Characteristic chromatogram construction method of substance reference of Kaixin powder and application of characteristic chromatogram construction method

Abstract

The invention provides a construction method and application of a Kaixin powder substance-based characteristic chromatogram. The construction method comprises the following steps: acquiring a characteristic chromatogram of a to-be-detected substance by adopting ultra-high performance liquid chromatography; chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a chromatographic column of a filler; acetonitrile is used as a mobile phase A, 0.025%-0.1% phosphoric acid aqueous solution is used as a mobile phase B, and elution is carried out according to a specific gradient program; the method comprises the following steps: respectively carrying out detection by using detection wavelengths of 198-208 nm and 315-325 nm, and switching the detection wavelength to 237-247 nm within t min when the detection wavelength of 315-325 nm is used for detection, and t is 100-110. The characteristic chromatogram obtained by the construction method comprises at least 52 characteristic peaks, the material information of the characteristic chromatogram is more comprehensive, and the construction method has important significance on the quality control of the Kaixin powder.

Description

Technical Field

[0001] The present invention relates to the field of identification of traditional Chinese medicines, and specifically relates to a method for constructing a characteristic chromatogram of the reference substance of KaiXinSan and its application. Background Art

[0002] The basic composition of KaiXinSan is: ginseng, polygala root, acorus tatarinowii rhizome, poria cocos, which has the effects of antidepressant, anti-dementia, improving learning and memory, anti-fatigue, etc., and is often clinically used to treat Alzheimer's disease, vascular dementia, cognitive impairment and other diseases mainly manifested by memory dysfunction. The chemical components of KaiXinSan are complex, mainly including: polygala oligosaccharide esters, polygala ketones, polygala saponins, ginsenosides, ginseng polysaccharides, poria cocos polysaccharides, poria triterpenic acids, asarum ether of acorus tatarinowii rhizome, phenolic acids, etc. Among them, the main active ingredients for calming the nerves and strengthening the mind are polygala oligosaccharide esters, ginsenosides, asarum ether of acorus tatarinowii rhizome and poria triterpenic acids. Various components play their medicinal effects synergistically. Therefore, it is particularly important to be able to comprehensively and efficiently control the quality of various active ingredients.

[0003] At present, the quality research on the reference substance of KaiXinSan mostly uses HPLC to establish characteristic chromatograms of one type or multiple types of components for combined quality control. The defect of these quality control methods is that the control of active ingredients is not comprehensive enough, the operation is complicated or the cost is higher. Therefore, for this classical prescription with multiple medicinal flavors, complex types of active ingredients and a large polarity span of various components, a quality control method that is comprehensive, fast, accurate and low-cost is needed. Summary of the Invention

[0004] Regarding the problem that the types of active ingredients in the characteristic chromatogram of KaiXinSan obtained by the characteristic chromatogram construction method disclosed in the prior art are not comprehensive enough; the present invention provides a method for constructing a characteristic chromatogram with more comprehensive types of active ingredients, which is fast, accurate and low-cost.

[0005] A method for constructing a characteristic chromatogram of the reference substance of KaiXinSan, which uses ultra-high performance liquid chromatography to obtain the characteristic chromatogram of the analyte; the chromatographic conditions of the ultra-high performance liquid chromatography are as follows:

[0006] A chromatographic column filled with octadecylsilane-bonded silica gel; acetonitrile as mobile phase A, and an aqueous solution of 0.025%-0.1% phosphoric acid as mobile phase B, and elution is carried out according to the following gradient program:

[0007]

[0008] Detection is carried out at detection wavelengths of 198-208 nm and 315-325 nm respectively. When detecting at a detection wavelength of 315-325 nm, the detection wavelength is switched to 237-247 nm at t min, and t is 100-110.

[0009] In the chromatographic conditions of the ultra-high performance liquid chromatography method, the column length of the chromatographic column is 150 mm, the inner diameter of the column is 2.1 mm, and the particle size is 1.6 μm;

[0010] and / or, the column temperature is 32 - 38 °C;

[0011] and / or, the flow rate is 0.23 - 0.27 ml / min;

[0012] and / or, the theoretical plate number calculated by the 3,6'-di-sinapoyl sucrose peak should be not less than 5000.

[0013] In the ultra-high performance liquid chromatography method, the chromatographic column is Phenomenex Luna Omega, Waters CORTECS UPLC T3, or Yuexu Ultimate UHPLC LP-C18.

[0014] When the analyte is the test sample, the preparation process of the test sample solution is as follows: Take the reference substance of KaiXinSan, accurately weigh it, place it in a stoppered conical flask, add the solvent, weigh it, perform extraction treatment, let it cool, make up the weight, filter, and obtain the solution;

[0015] When the analyte is the reference substance, the preparation process of the reference substance solution is as follows: Take the reference substance, accurately weigh it, and dissolve it in the solvent to prepare the reference substance solution.

