Method for constructing HPLC (High Performance Liquid Chromatography) contents of multi-index components of reference sample of traditional Chinese medicine powder

Abstract

The invention relates to a method for constructing HPLC (High Performance Liquid Chromatography) contents of multi-index components of a reference sample of traditional Chinese medicine powder, which comprises the following steps: step 1, preparation of a reference sample of Kaixin powder: taking polygala tenuifolia, ginseng, poria cocos and rhizoma acori graminei, and crushing into fine powder; 2, preparing a Kaixin powder reference sample and a reference substance detection solution; 3, detecting the Kaixin powder reference sample and the reference substance detection liquid by adopting an HPLC (High Performance Liquid Chromatography) instrument to obtain a chromatogram; and step 4, calculating the content of the effective components in the Kaixin powder reference sample according to the chromatogram. Wherein in the step 3, an HPLC (High Performance Liquid Chromatography) instrument is adopted for detection, and a chromatogram is obtained; comprising the following steps: step A, detecting the contents of polygala # imgabs0 # ketone III, 3, 6 '-dimustard acyl sucrose and beta-asarone in rhizoma acori graminei in polygala tenuifolia; step B, detecting the contents of ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1 in the ginseng; and step C, detecting the content of tenuifolin in the polygala tenuifolia.

Description

Technical Field

[0001] The invention relates to a method for detecting the content of a traditional Chinese medicine powder, and in particular to a method for constructing the HPLC content of multiple index components of a Kaixin powder reference sample. Background Art

[0002] Most of the classic prescriptions in the "Catalogue of Ancient Classic Prescriptions (First Batch)" are decoction varieties, and "Kaixin Powder" is one of only three powder varieties among the 100 prescriptions.

[0003] Kaixinsan originated from the book "Prescriptions for Emergencies" written by Sun Simiao in the Tang Dynasty. It is made of four Chinese medicines: Polygala, Ginseng, Acorus calamus, and Poria. It has the effects of nourishing qi and nourishing the heart, calming the mind and settling the mind. In the formula, ginseng greatly replenishes vital energy, calms the mind and improves intelligence; Poria strengthens the spleen and calms the mind, promotes diuresis and eliminates dampness; Acorus calamus resolves phlegm, opens the mind and awakens the mind; Polygala calms the mind and calms the mind, removes phlegm and opens the mind. It can be combined with Acorus calamus to enhance the effect of resolving phlegm and opening the mind, and can be combined with Poria to enhance the function of connecting the heart and kidney. In vivo and in vitro pharmacological studies have shown that Kaixinsan has anti-depression, anti-dementia, improves learning and memory, and resists fatigue. In modern clinical practice, it is often used to treat diseases with memory dysfunction as the main manifestation, such as Alzheimer's disease, vascular dementia, and cognitive impairment.

[0004] The invention patent application with application number "202211488997.0" provides a method for determining the HPLC characteristic spectrum of Kaixinsan reference sample and establishes the characteristic spectrum of Kaixinsan reference sample. Although this method can comprehensively and accurately evaluate the overall and intrinsic quality of Kaixinsan reference sample, it does not study the content of the reference sample, especially the content of effective active ingredients.

[0005] The invention patent application with application number "202210950855.5" provides a UPLC content determination method for Kaixin Powder. Although this method can accurately evaluate the content of Polygala tenuifolia in Kaixin Powder, The contents of ketone III, 3,6′-dienasinoylsucrose, ginsenoside Rg1, pachymic acid, and dehydropachymic acid were measured, but the main active ingredients, polygala tenuifolia saponins (Polygala tenuifolia), ginsenoside Re, ginsenoside Rb1 (Ginseng), and β-asarone (Acorus calamus) were not studied. At the same time, this method uses UPLC method, and UPLC instrument is expensive, with high production cost and maintenance cost, and is not as widely used as HPLC instrument.

[0006] The invention patent application with application number "202311148050.X" provides a method for determining the content of Kaixin Powder. Although this method can accurately evaluate the content of Polygala in Kaixin Powder, The contents of ketone III, 3,6′-dienasinoylsucrose, ginsenoside Re, ginsenoside Rb1, β-asarone and α-asarone were determined, but the main active ingredients, polygala tenuifolia saponins (Polygala tenuifolia) and ginsenoside Rg1 (Ginseng) were not studied. This method makes it difficult to comprehensively analyze the contents of polygala tenuifolia saponins and ginsenoside components.

[0007] The inventors searched the existing research data on Kaixin Powder and its raw materials and found that the prior art has not yet established a comprehensive evaluation method of the polygala in Kaixin Powder using HPLC instrument. The applicant has not found any method for detecting the contents of ketone III, 3,6′-dienasinoylsucrose, Polygala tenuifolia saponins, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, and β-asarone in other patents. Summary of the invention

[0008] Purpose of the Invention

[0009] Kaixinsan is one of the ancient classic prescriptions encouraged by the state to develop (the prescription is 13.80g of Polygala tenuifolia slices, 13.80g of Ginseng slices, 27.60g of Poria cocos slices, and 13.80g of Acorus calamus slices). Practice has shown that its effectiveness and safety are beyond doubt. For quality controllability, the key lies in how to establish quality standards based on pharmacodynamics and strictly control the quality during the preparation process. For Chinese medicine prescriptions with complex ingredients, fingerprints and multi-index component content determination are an important means to evaluate their quality. Based on this, this study developed an HPLC content detection method for multi-index components in the benchmark sample of the classic Chinese medicine prescription Kaixinsan. After methodological verification, it is simple and feasible, easy to operate, and applied to the quality evaluation of Kaixinsan benchmark samples, providing a content detection method for the quality standard research of Kaixinsan benchmark samples.

[0010] Technical Solutions

[0011] The existing methods for testing Kaixin Powder are not representative enough and cannot truly reflect the content of ingredients in traditional Chinese medicine. In order to promote the development of Kaixin Powder and ensure that enterprises can fully control the quality of finished medicines, it is urgent to establish analytical methods with high sensitivity, good specificity, accurate results and good reproducibility for the various index ingredients contained in Kaixin Powder, so as to provide a basis for the selection of raw materials for Chinese patent medicines, ensure product quality, provide a basis for the quality control and evaluation of Kaixin Powder, and provide a reference for the establishment of content determination methods for Chinese medicine formula preparations containing Polygala, Ginseng and Acorus calamus.

[0012] Polygala tenuifolia Ketone III, 3,6′-dienasinoylsucrose, Polygala tenuifolia saponins, ginsenoside Rg1 in ginseng, ginsenoside Re, ginsenoside Rb1, and β-asarone in Acorus calamus were studied as content detection items. The main reason is that (1) Polygala tenuifolia is the dried root of Polygala tenuifolia or Polygala ovalifolia of the Polygalaceae family. It is pungent, bitter, and warm in nature. It enters the heart, kidney, and lung meridians. It has the effects of calming the mind, improving intelligence, connecting the heart and kidney, removing phlegm, and reducing swelling. It is used for insomnia, dreaminess, forgetfulness, palpitations, confusion, coughing and expectoration, sores, swelling, and breast pain caused by disharmony between the heart and kidney. Polygala tenuifolia Ketone III, 3,6′-dienasinoylsucrose, and Polygala tenuifolia saponins are the main active ingredients in Polygala tenuifolia, which have strong anti-depressant, anti-senile dementia, intelligence-enhancing, and memory and learning ability-enhancing effects, which are consistent with the main functions of Kaixinsan. (2) Ginseng is the dried root and rhizome of the Araliaceae plant Panax ginseng, which is included in the 2020 edition of the Chinese Pharmacopoeia. It is sweet, slightly bitter, and slightly warm in nature. It enters the spleen, lung, heart, and kidney meridians. It has the effects of greatly replenishing vital energy, restoring the pulse and consolidating the body, replenishing the spleen and lungs, promoting fluid and nourishing blood, calming the mind and improving intelligence. It is mainly used for physical weakness and weakness, cold limbs and weak pulse, spleen deficiency and poor appetite, lung deficiency and wheezing and coughing, thirst due to fluid loss, internal heat and thirst, qi and blood deficiency, long-term illness and weakness, palpitations and insomnia, impotence and cold uterus. Ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 are the main active ingredients in the traditional Chinese medicine ginseng. (3) Acorus tatarinowii is the dried rhizome of Acorus tatarinowii, a plant of the Araceae family. It is pungent, bitter and warm in nature. It enters the heart and stomach meridians. It has the effects of opening the orifices and dispelling phlegm, refreshing the mind and improving intelligence, and removing dampness and stimulating appetite. It is used for coma, epilepsy, amnesia, insomnia, tinnitus, deafness, abdominal distension without hunger, and dysentery. β-Asarone is a major active ingredient in the traditional Chinese medicine Acorus tatarinowii and is included in the draft for the public notice of the revised draft of the national drug standard for Acorus tatarinowii.

[0013] Detailed description of the invention:

[0014] The invention discloses a method for constructing the HPLC content of multiple index components of Kaixinsan reference sample, which is based on the polygala root in the Kaixinsan formula. Ketone III, 3,6′-dienasinoylsucrose, Polygala tenuifolia saponins, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1 in ginseng, and β-asarone in Acorus calamus were taken as the detection objects, and a content detection method was provided.

[0015] To achieve the above purpose, according to the national requirements for the development and research of classic prescriptions, a reference sample was first prepared, and the reference sample was used as the test sample. The contents of ketone III, 3,6′-dienasinoylsucrose, β-asarone, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1 and polygala tenuifolia saponins were detected.

[0016] The specific technical solutions of the present invention are as follows:

[0017] Step 1, preparation of Kaixinsan reference sample:

[0018] Take Polygala, Ginseng, Poria, Acorus calamus, and crush them into fine powder;

[0019] Step 2, preparing Kaixinsan reference sample and reference substance test solution;

[0020] Step 3, using HPLC instrument to detect Kaixinsan reference sample and reference substance test solution to obtain chromatogram;

[0021] Step 4, calculate the content of the active ingredient in the Kaixinsan reference sample according to the chromatogram.

[0022] Wherein, in step 1, Polygala tenuifolia, ginseng, Poria cocos and Acorus calamus are weighed according to the prescribed amount.

[0023] Wherein, in step 2, the reference substance is Polygala tenuifolia Ketone III, 3,6′-dienasinoylsucrose, β-asarone, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, polygala tenuifolia saponin, these substances are all known substances and can be purchased from the market, for example, from the China Food and Drug Inspection Institutes.

[0024] Wherein, in step 3, the HPLC instrument is used for detection to obtain a chromatogram; comprising the following steps:

[0025] Step A: Detection of Polygala tenuifolia Ketone III, 3,6′-dicerinoylsucrose, and the content of β-asarone in Acorus calamus;

[0026] Step B, detecting the contents of ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1 in ginseng;

[0027] Step C, detecting the content of Polygala tenuifolia saponin in Polygala tenuifolia.

