A chromatographic method for identifying genuine and fake pyrola herb

Abstract

The present application belongs to the field of traditional Chinese medicine identification, and particularly relates to a chromatographic method for identifying true and false Pyrola calliantha. The present application primarily establishes a characteristic chromatogram of Pyrola calliantha reference substance through liquid chromatography-mass spectrometry technology, and then attributes characteristic peaks in the characteristic chromatogram according to the chromatographic peaks of the index component reference substance, and then detects and identifies the test sample. The method comprehensively reflects the characteristics of Pyrola calliantha and its counterfeit products, provides a new analysis method for the internal quality control of Pyrola calliantha medicinal materials, and is suitable for not only Pyrola calliantha and its counterfeit medicinal materials, but also medicinal slices, standard decoctions and / or formula granules. The method is simple, accurate and reliable, easy to operate, short in time, low in solvent consumption and small in environmental pollution. The method has the advantages of strong specificity, high stability and good repeatability, and is particularly suitable for detection and monitoring of large-scale production.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine identification, specifically involving a chromatographic method for identifying the authenticity of Sedum sarmentosum. Background Technology

[0002] Sedum sarmentosum Bunge, a plant in the Crassulaceae family, is the dried whole herb. It is a commonly used folk medicine, not explicitly recorded in ancient literature, and first included in the Chinese Pharmacopoeia in 1977. Sedum sarmentosum is cool in nature and sweet and bland in taste. It enters the liver, gallbladder, and small intestine meridians. It has the effects of promoting diuresis and relieving jaundice, clearing heat and detoxifying. It is used for damp-heat jaundice, difficulty urinating, carbuncles and sores (National Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China [M]. China Medical Science and Technology Press, 2015). Modern pharmacological studies have shown that Sedum sarmentosum has hepatoprotective, anti-tumor, immunomodulatory, estrogen-like, angiotensin-converting enzyme (ACE) inhibitory, and muscle-strengthening effects; in addition, its injection has antibacterial effects, and its extract has anti-inflammatory, antioxidant, and cell-aging-delaying effects. Clinically, Sedum sarmentosum is often used to treat various acute and chronic liver diseases. Granules are used for the preventive treatment of intrahepatic cholestasis of pregnancy, and injections are used to treat corneal ulcers. It can be used both internally and externally to treat boils and carbuncles. Fresh Sedum sarmentosum can be used to treat pressure sores and herpes zoster in the elderly. External application can also be used to treat burns, scalds, and snake bites (Li Huijuan et al. Research progress of Sedum sarmentosum [J]. Pharmaceutical Research, 2015, 34(11): 661-663).

[0003] Due to the increased market demand in recent years, the medicinal herb market has become somewhat chaotic, seriously affecting the quality of Sedum sarmentosum and causing a decline in clinical efficacy. Common adulterants of Sedum sarmentosum on the market include Eclipta prostrata, Portulaca oleracea, Sedum sarmentosum, and Sedum sarmentosum var. ... Lü Jiani et al. used microscopic identification to distinguish two representative plants of the Crassulaceae family, Sedum sarmentosum and Sedum concave-leaved, from the Portulacaceae family, Portulaca oleracea and Portulaca grandiflora. Through comparative research, the two groups of medicinal plants, Sedum sarmentosum and Portulaca grandiflora, were found to be similar in appearance (before flowering), but there were obvious differences in microstructure (Lü Jiani et al. Identification of two easily confused crude drugs of the Crassulaceae and Portulacaceae families [J]. Zhejiang Medical Education, 2008(2):4). Liu Meizi et al. obtained the ITS2 gene fragment by PCR amplification of Sedum sarmentosum DNA. The purified PCR product was subjected to bidirectional sequencing, and the sequence was assembled using CodonCode Aligner. The sequence was analyzed using MEGA5.0 software, and a K2P model NJ tree was constructed. The results showed that there was significant variation in the ITS2 sequence between Sedum sarmentosum and its adulterants. The intraspecific genetic distance was smaller than the interspecific genetic distance. From the NJ tree, it was clear that Sedum sarmentosum from different sources clustered together, which could be well distinguished from adulterants (Liu Meizi et al. Identification of Sedum sarmentosum and its adulterants using ITS2 sequence [J]. Modern Chinese Materia Medica, 2011).

[0004] However, there is currently limited research on the differentiation between *Sedum sarmentosum* and its adulterants, such as *Eclipta prostrata*, *Portulaca oleracea*, *Sedum aizoon*, and *Sedum dasycarpus*. Furthermore, the existing studies have several limitations in distinguishing *Sedum sarmentosum* from its adulterants: First, due to societal development and environmental changes, corresponding morphological identification markers have not yet been established. Additionally, morphological identification requires extensive practical experience and long-term knowledge accumulation from the appraiser, making its widespread application difficult. Second, microscopic identification suffers from low resolution and difficulty in identifying medicinal materials with multiple origins and diverse varieties. Finally, DNA barcoding identification has drawbacks such as insufficient sensitivity and limited operability.

[0005] Therefore, it is necessary to establish a method that can distinguish Sedum sarmentosum from its counterfeits, Eclipta prostrata, Portulaca oleracea, Sedum sarmentosum, and Sedum sarmentosum var. ... Summary of the Invention

[0006] The problem the invention aims to solve

[0007] To address the aforementioned problems in the existing technology, the present invention aims to provide a chromatographic method for identifying the authenticity of Sedum sarmentosum. This method is applicable to the medicinal materials, processed slices, standard decoctions, and / or formulation granules of Sedum sarmentosum and its adulterants. It can distinguish Sedum sarmentosum from adulterants including Eclipta prostrata, Portulaca oleracea, Sedum aizoon, and Sedum sarmentosum var. ...

[0008] Solution for solving the problem

[0009] [1]. This invention provides a chromatographic method for identifying the authenticity of Sedum sarmentosum, wherein the method includes the following steps:

[0010] (1) Preparation of Sedum sarmentosum reference solution: Sedum sarmentosum reference standard was extracted with extraction solvent, and the extract was collected to obtain Sedum sarmentosum reference solution.

[0011] The reference standard for Sedum sarmentosum is selected from one or more of the following: Sedum sarmentosum medicinal material, Sedum sarmentosum slices, Sedum sarmentosum standard decoction, and Sedum sarmentosum formula granules.