[0016] The reference substance of KaiXinSan includes KaiXinSan, the negative substances of KaiXinSan lacking each medicinal flavor (KaiXinSan lacking ginseng, KaiXinSan lacking polygala root, KaiXinSan lacking poria cocos, KaiXinSan lacking acorus tatarinowii).

[0017] The reference substances include sibiricose A6, polygala root ketone III, 3,6'-di-sinapoyl sucrose, β-asarone, dehydrotumulosic acid, pachymic acid, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, ginsenoside Rc.

[0018] and / or, the solvent is methanol or a methanol aqueous solution with a volume concentration of more than 70%;

[0019] and / or, the addition amount of the test sample in the test sample solution is 15 - 25 ml / g;

[0020] and / or, in the preparation of the test sample solution, the extraction treatment method includes one of reflux treatment, ultrasonic treatment, and shaking treatment; the extraction treatment time is 30 - 60 min.

[0021] At the detection wavelength of 320 nm - 242 nm, the characteristic spectrum includes characteristic peaks of peak 1 - peak 34; at the detection wavelength of 203 nm, the characteristic spectrum includes characteristic peaks of peak 35 - peak 52.

[0022] Among the 52 characteristic peaks, 31 belong to the characteristic peaks of Polygala tenuifolia Willd., and 11 are identified; 5 belong to the characteristic peaks of Acorus tatarinowii Schott, and 4 are identified; 5 belong to the characteristic peaks of Poria cocos (Schw.) Wolf, and 5 are identified; 9 belong to the characteristic peaks of Panax ginseng C. A. Mey., and 4 are identified; 1 belongs to the common peak. Among them, peak 1: sibiricose A5, peak 2: sibiricose A6, peak 4: tenuifolin ketone B, peak 5: sphaeradenia glycoside A, peak 6: tenuifolin ketone XI, peak 7: tenuifolin ketone III, peak 8: tenuifoliside B (tenuifoliside B), peak 9: 3,6'-di-O-sinapoyl-sucrose, peak 10: xanthopuccine A, peak 11: tenuifoliside A, peak 12: tenuifoliside C, peak 20: β-asarone, peak 21: α-asarone, peak 31: dehydrotumulosic acid, peak 32: polyporenic acid C, peak 33: 3-epi-dehydrotumulosic acid, peak 34: dehydroplicatic acid, peak 39: ginsenoside Rg1, peak 40: ginsenoside Re, peak 41: methyl eugenol, peak 42: γ-asarone, peak 43: ginsenoside Rb1, peak 44: ginsenoside Rc, peak 50: pachymic acid.

[0023] The peak corresponding to the sibiricose A6 reference substance peak is the S1 peak, and the relative retention times of peaks 1, 35-38 and the S1 peak are calculated; the peak corresponding to the tenuifolin ketone III reference substance peak is the S2 peak, and the relative retention times of peaks 3-8 and the S2 peak are calculated; the peak corresponding to the 3,6'-di-O-sinapoyl-sucrose reference substance peak is the S3 peak, and the relative retention times of peaks 10-19 and the S3 peak are calculated; the peak corresponding to the β-asarone reference substance peak is the S4 peak, and the relative retention times of peaks 21, 41, 42 and the S4 peak are calculated; the peak corresponding to the dehydrotumulosic acid reference substance peak is the S5 peak, and the relative retention times of peaks 22-30, 32-34 and the S5 peak are calculated; the peak corresponding to the pachymic acid reference substance peak is the S6 peak, and the relative retention times of peaks 45-52 and the S6 peak are calculated; the relative retention times of each characteristic peak are within the range of ±5% of the specified value;

[0024] The specified values are as follows:

[0025] Peak 1: 0.90, Peak 2: 1, Peak 3: 0.72, Peak 4: 0.85, Peak 5: 0.90, Peak 6: 0.96, Peak 7: 1, Peak 8: 1.09, Peak 9: 1, Peak 10: 1.02, Peak 11: 1.09, Peak 12: 1.29, Peak 13: 1.41, Peak 14: 1.46, Peak 15: 1.58, Peak 16: 1.64, Peak 17: 1.88, Peak 18: 1.96, Peak 19: 2.04, Peak 20: 1, Peak 21: 1.12, Peak 22: 0.73, Peak 23: 0.74, Peak 24: 0.74, Peak 25: 0.75, Peak 26: 0.76, Peak 27: 0.77, Peak 28: 0.78, Peak 29: 0.79, Peak 30: 0.81, Peak 31: 1, Peak 32: 1.04, Peak 33: 1.05, Peak 34: 1.12, Peak 35: 0.37, Peak 36: 0.94, Peak 37: 1.16, Peak 38: 1.27, Peak 41: 0.96, Peak 42: 1.03, Peak 45: 0.60, Peak 46: 0.61, Peak 47: 0.62, Peak 48: 0.81, Peak 49: 0.96, Peak 50: 1, Peak 51: 1.01, Peak 52: 1.06.

