A method for constructing and detecting a UPLC characteristic spectrum of a persimmon calyx sample

Abstract

The present invention relates to a method for constructing and detecting a UPLC characteristic spectrum of a persimmon calyx sample. The present invention employs ultra-high performance liquid chromatography (ULC) and rationally controls the chromatographic conditions to identify ULLC conditions that are simultaneously applicable to persimmon calyx samples, such as the persimmon calyx medicinal material, its standard decoction, and its formulated granules, thereby constructing a characteristic spectrum of the persimmon calyx sample. The characteristic spectrum of the present invention comprises 10 characteristic peaks, which enables the quality control of the persimmon calyx to be upgraded from the original determination of the content of a single component or the determination of a few common peaks to the control of the intrinsic quality of the entire persimmon calyx, thereby providing an effective method for the quality detection of the persimmon calyx and its modern preparations.

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 and detecting a UPLC characteristic spectrum of a persimmon calyx sample. Background Art

[0002] Persimmon calyx is the dried persistent calyx of Diospyros kaki Thunb. of the Ebenaceae family. It is harvested in winter when the fruit is ripe. When eaten, it is collected, washed, and sun-dried. Traditional Chinese medicine believes that this product is bitter and astringent in taste, neutral in nature, enters the stomach meridian, has the effect of lowering qi and stopping hiccups, and is suitable for hiccups caused by stomach disharmony. At present, chemical components such as flavonoids, triterpenes, tannins, organic acids, coumarins, and sterols have been isolated from persimmon calyx. Persimmon calyx tastes bitter and astringent, and is warm in nature. It is often used to treat symptoms of lowering adverse qi and stopping vomiting. Persimmon calyx is often used together with cloves to treat hiccups. In addition, persimmon calyx and cloves can treat reflux esophagitis, functional dyspepsia, and chronic gastritis.

[0003] my country has abundant persimmon resources, with widespread production areas and complex growing environments. This results in varying quality of commercial medicinal materials from different origins and sources. Traditional Chinese medicine fingerprints, or characteristic chromatograms, are comprehensive, quantifiable identification methods used to evaluate the quality of traditional Chinese medicines, preparations, and semi-finished products. They can systematically, holistically, and exclusively characterize the intrinsic characteristics of traditional Chinese medicines and have been accepted by the World Health Organization as an effective method for evaluating the quality of traditional Chinese medicines. By extracting the chemical components of the traditional Chinese medicine or its preparations and employing specific traditional Chinese medicine analytical methods, a chromatogram is generated that reflects the characteristics of each component. This represents the overall information of the traditional Chinese medicine and is relatively difficult to implement. So far, there are few studies on the characteristic spectrum or fingerprint spectrum that can reveal the intrinsic quality of persimmon calyx: Zhou Benhong et al. used HPLC to determine the fingerprint spectrum of persimmon calyx medicinal materials (Zhou Benhong et al. Study on the fingerprint spectrum of persimmon calyx medicinal materials by high performance liquid chromatography [J]. Journal of Guangdong Pharmaceutical College, 2011, 27(02):147-150.), with a mobile phase of methanol-0.5% acetic acid, an elution gradient of 120 min, and a detection wavelength of 264 nm. A total of 10 batches of persimmon calyx medicinal materials were tested. Li Ye et al. established a characteristic spectrum method for persimmon calyx medicinal materials by HPLC, with a mobile phase of acetonitrile-0.01% phosphoric acid, an elution gradient of 120 min, and a detection wavelength of 313 nm. A total of 12 batches of persimmon calyx medicinal materials were tested. These studies focused solely on the medicinal material, ignoring the water-soluble active ingredients in the decoction. This makes it difficult to reflect the clinical efficacy of persimmon calyx, lacks a clear spectrum-effect relationship, and hinders a deep understanding of the transfer of the persimmon calyx's material basis from the medicinal material to the standardized decoction and then to the finished formulated granules. Furthermore, these studies all employed HPLC, which has a long detection time of 120 minutes and a small number of batches tested. Therefore, to ensure the consistency of chemical composition from the medicinal material to the standardized decoction and then to the formulated granules, it is necessary to develop a comprehensive, accurate, rapid, and highly specific detection method that can be used not only for the detection of persimmon calyx medicinal material, but also for the detection of persimmon calyx standardized decoctions and formulated granules. This method is crucial for the quality control of modern persimmon calyx preparations. Summary of the Invention

[0004] Problems to be solved by the invention

[0005] In order to solve the above problems in the prior art, the object of the present invention is to provide a method for constructing a UPLC characteristic spectrum of a persimmon calyx sample, wherein the characteristic spectrum constructed by this method can comprehensively control the quality of the persimmon calyx sample.

[0006] Furthermore, the present invention aims to provide a method for detecting persimmon calyx samples, which is simple and rapid, has objective results, is precise and reliable, has strong specificity and high sensitivity.

[0007] Solutions for solving problems

[0008] Through long-term research, the inventors found that the above technical problems can be solved by implementing the following technical solutions:

[0009] [1] A method for constructing a UPLC characteristic spectrum of a persimmon calyx sample, wherein the construction method comprises the following steps:

[0010] (a) Preparation of reference solution: Prepare reference solution by taking protocatechuic acid reference substance, hyperoside reference substance, isoquercetin reference substance, quercetin reference substance, and kaempferol reference substance respectively;

[0011] (b) Preparation of a test solution: extracting a persimmon calyx sample with an extraction solvent, collecting the extract, and preparing a test solution. The persimmon calyx sample is a persimmon calyx medicinal material, a persimmon calyx decoction piece, a persimmon calyx standard decoction, a preparation intermediate containing a persimmon calyx, and / or a finished preparation.

[0012] (c) Establishment of characteristic spectrum: The reference solution and the test solution are tested by ultra-high performance liquid chromatography to obtain a reference spectrum and a test spectrum, and a characteristic spectrum is established by using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system;

[0013] The chromatographic conditions of the ultra-high performance liquid chromatography method include:

[0014] Stationary phase: chromatographic column with octadecylsilane bonded silica as filler and particle size of 1.5-2 μm;

[0015] Mobile phase: acetonitrile as mobile phase A, 0.05-0.30 wt% phosphoric acid solution or water as mobile phase B;

[0016] Gradient elution program:

[0017] From 0 min to 5 min, the volume percentage of the mobile phase A increases from 10% to 14%; from 5 min to 20 min, the volume percentage of the mobile phase A increases from 14% to 25%; from 20 min to 30 min, the volume percentage of the mobile phase A increases from 25% to 40%.

[0018] [2] The construction method according to [1], wherein the construction method further comprises the following step: identifying the chromatographic characteristic peaks using liquid chromatography-mass spectrometry technology.

[0019] [3] The construction method according to [1] or [2], wherein the characteristic spectrum contains 10 peaks, wherein peak 1 is protocatechuic acid, peak 3 is hyperoside, peak 4 is isoquercetin, peak 6 is kaempferol-7-O-β-D-glucoside, peak 7 is trifolioside, peak 9 is quercetin, and peak 10 is kaempferol.

[0020] [4] According to the construction method described in [3], peak 4 is used as the reference peak to calculate the relative retention time of each peak. The relative retention time should be within ±10% of the specified value. The specified values ​​corresponding to peaks 1 to 10 are 0.19, 0.49, 0.95, 1.00, 1.10, 1.15, 1.25, 1.37, 1.90, and 2.3, respectively.

[0021] [5] The construction method according to any one of the technical solutions [1] to [4], wherein the extraction solvent is selected from methanol or a methanol aqueous solution containing 20% ​​to 80% methanol by volume.

[0022] [6] The construction method according to any one of the technical solutions [1] to [5], wherein the chromatographic conditions further include: a flow rate of 0.25 mL / min to 0.35 mL / min; and / or a column temperature of 33°C to 37°C; and / or a detection wavelength of 240 to 270 nm.