[0012] (2) Preparation of reference solution: Vanillic acid-4-β-D-glucoside and isorhamnetin-3,7-O-diglucoside, which are used as reference standards, are dissolved in solvent to obtain reference solution;

[0013] (3) Establishment of characteristic chromatograms: The Sedum sarmentosum reference solution in step (1) and the reference solution in step (2) were detected by ultra-high performance liquid chromatography. After obtaining the Sedum sarmentosum reference detection chromatogram and the reference detection chromatogram, the characteristic chromatogram of the Sedum sarmentosum reference was established, and the characteristic peaks in the characteristic chromatogram were assigned according to the chromatographic peaks of the reference.

[0014] (4) Processing and identification of test samples: Take the test sample, obtain the test sample solution according to the method in step (1), and then establish the characteristic spectrum of the test sample according to the method in step (3); compare the characteristic spectrum of the test sample with the characteristic spectrum of the Sedum sarmentosum reference standard in step (3) to identify whether the test sample is Sedum sarmentosum or a counterfeit.

[0015] The test sample is selected from one or more of the following: medicinal materials to be tested, processed medicinal slices, standard decoctions, and formula granules.

[0016] [2]. According to the method described in [1], in step (1), the extraction solvent is methanol, water or a mixture of the two; preferably, the extraction solvent is a 30% to 70% v / v methanol aqueous solution;

[0017] And / or,

[0018] The extraction method is ultrasonic treatment, shaking extraction, or heating reflux extraction; preferably, the extraction method is ultrasonic extraction, the ultrasonic extraction power is 200-300W, the ultrasonic extraction frequency is 35-45kHz, and the ultrasonic extraction time is 30-120min.

[0019] And / or,

[0020] The mass-to-volume ratio of the *Sedum sarmentosum* reference standard to the extraction solvent is 1 g: (5-50 mL).

[0021] [3]. According to the method described in [1] or [2], wherein in step (2), the solvent is methanol or an aqueous solution thereof; preferably, the solvent is methanol or an aqueous solution of methanol of 50% v / v or higher.

[0022] And / or,

[0023] The concentration of the reference solution is 5–40 μg / mL.

[0024] [4]. The method according to any of the technical solutions [1] to [3], wherein, in step (3),

[0025] The chromatographic conditions for the ultra-high performance liquid chromatography method include:

[0026] Stationary phase: A chromatographic column packed with octadecylsilane-bonded silica gel;

[0027] Mobile phase: Acetonitrile as mobile phase A, and formic acid aqueous solution as mobile phase B;

[0028] Elution method: gradient elution.

[0029] [5]. According to the method described in [4], in step (3), a 0.05% to 0.20% v / v aqueous solution of formic acid is used as the mobile phase B;

[0030] And / or,

[0031] The gradient elution procedure is as follows:

[0032]

[0033] [6]. According to the method described in [4] or [5], the chromatographic conditions of the ultra-high performance liquid chromatography method further include: a column temperature of 20 to 35°C, and / or a flow rate of 0.25 to 0.30 mL / min, and / or a detection wavelength of 220 to 300 nm.

[0034] [7]. The method according to any of the technical solutions [1] to [6], wherein, in step (3),

[0035] The characteristic chromatogram of the Sedum sarmentosum reference standard includes 9 characteristic peaks, of which characteristic peak 3 corresponds to the chromatographic peak of vanillic acid-4-β-D-glucoside reference standard, and characteristic peak 9 corresponds to the chromatographic peak of isorhamnetin-3,7-O-diglucoside reference standard.

[0036] Preferably, using characteristic peak 3 as a reference peak, the relative retention times of characteristic peak 1 and characteristic peak 2 are calculated, and their relative retention times should be within ±10% of a specified value, which are 0.46 and 0.64 respectively.

[0037] Using characteristic peak 9 as a reference peak, calculate the relative retention times of characteristic peaks 4, 5, 6, 7, and 8. Their relative retention times should be within ±10% of the specified values, which are 0.56, 0.71, 0.77, 0.82, and 0.97, respectively.

[0038] [8]. The method according to any of the technical solutions [1] to [7], wherein, in step (4),

[0039] The identification criteria include: if the characteristic spectrum of the test sample contains characteristic peak 9, then the test sample is *Sedum sarmentosum*; otherwise, it is a counterfeit.

[0040] Preferably, the criteria for identification further include:

[0041] If the characteristic spectrum of the test sample does not contain characteristic peak 1, then the test sample is purslane or Eclipta prostrata;

[0042] If the characteristic spectrum of the test sample contains characteristic peak 1 and characteristic peak 3, then the test sample is Sedum concave-leaf;

[0043] If the characteristic spectrum of the test sample contains characteristic peak 1 and characteristic peak 5, then the test sample is Sedum aizoon.

[0044] [9]. The method according to any of the technical solutions [1] to [8], wherein the counterfeit product includes one or more of purslane, Eclipta prostrata, Sedum aizoon and Sedum sarmentosum.

[0045]

[10] . This invention also provides the application of the method described in any of the technical solutions [1] to [9] in the quality detection of Sedum sarmentosum and its counterfeits.

[0046] The effects of the invention

[0047] This invention utilizes liquid chromatography-mass spectrometry (LC-MS) to establish a characteristic chromatogram of *Sedum sarmentosum* reference standard, providing a chromatographic method for identifying genuine *Sedum sarmentosum*. By employing ultra-high performance liquid chromatography (UHPLC) with carefully controlled chromatographic conditions, nine characteristic peaks of the *Sedum sarmentosum* reference standard were determined, forming a comprehensive characteristic chromatogram of the medicinal material and its preparations. Simultaneously, comparative studies were conducted on preparations of adulterants such as *Eclipta prostrata*, *Portulaca oleracea*, *Sedum aizoon*, and *Sedum dasycarpus*, etc. This method comprehensively reflects the characteristics of the medicinal material and its adulterants, achieving the goal of identifying genuine *Sedum sarmentosum*.

[0048] This invention controls the intrinsic overall quality of Sedum sarmentosum, comprehensively reflects the characteristics of Sedum sarmentosum and its adulterants, achieves the purpose of simultaneously identifying Sedum sarmentosum and its adulterants, and provides a new analytical method for the intrinsic quality control of Sedum sarmentosum, processed medicinal slices, standard decoctions and / or formula granules, etc.

[0049] The method of this invention is simple, accurate, reliable, easy to operate, quick, consumes little solvent, and causes minimal environmental pollution. It also boasts advantages such as high specificity, high stability, and good reproducibility, making it particularly suitable for detection and monitoring in large-scale production.