[0026] The technical solution of the present invention has the following advantages:

[0027] 1. A method for constructing a characteristic chromatogram of the material basis of KaiXinSan. By using UPLC means and a characteristic chromatogram analysis method with one chromatographic condition and multiple wavelengths, it comprehensively characterizes the characteristics of the effective components of the four medicinal flavors in the material basis of KaiXinSan. The characteristic peaks in the obtained characteristic chromatogram include at least 52 characteristic peaks, including oligosaccharides, ketones, saponins in Polygala tenuifolia, ginsenosides in Panax ginseng, asarone in the volatile oil of Acorus tatarinowii, and triterpenic acids in Poria cocos. The material information of the characteristic chromatogram is more comprehensive, which has important significance for the quality control of KaiXinSan preparations.

[0028] 2. In the construction method of the present invention, the preparation method of the test sample is simple, which improves the inspection efficiency and saves costs.

[0029] 3. The construction method of the present invention has the advantages of simplicity, stability, high precision, and good reproducibility. It can quickly and accurately identify the quality of products, providing a scientific basis for comprehensively establishing the quality control standards of KaiXinSan preparations. Description of the Drawings

[0030] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0031] Figure 1 It is the characteristic spectrum of the test sample solution in Example 1 of the present invention under the detection wavelength of 320 - 242 nm.

[0032] Figure 2 It is the characteristic spectrum of the test sample solution in Example 1 of the present invention under the detection wavelength of 203 nm.

[0033] Figure 3 It is the control characteristic spectrum of the Kāixīn Sàn and the negative reference substance of Kāixīn Sàn in Example 1 of the present invention under the detection wavelength of 320 - 242 nm.

[0034] Figure 4 It is the control characteristic spectrum of the Kāixīn Sàn and the negative reference substance of Kāixīn Sàn in Example 1 of the present invention under the detection wavelength of 203 nm. Specific Embodiments

[0035] For those not specifying the specific experimental steps or conditions in the embodiments, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For the reagents or instruments without indicating the manufacturer, they are all conventional reagent products that can be obtained through commercial purchase.

[0036] Instrument: Waters ACQUITY H-Class ultra-high performance liquid chromatograph, TUV Detector ultraviolet detector, Empower 3 chromatographic workstation; ME104E electronic balance (Mettler Toledo), JY2002 electronic balance (Mettler Toledo), KQ-500DB ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); electronic constant temperature water bath DZKW-4 (Beijing Zhongxing Weiye Instrument Co., Ltd.).

[0037] The test drugs are shown in Table 1 below:

[0038] Table 1

[0039]

[0040]

[0041] Preparation of Kāixīn Sàn: Take the fine powders of Polygala tenuifolia, Panax ginseng, Poria cocos, and Acorus tatarinowii, and mix them evenly according to the prescription requirements in a ratio of 1:1:2:1 to obtain it.

[0042] Preparation of Kāixīn Sǎn without ginseng: Take the fine powders of Polygala tenuifolia, Poria cocos, and Acorus tatarinowii, mix them evenly according to the ratio of 1:2:1 as required by the prescription, and you will get it.

[0043] Preparation of Kāixīn Sǎn without Polygala tenuifolia: Take the fine powders of ginseng, Poria cocos, and Acorus tatarinowii, mix them evenly according to the ratio of 1:2:1 as required by the prescription, and you will get it.

[0044] Preparation of Kāixīn Sǎn without Poria cocos: Take the fine powders of Polygala tenuifolia, ginseng, and Acorus tatarinowii, mix them evenly according to the ratio of 1:1:1 as required by the prescription, and you will get it.

[0045] Preparation of Kāixīn Sǎn without Acorus tatarinowii: Take the fine powders of Polygala tenuifolia, ginseng, and Poria cocos, mix them evenly according to the ratio of 1:1:2 as required by the prescription, and you will get it.

[0046] Reagents: Acetonitrile and phosphoric acid (Fisher Chemical) are of chromatographic grade, methanol and ethanol are of analytical grade, and water is purified water from Watson.

[0047] Example 1

[0048] A method for constructing the characteristic chromatogram of the reference substance of Kāixīn Sǎn is as follows:

[0049] 1. Preparation of the test solution

[0050] Preparation of the test solution: Take an appropriate amount of the test sample (Kāixīn Sǎn), grind it finely, take about 2.0 g, weigh it accurately, place it in a stoppered conical flask, accurately add 25 ml of methanol, weigh it, ultrasonicate for 30 min, let it cool, replenish the weight, filter, and you will get it.

