Construction method of a sample uplc characteristic map of evolvulus alsinoides and detection method thereof

Abstract

The present application relates to a kind of construction method and detection method of UPLC characteristic map of Evodia rutaecarpa sample, comprising the following steps: the present application selects the high performance liquid chromatography condition suitable for Evodia rutaecarpa medicinal materials and its standard decoction and traditional Chinese medicine formula granules, and the characteristic map is constructed by detecting with medicinal materials, standard decoction or / and traditional Chinese medicine formula granules as test sample, the characteristic map includes 7 characteristic peaks, peak 1 is gallic acid, peak 2 is protocatechuic acid, peak 3 is brevifolin carboxylic acid, peak 5 is ellagic acid, peak 6 is cinnamic acid.The method of the present application is strong in specificity, good in precision, high in accuracy, and can be widely applied to quality detection of various Evodia rutaecarpa samples, provides technical support for quality supervision of each production link of Evodia rutaecarpa and its preparation.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of traditional Chinese medicine analysis and quality control, and particularly relates to a construction method and detection method of a UPLC characteristic spectrum of a Lullutong sample. BACKGROUND

[0002] Lullutong is the dried mature fruit of Liquidambar formosana Hance in Hamamelidaceae, which has the effects of dispelling wind and activating collaterals, promoting water and unblocking channels, and is used for joint pain, numbness, spasm, edema, oligomenorrhea and other symptoms. Modern pharmacological studies have shown that the methanol extract of Lullutong, Lullutong acid, has anti-hepatocyte toxicity activity. Pharmacological experiments have also shown that Lullutong decoction can significantly inhibit the swelling of egg albumin arthritis. In clinical practice, the drug is mainly used for pain relief, diuresis, antispasticity and treatment of urticaria, dermatitis, tinea, allergic rhinitis and other diseases.

[0003] Lullutong is mainly used in the form of decoction in clinical practice, and the content of effective components in the decoction directly affects the clinical effectiveness of traditional Chinese medicine. The traditional Chinese medicine formula granules produced by modern equipment are produced based on standard decoction.

[0004] To ensure the consistency of chemical components from medicinal materials to standard decoction to formula granules, a detection method is urgently needed, which can not only detect Lullutong medicinal materials, but also be suitable for the detection of Lullutong standard decoction and traditional Chinese medicine formula granules, and is crucial for the quality detection of Lullutong modern preparations. At present, Lullutong acid is used as the content determination index to evaluate the quality of Lullutong. Lullutong acid is a pentacyclic triterpenoid compound, which has poor solubility in water, and therefore is not suitable as an evaluation index for Lullutong standard decoction and Lullutong formula granules.

[0005] High-performance liquid chromatography (HPLC) can provide more comprehensive and rich information, and is a commonly used evaluation method in the quality standards of traditional Chinese medicine. The literature (Yang Xin, Zhang Yun, Wang Shuai, Li Tianjiao, Bao Yongrui, Meng Xiansheng. In vitro anti-hepatoma effect of Lullutong from different producing areas and its spectrum-effect relationship [J]. Chinese patent medicine, 2021, 43(07): 1799-1804.) discloses a characteristic spectrum study of Lullutong by high-performance liquid chromatography (HPLC). This method takes a long time, has low sensitivity, and is only suitable for the ethyl acetate fraction of Lullutong medicinal materials, while ignoring the water-soluble active ingredients in the decoction, and cannot reflect the characteristics of the material basis of the clinical decoction. Therefore, it is necessary to establish a comprehensive, accurate and rapid characteristic spectrum method that can be used for the detection of various Lullutong samples such as medicinal materials, standard decoction and formula granules. SUMMARY

[0006] Problem to be solved by the invention

[0007] In order to solve the above problems, the application provides a construction method of UPLC characteristic spectrum of radix rubeae, which has short analysis time, good separation degree, stable baseline, rich information, and many identified characteristic peaks.

[0008] Further, the application also provides a detection method of a sample to be detected, which is simple and fast, objective, precise and reliable, specific, and high in sensitivity.

[0009] Solution for solving the problem

[0010] Through long-term research of the inventor, it is found that the above technical problems can be solved by implementing the following technical solutions.

[0011] [1] A construction method of UPLC characteristic spectrum of radix rubeae, wherein the construction method comprises the following steps:

[0012] (a) providing a test solution: extracting radix rubeae samples with an extraction solvent, collecting the extraction solution, and preparing the test solution, wherein the radix rubeae samples are radix rubeae medicinal materials, radix rubeae decoction pieces, radix rubeae standard decoction, intermediates containing radix rubeae, and / or finished products containing radix rubeae;

[0013] (b) providing a reference solution: the reference solution comprises gallic acid and ellagic acid;

[0014] (c) establishing a characteristic spectrum: detecting the test solution and the reference solution by using ultra-high performance liquid chromatography, obtaining a detection spectrum and a reference spectrum, and analyzing and establishing a UPLC characteristic spectrum by using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system;

[0015] The ultra-high performance liquid chromatography conditions comprise:

[0016] a stationary phase: a chromatographic column filled with octadecylsilane-bonded silica gel,

[0017] a mobile phase: the mobile phase A is methanol or a methanol aqueous solution with a volume percentage of more than 70%, and the mobile phase B is a formic acid aqueous solution with a mass percentage of 0.05%-0.20%,

[0018] a gradient elution program: 0 min-8 min, the volume percentage of the mobile phase A is increased from 2% to 14%; 8 min-18 min, the volume percentage of the mobile phase A is increased from 14% to 37%; 18 min-26 min, the volume percentage of the mobile phase A is increased from 37% to 46%; and 26 min-40 min, the volume percentage of the mobile phase A is increased from 46% to 100%.

[0019] [2] The construction method according to [1], wherein the extraction solvent is selected from water or an alcohol solvent, preferably the alcohol solvent is selected from a lower alcohol solvent with carbon atoms C1-C5 or an aqueous solution thereof, further preferably the lower alcohol solvent with carbon atoms C1-C5 is selected from methanol, ethanol or isopropanol.

[0020] [3] The construction method according to [1] or [2], wherein the sample of Radix Rubrogeijertianae is Radix Rubrogeijertianae medicinal material, the extraction method is heating reflux, and the heating reflux extraction time is 30 min-150 min.

[0021] [4] The construction method according to [1] or [2], wherein the sample of Radix Rubrogeijertianae is selected from Radix Rubrogeijertianae standard decoction or Radix Rubrogeijertianae traditional Chinese medicine dispensing granules, the extraction method is ultrasonic extraction, and the ultrasonic extraction time is 15 min-60 min.

[0022] [5] The construction method according to any one of [1]-[4], wherein the particle size of the chromatographic column is 1.5 μm-2.0 μm, and the column length is 100-200 mm.

[0023] [6] The construction method according to any one of [1]-[5], wherein 1 mL of the reference solution contains 10-50 μg of gallic acid reference substance and 10-50 μg of ellagic acid reference substance; and the dissolving solvent in the reference solution is selected from an alcohol solvent.

[0024] [7] The construction method according to any one of [1]-[6], wherein the characteristic spectrum contains 7 peaks, peak 1 is gallic acid, peak 2 is protocatechuic acid, peak 3 is breynin, peak 5 is ellagic acid, and peak 6 is cinnamic acid; taking peak 5 as a reference peak, the relative retention times of peaks 2, 3, 4, 6 and 7 are calculated, which should be within ±10% of the specified values, and the specified values corresponding to peaks 2, 3, 4, 6 and 7 are 0.31, 0.65, 0.85, 1.11 and 1.31, respectively.