[0023] [7] The construction method according to any one of the technical solutions [1] to [6], wherein the persimmon calyx sample is a persimmon calyx medicinal material, the extraction solvent is a methanol aqueous solution with a methanol volume percentage of 60% to 80%, and the extraction method is heating under reflux for 40 to 80 minutes.

[0024] [8] The construction method according to any one of the technical solutions [1] to [6], wherein the persimmon calyx sample is a standard decoction of persimmon calyx or a formula granule of persimmon calyx, the extraction solvent is a methanol aqueous solution with a methanol volume percentage of 60% to 80%, the extraction method is ultrasonic treatment, the ultrasonic extraction time is 20 min to 60 min, the power is 220 W to 300 W, and the frequency is 35 kHz to 45 kHz.

[0025] [9] A method for detecting a persimmon calyx sample, wherein the detection method comprises the following steps:

[0026] Extracting a persimmon calyx sample to be tested with an extraction solvent, collecting the extract, and preparing a test solution; the persimmon calyx sample is a persimmon calyx medicinal material, persimmon calyx decoction piece, persimmon calyx standard decoction, a preparation intermediate containing persimmon calyx, and / or a preparation finished product;

[0027] The test solution is tested by ultra-high performance liquid chromatography, and the detection spectrum obtained is compared with the characteristic spectrum constructed by any one of the construction methods of the technical solutions [1] to [8] to detect the quality and / or authenticity of the persimmon calyx sample;

[0028] The chromatographic conditions of the ultra-high performance liquid chromatography method include:

[0029] Stationary phase: chromatographic column with octadecylsilane bonded silica as filler and particle size of 1.5-2 μm;

[0030] Mobile phase: acetonitrile as mobile phase A, 0.05-0.30 wt% phosphoric acid solution or water as mobile phase B;

[0031] Gradient elution program:

[0032] From 0 min to 5 min, the volume percentage of the mobile phase A increases from 10% to 14%; from 5 min to 20 min, the volume percentage of the mobile phase A increases from 14% to 25%; from 20 min to 30 min, the volume percentage of the mobile phase A increases from 25% to 40%.

[0033]

[10] The detection method according to [9], wherein the extraction solvent is selected from methanol or a methanol aqueous solution containing 20% ​​to 80% methanol by volume.

[0034] Effects of the Invention

[0035] The present invention adopts ultra-high performance liquid chromatography and reasonably controls the chromatographic conditions to find out the ultra-high performance liquid chromatography conditions that are applicable to persimmon calyx samples such as persimmon calyx medicinal materials and their standard decoctions and formula granules, thereby constructing the characteristic spectrum of persimmon calyx samples. The characteristic spectrum of the present invention comprises 10 characteristic peaks, which makes the quality control of persimmon calyx rise to the control of the intrinsic quality of the whole persimmon calyx from the content determination of the original single component or the determination of a few common peaks. In some specific embodiments of the present invention, the characteristics of persimmon calyx can be more comprehensively reflected by determining the medicinal materials, standard decoctions and granules, avoiding the defects of single or a few peak determinations, and can provide new analytical means for the intrinsic quality control of persimmon calyx, provide an effective method for the quality detection of persimmon calyx and its modern preparations, and provide a scientific experimental basis for the comprehensive quality evaluation of persimmon calyx and its modern preparations.

[0036] The method of the present invention is simple, reproducible, accurate, reliable, easy to operate, and saves time compared to HPLC. By optimizing the chromatographic conditions, the detection time is shortened to 30 minutes, the solvent consumption is low, and the environmental pollution is small. The characteristic peaks of the spectrum of the present invention are well separated, and the chemical information of all characteristic peaks is confirmed by reference substances and mass spectrum information. The method has strong specificity, high analysis efficiency, high stability, good repeatability, and a wide range of applications. It is suitable for the detection of products such as standard decoctions and formula granules of persimmon calyx medicinal materials, and is particularly 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 invention and all advantages of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 :3D UPLC chromatogram of persimmon calyx medicinal material under full wavelength scanning

[0039] Figure 2 :UPLC chromatograms of persimmon calyx medicinal materials under different wavelength conditions

[0040] Figure 3 :UPLC chromatograms of persimmon calyx under different gradient elution conditions

[0041] Figure 4 :UPLC chromatograms of persimmon calyx under different extraction solvent conditions

[0042] Figure 5 :UPLC chromatograms of persimmon calyx under different extraction methods

[0043] Figure 6 :UPLC chromatograms of persimmon calyx at different extraction times

[0044] Figure 7 : UPLC overlay of persimmon calyx medicinal material

[0045] Figure 8 : UPLC comparison characteristic spectrum of persimmon calyx medicinal materials

[0046] Figure 9 : UPLC characteristic spectrum of the reference substance, where: Peak 1 is protocatechuic acid, Peak 3 is hyperoside, Peak 4 is isoquercetin, Peak 9 is quercetin, Peak 10 is kaempferol

[0047] Figure 10 :UPLC chromatograms of persimmon calyx medicinal materials under different chromatographic column conditions

[0048] Figure 11 :UPLC chromatograms of persimmon calyx under different column temperature conditions

[0049] Figure 12 :UPLC chromatograms of persimmon calyx under different flow rate conditions

[0050] Figure 13 UPLC overlay of persimmon calyx standard decoction

[0051] Figure 14 : UPLC comparison characteristic spectrum of persimmon calyx standard decoction

[0052] Figure 15 :UPLC chromatograms of persimmon calyx standard decoction under different chromatographic column conditions

[0053] Figure 16 :UPLC chromatograms of persimmon calyx standard decoction under different column temperature conditions

[0054] Figure 17 :UPLC chromatograms of persimmon calyx standard decoction under different flow rate conditions

[0055] Figure 18UPLC overlay of persimmon calyx formula granules

[0056] Figure 19 : UPLC comparison characteristic spectrum of persimmon calyx formula granules

[0057] Figure 20 UPLC chromatograms of persimmon calyx formula granules under different column conditions

[0058] Figure 21 UPLC chromatograms of persimmon calyx formula granules under different column temperature conditions

[0059] Figure 22 UPLC chromatograms of persimmon calyx formula granules under different flow rate conditions

[0060] Figure 23 UPLC chromatograms of persimmon calyx medicinal materials, standard decoctions, and formula granules. DETAILED DESCRIPTION

[0061] The following describes embodiments of the present invention, but the present invention is not limited thereto. The present invention is not limited to the various configurations described below; various modifications may be made 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 within the technical scope of the present invention. In addition, all documents listed in this specification are cited as references in this specification.

[0062] Unless defined otherwise, technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] Unless otherwise specified, the term "test article" used herein refers to an experimental sample used for detection or identification.

[0070] 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, and the term "precision aspiration" refers to the operation method of accurately measuring samples through a microinjector.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] Unless otherwise noted, percentages (%) in solution concentrations are by volume. For example, a 0.05% phosphoric acid aqueous solution is equivalent to adding 0.5 mL of phosphoric acid to 1000 mL of pure water. Since graduated cylinders are not recommended for mixing solutions, a 0.05% phosphoric acid aqueous solution is also equivalent to mixing 1000 mL of pure water with 0.5 mL of phosphoric acid.