[0051] Reference substance solution of the reference standard: Take appropriate amounts of the reference standards of polygalaxside A5, tenuifolin, 3,6'-di-O-sinapoyl-sucrose, β-asarone, dehydrotumulosic acid, pachymic acid, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, and ginsenoside Rc, weigh them accurately, and dissolve them in methanol to prepare a solution containing 640 μg of polygalaxside A6, 40 μg of tenuifolin, 85 μg of 3,6'-di-O-sinapoyl-sucrose, 280 μg of β-asarone, 12 μg of dehydrotumulosic acid, 20 μg of pachymic acid, 100 μg of ginsenoside Rg1, 100 μg of ginsenoside Re, 100 μg of ginsenoside Rb1, and 100 μg of ginsenoside Rc in 1 ml, and you will get it.

[0052]

[0053] 2. Chromatographic method

[0054] The following chromatographic conditions are used for detection:

[0054] Using octadecylsilyl-bonded silica gel as the filler (Waters CORTECS UPLC T3, column length 150 mm, column inner diameter 2.1 mm, particle size 1.6 μm); using acetonitrile as mobile phase A and 0.05% phosphoric acid as mobile phase B, perform gradient elution according to the regulations in Table 2 below; the flow rate is 0.25 mL per minute, the column temperature is 35 °C, the detection wavelengths are 320 nm - 242 nm (the wavelength is switched from 320 nm to 242 nm at 110 min) and 203 nm, and the number of theoretical plates calculated based on 3,6'-disesamoyl sucrose shall not be less than 5000.

[0055] Table 2

[0056]

[0057]

[0058] Precisely pipette 1 μL each of the reference substance solution and the test solution, inject into the ultra-high performance liquid chromatograph, and determine to obtain.

[0059] The characteristic chromatogram obtained from the test solution is as Figure 1 and Figure 2 shown. Figure 1 is the characteristic chromatogram of the test solution in Example 1 at the detection wavelength of 320 - 242 nm, Figure 2 is the characteristic chromatogram of the test solution in Example 1 at the detection wavelength of 203 nm.

[0060] Collect the negative substance reference of Kaixin Powder without each medicinal material flavor, obtain its liquid chromatogram according to the above method, as Figure 3 and Figure 4 shown, and assign the common peaks to the medicinal material flavors to determine the characteristic peaks. According to the negative substance reference of Kaixin Powder for peak assignment, among them, peaks 1 - 19, 23 - 30, 36 - 38, 52 are attributed to Polygala tenuifolia Willd. pieces, peaks 20, 21, 41, 42, 48 are attributed to Acorus tatarinowii Schott pieces, peaks 31 - 34, 50 are attributed to Poria cocos Wolf pieces, peaks 35, 39, 40, 43 - 47, 49 are attributed to Panax ginseng C. A. Mey, and peak 51 is a common peak.

[0061] Take the test solution and the reference solution of the main chemical components, perform detection and comparison according to the above chromatographic conditions, determine each characteristic peak in the characteristic chromatogram of the test solution, and the identification results are as follows:

[0062] Peak 1 is sibiricose A5; peak 2 is sibiricose A6; peak 4 is tenuigenin; peak 5 is globularicoside A; peak 6 is polygalaxanthone XI; peak 7 is polygalaxanthone Peak Ⅲ; Peak 8 is polygala saponin B (tenuifolin B); Peak 9 is 3,6'-di-O-sinapoyl-sucrose; Peak 10 is polygalaflavone A; Peak 11 is polygala saponin A; Peak 12 is polygala saponin C; Peak 20 is β-asarone; Peak 21 is α-asarone; Peak 31 is dehydrotumulosic acid; Peak 32 is polyporenic acid C; Peak 33 is 3-epi-dehydrotumulosic acid; Peak 34 is dehydro-pachymic acid; Peak 39 is ginsenoside Rg1; Peak 40 is ginsenoside Re; Peak 41 is methyl eugenol; Peak 42 is γ-asarone; Peak 43 is ginsenoside Rb1; Peak 44 is ginsenoside Rc; Peak 50 is pachymic acid.

