A method for constructing a characteristic spectrum of volatile oil in Qingshang Juantong Decoction and a method for detecting Qingshang Juantong Decoction samples

Abstract

The present invention relates to a method for constructing a characteristic spectrum of volatile oil in Qingshang Juantong Decoction and a method for detecting Qingshang Juantong Decoction samples. By using an ultra-high performance liquid chromatography (ULPC) detection method, the chromatographic conditions are rationally controlled to confirm the common peaks of the Qingshang Juantong Decoction standard decoction, and to construct a characteristic spectrum of volatile oil in Qingshang Juantong Decoction. The characteristic spectrum of the present invention comprises 12 characteristic peaks, wherein peak 8, peak 9, and peak 11 are reference substances methyl eugenol, ligustilide A, and ligustilide, respectively. The method is widely applicable to the quality inspection of Qingshang Juantong Decoction benchmark samples, preparation intermediates, and / or finished preparations, can more comprehensively reflect product quality, has high analysis efficiency, is convenient to operate, and is suitable for quality monitoring of continuous production.

Description

Technical Field

[0001] The present invention relates to the fields of traditional Chinese medicine analysis and quality control, and in particular to a method for constructing a characteristic spectrum of volatile oil in Qingshangjuantong Decoction, and a method for detecting Qingshangjuantong Decoction samples using the characteristic spectrum. Background Art

[0002] Qingshang Quntong Decoction, recorded in the "Shoushibaoyuan" (author Gong Tingxian of the Ming Dynasty), is the first prescription in the headache section of Volume 6 and a famous prescription for treating headaches. In 2018, China published the first batch of "Catalogue of Ancient Classic Prescriptions," and this prescription was listed as number 66. The full formula consists of thirteen ingredients: angelica sinensis (wine-scented), ligusticum chuanxiong (Chuanxiong), angelica dahurica (Angelica dahurica), asarum, notopterygium wilfordii (Rhizoma Angelicae Dahuricae), duhuo (Puliflora pubescens), siler (Saposhnikovia divaricata), chrysanthemum (Flos Chrysanthemi), vitex rotundifolia (Vitex mandshuricae), atractylodes lancea (Atractylodes macrocephala), scutellaria baicalensis (wine-scented), ophiopogon japonicus (Ophiopogon japonicus), and liquorice. It has the effects of dispelling wind, cold, and dampness, promoting qi and blood circulation, and dredge meridians to relieve pain. It is commonly used clinically to treat various headaches.

[0003] There are many volatile ingredients in Qingshang Quntong Decoction, among which: Angelica dahurica volatile oil has the pharmacological effects of lowering blood pressure, improving cerebral ischemia, anti-inflammatory and analgesic; Chuanxiong volatile oil has the pharmacological effects of protecting cardiovascular and cerebrovascular, relieving spasms, relieving fever, relieving pain, anti-inflammatory and anti-cerebral ischemia; Asarum volatile oil has the pharmacological effects of anesthesia, anti-inflammatory, anticancer, anticonvulsant, antidepressant, antipyretic and analgesic; Notopterygium incisum volatile oil has the pharmacological effects of anti-inflammatory and analgesia. For example, the research of Zhou Yi et al. showed that the substance with analgesic effect in Notopterygium incisum is not the water-soluble component but the volatile oil (Zhou Yi et al., Study on the Origin and Analgesic Effect of Notopterygium in Sichuan [J], Pharmacology and Clinic of Chinese Medicine, 2003(06):22-23).

[0004] Currently, research on Qingshang Juantong Decoction (QSJT) has primarily focused on its clinical use. Studies have also examined the fingerprints, characteristic profiles, decoction processes, content determination, and quality control of its water-soluble components. For example, Reference 1 discloses a method for constructing a characteristic profile for QSJT, using chlorogenic acid, ferulic acid, peucedanum praeruptoside, 3,5-O-dicaffeoylquinic acid, baicalin, wogonin, baicalein, vitexin, and ligustilide as characteristic components, but only ligustilide is a volatile component. Reference 2 also addresses a quality control method for QSJT, but its reference substances still use non-volatile components such as baicalin, wogonin, vitexin, ammonium glycyrrhizate, and cimicifuga glycosides. However, research on the characteristic profiles, content determination, and quality control of the volatile oil components that contribute to the efficacy and play a major role in QSJT has yet to be reported.

[0005] In summary, current research on the volatile oils in Qingshang Juantong Decoction is far from in-depth. Problems include an incomplete qualitative evaluation system for medicinal flavors and a lack of quality control for volatility indicators. Because volatile oils are the primary active ingredients in Qingshang Juantong Decoction that contribute to its clinical efficacy, such as promoting qi flow and relieving pain, it is necessary to establish a characteristic profile for the volatile oil components in Qingshang Juantong Decoction.

[0006] References:

[0007] Reference 1: CN114646707A

[0008] Reference 2: CN114965734A Summary of the Invention

[0009] Problems to be solved by the invention

[0010] In order to solve the above-mentioned problems of the prior art, the object of the present invention is to provide a method for constructing a characteristic spectrum of volatile oil in Qingshang Juantong Decoction. The characteristic spectrum constructed by this method can be used for quality evaluation of Qingshang Juantong Decoction benchmark samples, preparation intermediates and / or preparation finished products, etc., and is particularly suitable for quality control and evaluation of Qingshang Juantong Decoction standard decoction.

[0011] Solutions for solving problems

[0012] Through long-term research, the inventors found that the above technical problems can be solved by implementing the following technical solutions:

[0013] [1] The present invention provides a method for constructing a characteristic spectrum of volatile oil in Qingshang Juantong Decoction, which comprises the following steps:

[0014] (1) preparing a volatile oil test solution and a reference solution, wherein the test sample is a reference sample of Qingshang Juantong Decoction, a preparation intermediate, and / or a preparation finished product; and the reference solution comprises at least one of methyl eugenol, ligustilide A, and ligustilide;

[0015] (2) a step of detecting and establishing a characteristic spectrum, wherein the volatile oil test solution and the reference solution are detected by ultra-high performance liquid chromatography, and the results are analyzed by a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to establish a characteristic spectrum;

[0016] Wherein, the chromatographic conditions of the ultra-high performance liquid chromatography method include:

[0017] The stationary phase is a chromatographic column filled with alkylsilane bonded silica gel.

[0018] Mobile phase A is acetonitrile, mobile phase B is water or 0.01% to 0.1% phosphoric acid aqueous solution by volume,

[0019] Elution was performed according to the following gradient elution program:

[0020]

[0021] [2] The construction method according to [1], wherein the characteristic spectrum comprises at least 3, preferably at least 6, more preferably at least 9, and further preferably 12 characteristic peaks;

[0022] When the characteristic spectrum contains 12 characteristic peaks, peak 8, peak 9 and peak 11 correspond to the chromatographic peaks of methyl eugenol, ligustilide A and ligustilide reference substances, respectively; taking peak 8 as the S peak, the relative retention times of the other characteristic peaks and the S peak are calculated, and the relative retention times should be within ±10% of the specified values, and the specified values ​​are: peak 1: 0.409, peak 2: 0.491, peak 3: 0.629, peak 4: 0.698, peak 5: 0.774, peak 6: 0.889, peak 7: 0.955, peak 8: 1, peak 9: 1.079, peak 10: 1.228, peak 11: 1.868, peak 12: 2.016.