[0025] [8] A detection method of a sample to be detected, wherein the detection method comprises the following steps:

[0026] extracting the sample to be detected with an extraction solvent, collecting the extraction solution, and preparing a sample to be detected solution;

[0027] detecting the sample to be detected solution by using ultra-high performance liquid chromatography, and comparing the detected detection spectrum with the UPLC characteristic spectrum of the sample of Radix Rubrogeijertianae constructed by the construction method according to any one of [1]-[7] to detect the quality and / or authenticity of the sample to be detected;

[0028] the ultra-high performance liquid chromatography conditions include:

[0029] Stationary phase: a chromatographic column with octadecylsilane bonded silica as the packing material,

[0030] Mobile phase: the mobile phase A is methanol or a methanol aqueous solution with a volume percentage of 70% or more, and the mobile phase B is a formic acid aqueous solution with a mass percentage of 0.05%-0.20%,

[0031] The gradient elution program is as follows: 0 min-8 min, the volume percentage of the mobile phase A is increased from 2% to 14%; 8 min-18 min, the volume percentage of the mobile phase A is increased from 14% to 37%; 18 min-26 min, the volume percentage of the mobile phase A is increased from 37% to 46%; 26 min-40 min, the volume percentage of the mobile phase A is increased from 46% to 100%.

[0032] [9] The detection method according to [8], wherein the extraction solvent is selected from alcohol solvents, preferably the alcohol solvents are selected from low alcohol solvents with carbon atoms of C1-C5 or aqueous solutions thereof, further, the low alcohol solvents with carbon atoms of C1-C5 are selected from methanol, ethanol or isopropyl alcohol.

[0033]

[10] The detection method according to [8] or 9, wherein the packing material particle size of the chromatographic column is 1.5-2.0 μm, and the column length is 100-200 mm.

[0034] Effects of the invention

[0035] The present application finds out the ultra-high performance liquid chromatography conditions suitable for the traditional Chinese medicinal material radix et rhizoma rhei, the radix et rhizoma rhei standard decoction and the radix et rhizoma rhei granules by adopting the ultra-high performance liquid chromatography method and reasonably controlling the chromatographic conditions, so as to construct the characteristic chromatogram of the radix et rhizoma rhei sample. The method has the advantages of short analysis time, good separation degree, smooth baseline, rich information of characteristic peaks, and many identified structure characteristic peaks. The characteristic chromatogram of the present application contains at least 7 characteristic peaks, which can not only reflect the quality of the radix et rhizoma rhei, but also reflect the quality of the radix et rhizoma rhei standard decoction and the radix et rhizoma rhei granules, and can be simultaneously used for the quality detection of the radix et rhizoma rhei, the radix et rhizoma rhei standard decoction and the radix et rhizoma rhei granules, and provides an effective method for the quality detection of the radix et rhizoma rhei and modern preparations thereof, and can also reflect the material basis characteristics of the clinical decoction, and provides a scientific experimental basis for the comprehensive quality evaluation of the radix et rhizoma rhei and modern preparations thereof.

[0036] The method is simple, reproducible, accurate, reliable and convenient to operate, compared with HPLC, time is saved, the detection time is shortened to 40 min through optimization of chromatographic conditions, solvent consumption is small, and environmental pollution is small. The spectrum has good separation degree of each characteristic peak, high analysis efficiency, high stability, good repeatability, wide application range, and is suitable for detection of standard decoction and formula granules of road rutong medicinal materials, and is especially suitable for detection and monitoring of large-scale production.

[0037] It should be noted that the above description does not disclose all embodiments of the present application and all advantages of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0038] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the specific embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0039] Figure 1 is the characteristic spectrum of the reference substance in Example 1 of the present application.

[0040] Figure 2 is the superimposed characteristic spectrum of 19 batches of road rutong medicinal materials in Example 1 of the present application.

[0041] Figure 3 is the superimposed characteristic spectrum of 19 batches of standard decoction of road rutong in Example 1 of the present application.

[0042] Figure 4 is the superimposed characteristic spectrum of 3 batches of formula granules of road rutong in Example 1 of the present application.

[0043] Figure 5 is the control characteristic spectrum of road rutong medicinal materials in Example 1 of the present application.

[0044] Figure 6 is the control characteristic spectrum of standard decoction of road rutong in Example 1 of the present application.

[0045] Figure 7 is the control characteristic spectrum of formula granules of road rutong in Example 1 of the present application.

[0046] Figure 8 is the investigation of different column temperatures of the characteristic spectrum of road rutong in Example 1 of the present application.

[0047] Figure 9 is the investigation of different flow rates of the characteristic spectrum of road rutong in Example 1 of the present application.

[0048] Figure 10is the characteristic spectrum of Luolutong in Example 1 of the present application, which is obtained by using different chromatographic columns.

[0049] Figure 11 is the characteristic spectrum of Luolutong medicinal materials when the gradient is inconsistent.

[0050] Figure 12 is the characteristic spectrum of Luolutong formula granules when the mobile phase and the gradient are inconsistent.

[0051] Figure 13 is the characteristic spectrum of Luolutong formula granules when the chromatographic column and the gradient are inconsistent.

[0052] Figure 14 is the comparison chart of the characteristic spectrum of Luolutong medicinal materials, standard decoction and formula granules in Example 5 of the present application. DETAILED DESCRIPTION

[0053] In order to facilitate the understanding of the present application, the present application will be described in more detail below. However, it should be understood that the present application can be realized in many different forms and is not limited to the embodiments or examples described herein. On the contrary, the purpose of providing these embodiments or examples is to make the disclosure of the present application more thorough and comprehensive.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terminology used in the specification of the present application herein is only for the purpose of describing specific embodiments or examples and is not intended to limit the present application. The optional scope of the term "and / or" used herein includes any one of two or more related listed items, and also includes any and all combinations of related listed items, which includes any two related listed items, any more related listed items, or a combination of all related listed items.

[0055] In the present application, the technical features described in an open form include both the closed technical solution consisting of listed features and the open technical solution containing listed features.

[0056] The percentage content involved in the present application, if not specifically stated, refers to mass percentage for solid-liquid mixing and solid-solid mixing, and refers to volume percentage for liquid-liquid mixing.

[0057] The percentage concentration involved in the present application, if not specifically stated, refers to the final concentration. The final concentration refers to the proportion of the added ingredient in the system after the ingredient is added.

[0058] The temperature parameter in the present application, unless otherwise specified, allows for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows for fluctuations within the accuracy range of the instrument control.

[0059] In the context of describing the present specification (especially in the context of the appended claims), the terms "one", "a", and "the" and similar language will be interpreted to cover both the singular and the plural unless otherwise indicated by the context or clearly contradicted by the specification.

[0060] In the present specification, the numerical range expressed using "numerical value A to numerical value B" or "numerical value A - numerical value B" means a range including the end point values A, B.

[0061] In the present specification, the meaning expressed using "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process. In the present specification, "optional" or "optionally" means that the event or circumstance described next can occur or not occur, and the description includes the case where the event occurs and the case where the event does not occur.

[0062] In the present specification, the phrases "some specific / preferred embodiments", "other specific / preferred embodiments", "some specific / preferred technical solutions", "other specific / preferred technical solutions", and the like mean that the specific elements (for example, features, structures, properties, and / or characteristics) described in relation to the embodiments are included in at least one embodiment described herein, and can or can not be present in other embodiments. In addition, it should be understood that the described elements can be combined in various embodiments in any suitable manner.

[0063] The term "comprising" and its variants, such as "comprise" and "comprises", in the specification and claims of the present application are intended to cover not exclusively including. For example, a process, method or system, product or device comprising a series of steps or units is not limited to the listed steps or units, but optionally also includes steps or units not listed, or optionally also includes other steps or units inherent to these processes, methods, products or devices.

[0064] Unless otherwise specified, the term "control" used herein refers to a standard substance used for identification, examination, assay, and calibration of the performance of a testing instrument, which is usually reviewed and approved by a 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 sample" used herein refers to an experimental sample used for detection or identification.

[0066] Unless otherwise stated, the term "precisely weighed or precisely measured" as used herein means that the weight taken should be accurate to one part in one thousand of the weight taken, the term "weighed" means that the weight taken should be accurate to one part in one hundred of the weight taken, and the term "precisely pipetted" means the operation mode of accurately measuring the sample by means of a micro pipette.