[0028] Wherein, the step A, detecting the polygala root in Ketone III, 3,6′-dicerinoyl sucrose, and the content of β-asarone in Acorus tatarinowii, the steps are as follows:

[0029] Preparation of reference solution: Take Polygala tenuifolia An appropriate amount of ketone III reference substance, 3,6′-dicorinoyl sucrose reference substance, and β-asarone reference substance were accurately weighed and added with 70% methanol to make a 1 ml solution containing Polygala tenuifolia. A mixed solution of 50 μg of ketone III, 150 μg of 3,6′-dicerinoyl sucrose, and 100 μg of β-asarone is obtained;

[0030] Preparation of test solution: Take 2.5g of the standard sample of Xinsan, weigh it accurately, put it in a stoppered conical flask, add 25ml of 70% methanol accurately, weigh it, heat and reflux for 30 minutes, cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the solution;

[0031] Determination method: Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0032] The chromatographic conditions are as follows: octadecylsilane bonded silica gel is used as filler ( AQ-C18 4.6×250mm, 5μm); acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution as specified in the following table; flow rate 1.0ml per minute; column temperature 30℃; detection wavelength 240nm, theoretical plate number calculated based on 3,6′-diesinapoylsucrose peak should be not less than 3000.

[0033]

[0034] Wherein, step B, detecting the content of ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 in ginseng; the steps are as follows:

[0035] Preparation of reference solution: Accurately weigh ginsenoside Rg1 reference, ginsenoside Re reference and ginsenoside Rb1 reference, add methanol to make a mixed solution containing 0.3 mg of each per 1 ml, shake well, and obtain;

[0036] Preparation of test solution: Take 5.0g of the standard sample of Xinsan, accurately weigh it, put it in a stoppered conical flask, accurately add 50ml of water-saturated n-butanol, weigh it, heat and reflux for 90 minutes, cool it, weigh it again, make up the lost weight with water-saturated n-butanol, shake it well, filter it, accurately measure 25ml of the filtrate, evaporate it to dryness in an evaporating dish, dissolve the residue in methanol and transfer it to a 5ml volumetric flask, dilute it to the mark with methanol, shake it well, filter it, and take the filtrate to obtain the solution;

[0037] Determination method: Accurately pipette 10 and 15 μl of reference solution and 10 μl of test solution respectively, inject into liquid chromatograph, and determine;

[0038] The chromatographic conditions are as follows: octadecylsilane bonded silica gel as filler (Waters Xselect HSS T34.6×250mm, 5μm); acetonitrile as mobile phase A, water as mobile phase B, gradient elution as specified in the following table; flow rate of 1.0 ml per minute; column temperature of 30°C; evaporative light scattering detector detection. The theoretical plate number calculated based on the ginsenoside Rg1 peak should be no less than 6000.

[0039]

[0040] Wherein, step C, detecting the content of Polygala tenuifolia saponin in Polygala tenuifolia, comprises the following steps:

[0041] Preparation of reference solution: Take an appropriate amount of Polygala tenuifolia saponin reference, weigh accurately, and add methanol to make a solution containing 0.5 mg per 1 ml.

[0042] Preparation of test solution: Take 2.5g of the standard sample of Xinsan, accurately weigh it, put it in a stoppered conical flask, accurately add 50ml of 70% methanol, weigh it, ultrasonically treat it (power 400W, frequency 40kHz) for 1 hour, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, accurately measure 25ml of the filtrate, put it in an evaporating dish and evaporate it to dryness, add 50ml of 10% sodium hydroxide solution to the residue and dissolve it and transfer it to a stoppered conical flask, heat and reflux for 2 hours, let it cool, adjust the pH value to 4-5 with hydrochloric acid, shake it with water-saturated n-butanol to extract it 3 times, 50ml each time, combine the n-butanol solution, recover the solvent to dryness, add an appropriate amount of methanol to dissolve the residue, transfer it to a 25ml volumetric flask, add methanol to the scale, shake it well, filter it, and obtain it;

[0043] Determination method: Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine;

[0044] The chromatographic conditions are as follows: octadecylsilane bonded silica gel is used as filler (JADE ODS-AQ C184.6×250mm, 5μm); acetonitrile: 0.05% phosphoric acid (33:67) as mobile phase; flow rate of 1.0 ml per minute; column temperature of 30°C; detection wavelength of 210nm. The theoretical plate number calculated based on the Polygala tenuifolia saponin peak should not be less than 3000.

[0045] The inventors screened and studied the content detection method of the present invention, conducted chromatographic conditions and system suitability tests, and studied the necessary linear relationship, reproducibility, and stability of the preparation of reference solution and test solution.

[0046] The method validation includes specificity, injection precision, linearity, sample stability, repeatability, intermediate precision, accuracy and durability (including investigations on different chromatographic columns, different flow rates, different column temperatures, and different wavelengths) to ensure that the detection method is complete and effective.

[0047] The specificity of the method was verified by examining whether the blank interfered with the determination of the main component.

[0048] The precision of the instrument was investigated by continuous sampling.

[0049] Investigate whether the peak area of ​​each component is linearly related to its concentration within the designed range.

[0050] Investigate the stability of the test solution over a certain period of time.

[0051] Six samples of the test solution were measured repeatedly to investigate the repeatability of the method.

[0052] In the same laboratory, different times, different analysts, and different equipment were used to examine the intermediate precision.

[0053] The accuracy of this method was investigated by adding a control substance of known concentration to the test sample.

[0054] Investigate the robustness of the experimental method under slight changes in measurement conditions.

[0055] The following is the method research process of the present invention:

[0056] 3.1 Polygala tenuifolia Study on the determination of ketone Ⅲ, 3,6′-dicansinoylsucrose and β-asarone

[0057] 3.1.1. Exclusivity

[0058] 3.1.1.1. Solution

[0059] (1) Blank solvent interference test: Take 70% methanol solution and inject it according to the proposed method.

[0060] (2) Reference article positioning test: Polygala tenuifolia The mixed reference solution of ketone III, 3,6′-dicorinoylsucrose and β-asarone was injected according to the proposed method.

[0061] (3) Test sample: Take the sample for method verification, prepare the test sample solution, and perform the injection test according to the proposed method.

[0062] 3.1.1.2. Acceptance criteria

[0063] (1) The blank solvent should have no interference at each chromatographic peak.

[0064] (2) The retention time of the chromatogram of the test sample corresponds to that of the chromatogram of the corresponding reference sample.

[0065] 3.1.1.3. Results Figure 1

[0066] The results showed that the chromatogram of the test sample had the same chromatographic peak at the corresponding retention time as the chromatogram of the reference sample, and there was no interference from the blank solution, indicating that the method had good specificity. The resolution of ketone III is 26.65 and that of 3,6′-dicerinoylsucrose is 65.06.

[0067] 3.1.2. Injection precision

[0068] 3.1.2.1. Solution

[0069] Polygala tenuifolia The mixed reference solutions of ketone III, 3,6′-dicorinoylsucrose and β-asarone were prepared according to the proposed method, and the samples were continuously injected and determined according to the proposed analytical method.

[0070] 3.1.2.2. Acceptance criteria

[0071] (1) Polygala tenuifolia The RSD of the peak areas of ketone III, 3,6′-diesinapoylsucrose and β-asarone shall not be greater than 2.0%, and the RSD of the retention time shall not be greater than 1.0%.

[0072] (2) The theoretical plate number of the 3,6′-dicerinoylsucrose peak should not be less than 3000.

[0073] Results

[0074] Table 1 Results of injection precision investigation

[0075]

[0076]

[0077] The results showed that Polygala The RSD value of ketone III retention time was 0.5%, and the RSD value of peak area was 0.8%; the RSD value of 3,6′-dicerucyl sucrose retention time was 0.2%, and the RSD value of peak area was 0.7%; the RSD value of β-asarone retention time was 0.1%, and the RSD of peak area was 0.7%. The RSD of retention time of each component was less than 1.0%, and the RSD of peak area was less than 2.0%. The number of theoretical plates of each component was not less than 3000, indicating that the instrument had good precision.

[0078] 3.1.3. Linear

[0079] 3.1.3.1. Solution

[0080] Polygala tenuifolia An appropriate amount of ketone III, 3,6′-dicorinoyl sucrose and β-asarone reference substances were accurately weighed and prepared according to 400% of the reference substance solution concentration, and dissolved in methanol solution and quantitatively diluted step by step to prepare 200%, 100%, 50%, 25%, and 10% linear test solutions. According to the proposed analytical method, 10%, 25%, 50%, 100%, 200%, and 400% linear solutions were determined respectively. The concentrations of ketone III, 3,6′-diesinapoylsucrose and β-asarone (μg / ml) were taken as the abscissa and the peak area was taken as the ordinate for linear regression calculation.

[0081] 3.1.3.2. Acceptance criteria

[0082] (1) Draw a linear graph of concentration-peak area, and there is a linear relationship.

[0083] (2) Report the linear equation, residual sum of squares, and the linear correlation coefficient (r) must not be less than 0.999.

[0084] Results

[0085] Table 2 Results of linearity test on the content of Kaixinsan reference samples

[0086]

[0087]

[0088] The results are shown in the following figure

[0089] Figure 2 Polygala tenuifolia Standard curve of ketone Ⅲ content determination

[0090] Figure 3 Standard curve for determination of 3,6′-dicerinoylsucrose content in Kaixinsan reference sample

[0091] Figure 4 Standard curve of β-asarone content determination in Kaixinsan reference sample

[0092] The experimental results show that the correlation coefficients of each component are not less than 0.999, indicating that Ketone III was in the range of 2.030-81.19 μg / ml, 3,6′-diesinapoylsucrose was in the range of 15.21-608.2 μg / ml, and β-asarone was in the range of 25.36-1014.3 μg / ml, and the linear relationships were all good.

[0093] 3.1.4. Sample stability

[0094] 3.1.4.1. Solution

[0095] Prepare the reference solution according to the proposed concentration, take the sample of the method validation batch, prepare the test solution according to the proposed method, and inject the sample at 0h, 2h, 4h, 8h, 12h, 24h, 36h, 48h, and 72h at room temperature to examine the stability of the reference and test solutions. If the stability at room temperature cannot meet the required experimental time, examine the stability of the sample under refrigeration (2-10℃). The time point can be fine-tuned according to the actual situation.

[0096] 3.1.4.2. Acceptance criteria

[0097] Polygala tenuifolia The detection of ketone III, 3,6′-dicorinoylsucrose and β-asarone peaks should remain stable. Compared with 0h, The peak area RAD of ketone III, 3,6′-diesinarosylsucrose and β-asarone shall not be greater than 2.0%.

[0098] Results

[0099] Table 3 Results of stability study on the content determination of Kaixinsan reference sample

[0100]

[0101] The results showed that within 72 hours, The RSDs of the peak areas of ketone III, 3,6′-diesinapoylsucrose, and β-asarone were all less than 2.0%, indicating that the test solution was stable within 72 h.

[0102] 3.1.5. Repeatability

[0103] 3.1.5.1. Solution

[0104] Take samples from the methodology validation batch and prepare 6 test sample solutions in parallel according to the proposed method, and then inject and measure them according to the proposed analytical method.

[0105] 3.1.5.2. Acceptance criteria

[0106] (1) The retention time of the chromatogram of the test sample corresponds to that of the chromatogram of the corresponding reference sample.

[0107] (2) Polygala in the test solution The detection of ketone III, 3,6′-dicorinoylsucrose and β-asarone should be consistent. The RSD of ketone III detection amount shall not be greater than 3.0%, and the RSD of 3,6′-dicerinoylsucrose and β-asarone content shall not be greater than 2.0%.