[0076] <Construction Method of UPLC Characteristic Chromatogram of Persimmon Calyx Sample>

[0077] The method for constructing the UPLC characteristic spectrum of the persimmon calyx sample of the present invention mainly comprises the following steps:

[0078] (a) Preparation of reference solution: prepare reference solution by taking protocatechuic acid reference, hyperoside reference, isoquercetin reference, quercetin reference, and kaempferol reference respectively; further, dissolve the reference in water, alcohol, or alcohol-water solution. In some specific embodiments of the present invention, the preparation method of the reference solution is as follows: take an appropriate amount of reference, accurately weigh it, add the aforementioned solvent, and mix until the solution is clear. Furthermore, the extraction solvent is an alcohol or an alcohol-water solution, such as methanol or a methanol-water solution. Furthermore, the methanol volume fraction of the methanol-water solution is not less than 30%, and preferably the concentration of the extraction solvent is 50% to 80% in terms of the volume fraction of methanol. More specifically, the concentration of the protocatechuic acid reference substance is 40-70 μg / mL, the concentration of the hyperoside reference substance is 40-70 μg / mL, the concentration of the isoquercetin reference substance is 30-50 μg / mL, the concentration of the quercetin reference substance is 5-15 μg / mL, and the concentration of the kaempferol reference substance is 5-15 μg / mL.

[0079] (b) Preparation of test solution: Extract the persimmon calyx sample with an extraction solvent, collect the extract, and prepare the test solution. The persimmon calyx sample is a persimmon calyx medicinal material, a persimmon calyx decoction piece, a persimmon calyx standard decoction, a preparation intermediate containing the persimmon calyx, and / or a finished preparation. In some preferred embodiments of the present invention, the persimmon calyx sample is a persimmon calyx medicinal material, a persimmon calyx standard decoction, and / or a persimmon calyx formula granule; wherein, "medicinal material" or "raw drug" refers to a Chinese medicine raw material that has not been processed or made into a finished product; "decoction piece" refers to a Chinese medicine for formulation formed by processing according to needs, or a Chinese medicine that can be directly used in Chinese medicine clinical practice; "standard decoction" refers to a single-flavor Chinese medicine decoction piece water decoction prepared by a standardized process based on Chinese medicine theory and clinical application, with reference to modern extraction methods; "formulated granules" are granules prepared by extracting and concentrating a single-flavor Chinese medicine decoction piece according to traditional standards for use in Chinese medicine clinical formulation. In the present invention, "preparation intermediate" includes extracts (water extracts), etc.; "finished preparation" includes formula granules. In some specific embodiments of the present invention, the extraction solvent is selected from methanol or a methanol aqueous solution containing 20% ​​to 80% methanol by volume. In order to obtain a better peak shape (or peak shape) and comprehensively consider the total extraction efficiency, the extraction reagent is preferably a methanol aqueous solution with a methanol volume percentage of 60% to 80%. Regarding the extraction method, for the persimmon calyx medicinal material, it is preferred to first sieve the medicinal material powder. In order to obtain richer chromatographic peak information, it is preferred to adopt a heating reflux extraction method. In some preferred embodiments of the present invention, a methanol aqueous solution with a methanol volume percentage of 60% to 80% is used to extract the sieved persimmon calyx medicinal material powder. The extraction method is heating reflux for 30 to 120 minutes. Furthermore, considering the extraction efficiency of each chromatographic peak, the extraction time is 40 to 70 minutes. For the standard decoction of persimmon calyx or persimmon calyx formula granules, it is preferred that the sample be first ground into a fine powder, and then extracted with a methanol aqueous solution with a volume percentage of 60% to 80%. The extraction can be carried out by ultrasound, heating reflux or shaking. In order to simplify the extraction operation, it is preferred to use an ultrasonic treatment method. The power and frequency of the ultrasound have little effect on the present invention. Furthermore, the duration of the ultrasonic extraction is 20min to 60min, the power is 220W to 300W, and the frequency is 35kHz to 45kHz. In some specific embodiments of the present invention, the amount of the extraction solvent added relative to the persimmon calyx sample is 5 to 25mL / g, further 6 to 20mL / g. Furthermore, for persimmon calyx powder, the amount of the extraction solvent added relative to the persimmon calyx powder is 6 to 10mL / g; for the standard decoction of persimmon calyx or persimmon calyx formula granules, the relative amount of the extraction solvent added is 16 to 20mL / g. If the test sample concentration is too low, it is likely to be detrimental to the detection of the chromatographic peak. If the concentration is too high, the chromatographic column will be overloaded, which can easily lead to poor chromatographic peak shape.In some specific embodiments of the present invention, the preparation method of the test solution is as follows: take persimmon calyx medicinal material powder, accurately weigh it, place it in a stoppered conical flask, accurately add 60-80% methanol, seal it, weigh it, heat and reflux for 40-70 minutes, let it cool, weigh it again, make up the lost weight with 60-80% methanol, shake it well, filter it, and take the filtrate to obtain; or take an appropriate amount of persimmon calyx standard decoction or persimmon calyx formula granules, grind it into powder, accurately weigh it, place it in a stoppered conical flask, accurately add 60-80% methanol, seal it, ultrasonically treat it for 20-60 minutes, let it cool, shake it well, filter it, and take the filtrate to obtain. Wherein, the mass volume ratio of persimmon calyx medicinal material, persimmon calyx standard decoction or persimmon calyx formula granules to 60-80% methanol is 1g:(5-25mL).

[0080] (c) Establishment of characteristic spectrum: The reference solution and the test solution are tested by ultra-high performance liquid chromatography to obtain a reference spectrum and a test spectrum respectively, and a characteristic spectrum is established by using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system.

[0081] Through in-depth research, the present invention has determined ultra-high performance liquid chromatography conditions that are applicable to both persimmon calyx medicinal materials and their standard decoctions and formulated granules, which include:

[0082] Stationary phase: A chromatographic column with octadecylsilane bonded silica as a filler and a particle size (filler particle size) of 1.5 to 2 μm; further, to obtain better column efficiency, the particle size is 1.6 to 1.8 μm; further, the chromatographic column has a length of 100 to 150 mm and an inner diameter of 2.1 mm. In some specific embodiments of the present invention, the chromatographic column is selected from one of ZORBAX Eclipse Plus C18 (Agilent, 100 mm × 2.1 mm, 1.8 μm), ACQUITY UPLC HSS T3 (Waters Corporation, 100 mm × 2.1 mm, 1.8 μm), and ACQUITY UPLC BEH C18 (Waters Corporation, 100 mm × 2.1 mm, 1.7 μm); further, for better separation, the chromatographic column is preferably a ZORBAX Eclipse Plus C18 (Agilent, 100 mm × 2.1 mm, 1.8 μm).

[0083] Mobile phase: acetonitrile is used as mobile phase A, and 0.05-0.30 wt% phosphoric acid solution or water is used as mobile phase B. In some preferred embodiments of the present invention, 0.08-0.15% phosphoric acid solution is used as mobile phase B to help obtain a good peak shape.

[0084] The gradient elution procedure of the present invention is as follows:

[0085] From 0 to 5 minutes, the volume percentage of mobile phase A increases from 10% to 14%; from 5 to 20 minutes, the volume percentage of mobile phase A increases from 14% to 25%; and from 20 to 30 minutes, the volume percentage of mobile phase A increases from 25% to 40%. These conditions ensure that all chromatographic peaks are fully separated and have good peak shapes, regardless of whether the sample is a persimmon calyx medicinal material, a standard decoction, or a formulated granule. This also shortens the analysis cycle and increases separation efficiency and resolution.

[0086] The detector used in the present invention is an ultraviolet detector. Furthermore, the chromatographic conditions of the present invention also include: a detection wavelength of 240 to 270 nm. In some preferred embodiments of the present invention, the detection wavelength is 240 to 260 nm. 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. Furthermore, the chromatographic conditions of the present invention also include: a flow rate of 0.25 mL / min to 0.35 mL / min; and / or, a column temperature of 33°C to 37°C. Within the column temperature or flow rate range, 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 is 0.28 mL / min to 0.32 mL / min; and / or, the column temperature is 34°C to 36°C.