[0050] It should be noted that the above description does not disclose all embodiments of the present invention or all advantages of the present invention. Attached Figure Description

[0051] Figure 1 The image shown is a DAD-3D representation of the Sedum sarmentosum herb in Example 1.

[0052] Figure 2 Chromatograms of Sedum sarmentosum at different wavelengths in Example 1

[0053] Figure 3 Chromatogram of Sedum sarmentosum during elution using gradient 1

[0054] Figure 4 Chromatogram of Sedum sarmentosum during elution according to gradient 2

[0055] Figure 5 Chromatogram of Sedum sarmentosum during elution using gradient 3.

[0056] Figure 6 Chromatogram of Sedum sarmentosum during elution using gradient 4

[0057] Figure 7 Chromatogram of Sedum sarmentosum during elution using gradient 5.

[0058] Figure 8 Chromatogram of Sedum sarmentosum during elution using gradient 6.

[0059] Figure 9Chromatogram of Sedum sarmentosum during elution using gradient 7.

[0060] Figure 10 Chromatograms of Sedum aizoon under different chromatographic columns

[0061] Figure 11 Chromatograms of Sedum aizoon at different column temperatures

[0062] Figure 12 Chromatograms of *Sedum aizoon* herb at different flow rates

[0063] Figure 13 Characteristic atlas of *Sedum sarmentosum* control standard in Example 1

[0064] Figure 14 Comparative characteristic maps of the *Sedum sarmentosum* control and its adulterants from Example 1.

[0065] Figure 15 The identification feature map of the reference standard in Example 1

[0066] Figure 16 Characteristic spectra of Sedum sarmentosum and its related preparations in Example 2

[0067] Figure 17 Characteristic chromatogram of the test sample in Example 3 Detailed Implementation

[0068] The following describes embodiments of the present invention, but the invention is not limited thereto. The present invention is not limited to the configurations described below; various modifications can be made within the scope of the claims. Embodiments and examples obtained by appropriately combining the disclosed technical means in different embodiments and examples are also included within the technical scope of the present invention. Furthermore, all documents described in this specification are incorporated herein by reference.

[0069] Unless otherwise defined, the 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 pertains.

[0070] In the context of describing this specification (especially in the context of the appended claims), the terms “a,” “an,” and “the,” and similar language will be interpreted to cover both the singular and plural, unless otherwise indicated herein or clearly contradicted by the context.

[0071] In this specification, the range of values ​​referred to as “value A ~ value B” or “value A - value B” refers to the range including the endpoint values ​​A and B.

[0072] In this specification, the word "may" has 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 the case where the event occurs and the case where the event does not occur.

[0073] In this specification, the terms "some specific / preferred embodiments," "other specific / preferred embodiments," "some specific / preferred technical solutions," and "other specific / preferred technical solutions" refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to the described embodiment that are included in at least one of the embodiments described herein, and may or may not exist in other embodiments. Furthermore, it should be understood that these elements can be combined in any suitable manner in various embodiments.

[0074] The term "comprising" and any variations thereof in the specification and claims of this invention are intended to cover a non-exclusive inclusion. For example, a process, method, system, product, or apparatus that comprises a series of steps or units is not limited to the steps or units listed, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to such processes, methods, products, or apparatus.

[0075] Unless otherwise specified, the percentage content involved in this invention refers to mass percentage for solid-liquid mixtures and solid-phase-solid mixtures, and volume percentage for liquid-phase-liquid mixtures.

[0076] Unless otherwise specified, all percentage concentrations mentioned in this invention refer to the final concentration. The final concentration refers to the proportion of the added component in the system after the addition of that component.

[0077] Unless otherwise specified, the temperature parameters in this invention can be either constant temperature processing or processing within a certain temperature range. The constant temperature processing allows temperature fluctuations within the precision range controlled by the instrument.

[0078] Unless otherwise stated, the terms “precise weighing” or “precise aliquoting” as used herein mean that the weight should be accurate to one-thousandth of the weight taken, the term “weigh” means that the weight should be accurate to one-hundredth of the weight taken, the term “precise addition” means that the volume should be accurate to one-thousandth of the volume taken, and the term “precise aliquoting” means the operation of accurately measuring the sample using a microsyringe.

[0079] Unless otherwise stated, the term "follow-up filtrate" as used herein refers to the filtrate collected after the initial filtrate has been discarded during filtration. Compared to the initial filtrate, the follow-up filtrate is closer to the true concentration of the sample because the filter medium (such as a filter membrane or filter paper) may adsorb solutes, resulting in a lower sample concentration in the initial filtrate. In addition, the follow-up filtrate is cleaner because the solutes adsorbed by the filter medium can form a filter cake, reducing the pore size and thus trapping smaller particles.

[0080] Unless otherwise stated, the term “reference peak” (or “comparison peak”) as used herein refers to the chromatographic peak corresponding to the standard sample when calculating relative retention time.

[0081] Unless otherwise stated, the percentage of solution concentration referred to in this article is a volume percentage.

[0082] Unless otherwise stated, the term "reference standard" as used herein refers to a standard substance used for identification, inspection, content determination and calibration of instrument performance, which is usually reviewed and approved by the national drug testing agency and its standard should be no lower than the quality standard of the product.

[0083] Unless otherwise stated, the term “test sample” as used herein refers to an experimental sample used for testing or identification.

[0084] Unless otherwise stated, "sample" in this invention refers to medicinal material that has undergone crude drug identification (e.g., identification of the original plant (animal), morphological identification, microscopic identification, physicochemical identification, etc.) and is used to establish corresponding characteristic spectra. For example, "purslane sample" in this invention refers to purslane medicinal material that has undergone crude drug identification and is used to establish corresponding characteristic spectra.

[0085] First aspect

[0086] This invention provides a chromatographic method for identifying the authenticity of Sedum sarmentosum, which includes the following steps:

[0087] (1) Preparation of Sedum sarmentosum reference solution: Sedum sarmentosum reference standard was extracted with extraction solvent, and the extract was collected to obtain Sedum sarmentosum reference solution.

[0088] In some specific embodiments of the present invention, in step (1), the Sedum sarmentosum reference standard is selected from one or more of Sedum sarmentosum medicinal materials, Sedum sarmentosum slices, Sedum sarmentosum standard decoction and Sedum sarmentosum formula granules.