[0108] (3) In analyses with complex matrices, component content less than 0.01%, and multiple components, the precision limit can be appropriately relaxed, and the acceptable range can be 0.5 to 2 times the value given in the 9101 Validation of Analytical Methods Guidelines of the Chinese Pharmacopoeia (2020 Edition, Part Four Guidelines).

[0109] 3.1.5.3. Results

[0110] Table 4 Results of repeatability study on the content determination of Kaixinsan reference sample

[0111]

[0112] The experimental results showed that the RSD values ​​of the contents of each component in the six test solutions prepared from the same batch of samples were all less than 2.0%, indicating that the analytical method had good repeatability.

[0113] 3.1.6. Intermediate precision

[0114] 3.1.6.1. Solution

[0115] Six test sample solutions were prepared by different analysts at different times and measured using the same method on HPLCs of different brands. The intermediate precision of the method was evaluated using the test results of the 12 samples.

[0116] 3.1.6.2. Acceptance criteria

[0117] (1) The retention time of the chromatogram of the test sample corresponds to that of the chromatogram of the corresponding reference sample.

[0118] (2) Polygala in the test solution The detection of ketone III, 3,6′-dicorinoylsucrose and β-asarone should be consistent. The RSD of ketone III detection amount shall not be greater than 3.0%, and the RSD of 3,6′-dicorinoylsucrose and β-asarone detection amount shall not be greater than 2.0%.

[0119] (3) In analyses with complex matrices, component content less than 0.01%, and multiple components, the precision limit can be appropriately relaxed, and the acceptable range can be 0.5 to 2 times the value given in the 9101 Validation of Analytical Methods Guidelines of the Chinese Pharmacopoeia (2020 Edition, Part Four Guidelines).

[0120] 3.1.6.3. Results

[0121] Table 5 Results of intermediate precision study on the content determination of Kaixinsan reference sample

[0122]

[0123]

[0124] Note: Personnel 1 is the reproducible result, instrument: high performance liquid chromatograph, Thermo Vanquish, number: JS-01-166; experimental date: 2024.05.07; Personnel 2, instrument: high performance liquid chromatograph, Thermo Vanquish, number: JS-01-169; experimental date: 2024.05.13

[0125] The experimental results showed that different analysts used different chromatographs to measure samples on different dates, with a total of 12 samples. The RSD value of ketone III was less than 3.0%, and the RSD values ​​of 3,6′-dicorinoylsucrose and β-asarone were both less than 2.0%. The intermediate precision of this analytical method was good.

[0126] 3.1.7. Accuracy

[0127] 3.1.7.1. Solution

[0128] Take 1.25g of the sample from the method validation batch, weigh it accurately, and then add a certain amount of the reference substance with a known content of the tested component accurately, prepare it according to 100% of the measured concentration, and repeat 6 times. The recoveries of ketone III, 3,6′-diesinaroylsucrose and β-asarone were used to evaluate the accuracy of the method.

[0129] 3.1.7.2. Acceptance criteria

[0130] (1) Polygala tenuifolia The recovery rate of ketone III should be between 90% and 108%, and the RSD should not be greater than 3.0%; the recovery rates of 3,6′-dicercosylsucrose and β-asarone should be between 92% and 105%, and the RSD should not be greater than 2.0%.

[0131] (2) In the analysis of complex matrices, component content less than 0.01%, and multiple components, the recovery limit can be appropriately relaxed.

[0132] 3.1.7.3. Results

[0133] Table 6 Kaixinsan standard sample content determination (Polygala Results of sample recovery of ketone III

[0134]

[0135]

[0136] Table 7 Results of sample recovery rate investigation of Kaixinsan standard sample content determination (3,6' dierucyl sucrose)

[0137]

[0138] Table 8 Kaixinsan standard sample content determination (β-asarone) sample recovery rate investigation results

[0139]

[0140] The results showed that Polygala The recovery rates of ketone III ranged from 96.17% to 99.17%, with an average recovery rate of 97.3% and an RSD value of 1.2%; the recovery rates of 3,6′-dicansinoylsucrose ranged from 102.30% to 103.42%, with an average recovery rate of 102.8% and an RSD value of 0.4%; the recovery rates of β-asarone ranged from 92.30% to 93.17%, with an average recovery rate of 92.6% and an RSD value of 0.5%, indicating that the method has good accuracy.

[0141] Durability

[0142] 3.1.1.1. Solution

[0143] Take the samples from the methodology validation batch and prepare the test solution according to the proposed method. Inject the samples according to the chromatographic conditions after fine-tuning as shown in the table below:

[0144] Table 9 Variation degree of each parameter in durability test

[0145]

[0146] 3.1.1.2. Acceptance criteria

[0147] (1) The retention time of the chromatogram of the test sample corresponds to that of the chromatogram of the corresponding reference sample.

[0148] (2) Polygala in the test solution The detection of chromatographic peaks of ketone III, 3,6′-dicorinoylsucrose and β-asarone should be consistent. The RSD of the detection amounts of ketone III, 3,6′-dicorinoylsucrose and β-asarone shall not be greater than 2.0%.

[0149] Results

[0150] (1) Investigation of different column temperatures

[0151] Comparison of the effects of different column temperatures (25℃ and 35℃) on the polygala in Kaixinsan standard sample Take the standard sample of Kaixinsan, prepare the test solution according to the proposed method, and perform the determination according to the proposed chromatographic conditions. Set the column temperature to 25℃ and 35℃ respectively, and the other conditions are the same as the proposed chromatographic conditions. Determine the content of Polygala tenuifolia at different column temperatures. The contents of ketone III, 3,6′-diesinaroylsucrose and β-asarone.

[0152] Table 10 Results of investigation at different column temperatures

[0153]

[0154] Experimental results: Polygala tenuifolia at different column temperatures ±5℃ The RSD values ​​of ketone III and β-asarone content were within 2.0%, and the RSD value of 3,6′-dicansinoyl sucrose was 4.7%, which did not meet the standard, indicating that when the column temperature changes within the range of ±5°C, the analytical method has a greater impact on 3,6′-dicansinoyl sucrose and has poor durability. Therefore, the column temperature needs to be fixed at 30°C during detection.

[0155] (2) Investigation of different flow rates

[0156] Comparison of the effects of different flow rates of 0.9ml / min and 1.1ml / min on the performance of Polygala tenuifolia in Kaixinsan standard sample The "repeatability test" test solution was used as the durability test solution, and the flow rates were set to 0.9ml / min and 1.1ml / min respectively. The other conditions were the same as the proposed chromatographic conditions. The content of Polygala tenuifolia at different flow rates was determined. The contents of ketone III, 3,6′-diesinaroylsucrose and β-asarone.

[0157] Table 11 Investigation results of different flow rates

[0158]

[0159] Experimental results: Polygala tenuifolia at different flow rates The RSD values ​​of ketone III, 3,6′-diesinapoylsucrose, and β-asarone contents were all less than 2.0%, indicating that the analytical method had good durability within the flow rate range of 0.9 ml / min to 1.1 ml / min.

[0160] (3) Investigation at different wavelengths

[0161] Comparison of the polygala in the standard samples with different wavelengths of 235nm and 245nm The "repeatability test" test solution was used as the test solution for durability test, and the wavelengths were set to 235nm and 245nm. The other conditions were determined according to the chromatographic conditions at different wavelengths. The contents of ketone III, 3,6′-diesinaroylsucrose and β-asarone.

[0162] Table 12 Results of investigation at different wavelengths

[0163]

[0164]

[0165] Experimental results: Polygala tenuifolia at different wavelengths The RSD values ​​of ketone III, 3,6′-diesinapoylsucrose, and β-asarone contents were all less than 2.0%, indicating that the analytical method had good durability within the range of slight wavelength changes.

[0166] (4) Investigation of different chromatographic columns

[0167] Comparison of different chromatographic columns on the performance of Polygala tenuifolia in Kaixinsan standard sample Take the standard sample of Kaixinsan, prepare the test solution according to the proposed method, and perform the determination according to the proposed chromatographic conditions. Use the chromatographic column A-109 to determine the content of Polygala tenuifolia. The contents of ketone III, 3,6′-diesinaroylsucrose and β-asarone.

[0168] Column 1: Agilent 5TC-C18 4.6mm×250mm 5μm SN: 722594A-109

[0169] Table 13 Results of investigation on different chromatographic columns

[0170]

[0171] Experimental results: The RSD value of the 3,6′-dicerinoylsucrose content measured by the chromatographic column (different manufacturer) was greater than 2.0%, which did not meet the standard, so the chromatographic column was subsequently replaced and retested.

[0172] Column 2: ODS-AQ 4.6mm×250mm 5μm US2137AJUD73(A-085)

[0173] Column 3: AQ-C18 4.6mm×250mm 5μm SN: 60230502488(A-106)

[0174] Table 14 Results of investigation on different chromatographic columns

[0175]

[0176] Experimental results: When the chromatographic columns of different manufacturers were replaced, the RSD value of β-asarone content was greater than 2.0%, which did not meet the standard. The RSD values ​​of ketone III content, 3,6′-diesinapoylsucrose, and β-asarone content were all less than 2.0%. Combined with the results of different manufacturers, it is recommended that this analysis method use the chromatographic column of the same manufacturer.

[0177] (5) Investigation of different phosphoric acid concentrations

[0178] Comparison of the effect of different phosphoric acid concentrations on the performance of polygala in the standard sample Take the standard sample of Kaixinsan, prepare the test solution according to the proposed method, and determine it according to the proposed chromatographic conditions. Except for using different phosphoric acid concentration mobile phases (the phosphoric acid concentrations are 0.09% and 0.11%), the other conditions are determined according to the proposed chromatographic conditions. The contents of ketone III, 3,6′-diesinaroylsucrose and β-asarone.

[0179] Table 15 Results of investigation of different phosphoric acid concentrations

[0180]

[0181] Experimental results: Polygala tenuifolia with different phosphate concentrations The RSD values ​​of ketone III, 3,6′-diesinapoylsucrose, and β-asarone contents were all less than 2.0%, indicating that the analytical method had good durability in the range of 0.09% to 0.11% of phosphoric acid concentration.

[0182] 3.1.9. Summary of methodological validation

[0183] Polygala tenuifolia In the methodological verification test of ketone III, 3,6′-dienasinoylsucrose and β-asarone content, the blank had no interference and good specificity; The theoretical plate numbers of ketone III, β-asarone, and 3,6′-dicerinoylsucrose are not less than 3000; Ketone III had good linearity in the concentration range of 2.030-81.19 μg / ml, 3,6′-dienasoylsucrose had good linearity in the concentration range of 15.21-608.2 μg / ml, and β-asarone had good linearity in the concentration range of 25.36-1014.3 μg / ml. The injection precision, repeatability and intermediate precision were good. The test solution was stable at room temperature for 72 hours. The results showed that Polygala tenuifolia The recovery rate of ketone III ranged from 96.17% to 99.17%, with an average recovery rate of 97.3%; the recovery rate of 3,6′-dienasinoylsucrose ranged from 102.30% to 103.42%, with an average recovery rate of 102.8%; the recovery rate of β-asarone ranged from 92.30% to 93.17%, with an average recovery rate of 92.6%, indicating that the method has good accuracy; except for the poor durability of different column temperatures (±5°C) and different brands of chromatographic columns, the durability results for different flow rates (±0.1ml / min), different wavelengths (±5nm), and different phosphoric acid concentrations (±10%) are all good. After verification, this method can be accurately used for the determination of Polygala tenuifolia in Kaixinsan benchmark samples. Quantitative determination of ketone III, 3,6′-diesinaroylsucrose and β-asarone contents.