[0023] [3] The construction method according to [1] or [2], wherein the step of preparing the volatile oil test solution comprises preparing a standard decoction, extracting the volatile oil in the standard decoction, removing the volatile oil and diluting it with methanol to a concentration of 3% to 8%.

[0024] [4]. The construction method according to any one of the technical solutions [1] to [3], wherein the reference substance solution is a solution of the reference substance in alcohol or an alcohol-water mixed solvent; preferably, the alcohol or alcohol-water mixed solvent is methanol or a methanol-water mixed solvent, and the methanol-water mixed solvent is a methanol-water mixed solvent having a volume fraction of methanol of 70% to 90%.

[0025] [5] The construction method according to any one of the technical solutions [1] to [4], wherein the concentrations of the reference solution are: methyl eugenol: 2.8-3.7 mg / mL, ligustilide A: 1.5-2.3 mg / mL, and ligustilide: 0.05-0.2 mg / mL;

[0026] Preferably, the concentrations of the reference solution are: methyl eugenol: 3.340 mg / mL, ligustilide A: 1.960 mg / mL, and ligustilide: 0.1 mg / mL.

[0027] [6]. The construction method according to any one of the technical solutions [1] to [5], wherein the ultra-high performance liquid chromatography conditions further include: a detection wavelength of 210 to 280 nm, a flow rate of 0.28 to 0.32 mL / min, and a column temperature of 28 to 32°C; and the injection volume of the detection is 0.5 μL.

[0028] [7] The construction method according to any one of the technical solutions [1] to [6], wherein the chromatographic column is a C18 chromatographic column with a column length of 100 mm and a filler particle size of 1.6 to 1.9 μm;

[0029] Preferably, the C18 chromatographic column is selected from any one of Waters ACQUITY (BEH C18 2.1×100mm1.7μm), Waters CORTECS C18 (2.1×100mm, 1.6μm), and Thermo GOLD (Hypersil GOLD 2.1×100mm, 1.9μm).

[0030] [8]. The construction method according to any one of the technical solutions [1] to [7], wherein the test sample is the standard decoction of Qingshang Juantong Decoction.

[0031] [9]. In addition, the present invention also provides a method for detecting a sample of Qingshang Juantong Decoction, which comprises the following steps:

[0032] The sample of Qingshang Juantong Decoction to be tested is extracted using water as a solvent, the extract is collected, and a volatile oil test solution is further prepared by extraction; the sample of Qingshang Juantong Decoction is a reference sample, an intermediate product of a preparation, and / or a finished preparation of Qingshang Juantong Decoction; further, the sample of Qingshang Juantong Decoction is a standard decoction of Qingshang Juantong Decoction;

[0033] The volatile oil sample solution is tested by ultra-high performance liquid chromatography, and the detection spectrum obtained is compared with the characteristic spectrum constructed by the construction method according to any one of claims 1 to 8 to detect the quality of the Qingshang Quntong Decoction sample;

[0034] UPLC conditions included:

[0035] The stationary phase is a chromatographic column filled with alkylsilane bonded silica gel.

[0036] Mobile phase A is acetonitrile, mobile phase B is water or 0.01% to 0.1% phosphoric acid aqueous solution by volume,

[0037] Elution was performed according to the following gradient elution program:

[0038]

[0039]

[10] The detection method according to [9], wherein the extraction solvent is methanol or a methanol-water solution with a methanol volume percentage of not less than 70%;

[0040] The quality of the Qingshang Juantong Decoction sample is detected by comparing the detection spectrum with the characteristic spectrum. If the two are consistent, the Qingshang Juantong Decoction sample is qualified.

[0041] If the two are inconsistent, the Qingshang Quntong Decoction sample is unqualified.

[0042] Effects of the Invention

[0043] 1. The construction method of the present invention identified 12 common peaks as the characteristic peaks of volatile oil in the standard decoction of Qingshang Juantong Decoction, and analyzed the chemical information of all characteristic peaks by mass spectrometry. Among them, the reference substance identified 3 peaks, and the medicinal sources of the characteristic peaks were attributed and studied, providing a scientific experimental basis for tracing the source of product quality.

[0044] 2. The present invention adopts ultra-high performance liquid chromatography for detection and analysis, with a detection time of 32 minutes. Compared with HPLC, it has high analysis efficiency, saves time, consumes less solvent, and has little pollution to the environment.

[0045] 3. This method is simple to operate, accurate, and reliable. The established spectrum shows good resolution of characteristic peaks, strong component specificity, and good reproducibility. It is suitable for the quality evaluation of Qingshang Juantong Decoction reference samples, intermediates, and / or finished products, and is particularly suitable for the quality control and evaluation of Qingshang Juantong Decoction standard decoctions. It is also suitable for the detection and monitoring of large-scale production.

[0046] 4. The present invention uses the extracted volatile oil from the standard decoction of Qingshang Juantong Decoction as the test sample for detection. The characteristic spectrum constructed by the method can comprehensively characterize the characteristics and information of the volatile active ingredients of the four medicinal flavors of Angelica sinensis, Chuanxiong, Notopterygium wilfordii, and Asarum in Qingshang Juantong Decoction, which makes up for the defects of the complex properties of the volatile oil components in the traditional Chinese medicine compound preparations, the difficulty in collecting them, and the insufficient detection methods. It provides scientific guidance for the research on the preparation process of Qingshang Juantong Decoction, conducts more comprehensive control and evaluation of product quality, and ensures the consistency of the quality and clinical efficacy of the medicine and clinical decoction.

[0047] It should be noted that the above description does not disclose all embodiments of the present invention and all advantages of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is the result of investigating the detection wavelength in Example 1 of the present invention.

[0049] Figure 2 This is the result of investigating the mobile phase in Example 1 of the present invention.

[0050] Figure 3 This is the investigation result of the chromatographic column in Example 1 of the present invention.

[0051] Figure 4 This is the result of investigating the elution time gradients (A) to (G) in Example 1 of the present invention.

[0052] Figure 5 This is the superimposed characteristic spectrum of 15 batches of standard decoctions of Qingshang Quntong Decoction in Example 1 of the present invention.

[0053] Figure 6 This is the characteristic spectrum of the volatile oil of the standard decoction of Qingshangjuantong Decoction in Example 1 of the present invention. DETAILED DESCRIPTION

[0054] The following describes the embodiments of the present invention in detail, but the present invention is not limited thereto. It should be understood by those skilled in the art that the present invention can be implemented without certain specific details. In other examples, methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present invention. Unless otherwise defined, the technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art in the art to which the present invention belongs.

[0055] The present invention is not limited to the various configurations described below. Various modifications are possible within the scope of the invention. Embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included in the technical scope of the present invention. In addition, all documents listed in this specification are cited as references in this specification.

[0056] In the context of describing this specification (especially in the context of the following claims), the terms "a," "an," and "the" and similar language are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0057] In this specification, a numerical range expressed using "numerical value A to numerical value B" or "numerical value A - numerical value B" means a range including the endpoints numerical values ​​A and B.

