A method for constructing and applying the characteristic spectrum of an open-heart powder material reference

Abstract

This invention provides a method and application for constructing characteristic spectra of Kaixin San (a traditional Chinese medicine) as a reference material. The method employs ultra-high performance liquid chromatography (UHPLC) to obtain the characteristic spectra of the analyte. The chromatographic conditions are as follows: a column packed with octadecylsilane-bonded silica gel; acetonitrile as mobile phase A; and 0.025%–0.1% phosphoric acid aqueous solution as mobile phase B, eluted according to a specific gradient program. Detection is performed at wavelengths of 198–208 nm and 315–325 nm, respectively. When detecting at 315–325 nm, the detection wavelength is switched to 237–247 nm at t min, with t being 100–110. The characteristic spectra obtained by this method include at least 52 characteristic peaks, providing more comprehensive material information and playing a significant role in the quality control of Kaixin San.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of traditional Chinese medicine identification, and particularly relates to a characteristic spectrum construction method of KaiXinSan material reference and application. BACKGROUND

[0002] The basic components of KaiXinSan are ginseng, polygala, atractylodes, and poria. KaiXinSan has the effects of anti-depression, anti-dementia, improving learning and memory, and anti-fatigue. KaiXinSan is often used in the clinical treatment of Alzheimer's disease, vascular dementia, cognitive impairment, and other diseases mainly manifested as memory dysfunction. KaiXinSan has complex chemical components, mainly including polygala oligosaccharide ester, polygala ketone, polygala saponin, ginsenoside, ginseng polysaccharide, poria triterpene acid, atractylodes asarum ether, and phenolic acid. The main effective components for soothing the nerves and improving intelligence are polygala oligosaccharide ester, ginsenoside, atractylodes asarum ether, and poria triterpene acid. The various components synergistically exert the pharmacological effects. Therefore, it is particularly important to comprehensively and efficiently control the various effective components.

[0003] At present, the quality research in the KaiXinSan material reference mainly adopts HPLC to establish a characteristic spectrum of one type of component or multiple types of components and to perform joint quality control. However, the defects of these quality control methods are that the control of the effective components is not comprehensive, the operation is complicated, or the cost is higher. Therefore, based on the classic famous prescription with multiple medicinal ingredients, complex types of effective components, and large polarity span of various components, a comprehensive, rapid, accurate, and low-cost quality control method is needed. SUMMARY

[0004] The characteristic spectrum of KaiXinSan obtained by the characteristic spectrum construction method disclosed in the prior art does not have a comprehensive type of effective component; the present application provides a characteristic spectrum construction method with a more comprehensive type of effective component, which is rapid, accurate, and low-cost.

[0005] A characteristic spectrum construction method of KaiXinSan material reference, which adopts ultra-high performance liquid chromatography to obtain the characteristic spectrum of a test object; the chromatographic conditions of the ultra-high performance liquid chromatography are as follows:

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

[0007]

[0008] The detection wavelengths are 198-208 nm and 315-325 nm, respectively; when the detection wavelength is 315-325 nm, the detection wavelength is switched to 237-247 nm at t min, and t is 100-110. ​

[0009] The column length of the chromatographic column in the chromatographic conditions of the ultra-high performance liquid chromatography 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℃;

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

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

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

[0014] When the test substance is a test sample, the preparation process of the test sample solution is as follows: take the KaiXinSan reference material, accurately weigh, place in a conical flask with a plug, add solvent, weigh, extract and process, cool, supplement weight, filter, and obtain.

[0015] When the test substance is a control sample, the preparation process of the control sample reference solution is as follows: take the control sample, accurately weigh, add solvent to prepare the control sample reference solution.

[0016] The KaiXinSan reference material includes KaiXinSan, KaiXinSan negative material lacking each medicinal decoction piece (KaiXinSan-lacking ginseng, KaiXinSan-lacking polygala, KaiXinSan-lacking poria, and KaiXinSan-lacking acorus calamus).

[0017] The control sample includes Siberian polygala glycoside A6, polygala ketone III, 3,6'-dijessyl sucrose, β-asarone, dehydrotrichosanic acid, pachymic acid, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, and ginsenoside Rc.