[0184] 3.2. Study on the content determination of ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1

[0185] 3.2.1. [Assay (II)] Specificity

[0186] 3.2.1.1. Solution

[0187] (1) Blank solvent interference test: Take methanol solution and inject it according to the proposed method.

[0188] (2) Reference substance positioning test: Take a mixed reference substance solution of ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 and perform sampling inspection according to the proposed method.

[0189] (3) Test sample: Take the sample for method verification, prepare the test sample solution, and perform the injection test according to the proposed method.

[0190] 3.2.1.2. Acceptance criteria

[0191] (1) The blank solvent should have no interference at each chromatographic peak.

[0192] (2) The retention time of the chromatogram of the test sample corresponds to that of the chromatogram of the corresponding reference sample.

[0193] 3.2.1.3. Results Figure 5

[0194] The results showed that the test sample chromatogram had the same chromatographic peak at the corresponding retention time as the reference sample chromatogram, and there was no interference from the blank solution, indicating that the method had good specificity.

[0195] 3.2.2. Injection precision

[0196] 3.2.2.1. Solution

[0197] Take ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1 to prepare a mixed reference solution according to the proposed method, and perform continuous sampling and determination according to the proposed analytical method.

[0198] 3.2.2.2. Acceptance criteria

[0199] (1) The RSD of the peak areas of ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 shall not be greater than 4.0%, and the RSD of the retention times shall not be greater than 1.0%.

[0200] (2) The theoretical plate number of ginsenoside Rg1 peak shall not be less than 6000.

[0201] Results

[0202] Table 16 Results of the injection precision test of Kaixinsan reference sample content determination

[0203]

[0204]

[0205] The results showed that the RSD value of the retention time of ginsenoside Rg1 was 0.5%, the RSD value of the peak area was 2.0%, the RSD value of the retention time of ginsenoside Re was 0.5%, the RSD value of the peak area was 1.8%; the RSD value of the retention time of ginsenoside Rb1 was 0.3%, and the RSD of the peak area was 1.6%. The RSD of the retention time of each component was less than 1.0%, the RSD of the peak area was less than 4.0%, and the number of theoretical plates was not less than 5000. This shows that the instrument has good precision.

[0206] 3.2.3. Linear

[0207] 3.2.3.1. Solution

[0208] Take ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1 to prepare a mixed reference solution according to the proposed method, and inject 2 μl, 5 μl, 10 μl, 15 μl and 20 μl of the linear solution respectively according to the proposed analysis method, and perform linear regression calculation with the logarithm of the absolute injection amount (μg) of ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1 as the abscissa and the logarithm of the peak area as the ordinate.

[0209] 3.2.3.2. Acceptance criteria

[0210] (1) Draw a linear graph of concentration-peak area, and there is a linear relationship.

[0211] (2) Report the linear equation, residual sum of squares, and the linear correlation coefficient (r) must not be less than 0.999.

[0212] Results

[0213] Table 17 Results of linearity test on the content of Kaixinsan reference sample

[0214]

[0215]

[0216] The results are shown in the figure below

[0217] Figure 6 Standard curve of ginsenoside Rg1 content determination in Kaixinsan reference sample

[0218] Figure 7 Standard curve of ginsenoside Re content determination in Kaixinsan reference sample

[0219] Figure 8 Standard curve of ginsenoside Rb1 content determination in Kaixinsan reference sample

[0220] The experimental results show that the linear regression equation of ginsenoside Rg1 is y=1.6596x-0.0565; the linear regression equation of ginsenoside Re is y=1.6698x-0.0718; the linear regression equation of ginsenoside Rb1 is y=1.6514x+0.061, and the correlation coefficient r of each component is not less than 0.999. This shows that the linear relationship is good in the range of 0.615-6.146μg for ginsenoside Rg1, 0.602-6.018μg for ginsenoside Re, and 0.604-6.038μg for ginsenoside Rb1.

[0221] 3.2.4. Sample stability

[0222] 3.2.4.1. Solution

[0223] Prepare the reference solution according to the proposed concentration, take the sample of the method validation batch, prepare the test solution according to the proposed method, and inject the sample at 0h, 2h, 4h, 8h, 12h, 24h, 36h, 48h, 72h, and 94h at room temperature to examine the stability of the reference and test solutions. If the stability at room temperature cannot meet the required experimental time, examine the stability of the sample under refrigeration (2-10°C). The time point can be fine-tuned according to the actual situation.

[0224] 3.2.4.2. Acceptance criteria

[0225] The detection of chromatographic peaks of ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1 in the test solution should remain stable. Compared with 0h, the peak area RAD of ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1 shall not be greater than 4.0%.

[0226] 3.2.4.3. Results

[0227] Table 18 Results of stability study on the content of Kaixinsan reference sample

[0228]

[0229]

[0230] The chromatographic peaks of the test sample were detected stably within 48 hours. The RAD of the chromatographic peaks of ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 were all less than 4.0% compared with 0h. After 48 hours, the peak area gradually decreased, so a sample was added for 142 hours under refrigerated conditions (2-10°C) from the time the sample preparation was completed. The results showed that under refrigerated conditions, the chromatographic peaks of the test sample were detected stably. The RAD of the chromatographic peaks of ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 were all less than 4.0% compared with 0h. Therefore, the chromatographic peaks of the test sample solution were stable at room temperature for 48 hours and refrigerated for 142 hours.

[0231] 3.2.5. Repeatability

[0232] 3.2.5.1. Solution

[0233] Take samples from the methodology validation batch and prepare 6 test sample solutions in parallel according to the proposed method, and then inject and measure them according to the proposed analytical method.

[0234] 3.2.5.2. Acceptance criteria

[0235] (1) The retention time of the chromatogram of the test sample corresponds to that of the chromatogram of the corresponding reference sample.

[0236] (2) The detection levels of ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1 in the test solution should be consistent, and the RSD of the detection amounts of ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1 should not be greater than 4.0%.

[0237] (3) In analyses with complex matrices, component content less than 0.01%, and multiple components, the precision limit can be appropriately relaxed, and the acceptable range can be 0.5 to 2 times the value given in the 9101 Validation of Analytical Methods Guidelines of the Chinese Pharmacopoeia (2020 Edition, Part Four Guidelines).

[0238] 3.2.5.3. Results

[0239] Table 19 Results of repeatability study on the content determination of Kaixinsan reference sample

[0240]

[0241]

[0242] The experimental results showed that the RSD values ​​of the contents of each component in the six test solutions prepared from the same batch of samples were all less than 4.0%, indicating that the analytical method had good repeatability.

[0243] 3.2.6. Intermediate precision

[0244] 3.2.6.1. Solution

[0245] Six test sample solutions were prepared by different analysts at different times and measured using the same method on HPLCs of different brands. The intermediate precision of the method was evaluated using the test results of the 12 samples.

[0246] 3.2.6.2. Acceptance criteria

[0247] (1) The retention time of the chromatogram of the test sample corresponds to that of the chromatogram of the corresponding reference sample.

[0248] (2) The detection levels of ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1 in the test solution should be consistent, and the RSD of the detection amounts of ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1 should not be greater than 4.0%.

[0249] (3) In analyses with complex matrices, component content less than 0.01%, and multiple components, the precision limit can be appropriately relaxed, and the acceptable range can be 0.5 to 2 times the value given in the 9101 Validation of Analytical Methods Guidelines of the Chinese Pharmacopoeia (2020 Edition, Part Four Guidelines).

[0250] 3.2.6.3. Results

[0251] Table 20 Results of intermediate precision study of Kaixinsan standard sample content determination

[0252]

[0253]

[0254] Note: Personnel 1 is for repeatability results, instrument: High Performance Liquid Chromatograph Waters Arc, No.: JS-01-170; Experimental date: 2024.06.05

[0255] Personnel 2, Instrument: High Performance Liquid Chromatograph Vanquish, No.: JS-01-166; Experiment Date: 2024.06.14

[0256] The experimental results show that different analysts used different chromatographs to measure samples on different dates, for a total of 12 samples, and the RSD value of each component was less than 4.0%, which met the standard, indicating that the intermediate precision of this analytical method was good.

[0257] 3.2.7. Accuracy

[0258] 3.2.7.1. Solution

[0259] About 2.5 g of the method validation sample was taken and accurately weighed, and then a certain amount of the reference substance with a known content of the measured component was accurately added, and prepared according to 100% of the measured concentration, and 6 parallels were made. According to the proposed analytical method, continuous sampling was performed, and the accuracy of the method was evaluated by the recovery rate of ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1.

[0260] 3.2.7.2. Acceptance criteria

[0261] (1) The recoveries of ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 should be between 90% and 108%, and the RSD should not be greater than 4.0%.

[0262] (2) In the analysis of complex matrices, component content less than 0.01%, and multiple components, the recovery limit can be appropriately relaxed.

[0263] 3.2.7.3. Results

[0264] Table 21 Results of sample recovery rate determination of Kaixinsan standard sample (ginsenoside Rg1)

[0265]

[0266]

[0267] Table 22 Results of sample recovery rate of Kaixinsan standard sample content determination (ginsenoside Re)

[0268]

[0269] Table 23 Results of sample recovery rate of Kaixinsan standard sample content determination (ginsenoside Rb1)

[0270]

[0271] The results showed that the recovery rate of ginsenoside Rg1 ranged from 105.1% to 107.9%, with an average recovery rate of 106.0% and an RSD value of 1.1%; the recovery rate of ginsenoside Re1 ranged from 97.5% to 106.4%, with an average recovery rate of 101.8% and an RSD value of 3.3%; the recovery rate of ginsenoside Rb1 ranged from 96.8% to 99.8%, with an average recovery rate of 98.7% and an RSD value of 1.2%. All components were within the standard range, indicating that the method had good accuracy.

[0272] 3.2.8. Durability

[0273] 3.2.8.1. Solution

[0274] Take the samples from the methodology validation batch and prepare the test solution according to the proposed method. Inject the samples according to the chromatographic conditions after fine-tuning as shown in the table below:

[0275] Table 24 Variation degree of each parameter in durability test

[0276]

[0277] 3.2.8.2. Acceptance criteria

[0278] (1) The retention time of the chromatogram of the test sample corresponds to that of the chromatogram of the corresponding reference sample.

[0279] (2) The detection of chromatographic peaks of ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 in the test solution should be consistent, and the RSD of the detection amounts of ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 should not be greater than 4.0%.

[0280] 3.2.8.3. Results

[0281] (1) Investigation of different column temperatures

[0282] Compare the effects of different column temperatures of 25℃ and 35℃ on the determination of ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1 in Kaixinsan reference samples. Take Kaixinsan reference samples, prepare the test solution according to the proposed method, and determine it according to the proposed chromatographic conditions. Set the column temperatures to 25℃ and 35℃ respectively, and the other conditions are the same as the proposed chromatographic conditions to determine the contents of ginsenoside Rg1, ginsenoside Re and ginsenoside Rg1 at different column temperatures.