[0087] Furthermore, the present invention detects at least 15 batches of test solution, analyzes and compares them, selects common peaks to establish UPLC characteristic spectrum, and refers to the chromatographic peaks of the reference sample and uses liquid chromatography-mass spectrometry technology to identify the common peaks. Furthermore, the present invention uses liquid chromatography-mass spectrometry technology to collect high-resolution mass spectrometry data according to the liquid chromatography conditions of the characteristic spectrum of the persimmon calyx sample, combines isotope analysis to infer the molecular formula of the compound in the persimmon calyx sample, combines reference literature and relevant reference sample comparison, and attributes its common peaks. In some specific embodiments of the present invention, the mass spectrometry conditions include: ion source: electrospray positive ion; detection mode: positive ion mode, and the remaining conditions are the same as the liquid chromatography conditions described above in the present invention. The characteristic spectrum of the persimmon calyx sample obtained by the aforementioned construction method of the present invention contains at least 10 peaks. Furthermore, taking peak 4 as the reference peak, the relative retention times of peak 1, peak 2, peak 3, peak 5, peak 6, peak 7, peak 8, peak 9 and peak 10 are 0.19 (±10%), 0.49 (±10%), 0.95 (±10%), 1.10 (±10%), 1.15 (±10%), 1.25 (±10%), 1.37 (±10%), 1.90 (±10%), and 2.39 (±10%), respectively. The retention times and UV spectra of peaks 1, 3, 4, 9, and 10 matched those of reference solutions of protocatechuic acid, hyperoside, isoquercetin, quercetin, and kaempferol, respectively. Therefore, peaks 1, 3, 4, 9, and 10 in the characteristic spectrum of the persimmon calyx sample were identified as protocatechuic acid, hyperoside, isoquercetin, quercetin, and kaempferol, respectively. Liquid chromatography-mass spectrometry (LC-MS / MS) identified three common peaks: peaks 6, 7, and 8 as kaempferol-7-O-β-D-glucoside, trilobatin, and kaempferol-3-O-(6-galloyl)-BD-glucopyranoside, respectively. These 10 common peaks consistently appeared in all medicinal materials, standard decoctions, and formulated granules, with good agreement.

[0088] The present invention shortens the analysis time and improves the analysis efficiency by optimizing the test sample preparation and chromatographic analysis conditions, establishes ultra-high performance liquid chromatography conditions that are simultaneously applicable to persimmon calyx medicinal materials and their standard decoctions and formula granules, and truly reflects the original chemical composition of persimmon calyx samples, especially persimmon calyx medicinal materials, standard decoctions and formula granules.

[0089] <Testing Method for Persimmon Calyx Samples>

[0090] On the other hand, the characteristic spectrum of the present invention can be used for the detection of quality control of persimmon calyx samples (such as persimmon calyx medicinal materials and preparations thereof), and the detection method comprises the following steps:

[0091] Extracting a persimmon calyx sample to be tested with an extraction solvent, collecting the extract, and preparing a test solution; the persimmon calyx sample is a persimmon calyx medicinal material, persimmon calyx decoction piece, persimmon calyx standard decoction, a preparation intermediate containing persimmon calyx, and / or a preparation finished product;

[0092] The test solution is tested using ultra-high performance liquid chromatography, and the resulting test pattern is compared with the characteristic pattern constructed using the construction method of the present invention to detect the quality and / or authenticity of the persimmon calyx sample; further, if the UPLC characteristic patterns of the two are consistent, the sample is qualified; if the UPLC characteristic patterns of the two are inconsistent, the sample is unqualified. In some specific embodiments of the present invention, the test is performed using the test solution preparation method and chromatographic conditions described above.

[0093] In some specific embodiments of the present invention, by using characteristic spectra of persimmon calyx medicinal materials, standard decoctions, and formula granules to detect the test samples, a new analytical means is provided for the intrinsic quality control of persimmon calyx from raw materials to finished products. The method is simple to operate, has high precision, good stability, good repeatability, and high accuracy, maintains the consistency of the quality of persimmon calyx medicinal materials, standard decoctions and formula granules, and provides an important multi-index parameter basis for evaluating process stability.

[0094] Example

[0095] The present invention will be further described below through specific embodiments:

[0096] Laboratory instruments, reagents and medicines

[0097] A Waters Acquity UPLC ultra-high-performance liquid chromatograph (Waters); an Empower 3 workstation (Waters); an Agilent G6530 Accurate-Mass Q-TOF mass spectrometer (Agilent); Agilent Mass Hunter Workstation data acquisition and qualitative analysis software (Agilent); an electronic analytical balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); a temperature-controlled water bath (Nantong Huatai Experimental Instrument Co., Ltd.); a KQ-250B ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); a pure water system (Sartorius); and an AS165W centrifuge (Azov (Shanghai) Trading Co., Ltd.) were used. Acetonitrile (chromatographic grade, Thermo Fisher); water was ultrapure; phosphoric acid (chromatographic grade, Aladdin); all other reagents were of analytical grade.

[0098] Protocatechuic acid reference substance, hyperoside reference substance, isoquercetin reference substance, quercetin reference substance, and kaempferol reference substance were all purchased from the China Food and Drug Inspection Institute.

[0099] Persimmon calyx was provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd. and identified as the dried persistent calyx of Diospyros kaki Thunb. Standardized decoction and granules of persimmon calyx were also prepared and provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0100] Example 1: Construction of UPLC characteristic spectrum of persimmon calyx medicinal material

[0101] 1. Preparation of reference solution: Take appropriate amount of protocatechuic acid reference substance, hyperoside reference substance, isoquercetin reference substance, quercetin reference substance, and kaempferol reference substance, accurately weigh them, and add methanol to prepare solutions containing 50 μg of protocatechuic acid, 50 μg of hyperoside, 40 μg of isoquercetin, 10 μg of quercetin, and 10 μg of kaempferol per 1 mL, which are used as reference solution.

[0102] 2. Preparation of test solution:

[0103] Take about 2.5 g of persimmon calyx powder (passed through No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 20 mL of 70% methanol, stopper it, weigh it, heat and reflux it for 60 minutes, cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0104] 3. Determination of chromatographic conditions

[0105] 3.1 Determination of detection wavelength

[0106] Take the above test solution and use octadecylsilane bonded silica gel as the filler (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm); use acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and perform gradient elution according to the following gradient at a flow rate of 0.3 mL per minute; the column temperature is 35°C; record the absorption spectrum in the range of 190-400 nm, see Figure 1 and Figure 2 .

[0107]

[0108] It can be seen that the chromatographic peak information at 254 nm is relatively rich, the separation effect of each chromatographic peak is good, and the baseline is relatively stable, so 254 nm is selected as the detection wavelength.

[0109] 3.2 Optimization of chromatographic conditions

[0110] An ACQUITY UPLC CORTECS T3 (column length: 100 mm, inner diameter: 2.1 mm, particle size: 1.8 μm) was used as the chromatographic column; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, with gradient elution performed as specified in the table below; the flow rate was 0.3 mL / min; the column temperature was 30°C; and the detection wavelength was 320 nm. The resulting chromatogram is shown in the table below. Figure 3 .

[0111] Gradient 1:

[0112]

[0113] Gradient 2:

[0114]

[0115] Replace the chromatographic column: Use ZORBAX Eclipse Plus C18 (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm) as the chromatographic column; use acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and perform gradient elution as specified in the table below; flow rate 0.3 mL / min; column temperature 30°C; detection wavelength 320 nm. The resulting chromatogram is shown in the table below. Figure 3 .

[0116] Gradient 3:

[0117]

[0118] Gradient 4:

[0119]

[0120] The chromatographic conditions for the characteristic spectrum of Persimmon Calyx Granules were as follows: octadecylsilane bonded silica gel as the filler (column length, 100 mm, inner diameter, 2.1 mm, particle size, 1.8 μm); acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution as specified in Table 1; flow rate, 0.3 mL / min; column temperature, 35°C; detection wavelength, 254 nm. The number of theoretical plates, calculated based on the isoquercetin peak, should be no less than 3000.