[0089] Among them, "medicinal materials" refers to unprocessed or unfinished Chinese medicinal raw materials; "processed slices" refers to Chinese medicinal materials that have been processed according to needs and are used for prescriptions, or Chinese medicinal materials that can be directly used in clinical practice; "standard decoction" refers to a single-herb Chinese medicinal decoction prepared by standardized processes with reference to modern extraction methods and guided by Chinese medicine theory and clinical application; "prescription granules" are granules made by extracting and concentrating single-herb Chinese medicinal slices according to traditional standards and used for prescriptions in clinical practice.

[0090] In addition, in some specific embodiments of the present invention, the extraction solvent is methanol, water, or a mixture of the two; in some preferred embodiments, the extraction solvent is a 30% to 70% v / v methanol aqueous solution. Regarding the amount of extraction solvent, in some specific embodiments of the present invention, the mass-to-volume ratio of the *Sedum sarmentosum* reference standard powder to the extraction solvent is 1 g:(5-50 mL), specifically 1 g:5 mL, 1 g:10 mL, 1 g:12.5 mL, 1 g:15 mL, 1 g:25 mL, 1 g:30 mL, 1 g:40 mL, 1 g:45 mL, 1 g:50 mL, etc.; to further optimize the extraction effect and material dosage, the mass-to-volume ratio of the medicinal material, processed slices, standard decoction, and / or formulation granules powder to the extraction solvent is preferably 1 g:(5-15) mL; too low a concentration of the reference standard is not conducive to the detection of chromatographic peaks, while too high a concentration may lead to concerns about poor peak shape due to column overload.

[0091] Regarding the extraction method, to obtain richer chromatographic peak information, extraction can be performed using ultrasonic treatment, shaking extraction, or heating and reflux extraction. Ultrasonic treatment, which is simpler to operate, is preferred. This invention does not impose particular limitations on the power, frequency, or other conditions of the ultrasonic treatment. In some preferred embodiments, the ultrasonic power can be 200W–300W, and the frequency can be 35kHz–45kHz; a further preferred power is 250W and the frequency is 40kHz. The extraction time is 30–120 min, specifically 30 min, 45 min, 55 min, 60 min, 70 min, 80 min, 90 min, 100 min, or 120 min. To further optimize the extraction effect and processing cycle, considering the extraction efficiency of each chromatographic peak, the preferred extraction time is 35–60 min, and more preferably 45 min.

[0092] For Sedum sarmentosum medicinal materials, it is preferable to first sieve the powder, such as using a No. 2 sieve (24 mesh), a No. 3 sieve (50 mesh), or a No. 4 sieve (65 mesh), with the No. 2 sieve being the most preferred. For Sedum sarmentosum standard decoction, the medicinal materials undergo pretreatment, decoction, low-temperature concentration, and freeze-drying to obtain freeze-dried powder, and the freeze-dried powder is preferably finely ground. For Sedum sarmentosum formula granules, the medicinal materials are pretreated, extracted, concentrated, spray-dried, granulated, and packaged to obtain traditional Chinese medicine formula granules, which are then extracted. In some specific embodiments of the present invention, the preparation method of the reference solution is as follows: Take an appropriate amount of Sedum sarmentosum herb and Sedum sarmentosum slices, crush them, pass them through a No. 2 sieve (24 mesh), accurately weigh 2g, place them in a stoppered conical flask, accurately add 25mL of 30% methanol aqueous solution, seal tightly, weigh, sonicate (power 250W, frequency 40kHz) for 45 minutes, cool, weigh again, replenish the lost weight with 30% methanol aqueous solution, shake well, filter, and take the filtrate to obtain the reference solution;

[0093] Alternatively, in some specific embodiments of the present invention, take an appropriate amount of standard decoction of Sedum sarmentosum or granulated formula of Sedum sarmentosum, grind it into a fine powder, take 0.8g, place it in a stoppered conical flask, accurately add 25ml of methanol aqueous solution with a volume fraction of 30%, seal tightly, sonicate (power 250W, frequency 40kHz) for 45 minutes, cool, shake well, filter, and take the filtrate to obtain the product.

[0094] (2) Preparation of reference solution: Vanillic acid-4-β-D-glucoside reference standard and isorhamnetin-3,7-O-diglucoside reference standard were dissolved in solvent to obtain reference solution;

[0095] The obtained reference solution comprises dissolving the reference standard in methanol or its aqueous solution to prepare a vanillic acid-4-β-D-glucoside reference solution and an isorhamnetin-3,7-O-diglucoside reference solution with a concentration of 5-40 μg / mL. In some specific embodiments of the present invention, preferably, a vanillic acid-4-β-D-glucoside reference solution with a concentration of 20 μg / mL is prepared using a methanol aqueous solution of 50% v / v or higher, and an isorhamnetin-3,7-O-diglucoside reference solution with a concentration of 15 μg / mL is prepared using methanol.

[0096] (3) Establishment of characteristic chromatograms: The Sedum sarmentosum reference solution in step (1) and the reference solution in step (2) were detected by ultra-high performance liquid chromatography. After obtaining the Sedum sarmentosum reference detection chromatogram and the reference detection chromatogram, the characteristic chromatogram of the Sedum sarmentosum reference was established, and the characteristic peaks in the characteristic chromatogram were assigned according to the chromatographic peaks of the reference.

[0097] This invention, through in-depth research, has determined ultra-high performance liquid chromatography (UHPLC) conditions applicable to both raw Sedum sarmentosum herbs, processed Sedum sarmentosum slices, standard Sedum sarmentosum decoctions, and Sedum sarmentosum granule formulations, which include:

[0098] Stationary phase: A chromatographic column with octadecylsilane-bonded silica gel as the packing material and a particle size (packing particle size) of 1.6–1.8 μm; more specifically, the column length is 100–150 mm and the inner diameter is 2.1 mm; preferably, the column length is 100 mm, the inner diameter is 2.1 mm, and the packing particle size is 1.8 μm; in some specific embodiments of the present invention, the chromatographic column is selected from any one of Eclipse Plus C18 (Agilent, 100 mm × 2.1 mm × 1.8 μm), CORTECS UPLC T3 (Waters, 2.1 mm × 100 mm × 1.6 μm), and ACQUITY BEH C18 (Waters, 2.1 × 100 mm × 1.7 μm), and more specifically, the Eclipse Plus C18 (Agilent, 100 mm × 2.1 mm × 1.8 μm) column is used. Using this column for separation results in better resolution of characteristic peaks and a more aesthetically pleasing overall chromatogram.

[0099] Mobile phase: Acetonitrile is used as mobile phase A, and an aqueous solution of formic acid is used as mobile phase B. In some specific embodiments of the present invention, a 0.05% to 0.20% v / v aqueous solution of formic acid is preferred, and a 0.1% v / v aqueous solution of formic acid is more preferred as mobile phase B.