[0283] Table 25 Results of investigation at different column temperatures 1

[0284]

[0285] Experimental results: When the chromatographic conditions were slightly changed, the RSD values ​​of ginsenoside Rg1 and ginsenoside Re at different column temperatures (±5°C) were less than 4.0%, and the RSD value of ginsenoside Rb1 was 4.9%, which did not meet the standard. The analysis data showed that the analytical method had a greater impact on ginsenoside Rb1 at a column temperature of 35°C, so the column temperature was reduced and the investigation was continued under the original conditions.

[0286] Table 26 Results of investigation at different column temperatures 2

[0287]

[0288] Experimental results: After reducing the column temperature, the RSD values ​​of ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 were all less than 4.0%, indicating that the analytical method has good durability within the column temperature range of 25-32°C.

[0289] (2) Investigation of different flow rates

[0290] Compare the effects of different flow rates of 0.9ml / min and 1.1ml / min on the determination of ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 in Kaixinsan reference samples. Take the "repeatability test" test solution as the durability test solution, set the flow rates to 0.9ml / min and 1.1ml / min respectively, and the other conditions follow the proposed chromatographic conditions to determine the contents of ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 at different flow rates.

[0291] Table 27 Results of investigation at different flow rates

[0292]

[0293] Experimental results: At different flow rates (±10%), the RSD values ​​of ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 were all less than 4.0%, indicating that the analytical method has good durability within the flow rate range of 0.9 ml / min to 1.1 ml / min.

[0294] (3) Investigation of different chromatographic columns

[0295] Compare the effects of different chromatographic columns on the determination of ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 in Kaixinsan reference samples. Take Kaixinsan reference samples, prepare the test solution according to the proposed method, and determine the contents of ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 under different chromatographic columns according to the proposed chromatographic conditions.

[0296] Column 1: YMC-Pack ODS-A C18 4.6mm×250mm, 5μm SN:102BB10195 A-100

[0297] Column 2: HSS T3C18 4.6mm×250mm, 5μm SN:01983329912439A-132

[0298] Table 28 Investigation results of different chromatographic columns 2

[0299]

[0300] Experimental results: When using chromatographic columns of different manufacturers and different models of the same manufacturer for durability inspection, the durability is better when using chromatographic columns of different models of the same manufacturer. Combined with the content data analysis, when using chromatographic columns of different manufacturers, the RSD value is greater than the specified range, so it is recommended that this analysis method be determined using chromatographic columns of different models of the same manufacturer.

[0301] 3.2.9. Summary of methodological validation

[0302] In the methodological verification test of the content of ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1 in Kaixinsan benchmark samples, the blank had no interference and good specificity; the theoretical plate number of ginsenoside Rg1 was not less than 6000; ginsenoside Rg1 had good linearity in the range of 0.615-6.146μg, ginsenoside Re had good linearity in the range of 0.602-6.018μg, and ginsenoside Rb1 had good linearity in the range of 0.604-6.038μg. The results showed that the ginsenoside Rg1 recovery rate ranged from 105.1% to 107.9%, with an average recovery rate of 106.0%; the ginsenoside Re recovery rate ranged from 97.5% to 106.4%, with an average recovery rate of 101.8%; the ginsenoside Rb1 recovery rate ranged from 96.8% to 99.8%, with an average recovery rate of 98.7%. Except for the poor durability of chromatographic columns of different brands, the other durability conditions were good. After verification, this method can be accurately used for the quantitative determination of ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 in Kaixinsan reference samples.

[0303] 3.3. Polygala tenuifolia saponins

[0304] 3.3.1.1. Exclusivity

[0305] 3.3.1.2. Solution

[0306] (1) Blank solvent interference test: Take methanol solution and inject it according to the proposed method.

[0307] (2) Reference substance positioning test: Take the reference substance solution of Polygala tenuifolia saponin and conduct sampling inspection according to the proposed method.

[0308] (3) Test sample: Take samples from the methodology validation batch, prepare the test sample solution, and perform sample injection inspection according to the proposed method.

[0309] 3.3.1.3. Acceptance criteria

[0310] (1) The blank solvent should have no interference at each chromatographic peak.

[0311] (2) The retention time of the chromatogram of the test sample corresponds to that of the chromatogram of the corresponding reference sample.

[0312] 3.3.1.4. Results Figure 8

[0313] Figure 8 Investigation on the specificity of content determination of Kaixinsan reference sample

[0314] The results showed that the test sample chromatogram had the same chromatographic peak at the corresponding retention time as the reference sample chromatogram, and there was no interference from the blank solution, indicating that the method had good specificity.

[0315] 3.3.1. Injection precision

[0316] 3.3.2.1. Solution

[0317] Take Polygala tenuifolia saponins and prepare the reference solution according to the proposed method, and perform continuous sampling and determination according to the proposed analytical method.

[0318] 3.3.2.2. Acceptance criteria

[0319] (1) The RSD of the peak area of ​​Polygala tenuifolia saponin should not be greater than 2.0%, and the RSD of the retention time should not be greater than 1.0%.

[0320] (2) The theoretical plate number of the Polygala tenuifolia saponin peak shall not be less than 3000.

[0321] Results

[0322] Table 29 Results of the injection precision test of Kaixinsan reference sample content determination

[0323]

[0324] The results showed that the RSD of the retention time of Polygala tenuifolia saponins was 0.7%, the RSD of the peak area was 0.5%, and the theoretical plate number was not less than 3000, indicating that the instrument had good precision.

[0325] 3.3.2. Linear

[0326] 3.3.3.1. Solution

[0327] Take an appropriate amount of Polygala tenuifolia saponin reference substance, weigh it accurately, prepare it at 400% of the concentration of the reference substance solution, dissolve it with methanol solution and quantitatively dilute it step by step to make 200%, 100%, 50%, 25%, and 10% linear test solutions. According to the proposed analytical method, measure 10%, 25%, 50%, 100%, 200%, and 400% linear solutions respectively, and perform linear regression calculation with the concentration of Polygala tenuifolia saponin (mg / ml) as the horizontal axis and the peak area as the vertical axis.

[0328] 3.3.3.2. Acceptance criteria

[0329] (1) Draw a linear graph of concentration-peak area, and there is a linear relationship.

[0330] (2) Report the linear equation, residual sum of squares, and the linear correlation coefficient (r) must not be less than 0.999.

[0331] Results

[0332] Table 30 Results of linearity test on the content of Kaixinsan reference sample

[0333]

[0334] Results Fig. 9 Standard curve for determination of saponin content in Polygala tenuifolia in Kaixinsan reference sample

[0335] The experimental results show that within the range of 0.05005-2.002 mg / ml, the linear equation of Polygala tenuifolia saponin is: y=57.8886x+0.3380, the correlation coefficient r=0.9999, and the linear relationship is good.

[0336] Sample stability

[0337] 3.3.4.1. Solution

[0338] Prepare the reference solution according to the proposed concentration, take the sample of the method validation batch, prepare the test solution according to the proposed method, and inject the sample at 0h, 2h, 4h, 8h, 12h, 24h, 36h, 48h, and 72h at room temperature to examine the stability of the test solution. If the stability at room temperature cannot meet the required experimental time, examine the stability of the sample under refrigeration (2-10℃). The time point can be fine-tuned according to the actual situation.

[0339] 3.3.4.2. Acceptance criteria

[0340] The detection of the Polygala tenuifolia saponin peak in the test solution should remain stable. Compared with 0h, the Polygala tenuifolia saponin peak area RAD should not be greater than 2.0%.

[0341] Results

[0342] Table 31 Kaixinsan reference sample content determination sample stability investigation results

[0343]

[0344] The results showed that the RSD of the peak area of ​​Polygala tenuifolia saponins was less than 2.0% within 72 hours, indicating that the test solution was stable within 72 hours.

[0345] 3.3.4. Repeatability

[0346] 3.3.5.1. Solution

[0347] Take samples from the methodology validation batch and prepare 6 test sample solutions in parallel according to the proposed method, and then inject and measure them according to the proposed analytical method.

[0348] 3.3.5.2. Acceptance criteria

[0349] (1) The retention time of the chromatogram of the test sample corresponds to that of the chromatogram of the corresponding reference sample.

[0350] (2) The detection of Polygala tenuifolia saponins in the test solution should be consistent, and the RSD of the detection amount should not be greater than 2.0%.

[0351] (3) In analyses with complex matrices, component content less than 0.01%, and multiple components, the precision limit can be appropriately relaxed, and the acceptable range can be 0.5 to 2 times the value given in the 9101 Validation of Analytical Methods Guidelines of the Chinese Pharmacopoeia (2020 Edition, Part Four Guidelines).

[0352] 3.3.5.3. Results

[0353] Table 32 Results of repeatability study on the content determination of Kaixinsan reference sample

[0354]

[0355]

[0356] The experimental results showed that the average saponin content of Polygala tenuifolia in 6 test solutions prepared from the same batch of samples was 7.2944 mg / g, and the RSD values ​​of the 6 repeatable samples were less than 2.0%, indicating that the analytical method had good repeatability.

[0357] 3.3.5. Intermediate precision

[0358] 3.3.6.1. Solution

[0359] Six test sample solutions were prepared by different analysts at different times and measured using the same method on HPLCs of different brands. The intermediate precision of the method was evaluated using the test results of the 12 samples.

[0360] 3.3.6.2. Acceptance criteria

[0361] (1) The retention time of the chromatogram of the test sample corresponds to that of the chromatogram of the corresponding reference sample.

[0362] (2) The detection of Polygala tenuifolia saponins in the test solution should be consistent, and the RSD of the detection amount should not be greater than 3.0%.

[0363] (3) In analyses with complex matrices, component content less than 0.01%, and multiple components, the precision limit can be appropriately relaxed, and the acceptable range can be 0.5 to 2 times the value given in the 9101 Validation of Analytical Methods Guidelines of the Chinese Pharmacopoeia (2020 Edition, Part Four Guidelines).

[0364] 3.3.6.3. Results

[0365] Table 33 Results of intermediate precision study of Kaixinsan standard sample content determination

[0366]

[0367]

[0368] Personnel 1 is for repeatability results, instrument: High Performance Liquid Chromatography, Thermo Vanquish, No.: JS-01-166; Experimental date: 2024.05.24

[0369] Personnel 2, Instrument: High Performance Liquid Chromatograph, Thermo Vanquish, No.: JS-01-165; Experiment Date: 2024.05.28

[0370] The experimental results showed that different analysts used different chromatographs to measure samples on different dates, for a total of 12 samples, the RSD of Polygala tenuifolia saponins was less than 3.0%, and the intermediate precision of this analytical method was good.

[0371] 3.3.6. Accuracy

[0372] 3.3.7.1. Solution

[0373] Take 1.25g of the method validation batch sample, weigh it accurately, and then add a certain amount of the reference substance with a known content of the measured component accurately, prepare 6 copies according to 100% of the measured concentration. Continuously inject samples according to the proposed analytical method, and evaluate the accuracy of the method by the recovery rate of Polygala tenuifolia saponins.

[0374] 3.3.7.2. Acceptance criteria

[0375] (1) The recovery rate of Polygala tenuifolia saponins should be between 92% and 105%, and the RSD should not be greater than 4.0%.

[0376] (2) In the analysis of complex matrices, component content less than 0.01%, and multiple components, the recovery limit can be appropriately relaxed.