[0018] And / or, the solvent is methanol or 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 and processing method includes one of reflux processing, ultrasonic processing, and shaking processing; the extraction and processing time is 30-60 min.

[0021] Under the detection wavelength of 320 nm-242 nm, the characteristic peaks of peaks 1-34 are included in the characteristic spectrum; under the detection wavelength of 203 nm, the characteristic peaks of peaks 35-52 are included in the characteristic spectrum.

[0022] 31 peaks were attributed to the characteristic peaks of the medicinal flavor of S. chinensis, and 11 peaks were identified; 5 peaks were attributed to the characteristic peaks of the medicinal flavor of A. griffithii, and 4 peaks were identified; 5 peaks were attributed to the characteristic peaks of the medicinal flavor of P. lucidum, and 5 peaks were identified; 9 peaks were attributed to the characteristic peaks of the medicinal flavor of P. ginseng, and 4 peaks were identified; and 1 peak was attributed to the common peaks. Among them, peak 1: Siberian milkwort sugar A5, peak 2: Siberian milkwort sugar A6, peak 4: S. chinensis ketone B, peak 5: globoside A, peak 6: S. chinensis ketone XI, peak 7: S. chinensis ketone III, peak 8: S. chinensis glycoside B (sophoradiol B), peak 9: 3,6'-diacetyl sucrose, peak 10: K. chinensis A, peak 11: S. chinensis glycoside A, peak 12: S. chinensis glycoside C, peak 20: β-asarone, peak 21: α-asarone, peak 31: dehydrotrum acid, peak 32: polyporus acid C, peak 33: 3-epidehydrotrum acid, peak 34: dehydrotrametenolic 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 Siberian milkwort sugar A6 reference peak is S1 peak, and the relative retention times of peaks 1, 35-38 and S1 peak are calculated; the peak corresponding to the S. chinensis ketone III reference peak is S2, and the relative retention times of peaks 3-8 and S2 peak are calculated; the peak corresponding to the 3,6'-diacetyl sucrose reference peak is S3 peak, and the relative retention times of peaks 10-19 and S3 peak are calculated; the peak corresponding to the β-asarone reference peak is S4 peak, and the relative retention times of peaks 21, 41, 42 and S4 peak are calculated; the peak corresponding to the dehydrotrum acid reference peak is S5 peak, and the relative retention times of peaks 22-30, 32-34 and S5 peak are calculated; the peak corresponding to the pachymic acid reference peak is S6 peak, and the relative retention times of peaks 45-52 and S6 peak are calculated; the relative retention times of each characteristic peak are within ±5% of the specified value;

[0024] The specified value is 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 scheme of the present application has the following advantages:

[0027] 1. The present application provides a method for constructing a characteristic spectrum of the substance basis of Kaixin San, which uses UPLC to analyze the characteristic spectrum at multiple wavelengths under a chromatographic condition, and comprehensively represents the characteristics of four effective components in the substance basis of Kaixin San. The characteristic spectrum obtained by the method includes at least 52 characteristic peaks, including oligosaccharides, ketones and saponins in Radix Polygalae, ginsenosides in Radix et Rhizoma Ginseng, asarone in volatile oil in Rhizoma Pinelliae, and triterpenoid acids in Poria cocos, and the substance information of the characteristic spectrum is more comprehensive, which is of great significance for the quality control of Kaixin San preparations.

[0028] 2. The method of the present application, wherein the preparation method of the test sample is simple, which improves the test efficiency and saves costs.

[0029] 3. The method of the present application has the advantages of simplicity, stability, high precision and good reproducibility, and can quickly and accurately identify the quality of the product, providing a scientific basis for the comprehensive establishment of the quality control standard of Kaixin San preparations. BRIEF DESCRIPTION OF DRAWINGS

[0030] ​To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0031] Figure 1 This is a characteristic spectrum of the test solution in Example 1 of the present invention at a detection wavelength of 320-242nm.

[0032] Figure 2 This is the characteristic spectrum of the test solution in Example 1 of the present invention at a detection wavelength of 203 nm.

[0033] Figure 3 The comparison feature spectrum of Kaixin San and Kaixin San negative substance reference in Example 1 of the present invention is shown at a detection wavelength of 320-242nm.