[0100] The elution method of this invention employs gradient elution, and the gradient elution procedure is as follows:

[0101]

[0102] Considering the chromatographic peak separation effect, the preferred flow rate of this invention is 0.28 mL / min to 0.32 mL / min; and / or, the column temperature is 15℃ to 35℃. Within this column temperature or flow rate range, the construction method of this invention allows for relatively small variations in either the column temperature or flow rate to meet the system suitability requirements. In some preferred embodiments of this invention, the flow rate can be 0.25 mL / min to 0.30 mL / min; and / or, the column temperature can be 20℃ to 32℃; in some specific embodiments of this invention, the flow rate is 0.3 mL / min, the column temperature is 30℃, and the injection volume is 2 μL.

[0103] The detector used in this invention can be an ultraviolet detector, an evaporative light scattering detector, or an electro-fogging detector. In some preferred embodiments of this invention, an ultraviolet detector is preferably used, with a detection wavelength of 220 nm to 300 nm. In some preferred embodiments of this invention, the detection wavelength can be 254 nm. At this detection wavelength range, the chromatographic peak information is relatively rich, the separation effect of each chromatographic peak is good, and the baseline is relatively stable.

[0104] This invention establishes a characteristic chromatogram of Sedum sarmentosum reference standards by testing at least 17 batches of Sedum sarmentosum reference standards from different origins, and assigns the characteristic peaks in the characteristic chromatogram of Sedum sarmentosum reference standards to the chromatograms based on the chromatographic peaks of the reference standards, thereby establishing a standard for identifying Sedum sarmentosum and its adulterants.

[0105] The characteristic spectrum of the *Sedum sarmentosum* reference standard established using the above method includes 9 characteristic peaks, among which peak 3 is the peak of vanillic acid-4-β-D-glucoside and peak 9 is the peak of isorhamnetin-3,7-O-diglucoside.

[0106] (4) Processing and identification of test samples: Take the test sample, obtain the test sample solution according to the method in step (1), and then establish the characteristic spectrum of the test sample according to the method in step (3); compare the characteristic spectrum of the test sample with the characteristic spectrum of the Sedum sarmentosum reference standard in step (3) to identify whether the test sample is Sedum sarmentosum or a counterfeit.

[0107] The identification criteria include: if the characteristic spectrum of the test sample contains characteristic peak 9, then the test sample is *Sedum sarmentosum*; otherwise, it is a counterfeit.

[0108] Furthermore, the criteria for identification also include:

[0109] If the characteristic spectrum of the test sample does not contain characteristic peak 1, then the test sample is purslane or Eclipta prostrata.

[0110] If the characteristic spectrum of the test sample contains characteristic peak 1 and characteristic peak 3, then the test sample is Sedum concave-leaf;

[0111] If the characteristic spectrum of the test sample contains characteristic peak 1 and characteristic peak 5, then the test sample is Sedum sarmentosum.

[0112] If the characteristic spectrum of the test sample contains characteristic peak 1 and characteristic peak 9, then the test sample is *Sedum sarmentosum*.

[0113] This invention improves analytical efficiency by optimizing the preparation method of Sedum sarmentosum reference standard, the reference standard preparation method, and the chromatographic analysis conditions. It establishes ultra-high performance liquid chromatography conditions applicable to Sedum sarmentosum medicinal materials, Sedum sarmentosum slices, Sedum sarmentosum standard decoctions, and / or Sedum sarmentosum formulation granules and their adulterants. This method can distinguish Sedum sarmentosum from adulterants including Portulaca oleracea, Eclipta prostrata, Sedum aizoon and / or Panax notoginseng, providing analytical means and basis for quality control of Sedum sarmentosum and its adulterants.

[0114] Second aspect

[0115] This invention also provides the application of the method in identifying any two or more of Sedum sarmentosum, Portulaca oleracea, Eclipta prostrata, Sedum aizoon and / or Sedum sarmentosum, and provides the application of the method in the quality detection of Sedum sarmentosum and its adulterants.

[0116] In one specific embodiment of the present invention, the method can identify *Sedum sarmentosum* and *Portulaca oleracea* or *Eclipta prostrata*; in one specific embodiment of the present invention, the method can identify *Sedum sarmentosum* and *Sedum aizoon*; in one specific embodiment of the present invention, the method can identify *Sedum sarmentosum* and *Sedum aizoon*.

[0117] Furthermore, in one specific embodiment of the present invention, the method is capable of identifying Portulaca oleracea or Eclipta prostrata and Sedum dasycarpus; in one specific embodiment of the present invention, the method is capable of identifying Portulaca oleracea or Eclipta prostrata and Sedum dasycarpus; in one specific embodiment of the present invention, the method is capable of identifying Sedum dasycarpus and Sedum dasycarpus.

[0118] The method provided by the present invention will be described in detail below with reference to the embodiments. However, the specific experimental conditions and results described in the embodiments are only for illustrating the present invention and should not be construed as limiting the scope of protection of the present invention.

[0119] Example

[0120] Instruments, reagents and samples

[0121] Waters Acquity UPLC (Ultra-High Performance Liquid Chromatograph) (Waters Corporation); Empower 3 workstation (Waters Corporation); Agilent G6530 Accurate-Mass Q-TOF mass spectrometer (Agilent Corporation); Agilent Mass Hunter Workstation data acquisition and qualitative analysis software (Agilent Corporation); electronic analytical balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); temperature-controlled water bath (Nantong Huatai Experimental Instruments Co., Ltd.); pure water system (Sartorius Corporation); AS165W centrifuge (Asia Pacific (Shanghai) Trading Co., Ltd.).

[0122] Acetonitrile (chromatographic grade, Thermo Fisher Scientific); water was ultrapure water; formic acid (chromatographic grade, Aladdin Scientific); all other reagents were of analytical grade.

[0123] Vanillic acid-4-β-D-glucoside (CAS NO.32142-31-7) was purchased from Shanghai Zhenzhun Biotechnology Co., Ltd., with the product number 21Z017-Q1. Isorhamnetin-3,7-O-diglucoside reference standard (CAS NO.6758-51-6) was purchased from Shanghai Shidande Biotechnology Co., Ltd., with the product number 10502. A total of 29 batches of experimental samples, including 17 batches of Sedum sarmentosum, 3 batches of Eclipta prostrata, 3 batches of Portulaca oleracea, 3 batches of Sedum aizoon, and 3 batches of Sedum dasycarpus, were sourced from major producing areas and authentic producing regions in China, and all met the requirements of the 2020 edition of the Chinese Pharmacopoeia.