[0377] 3.3.7.3. Results

[0378] Table 34

[0379]

[0380] Table 6 Results of sample recovery rate of Kaixinsan standard sample content determination (polygala tenuifolia saponins)

[0381] The results showed that the average recovery rate of Polygala tenuifolia saponins was 100.1% and the RSD was 3.7%, indicating that the method had good accuracy.

[0382] 3.3.7. Durability

[0383] 3.3.8.1. Solution

[0384] Take the samples from the methodology validation batch and prepare the test solution according to the proposed method. Inject the samples according to the chromatographic conditions after fine-tuning as shown in the table below:

[0385] Table 35 Variation of each parameter in durability test

[0386]

[0387] 3.3.8.2. Acceptance criteria

[0388] (1) The retention time of the chromatogram of the test sample corresponds to that of the chromatogram of the corresponding reference sample.

[0389] (2) The detection of Polygala tenuifolia saponins in the test solution should be consistent, and the RSD of the detection amount should not be greater than 2.0%.

[0390] 3.3.8.3. Results

[0391] (1) Investigation of different column temperatures

[0392] Compare the effects of different column temperatures of 25℃ and 35℃ on the determination of Polygala tenuifolia saponin content in Kaixinsan reference samples. Take Kaixinsan reference samples, prepare the test solution according to the proposed method, and determine it according to the proposed chromatographic conditions. Set the column temperatures to 25℃ and 35℃ respectively, and the other conditions follow the proposed chromatographic conditions to determine the content of Polygala tenuifolia saponin at different column temperatures.

[0393] Table 36 Results of investigation at different column temperatures

[0394]

[0395]

[0396] Experimental results: The RSD values ​​of the saponin content of Polygala tenuifolia at different column temperatures (±5°C) were within 2.0%, which was in line with the standard, indicating that the analytical method had good durability within the column temperature range of ±5°C.

[0397] (2) Investigation of different flow rates

[0398] Compare the effects of different flow rates of 0.9ml / min and 1.1ml / min on the determination of the content of Polygala tenuifolia saponins in Kaixinsan benchmark samples. Set the flow rates to 0.9ml / min and 1.1ml / min respectively, and follow the other conditions of the proposed chromatographic conditions to determine the content of Polygala tenuifolia saponins at different flow rates.

[0399] Table 37 Results of investigation at different flow rates

[0400]

[0401] Experimental results: At different flow rates, the RSD values ​​of the saponin content of Polygala tenuifolia were all less than 2.0%, indicating that the analytical method had good durability within the range of 0.9 ml / min to 1.1 ml / min.

[0402] (3) Investigation at different wavelengths

[0403] Compare the effects of different wavelengths of 205nm and 215nm on the determination of Polygala tenuifolia saponin content in Kaixinsan reference samples. Set the wavelengths to 205nm and 215nm, and follow the proposed chromatographic conditions for the other conditions to determine the content of Polygala tenuifolia saponin at different wavelengths.

[0404] Table 38 Investigation results of different wavelengths

[0405]

[0406] Experimental results: The RSD values ​​of saponin content in Polygala tenuifolia at different wavelengths were all less than 2.0%, indicating that the analytical method had good durability within the wavelength range of 205nm to 215nm.

[0407] (4) Investigation of different chromatographic columns

[0408] Compare the effects of different chromatographic columns on the determination of Polygala tenuifolia saponin content in Kaixinsan reference samples. Take the "repetitive solution" sample and determine it according to the proposed chromatographic conditions. Use chromatographic columns A-109 and A-085 to determine the content of Polygala tenuifolia saponin.

[0409] Table 39 Results of investigation on different chromatographic columns

[0410]

[0411] Experimental results: The RSD values ​​of the saponin content of Polygala tenuifolia using different chromatographic columns were all less than 2.0%, indicating that the analytical method has good durability when using chromatographic columns of the same model from different manufacturers or chromatographic columns of different models from the same manufacturer.

[0412] (5) Investigation of different phosphoric acid concentrations

[0413] The effects of different phosphate concentration mobile phases on the determination of Polygala tenuifolia saponin content in Kaixinsan reference samples were compared. Kaixinsan reference samples were taken, and the test solution was prepared according to the proposed method. The determination was carried out according to the proposed chromatographic conditions, except that different phosphate concentration mobile phases (phosphate concentrations were 0.045% and 0.055%, respectively) were used. The rest of the conditions were the same as the proposed chromatographic conditions to determine the content of Polygala tenuifolia saponin.

[0414] Table 40 Results of investigation of different phosphoric acid concentrations

[0415]

[0416] Experimental results: The RSD values ​​of saponin content in Polygala tenuifolia at different phosphate concentrations were all less than 2.0%, indicating that the analytical method had good durability within the range of phosphate concentration of 0.045% to 0.055%.

[0417] 3.3.8. Summary of methodological validation

[0418] In the methodological verification test of the content of Polygala tenuifolia saponin in the Kaixinsan reference sample, the blank had no interference and good specificity; the theoretical plate number of Polygala tenuifolia saponin was not less than 3000; the linearity of Polygala tenuifolia saponin was good in the concentration range of 0.05005-2.002mg / ml; the injection precision, repeatability and intermediate precision were good; the test solution was stable at room temperature for 72 hours; the recovery rate of Polygala tenuifolia saponin was between 94.2% and 103.6%, and the average recovery rate was 100.1%; the durability results for different column temperatures (±5℃), different flow rates (±0.1ml / min), different wavelengths (±5nm), different phosphoric acid concentrations (±10%) and different chromatographic columns were good. After verification, this method can be accurately used for the quantitative determination of the content of Polygala tenuifolia saponin in Kaixinsan reference samples.

[0419] The inventors have further studied the relevant literature and improved the methods in these literatures. The main improvements are as follows:

[0420] Prior art 1, CN202311148050.X A method for detecting the content of ingredients in Kaixinsan

[0421] Existing technology defects:

[0422] 1. The content of the main active ingredient, Polygala tenuifolia saponin (Polygala), was not controlled. Polygala tenuifolia saponin is the active ingredient that is focused on controlling under the Polygala item in Part I of the Pharmacopoeia. This method makes it difficult to comprehensively analyze the content of Polygala tenuifolia saponin components.

[0423] 2. The content of the main active ingredient, ginsenoside Rg1 (ginseng), is not controlled. Ginsenoside Rg1 is the active ingredient that is focused on controlling under the ginseng item in Part I of the Pharmacopoeia (the number of theoretical plates is calculated based on ginsenoside Rg1). This method makes it difficult to comprehensively analyze the content of ginsenoside components.

[0424] 3 The HPLC method is a dual-wavelength detection method, which requires analyzing the contents of six components at two wavelengths of 203nm and 245nm. The analysis method is not simple enough.

[0425] Improvements of this patent:

[0426] 1. Control the content of Polygala tenuifolia (the main active ingredient is Polygala tenuifolia saponin) in the prescription.

[0427] 2. Control the content of ginseng (main active ingredients ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1) in the prescription. 3. Use HPLC method, which is a single wavelength detection method and the analysis method is relatively simple.

[0428] Prior art 2, CN202210950855.5 A method for detecting the content of multiple index components in Kaixin powder

[0429] Existing technology defects:

[0430] 1. The content of the main active ingredient, Polygala tenuifolia saponin (Polygala), was not controlled. Polygala tenuifolia saponin is the active ingredient that is focused on controlling under the Polygala item in Part I of the Pharmacopoeia. This method makes it difficult to comprehensively analyze the content of Polygala tenuifolia saponin components.

[0431] 2. The content of the main active ingredients, ginsenoside Re and ginsenoside Rb1 (ginseng), was not controlled. Ginsenoside Re and ginsenoside Rb1 are the active ingredients that are controlled under the ginseng item in the Pharmacopoeia. This method makes it difficult to fully analyze the content of ginsenoside ingredients. 3. The content of the main active ingredient, β-asarone (Acorus tatarinowii), was not controlled. β-asarone is the main active ingredient of Acorus tatarinowii and is included in the public notice of the revised draft of the national drug standard for Acorus tatarinowii.

[0432] 4. Using the UPLC method, UPLC instruments are expensive, both in production cost and maintenance cost. The price of UPLC is twice that of HPLC, and is not suitable for widespread application.

[0433] 5 This detection method is a four-wavelength detection, which requires analyzing the contents of five components at four wavelengths of 203nm, 210nm, 242nm, and 320nm. The analysis method is not simple enough.

[0434] Advantages of this patent:

[0435] 1. Control the content of Polygala tenuifolia (the main active ingredient is Polygala tenuifolia saponin) in the prescription.

[0436] 2. Control the content of ginseng (main active ingredients ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1) in the prescription. 3. Control the content of the main active ingredient β-asarone (Acorus calamus).

[0437] 4 Using HPLC method, HPLC instruments are widely used.

[0438] 4 The HPLC method is used, which is a single wavelength detection method and the analysis method is relatively simple.

[0439] Prior art 3, CN202211488997.0 A method for constructing a HPLC characteristic spectrum of a Kaixinsan reference sample

[0440] Existing technology defects:

[0441] 1. The characteristic spectrum is a qualitative and semi-quantitative method. 2. Although this method can comprehensively and accurately evaluate the overall and intrinsic quality of Kai Xin San San reference samples, no research on the detection method of the reference samples, especially the content of effective active ingredients, has been conducted.

[0442] Advantages of this patent: The contents of ketone III, 3,6′-dienasinoylsucrose, Polygala tenuifolia saponins), ginseng (ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1), and Acorus tatarinowii (β-asarone) were all controlled.

[0443] Prior art 4, CN202111525386.4 A quality marker group of Kaixinsan

[0444] Deficiencies of the prior art: No quantitative method for its components is involved.

[0445] Advantages of this patent: The contents of ketone III, 3,6′-dienasinoylsucrose, Polygala tenuifolia saponins), ginseng (ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1), and Acorus tatarinowii (β-asarone) were all controlled.

[0446] In summary, compared with the prior art, the present invention aims to provide a quality control method for Kaixinsan that is more accurate and strictly follows the philosophy and theory of Chinese medicine for Chinese medicine compound preparations. Specifically, the present invention achieves high-precision determination and analysis of the content, proportion and pharmacodynamic characteristics of each component of Kaixinsan through innovative technical means, thereby ensuring that the quality control level of the compound preparation in preparation and clinical application is significantly improved.

[0447] Furthermore, the present invention not only meets the actual needs of modern production of traditional Chinese medicine, but also effectively reduces the cost of quality control. Through a more accurate quality control method, multi-component quantitative analysis of the content, proportion and efficacy characteristics of each component of the prescription is carried out, which greatly improves the detection efficiency and accuracy, and through repeated scientific verification, ensures the reliability and repeatability of the test results.

[0448] The prior art cannot achieve the object of the present invention, namely, to treat Polygala tenuifolia (Polygala Ketone III, 3,6′-dienasinoylsucrose, Polygala tenuifolia saponins), ginseng (ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1), and Acorus calamus (β-asarone) are all fully tested, and the present invention has made significant improvements in chromatographic conditions, which is completely different from the existing technology, so as to complete the detection of the three, and the precision, accuracy, and sensitivity are greatly improved. The details are as follows:

[0449] Prior art 1. Application No. CN202311148050.X A method for detecting the content of ingredients in Kaixinsan

[0450] Advantages and disadvantages of this patent application and prior art

[0451] Existing technology defects:

[0452] 1. The content of the main active ingredient, Polygala tenuifolia saponin (Polygala), was not controlled. Polygala tenuifolia saponin is the active ingredient that is focused on controlling under the Polygala item in Part I of the Pharmacopoeia. This method makes it difficult to comprehensively analyze the content of Polygala tenuifolia saponin components.

