Construction of HPLC-ELSD characteristic spectrum of standard decoction of Jijizi slices and its identification application

Abstract

The present invention utilizes a high performance liquid chromatograph and an evaporative light scattering detector for detection, and provides a method for establishing an HPLC-ELSD characteristic spectrum of a standard decoction of Jinjizi decoction pieces. The characteristic spectrum constructed by this method presents 10 characteristic peaks, which can fully reflect the characteristic peak information of the sample, demonstrate the chemical composition characteristics of the standard decoction of Jinjizi decoction pieces, and provide a more comprehensive and effective evaluation method for the quality control of the standard decoction of Jinjizi decoction pieces. Furthermore, the established HPLC-ELSD characteristic spectrum can be used to identify the standard decoction of confused products that have lost their medicinal properties, providing a basis for the formulation of quality standards for the standard decoction of Jinjizi decoction pieces. Moreover, the construction method of the present application also has the advantages of being fast, stable, reproducible, and highly specific.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine identification and drug analysis, and particularly relates to the construction of an HPLC-ELSD characteristic spectrum of a standard decoction of Jijizi decoction pieces and its identification application. Background Art

[0002] Standard decoctions of Chinese herbal medicine slices are single-ingredient decoctions prepared using standardized processes, guided by Traditional Chinese Medicine theory and based on clinical application, with reference to modern extraction methods. As a reference for quality control of related Chinese medicine products, standard decoctions of Chinese herbal medicine slices serve as a key link between Chinese herbal medicine slices and modern Chinese medicine preparations. Therefore, research on the quality standards of standard decoctions of Chinese herbal medicine slices can lay the foundation for the development of standards for formulated granules derived from decoctions of these slices, as well as other finished Chinese medicine products.

[0003] Impatiens balsamina L. is the dried mature seed of Impatiens balsamina L., a plant of the Impatiens family. The fruit is harvested in summer and autumn when it is about to mature, sun-dried, and the peel and impurities are removed. It has a pungent and bitter taste and a warm nature. It enters the spleen and liver meridians, and has the effects of promoting blood circulation and promoting qi, promoting menstruation and relieving pain. It is used for chest and flank pain, chest pain and heart pain, dysmenorrhea and amenorrhea, scars, rheumatic shoulder and arm pain, and swelling and pain caused by falls. Studies have shown that the main chemical components of Impatiens balsamina include fatty oils, diterpenes, flavonoids, triterpenoid saponins, sterols, polypeptide compounds, and proteins. It has the effects of promoting skin penetration, anti-fertility, anti-tumor, anti-cancer, and antibacterial. Among them, flavonoids and triterpenoid saponins are the material basis of the main pharmacological effects of Impatiens balsamina. The Chinese Pharmacopoeia (2020 edition) lacks fingerprint / characteristic map items for the medicinal material of Impatiens balsamina, making it difficult to control its quality from a holistic perspective. Therefore, the quality standards need to be further improved.

[0004] Fingerprints / characteristics, due to their holistic control concept, can comprehensively reflect the types and quantities of chemical components contained in traditional Chinese medicines and their preparations. They can identify differences between medicinal samples and identify related counterfeits and adulterants, thereby comprehensively assessing the quality of traditional Chinese medicines. Due to the limited research on the material basis of jizizi, there is very little research on its fingerprints / characteristics. Reported fingerprints / characteristics of jizizi have poor separation of chromatographic peaks, focusing on only one class of compounds (flavonoids), and the number of identified compounds is very small. There are no reports on research related to the standard decoction of jizizi medicinal slices.

[0005] In relevant literature reports and the catalog of 130 commonly used counterfeit and confusing Chinese medicines, it is mentioned that Perilla seed, Chrysanthemum villosum seed, Wangjiangnan seed, Radish seed, Astragalus seed, and Winter Melon seed are similar to Jinjizi in properties and are confusing products of Jinjizi. In addition, due to the loss of medicinal properties, Jinjizi standard decoctions and related preparations cannot be identified by traditional medicinal identification methods such as properties and smell, and thus different confusing products cannot be distinguished. The difference in efficacy seriously affects their medication safety. Therefore, this study established a characteristic spectrum of the standard decoction of Jinjizi slices, and simultaneously characterized as many of the main chemical components (flavonoids and saponins) in the standard decoction of Jinjizi as possible, which can effectively qualitatively identify related confusing products. In addition, the establishment of this characteristic spectrum not only provides a basis for the establishment of quality standards for standard decoctions of Jinjizi slices, but also provides an important reference for the establishment of quality standards for Jinjizi formula granules and related Chinese medicine preparations. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for establishing an HPLC-ELSD characteristic spectrum of a standard decoction of Jinjizi medicinal slices and identifying characteristic components. The method is rapid, stable and highly specific. The established HPLC-ELSD characteristic spectrum can be used to identify standard decoctions of confused products that have lost their medicinal properties, providing a more comprehensive and effective evaluation method for the quality control of standard decoctions of Jinjizi medicinal slices.