[0058] In this specification, the use of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process. In this specification, "optional" or "optionally" means that the event or situation described below may or may not occur, and the description includes both cases where the event occurs and cases where the event does not occur.

[0059] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "some specific / preferred technical solutions," "other specific / preferred technical solutions," etc., mean that the specific elements (e.g., features, structures, properties, and / or characteristics) described in connection with the embodiment are included in at least one embodiment described herein and may or may not be present in other embodiments. Furthermore, it should be understood that the elements may be combined in various embodiments in any appropriate manner.

[0060] The term "comprises" and any variations thereof in the description and claims of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, system, product, or apparatus comprising a series of steps or elements is not limited to the listed steps or elements, but may optionally include steps or elements not listed, or may optionally include other steps or elements inherent to the process, method, product, or apparatus.

[0061] Unless otherwise specified, the percentage contents mentioned in the present invention refer to mass percentage for solid-liquid mixture and solid-solid mixture, and refer to volume percentage for liquid-liquid mixture.

[0062] The percentage concentrations mentioned in the present invention, unless otherwise specified, refer to the final concentration, which refers to the percentage of the added component in the system after the addition of the component.

[0063] The temperature parameters in the present invention, unless otherwise specified, allow for either constant temperature treatment or treatment within a certain temperature range. The constant temperature treatment allows for temperature fluctuations within the precision range controlled by the instrument.

[0064] Unless otherwise specified, the term "reference substance" used in this article refers to a standard substance used for identification, inspection, content determination and calibration of instrument performance. It is usually reviewed and approved by the national drug testing agency, and its standard should not be lower than the quality standard of the product.

[0065] Unless otherwise specified, the term "test article" used herein refers to an experimental sample used for detection or identification.

[0066] Unless otherwise specified, the terms "precision weighing or precision weighing" used in this article mean that the weight should be weighed accurately to one thousandth of the weight taken, the term "weighing" means that the weight should be weighed accurately to one hundredth of the weight taken, the term "precision addition" means that the volume should be measured accurately to one thousandth of the volume taken, and the term "precision aspiration" refers to the operation method of accurately measuring samples through a microinjector.

[0067] Unless otherwise specified, the term "subsequent filtrate" as used herein refers to the filtrate collected after the primary filtrate is discarded during filtration. Compared to the primary filtrate, the subsequent filtrate is closer to the true concentration of the sample because the filter medium (such as the membrane or filter paper) may adsorb solutes, resulting in a lower sample concentration in the primary filtrate. Furthermore, the subsequent filtrate is cleaner because solutes adsorbed by the filter medium form a filter cake, reducing the filter pore size and thus retaining smaller particles.

[0068] Unless otherwise specified, the term "ultra-performance liquid chromatography" (or "UPLC") used in this article refers to a new technology developed based on high-performance liquid chromatography (HPLC) that has the characteristics of small filler particles, fast detection speed, large analytical throughput, and high sensitivity.

[0069] Unless otherwise specified, the term "retention time" used in this article refers to the time from the start of injection to the appearance of the maximum concentration of the separated component after the column, that is, the time from the start of injection to the appearance of the apex of the chromatographic peak of the separated component; the term "relative retention time" refers to the ratio of the calibrated retention time of the separated component to the calibrated retention time of the standard sample.

[0070] Unless otherwise specified, the term "reference peak" (or "control peak") used herein refers to the chromatographic peak corresponding to the standard sample when calculating the relative retention time.

[0071] Unless otherwise noted, percentages (%) in solution concentrations are by volume. For example, a 0.01% phosphoric acid aqueous solution is equivalent to adding 0.1 mL of phosphoric acid to 1000 mL of pure water. Since graduated cylinders are not recommended for mixing solutions, a 0.01% phosphoric acid aqueous solution is also equivalent to mixing 1000 mL of pure water with 0.1 mL of phosphoric acid.

[0072] The present invention provides a method for constructing a characteristic spectrum of volatile oil in Qingshangjuantong Decoction. The constructed characteristic spectrum can be used for quality detection of Qingshangjuantong Decoction (it can also be used as a quality control method or a quality evaluation method, mainly a qualitative identification method). That is, the present invention also provides a method for extracting, preparing and detecting volatile components in Qingshangjuantong Decoction (i.e., a sample to be tested). The method comprises: comparing a liquid chromatogram of the sample to be tested with a characteristic spectrum of the volatile oil in Qingshangjuantong Decoction (including studying the characteristic spectrum of the volatile oil in a standard decoction of Qingshangjuantong Decoction by using ultra-high performance liquid chromatography).

[0073] (Construction method of characteristic spectrum of volatile oil in Qingshang Juantong Decoction)

[0074] The method for constructing a characteristic spectrum of volatile oil in Qingshang Juantong Decoction provided by the present invention mainly comprises the steps of preparing a test solution and a reference solution, and the steps of detecting and establishing the characteristic spectrum.

[0075] Prepare test solution

[0076] A standard decoction is prepared according to 6 times the original prescription amount, and then the volatile oil in the standard decoction is extracted. The volatile oil in the extractor is then collected with 0.5-2 mL of ethyl acetate solution, and 100 uL of the volatile oil is then transferred and diluted with methanol in a 2 mL volumetric flask to a concentration of 3% to 8%, preferably, 3.5% to 7.2%; more preferably, the concentration is 4% to 6.5%; more preferably, the concentration is 4.5% to 5.5%.

[0077] In some specific embodiments of the present invention, the steps of preparing the test solution are specifically as follows:

[0078] A standard decoction was prepared according to 6 times the original prescription amount, i.e., 22.38 g of wine-soaked angelica, 22.38 g of Chuanxiong, 22.38 g of Angelica dahurica, 6.72 g of Asarum, 22.38 g of Notopterygium wilfordii, 22.38 g of Pubescent Angelica pubescent, 22.38 g of Saposhnikovia divaricata, 11.22 g of Chrysanthemum Flos, 11.22 g of Vitex Mangifera Indica, 22.38 g of Atractylodes lancea, 33.60 g of wine-soaked Scutellaria baicalensis, 22.38 g of Ophiopogon japonicus, 6.72 g of Licorice, and 22.38 g of ginger were mixed, crushed and sieved, added with appropriate amount of water, soaked for 30 min, boiled and decocted for 20 min, filtered while hot, added appropriate amount of water to the residue, boiled and decocted for 15 min, filtered while hot, and the two filtrates were combined to obtain the standard decoction of Qingshang Quntong Decoction.

[0079] Transfer the prepared Qingshang Juantong Decoction standard decoction while hot to a round-bottom flask, add a few glass beads, connect the volatile oil extraction device, add water from the upper end of the collector to fill the scale part of the volatile oil analyzer and overflow into the flask, add 0.5-2 mL of ethyl acetate from the upper end of the extractor, preferably 1 mL of ethyl acetate, heat with an electric heating mantle and maintain a slight boiling state, reflux for 4 hours and then cool, collect the ethyl acetate layer into a 2 mL volumetric flask, wash the condenser and the inner wall of the volatile oil extractor with ethyl acetate, combine the washing liquid into the same volumetric flask, add ethyl acetate to the scale, shake well, and obtain the Qingshang Juantong Decoction standard decoction mixed oil.