[0453] 2. The content of the main active ingredient, ginsenoside Rg1 (ginseng), is not controlled. Ginsenoside Rg1 is the active ingredient that is focused on controlling under the ginseng item in Part I of the Pharmacopoeia (the number of theoretical plates is calculated based on ginsenoside Rg1). This method makes it difficult to comprehensively analyze the content of ginsenoside components.

[0454] 3 The HPLC method is a dual-wavelength detection method, which requires analyzing the contents of six components at two wavelengths of 203nm and 245nm. The analysis method is not simple enough.

[0455] Improvements of this patent:

[0456] 1. Control the content of Polygala tenuifolia (the main active ingredient is Polygala tenuifolia saponin) in the prescription.

[0457] 2. The content of ginseng (main active ingredients ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1) in the prescription is controlled. 3. HPLC method is used, which is a single wavelength detection method and the analysis method is relatively simple.

[0458] Chromatographic conditions 1 are as follows:

[0459] (1) Chromatographic conditions and systematic tests: Octadecylsilane bonded silica gel was used as filler ( AQ-C184.6×250mm, 5μm); acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution as specified in the following table; flow rate 1.0ml per minute; column temperature 30℃; detection wavelength 240nm. The number of theoretical plates calculated based on the 3,6′-diesinapoylsucrose peak should not be less than 3000. Gradient program: 0~30min, 86%→84%B; 30~31min, 84%→78%B; 31~45min, 78~77%B; 45~50min, 77%→64%B; 50~51min, 64%→57%B; 69~90min, 17%B.

[0460] (2) Preparation of reference solution: Take Polygala tenuifolia An appropriate amount of ketone III reference substance, 3,6′-dicorinoyl sucrose reference substance, and β-asarone reference substance were accurately weighed and added with 70% methanol to make a 1 ml solution containing Polygala tenuifolia. A mixed solution of 50 μg of ketone III, 150 μg of 3,6′-dicorinoylsucrose and 100 μg of β-asarone is obtained.

[0461] (3) Preparation of test solution: Take about 2.5 g of the product, weigh it accurately, place it in a stoppered conical flask, add 25 ml of 70% methanol accurately, weigh it, heat and reflux for 30 minutes, cool, weigh it again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate.

[0462] (4) Determination: Accurately pipette 10 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, and determine. Chromatographic conditions 2 are as follows:

[0463] (1) Chromatographic conditions and systematic tests: Octadecylsilane bonded silica gel was used as the filler (Waters XselectHSS T34.6×250mm, 5μm); acetonitrile was used as the mobile phase A and water was used as the mobile phase B, and gradient elution was performed as specified in the following table; the flow rate was 1.0 ml per minute; the column temperature was 30°C; and the evaporative light scattering detector was used for detection. The number of theoretical plates calculated based on the ginsenoside Rg1 peak should not be less than 6000. Gradient program: 0-10 min, 80% B; 10-38 min, 80%→79% B; 38-43 min, 79-70% B; 43-71 min, 70% B; 71-72 min, 70%→65% B; 72-90 min, 65% B.

[0464] (2) Preparation of reference solution: Accurately weigh ginsenoside Rg1 reference, ginsenoside Re reference and ginsenoside Rb1 reference, add methanol to make a mixed solution containing 0.3 mg of each per 1 ml, shake well, and obtain.

[0465] (3) Preparation of test solution: Take about 5.0 g of the product, accurately weigh it, place it in a stoppered conical flask, accurately add 50 ml of water-saturated n-butanol, weigh it, heat and reflux for 90 minutes, cool, weigh it again, make up the lost weight with water-saturated n-butanol, shake well, filter, accurately measure 25 ml of the filtrate, place it in an evaporating dish and evaporate to dryness, dissolve the residue in methanol and transfer it to a 5 ml volumetric flask, add methanol to dilute to the scale, shake well, filter, and take the filtrate.

[0466] (4) Determination: Accurately pipette 10 and 15 μl of the reference solution and 10 μl of the test solution respectively, inject into the liquid chromatograph, and determine.

[0467] Chromatographic conditions 3 are as follows:

[0468] (1) Chromatographic conditions and systematic tests: Octadecylsilane bonded silica gel was used as filler (JADE ODS-AQ C184.6×250mm, 5μm); acetonitrile: 0.05% phosphoric acid (33:67) as mobile phase; flow rate of 1.0 ml per minute; column temperature of 30°C; detection wavelength of 210nm. The theoretical plate number calculated based on the Polygala tenuifolia saponin peak should not be less than 3000.

[0469] (2) Preparation of reference solution: Take an appropriate amount of Polygala tenuifolia saponin reference standard, accurately weigh it, and add methanol to make a solution containing 0.5 mg per 1 ml.

[0470] (3) Preparation of test solution: Take about 2.5 g of the test sample, accurately weigh it, place it in a stoppered conical flask, accurately add 50 ml of 70% methanol, weigh it, ultrasonicate it (power 400 W, frequency 40 kHz) for 1 hour, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, accurately measure 25 ml of the filtrate, place it in an evaporating dish and evaporate it to dryness, dissolve the residue in 50 ml of 10% sodium hydroxide solution and transfer it to a stoppered conical flask, heat and reflux for 2 hours, let it cool, adjust the pH value to 4-5 with hydrochloric acid, extract it three times with saturated n-butanol with water, shake it each time, recover the solvent to dryness, add an appropriate amount of methanol to dissolve the residue, transfer it to a 25 ml volumetric flask, add methanol to the mark, shake it well, filter it, and obtain the product.

[0471] (4) Determination: Accurately pipette 10 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, and determine.

[0472] The results of this patent application

[0473] Chromatographic conditions 1:

[0474] 1Blank has no interference and good specificity.

[0475] The theoretical plate number of 3,6′-dicerucyl sucrose is not less than 3,000.

[0476] 2 Polygala Ketone III showed good linearity in the concentration range of 2.030-81.19 μg / ml, with R 2 of 1.0000 and correlation coefficient r=1.0000.

[0477] 3,6′-bisinarosyl sucrose has good linearity in the concentration range of 15.21-608.2 μg / ml, R 2 is 1.0000, and the correlation coefficient is r = 1.0000. β-asarone has good linearity in the concentration range of 25.36-1014.3 μg / ml, R 2 is 0.9999, and the correlation coefficient is r = 0.9999.

[0478] 3. Injection precision RSD≤1.0%, repeatability (Polygala The RSD of ketone III content was ≤1.5%, the RSD of 3,6′-bisinacoylsucrose and β-asarone content was ≤0.5%), and the intermediate precision was good (Polygala tenuifolia The RSD of the content of ketone III was ≤2.1%, and the RSD of the content of 3,6′-dicorinoylsucrose and β-asarone was ≤2.0%).

[0479] 4 The test solution is stable at room temperature for 72 hours, and the peak area and 0h RAD ≤ 1.5%.

[0480] 5 Polygala The recovery rate of ketone III is between 96.17% and 99.17%, RSD≤1.5%. The recovery rate of 3,6' dierucyl sucrose is between 102.30% and 103.42%, RSD≤0.5%. The recovery rate of β-asarone is between 92.30% and 93.17%, RSD≤0.5%. The accuracy is high.

[0481] 6 Durability: RSD of different flow rates (±0.1ml / min) ≤ 2.0%, RSD of different wavelengths (±5nm) ≤ 2.0%, RSD of different phosphoric acid concentrations (±10%) ≤ 2.0%, RSD of different column temperatures (±5℃) ≤ 4.7% and RAD of different chromatographic columns ≤ 2.0%. The durability results are all good.

[0482] Chromatographic conditions 2:

[0483] 1Blank has no interference and good specificity.

[0484] The theoretical plate number of ginsenoside Rg1 peak shall not be less than 6000.

[0485] 2 Ginsenoside Rg1 has good linearity in the range of 0.615-6.146 μg, R2 is 0.9995, and the correlation coefficient is r = 0.9997. Ginsenoside Re has good linearity in the range of 0.602-6.018 μg, R2 is 0.9990, and the correlation coefficient is r = 0.9995. Ginsenoside Rb1 has good linearity in the range of 0.604-6.038 μg, R2 is 0.9995, and the correlation coefficient is r = 0.9997.

[0486] 3. The injection precision RSD≤2.0%, repeatability (RSD of ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 content≤1.5%), and intermediate precision were good (RSD of ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 content≤4.0%).

[0487] 4 The test solution is stable at room temperature for 48 hours, and the peak area and 0h RAD ≤ 3.0%.

[0488] 5. The recovery rate of ginsenoside Rg1 is between 105.1% and 107.9%, with RSD ≤ 1.5%. The recovery rate of ginsenoside Re is between 97.5% and 106.4%, with RSD ≤ 3.3%. The recovery rate of ginsenoside Rb1 is between 96.8% and 99.8%, with RSD ≤ 1.5%. The accuracy is relatively high.

[0489] 6 Durability: RSD of different flow rates (±0.1ml / min) ≤4.0%, RSD of different column temperatures (25-32°C) ≤3.7% and RAD of different chromatographic columns ≤4.0%. The durability results are all good.

[0490] Chromatographic conditions 3:

[0491] 1Blank has no interference and good specificity.

[0492] The theoretical plate number of Polygala tenuifolia saponin is not less than 3000.

[0493] 2 The linearity of Polygala tenuifolia saponins was good in the concentration range of 0.05005-2.002 mg / ml, with R 2 of 0.9999 and correlation coefficient r=0.9999.

[0494] 3. The injection precision RSD≤1.0%, repeatability (RSD of saponin content in Polygala tenuifolia ≤1.0%) and intermediate precision (RSD of saponin content in Polygala tenuifolia ≤2.2%) were good.

[0495] 4 The test solution is stable at room temperature for 72 hours, and the peak area and 0h RAD ≤ 1.0%.

[0496] 5 The recovery rate of Polygala tenuifolia saponins was between 94.2% and 103.6%, with RSD ≤ 3.7%. The accuracy was good.