[0034] Figure 4 This is a comparative characteristic spectrum of Kaixin San and Kaixin San negative substance reference in Embodiment 1 of the present invention at a detection wavelength of 203nm. Detailed Implementation

[0035] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

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

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

[0038] Table 1

[0039]

[0040]

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

[0042] Preparation of Kaixin San (without ginseng): Take fine powders of Polygala tenuifolia, Poria cocos, and Acorus tatarinowii, and mix them evenly according to the prescription requirements in a ratio of 1:2:1.

[0043] Preparation of Kaixin San - without Polygala tenuifolia: Take ginseng, Poria cocos and Acorus tatarinowii powder, mix them evenly in a ratio of 1:2:1 according to the prescription requirements, and the product is ready.

[0044] Preparation of Kaixin San (without Poria cocos): Take fine powders of Polygala tenuifolia, ginseng, and Acorus tatarinowii, and mix them evenly in a 1:1:1 ratio according to the prescription requirements.

[0045] Preparation of Kaixin San - without Acorus tatarinowii: Take the fine powders of Polygala tenuifolia, ginseng, and Poria cocos, and mix them evenly in a ratio of 1:1:2 according to the prescription requirements.

[0046] Reagents: Acetonitrile and phosphoric acid (Fisher Chemical) were chromatographic grade; methanol and ethanol were analytical grade; and water was Watson's purified water.

[0047] Example 1

[0048] A method for constructing a reference feature map of an open-heart powder substance is described below:

[0049] 1. Preparation of the analyte solution

[0050] Preparation of the test solution: Take an appropriate amount of the test sample (Kaixin San), grind it into a fine powder, take about 2.0 g, weigh it accurately, place it in a stoppered conical flask, accurately add 25 ml of methanol, weigh it, sonicate for 30 min, cool it, add the weight, filter it, and the solution is obtained.

[0051] Reference solution: Take Siberian polygalactosidase A5 and polygalactosidase... Ketone III, 3,6'-disinyl sucrose, β-asarone, dehydrotomonic acid, pamoic acid, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, and ginsenoside Rc reference standards were accurately weighed and dissolved in methanol to prepare a solution containing 40 μg of Siberian polygalactosidase A6 and polygalactosidase per 1 ml. A solution containing 40 μg of ketone III, 85 μg of 3,6'-disinyl sucrose, 280 μg of β-asarone, 12 μg of dehydrotomonic acid, 20 μg of pachymic acid, 1100 μg of ginsenoside Rg1, 100 μg of ginsenoside Re, 100 μg of ginsenoside Rb1, and 100 μg of ginsenoside Rc is obtained.

[0052] 2. Chromatographic methods

[0053] The following chromatographic conditions were used for detection:

[0054] The column was packed with octadecylsilane and silica gel (Waters CORTECS UPLC T3, column length 150 mm, column inner diameter 2.1 mm, particle size 1.6 μm); acetonitrile was used as mobile phase A and 0.05% phosphoric acid was used as mobile phase B, and gradient elution was performed according to the specifications in Table 2; the flow rate was 0.25 mL per minute, the column temperature was 35 °C, and the detection wavelengths were 320 nm-242 nm (320 nm was switched to 242 nm at 110 min) and 203 nm, respectively. The theoretical plate number, calculated based on 3,6'-disinyl sucrose, should not be less than 5000.

[0055] Table 2

[0056]

[0057]

[0058] Accurately pipette 1 μl of the reference solution and 1 μl of the test solution into an ultra-high performance liquid chromatograph and measure them to obtain the results.

[0059] The characteristic chromatograms obtained from the test solution are as follows: Figure 1 and Figure 2 As shown. Figure 1 The image shows the characteristic spectrum of the test solution in Example 1 at a detection wavelength of 320-242 nm. Figure 2 The image shows the characteristic spectrum of the test solution in Example 1 at a detection wavelength of 203 nm.