[0080] If the concentration of the test sample is too low, it will be detrimental to the detection of the chromatographic peak. If the concentration is too high, there may be concerns about poor chromatographic peak shape due to overloading of the chromatographic column. In the present invention, 100 μL of the collected mixed oil is preferably transferred to a 2 mL volumetric flask, diluted with methanol and fixed to volume, shaken, filtered, and the filtrate is obtained.

[0081] In some specific embodiments of the present invention, the test sample can be a Qingshang Quntong Decoction benchmark sample, a preparation intermediate product and / or a finished preparation. Wherein, the "benchmark sample" refers to a Chinese medicinal substance prepared based on the preparation method of ancient classic prescriptions recorded in ancient medical books. Except for the molding process, the other preparation methods should be basically consistent with the records in ancient medical books. "Preparation intermediate product" refers to an intermediate product made from raw materials and auxiliary materials into a finished preparation, such as a thick draft of Chinese medicine extract that has not been made into granules, a dry extract powder, etc., wherein the preparation intermediate also includes a standard decoction, etc., and a "standard decoction" refers to a standard decoction that meets certain quality standards and is prepared according to a standardized production process, for example, an alcohol (such as methanol, ethanol, etc.) extract, an aqueous extract, etc. "Finished preparation" refers to a final product that can be directly supplied to the terminal. Preparations generally refer to various dosage forms, such as pastes, pills, pills, powders, tablets or injections and finished products for special purposes.

[0082] Preparation of reference solution

[0083] Accurately weigh appropriate amounts of reference substances methyl eugenol, ligustilide A and ligustilide, and prepare reference substance solutions with concentrations of 2.8-3.7 mg / mL, 1.5-2.3 mg / mL and 0.05-0.2 mg / mL, respectively, with alcohol or alcohol-water solution. In order to obtain a better peak type (or peak shape), taking into account the total extraction efficiency, it is preferably prepared with methanol or methanol-water solution; more preferably, methanol or a methanol-water solution with a volume fraction of methanol of 70% to 90% is used, and further preferably, methanol is used to prepare the reference substance solution.

[0084] In order to further optimize the extraction effect and material dosage, the concentrations of methyl eugenol, ligustilide A and ligustilide reference solutions were 3.340 mg / mL, 1.960 mg / mL and 0.1 mg / mL, respectively.

[0085] Detection

[0086] The present invention has determined, through in-depth research, ultra-high performance liquid chromatography conditions that are applicable to Qingshang Juantong Decoction reference samples, preparation intermediates and / or finished preparations, especially the standard decoction of Qingshang Juantong Decoction, which include:

[0087] Stationary phase: A chromatographic column with an alkylsilane bonded silica gel as a filler, preferably a C18 chromatographic column with a column length of 100 mm and a filler particle size of 1.6 to 1.9 μm. In some specific embodiments of the present invention, the C18 chromatographic column is selected from any one of Waters ACQUITY (BEH C18 2.1×100 mm 1.7 μm), Waters CORTECS C18 (2.1×100 mm, 1.6 μm), and ThermoGOLD (Hypersil GOLD 100×2.1 mm 1.9 μm). Furthermore, the present invention uses a Waters CORTECS C18 (2.1×100 mm, 1.6 μm) chromatographic column. Using this chromatographic column for separation results in better separation of characteristic peaks and a more beautiful overall spectrum.

[0088] Mobile phase: Mobile phase A is acetonitrile, and mobile phase B is water or a 0.01% to 0.1% by volume phosphoric acid aqueous solution; preferably, water is used as mobile phase B;

[0089] The gradient elution procedure of the present invention is as follows:

[0090]

[0091] The use of these conditions can ensure that all chromatographic peaks can be fully separated and have good peak shapes, high responsiveness, short analysis cycles, and higher separation efficiency and resolution, regardless of whether it is a Qingshang Quntong Decoction benchmark sample, a preparation intermediate, and / or a preparation finished product.

[0092] The detector used in the present invention can be an ultraviolet detector, an evaporative light scattering detector or a charged aerosol detector; in some preferred embodiments of the present invention, an ultraviolet detector is preferably used, and the detection wavelength is 210nm to 280nm. In some preferred embodiments of the present invention, the detection wavelength is 235nm. At this detection wavelength, the chromatographic peak information is relatively rich, the separation effect of each chromatographic peak is good, and the baseline is relatively stable. Furthermore, the chromatographic conditions of the present invention also include: a flow rate of 0.25mL / min to 0.35mL / min; and / or, a column temperature of 25℃ to 35℃. Within the range of column temperature or flow rate, the construction method of the present invention can meet the system applicability requirements with smaller column temperature changes or flow rate changes. In some preferred embodiments of the present invention, the flow rate can be 0.28mL / min to 0.32mL / min; and / or, the column temperature can be 28℃ to 32℃.

[0093] In some specific embodiments of the present invention, 0.5 μL of the test solution and the reference solution are accurately aspirated and injected into an ultra-high performance liquid chromatograph for detection.

[0094] Building a feature map

[0095] The present invention uses the "Chinese Herbal Medicine Chromatographic Characteristic Spectrum Similarity Evaluation System (2012 Edition)" recommended by the National Pharmacopoeia Committee for result analysis. Based on the peak attribution, the common peaks are selected as characteristic peaks. Peak 8, methyl eugenol, is used as the reference peak. The relative retention time and relative peak area of ​​each characteristic peak are calculated. The characteristic peak superposition spectrum is fitted to generate a characteristic reference spectrum of the common pattern and establish a UPLC characteristic spectrum. Based on practical feasibility, the present invention determines 12 of the common peaks as the characteristic peaks of the volatile oil of the Qingshang Juantong Decoction standard decoction. Among them, peaks 8, 9, and 11 correspond to the retention times of the reference peaks of methyl eugenol, ligustilide A, and ligustilide, respectively. Taking Peak 8 with good peak shape and large separation as the reference peak (S peak), the relative retention times of each peak were calculated. The relative retention times were: Peak 1: 0.35-0.47, Peak 2: 0.42-0.57, Peak 3: 0.54-0.72, Peak 4: 0.59-0.80, Peak 5: 0.65-0.89, Peak 6: 0.76-1.02, Peak 7: 0.82-1.10, Peak 8: 1.00, Peak 9: 0.92-1.24, Peak 10: 1.05-1.41, Peak 11: 1.59-2.15, and Peak 12: 1.87-2.32.

[0096] Since the relative retention time of each peak should be within the range of ±10% of the specified value, the specified values ​​are Peak 1: 0.409, Peak 2: 0.491, Peak 3: 0.629, Peak 4: 0.698, Peak 5: 0.774, Peak 6: 0.889, Peak 7: 0.955, Peak 8: 1, Peak 9: 1.079, Peak 10: 1.228, Peak 11: 1.868, Peak 12: 2.016.