[0124] Example 1: Establishment of a chromatographic method for identifying the authenticity of Sedum sarmentosum.

[0125] 1. Preparation of *Sedum sarmentosum* reference solution

[0126] Take an appropriate amount of Sedum sarmentosum herb, crush it, pass it through a No. 2 sieve (24 mesh), accurately weigh 2g, place it in a stoppered conical flask, accurately add 25mL of 30% methanol aqueous solution, seal tightly, weigh, sonicate (power 250W, frequency 40kHz) for 45 minutes, cool, weigh again, replenish the lost weight with 30% v / v methanol aqueous solution, shake well, filter, and collect the filtrate to obtain the product.

[0127] 2. Preparation of reference solution

[0128] Prepare a solution containing 20 μg per ml by dissolving vanillic acid-4-β-D-glucoside in 50% v / v methanol; prepare a solution containing 15 μg per ml by dissolving isorhamnetin-3,7-O-diglucoside in methanol.

[0129] 3. Determination of Ultra-High Performance Liquid Chromatography Conditions

[0130] 3.1 Determination of detection wavelength

[0131] Accurately pipette 2 μL of the *Sedum sarmentosum* reference solution and inject it into the ultra-high performance liquid chromatograph. Measure and record the absorption spectrum in the range of 210–400 nm. Figure 1 Chromatograms of Sedum sarmentosum were determined at different wavelengths (240 nm, 254 nm, 280 nm), such as... Figure 2 The results show 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.

[0132] 3.2 Optimization of chromatographic conditions

[0133] An ACQUITY UPLC BEH C18 column (100 mm in length, 2.1 mm in inner diameter, and 1.8 μm in particle size) was used as the chromatographic column; acetonitrile was used as the mobile phase A, and 0.1% v / v phosphoric acid solution was used as the mobile phase B. Elution was performed according to gradient 1 specified in Table 1, with a flow rate of 0.3 mL / min, a column temperature of 30 °C, and a detection wavelength of 254 nm.

[0134] Table 1:

[0135]

[0136] Get as Figure 3 The chromatogram shown is of Sedum sarmentosum when eluted according to gradient 1.

[0137] Next, elution was performed according to gradient 2 as specified in Table 2, with other conditions remaining unchanged.

[0138] Table 2:

[0139]

[0140] Get as Figure 4 The chromatogram shown is of Sedum sarmentosum when eluted according to gradient 2.

[0141] The chromatographic column was changed to an ACQUITY UPLC HSS T3 column (100 mm in length, 2.1 mm in inner diameter, and 1.8 μm in particle size); acetonitrile was used as mobile phase A, and 0.1% v / v phosphoric acid solution was used as mobile phase B. Elution was performed according to gradient 3 specified in Table 3, with a flow rate of 0.3 ml / min, a column temperature of 30 °C, and a detection wavelength of 254 nm.

[0142] Table 3:

[0143]

[0144] Get as Figure 5 The chromatogram of Sedum sarmentosum shown is obtained by elution according to gradient 3.

[0145] Next, elution was performed according to gradient 4 as specified in Table 4, with other conditions remaining unchanged.

[0146] Table 4:

[0147]

[0148] Get as Figure 6 The chromatogram of Sedum sarmentosum shown is obtained by elution according to gradient 4.

[0149] Continue changing the chromatographic column to a ZORBAX Eclipse Plus C18 column (100 mm in length, 2.1 mm in inner diameter, and 1.8 μm in particle size); use acetonitrile as mobile phase A and 0.1% v / v phosphoric acid solution as mobile phase B, and elute according to gradient 5 specified in Table 5, with a flow rate of 0.3 ml / min, a column temperature of 30 °C, and a detection wavelength of 254 nm.

[0150] Table 5:

[0151]

[0152] Get as Figure 7 The chromatogram shown is of Sedum sarmentosum when eluted according to gradient 5.

[0153] The mobile phase was changed to a ZORBAX Eclipse Plus C18 column (100 mm in length, 2.1 mm in inner diameter, and 1.8 μm in particle size). Acetonitrile was used as mobile phase A, and 0.1% v / v formic acid solution was used as mobile phase B. Elution was performed according to gradient 6 specified in Table 6, with a flow rate of 0.3 mL / min, a column temperature of 30 °C, and a detection wavelength of 254 nm.

[0154] Table 6:

[0155]

[0156] Get as Figure 8 The chromatogram shown is of Sedum sarmentosum when eluted according to gradient 6.

[0157] The gradient was adjusted again, using octadecylsilane-bonded silica gel as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm); acetonitrile as mobile phase A, and 0.1% v / v formic acid solution as mobile phase B, eluted according to gradient 7 specified in Table 7; the flow rate was 0.3 ml / min, the column temperature was 30 °C, and the detection wavelength was 254 nm.

[0158] Table 7:

[0159]

[0160] Get as Figure 9 The chromatogram of Sedum sarmentosum shown is obtained by eluting according to gradient 7, and the final elution gradient is determined.

[0161] 3.3 Determination of the mobile phase

[0162] Acetonitrile was chosen as mobile phase A, and a 0.1% v / v formic acid solution was chosen as mobile phase B.

[0163] 3.4 Examination of the chromatographic column

[0164] Take the *Sedum sarmentosum* reference solution prepared according to section "1. Preparation of *Sedum sarmentosum* reference solution" above, and select commonly used chromatographic columns, namely Eclipse Plus C18 (Agilent, 100mm×2.1mm×1.8μm), CORTECS UPLC T3 (Waters, 2.1mm×100mm×1.6μm), and ACQUITY BEH C18 (Waters, 2.1×100mm×1.7μm), with acetonitrile as mobile phase A and 0.1% v / v formic acid solution as mobile phase B, and perform injection and determination according to the elution gradient described in Table 7 above. Compare the separation effects of different chromatographic columns, such as... Figure 10 As shown.

[0165] The results showed that the Eclipse Plus C18 RRHD column (2.1 mm × 100 mm × 1.8 μm) provided the best sample separation performance and had a stable baseline.

[0166] 3.5 Investigation of column temperature

[0167] Take the *Sedum sarmentosum* reference solution prepared according to section "1. Preparation of *Sedum sarmentosum* reference solution" above, use the same chromatographic column, and inject the solution under the above chromatographic conditions, but at different column temperatures (25℃, 30℃, 35℃). Record the chromatograms, as shown below. Figure 11 As shown.