[0121] Table 1: Gradient elution conditions

[0122]

[0123] 4. Preparation of Test Solution

[0124] (1) Investigation of extraction solvent

[0125] This experiment investigated the effects of different extraction solvents on the characteristic spectra of persimmon calyx medicinal materials. 30% methanol, 50% methanol, 70% methanol, methanol and water were selected as extraction solvents. The characteristic spectra of different extraction solvents were compared by the total peak area / sample weight and chromatograms of 10 temporarily identified chromatographic peaks.

[0126] Take about 2.5 g of persimmon calyx powder, accurately weigh it, place it in a stoppered conical flask, and accurately add water, 30% methanol, 50% methanol, 70% methanol, and 20 mL of methanol respectively in 5 parallel portions, stopper it, weigh it, and treat it ultrasonically (power 250 W, frequency 40 kHz) for 60 minutes. Let it cool, weigh it again, make up the lost weight with each extraction solvent, shake it well, filter it, and take the filtrate as the test solution.

[0127] According to the chromatographic conditions under "3.2", the characteristic spectrum of the persimmon calyx medicinal material was obtained. The results of the investigation of different extraction solvents are shown in Table 2. Figure 4 .

[0128] Table 2 Characteristic spectrum of persimmon calyx medicinal material extraction solvent investigation (peak area / sample weight)

[0129]

[0130] from Figure 4 It can be seen that when water is used as the extraction solvent, the peak shape is poor and few peaks appear. When 30%, 50%, 70%, and 10 other methanol are used as the extraction solvent, 10 characteristic peaks are well separated. Taking into account the overall extraction efficiency, 70% methanol was finally selected as the extraction solvent for the characteristic spectrum of the persimmon calyx medicinal material.

[0131] (2) Investigation of extraction methods

[0132] This experiment investigated the effects of different extraction methods on the characteristic spectrum of persimmon calyx medicinal materials. Ultrasonic treatment, heating reflux and shaking extraction were selected as the extraction methods. The characteristic spectrum results of different extraction methods were compared through the total peak area / sample weight of 10 temporarily identified chromatographic peaks and chromatograms.

[0133] Take about 2.5 g of persimmon calyx powder, accurately weigh it, place it in a stoppered conical flask, accurately add 20 mL of 70% methanol, stopper it, weigh it, and ultrasonically treat it (power 250 W, frequency 40 kHz) for 60 minutes, shake it for 60 minutes, and heat it under reflux for 60 minutes. Let it cool, weigh it again, make up the lost weight with 70% methanol, shake it evenly, filter it, and take the filtrate as the test solution.

[0134] According to the chromatographic conditions under "3.2", the characteristic spectrum of the persimmon calyx medicinal material was obtained. The results of the investigation of different extraction methods are shown in Table 3. Figure 5 .

[0135] Table 3 Investigation of extraction methods for characteristic spectrum of persimmon calyx medicinal materials (peak area / sample weight)

[0136]

[0137] from Figure 5 It can be seen that the chromatographic peak information of the medicinal materials extracted by heating and reflux is rich and the peak area is large, while the chromatographic peak information of ultrasonic and shaking extraction is relatively incomplete, so heating and reflux extraction is selected as the final extraction method.

[0138] (3) Investigation of extraction time

[0139] This invention investigated the effects of different extraction times on the characteristic spectrum of persimmon calyx medicinal materials. Four different extraction times of 30 minutes, 60 minutes, 90 minutes and 120 minutes were selected for investigation. The characteristic spectrum results of different extraction times were compared through the total peak area / sample weight of 10 temporarily identified chromatographic peaks and chromatograms.

[0140] Take about 2.5 g of persimmon calyx powder, accurately weigh it, place it in a stoppered conical flask, accurately add 20 mL of 70% methanol, stopper it tightly, weigh it, heat and reflux and extract for 30 minutes, 60 minutes, 90 minutes and 120 minutes respectively, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate as the test solution.

[0141] According to the chromatographic conditions under "3.2", the characteristic spectrum of the persimmon calyx medicinal material was obtained. The results of the investigation at different extraction times are shown in Table 4. Figure 6 .

[0142] Table 4 Characteristic spectrum of persimmon calyx medicinal material extraction time investigation (peak area / sample weight)

[0143]

[0144] from Figure 6 It can be seen that the number of chromatographic peaks obtained at each extraction time is consistent. Taking into account the extraction efficiency of each chromatographic peak, the final extraction time was selected as 60 minutes.

[0145] 5. Establishment of feature maps

[0146] 5.1 Accurately pipette 1 μL of the reference solution and 2 μL of the test solution into the liquid chromatograph;

[0147] 5.2 18 batches of persimmon calyx medicinal materials were analyzed and compared, and a UPLC characteristic spectrum consisting of their common characteristic peaks was obtained, such as Figure 7As shown in Figure 1, it contains 10 common characteristic peaks, peak 1 is protocatechuic acid, peak 3 is hyperoside, peak 4 is isoquercetin, peak 6 is kaempferol-7-O-β-D-glucoside, peak 7 is trifolioside, peak 9 is quercetin, and peak 10 is kaempferol. 18 batches of medicinal materials were selected and introduced into the "Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System (2012.0 Version)" promulgated by the State Pharmacopoeia Committee. The time window width was selected as 0.1 min, and the reference spectrum was generated using the median. After multi-point correction, the Mark peaks were matched to generate the common pattern of the characteristic spectrum of the persimmon calyx medicinal materials (i.e., the reference characteristic spectrum of the persimmon calyx medicinal materials) as shown in Figure 1. Figure 8 Taking peak 4 as the reference peak, the relative retention times of the common peaks in the characteristic spectrum were calculated and shown in Tables 5 and 6.

[0148] Table 5 Relative retention time of 18 batches of persimmon calyx medicinal materials

[0149]

[0150] Table 6 Relative peak areas of 18 batches of persimmon calyx medicinal materials

[0151]

[0152]

[0153] The results showed that the retention times of the characteristic peaks in the above medicinal materials were relatively stable, with RSDs of less than 0.2%; the peak area ratios had a large fluctuation range, with RSD values ​​between 12 and 87%, indicating that there were large differences in the relative amounts of individual components in different batches of products.

[0154] 5.3 Similarity evaluation of persimmon calyx medicinal materials The similarity evaluation system of Chinese medicine chromatographic fingerprint (2012.0 version) issued by the Chinese Pharmacopoeia Committee was used to overlay the characteristic spectra of 18 batches of persimmon calyx medicinal materials. Figure 7 , the consistency of chromatographic peak similarity was investigated and similarity evaluation was performed. The results showed that S1 was 0.818, S2 was 0.881, S3 was 0.850, S4 was 0.979, S5 was 0.983, S6 was 0.980, S7 was 0.993, S8 was 0.992, S9 was 0.988, S10 was 0.994, S11 was 0.995, S12 was 0.995, S13 was 0.994, S14 was 0.994, S15 was 0.995, S16 was 0.987, S17 was 0.995, and S18 was 0.988, all of which were greater than 0.8, indicating that the similarity between the characteristic spectrum of the medicinal material and the reference spectrum was high. The 10 common peaks can all appear stably in each medicinal material, and the degree of agreement is good. Therefore, this spectrum can be used as the characteristic spectrum of Persimmon Calyx medicinal material.