[0097] The present invention shortens analysis time and improves analysis efficiency by optimizing the preparation of a test solution, the preparation of a reference solution, chromatographic analysis conditions, etc., establishes ultra-high performance liquid chromatography conditions that are applicable to a Qingshang Juantong Decoction benchmark sample, a preparation intermediate product, and / or a preparation finished product, and are particularly applicable to a standard decoction of Qingshang Juantong Decoction, truly reflects the quality of volatile oil in the preparation process of Qingshang Juantong Decoction, and can be used for establishing the pharmaceutical quality standard and quality control of Qingshang Juantong Decoction.

[0098] (Testing Method for Qingshang Juantong Decoction Samples)

[0099] On the other hand, the characteristic spectrum constructed by the present invention can be used for the detection of Qingshang Juantong Decoction reference samples, preparation intermediates and / or preparation finished products; in particular, the detection of Qingshang Juantong Decoction standard decoction. Therefore, the present invention also provides a method for detecting Qingshang Juantong Decoction samples, the detection method comprising the following steps:

[0100] The Qingshang Juantong Decoction sample to be tested is extracted using water as a solvent, the extract is collected, and a volatile oil test solution is further prepared by extraction; the Qingshang Juantong Decoction sample is a Qingshang Juantong Decoction benchmark sample, a preparation intermediate product and / or a preparation finished product; further, it is a Qingshang Juantong Decoction standard decoction; in some specific embodiments of the present invention, the extraction solvent is methanol or a methanol aqueous solution with a methanol volume percentage of not less than 70%; preferably, the extraction solvent is methanol.

[0101] The volatile oil test solution is detected by ultra-high performance liquid chromatography, and the detection spectrum obtained by the detection is compared with the characteristic spectrum constructed by the construction method of the present invention to detect the quality of the Qingshang Juantong Decoction sample; further, if the two are consistent, the Qingshang Juantong Decoction sample is a qualified product; if the two are inconsistent, the Qingshang Juantong Decoction sample is an unqualified product.

[0102] The ultra-high performance liquid chromatography conditions are basically the same as those used in the aforementioned construction method, including: the stationary phase is a chromatographic column filled with alkylsilane bonded silica gel, the mobile phase A is acetonitrile, and the mobile phase B is water or a 0.01% to 0.1% by volume phosphoric acid aqueous solution, preferably water is used as the mobile phase B, and elution is performed according to the following gradient elution program:

[0103]

[0104]

[0105] The construction and detection method of the present invention provides a new analytical means for the intrinsic quality control of Qingshang Juantong Decoction reference samples, preparation intermediates and / or preparation finished products. The method is simple to operate, has high precision, good stability, good repeatability and high accuracy, and provides an important multi-index parameter basis for evaluating process stability and quality control of Qingshang Juantong Decoction.

[0106] Example

[0107] The present invention will be further described below through specific examples. It should be understood that the following examples are only used to explain and illustrate the present invention, and are not used to limit the scope of protection of the present invention.

[0108] Laboratory instruments, reagents and medicines

[0109] Agilent 1290 ultra-high performance liquid chromatograph (Agilent, USA); Agilent 6530 quadrupole tandem time-of-flight mass spectrometer with electrospray ionization (ESI); MassHunter Data Acquisition workstation and MassHunter Qualiative Analysis workstation (Agilent, USA); JJ500 electronic balance (Changshu Shuangjie Testing Instrument Factory); Mettler Toledo ME204 1 / 10,000 electronic analytical balance (Shanghai Mettler-Toledo Instrument Co., Ltd.); POMEX volatile oil extractor (Beijing North Glass Bomei Glass Co., Ltd.); ZNHW5000ML intelligent digital display constant temperature electric heating mantle (Zhengzhou Ketai Experimental Equipment Co., Ltd.); YMW 3L 500W ceramic health pot (Shenzhen Yimeiwang E-commerce Co., Ltd.); methanol and acetonitrile were chromatographic grade (Thermo Fisher Scientific). Fisher Company); water was ultrapure water; formic acid, phosphoric acid, and glacial acetic acid were chromatographically pure (Aladdin Company); and other reagents were of analytical grade.

[0110] The standard decoction of Qingshang Quntong Decoction was prepared and provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0111] The reference substance methyl eugenol (batch number: 111642-200301, for content determination) was purchased from the China Food and Drug Inspection Institute; ligustilide A (batch number: ST10580120, purity ≥98.0%) and ligustilide (batch number: RS04251081, purity ≥98.0%) were purchased from Stend Biotechnology Co., Ltd.

[0112] Example 1: Construction of characteristic spectrum of volatile oil of Qingshang Juantong Decoction

[0113] 1. Prepare the test solution

[0114] Take 22.38g of wine-soaked angelica, 22.38g of Chuanxiong, 22.38g of Angelica dahurica, 6.72g of Asarum, 22.38g of Notopterygium wilfordii, 22.38g of Duhuo, 22.38g of Saposhnikovia divaricata, 11.22g of Chrysanthemum, 11.22g of Vitex rotundifolia, 22.38g of Atractylodes macrocephala, 33.60g of wine-soaked Scutellaria baicalensis, 22.38g of Ophiopogon japonicus, 6.72g of Licorice, and 22.38g of ginger, grind and sieve, add appropriate amount of water, soak for 30min, boil and then decoct for 20min, filter while hot, add appropriate amount of water to the residue, boil and then decoct for 15min, filter while hot, combine the two filtrates to obtain the standard decoction of Qingshang Quntong Decoction.

[0115] Transfer the prepared Qingshang Juantong Decoction standard decoction to a round-bottom flask while hot, add a few glass beads, connect the volatile oil extraction device, add water from the upper end of the collector to fill the scale part of the volatile oil measuring device and overflow into the flask, add 1 mL of ethyl acetate from the upper end of the extractor, heat with an electric heating mantle and keep it in a slightly boiling state, reflux for 4 hours and then cool, collect the ethyl acetate layer into a 2 mL volumetric flask, wash the condenser and the inner wall of the volatile oil extractor with ethyl acetate, combine the washing liquid into the same volumetric flask, add ethyl acetate to the scale, shake well, and the Qingshang Juantong Decoction standard decoction mixed oil is obtained.

[0116] Pipette 100 μL of the collected mixed oil into a 2 mL volumetric flask, dilute with methanol and make up to volume, shake well, filter, and take the filtrate.

[0117] 2. Prepare reference solution

[0118] Accurately weigh appropriate amounts of reference substances methyl eugenol, ligustilide A, and ligustilide, and add methanol to prepare solutions containing 3.340 mg, 1.960 mg, and 0.1 mg per mL, respectively.

[0119] 3. Chromatographic conditions

[0120] 3.1 Investigation of detection wavelength

[0121] A Waters CORTECS C18 (2.1×100 mm, 1.6 μm) column was used; acetonitrile was used as mobile phase A and water was used as mobile phase B. Elution was performed according to the following gradient elution program:

[0122] From 0 to 2 minutes, the volume percentage of mobile phase A increased from 25% to 34%; from 2 to 13 minutes, the volume percentage of mobile phase A was maintained at 34%; from 13 to 15 minutes, the volume percentage of mobile phase A increased from 34% to 35%; from 15 to 27 minutes, the volume percentage of mobile phase A was maintained at 35%; from 27 to 28 minutes, the volume percentage of mobile phase A decreased from 35% to 25%; from 28 to 32 minutes, the volume percentage of mobile phase A was maintained at 25%. The flow rate was 0.30 ml / min; the column temperature was 30°C; and the detection wavelengths were 235 nm, 245 nm, and 255 nm, respectively.