[0063] The test solution characteristic chromatogram should show 52 characteristic peaks. Among them, Peak 2, Peak 7, Peak 9, Peak 20, Peak 31, Peak 39, Peak 40, Peak 43, Peak 44, and Peak 50 should be consistent with the retention times of the reference chromatographic peaks of sibiricose A6, tenuigenin Ⅲ, 3,6'-di-O-sinapoyl-sucrose, β-asarone, dehydrotumulosic acid, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, ginsenoside Rc, and pachymic acid, respectively. At the detection wavelength of 320 - 242 nm, the peak corresponding to the sibiricose A6 reference peak is Peak S1, and calculate the relative retention times of Peak 1, Peak 35 - Peak 38 with respect to Peak S1; the peak corresponding to the tenuigenin Ⅲ reference peak is Peak S2, and calculate the relative retention times of Peak 3 - Peak 8 with respect to Peak S2; the peak corresponding to the 3,6'-di-O-sinapoyl-sucrose reference peak is Peak S3, and calculate the relative retention times of Peak 10 - Peak 19 with respect to Peak S3; the peak corresponding to the β-asarone reference peak is Peak S4, and calculate the relative retention times of Peak 21, Peak 41, Peak 42 with respect to Peak S4; the peak corresponding to the dehydrotumulosic acid reference peak is Peak S5, and calculate the relative retention times of Peak 22 - Peak 30, Peak 32 - Peak 34 with respect to Peak S5; the peak corresponding to the pachymic acid reference peak is Peak S6, and calculate the relative retention times of Peak 45 - Peak 52 with respect to Peak S6; the relative retention times of each characteristic peak are within the range of ±5% of the specified value;

[0064] The specified values are shown in Table 3 below:

[0065] Table 3

[0066]

[0067]

[0068] Example 2

[0069] This example is used to conduct a methodological verification of the chromatographic conditions in Example 1.

[0070] 1. Precision investigation

[0071] According to the characteristic chromatogram method established above, a test solution was prepared from the reference sample of KaiXinSan, and injected continuously for 6 times to obtain the characteristic chromatogram. The retention times and relative retention times of peaks 1 - 52 were obtained, and the RSD and the range from the specified value were calculated.

[0072] The results of the precision experiment showed that the RSD of the relative retention times of the characteristic peaks of the six samples was between 0% and 0.5%, indicating that the precision of this characteristic chromatogram was good.

[0073] 2. Repeatability investigation

[0074] According to the characteristic chromatogram method established above, 6 test solutions were prepared from the reference sample of KaiXinSan for injection analysis to obtain the characteristic chromatogram. The retention times and relative retention times of peaks 1 - 52 were obtained, and the RSD and the range from the specified value were calculated.

[0075] The results of the repeatability experiment showed that the RSD of the relative retention times of the characteristic peaks was between 0% and 0.5%, indicating that the repeatability of this characteristic chromatogram was good.

[0076] 3. Intermediate precision investigation

[0077] Using Shimadzu LC - 30AD and TUV detector, according to the characteristic chromatogram method established above, 6 test solutions were prepared from the reference sample of KaiXinSan for injection analysis to obtain the characteristic chromatogram. The retention times and relative retention times of each characteristic peak were obtained, and the RSD value was calculated.

[0078] The results of the intermediate precision showed that the RSD of the relative retention times of the characteristic peaks was between 0% and 0.9%. Comparing with the data of the relative retention times of the characteristic peaks of the 6 samples in the repeatability investigation, the RSD range of the relative retention times was 0% - 3.0%, indicating that the relative retention times of this characteristic chromatogram met the analysis requirements among different instruments.

[0079] 4. Stability investigation

[0080] Taking the same test solution, determinations were carried out at 0, 3, 6, 9, 15, 18, and 24 h respectively to obtain the characteristic chromatogram, and the retention times and relative retention times of each characteristic peak were obtained, and the RSD was calculated.

[0081] The results of the stability showed that the RSD of the relative retention times of the characteristic peaks was between 0% and 0.6%, indicating that the chemical components in the solution had good stability within 24 hours.

[0082] Example 3

[0083] A method for constructing the characteristic chromatogram of the reference substance of KaiXinSan, which is different from Example 1 in that the preparation of the test solution is different.

[0084] (1) Selection of extraction solvent: Appropriate amount of reference substance of KaiXinSan, about 2.0 g for each portion, was accurately weighed and placed in a stoppered conical flask. Appropriate solvents (methanol, 70% ethanol, 50% ethanol, 30% ethanol, 70% methanol, 50% methanol, 30% methanol, water) were accurately added to each flask, 25 ml for each, and the flasks were stoppered and weighed. After ultrasonic treatment (power 500 W, frequency 40 KHz) for 30 minutes, the flasks were taken out, cooled to room temperature, and weighed again. The lost weight was made up with the corresponding solvent, and the mixtures were shaken well and filtered to obtain the solutions.

[0085] 1 μl of the reference solution and the test solution were accurately pipetted and injected into an ultra-high performance liquid chromatograph for determination.