[0155] 6. Attribution of common peaks and determination of characteristic peaks

[0156] Liquid chromatography-mass spectrometry (LC-MS) was used to collect high-resolution mass spectrometry data according to the liquid chromatography conditions of the characteristic spectrum of the persimmon calyx medicinal material. The molecular formula of the compounds in the persimmon calyx medicinal material was inferred by combining isotope analysis. The common peaks were attributed by combining references and relevant reference substances. The retention time and UV spectrum of peak 1, peak 3, peak 4, peak 9, and peak 10 were consistent with those of the reference substances protocatechuic acid, hyperoside, isoquercetin, quercetin, and kaempferol. Therefore, peak 1, peak 3, peak 4, peak 9, and peak 10 in the fingerprint of the test sample were identified as protocatechuic acid, hyperoside, isoquercetin, quercetin, and kaempferol, respectively. Figure 9 In addition, three common peaks were identified by LC-MS / MS, and the results are shown in Table 7.

[0157] Table 7 LC / MS analysis results of peak 6, peak 7 and peak 8

[0158]

[0159] 7. Validation of Feature Mapping Methodology

[0160] (1) Overall investigation

[0161] Under the same chromatographic conditions, maintaining the same gradient, the elution time was doubled, and the chromatogram was recorded. No obvious chromatographic peak eluted after 30 minutes, indicating that the chromatographic conditions basically met the principle of maximum information content.

[0162] (2) Precision inspection

[0163] The persimmon calyx medicinal material powder was taken and the test solution was prepared according to the method under "2. Preparation of test solution" in Example 1. 2 μL of the same test solution was accurately aspirated and injected 6 times continuously. Peak 4 was used as the reference peak, and the relative retention time and relative peak area RSD values ​​of the common fingerprint peaks 1-10 were calculated to be less than 1.3%. At the same time, the similarity of each chromatographic fingerprint calculated by the similarity evaluation software was greater than 0.9, indicating that the instrument method was stable and the precision was good.

[0164] (3) Repeatability study

[0165] Take 6 portions of the same batch of persimmon calyx medicinal powder, accurately weigh them, and prepare 6 test solutions according to the method under "2. Preparation of test solution" in Example 1. 2 μL of each solution was injected, and peak 4 was used as the reference peak. The relative retention time and relative peak area RSD values ​​of the common peaks 1-10 were calculated to be less than 2.0%, indicating that the construction method has good reproducibility.

[0166] (4) Stability investigation

[0167] Take the same test solution and inject it at 0, 2, 4, 6, 8, and 12 hours. Taking peak 4 as the reference peak, the RSD values ​​of the relative retention times and relative peak areas of common peaks 1-10 are calculated to be less than 0.9%, indicating that the test solution is stable for at least 12 hours.

[0168] (5) Durability inspection

[0169] 1) Investigation of different chromatographic columns

[0170] The effects of three chromatographic columns, namely, ZORBAX Eclipse Plus C18 (Agilent, 100mm×2.1mm, 1.8μm), ACQUITY UPLC HSS T3 (Waters Corporation, 2.1×100mm, 1.8μm), and ACQUITY UPLCBEH C18 (Waters Corporation, 2.1×100mm, 1.7μm), on the durability of the characteristic spectrum of persimmon calyx were compared.

[0171] See the results Figure 10 Persimmon calyx can be well separated on the ZORBAX Eclipse Plus C18, ACQUITY HSS T3, and ACQUITY BEH C18 columns. The ZORBAX Eclipse Plus C18 (Agilent, 2.1 × 100 mm, 1.8 μm) column is recommended for characterization of persimmon calyx.

[0172] 2) Investigation at different temperatures

[0173] A ZORBAX Eclipse Plus C18 (Agilent, 2.1×100 mm, 1.8 μm) column was used to investigate the separation effects of medicinal materials at column temperatures of 33°C, 35°C, and 37°C.

[0174] See the results Figure 11 , this analysis method can meet the system applicability requirements within the column temperature range of 33℃~37℃, and the smaller column temperature fluctuation can meet the system applicability requirements.

[0175] 3) Investigation of different flow rates

[0176] A ZORBAX Eclipse Plus C18 (Agilent, 2.1×100 mm, 1.8 μm) column was used to investigate the separation effects of medicinal materials at flow rates of 0.25 mL / min, 0.30 mL / min, and 0.35 mL / min.

[0177] See the results Figure 12, this analysis method can meet the system applicability requirements with a small flow rate change within the flow rate range of 0.25mL / min to 0.35mL / min.

[0178] Example 2: Construction of UPLC Characteristic Spectrum of Persimmon Calyx Standard Decoction

[0179] 1. Preparation of reference solution

[0180] Take appropriate amounts of protocatechuic acid reference substance, hyperoside reference substance, isoquercetin reference substance, quercetin reference substance, and kaempferol reference substance, accurately weigh them, and add methanol to prepare solutions containing 50 μg of protocatechuic acid, 50 μg of hyperoside, 40 μg of isoquercetin, 10 μg of quercetin, and 10 μg of kaempferol per 1 mL, as reference substance solutions.

[0181] 2. Preparation of test solution

[0182] Take an appropriate amount of standard decoction of persimmon calyx, grind it into powder, take 0.8g, weigh it accurately, put it into a stoppered conical flask, accurately add 15mL of 70% methanol, stopper it tightly, and ultrasonically treat it (power 250W, frequency 40kHz) for 45 minutes. Let it cool, shake it well, filter it, and take the filtrate to obtain the product.

[0183] 3. Chromatographic conditions

[0184] An Eclipse Plus C18 column (column length: 100 mm, inner diameter: 2.1 mm, particle size: 1.8 μm) was used; acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to Table 1; the column temperature was 35° C.; the flow rate was 0.30 mL / min; and the detection wavelength was 254 nm.

[0185] 4. Establishment of feature maps

[0186] 4.1 Accurately pipette 1 μL of reference solution and 2 μL of test solution into the liquid chromatograph.

[0187] 4.2 18 batches of persimmon calyx standard decoction were analyzed and compared to obtain a UPLC characteristic spectrum consisting of their common characteristic peaks, such as Figure 13 As shown in FIG, it contains 10 common characteristic peaks, peak 1 is protocatechuic acid, peak 3 is hyperoside, peak 4 is isoquercetin, peak 6 is kaempferol-7-O-β-D-glucoside, peak 7 is trifolioside, peak 9 is quercetin, and peak 10 is kaempferol. 18 batches of standard decoctions were selected and introduced into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012.0 Version)" promulgated by the State Pharmacopoeia Committee. The time window width was selected as 0.1 min, and the reference spectrum was generated with the median. After multi-point correction, the Mark peaks were matched to generate the common pattern of the characteristic spectrum of the persimmon calyx standard decoction (i.e., the reference characteristic spectrum of the persimmon calyx standard decoction) as shown in FIG. Figure 14Taking peak 4 as the reference peak, the relative retention time and relative peak area of ​​the common peaks in the characteristic spectrum were calculated and shown in Tables 8 and 9.

[0188] Table 8 Relative retention time of 18 batches of persimmon calyx standard decoction

[0189]

[0190] Table 9 Relative peak areas of 18 batches of persimmon calyx standard decoction

[0191]

[0192]

[0193] The results showed that the retention times of the characteristic peaks in the above standard decoctions were relatively stable, with RSDs of less than 0.6%; the peak area ratios fluctuated in a large range, with RSD values ​​between 22 and 91%, indicating that there were large differences in the relative amounts of individual components in different batches of products.

[0194] 4.3 Similarity evaluation of persimmon calyx standard decoction The characteristic spectra of 18 batches of persimmon calyx standard decoction were superimposed using the “Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012.0 Version)” issued by the Chinese Pharmacopoeia Committee. Figure 13 The consistency of the chromatographic peak similarity was examined and similarity evaluation was performed. The results showed that all the results were greater than 0.8, indicating that the similarity between the characteristic spectrum of the standard decoction and the control spectrum was high. The 10 common peaks appeared stably in each standard decoction and the degree of agreement was good. Therefore, this spectrum can be used as the characteristic spectrum of the standard decoction of persimmon calyx.