[0123] The chromatograms obtained with detection wavelengths of 235 nm, 245 nm, and 255 nm are as follows Figure 1 As shown in the figure, by comparison, it is found that the maximum absorption peaks of each compound are the most at the detection wavelength of 235nm, and the chromatographic peaks are better separated.

[0124] 3.2 Investigation of mobile phase

[0125] A Waters CORTECS C18 column (2.1×100 mm, 1.6 μm) was used; mobile phases A and B were methanol-0.05% phosphoric acid aqueous solution, acetonitrile-0.05% phosphoric acid aqueous solution, acetonitrile-0.1% phosphoric acid aqueous solution, acetonitrile-0.1% formic acid aqueous solution, acetonitrile-0.1% glacial acetic acid aqueous solution, acetonitrile-0.01% phosphoric acid aqueous solution, and acetonitrile-water, respectively; and elution was performed according to the following gradient elution program:

[0126] From 0 to 2 minutes, the volume percentage of mobile phase A increased from 25% to 34%; from 2 to 13 minutes, the volume percentage of mobile phase A was maintained at 34%; from 13 to 15 minutes, the volume percentage of mobile phase A increased from 34% to 35%; from 15 to 27 minutes, the volume percentage of mobile phase A was maintained at 35%; from 27 to 28 minutes, the volume percentage of mobile phase A decreased from 35% to 25%; from 28 to 32 minutes, the volume percentage of mobile phase A was maintained at 25%. The flow rate was 0.30 ml / min; the column temperature was 30°C; and the detection wavelength was 235 nm.

[0127] The chromatograms obtained using the above 6 mobile phases are as follows Figure 2 As shown in the figure, by comparing the separation of each chromatographic peak under different mobile phase systems, it was found that the chromatographic peak separation effect was not much different. After comprehensive comparison of the chromatographic peaks, acetonitrile-water was finally selected as the mobile phase.

[0128] 3.3 Investigation of chromatographic columns

[0129] BEH C 18 (2.1 mm × 100 mm, 1.7 μm), Waters CORTECS C18 (2.1 mm × 100 mm, 1.6 μm), HSS T3 (2.1 mm × 100 mm, 1.8 μm), and Hypersil GOLD (2.1 mm × 100 mm, 1.9 μm) columns were used; mobile phase A was acetonitrile, and mobile phase B was water; and elution was performed according to the following gradient elution program:

[0130] From 0 to 2 minutes, the volume percentage of mobile phase A increased from 25% to 34%; from 2 to 13 minutes, the volume percentage of mobile phase A was maintained at 34%; from 13 to 15 minutes, the volume percentage of mobile phase A increased from 34% to 35%; from 15 to 27 minutes, the volume percentage of mobile phase A was maintained at 35%; from 27 to 28 minutes, the volume percentage of mobile phase A decreased from 35% to 25%; from 28 to 32 minutes, the volume percentage of mobile phase A was maintained at 25%. The flow rate was 0.30 ml / min; the column temperature was 30°C; and the detection wavelength was 235 nm.

[0131] The chromatograms obtained using the above four chromatographic columns are as follows: Figure 3 As shown, the results show that the best peak separation and peak shape were obtained using the Waters CORTECS C18 column.

[0132] 3.4 Investigation of elution time gradient

[0133] A Waters CORTECS C18 (2.1×100 mm, 1.6 μm) column was used; mobile phase A was acetonitrile and mobile phase B was water. First, elution was performed according to the following elution gradient A:

[0134] (A) From 0 to 5 minutes, the volume percentage of mobile phase A is maintained at 55%; from 5 to 10 minutes, the volume percentage of mobile phase A increases from 55% to 80%; from 10 to 11 minutes, the volume percentage of mobile phase A decreases from 80% to 55%; from 11 to 15 minutes, the volume percentage of mobile phase A is maintained at 55%. The results of elution gradient A are shown in Figure 1. Figure 4 As shown in the figure, under this time gradient, multiple chromatographic peaks appear to be overlapping, and the separation is poor.

[0135] (B) While keeping the original time unchanged, reduce the proportion of organic phase and adjust to elution gradient B: from 0 to 5 minutes, the volume percentage of mobile phase A increases from 25% to 30%; from 5 to 10 minutes, the volume percentage of mobile phase A increases from 30% to 60%; from 10 to 11 minutes, the volume percentage of mobile phase A decreases from 60% to 25%; from 11 to 15 minutes, the volume percentage of mobile phase A remains at 25%. The results of elution gradient B are shown in Figure 1. Figure 4 As shown in the figure, under this time gradient, the peak spacing of the chromatographic peaks from 0 to 8 min is large, and the separation of the chromatographic peaks from 8 to 12 min is poor.

[0136] (C) The elution gradient was adjusted to elution gradient C: 0-3 min, the volume percentage of mobile phase A was maintained at 30%; 3-10 min, the volume percentage of mobile phase A was increased from 30% to 35%; 10-15 min, the volume percentage of mobile phase A was increased from 35% to 50%; 15-16 min, the volume percentage of mobile phase A was decreased from 50% to 30%; 16-20 min, the volume percentage of mobile phase A was maintained at 30%. The results of elution gradient C are shown in Figure 1. Figure 4 As shown in the figure, under this time gradient, the separation of the chromatographic peaks from 5 to 10 minutes is poor and the response value is low. Although the separation of the chromatographic peaks from 10 to 16 minutes is improved, the subsequent peaks are not completely eluted, so the elution time needs to be appropriately extended.

[0137] (D) The elution gradient was adjusted to elution gradient D: 0-2 min, the volume percentage of mobile phase A increased from 25% to 35%; 2-8 min, the volume percentage of mobile phase A was maintained at 35%; 8-13 min, the volume percentage of mobile phase A increased from 35% to 37%; 13-15 min, the volume percentage of mobile phase A was maintained at 37%; 15-20 min, the volume percentage of mobile phase A increased from 37% to 40%; 20-21 min, the volume percentage of mobile phase A decreased from 40% to 25%; 21-25 min, the volume percentage of mobile phase A was maintained at 25%. The results of elution gradient D are shown in Figure 1. Figure 4 As shown in the figure, under this time gradient, the response values ​​of each chromatographic peak increase and the resolution is significantly improved. However, the peak spacing between 8 and 13 minutes is large, and the resolution of the chromatographic peak at 19.5 minutes needs to be improved.

[0138] (E) The elution gradient was adjusted to elution gradient E: from 0 to 2 minutes, the volume percentage of mobile phase A increased from 25% to 35%; from 2 to 13 minutes, the volume percentage of mobile phase A was maintained at 35%; from 13 to 15 minutes, the volume percentage of mobile phase A increased from 35% to 37%; from 15 to 20 minutes, the volume percentage of mobile phase A was maintained at 37%; from 20 to 21 minutes, the volume percentage of mobile phase A decreased from 37% to 25%; from 21 to 25 minutes, the volume percentage of mobile phase A was maintained at 25%. The results of elution gradient E are shown in Figure 1. Figure 4 As shown in the figure, under this time gradient, the peak separation between 4 and 11 min needs to be further improved, and the peak separation at 20.5 min has no obvious improvement.