[0168] The results showed that the chromatographic peaks were well separated and the column exhibited good robustness within the column temperature range of 25℃ to 35℃. Based on the actual analytical conditions, a column temperature of 30℃ was selected for analysis.

[0169] 3.6 Examination of Flow Velocity

[0170] Take the *Sedum sarmentosum* reference solution prepared according to section "1. Preparation of *Sedum sarmentosum* reference solution" above, use the same chromatographic column, and determine the chromatograms at different flow rates of 0.25 ml / min, 0.30 ml / min, and 0.35 ml / min under the chromatographic conditions described above. Record the chromatograms. Figure 12 As shown.

[0171] The results showed that small flow rate variations within the range of 0.25 ml / min to 0.30 ml / min could meet the system applicability requirements, with a fixed flow rate of 0.30 ml / min.

[0172] 4. Establishment of characteristic chromatograms of *Sedum sarmentosum* reference standard and its adulterants.

[0173] Seventeen batches of Sedum sarmentosum medicinal materials, three batches of Eclipta prostrata medicinal materials, three batches of Portulaca oleracea medicinal materials, three batches of Sedum aizoon medicinal materials, and three batches of Sedum dasycarpus medicinal materials, each sample weighing 2.0 g, were prepared according to the above preparation method. 2 μL of each sample solution and reference solution were precisely pipetted into an ultra-high performance liquid chromatograph (UHPLC). The characteristic chromatograms of the samples were determined under the above chromatographic conditions. The characteristic chromatograms of the 17 batches of Sedum sarmentosum reference standards were then overlaid using the "Similarity Evaluation System for Chromatographic Characteristic Chromatographic Features of Traditional Chinese Medicine (Version 2012.0)" issued by the National Pharmacopoeia Commission. Figure 13 ; UPLC comparison atlas of *Sedum sarmentosum*, *Eclipta prostrata*, *Portulaca oleracea*, *Sedum aizoon*, and *Sedum davidii* can be found in [link to UPLC atlas]. Figure 14 See the reference identification chart. Figure 15 .

[0174] 5. Identification and research of Sedum sarmentosum and its adulterants

[0175] The characteristic spectra of common adulterants of Sedum sarmentosum, such as Eclipta prostrata, Portulaca oleracea, Sedum aizoon, and Sedum dasycarpus, differ significantly from those of the Sedum sarmentosum reference standard. The Sedum sarmentosum reference standard exhibits nine characteristic peaks. Peak 3 is vanillic acid-4-β-D-glucoside, and peak 9 is isorhamnetin-3,7-O-diglucoside. Using peak 3 as a reference, the relative retention times of peaks 1 and 2 are calculated; these relative retention times should be within ±10% of the specified values, which are 0.46 and 0.64, respectively. Using peak 9 as a reference, the relative retention times of peaks 4, 5, 6, 7, and 8 are calculated; these relative retention times should also be within ±10% of the specified values, which are 0.56, 0.71, 0.77, 0.82, and 0.97, respectively. Purslane and Eclipta prostrata are plants from different families, and under the same characteristic chromatogram conditions as Sedum sarmentosum, they showed almost no corresponding chromatographic peaks. Sedum concave-leaved has characteristic peaks 1 and 3, but other peaks are missing. Sedum aizoon has characteristic peaks 1 and 5, but other peaks are missing.

[0176] The characteristic spectra of the above-mentioned Sedum sarmentosum and its counterfeits are compared, the differences in characteristic peaks in each spectra are compared, and the differences are recorded to obtain the identification criteria.

[0177] In some specific embodiments of the present invention, the differences include characteristic peak 1 which can distinguish purslane and Eclipta prostrata, characteristic peak 3 which can distinguish Sedum concave-leaved, characteristic peak 5 which can distinguish Sedum sarmentosum, and characteristic peak 9 which can distinguish Sedum sarmentosum.

[0178] Example 2: Chromatographic method for identifying the authenticity of Sedum sarmentosum slices, standard Sedum sarmentosum decoction, and Sedum sarmentosum granules.

[0179] Preparation of Sedum sarmentosum reference solution

[0180] For Sedum sarmentosum slices: Take an appropriate amount of Sedum sarmentosum slices, crush them, pass them through a No. 2 sieve (24 mesh), accurately weigh 2g, place them in a stoppered conical flask, accurately add 25mL of 30% methanol aqueous solution, seal tightly, weigh, sonicate (power 250W, frequency 40kHz) for 45 minutes, cool, weigh again, replenish the lost weight with 30% v / v methanol aqueous solution, shake well, filter, and take the filtrate to obtain the product.

[0181] For the standard decoction of Sedum sarmentosum: Take an appropriate amount of freeze-dried powder of the standard decoction of Sedum sarmentosum prepared by pretreatment, decoction, low-temperature concentration and freeze-drying of Sedum sarmentosum, grind it into a fine powder, take 0.8g, put it into a stoppered conical flask, accurately add 25ml of methanol aqueous solution with a volume fraction of 30%, stopper tightly, sonicate (power 250W, frequency 40kHz) for 45 minutes, cool, shake well, filter, and take the filtrate to obtain the decoction.

[0182] For Sedum sarmentosum formula granules: Take an appropriate amount of Sedum sarmentosum formula granules obtained by pretreatment, extraction, concentration, spray drying, granulation and packaging, grind them into a fine powder, take 0.8g, place them in a stoppered conical flask, accurately add 25ml of methanol aqueous solution with a volume fraction of 30%, seal tightly, sonicate (power 250W, frequency 40kHz) for 45 minutes, cool, shake well, filter, and take the filtrate to obtain the final product.

[0183] In addition, the preparation of the reference solution was carried out according to the method described in "2. Preparation of the reference solution" of Example 1 above. An Eclipse Plus C18 RRHD column was used as the chromatographic column, acetonitrile was used as mobile phase A, and 0.1% v / v formic acid aqueous solution was used as mobile phase B. Elution was performed according to the gradient in Table 7 of Example 1 above, with a flow rate of 0.3 ml / min, a column temperature of 30℃, and a detection wavelength of 254 nm. Characteristic chromatograms of *Sedum sarmentosum* and its related preparations were obtained, such as... Figure 16 Among them, after identification by reference standards, peak 3 (S1) is the peak of vanillic acid-4-β-D-glucoside, and peak 9 (S2) is the peak of isorhamnetin-3,7-O-diglucoside.