[0195] 5. Validation of Feature Mapping Methodology

[0196] (1) Overall investigation

[0197] Under the same chromatographic conditions, maintaining the same gradient, the elution time was doubled, and the chromatogram was recorded. No obvious chromatographic peak eluted after 30 minutes, indicating that the chromatographic conditions basically met the principle of maximum information content.

[0198] (2) Precision inspection

[0199] The test solution was prepared from the standard decoction of persimmon calyx according to the method under "2. Preparation of test solution" in Example 2. 1 μL of the same test solution was accurately aspirated and injected 6 times continuously. Peak 4 was used as the reference peak, and the relative retention time and relative peak area RSD values ​​of the common fingerprint peaks 1-10 were calculated to be less than 1.7%. At the same time, the similarity of each chromatographic fingerprint calculated by the similarity evaluation software was greater than 0.9, indicating that the instrument method was stable and the precision was good.

[0200] (3) Repeatability study

[0201] Take 6 portions of the same batch of persimmon calyx standard decoction, accurately weigh them, and prepare 6 test solutions according to the method under "2. Preparation of test solution" in Example 2. Samples were injected separately, and peak 4 was used as the reference peak. The relative retention time and relative peak area RSD values ​​of the common peaks 1-10 were calculated to be less than 1.8%, indicating that the construction method has good reproducibility.

[0202] (4) Stability investigation

[0203] Take the same test solution and inject it at 0, 2, 4, 6, 8, and 12 hours. Taking peak 4 as the reference peak, the RSD values ​​of the relative retention times and relative peak areas of common peaks 1-10 are calculated to be less than 1.7%, indicating that the test solution is stable for at least 12 hours.

[0204] (5) Durability inspection

[0205] 1) Investigation of different chromatographic columns

[0206] The effects of three chromatographic columns, namely, ZORBAX Eclipse Plus C18 (Agilent, 100mm×2.1mm, 1.8μm), ACQUITY UPLC HSS T3 (Waters Corporation, 2.1×100mm, 1.8μm), and ACQUITY UPLCBEH C18 (Waters Corporation, 2.1×100mm, 1.7μm), on the durability of the characteristic spectrum of the standard decoction of Persimmon Calyx were compared.

[0207] See the results Figure 15 The standard decoction of persimmon calyx was well separated on the ZORBAX Eclipse Plus C18, ACQUITY HSS T3, and ACQUITY BEH C18 columns. The ZORBAX Eclipse Plus C18 (Agilent, 2.1 × 100 mm, 1.8 μm) column is recommended for characterization of the standard decoction of persimmon calyx.

[0208] 2) Investigation at different temperatures

[0209] A ZORBAX Eclipse Plus C18 (Agilent, 2.1×100 mm, 1.8 μm) column was used to investigate the separation effects of standard decoctions at column temperatures of 33°C, 35°C, and 37°C.

[0210] See the results Figure 16 , this analysis method can meet the system applicability requirements within the column temperature range of 33℃~37℃, and the smaller column temperature fluctuation can meet the system applicability requirements.

[0211] 3) Investigation of different flow rates

[0212] A ZORBAX Eclipse Plus C18 (Agilent, 2.1×100 mm, 1.8 μm) column was used to investigate the separation effect of the standard decoction at flow rates of 0.25 mL / min, 0.30 mL / min, and 0.35 mL / min.

[0213] See the results Figure 17 , this analysis method can meet the system applicability requirements with a small flow rate change within the flow rate range of 0.25mL / min to 0.35mL / min.

[0214] Example 3: Construction of UPLC Characteristic Spectrum of Persimmon Calyx Formula Granules

[0215] 1. Preparation of reference solution

[0216] Take appropriate amounts of protocatechuic acid reference substance, hyperoside reference substance, isoquercetin reference substance, quercetin reference substance, and kaempferol reference substance, accurately weigh them, and add methanol to prepare solutions containing 50 μg of protocatechuic acid, 50 μg of hyperoside, 40 μg of isoquercetin, 10 μg of quercetin, and 10 μg of kaempferol per 1 mL, as reference substance solutions.

[0217] 2. Preparation of test solution

[0218] Take an appropriate amount of persimmon calyx formula granules, grind them into powder, take 0.8g, accurately weigh, place in a stoppered conical flask, accurately add 15mL of 70% methanol, seal it tightly, and ultrasonically treat (power 250W, frequency 40kHz) for 45 minutes. Let cool, shake well, filter, and take the filtrate to obtain the product.

[0219] 3. Chromatographic conditions

[0220] An Eclipse Plus C18 column (column length: 100 mm, inner diameter: 2.1 mm, particle size: 1.8 μm) was used; acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to Table 1; the column temperature was 35° C.; the flow rate was 0.30 mL / min; and the detection wavelength was 254 nm.

[0221] 4. Establishment of feature maps

[0222] 4.1 Accurately pipette 1 μL of the reference solution and 2 μL of the test solution into the liquid chromatograph.

[0223] 4.2 Three batches of persimmon calyx formula granules were analyzed and compared to obtain a UPLC characteristic spectrum consisting of their common characteristic peaks, such as Figure 18As shown in Figure 1, it contains 10 common characteristic peaks, peak 1 is protocatechuic acid, peak 3 is hyperoside, peak 4 is isoquercetin, peak 6 is kaempferol-7-O-β-D-glucoside, peak 7 is trifolioside, peak 9 is quercetin, and peak 10 is kaempferol. Three batches of granules were selected and imported into the "Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System (2012.0 Version)" promulgated by the State Pharmacopoeia Commission. The time window width was selected as 0.1 min, and the reference spectrum was generated using the median. After multi-point correction, the Mark peaks were matched to generate the common pattern of the characteristic spectrum of Persimmon Calyx Granules (i.e., the reference characteristic spectrum of Persimmon Calyx Formula Granules). Figure 19 Taking peak 4 as the reference peak, the relative retention time and relative peak area of ​​the common peaks in the characteristic spectrum were calculated and shown in Tables 10 and 11.

[0224] Table 10 Relative retention time of three batches of persimmon calyx granules

[0225]

[0226] Table 11 Relative peak areas of three batches of persimmon calyx granules

[0227]

[0228] The results showed that the retention time of the characteristic peaks in the above particles was relatively stable, with RSDs of less than 0.2%; the peak area ratios fluctuated in a large range, with RSD values ​​between 0 and 3%, indicating that there were large differences in the relative amounts of the individual components in different batches of products.

[0229] 4.3 Similarity evaluation of persimmon pedicle granules The characteristic spectra of three batches of persimmon pedicle granules were superimposed using the “Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012.0 Version)” issued by the Chinese Pharmacopoeia Committee. Figure 18 The consistency of the chromatographic peak similarity was examined and similarity evaluation was performed. The results showed that they were all greater than 0.9, indicating that the similarity between the characteristic spectrum of the particles and the control spectrum was high. The 10 common peaks can appear stably in each particle and the degree of agreement is good. Therefore, this spectrum can be used as the characteristic spectrum of Persimmon Calyx Granules.

[0230] 5. Validation of Feature Mapping Methodology

[0231] (1) Overall investigation

[0232] Under the same chromatographic conditions, maintaining the same gradient, the elution time was doubled, and the chromatogram was recorded. No obvious chromatographic peak eluted after 30 minutes, indicating that the chromatographic conditions basically met the principle of maximum information content.

[0233] (2) Precision inspection

[0234] The test solution was prepared from the persimmon calyx formula granules according to the method under "2. Preparation of test solution" in Example 3. 1 μL of the same test solution was accurately aspirated and injected 6 times continuously. Peak 4 was used as the reference peak, and the relative retention time and relative peak area RSD values ​​of the common fingerprint peaks 1-10 were calculated to be less than 1.5%. At the same time, the similarity of each chromatographic fingerprint calculated by the similarity evaluation software was greater than 0.9, indicating that the instrument method was stable and the precision was good.