[0067] Unless otherwise stated, the term "filtrate" as used herein means the filtrate collected after the initial filtrate is discarded during filtration. Compared with the initial filtrate, the filtrate is closer to the true concentration of the sample, because the filter medium (such as filter membrane, filter paper, etc.) can adsorb the solute, thus resulting in a lower sample concentration in the initial filtrate; in addition, the filtrate is cleaner, because the solute adsorbed by the filter medium can form a filter cake, thus reducing the filter aperture and being able to trap smaller particles.

[0068] Unless otherwise stated, the term "ultra performance liquid chromatography" (or "UPLC") as used herein means a new technology developed on the basis of high performance liquid chromatography (HPLC), which has the characteristics of small filler particle size, fast detection speed, large analysis flux, and high sensitivity.

[0069] Unless otherwise stated, the term "retention time" as used herein means the time experienced by the separated component from the start of injection to the appearance of the maximum concentration of the component after the column, i.e. the time experienced from the start of injection to the appearance of the vertex of the chromatographic peak of the separated component; the term "relative retention time" means the ratio of the corrected retention time of the separated component to the corrected retention time of the standard.

[0070] Unless otherwise stated, the term "reference peak" (or "control peak") as used herein means the chromatographic peak corresponding to the standard in the calculation of the relative retention time.

[0071] <Construction method of UPLC characteristic pattern of Rulangtong sample>

[0072] The construction method of the UPLC characteristic pattern of the Rulangtong sample of the present application mainly comprises the following steps:

[0073] (a) providing a test sample solution;

[0074] (b) providing a reference solution;

[0075] (c) establishing the characteristic pattern.

[0076] Step (a)

[0077] In step (a), the sample of Radix Rhei is extracted with an extraction solvent, and the extract is collected to prepare a sample solution. The sample of Radix Rhei is selected from the group consisting of Radix Rhei, Radix Rhei decoction pieces, Radix Rhei standard decoction, a preparation intermediate containing Radix Rhei, and / or a preparation final product containing Radix Rhei. "Medicinal material" refers to a raw material of traditional Chinese medicine that has not been processed or made into a final product. "Decoction piece" refers to a piece of traditional Chinese medicine that has been processed according to need and is used for prescription or can be directly used in clinical treatment. "Standard decoction" refers to a single-ingredient traditional Chinese medicine decoction piece prepared by a standardized process based on the theory of traditional Chinese medicine and clinical application and referring to modern extraction methods. "Preparation intermediate" refers to an intermediate product of a preparation final product, which includes an extract and the like. "Extract" refers to an extract prepared according to a standardized production process and meeting certain quality standards, such as an alcohol (e.g., methanol, ethanol, etc.) extract, a water-extracted extract of traditional Chinese medicine that has not been made into a prescription granule, etc. "Preparation final product" refers to a final product that can be directly supplied to a terminal. "Preparation" refers to various dosage forms, such as paste, pill, pellet, powder, tablet, injection, and special-use final product. In the present application, "preparation final product" includes traditional Chinese medicine prescription granule, and "prescription granule" refers to a granule prepared from a single-ingredient traditional Chinese medicine decoction piece according to a traditional standard processing method and used for clinical prescription. In some specific embodiments of the present application, the sample of Radix Rhei is selected from the group consisting of Radix Rhei, Radix Rhei decoction piece, Radix Rhei standard decoction, and / or Radix Rhei prescription granule.

[0078] In some specific embodiments of the present application, the extraction solvent is selected from water or an alcohol solvent. In order to obtain a better peak shape (or peak form) and considering the overall extraction efficiency, the alcohol solvent is preferably selected from a lower alcohol solvent with C1-C5 carbon atoms or an aqueous solution thereof. Further, the lower alcohol solvent with C1-C5 carbon atoms is selected from methanol, ethanol, or isopropyl alcohol. In some preferred embodiments of the present application, in order to obtain a higher extraction efficiency, the extraction solvent for Radix Rhei medicinal material is preferably 40%-60% methanol aqueous solution in terms of volume percentage of methanol, and the extraction solvent for Radix Rhei standard decoction and Radix Rhei prescription granule is preferably 20%-35% methanol aqueous solution. The amount of the extraction solvent is not particularly limited in the present application. In some specific embodiments of the present application, the mass-to-volume ratio of Radix Rhei medicinal material to the extraction solvent is 1 g:(5-25) mL, which can be specifically 1 g:7.5 mL, 1 g:10 mL, or 1 g:20 mL, etc. The mass-to-volume ratio of Radix Rhei standard decoction to the extraction solvent is preferably 1 g:(30-60) mL. The volume-to-mass ratio of Radix Rhei prescription granule to the extraction solvent is preferably 1 g:(15-40) mL. Too small a concentration of the sample solution can be unfavorable for the detection of chromatographic peaks, and too large a concentration can cause a concern about poor peak shape due to overloading of the chromatographic column.

[0079] As to the extraction method, in order to obtain more abundant chromatographic peak information, the extraction can be carried out in the manner of ultrasonic, shaking or heating reflux. In some specific embodiments of the present application, according to the extraction efficiency, the extraction method of radix rhei is preferably heating reflux extraction. The heating reflux extraction can be carried out in the manner commonly used in the art. The heating reflux extraction time can be 30 min to 150 min, and further preferably 60 min to 100 min. In other specific embodiments of the present application, the extraction method of radix rhei standard decoction or radix rhei formula granules is preferably ultrasonic treatment. The present application does not have special limitations on the power and frequency of ultrasonic treatment. In some preferred embodiments, the power of ultrasonic treatment can be 250 W to 300 W, and the frequency can be 35 kHz to 45 kHz. Further preferably, the power is 250 W, and the frequency is 40 kHz. The ultrasonic extraction treatment time is 15 min to 60 min, and further can be 20 min to 40 min. In addition, the present application does not have special limitations on the extraction temperature, and room temperature can be used, which is convenient to operate.

[0080] In the process of preparing the sample solution, in some specific embodiments of the present application, for radix rhei, a pulverizing method can be used to obtain a powder sample. In some specific embodiments of the present application, the medicinal material powder can be sieved first. The mesh number of the sieves can be listed as follows: No. 2 sieve (24 mesh), No. 3 sieve (50 mesh) and No. 4 sieve (65 mesh), which have little effect on the technical effect of the present application. Then, the powder is accurately weighed and placed in a conical flask with a plug. Methanol aqueous solution with a methanol volume percentage of 40% to 60% is accurately added. The flask is tightly plugged, and the weight is determined. The sample is heated and refluxed for 60 min to 100 min. After cooling, the weight is determined again. The lost weight is made up with methanol aqueous solution with a methanol volume percentage of 40% to 60%. After shaking, the sample is filtered, and the filtrate is obtained.

[0081] Step (b)

[0082] The step of providing the reference solution of the present application at least includes preparing a control reference solution of gallic acid and a control reference solution of ellagic acid, respectively.

[0083] Gallic acid is a kind of polyphenol organic compound with the chemical name of 3,4,5-trihydroxybenzoic acid and the molecular formula of C7H6O5, and has the effects of reducing blood sugar, antioxidation, antibiosis and the like. Ellagic acid has the chemical formula of C 14 H6O8, is a kind of polyphenol dilactone, and is a dimeric derivative of gallic acid. Ellagic acid has a moderate retention time, and is suitable for being used as a reference peak S to calculate the relative retention time of each peak. In some specific embodiments of the present application, 10-50 μg of gallic acid reference substance and 10-50 μg of ellagic acid reference substance are contained in 1 mL of the reference solution. In some other specific embodiments of the present application, the dissolving solvent in the reference solution is selected from alcohol solvents, and further, the alcohol solvents are selected from low alcohol solvents with carbon atoms of C1-C5 or aqueous solutions thereof, and further, the low alcohol solvents with carbon atoms of C1-C5 are selected from methanol, ethanol or isopropanol. In some preferred embodiments of the present application, the gallic acid reference substance, the ellagic acid reference substance, the protocatechuic acid reference substance, the brevifolin carboxylic acid reference substance and the cinnamic acid reference substance are appropriately taken, and methanol is used to prepare a solution containing 10-50 μg of gallic acid, 10-50 μg of ellagic acid, 40-80 μg of protocatechuic acid, 40-80 μg of brevifolin carboxylic acid and 40-80 μg of cinnamic acid in 1 mL, as the reference solution of the reference substance.