[0060] Collect negative substance references for Kaixin San (a traditional Chinese medicine formula) and other medicinal slices lacking certain ingredients, and obtain their liquid chromatography spectra according to the methods described above. Figure 3 and Figure 4 As shown, the common peaks were assigned medicinal properties to determine the characteristic peaks. Based on the negative substance standard of Kaixin San, the peaks were assigned as follows: peaks 1-19, 23-30, 36-38, and 52 were assigned to Polygala tenuifolia slices; peaks 20, 21, 41, 42, and 48 were assigned to Acorus tatarinowii slices; peaks 31-34 and 50 were assigned to Poria cocos slices; peaks 35, 39, 40, 43-47, and 49 were assigned to Ginseng; and peak 51 was a common peak.

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

[0062] Peak 1 is Siberian polygalactosyl sugar A5; Peak 2 is Siberian polygalactosyl sugar A6; Peak 4 is polygalactosyl sugar. Ketone B; Peak 5 is globulin A; Peak 6 is Polygala tenuifolia. Ketone XI; Peak 7 is Polygala tenuifolia. Ketone III; Peak 8 is Polygala glycoside B (Polygala tenuifolia saponin B); Peak 9 is 3,6'-disinoyl sucrose; Peak 10 is Polygala tenuifolia A; Peak 11 is Polygala glycoside A; Peak 12 is Polygala glycoside C; Peak 20 is β-asarone; Peak 21 is α-asarone; Peak 31 is dehydrothomoleic acid; Peak 32 is porphyric acid C; Peak 33 is 3-epidehydrothomoleic acid; Peak 34 is dehydroporphyric acid; Peak 39 is ginsenoside Rg1; Peak 40 is ginsenoside Re; Peak 41 is methyleugenol; Peak 42 is γ-asarone; Peak 43 is ginsenoside Rb1; Peak 44 is ginsenoside Rc; Peak 50 is porphyric acid.

[0063] The chromatogram of the test sample should show 52 characteristic peaks, among which peaks 2, 7, 9, 20, 31, 39, 40, 43, 44, and 50 should be associated with Siberian Polygala A6 and Polygala tenuifolia, respectively. The retention times of the chromatographic peaks of ketone III, 3,6'-disinanoyl sucrose, β-asarone, dehydrotomonic acid, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, ginsenoside Rc, and pamoate reference standards were consistent. At a detection wavelength of 320-242 nm, the peak corresponding to the Siberian polygalactosyl sugar A6 reference peak was peak S1. The relative retention times of peaks 1, 35-38, and S1 were calculated. The peak corresponding to the ketone III reference peak is S2. Calculate the relative retention times of peaks 3–8 with peak S2. The peak corresponding to the 3,6'-disinyl sucrose reference peak is S3. Calculate the relative retention times of peaks 10–19 with peak S3. The peak corresponding to the β-asarone reference peak is S4. Calculate the relative retention times of peaks 21, 41, and 42 with peak S4. The peak corresponding to the dehydrothomoleic acid reference peak is S5. Calculate the relative retention times of peaks 22–30 and 32–34 with peak S5. The peak corresponding to the pamoate reference peak is S6. Calculate the relative retention times of peaks 45–52 with peak S6. The relative retention times of each characteristic peak are within ±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 embodiment is used to validate the chromatographic conditions in Example 1.

[0070] 1. Precision test

[0071] According to the established characteristic spectrum method, the test solution was prepared by taking the Kaixin San reference sample, and the sample was injected 6 times in a row to obtain the characteristic spectrum. The retention time and relative retention time of peak 1 to peak 52 were obtained, and the RSD and the range of the specified value were calculated.

[0072] Precision test results showed that the relative retention time (RSD) of each characteristic peak of the six samples was between 0 and 0.5%, indicating that the precision of the characteristic spectrum was good.

[0073] 2. Repeatability testing

[0074] According to the established characteristic chromatogram method, six test solutions were prepared from the Kaixin San reference sample and analyzed to obtain characteristic chromatograms. The retention times and relative retention times of peaks 1-52 were obtained, and the RSD and its range with the specified value were calculated.

[0075] The repeatability test results showed that the relative retention time RSD of each characteristic peak was between 0 and 0.5%, indicating that the repeatability of the characteristic spectrum was good.

[0076] 3. Intermediate precision test

[0077] Using a Shimadzu LC-30AD with a TUV detector, and following the established characteristic spectral method, six test solutions were prepared from the Kaixin San reference sample and analyzed to obtain characteristic spectra. The retention times and relative retention times of each characteristic peak were obtained, and the RSD values ​​were calculated.