[0086] Table 4 Selection of extraction solvent

[0087]

[0088]

[0089] Result analysis: As can be seen from the table, only comparing the peak areas of the identified peaks, water, 50% methanol, 50% ethanol, 30% methanol, and 30% ethanol solvents showed incomplete extraction. Methanol and 70% methanol had good dissolution effects on each target component, and 70%-100% methanol could be selected as the extraction solvent.

[0090] (2) Selection of solvent dosage: Appropriate amount of reference substance of KaiXinSan, about 2.0 g for each portion, was accurately weighed and placed in a stoppered conical flask. Appropriate amounts of methanol (15 ml, 25 ml, and 50 ml) were accurately added to each flask, and the flasks were stoppered and weighed. After ultrasonic treatment (power 500 W, frequency 40 KHz) for 30 minutes, the flasks were taken out, cooled to room temperature, and weighed again. The lost weight was made up with the corresponding methanol, and the mixtures were shaken well and filtered to obtain the solutions.

[0091] 1 μl of the reference solution and the test solution were accurately pipetted and injected into an ultra-high performance liquid chromatograph for determination. The peak areas of each component after determination were converted to the same concentration for comparison.

[0092] Table 5 Selection of solvent dosage

[0093]

[0094]

[0095] Result analysis: As can be seen from the table, only comparing the peak areas of the identified peaks, although methanol had good dissolution effects on each peak, for some peaks, too much solvent that affected the response to the detection wavelength and the component concentration was not conducive to data reading. At the same time, there was little difference in the peaks after extraction with 15 ml and 25 ml of methanol. Therefore, it was determined that the solvent dosage of 15 ml - 25 ml was appropriate.

[0096] (3) Selection of extraction time: Appropriate amount of the reference substance of KaiXinSan, about 2.0 g for each portion, was accurately weighed and placed in a stoppered conical flask. An appropriate amount of 25 ml of methanol was accurately added respectively, the stopper was closed tightly, and the weight was weighed. It was ultrasonicated (power 500 W, frequency 40 KHz) for different times (30 minutes, 60 minutes, 90 minutes), taken out, cooled, and weighed again. The lost weight was made up with the corresponding methanol, shaken well, filtered, and thus obtained.

[0097] 1 μl of the reference substance solution and the test solution were accurately pipetted respectively, injected into an ultra-high performance liquid chromatograph for determination, and thus obtained.

[0098] Table 6 Selection of extraction time

[0099]

[0100]

[0101] Result analysis: It can be seen from the table that at different extraction times, there is no obvious difference in the peak areas of each peak in the characteristic chromatogram, and the extraction time was determined to be not less than 30 min.

[0102] (4) Selection of extraction method: The reference substance of KaiXinSan, about 2 g, was accurately weighed and placed in a stoppered conical flask. 25 ml of methanol was accurately added, the stopper was closed tightly, and the weight was weighed. It was shaken and refluxed for 60 minutes respectively, taken out, cooled, and weighed again. The lost weight was made up with methanol, shaken well, filtered, and thus obtained.

[0103] 1 μl of the reference substance solution and the test solution were accurately pipetted respectively, injected into an ultra-high performance liquid chromatograph for determination, and thus obtained.

[0104] Table 7 Selection of extraction method

[0105]

[0106]

[0107] Result analysis: It can be seen from the table that for different extraction methods, there is no obvious difference in the peak areas of each peak in the characteristic chromatogram, and all the above methods can be used. However, considering the simplicity of operation, ultrasonic extraction was determined to be used.

[0108] Example 4

[0109] A method for constructing the characteristic chromatogram of the reference substance of KaiXinSan, which is different from Example 1 in that the chromatographic conditions are as follows:

[0110] (1) Investigation of different column temperatures: Precisely pipette 1 μl of the test solution respectively, inject it into the ultra-high performance liquid chromatograph, and perform the determination at different column temperatures (32 °C, 35 °C, 38 °C).

[0111] Table 8 Relative retention times of each peak at different column temperatures

[0112]

[0113]

[0114]

[0115] Result analysis: It can be seen from the table that at different column temperatures, the relative retention times of each peak in the characteristic chromatogram meet the requirements of the specified value fluctuations, indicating good applicability to the above column temperatures.

[0116] (2) Investigation of different flow rates: Precisely pipette 1 μl of the test solution respectively, inject it into the ultra-high performance liquid chromatograph, and perform the determination at different flow rates (0.23 ml / min, 0.25 ml / min, 0.27 ml / min).

[0117] Table 9 Relative retention times of each peak at different flow rates

[0118]

[0119]

[0120] Result analysis: It can be seen from the table that at different flow rates, the relative retention times of each peak in the characteristic chromatogram meet the requirements of the specified value fluctuations, indicating good applicability to the above flow rates.