[0084] In some specific embodiments of the present application, in order to identify the characteristic peaks, the reference substance can further include protocatechuic acid reference substance, brevifolin carboxylic acid reference substance and cinnamic acid reference substance, and the aforementioned alcohol solvents are used to dissolve to prepare a solution containing 40-80 μg of protocatechuic acid, 40-80 μg of brevifolin carboxylic acid and 40-80 μg of cinnamic acid in 1 mL, respectively.

[0085] Step (c)

[0086] Step (c) of the present application relates to the establishment of a characteristic spectrum, specifically, the test solution and the reference solution are detected by using ultra-high performance liquid chromatography, and the detection spectrum and the reference spectrum are obtained correspondingly, and the traditional Chinese medicine chromatographic fingerprint similarity evaluation system is used for analysis to establish the UPLC characteristic spectrum. The present application determines the ultra-high performance liquid chromatography conditions suitable for the radix tinosporae, the radix tinosporae decoction pieces, the radix tinosporae standard decoction, the preparation intermediates containing the radix tinosporae and / or the preparation finished products containing the radix tinosporae, especially the radix tinosporae, the standard decoction and / or the formula granules, which includes:

[0087] The stationary phase is a chromatographic column with octadecylsilane-bonded silica gel as the filler. In some embodiments of the present application, the particle size of the filler of the chromatographic column used is 1.5-2.0 μm, further 1.6-1.8 μm; in some embodiments of the present application, the length of the chromatographic column used is 100-200 mm, further 100-150 mm, and the inner diameter is 2.1 mm. In some embodiments of the present application, the chromatographic column is selected from any one of Eclipse Plus C18 (Agilent, 2.1 x 100 mm, 1.8 μm), HSS T3 (Waters, 2.1 x 100 mm, 1.8 μm), and BEH C18 (Waters, 2.1 x 100 mm, 1.7 μm), and the peak shape of the three chromatographic columns is good and the resolution is good.

[0088] The mobile phase A is methanol or a methanol aqueous solution with a volume percentage of 70% or more, and the mobile phase B is a formic acid aqueous solution with a mass percentage of 0.05-0.20%. In some embodiments of the present application, the mobile phase A is methanol, and the mobile phase B is a formic acid aqueous solution with a mass percentage of 0.05-0.20%. Compared with other mobile phase combinations (such as acetonitrile as the mobile phase A and 0.1% formic acid solution as the mobile phase B), the mobile phase selected in the present application is helpful to obtain a good peak shape and improve the resolution of the chromatographic peak.

[0089] The elution procedure of the present application is gradient elution, and the gradient elution procedure is as follows: 0 min-8 min, the volume percentage of the mobile phase A is increased from 2% to 14%; 8 min-18 min, the volume percentage of the mobile phase A is increased from 14% to 37%; 18 min-26 min, the volume percentage of the mobile phase A is increased from 37% to 46%; and 26 min-40 min, the volume percentage of the mobile phase A is increased from 46% to 100%. The use of this gradient elution procedure has the advantages of short analysis time, wide sample applicability, improved resolution of the chromatographic peak, and effective solution to the problem of chromatographic peak tailing.

[0090] From the perspective of chromatographic peak separation effect, the preferred flow rate of the present application is 0.25-0.35 mL / min; and / or, the column temperature is 30-40℃. The construction method of the present application can meet the system applicability requirements within this column temperature or flow rate range with small changes in column temperature or flow rate. In some preferred embodiments of the present application, the flow rate can be 0.25-0.30 mL / min; and / or, the column temperature can be 30-35℃.

[0091] The detector used in the present application can be an ultraviolet detector; in some preferred embodiments of the present application, the detection wavelength is 220nm-280nm, and in some preferred embodiments of the present application, the detection wavelength can be 265nm; under the detection wavelength in this range, the chromatographic peak information is relatively rich, the separation effect of each chromatographic peak is relatively good, and the baseline is relatively stable.

[0092] Further, the present application detects and analyzes and compares multiple batches of the traditional Chinese medicine radix ophiopogonis, radix ophiopogonis standard decoction, radix ophiopogonis dispensing granules, and the control product solution, selects common peaks to establish a characteristic spectrum, and identifies the common peaks by referring to the positioning of the control product chromatographic peak. Finally, the chromatograms of the traditional Chinese medicine radix ophiopogonis, the standard decoction, and the dispensing granules are introduced into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition", and the median method is used to generate the superimposed characteristic spectrum and the control characteristic spectrum. By using the foregoing construction method of the present application, the characteristic spectrum of the radix ophiopogonis sample contains at least 7 characteristic peaks, further, peak 1 is gallic acid, peak 2 is protocatechuic acid, peak 3 is brevifolin carboxylic acid, peak 5 is ellagic acid, and peak 6 is cinnamic acid, taking peak 5 as the reference peak, the relative retention of peak 2, peak 3, peak 4, peak 6, and peak 7 is calculated, and the relative retention time thereof should be within ±10% of the specified value, and the specified value corresponding to peak 2, peak 3, peak 4, peak 6, and peak 7 is 0.31, 0.65, 0.85, 1.11, and 1.31, respectively. In some specific embodiments of the present application, considering that the retention time of peak 1 is short, the determination is greatly affected by different instruments, and the relative retention time is prone to large deviation, therefore, it is not suitable to specify the relative retention time, and the control product is directly positioned. The 7 common peaks can stably appear in the traditional Chinese medicine radix ophiopogonis, the standard decoction, and the dispensing granules, and have good coincidence.

[0093] The present application optimizes the preparation of the test product, the preparation of the control product, and the chromatographic analysis conditions, shortens the analysis time, improves the analysis efficiency, and establishes an ultra-high performance liquid chromatography condition suitable for the traditional Chinese medicine radix ophiopogonis, radix ophiopogonis decoction pieces, radix ophiopogonis standard decoction, intermediates containing radix ophiopogonis, and / or finished products containing radix ophiopogonis; especially the traditional Chinese medicine radix ophiopogonis and its standard decoction and / or dispensing granules, which provides an important multi-index parameter basis for evaluating the process stability. Moreover, the present application considers the water-soluble active ingredients in radix ophiopogonis, which can effectively reflect the material basis characteristics of the clinical decoction. The method of the present application is simple to operate, has high precision, and has good repeatability, stability, and durability.

[0094] <Detection method>

[0095] The present application also provides a detection method of a to-be-detected sample, which comprises the following steps:

[0096] The to-be-detected sample is extracted with an extraction solvent, the extract is collected, and the to-be-detected product solution is prepared;

[0097] The solution of the sample to be detected is detected by using the ultra-high performance liquid chromatography, and the detection spectrum obtained is compared with the UPLC characteristic spectrum of the sample of radix rhei prepared by the aforementioned preparation method of the application to detect the quality and / or authenticity of the sample to be detected;

[0098] The ultra-high performance liquid chromatography conditions include:

[0099] Stationary phase: a chromatographic column with octadecylsilane bonded silica as the filler,

[0100] Mobile phase: the mobile phase A is methanol or a methanol aqueous solution with a volume percentage of 70% or more, and the mobile phase B is a formic acid aqueous solution with a mass percentage of 0.05%-0.20%,

[0101] The gradient elution program is as follows: 0 min-8 min, the volume percentage of the mobile phase A is increased from 2% to 14%; 8 min-18 min, the volume percentage of the mobile phase A is increased from 14% to 37%; 18 min-26 min, the volume percentage of the mobile phase A is increased from 37% to 46%; and 26 min-40 min, the volume percentage of the mobile phase A is increased from 46% to 100%.

