Poria cocos peel are shown in Table 30.

[0070] Table 30 Ten Triterpenoid Components in Poria Cocos and Poria Cocos Peel

[0071]

[0072] Determination of relative retention time (RRT) and correction factor (f) Three portions each of pachymic acid B, dehydro-termoic acid, pachymic acid A, porphyric acid C, 3-epi-dehydro-termoic acid, dehydropachymic acid, pinocembrinic acid, termoic acid, 3-O-acetyl-16α-hydroxy-hydro-pinocembrinic acid, and pachymic acid reference standards were accurately weighed and dissolved in methanol to prepare a series of concentration solutions. These solutions were injected into an ultra-high performance liquid chromatograph (UHPLC) and the peak areas were measured, as shown in Table 31. The correction factor was calculated using the standard curve method, and the results are shown in Table 32. The relative retention times are shown in Table 33, where f = ks / ki, ks is the slope of the reference standard curve, and ki is the slope of the analyte standard curve.

[0073] Table 31 Standard Curve Measurement Data

[0074]

[0075]

[0076] Table 32 Results of Correction Factor (f) Measurement

[0077] Table 33 Results of relative retention time determination

[0078] Comparison of content determination by external standard method and content determination by correction factor-based multi-evaluation method The total amounts of pachymic acid B, dehydrotomonic acid, pachymic acid A, porphyric acid C, 3-epidehydrotomonic acid, dehydropachymic acid, pinocembrinic acid, tomonic acid, 3-O-acetyl-16α-hydroxy-hydropinepoetic acid, pachymic acid, and ten other compounds in Poria cocos calculated using the external standard method were compared with the results calculated using the correction factor method. The results showed that the determination results from the external standard method and the correction factor method were basically consistent. The results are as follows: Figures 5-7 As shown, Ⅰ is the external standard method, and Ⅱ is the one-measure-multiple-evaluation method.

[0079] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for determining the content of 10 triterpenoid components in Poria cocos, characterized in that, Includes the following steps: S1. Prepare a reference solution containing dehydropachymic acid and pachymic acid; S2. Prepare a test solution containing Poria cocos; S3. The contents of 10 triterpenoid components in the reference solution and the test solution were determined by high performance liquid chromatography. The triterpenoid components are pamoic acid B, dehydro-termoic acid, pamoic acid A, porphyric acid C, 3-epidehydro-termoic acid, dehydropamoic acid, pinocembrinic acid, termoic acid, 3-O-acetyl-16α-hydroxy-hydro-pinocembrinic acid, and pamoic acid. The detection conditions for the high-performance liquid chromatography (HPLC) method include: using a C18 column, with a mobile phase consisting of phase A and phase B, where phase A is acetonitrile and phase B is a 0.1% (v / v) phosphoric acid solution, and performing gradient elution; the gradient elution conditions are as follows: From 0 to 5 minutes, the volume fraction of phase A increased from 53% to 55%, while the volume fraction of phase B decreased from 47% to 45%. Within 5–10 minutes, the volume fraction of phase A increased from 55% to 65%, while the volume fraction of phase B decreased from 45% to 35%. Within 10-12 minutes, the volume fraction of phase A increased from 65% to 85%, while the volume fraction of phase B decreased from 35% to 15%. Over 12-15 minutes, the volume fraction of phase A increased from 85% to 90%, while the volume fraction of phase B decreased from 15% to 10%. The detection wavelengths for high performance liquid chromatography are as follows: 242 nm for pamoic acid B, dehydrotomonic acid, pamoic acid A, porphyric acid C, 3-epidemonic acid, dehydropamoic acid and pinocembrinic acid; and 210 nm for tomonic acid, 3-O-acetyl-16α-hydroxy-hydropinepoic acid and pamoic acid. The solvent for both the reference solution and the test solution is methanol.

2. The method for determining the content of 10 triterpenoid components in Poria cocos according to claim 1, characterized in that, In step S2, the preparation process employs one of the following methods: ultrasonic mixing, reflux mixing, or shaking mixing.

3. The method for determining the content of 10 triterpenoid components in Poria cocos according to claim 2, characterized in that, When the preparation is carried out by ultrasonic mixing, the ultrasonic mixing time is 15~45min, the power is 400~500W, and the frequency is 40~50kHz.

4. The method for determining the content of 10 triterpenoid components in Poria cocos according to claim 1, characterized in that, In step S2, the mass-to-volume ratio of Poria cocos to solvent in the test solution is 1g:10~15mL.

5. The method for determining the content of 10 triterpenoid components in Poria cocos according to claim 1, characterized in that, The detection conditions for the high-performance liquid chromatography method also include a flow rate of 0.4 mL / min and a column temperature of 30 °C.

6. The method for determining the content of 10 triterpenoid components in Poria cocos according to claim 1, characterized in that, The method for determining the content of 10 triterpenoid components in Poria cocos using the high performance liquid chromatography method is as follows: A1. Using the peak corresponding to dehydropamoic acid as peak S1, calculate the relative retention times of pamoic acid B, dehydropamoic acid, pamoic acid A, porphyrin C, 3-epi-dehydropamoic acid, and pinocembrinic acid, and then multiply them by correction factors to calculate the contents of pamoic acid B, dehydropamoic acid, pamoic acid A, porphyrin C, 3-epi-dehydropamoic acid, dehydropamoic acid, and pinocembrinic acid. A2. Using the corresponding peak of pamoic acid as the S2 peak, calculate the relative retention times of tomonic acid and 3-O-acetyl-16α-hydroxy-hydrogenated pamoic acid, and then multiply them by correction factors to calculate the contents of tomonic acid, 3-O-acetyl-16α-hydroxy-hydrogenated pamoic acid, and pamoic acid.

7. The method for determining the content of 10 triterpenoid components in Poria cocos according to claim 6, characterized in that, The relative retention time of pachymic acid B was 0.36, with a correction factor of 1.04; the relative retention time of dehydrotemoic acid was 0.38, with a correction factor of 1.02; the relative retention time of pachymic acid A was 0.48, with a correction factor of 1.09; the relative retention time of pachymic acid C was 0.57, with a correction factor of 0.94; the relative retention time of 3-epi-dehydrotemoic acid was 0.65, with a correction factor of 1.00; the relative retention time of dehydropachymic acid B was 0.36, with a correction factor of 1.04; the relative retention time of pachymic acid B was 0.36, with a correction factor of 1.04; the relative retention time of dehydrotemoic acid C was 0.57, with a correction factor of 0.94; the relative retention time of 3-epi-dehydrotemo The relative retention time of pamoic acid is 1.00, with a correction factor of 1.00; the relative retention time of pamoic acid is 1.15, with a correction factor of 0.93; the relative retention time of tumanoic acid is 0.39, with a correction factor of 0.98; the relative retention time of 3-O-acetyl-16α-hydroxy-hydrogenated pamoic acid is 0.90, with a correction factor of 0.82; and the relative retention time of pamoic acid is 1.00, with a correction factor of 1.00.

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