[0121] (3) Investigation of different acid concentrations: Precisely pipette 1 μl of the test solution respectively, inject it into the ultra-high performance liquid chromatograph, and perform the determination under mobile phase B with different acidities (0.025% phosphoric acid, 0.05% phosphoric acid, 0.10% phosphoric acid).

[0122] Table 10 Relative retention times of each peak at different acidities

[0123]

[0124]

[0125] Result analysis: It can be seen from the table that under the mobile phase with different acidities, the relative retention times of each peak in the characteristic chromatogram meet the requirements of the specified value fluctuations, indicating good applicability to the mobile phase with the above acidities.

[0126] (4) Investigation at different wavelengths: Precisely pipette 1 μL of the test solution respectively, inject it into the ultra-high performance liquid chromatograph, and perform the determination at the specified wavelengths ±5 nm (switch to 237 nm for 198 nm and 315 nm, switch to 247 nm for 208 nm and 325 nm).

[0127] Table 11 Relative retention times of each peak at different wavelengths

[0128]

[0129]

[0130] Result analysis: It can be seen from the table that under the detection of wavelengths with a ±5 nm fluctuation from the specified wavelength, the relative retention times of each peak in the characteristic chromatogram meet the requirements of the specified value fluctuation, indicating good applicability to the above wavelengths.

[0131] (5) Investigation of different types of chromatographic columns: Precisely pipette 1 μL of the test solution respectively, inject it into the ultra-high performance liquid chromatograph, and perform the determination using different chromatographic columns, namely chromatographic column 1 (Phenomenex Luna Omega, 1.6 μm 150 * 2.1 mm), chromatographic column 2 (Waters CORTECS UPLC T3, 1.6 μm 150 * 2.1 mm), and chromatographic column 3 (Yuexu Ultimate UHPLC LP-C18, 1.6 μm 2.1 * 150 mm).

[0132] Table 12 Relative retention times of each peak under different chromatographic columns

[0133]

[0134]

[0135] Result analysis: It can be seen from the table that for the characteristic chromatograms obtained using different chromatographic columns, the relative retention times of each peak in the characteristic chromatogram meet the requirements of the specified value fluctuation, indicating good applicability to the above chromatographic columns.

[0136] (6) Investigation of the wavelength switching time: Considering that there is a wavelength switch at 320 nm, the time for switching the wavelength was investigated, and the situations of the front and rear characteristic peaks in the corresponding characteristic chromatograms at 100 min and 110 min were investigated, mainly the influence on characteristic peaks 27 to 31.

[0137] Table 13 Retention times and peak areas of characteristic peaks when switching wavelengths at different times

[0138]

[0139] Result analysis: As can be seen from the table, there is no obvious change in the retention time and peak area of the characteristic peaks under different time switches, indicating good applicability to the above switching times.

[0140] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation manners here. And the obvious changes or modifications derived therefrom still fall within the protection scope of the present invention.

Claims

1. A method for constructing a characteristic spectrum of the reference substance of KaiXinSan, characterized in that Obtain the characteristic chromatogram of the analyte by ultra - high performance liquid chromatography; the chromatographic conditions of the ultra - high performance liquid chromatography are as follows: A chromatographic column filled with octadecylsilyl silica gel as the filler; acetonitrile as mobile phase A and an aqueous phosphoric acid solution with a concentration of 0.025% - 0.1% as mobile phase B, and perform elution according to the following gradient program: Detect at detection wavelengths of 198 - 208 nm and 315 - 325 nm respectively. When detecting at the detection wavelength of 315 - 325 nm, switch the detection wavelength to 237 - 247 nm at t min, where t is 100 - 110.

2. The construction method according to claim 1, characterized in that, In the chromatographic conditions of the ultra - high performance liquid chromatography, the column length of the chromatographic column is 150 mm, the column inner diameter is 2.1 mm, and the particle size is 1.6 μm; and / or, the column temperature is 32 - 38 °C; and / or, the flow rate is 0.23 - 0.27 ml / min; and / or, the theoretical plate number calculated based on the 3,6’ - di - sinapoyl - sucrose peak should be not less than 5000.

3. The construction method according to claim 1, characterized in that, In the ultra - high performance liquid chromatography, the chromatographic column is Phenomenex Luna Omega, Waters CORTECS UPLC T3, or Yuexu Ultimate UHPLC LP - C18.

4. The construction method according to any one of claims 1-3, characterized in that, When the analyte is the test sample, the preparation process of the test sample solution is as follows: Take the reference substance of KaiXinSan, weigh it precisely, place it in a stoppered conical flask, add the solvent, weigh it, perform extraction treatment, cool it, make up the weight, filter, and obtain the solution; When the analyte is the reference substance, the preparation process of the reference substance solution is as follows: Take the reference substance, weigh it precisely, and dissolve it in the solvent to prepare the reference substance solution.