[0102] In some specific embodiments of the application, the sample to be detected can be selected from radix rhei medicinal materials, radix rhei decoction pieces, radix rhei standard decoction, preparation intermediates containing radix rhei, preparation finished products containing radix rhei, and / or radix rhei counterfeit products.

[0103] In some specific embodiments of the application, if the detection spectrum of the sample to be detected is consistent with the UPLC characteristic spectrum of the sample of radix rhei prepared by the aforementioned preparation method of the application, it is a qualified product of radix rhei; if the characteristic spectra of the two are inconsistent, it is an unqualified product or a counterfeit product of radix rhei.

[0104] In some specific embodiments of the application, the sample to be detected is detected by using the aforementioned sample solution preparation method and chromatography conditions mentioned in the preparation method of the application. This method provides a new analysis means for the internal quality control of radix rhei from raw materials to finished products, and the method is simple to operate, high in precision, good in stability and repeatability.

[0105] Examples

[0106] The application will be further described below through specific examples:

[0107] Example 1: Construction of characteristic spectrum of radix rhei medicinal materials, standard decoction and traditional Chinese medicine formula granules

[0108] 1. Instruments and reagents

[0109] 1.1 Instruments

[0110] Waters Acquity UPLC ultra-high performance liquid chromatograph (Waters Corporation); Agilent 12920 ultra-high performance liquid chromatograph (Agilent Corporation); METTLER TOLEDO XP6 millionth balance (Metller-Toledo (Shanghai) Instrument Co., Ltd.); ME204E electronic analytical balance (Metller-Toledo Instrument (Shanghai) Co., Ltd.); KQ-250B ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); AS165W centrifuge (Avesta (Shanghai) Trading Co., Ltd.); GKC114 temperature-controlled water bath (Nantong Huatai Experimental Instrument Co., Ltd.); pure water system (Sartorius Corporation).

[0111] 1.2 Reagents

[0112] 19 batches of Luolutong medicinal materials were collected and identified by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0113] The 19 batches of Luolutong standard decoction were prepared from the raw medicinal materials according to the guidelines of “Preparation of Standard Decoction for Research and Characterization” in “Technical Requirements for Quality Control and Standard Development of Traditional Chinese Medicine Dispensing Granules”. The 3 batches of Luolutong dispensing granules were produced by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0114] Gallic acid reference substance, protocatechuic acid reference substance, ellagic acid reference substance, and cinnamic acid reference substance were purchased from China Institute for Drug Control, with batch numbers 110831-201906, 110809-201205, 111959-201903, and 110786-201604, respectively. Shortleaf acyl acid reference substance was purchased from Shanghai Shidand Co., Ltd., with batch number 9615.

[0115] 1.3 Reagents

[0116] Methanol (analytical pure, National Pharmaceutical Group Chemical Reagent Co., Ltd.); methanol (chromatographically pure, Thermo Fisher Corporation); formic acid (chromatographically pure, aladdin Corporation); water was ultrapure water.

[0117] 2. Methods and Results

[0118] 2.1 Chromatographic conditions

[0119] Chromatographic column: HSS T3 (2.1 mm x 100 mm, 1.8 μm);

[0120] Column temperature: 35℃;

[0121] Flow rate: 0.3 mL / min;

[0122] Detection wavelength: 265 nm;

[0123] Injection volume: 2 μL

[0124] Methanol was used as mobile phase A and 0.1% formic acid solution was used as mobile phase B. Gradient elution was performed according to the following table:

[0125] Table 1 Gradient elution table of mobile phase

[0126]

[0127] 2.2 Preparation of test solution

[0128] Medicinal material: Take a proper amount of Radix et Rhizoma Rauvolfiae, crush, take 2 g, put into a conical flask with plug, add 50% methanol 15 mL, tightly plug, heat reflux for 90 minutes, cool, shake well, filter, take the filtrate, and get it.

[0129] Standard decoction: Take a proper amount of Radix et Rhizoma Rauvolfiae standard decoction prepared by pretreatment, decoction, low-temperature concentration, and freeze-drying, grind finely, take 0.3 g, put into a conical flask with plug, add 30% methanol 15 mL, tightly plug, ultrasonic treatment (power 250 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, take the filtrate, and get it.

[0130] Traditional Chinese medicine formula granules: Take a proper amount of traditional Chinese medicine formula granules prepared by pretreatment, extraction, concentration, spray drying, granulation, and packaging of Radix et Rhizoma Rauvolfiae, grind finely, take 0.8 g, put into a conical flask with plug, add 30% methanol 15 mL, tightly plug, ultrasonic treatment (power 250 W, frequency 40 kHz) for 45 minutes, cool, shake well, filter, take the filtrate, and get it.

[0131] 2.3 Preparation of reference solution

[0132] Take a proper amount of gallic acid reference substance, protocatechuic acid reference substance, brevifolin carboxylic acid reference substance, ellagic acid reference substance, and cinnamic acid reference substance, respectively add methanol to prepare a solution containing 20 μg of gallic acid, 50 μg of protocatechuic acid, 50 μg of brevifolin carboxylic acid, 25 μg of ellagic acid, and 50 μg of cinnamic acid per 1 mL.

[0133] 2.4 Determination method

[0134] Precisely take 1 μL of reference solution and test solution respectively, inject into the ultra-high performance liquid chromatograph, record the chromatogram, and get the characteristic chromatogram of Radix et Rhizoma Rauvolfiae, standard decoction, and traditional Chinese medicine formula granules.

[0135] 2.5 Identification of chromatographic peaks

[0136] Gallic acid, protocatechuic acid, bryophylloidin, ellagic acid, and cinnamic acid were used for identification. The results showed that peak 1 was gallic acid; peak 2 was protocatechuic acid; peak 3 was bryophylloidin; peak 5 was ellagic acid; and peak 6 was cinnamic acid. (See...) Figure 1 .

[0137] 2.6 Selection of Reference Peak and Common Peak

[0138] Chromatograms of 19 batches of *Ligustrum lucidum* medicinal materials from different origins were analyzed, and 7 common peaks were identified. Figure 2 . Figure 2 Overlay feature maps of 19 batches of Lulutong medicinal materials (S1~S19).

[0139] Chromatograms of 19 batches of self-produced Lulutong standard decoction were analyzed, and 7 common peaks were identified. (See attached image) Figure 3 . Figure 3 The superimposed characteristic spectra of 19 batches of standard decoction of Lulutong (S1~S19) are presented.

[0140] The chromatograms of three batches of Lulutong (a traditional Chinese medicine) formula granules (S1-S3) produced in pilot-scale production were analyzed, and seven common peaks were identified. (See attached chromatogram). Figure 4 . Figure 4 The superimposed characteristic spectra of three batches of traditional Chinese medicine formula granules for Lulutong.

[0141] 2.7 Establishment of comparative feature maps and evaluation of similarity

[0142] The chromatograms of 19 batches of *Lycopodium clavatum* medicinal materials, 19 batches of *Lycopodium clavatum* standard decoctions, and 3 batches of *Lycopodium clavatum* formula granules were each imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition". The median method was used to generate their respective superimposed characteristic chromatograms (see...). Figure 2 , Figure 3 , Figure 4 ) and the control feature map (see Figure 5 , Figure 6 , Figure 7 ).

[0143] 3. Methodological Examination

[0144] 3.1 Precision Test

[0145] Take one sample each of *Lysimachia christinae* herbal material, standard decoction, and traditional Chinese medicine granules, and prepare them according to the method under "2.2 Preparation of Test Solution". Accurately pipette 1 μL of the same test solution and inject it 6 times consecutively. Measure according to the method under "2.1 Chromatographic Conditions" and record the chromatogram. The two peaks of gallic acid and ellagic acid should correspond to the retention times of the corresponding reference peaks. The peak corresponding to the ellagic acid reference peak is the S peak. Calculate the relative retention time and relative peak area of ​​each characteristic peak.