[0235] (3) Repeatability study

[0236] Take 6 portions of the same batch of persimmon calyx formula granules, accurately weigh them, and prepare 6 test solutions according to the method under "2. Preparation of test solution" in Example 3. Samples were injected separately, and peak 4 was used as the reference peak. The relative retention time and relative peak area RSD values ​​of common peaks 1-10 were calculated, which were all less than 1.7%, indicating that the construction method has good reproducibility.

[0237] (4) Stability investigation

[0238] Take the same test solution and inject it at 0, 2, 4, 6, 8, and 12 hours. Taking peak 4 as the reference peak, the RSD values ​​of the relative retention times and relative peak areas of common peaks 1-10 are calculated to be less than 3.1%, indicating that the test solution is stable for at least 12 hours.

[0239] (5) Durability inspection

[0240] 1) Investigation of different chromatographic columns

[0241] The effects of three chromatographic columns, namely, ZORBAX Eclipse Plus C18 (Agilent, 100mm×2.1mm, 1.8μm), ACQUITY UPLC HSS T3 (Waters Corporation, 2.1×100mm, 1.8μm), and ACQUITY UPLCBEH C18 (Waters Corporation, 2.1×100mm, 1.7μm), on the durability of the characteristic spectrum of persimmon calyx formula granules were compared.

[0242] See the results Figure 20 Persimmon Calyx Formula Granules were well separated on the ZORBAX Eclipse Plus C18, ACQUITY HSS T3, and ACQUITY BEH C18 columns. The ZORBAX Eclipse Plus C18 (Agilent, 2.1 × 100 mm, 1.8 μm) column is recommended for characterization of Persimmon Calyx Formula Granules.

[0243] 2) Investigation at different temperatures

[0244] A ZORBAX Eclipse Plus C18 (Agilent, 2.1×100 mm, 1.8 μm) column was used to investigate the separation effects of the formulated particles at column temperatures of 33°C, 35°C, and 37°C.

[0245] See the results Figure 21 , this analysis method can meet the system applicability requirements within the column temperature range of 33℃~37℃, and the smaller column temperature fluctuation can meet the system applicability requirements.

[0246] 3) Investigation of different flow rates

[0247] A ZORBAX Eclipse Plus C18 (Agilent, 2.1×100 mm, 1.8 μm) column was used to investigate the separation effects of the formulated particles at flow rates of 0.25 mL / min, 0.30 mL / min, and 0.35 mL / min.

[0248] See the results Figure 22 , this analysis method can meet the system applicability requirements with a small flow rate change within the flow rate range of 0.25mL / min to 0.35mL / min.

[0249] 6. Comparison of characteristic spectra of persimmon calyx medicinal material, standard decoction, and formula granules

[0250] Take the same batch of persimmon calyx medicinal materials, standard decoction, and formula granule powder in Examples 1, 2, and 3, and prepare 3 test sample solutions according to the test sample preparation method in point 2 of Examples 1, 2, and 3, respectively, and inject them separately.

[0251] See the results Figure 23 The characteristic spectra of persimmon calyx medicinal material, standard decoction, and formula granules are basically the same, with a total of 10 characteristic peaks.

Claims

1. A method for constructing a UPLC characteristic spectrum of a persimmon calyx sample, characterized in that: The construction method comprises the following steps: (a) Preparation of reference solution: Prepare reference solution by taking protocatechuic acid reference substance, hyperoside reference substance, isoquercetin reference substance, quercetin reference substance, and kaempferol reference substance respectively; (b) Preparation of a test solution: extracting a persimmon calyx sample with an extraction solvent, collecting the extract, and preparing a test solution, wherein the persimmon calyx sample is a persimmon calyx medicinal material, persimmon calyx decoction slices, persimmon calyx standard decoction, and / or persimmon calyx formula granules; the extraction solvent is selected from methanol or a methanol-water solution containing 20% ​​to 80% methanol by volume; (c) Establishing a characteristic spectrum: The reference solution and the test solution are tested using ultra-high performance liquid chromatography to obtain a reference spectrum and a test spectrum, and a characteristic spectrum is established using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system; The chromatographic conditions of the ultra-high performance liquid chromatography method include: Stationary phase: chromatographic column with octadecylsilane bonded silica as filler and particle size of 1.6~1.8 μm; Mobile phase: acetonitrile as mobile phase A, 0.05-0.30 wt% phosphoric acid solution as mobile phase B; Gradient elution program: From 0 min to 5 min, the volume percentage of the mobile phase A increases from 10% to 14%; from 5 min to 20 min, the volume percentage of the mobile phase A increases from 14% to 25%; from 20 min to 30 min, the volume percentage of the mobile phase A increases from 25% to 40%; The detection wavelength is 240~270nm; The construction method further comprises the following steps: identifying characteristic peaks of the chromatogram using liquid chromatography-mass spectrometry technology; The characteristic spectrum contains 10 peaks, wherein peak 1 is protocatechuic acid, peak 3 is hyperoside, peak 4 is isoquercetin, peak 6 is kaempferol-7-O-β-D-glucoside, peak 7 is trifolioside, peak 8 is kaempferol 3-O-(6''-galloyl)-BD-glucopyranoside, peak 9 is quercetin, and peak 10 is kaempferol; Taking Peak 4 as the reference peak, calculate the relative retention time of each peak. The relative retention time should be within ±10% of the specified value. The specified values ​​corresponding to Peaks 1 to 10 are 0.19, 0.49, 0.95, 1.00, 1.10, 1.15, 1.25, 1.37, 1.90, and 2.3, respectively.

2. The construction method according to claim 1, characterized in that The chromatographic conditions also include: a flow rate of 0.25 mL / min to 0.35 mL / min; and / or a column temperature of 33° C. to 37° C.

3. The construction method according to claim 1 or 2, characterized in that The persimmon calyx sample is a persimmon calyx medicinal material, the extraction solvent is a methanol aqueous solution with a methanol volume percentage of 60% to 80%, and the extraction method is heating reflux for 30 to 120 minutes.

4. The construction method according to claim 1 or 2, characterized in that The persimmon calyx sample is a standard decoction of persimmon calyx or a formula granule of persimmon calyx, the extraction solvent is a methanol aqueous solution with a methanol volume percentage of 60% to 80%, the extraction method is ultrasonic treatment, the ultrasonic extraction time is 20 minutes to 60 minutes, the power is 220W to 300W, and the frequency is 35kHz to 45kHz.

5. A method for detecting a persimmon calyx sample, characterized in that: The detection method comprises the following steps: Extracting the persimmon calyx sample to be tested with an extraction solvent, collecting the extract, and preparing a test solution; the persimmon calyx sample is persimmon calyx medicinal material, persimmon calyx decoction slices, persimmon calyx standard decoction and / or persimmon calyx formula granules; the extraction solvent is selected from methanol or a methanol aqueous solution containing 20% ​​to 80% methanol by volume; The test solution is tested using ultra-performance liquid chromatography, and the detection spectrum obtained is compared with the characteristic spectrum constructed by the construction method of any one of claims 1 to 4 to detect the quality and / or authenticity of the persimmon calyx sample; The chromatographic conditions of the ultra-high performance liquid chromatography method include: Stationary phase: chromatographic column with octadecylsilane bonded silica as filler and particle size of 1.6~1.8 μm; Mobile phase: acetonitrile as mobile phase A, 0.05-0.30 wt% phosphoric acid solution as mobile phase B; Gradient elution program: From 0 min to 5 min, the volume percentage of the mobile phase A increases from 10% to 14%; from 5 min to 20 min, the volume percentage of the mobile phase A increases from 14% to 25%; from 20 min to 30 min, the volume percentage of the mobile phase A increases from 25% to 40%; The detection wavelength is 240~270nm.

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