[0078] Intermediate precision results showed that the relative retention time RSD of each characteristic peak was between 0% and 0.9%. Compared with the relative retention time data of each characteristic peak of the six samples in the repeatability test, the relative retention time RSD ranged from 0% to 3.0%, indicating that the relative retention time of the characteristic spectrum met the analytical requirements across different instruments.

[0079] 4. Stability test

[0080] Take the same test solution and measure it at 0, 3, 6, 9, 15, 18 and 24 h respectively to obtain characteristic chromatograms, and obtain the retention time and relative retention time of each characteristic peak, and calculate RSD.

[0081] Stability results showed that the relative retention times (RSD) of each characteristic peak were between 0 and 0.6%, indicating that the chemical components in the solution had good stability over 24 hours.

[0082] Example 3

[0083] A method for constructing a reference characteristic spectrum of Kaixin San (a traditional Chinese medicine) differs from Example 1 in that the preparation of the test solution is different.

[0084] (1) Selection of extraction solvent: Take an appropriate amount of Kaixin San substance standard, about 2.0g per part, accurately weigh it, place it in a stoppered conical flask, and accurately add 25ml of each of the appropriate solvents (methanol, 70% ethanol, 50% ethanol, 30% ethanol, 70% methanol, 50% methanol, 30% methanol, and water), seal tightly, weigh, sonicate (power 500W, frequency 40KHz) for 30 minutes, take it out, let it cool, weigh it again, make up the lost weight with the corresponding solvent, shake well, filter, and the product is obtained.

[0085] Accurately pipette 1 μL of the reference solution and 1 μL of the test solution into an ultra-high performance liquid chromatograph and determine the result.

[0086] Table 4 Selection of Extraction Solvent

[0087]

[0088]

[0089] Results Analysis: As can be seen from the table, when comparing peak areas based solely on the confirmed peaks, water, 50% methanol, 50% ethanol, 30% methanol, and 30% ethanol solvents resulted in incomplete extraction. Methanol and 70% methanol showed better solubility for the various index components, and 70%-100% methanol can be selected as the extraction solvent.

[0090] (2) Selection of solvent amount: Take an appropriate amount of Kaixin San substance standard, about 2.0g per part, accurately weigh it, place it in a stoppered conical flask, accurately add an appropriate amount of methanol (15ml, 25ml and 50ml) respectively, stopper tightly, weigh it, sonicate (power 500W, frequency 40KHz) for 30 minutes, take it out, let it cool, weigh it again, make up the lost weight with the corresponding methanol, shake well, filter, and it is ready.

[0091] Accurately pipette 1 μL of the reference solution and the test solution into an ultra-high performance liquid chromatograph for determination. Calculate the peak areas of each measured peak to the equivalent concentration for comparison.

[0092] Table 5 Selection of Solvent Amount

[0093]

[0094]

[0095] Results Analysis: As can be seen from the table, when comparing peak areas based solely on the confirmed peaks, although methanol has a good solubility for each peak, excessive solvent can actually hinder data reading for some peaks due to their response to the detection wavelength and the influence of component concentration. Furthermore, the peaks extracted with 15 ml and 25 ml of methanol showed little difference. Therefore, it is recommended to use a solvent volume of 15 ml to 25 ml.

[0096] (3) Selection of extraction time: Take an appropriate amount of Kaixin San substance standard, about 2.0g per portion, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of methanol, seal tightly, weigh, and sonicate (power 500W, frequency 40KHz) for different times (30 minutes, 60 minutes, 90 minutes), take it out, let it cool, weigh it again, replenish the lost weight with the corresponding methanol, shake well, filter, and the product is obtained.

[0097] Accurately pipette 1 μL of the reference solution and 1 μL of the test solution into an ultra-high performance liquid chromatograph and determine the result.

[0098] Table 6 Selection of Extraction Time

[0099]

[0100]

[0101] Results analysis: The table shows that there is no significant difference in the peak area of ​​each peak in the feature map at different extraction times, and the extraction time is determined to be no less than 30 min.