[0146] The precision test results show that the relative retention time RSD values of each characteristic peak of the Radix Lonicerae are less than 0.50%, and the relative peak area RSD values are less than 1.18%; the relative retention time RSD values of each characteristic peak of the Radix Lonicerae standard decoction are less than 0.18%, and the relative peak area RSD values are less than 0.61%; the relative retention time RSD values of each characteristic peak of the Radix Lonicerae traditional Chinese medicine dispensing granules are less than 0.09%, and the relative peak area RSD values are less than 0.42%; indicating that the instrument precision is good.

[0147] 3.2 Reproducibility test

[0148] Take 6 portions of the Radix Lonicerae, Radix Lonicerae standard decoction and Radix Lonicerae traditional Chinese medicine dispensing granules samples respectively, prepare according to the method under the item “2.2 Preparation of test solution”, precisely take 1 μL of the test solution, determine according to the method under the item “2.1 Chromatographic conditions”, record the chromatogram, wherein the two peaks of gallic acid and ellagic acid should correspond to the retention time of the corresponding reference peak of the reference substance; the peak corresponding to the reference peak of ellagic acid is S peak, and the relative retention time and relative peak area of each characteristic peak are calculated.

[0149] The reproducibility test results show that the relative retention time RSD values of each characteristic peak of the Radix Lonicerae are less than 1.71%, and the relative peak area RSD values are less than 2.05%; the relative retention time RSD values of each characteristic peak of the Radix Lonicerae standard decoction are less than 0.12%, and the relative peak area RSD values are less than 1.65%; the relative retention time RSD values of each characteristic peak of the Radix Lonicerae dispensing granules are less than 1.31%, and the relative peak area RSD values are less than 1.79%; indicating that the method has good reproducibility.

[0150] 3.3 Stability test

[0151] Take 1 portion of the Radix Lonicerae, Radix Lonicerae standard decoction and Radix Lonicerae traditional Chinese medicine dispensing granules samples respectively, prepare according to the method under the item “2.2 Preparation of test solution”, determine at 0 h, 2 h, 4 h, 8 h, 12 h, 18 h and 24 h according to the method under the item “2.1 Chromatographic conditions”, record the chromatogram, wherein the two peaks of gallic acid and ellagic acid should correspond to the retention time of the corresponding reference peak of the reference substance; the peak corresponding to the reference peak of ellagic acid is S peak, and the relative retention time and relative peak area of each characteristic peak are calculated.

[0152] The stability test results show that the relative retention time RSD values of each characteristic peak of the Radix Lonicerae are less than 0.53%, and the relative peak area RSD values are less than 2.22%; the relative retention time RSD values of each characteristic peak of the Radix Lonicerae standard decoction are less than 0.12%, and the relative peak area RSD values are less than 2.58%; the relative retention time RSD values of each characteristic peak of the Radix Lonicerae dispensing granules are less than 0.19%, and the relative peak area RSD values are less than 2.82%; indicating that the method has good stability.

[0153] 3.4 Durability

[0154] Column temperature investigation: The chromatograms at 30℃, 35℃, 40℃ were compared, and the results showed that the column temperature variation in the range of 30℃-40℃ could meet the system suitability requirements, and the column temperature of 35℃ was preferred. See Figure 8 .

[0155] Flow rate investigation: The chromatograms at 0.25 mL / min, 0.30 mL / min, 0.35 mL / min were compared, and the results showed that the flow rate variation in the range of 0.25-0.35 mL / min could meet the system suitability requirements, and the flow rate of 0.30 mL / min was preferred. See Figure 9 .

[0156] Chromatographic column investigation: Three different types of chromatographic columns were compared, Eclipse Plus C18(Agilent, 2.1 x 100 mm, 1.8 μm), HSS T3(Waters, 2.1 x 100 mm, 1.8 μm), and BEH C18(Waters, 2.1 x 100 mm, 1.7 μm). The peak shapes of the three chromatographic columns were good, and the separation degrees were good. See Figure 10 .

[0157] 4. Investigation of preparation of test solution

[0158] 4.1 Investigation of extraction solvent

[0159] Take 2g of Lulutong medicinal materials, 0.5g of Lulutong standard decoction, and 0.8g of Lulutong dispensing granules, each in parallel for 5 times, and place them in a conical flask with a plug. Precisionly add 20mL of water, 30% methanol, 50% methanol, 70% methanol, and methanol respectively as five different extraction solvents, and weigh. Ultrasonic treatment (power 250W, frequency 40kHz) for 60 minutes, cool, reweigh, make up the weight loss with the extraction solvent, shake well, filter, and take the filtrate. According to the method under "2.1 Chromatographic conditions", record the chromatogram, and compare the peak area of 7 characteristic peaks / weight and the chromatogram to compare the effects of different solvents on the Lulutong characteristic chromatogram. The results showed that using water, 30% methanol, 50% methanol, 70% methanol, and methanol as the extraction solvent, 7 characteristic peaks can be presented. According to the extraction efficiency, the extraction solvent for Lulutong medicinal materials is preferably 50% methanol, and the extraction solvent for Lulutong standard decoction and Lulutong dispensing granules is preferably 30% methanol.

[0160] 4.2 Investigation of extraction method

[0161] Take 2 g of Lulutong medicinal materials, 0.5 g of Lulutong standard decoction, and 0.8 g of Lulutong dispensing granules, each in 3 parallel samples, and place them in a conical flask with a stopper. Precisely add 20 mL of 30% methanol, weigh, and separately ultrasonic treat (power 250 W, frequency 40 kHz) for 60 minutes, heat reflux extraction for 60 minutes, and shake extraction for 60 minutes. Cool, re-weigh, make up the weight loss with 30% methanol solution, shake well, filter, and take the filtrate. Determine by the method under "2.1 Chromatographic conditions", record the chromatogram, and compare the effects of different solvents on the characteristic spectrum of Lulutong through the peak area of 7 characteristic peaks / weight. The results show that using 3 different extraction methods, 7 characteristic peaks can be presented. According to the extraction efficiency, the preferred extraction method for Lulutong medicinal materials is heat reflux extraction; and the preferred extraction method for Lulutong standard decoction and Lulutong dispensing granules is ultrasonic treatment.

[0162] 4.3 Extraction time investigation

[0163] Take 2 g of Lulutong medicinal materials, 0.5 g of Lulutong standard decoction, and 0.8 g of Lulutong dispensing granules, each in 3 parallel samples, and place them in a conical flask with a stopper. Precisely add 20 mL of 30% methanol, weigh, and separately ultrasonic treat (power 250 W, frequency 40 kHz) for 60 minutes, heat reflux extraction for 60 minutes, and shake extraction for 60 minutes. Cool, re-weigh, make up the weight loss with 30% methanol solution, shake well, filter, and take the filtrate. Determine by the method under "2.1 Chromatographic conditions", record the chromatogram, and compare the effects of different solvents on the characteristic spectrum of Lulutong through the peak area of 7 characteristic peaks / weight. The results show that using 3 different extraction methods, 7 characteristic peaks can be presented. According to the extraction efficiency, the preferred extraction method for Lulutong medicinal materials is heat reflux extraction; and the preferred extraction method for Lulutong standard decoction and Lulutong dispensing granules is ultrasonic treatment.

[0164] Take 2 g of Lulutong medicinal materials, 0.5 g of Lulutong standard decoction, and 0.8 g of Lulutong dispensing granules, each in 3 parallel samples, and place them in a conical flask with a stopper. Precisely add 20 mL of 30% methanol, weigh, and separately ultrasonic treat (power 250 W, frequency 40 kHz) for 60 minutes, heat reflux extraction for 60 minutes, and shake extraction for 60 minutes. Cool, re-weigh, make up the weight loss with 30% methanol solution, shake well, filter, and take the filtrate. Determine by the method under "2.1 Chromatographic conditions", record the chromatogram, and compare the effects of different solvents on the characteristic spectrum of Lulutong through the peak area of 7 characteristic peaks / weight. The results show that using 3 different extraction methods, 7 characteristic peaks can be presented. According to the extraction efficiency, the preferred extraction method for Lulutong medicinal materials is heat reflux extraction; and the preferred extraction method for Lulutong standard decoction and Lulutong dispensing granules is ultrasonic treatment.