[0139] (F) The elution gradient was adjusted to elution gradient F: from 0 to 2 minutes, the volume percentage of mobile phase A increased from 25% to 34%; from 2 to 13 minutes, the volume percentage of mobile phase A was maintained at 34%; from 13 to 15 minutes, the volume percentage of mobile phase A increased from 34% to 35%; from 15 to 23 minutes, the volume percentage of mobile phase A was maintained at 35%; from 23 to 24 minutes, the volume percentage of mobile phase A decreased from 35% to 25%; from 24 to 28 minutes, the volume percentage of mobile phase A was maintained at 25%. The results of elution gradient F are shown in Figure 1. Figure 4 As shown in the figure, under this time gradient, the peak separation between 4 and 11 min is improved, and the chromatographic peak separation at 23.2 min is also improved.

[0140] (G) The elution gradient was adjusted to elution gradient F: from 0 to 2 minutes, the volume percentage of mobile phase A increased from 25% to 34%; from 2 to 13 minutes, the volume percentage of mobile phase A was maintained at 34%; from 13 to 15 minutes, the volume percentage of mobile phase A increased from 34% to 35%; from 15 to 27 minutes, the volume percentage of mobile phase A was maintained at 35%; from 27 to 28 minutes, the volume percentage of mobile phase A decreased from 35% to 25%; from 28 to 32 minutes, the volume percentage of mobile phase A was maintained at 25%. The results of elution gradient G are shown in Figure 1. Figure 4 As shown in FIG, under this time gradient, the separation of the chromatographic peak at 23.3 min is further improved, and the separation of each chromatographic peak is good, so this elution gradient is determined as the final elution time gradient.

[0141] 4. Determination

[0142] Accurately aspirate 0.5 μL of the test solution and reference solution respectively, inject them into the ultra-high performance liquid chromatography instrument for detection.

[0143] 5. Analysis

[0144] Chromatograms of 15 batches of Qingshang Juantong Decoction were obtained and imported into the "Similarity Evaluation System for Chromatographic Characteristic Spectra of Traditional Chinese Medicine (2012 Edition)" recommended by the Chinese Pharmacopoeia Committee for result analysis. Based on the peak attribution, the common peaks were selected as characteristic peaks. The peak No. 8 methyl eugenol was used as the reference peak. The relative retention time and relative peak area of ​​each characteristic peak were calculated, and the characteristic peak superposition spectrum was fitted. Figure 5 In view of the practical feasibility, the present invention determines 12 common peaks as characteristic peaks of the volatile oil of the standard decoction of Qingshang Juantong Decoction. The characteristic spectrum of the volatile oil of the standard decoction of Qingshang Juantong Decoction constructed by the present invention can be found in Figure 6 .

[0145] The present invention carries out peak attribution through the study of negative and single-herb characteristic spectra and identification of reference substances. Peak 1 is a common component of Notopterygium wilfordii, Angelica sinensis, and Chuanxiong rhizome; Peak 4 is a common component of Notopterygium wilfordii and Angelica sinensis; Peaks 10 to 12 are common components of Angelica sinensis and Chuanxiong rhizome; Peak 6 has no attribution information. Relevant literature indicates that Peak 6 is derived from Notopterygium wilfordii, and the remaining peaks are attributed to Asarum or Chuanxiong rhizome; Among them, Angelica dahurica, Angelica pubescens, Saposhnikovia divaricata, Chrysanthemum morifolium, Vitex mangifera fruit, and Atractylodes lancea have no peaks reflected in the compound, as shown in Table 1 for details.

[0146] Table 1: Attribution of each chromatographic peak to a single herb

[0147]

[0148] Peaks 8, 9, and 11 correspond to the retention times of the reference peaks of methyl eugenol, ligustilide A, and ligustilide, respectively. Peak 8, with its good peak shape and high resolution, was used as the reference peak (S peak) to calculate the relative retention times of the peaks. The relative retention times should be within ±10% of the specified values, which are: Peak 1: 0.409, Peak 2: 0.491, Peak 3: 0.629, Peak 4: 0.698, Peak 5: 0.774, Peak 6: 0.889, Peak 7: 0.955, Peak 8: 1, Peak 9: 1.079, Peak 10: 1.228, Peak 11: 1.868, and Peak 12: 2.016.

[0149] 6. Methodological Investigation

[0150] 6.1 Precision Investigation

[0151] Prepare the test solution using the same batch number of samples according to the method under "1. Prepare the Test Solution." Accurately pipette 0.5 μL of the same test solution and inject it six times continuously. Measure according to the chromatographic conditions under "3.4 (G)" and record the chromatogram. Using Peak 8, which has good stability and high resolution, as the reference peak, calculate the relative retention time and relative peak area RSDs for each characteristic peak. A relative retention time RSD of <0.05% and a relative peak area RSD of <0.02% for each characteristic peak indicate good instrument precision.

[0152] 6.2 Stability test

[0153] Prepare the test solution using the same batch number of samples according to the method under "1. Preparation of Test Solution". Prepare six replicates and accurately pipette 0.5 μL of the test solution after 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h, respectively. Measure the solution according to the chromatographic conditions under "3.4 (G)" and record the chromatogram. Using Peak 8 as the reference peak, calculate the relative retention time and relative peak area RSD of each characteristic peak. The results show that the relative retention time RSD of each characteristic peak is less than 0.17%, and the relative peak area RSD is less than 2.95%, indicating that the test solution has good stability within 24 h.

[0154] 6.3 Repeatability test

[0155] Prepare the test solution using the same batch of samples according to the method under "1. Prepare the Test Solution." Prepare six replicates and accurately pipette 0.5 μL of the test solution into each aliquot. Perform the assay according to the chromatographic conditions under "3.4 (G)" and record the chromatogram. Using Peak 8 as the reference peak, calculate the relative retention time and relative peak area RSDs for each characteristic peak. The results showed that the relative retention time RSDs for each characteristic peak were <0.03%, and the relative peak area RSDs were <0.95%, indicating good reproducibility of the method.

[0156] Example 2: Detection of Qingshang Juantong Decoction Samples

[0157] 1. Preparation of the sample solution to be tested

[0158] Take 22.38g of wine-soaked angelica, 22.38g of Chuanxiong, 22.38g of Angelica dahurica, 6.72g of Asarum, 22.38g of Notopterygium wilfordii, 22.38g of Duhuo, 22.38g of Saposhnikovia divaricata, 11.22g of Chrysanthemum, 11.22g of Vitex rotundifolia, 22.38g of Atractylodes macrocephala, 33.60g of wine-soaked Scutellaria baicalensis, 22.38g of Ophiopogon japonicus, 6.72g of Licorice, and 22.38g of ginger, grind and sieve, add appropriate amount of water, soak for 30min, boil and then decoct for 20min, filter while hot, add appropriate amount of water to the residue, boil and then decoct for 15min, filter while hot, combine the two filtrates to obtain the standard decoction of Qingshang Quntong Decoction.