[0497] 6 Durability: RSD of different flow rates (±0.1ml / min) ≤2.0%, RSD of different wavelengths (±5nm) ≤1.0%, RSD of different phosphoric acid concentrations (±10%) ≤1.0%, RSD of different column temperatures (±5℃) ≤1.5% and RAD of different chromatographic columns ≤2.0%. The durability results are all good. BRIEF DESCRIPTION OF THE DRAWINGS

[0498] Figure 1 Polygala Investigation of Ketone III Specificity

[0499] Figure 2 Polygala tenuifolia Standard curve of ketone Ⅲ content determination

[0500] Figure 3 Standard curve for determination of 3,6′-dicerinoylsucrose content in Kaixinsan reference sample

[0501] Figure 4 Standard curve of β-asarone content determination in Kaixinsan reference sample

[0502] Figure 5 Investigation on the specificity of ginsenoside content determination in Kaixinsan reference sample

[0503] Figure 6 Standard curve of ginsenoside Rg1 content determination in Kaixinsan reference sample

[0504] Figure 7 Standard curve of ginsenoside Re content determination in Kaixinsan reference sample

[0505] Figure 8 Standard curve of ginsenoside Rb1 content determination in Kaixinsan reference sample

[0506] Fig. 9 Investigation on the specificity of content determination of Kaixinsan reference sample

[0507] Fig.10 Standard curve for determination of saponin content in Polygala tenuifolia in Kaixinsan reference sample

[0508] Fig.11 HPLC spectrum of the reference solution

[0509] (Peak 1: Polygala Ketone III; Peak 2: 3,6′ dierucyl sucrose; Peak 3: β-asarone)

[0510] Fig.12HPLC spectrum of the test solution

[0511] (Peak 1: Polygala Ketone III; Peak 2: 3,6′ dierucyl sucrose; Peak 3: β-asarone)

[0512] Fig.13 HPLC spectrum of the reference solution

[0513] (Peak 1: Ginsenoside Rg1; Peak 2: Ginsenoside Re; Peak 3: Ginsenoside Rb1)

[0514] Fig.14 HPLC spectrum of the test solution

[0515] (Peak 1: Ginsenoside Rg1; Peak 2: Ginsenoside Re; Peak 3: Ginsenoside Rb1) Fig.15 HPLC spectrum of the reference solution (polygala tenuifolia saponin)

[0516] Fig.16 This is the HPLC spectrum of the test solution (polygala tenuifolia saponins) DETAILED DESCRIPTION

[0517] The present invention is further illustrated by the following examples, but is not intended to be limiting of the present invention.

[0518] Example 1

[0519] The test data of three batches of products in the production process using the method of the present invention are as follows:

[0520]

Claims

1. A method for determining the content of active ingredients in a reference sample of Kaixinsan, the method comprising the following steps: Step 1, preparation of Kaixinsan reference sample: taking Polygala, Ginseng, Poria, and Acorus calamus, and crushing them into fine powder; Step 2, preparing Kaixinsan reference sample and reference substance test solution; Step 3, using HPLC instrument to detect Kaixinsan reference sample and reference substance test solution to obtain chromatogram; Step 4, calculate the content of the active ingredient in the Kaixinsan reference sample according to the chromatogram.

2. The method according to claim 1, wherein: In step 1, Polygala tenuifolia, ginseng, Poria cocos, and Acorus calamus are weighed according to the prescribed amount.

3. The method according to claim 1, wherein: In step 2, the reference substance is Polygala tenuifolia Ketone III, 3,6′-dienasinoylsucrose, β-asarone, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, and polygala tenuifolia saponins.

4. The method according to claim 1, wherein: In step 3, the HPLC instrument is used for detection to obtain a chromatogram; the steps include: step A, detecting the Step B, detecting the content of ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1 in ginseng; Step C, detecting the content of polygala tenuifolia saponin in Polygala tenuifolia.

5. The method according to claim 1, wherein: Step A: detecting Polygala tenuifolia in Polygala tenuifolia Ketone III, 3,6′-dicerinoyl sucrose, and the content of β-asarone in Acorus tatarinowii, the steps are as follows: Preparation of reference solution: Take Polygala tenuifolia An appropriate amount of ketone III reference substance, 3,6′-dicorinoyl sucrose reference substance, and β-asarone reference substance were accurately weighed and added with 70% methanol to make a 1 ml solution containing Polygala tenuifolia. A mixed solution of 50 μg of ketone III, 150 μg of 3,6′-diesinapoylsucrose, and 100 μg of β-asarone is obtained; Preparation of test solution: Take 2.5g of the standard sample of Xinsan, weigh it accurately, put it in a stoppered conical flask, add 25ml of 70% methanol accurately, weigh it, heat and reflux for 30 minutes, cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the solution; Determination method: Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine. The chromatographic conditions are as follows: octadecylsilane bonded silica gel is used as filler ( AQ-C18 4.6×250mm, 5μm); acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution as specified in the following table; flow rate 1.0ml per minute; column temperature 30℃; detection wavelength 240nm, theoretical plate number calculated based on 3,6′-dicerinoylsucrose peak should be not less than 3000; 6. The method according to claim 1, wherein: Step B, detecting the content of ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 in ginseng; the steps are as follows: Preparation of reference solution: Accurately weigh ginsenoside Rg1 reference, ginsenoside Re reference and ginsenoside Rb1 reference, add methanol to make a mixed solution containing 0.3 mg of each per 1 ml, shake well, and obtain; Preparation of test solution: Take 5.0g of the standard sample of Xinsan, accurately weigh it, put it in a stoppered conical flask, accurately add 50ml of water-saturated n-butanol, weigh it, heat and reflux for 90 minutes, cool it, weigh it again, make up the lost weight with water-saturated n-butanol, shake it well, filter it, accurately measure 25ml of the filtrate, evaporate it to dryness in an evaporating dish, dissolve the residue in methanol and transfer it to a 5ml volumetric flask, dilute it to the mark with methanol, shake it well, filter it, and take the filtrate to obtain the solution; Determination method: Accurately pipette 10 and 15 μl of reference solution and 10 μl of test solution respectively, inject into liquid chromatograph, and determine; The chromatographic conditions are as follows: octadecylsilane bonded silica gel as filler (Waters Xselect HSS T3 4.6×250mm, 5μm); acetonitrile as mobile phase A, water as mobile phase B, gradient elution as specified in the following table; flow rate of 1.0 ml per minute; column temperature of 30°C; evaporative light scattering detector detection, the theoretical plate number calculated based on the ginsenoside Rg1 peak should be no less than 6000, 7. The method according to claim 1, wherein: Step C, detecting the content of Polygala tenuifolia saponin in Polygala tenuifolia, the steps are as follows: Preparation of reference solution: Take an appropriate amount of Polygala tenuifolia saponin reference, weigh accurately, and add methanol to make a solution containing 0.5 mg per 1 ml. Preparation of test solution: Take 2.5g of the standard sample of Xinsan, accurately weigh it, put it in a stoppered conical flask, accurately add 50ml of 70% methanol, weigh it, ultrasonically treat it (power 400W, frequency 40kHz) for 1 hour, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, accurately measure 25ml of the filtrate, put it in an evaporating dish and evaporate it to dryness, add 50ml of 10% sodium hydroxide solution to the residue and dissolve it and transfer it to a stoppered conical flask, heat and reflux for 2 hours, let it cool, adjust the pH value to 4-5 with hydrochloric acid, shake it with water-saturated n-butanol to extract it 3 times, 50ml each time, combine the n-butanol solution, recover the solvent to dryness, add an appropriate amount of methanol to dissolve the residue, transfer it to a 25ml volumetric flask, add methanol to the scale, shake it well, filter it, and obtain it; Determination method: Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine; The chromatographic conditions are as follows: octadecylsilane bonded silica gel is used as filler (JADE ODS-AQ C18 4.6×250mm, 5μm); acetonitrile: 0.05% phosphoric acid (33:67) as mobile phase; flow rate 1.0ml per minute; column temperature 30℃; detection wavelength 210nm. The theoretical plate number calculated based on the Polygala tenuifolia saponin peak should not be less than 3000.

8. The method according to claim 4, wherein: Step A: detecting Polygala tenuifolia in Polygala tenuifolia Ketone III, 3,6′-dicerinoyl sucrose, and the content of β-asarone in Acorus tatarinowii, the steps are as follows: Preparation of reference solution: Take Polygala tenuifolia An appropriate amount of ketone III reference substance, 3,6′-dicorinoyl sucrose reference substance, and β-asarone reference substance were accurately weighed and added with 70% methanol to make a 1 ml solution containing Polygala tenuifolia. A mixed solution of 50 μg of ketone III, 150 μg of 3,6′-diesinapoylsucrose, and 100 μg of β-asarone is obtained; Preparation of test solution: Take 2.5g of the standard sample of Xinsan, weigh it accurately, put it in a stoppered conical flask, add 25ml of 70% methanol accurately, weigh it, heat and reflux for 30 minutes, cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the solution; Determination method: Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine. The chromatographic conditions are as follows: octadecylsilane bonded silica gel is used as filler ( AQ-C18 4.6×250mm, 5μm); acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution as specified in the following table; flow rate 1.0ml per minute; column temperature 30℃; detection wavelength 240nm, theoretical plate number calculated based on 3,6′-dicerinoylsucrose peak should be not less than 3000; Among them, step B, detecting the content of ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 in ginseng; the steps are as follows: preparation of reference solution: accurately weigh ginsenoside Rg1 reference, ginsenoside Re reference, and ginsenoside Rb1 reference, add methanol to make a mixed solution containing 0.3 mg of each per 1 ml, shake well, and obtain; Preparation of test solution: Take 5.0g of the standard sample of Xinsan, accurately weigh it, put it in a stoppered conical flask, accurately add 50ml of water-saturated n-butanol, weigh it, heat and reflux for 90 minutes, cool it, weigh it again, make up the lost weight with water-saturated n-butanol, shake it well, filter it, accurately measure 25ml of the filtrate, evaporate it to dryness in an evaporating dish, dissolve the residue in methanol and transfer it to a 5ml volumetric flask, dilute it to the mark with methanol, shake it well, filter it, and take the filtrate to obtain the solution; Determination method: Accurately pipette 10 and 15 μl of reference solution and 10 μl of test solution respectively, inject into liquid chromatograph, and determine; The chromatographic conditions are as follows: octadecylsilane bonded silica gel as filler (Waters Xselect HSS T3 4.6×250mm, 5μm); acetonitrile as mobile phase A, water as mobile phase B, gradient elution as specified in the following table; flow rate of 1.0 ml per minute; column temperature of 30°C; evaporative light scattering detector detection, the theoretical plate number calculated based on the ginsenoside Rg1 peak should be no less than 6000, Wherein, step C, detecting the content of Polygala tenuifolia saponin in Polygala tenuifolia, comprises the following steps: Preparation of reference solution: Take an appropriate amount of Polygala tenuifolia saponin reference, weigh accurately, and add methanol to make a solution containing 0.5 mg per 1 ml. Preparation of test solution: Take 2.5g of the standard sample of Xinsan, accurately weigh it, put it in a stoppered conical flask, accurately add 50ml of 70% methanol, weigh it, ultrasonically treat it (power 400W, frequency 40kHz) for 1 hour, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, accurately measure 25ml of the filtrate, put it in an evaporating dish and evaporate it to dryness, add 50ml of 10% sodium hydroxide solution to the residue and dissolve it and transfer it to a stoppered conical flask, heat and reflux for 2 hours, let it cool, adjust the pH value to 4-5 with hydrochloric acid, shake it with water-saturated n-butanol to extract it 3 times, 50ml each time, combine the n-butanol solution, recover the solvent to dryness, add an appropriate amount of methanol to dissolve the residue, transfer it to a 25ml volumetric flask, add methanol to the scale, shake it well, filter it, and obtain it; Determination method: Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine; The chromatographic conditions are as follows: octadecylsilane bonded silica gel is used as filler (JADE ODS-AQ C18 4.6×250mm, 5μm); acetonitrile: 0.05% phosphoric acid (33:67) as mobile phase; flow rate 1.0ml per minute; column temperature 30℃; detection wavelength 210nm. The theoretical plate number calculated based on the Polygala tenuifolia saponin peak should not be less than 3000.

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