[0165] 5. Chromatographic condition investigation

[0166] 5.1 Gradient inconsistency

[0167] Take Luotong medicinal materials, crush, take 2g, put in a conical flask with a plug, add 50% methanol 15mL, tightly plug, heat reflux for 90 minutes, cool, shake well, filter, take the filtrate, and you get the test sample solution. Accurately take 1 μL of the test sample solution, measure according to the following conditions, and record the chromatogram.

[0168] Chromatographic column: HSS T3 (2.1mm x 100mm, 1.8 μm); column temperature: 35℃; flow rate: 0.3mL / min; detection wavelength: 265nm; injection volume: 2 μL; gradient elution was carried out according to the following table with methanol as mobile phase A and 0.1% formic acid solution as mobile phase B, and the chromatogram is shown in Figure 11 ;

[0169] Table 2 Gradient elution table of mobile phase

[0170]

[0171] Conclusion: The chromatographic peak at retention time 17 minutes has poor resolution.

[0172] 5.2 Inconsistent mobile phase and gradient

[0173] Take Luotong medicinal materials, crush, take 2g, put in a conical flask with a plug, add 50% methanol 15mL, tightly plug, heat reflux for 90 minutes, cool, shake well, filter, take the filtrate, and you get the test sample solution. Accurately take 1 μL of the test sample solution, measure according to the following conditions, and record the chromatogram.

[0174] Chromatographic column: HSS T3 (2.1mm x 100mm, 1.8 μm); column temperature: 35℃; flow rate: 0.3mL / min; detection wavelength: 265nm; injection volume: 2 μL; gradient elution was carried out according to the following table with methanol as mobile phase A and 0.1% formic acid solution as mobile phase B, and the chromatogram is shown in Figure 12 ;

[0175] Table 3 Gradient elution table of mobile phase

[0176]

[0177] Conclusion: The baseline is not flat, with a bump.

[0178] 5.3 Inconsistent chromatographic column and gradient

[0179] Take an appropriate amount of the Chinese herbal formula granules obtained from the pretreatment, extraction, concentration, spray drying, granulation, and packaging of *Lysimachia christinae* medicinal material, grind them into a fine powder, take 0.8 g, place them in a stoppered conical flask, add 15 mL of 30% methanol, seal tightly, and sonicate (power 250 W, frequency 40 kHz) for 45 minutes. Cool, shake well, filter, and collect the filtrate to obtain the test solution. Accurately pipette 1 μL of the test solution and determine the chromatogram under the following conditions.

[0180] Chromatographic column: HSS T3 (4.6 mm × 250 mm, 5 μm); column temperature: 25℃; flow rate: 1.2 mL / min; detection wavelength: 265 nm; injection volume: 10 μL; acetonitrile was used as mobile phase A, and 0.1% formic acid solution was used as mobile phase B. Gradient elution was performed according to the specifications in the table below. The chromatogram is shown in [reference needed]. Figure 13 ;

[0181] Table 4 Gradient Elution of Mobile Phase

[0182]

[0183] Conclusion: The analysis time was long, the chromatographic peak resolution was poor, and the chromatographic peaks were tailed.

[0184] Example 2: A method for detecting the medicinal material *Luffa cylindrica*

[0185] (1) Preparation of test sample

[0186] Take an appropriate amount of *Luffa cylindrica* herb, crush it, take about 2g, weigh it accurately, place it in a stoppered conical flask, accurately add 15mL of 50% methanol, seal tightly, weigh it, heat under reflux for 90 minutes, cool, weigh it again, make up the lost weight with 50% methanol, shake well, filter, and take the filtrate to obtain the final product.

[0187] (2) Detection

[0188] Accurately pipette the test solution 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 in "2.1 Chromatographic Conditions" in Example 1.

[0189] By comparing the test sample with Figure 5The control characteristic spectrum of Radix et Rhizoma Rhei can be known from the detection spectrum of the sample to be tested and the control characteristic spectrum of Radix et Rhizoma Rhei. The similarity of the detection spectrum of the sample to be tested and the control characteristic spectrum of Radix et Rhizoma Rhei is greater than 0.9, and 7 characteristic peaks can be detected. The peak 1 and the peak 5 correspond to the retention time of the reference peak of the gallic acid control and the reference peak of the ellagic acid control, respectively. The peak corresponding to the reference peak of ellagic acid is the S peak. The relative retention times of the peaks 2, 3, 4, 6 and 7 are 0.31, 0.64, 0.83, 1.07 and 1.30, respectively, which are within the range of ±10% of the specified value. The specified value is 0.31 (peak 2), 0.65 (peak 3), 0.85 (peak 4), 1.11 (peak 6) and 1.31 (peak 7). It can be considered that the sample to be tested meets the quality requirements and is a qualified medicinal material.

[0190] Example 3: A detection method of a standard decoction of Radix et Rhizoma Rhei

[0191] (1) Preparation of the test sample

[0192] A certain amount of the freeze-dried powder of the standard decoction of Radix et Rhizoma Rhei prepared by pretreatment, decoction, low-temperature concentration and freeze-drying was finely ground, about 0.3 g was accurately weighed, placed in a conical bottle with a plug, 15 mL of 30% methanol was accurately added, tightly sealed, the weight was determined, ultrasonic treatment (power 250 W, frequency 40 kHz) was performed for 30 minutes, the sample was cooled, the weight was determined again, the lost weight was made up with 30% methanol, shaken well, filtered, and the filtrate was obtained.

[0193] (2) Detection

[0194] The test sample solution was accurately taken and injected into the ultra-high performance liquid chromatograph for determination. The conditions of the ultra-high performance liquid chromatograph were the same as those in the item “2.1 Chromatographic conditions” in Example 1.

[0195] By comparing the sample to be tested with Figure 6 The control characteristic spectrum of Radix et Rhizoma Rhei can be known from the detection spectrum of the sample to be tested and the control characteristic spectrum of Radix et Rhizoma Rhei. The similarity of the detection spectrum of the sample to be tested and the control characteristic spectrum of Radix et Rhizoma Rhei is greater than 0.9, and 7 characteristic peaks can be detected. The peak 1 and the peak 5 correspond to the retention time of the reference peak of the gallic acid control and the reference peak of the ellagic acid control, respectively. The peak corresponding to the reference peak of ellagic acid is the S peak. The relative retention times of the peaks 2, 3, 4, 6 and 7 are 0.31, 0.65, 0.85, 1.09 and 1.31, respectively, which are within the range of ±10% of the specified value. The specified value is 0.31 (peak 2), 0.65 (peak 3), 0.85 (peak 4), 1.11 (peak 6) and 1.31 (peak 7). It can be considered that the sample to be tested meets the quality requirements and is a qualified standard decoction.

[0196] Example 4: A detection method of a Radix et Rhizoma Rhei dispensing granule

[0197] (1) Preparation of the test sample

[0198] A certain amount of the traditional Chinese medicine formula granules of radix et rhizoma rhei obtained by pretreatment, extraction, concentration, spray drying, granulation and packaging was finely ground, about 0.8 g was accurately weighed, placed in a conical flask with a plug, tightly sealed, the weight was determined, ultrasonic treatment (power 250 W, frequency 40 kHz) was performed for 45 minutes, allowed to cool, the weight was determined again, the lost weight was made up with 30% methanol, shaken well, filtered, and the filtrate was obtained.

[0199] (2) Detection

[0200] The test sample solution was accurately taken and injected into an ultra-high performance liquid chromatograph for determination. The conditions of the ultra-high performance liquid chromatograph were the same as those in item "2.1 Chromatographic conditions" in Example 1.