5. The construction method according to claim 4, characterized in that, The reference substances include polygalacin D6, polygala tenuifolia ketone III, 3,6'-di-O-sinapoyl-sucrose, β-asarone, dehydrotumulosic acid, pachymic acid, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, ginsenoside Rc.

6. The construction method according to claim 4, characterized in that The solvent is methanol or an aqueous methanol solution with a volume concentration of 70% or more; and / or, the addition amount of the test sample in the test sample solution is 15 - 25 ml / g; and / or, in the preparation of the test sample solution, the extraction treatment method includes one of reflux treatment, ultrasonic treatment, and shaking treatment; the extraction treatment time is 30 - 60 min.

7. The construction method according to any one of claims 1-6, characterized in that, At a detection wavelength of 320 nm - 242 nm, the characteristic chromatogram includes characteristic peaks from peak 1 to peak 34; at a detection wavelength of 203 nm, the characteristic chromatogram includes characteristic peaks from peak 35 to peak 52.

8. The construction method according to claim 7, characterized in that, Among the 52 characteristic peaks, peak 1: polygalacin A5, peak 2: polygalacin A6, peak 4: tenuifolin ketone B, peak 5: globular adenocardoside A, peak 6: tenuifolin ketone XI, peak 7: tenuifolin ketone III, peak 8: polyglacin B, peak 9: 3,6'-di-O-sinapoyl-sucrose, peak 10: polygalaflavone A, peak 11: polyglacin A, peak 12: polyglacin C, peak 20: β-asarone, peak 21: α-asarone, peak 31: dehydrotumulosic acid, peak 32: polyporenic acid C, peak 33: 3-epi-dehydrotumulosic acid, peak 34: dehydroplicatic acid, peak 39: ginsenoside Rg1, peak 40: ginsenoside Re, peak 41: methyl eugenol, peak 42: γ-asarone, peak 43: ginsenoside Rb1, peak 44: ginsenoside Rc, peak 50: pachymic acid.

9. The construction method according to claim 7 or 8, characterized in that, The peak corresponding to the reference peak of sibiricose A6 is the S1 peak, and the relative retention times of peak 1, peaks 35 to 38 and the S1 peak are calculated; for polygala The peak corresponding to the reference peak of tenuifolin is S2, and the relative retention times of peaks 3 to 8 and the S2 peak are calculated; the peak corresponding to the reference peak of 3,6'-di-O-sinapoyl-sucrose is the S3 peak, and the relative retention times of peaks 10 to 19 and the S3 peak are calculated; the peak corresponding to the reference peak of β-asarone is the S4 peak, and the relative retention times of peaks 21, 41, 42 and the S4 peak are calculated; the peak corresponding to the reference peak of dehydrotumulosic acid is the S5 peak, and the relative retention times of peaks 22 to 30, 32 to 34 and the S5 peak are calculated; the peak corresponding to the reference peak of pachymic acid is the S6 peak, and the relative retention times of peaks 45 to 52 and the S6 peak are calculated; the relative retention times of each characteristic peak are within the range of ±5% of the specified value; The specified values are as follows: Peak 1: 0.90, Peak 2: 1, Peak 3: 0.72, Peak 4: 0.85, Peak 5: 0.90, Peak 6: 0.96, Peak 7: 1, Peak 8: 1.09, Peak 9: 1, Peak 10: 1.02, Peak 11: 1.09, Peak 12: 1.29, Peak 13: 1.41, Peak 14: 1.46, Peak 15: 1.58, Peak 16: 1.64, Peak 17: 1.88, Peak 18: 1.96, Peak 19: 2.04, Peak 20: 1, Peak 21: 1.12, Peak 22: 0.73, Peak 23: 0.74, Peak 24: 0.74, Peak 25: 0.75, Peak 26: 0.76, Peak 27: 0.77, Peak 28: 0.78, Peak 29: 0.79, Peak 30: 0.81, Peak 31: 1, Peak 32: 1.04, Peak 33: 1.05, Peak 34: 1.12, Peak 35: 0.37, Peak 36: 0.94, Peak 37: 1.16, Peak 38: 1.27, Peak 41: 0.96, Peak 42: 1.03, Peak 45: 0.60, Peak 46: 0.61, Peak 47: 0.62, Peak 48: 0.81, Peak 49: 0.96, Peak 50: 1, Peak 51: 1.01, Peak 52: 1.

06.

10. Use of the characteristic fingerprint spectrum constructed by the construction method according to any one of claims 1-9 in the overall quality control of the material basis of KaiXinSan.

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