[0184] Example 3: Application of chromatographic methods for identifying genuine and counterfeit Sedum sarmentosum

[0185] The test solution was prepared according to the method in "1. Preparation of Sedum sarmentosum reference solution" in Example 1. An Eclipse Plus C18 RRHD column was used as the chromatographic column, acetonitrile was used as mobile phase A, and 0.1% v / v formic acid aqueous solution was used as mobile phase B. Elution was performed according to the gradient in Table 7 of Example 1 above. The flow rate was 0.3 ml / min, the column temperature was 30℃, and the detection wavelength was 254 nm to obtain the characteristic chromatogram of the test sample.

[0186] Next, the obtained characteristic chromatogram of the test sample was compared with the characteristic chromatogram of the *Sedum sarmentosum* control established in Example 1 to identify whether the test sample was *Sedum sarmentosum* or a counterfeit. The characteristic chromatogram of the test sample is shown below. Figure 17 As shown.

[0187] The identification criteria are as follows: if the characteristic spectrum of the test sample contains characteristic peak 9, then the test sample is *Sedum sarmentosum*; otherwise, it is a counterfeit.

[0188] Further identification was performed based on the characteristic peaks of the sample's characteristic spectrum.

[0189] If the characteristic spectrum of the test sample does not contain characteristic peak 1, then the test sample is purslane or Eclipta prostrata.

[0190] If the characteristic spectrum of the test sample contains characteristic peak 1 and characteristic peak 3, then the test sample is Sedum concave-leaf;

[0191] If the characteristic spectrum of the test sample contains characteristic peak 1 and characteristic peak 5, then the test sample is Sedum sarmentosum.

[0192] If the characteristic spectrum of the test sample contains characteristic peak 1 and characteristic peak 9, then the test sample is *Sedum sarmentosum*.

[0193] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0194] The embodiments described above are merely illustrative of several implementations of the present invention, designed to facilitate a detailed understanding of the technical solutions of the present invention, but should not be construed as limiting the scope of patent protection. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

[0195] Industrial availability

[0196] The method described in this invention is applicable to the industrial identification of Sedum sarmentosum and its adulterants.

Claims

1. A chromatographic method for identifying the authenticity of Sedum sarmentosum, characterized in that, The method includes the following steps: (1) Preparation of Sedum sarmentosum reference solution: Sedum sarmentosum reference standard was extracted with an extraction solvent, and the extract was collected to obtain Sedum sarmentosum reference solution. The extraction solvent was a 30%~70% v / v methanol aqueous solution. The reference standard for Sedum sarmentosum is selected from one or more of the following: Sedum sarmentosum medicinal material, Sedum sarmentosum slices, Sedum sarmentosum standard decoction, and Sedum sarmentosum formula granules. (2) Preparation of reference solution: Vanillic acid-4-β-D-glucoside and isorhamnetin-3,7-O-diglucoside, which are used as reference standards, are dissolved in solvent to obtain reference solution; (3) Establishment of characteristic chromatograms: The reference solution of Sedum sarmentosum in step (1) and the reference solution in step (2) were detected by ultra-high performance liquid chromatography. After obtaining the detection chromatograms of Sedum sarmentosum and the reference solution, the characteristic chromatograms of Sedum sarmentosum were established, and the characteristic peaks in the characteristic chromatograms were assigned according to the chromatographic peaks of the reference solution. The chromatographic conditions for the ultra-high performance liquid chromatography method include: Stationary phase: Eclipse Plus C18 RRHD column with a length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.8 μm was used. Mobile phase: Acetonitrile was used as mobile phase A, and a 0.05%~0.20% v / v aqueous solution of formic acid was used as mobile phase B; Elution method: gradient elution, The gradient elution procedure is as follows: ， The detection wavelength is 254nm. The characteristic chromatogram of the *Sedum sarmentosum* reference standard includes nine characteristic peaks. Among them, characteristic peak 3 corresponds to the chromatographic peak of vanillic acid-4-β-D-glucoside reference standard, and characteristic peak 9 corresponds to the chromatographic peak of isorhamnetin-3,7-O-diglucoside reference standard. Using characteristic peak 3 as a reference peak, calculate the relative retention times of characteristic peak 1 and characteristic peak 2. Their relative retention times should be within ±10% of the specified values, which are 0.46 and 0.64 respectively. Using characteristic peak 9 as a reference peak, calculate the relative retention times of characteristic peaks 4, 5, 6, 7, and 8. Their relative retention times should be within ±10% of the specified values, which are 0.56, 0.71, 0.77, 0.82, and 0.97, respectively. (4) Processing and identification of test sample: Take the test sample, obtain the test sample solution according to the method in step (1), and then establish the characteristic spectrum of the test sample according to the method in step (3); compare the characteristic spectrum of the test sample with the characteristic spectrum of the Sedum sarmentosum reference standard in step (3) to identify whether the test sample is Sedum sarmentosum or a counterfeit. The test sample is selected from one or more of the following: medicinal materials to be tested, processed medicinal pieces, standard decoctions, and formula granules; The counterfeit products are one or more of the following: purslane, eclipta prostrata, sedum concave-leaf, and sedum sarmentosum. The criteria for identification include: If the characteristic spectrum of the test sample contains characteristic peak 9, then the test sample is *Sedum sarmentosum*; otherwise, it is a counterfeit.

2. The method according to claim 1, characterized in that, In step (1), The extraction methods include ultrasonic treatment, shaking extraction, and heating reflux extraction.

3. The method according to claim 2, characterized in that, The extraction method is ultrasonic extraction, the ultrasonic extraction power is 200-300W, the ultrasonic extraction frequency is 35-45kHz, and the ultrasonic extraction time is 30-120min. And / or, The mass-to-volume ratio of the *Sedum sarmentosum* reference standard to the extraction solvent is 1 g: (5-50 mL).

4. The method according to any one of claims 1 to 3, characterized in that, In step (2), the solvent is methanol or an aqueous solution thereof; And / or, The concentration of the reference solution is 5~40 μg / mL.

5. The method according to claim 4, characterized in that, In step (2), the solvent is methanol or an aqueous methanol solution with a content of 50% v / v or higher.

6. The method according to any one of claims 1 to 3, characterized in that, The chromatographic conditions for the ultra-high performance liquid chromatography method also include: a column temperature of 20~35℃ and / or a flow rate of 0.25~0.30mL / min.

7. The application of the method according to any one of claims 1 to 6 in the quality inspection of Sedum sarmentosum and its adulterants.

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