[0201] By comparing the sample to be tested with Figure 7 The detection spectrum of the sample to be tested was compared with the control characteristic spectrum of the radix et rhizoma rhei formula granules. The similarity of the two was greater than 0.9, and 7 characteristic peaks were detected, wherein peak 1 and peak 5 corresponded to the retention time of the reference peak of the gallic acid control and the ellagic acid control, respectively. The peak corresponding to the ellagic acid reference peak was the S peak. The relative retention times of peak 2, peak 3, peak 4, peak 6 and peak 7 were 0.31, 0.65, 0.85, 1.10 and 1.31, respectively, all of which were within the range of ±10% of the specified value. The specified value was 0.31 (peak 2), 0.65 (peak 3), 0.85 (peak 4), 1.11 (peak 6) and 1.31 (peak 7). It can be considered that the sample to be tested meets the quality requirements and is qualified radix et rhizoma rhei formula granules.

[0202] Example 5: Consistency evaluation of radix et rhizoma rhei, standard decoction and traditional Chinese medicine formula granules

[0203] In this example, the characteristic spectrum method constructed for radix et rhizoma rhei, standard decoction and formula granules was applied to the sample detection and quality evaluation of each link in the production process of radix et rhizoma rhei formula granules. The medicinal materials, standard decoction and formula granules in the production process of one batch were taken for testing.

[0204] (1) Preparation of the test sample solution

[0205] The preparation method of the test sample solution was the same as that in Examples 2, 3 and 4.

[0206] (2) Detection

[0207] The test sample solution was accurately taken and injected into an ultra-high performance liquid chromatograph for determination. The conditions of the ultra-high performance liquid chromatograph were the same as those in item "2.1 Chromatographic conditions" in Example 1.

[0208] The chromatogram of one batch of trial production of radix rhei, corresponding standard decoction and traditional Chinese medicine formula granules is introduced into "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition", and the median method is used to generate the superimposed characteristic spectrum, taking the standard decoction of one batch of trial production as the reference spectrum, and the characteristic spectra are compared, and the comparison results are as follows Table 5, Figure 14 .

[0209] Table 5 Similarity consistency analysis of one batch of raw materials, standard decoction and formula granules

[0210] Number Similarity Medicinal material 0.92 Standard decoction 1.00 Dispensing granules 0.99

[0211] According to the Figure 14 It can be known that the seven characteristic peaks defined by the present application are stably present in radix rhei, standard decoction (referred to as standard decoction) and formula granules, and the quantity value transmission is stable.

[0212] According to Table 5, the chromatographic similarity of radix rhei, its standard decoction and traditional Chinese medicine formula granules is very high, which shows that the characteristic spectrum constructed by using the medicinal material as the test sample can not only reflect the quality of the medicinal material and thus be used for the detection of the medicinal material, but also reflect the quality of the standard decoction and traditional Chinese medicine formula granules and thus be used for the detection of the standard decoction and traditional Chinese medicine formula granules, and vice versa. It is used for quality evaluation of each link of formula granule production, and it is ensured that each chromatographic peak corresponding to the chemical component is well transmitted from the raw material to the final product in the production process.

[0213] The above-described embodiments only express several embodiments of the present application, facilitate specific and detailed understanding of the technical solutions of the present application, but cannot be understood as a limitation on the protection scope of the invention patent. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are all within the protection scope of the present application.

Claims

1. A method for constructing an UPLC profile of a sample of Uncaria rhynchophylla, characterized in that, The constructed method comprises the following steps: (a) providing a test sample solution: extracting a luffa cylindrical sample with an extraction solvent, collecting the extract, and preparing a test sample solution, wherein the luffa cylindrical sample is a luffa cylindrical medicinal material, a luffa cylindrical decoction piece, a luffa cylindrical standard decoction or a luffa cylindrical dispensing granule, the extraction solvent is selected from water or an alcohol solvent, and the alcohol solvent is selected from methanol, ethanol, a methanol aqueous solution or an ethanol aqueous solution; (b) providing a reference solution: the reference solution comprises a gallic acid control and a tannic acid control; (c) establishing a characteristic chromatogram: detecting the test sample solution and the reference solution by using an ultra-high performance liquid chromatography method, and correspondingly obtaining a detection chromatogram and a control chromatogram, analyzing and establishing an UPLC characteristic chromatogram by using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, wherein the characteristic chromatogram comprises seven peaks, peak 1 is gallic acid, peak 2 is protocatechuic acid, peak 3 is breynol, peak 5 is tannic acid, and peak 6 is cinnamic acid; The ultra-high performance liquid chromatography conditions comprise: a stationary phase: a chromatographic column with octadecylsilane-bonded silica gel as a filler, wherein the particle size of the chromatographic column is 1.7 μm-1.8 μm, and the column length is 100 mm, a mobile phase: the mobile phase A is methanol, and the mobile phase B is a 0.05%-0.20% formic acid aqueous solution, a detection wavelength is 265 nm, a gradient elution program: 0 min-8 min, the volume percentage of the mobile phase A is increased from 2% to 14%; 8 min-18 min, the volume percentage of the mobile phase A is increased from 14% to 37%; 18 min-26 min, the volume percentage of the mobile phase A is increased from 37% to 46%; and 26 min-40 min, the volume percentage of the mobile phase A is increased from 46% to 100%.

2. The construction method according to claim 1, characterized in that, The luffa cylindrical sample is a luffa cylindrical medicinal material, and the extraction method is heating reflux, and the heating reflux extraction time is 30 min-150 min.

3. The construction method according to claim 1 or 2, characterized in that, The luffa cylindrical sample is selected from a luffa cylindrical standard decoction or a luffa cylindrical dispensing granule, and the extraction method is ultrasonic extraction, and the ultrasonic extraction time is 15 min-60 min.

4. The construction method according to claim 1 or 2, characterized in that, Each 1 mL of the reference solution comprises 10-50 μg of a gallic acid control and 10-50 μg of a tannic acid control; and the dissolving solvent in the reference solution is selected from an alcohol solvent.

5. The construction method according to claim 1 or 2, characterized in that, Taking peak 5 as a reference peak, the relative retention times of peaks 2, 3, 4, 6 and 7 are calculated, and the relative retention times of the peaks 2, 3, 4, 6 and 7 should be within ±10% of the specified values, and the specified values corresponding to the peaks 2, 3, 4, 6 and 7 are 0.31, 0.65, 0.85, 1.11 and 1.31 respectively.

6. A method of detecting a sample of guggal, characterized by, The detection method comprises the following steps: extracting a luffa cylindrical sample with an extraction solvent, collecting the extract, and preparing a test sample solution, wherein the luffa cylindrical sample is a luffa cylindrical medicinal material, a luffa cylindrical decoction piece, a luffa cylindrical standard decoction or a luffa cylindrical dispensing granule, the extraction solvent is selected from water or an alcohol solvent, and the alcohol solvent is selected from methanol, ethanol, a methanol aqueous solution or an ethanol aqueous solution; The solution of the to-be-tested product is detected by using the ultra-high performance liquid chromatography, and the detection spectrum obtained by the detection is compared with the UPLC characteristic spectrum of the sample of the road road tunnel constructed by the construction method of any one of claims 1 to 5 to detect the quality of the to-be-tested sample; The ultra-high performance liquid chromatography conditions include: The stationary phase is a chromatographic column with octadecylsilane bonded silica as the filler, the particle size of the chromatographic column is 1.7 μm~1.8 μm, the column length is 100 mm, The mobile phase A is methanol, and the mobile phase B is a 0.05%~0.20% formic acid aqueous solution, The detection wavelength is 265 nm, The gradient elution program is: 0 min~8 min, the volume percentage of the mobile phase A is increased from 2% to 14%; 8 min~18 min, the volume percentage of the mobile phase A is increased from 14% to 37%; 18 min~26 min, the volume percentage of the mobile phase A is increased from 37% to 46%; 26 min~40 min, the volume percentage of the mobile phase A is increased from 46% to 100%.

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