[0159] Transfer the prepared Qingshang Juantong Decoction standard decoction to a round-bottom flask while hot, add a few glass beads, connect the volatile oil extraction device, add water from the upper end of the collector to fill the scale part of the volatile oil measuring device and overflow into the flask, add 1 mL of ethyl acetate from the upper end of the extractor, heat with an electric heating mantle and keep it in a slightly boiling state, reflux for 4 hours and then cool, collect the ethyl acetate layer into a 2 mL volumetric flask, wash the condenser and the inner wall of the volatile oil extractor with ethyl acetate, combine the washing liquid into the same volumetric flask, add ethyl acetate to the scale, shake well, and the Qingshang Juantong Decoction standard decoction mixed oil is obtained.

[0160] Pipette 100 μL of the collected mixed oil into a 2 mL volumetric flask, dilute with methanol and make up to volume, shake well, filter, and take the filtrate.

[0161] 2. Detection

[0162] Accurately pipette 0.5 μL of the sample solution to be tested and inject it into an ultra-high performance liquid chromatograph for determination. The conditions of the ultra-high performance liquid chromatograph are the same as those under "3.4 (G)" in Example 1.

[0163] By comparing the sample to be tested with Figure 6The characteristic spectrum of the standard decoction of Qingshang Juantong Decoction shows 12 characteristic peaks. Peaks 8, 9, and 11 correspond to the retention times of the reference peaks of methyl eugenol, ligustilide A, and ligustilide, respectively. Using the methyl eugenol peak of Peak 8 as the reference peak (S peak), the relative retention times of the remaining peaks relative to the S peak were calculated. These relative retention times should be within ±10% of the specified values. The specified values ​​are: Peak 1: 0.409, Peak 2: 0.491, Peak 3: 0.629, Peak 4: 0.698, Peak 5: 0.774, Peak 6: 0.889, Peak 7: 0.955, Peak 8: 1, Peak 9: 1.079, Peak 10: 1.228, Peak 11: 1.868, and Peak 12: 2.016. Therefore, it can be considered that the quality of the sample to be tested is stable, meets the quality requirements, and is a qualified standard decoction.

[0164] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0165] The above-described embodiments merely illustrate several implementations of the present invention, facilitating a specific and detailed understanding of the technical solutions of the present invention, but should not be construed as limiting the scope of protection of the invention patent. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the scope of the present invention, all of which fall within the scope of protection of the present invention.

[0166] Industrial applicability

[0167] Both the construction method and the detection method of the present invention can be used for quality control of industrial Shangqing Shangjuantong Decoction.

Claims

1. A method for constructing a characteristic spectrum of volatile oil in Qingshang Juantong Decoction, comprising the following steps: (1) The steps of preparing a volatile oil test solution and a reference solution, wherein the test solution is a standard decoction of Qingshang Juantong Decoction; the reference solution comprises methyl eugenol, ligustilide A and ligustilide, The steps of preparing the volatile oil test solution include preparing a standard decoction, extracting the volatile oil from the standard decoction, removing the volatile oil and diluting it with methanol to a concentration of 3% to 8%; (2) a step of detecting and establishing a characteristic spectrum, wherein the volatile oil test solution and the reference solution are detected by ultra-high performance liquid chromatography, and the results are analyzed by a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to establish a characteristic spectrum; Wherein, the chromatographic conditions of the ultra-high performance liquid chromatography method include: The chromatographic column is Waters CORTECS C18, with an inner diameter of 2.1 mm, a length of 100 mm, and a filler particle size of 1.6 μm. Mobile phase A is acetonitrile, mobile phase B is water, The detection wavelength is 235nm, Elution was performed according to the following gradient elution program: 。 2. The construction method according to claim 1, characterized in that The characteristic spectrum contains 12 characteristic peaks; Among them, peak No. 8, peak No. 9 and peak No. 11 correspond to the chromatographic peaks of methyl eugenol, ligusticum lactone A and ligusticum lactone reference substances, respectively; taking peak No. 8 as the S peak, the relative retention time of other characteristic peaks and the S peak is calculated, and the relative retention time should be within ±10% of the specified value, and the specified values ​​are: peak No. 1: 0.409, peak No. 2: 0.491, peak No. 3: 0.629, peak No. 4: 0.698, peak No. 5: 0.774, peak No. 6: 0.889, peak No. 7: 0.955, peak No. 8: 1, peak No. 9: 1.079, peak No. 10: 1.228, peak No. 11: 1.868, peak No. 12: 2.

016.

3. The construction method according to claim 1 or 2, characterized in that The reference substance solution is a solution of the reference substance in alcohol or an alcohol-water mixed solvent.

4. The construction method according to claim 3, characterized in that The alcohol is methanol, and the alcohol-water mixed solvent is a methanol-water mixed solvent.

5. The construction method according to claim 4, characterized in that The methanol-water mixed solvent is a methanol-water mixed solvent with a volume fraction of methanol of 70% to 90%.

6. The construction method according to claim 1 or 2, characterized in that: The concentrations of the reference solution are: methyl eugenol: 2.8~3.7 mg / mL, ligustilide A: 1.5~2.3 mg / mL, and ligustilide: 0.05~0.2 mg / mL.

7. The construction method according to claim 6, characterized in that: The concentrations of the reference solution are: methyl eugenol: 3.340 mg / mL, ligustilide A: 1.960 mg / mL, and ligustilide: 0.1 mg / mL.

8. The construction method according to claim 1 or 2, characterized in that: The ultra-high performance liquid chromatography conditions also include: a flow rate of 0.28-0.32 mL / min, a column temperature of 28-32° C.; and an injection volume of 0.5 μL.

9. A method for detecting a sample of Qingshang Juantong Decoction, comprising the following steps: The Qingshang Juantong Decoction sample to be tested is extracted with water as a solvent, the extract is collected, and the volatile oil to be tested is further extracted with ethyl acetate to prepare a volatile oil to be tested; the volatile oil to be tested is diluted with methanol to a concentration of 3% to 8% to obtain a volatile oil to be tested solution; the Qingshang Juantong Decoction sample is a standard decoction of Qingshang Juantong Decoction; The volatile oil test solution is tested by ultra-high performance liquid chromatography, and the detection spectrum obtained by the detection is compared with the characteristic spectrum constructed by the construction method according to any one of claims 1 to 8 to detect the quality of the Qingshang Quntong Decoction sample; UPLC conditions included: The chromatographic column is Waters CORTECS C18, with an inner diameter of 2.1 mm, a length of 100 mm, and a filler particle size of 1.6 μm. Mobile phase A is acetonitrile, mobile phase B is water, The detection wavelength is 235nm, Elution was performed according to the following gradient elution program: 。 10. The detection method according to claim 9, characterized in that: The quality of the Qingshang Juantong Decoction sample is detected by comparing the detection spectrum with the characteristic spectrum. If the two are consistent, the Qingshang Juantong Decoction sample is qualified. If the two are inconsistent, the Qingshang Quntong Decoction sample is unqualified.

Images