[0102] (4) Selection of extraction method: Take about 2g of Kaixin San substance as a reference, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of methanol, seal tightly, weigh it, shake and reflux for 60 minutes respectively, take it out, cool it, weigh it again, make up the weight loss with methanol, shake well, filter it, and the product is obtained.

[0103] Accurately pipette 1 μL of the reference solution and 1 μL of the test solution into an ultra-high performance liquid chromatograph and determine the result.

[0104] Table 7 Selection of Extraction Method

[0105]

[0106]

[0107] Results analysis: The table shows that there is no significant difference in the peak area of ​​each peak in the feature map under different extraction methods, and all of them can be used. However, considering the simplicity of operation, ultrasonic extraction was selected.

[0108] Example 4

[0109] A method for constructing a reference characteristic spectrum of Kaixin San (a traditional Chinese medicine formula) differs from Example 1 in that the chromatographic conditions are different, as detailed below:

[0110] (1) Observation at different column temperatures: 1 μL of the test solution was accurately pipetted into the ultra-high performance liquid chromatograph and measured at different column temperatures (32℃, 35℃, 38℃).

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

[0112]

[0113]

[0114]

[0115] Results analysis: As can be seen from the table, the relative retention times of each peak in the characteristic spectrum meet the specified fluctuation requirements under different column temperatures, indicating that the above column temperatures have good applicability.

[0116] (2) Detection at different flow rates: 1 μL of the test solution was precisely pipetted into the ultra-high performance liquid chromatograph and measured at different flow rates (0.23 ml / min, 0.25 ml / min, and 0.27 ml / min).

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

[0118]

[0119]

[0120] Results analysis: As can be seen from the table, the relative retention times of each peak in the characteristic spectrum meet the specified fluctuation requirements under different flow rates, indicating that it has good applicability to the above flow rates.

[0121] (3) Investigation of different acid concentrations: 1 μL of the test solution was precisely pipetted into the ultra-high performance liquid chromatograph and measured under mobile phase B (0.025% phosphoric acid, 0.05% phosphoric acid, 0.10% phosphoric acid) with different acidities.

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

[0123]

[0124]

[0125] Results analysis: As can be seen from the table, under mobile phases with different acidities, the relative retention times of each peak in the characteristic spectrum meet the specified fluctuation requirements, indicating that it has good applicability to mobile phases with the above acidities.

[0126] (4) Examination of different wavelengths: 1 μL of the test solution was precisely pipetted into the ultra-high performance liquid chromatograph and measured at the specified wavelength ±5 nm (198 nm and 315 nm were switched to 237 nm, and 208 nm and 325 nm were switched to 247 nm).

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

[0128]

[0129]

[0130] Results analysis: As can be seen from the table, under wavelength detection with a specified wavelength fluctuation of ±5nm, the relative retention times of each peak in the characteristic spectrum meet the specified fluctuation requirements, indicating that it has good applicability to the above wavelength.

[0131] (5) Investigation of different chromatographic columns: 1 μL of the test solution was accurately pipetted into the ultra-high performance liquid chromatograph and measured using different chromatographic columns: column 1 (Phenomenex Luna Omega, 1.6 μm 150*2.1 mm), column 2 (Waters CORTECS UPLC T3, 1.6 μm 150*2.1 mm), and column 3 (Yuxu 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] Results Analysis: The table shows that the characteristic chromatograms obtained using different chromatographic columns all meet the specified fluctuation requirements for the relative retention times of each peak, indicating that the above chromatographic columns have good applicability.

[0136] (6) Examination of wavelength switching time: Considering that there is wavelength switching at a wavelength of 320nm, the time of wavelength switching was examined. The situation of the characteristic peaks before and after the corresponding characteristic spectrum at 100min and 110min was examined, mainly the influence on characteristic peaks 27 to 31.

[0137] Table 13 Retention time and peak area of ​​characteristic peaks under different time-switched wavelengths

[0138]

[0139] Results analysis: The table shows that the retention time and peak area of ​​the characteristic peaks did not change significantly under different switching times, indicating that the above switching times have good applicability.

[0140] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for constructing a characteristic map of a kaempferia galanga material reference, characterized by, The characteristic spectrum of the test substance is obtained by using ultra-high performance liquid chromatography; the chromatographic conditions of the ultra-high performance liquid chromatography are as follows: The chromatographic column is filled with octadecylsilane-bonded silica gel; acetonitrile is used as the mobile phase A, and 0.025%-0.1% phosphoric acid aqueous solution is used as the mobile phase B, and elution is carried out according to the following gradient program: The detection wavelength is 198-208 nm to obtain the characteristic spectrum of 198-208 nm, and the detection wavelength is 315-325 nm to obtain the characteristic spectrum of 315-325 nm, wherein the detection wavelength is switched to 237-247 nm at t min when the detection wavelength is 315-325 nm, t is 100-110; 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; The preparation process of the test solution is as follows: the reference substance of kai heart powder is accurately weighed, placed in a conical flask with a plug, solvent is added, weighed, extracted and treated, cooled, weighed, filtered, and obtained. The preparation process of the reference solution of the control substance is as follows: the control substance is accurately weighed, and the solvent is added to prepare the reference solution of the control substance. The control includes shanzhiside A6, polygalae radix ketonic III, 3,6'-dihydroxybenzylsucrose, β-asarone, dehydrotoacumic acid, pachymic acid, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1 and ginsenoside Rc; The solvent is methanol or methanol aqueous solution with a volume concentration of more than 70%.

2. The construction method according to claim 1, characterized in that, The column temperature is 32-38℃; And / or, the flow rate is 0.23-0.27 ml / min; And / or, the theoretical plate number calculated according to the 3,6'-dijeracyl sucrose peak should be not less than 5000.

3. The construction method of claim 1, wherein, In the ultra-high performance liquid chromatography, the chromatographic column is Phenomenex Luna Omega, Waters CORTECS UPLC T3, or Yue Xiu Ultimate UHPLC LP-C18.

4. The construction method of claim 1, wherein, The extraction treatment mode includes one of reflux treatment, ultrasonic treatment, and shaking treatment; the extraction treatment time is 30-60 min.

5. The construction method according to any one of claims 1 to 4, characterized in that, The characteristic spectrum is obtained by using the detection wavelength of 320 nm switched to 242 nm, and the characteristic spectrum includes the characteristic peaks of peaks 1-34; the characteristic spectrum is obtained by using the detection wavelength of 203 nm, and the characteristic spectrum includes the characteristic peaks of peaks 35-52.

6. The construction method of claim 5, wherein, 52 characteristic peaks, peak 1: polygalaxyl A5, peak 2: polygalaxyl A6, peak 4: polygala ketone B, peak 5: globularoside A, peak 6: polygala ketone XI, peak 7: polygala ketone III, peak 8: polygala glucoside B, peak 9: 3,6'-diacylsucrose, peak 10: polygalacin A, peak 11: polygala glucoside A, peak 12: polygala glucoside C, peak 20: β-asarone, peak 21: α-asarone, peak 31: dehydrotram-10-acetic acid, peak 32: poria acid C, peak 33: 3-epidehydrotram-10-acetic acid, peak 34: dehydrotram-10-acetic 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.

7. The construction method of claim 6, wherein, The peak corresponding to the reference peak of Siberian milkwort sugar A6 is S1 peak, and the relative retention time of peak 1, peak 35 to peak 38 and S1 peak is calculated; the peak corresponding to the reference peak of milkwort The peak corresponding to the reference peak of ketone III is S2, and the relative retention time of peak 3 to peak 8 and S2 peak is calculated; the peak corresponding to the reference peak of 3,6'-dithioglucoside is S3 peak, and the relative retention time of peak 10 to peak 19 and S3 peak is calculated; the peak corresponding to the reference peak of β-asarone is S4 peak, and the relative retention time of peak 21, peak 41 and peak 42 and S4 peak is calculated; the peak corresponding to the reference peak of dehydrogenated tomosic acid is S5 peak, and the relative retention time of peak 22 to peak 30, peak 32 to peak 34 and S5 peak is calculated; the peak corresponding to the reference peak of Poria cocos acid is S6 peak, and the relative retention time of peak 45 to peak 52 and S6 peak is calculated; the relative retention time of each characteristic peak is within ±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.

8. The use of the characteristic pattern constructed by the construction method of any one of claims 1-7 in the overall quality control of the material basis of KaiXinSan.

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