[0007] The first aspect of the present invention provides a method for establishing an HPLC-ELSD characteristic spectrum of a standard decoction of Jijizi decoction pieces, comprising the following steps:

[0008] Prepare a reference solution by taking one or more of the reference substances impatiens tetraol saponin K, impatiens tetraol saponin A, impatiens tetraol saponin G, impatiens tetraol saponin L, impatiens tetraol saponin B, and quercetin-3-O-[α-L-rhamnosyl-(1→2)-β-D-glucosyl]-5-O-β-D-glucoside;

[0009] Prepare the test solution from the standard decoction of Jijizi slices;

[0010] The reference solution and the test solution were respectively detected by high performance liquid chromatography-evaporative light scattering detection;

[0011] The chromatographic conditions of the high performance liquid chromatography-evaporative light scattering detection method include: acetonitrile as mobile phase A, water as mobile phase B, and gradient elution;

[0012] The gradient elution procedure includes:

[0013] From 0 to 25 minutes, the volume percentage of the mobile phase A increased from 16% to 32%, and the volume percentage of the mobile phase B decreased from 84% to 68%;

[0014] 25min~30min, the volume percentage of the mobile phase A is 32%, and the volume percentage of the mobile phase B is 68%;

[0015] The detection conditions of the high performance liquid chromatography-evaporative light scattering detection method include: a gain of 1 to 3; a carrier gas flow rate of 2.5 L / min to 3.5 L / min; and a temperature of 110° C. to 120° C.

[0016] In some embodiments of the present invention, the chromatographic conditions of the high performance liquid chromatography-evaporative light scattering detection method further include: using an Agilent Poroshell 120EC-C18 chromatographic column.

[0017] In some embodiments of the present invention, the chromatographic conditions of the high performance liquid chromatography-evaporative light scattering detection method further include: a flow rate of 0.8 mL / min to 1.2 mL / min.

[0018] In some embodiments of the present invention, the chromatographic conditions of the high performance liquid chromatography-evaporative light scattering detection method further include: a column temperature of 33°C to 37°C.

[0019] In some embodiments of the present invention, methanol is used as a dissolving reagent to prepare the reference solution; the concentration of the impatiens tetraol saponin K reference solution is 280 μg / mL to 320 μg / mL; the concentration of the impatiens tetraol saponin A reference solution is 260 μg / mL to 300 μg / mL; the concentration of the impatiens tetraol saponin G reference solution is 270 μg / mL to 310 μg / mL; the concentration of the impatiens tetraol saponin L reference solution is 290 μg / mL to 330 μg / mL; the concentration of the impatiens tetraol saponin B reference solution is 270 μg / mL to 310 μg / mL; and the concentration of the quercetin-3-O-[α-L-rhamnosyl-(1→2)-β-D-glucosyl]-5-O-β-D-glucoside reference solution is 140 μg / mL to 180 μg / mL.

[0020] In some embodiments of the present invention, the step of preparing the test solution includes: mixing the standard decoction of Jijizi slices with a first alcohol-containing aqueous solution having a volume fraction of 50% to 80% for extraction, concentrating, dissolving the residue, filtering, and obtaining a filtrate;

[0021] The alcohol in the first alcohol-containing aqueous solution is selected from methanol or ethanol;

[0022] The extraction method is ultrasonic extraction or heating reflux extraction, and the conditions of ultrasonic extraction include: power of 200W to 300W, frequency of 35kHz to 45kHz, and time of 30min to 60min.

[0023] In some embodiments of the present invention, in the step of preparing the test solution, the amount of ethanol solution corresponding to every 0.1 g of the standard decoction of the Jijizi medicinal slices is 15 mL to 25 mL.

[0024] It can be understood that in the present invention, the test sample of the standard decoction of Jijizi medicinal pieces is in the form of freeze-dried powder.

[0025] It is understandable that before extraction with the extraction solvent, the step of grinding the standard decoction sample of the Jinjizi decoction slices to prepare a powder sample of the standard decoction sample of the Jinjizi decoction slices is also included. Furthermore, the standard decoction sample of the Jinjizi decoction slices in the phrase "the amount of ethanol solution corresponding to each 0.1g of the standard decoction sample of the Jinjizi decoction slices is 15mL to 25mL" refers to the powder sample of the standard decoction sample of the Jinjizi decoction slices.

[0026] In some embodiments of the present invention, 10 characteristic peaks are presented in the characteristic spectrum, and the peak corresponding to the peak of the reference substance of impatiens tetraol saponin K is the S peak. The relative retention times of peaks 1 to 3, peaks 5 to 8, and peak 10 and the S peak are calculated, and the relative retention times are within the range of ±10% of the specified values, and the specified values ​​are: peak 1 is 0.21, peak 2 is 0.78, peak 3 is 0.89, peak 5 is 1.04, peak 6 is 1.07, peak 7 is 1.14, peak 8 is 1.24, and peak 10 is 1.59.

[0027] In some embodiments of the present invention, the establishment method further comprises the step of performing high-resolution mass spectrometric identification of characteristic peaks in the characteristic spectrum in conjunction with mass spectrometry;

[0028] The mass spectrometry conditions include:

[0029] HESI ion source parameters include: sheath gas flow rate of 30 arb to 40 arb, auxiliary gas flow rate of 8 arb to 12 arb, spray voltage of 3.5 kV to 4.0 kV, S-lens voltage of 45 V to 55 V, heating temperature of 320°C to 380°C, and capillary temperature of 320°C to 380°C;

[0030] The mass spectrometry scanning parameters include: scanning mode is positive ion mode and / or negative ion mode, scanning range is 120m / z to 1200m / z, normalized collision energy is 40V, and mass spectrum type is peak shape diagram.

[0031] A second aspect of the present invention further provides a method for identifying a standard decoction of Jijizi decoction pieces, comprising the following steps:

[0032] Prepare a test solution by taking the test product;

[0033] The test solution is detected by high performance liquid chromatography-evaporative light scattering detection, and the obtained chromatogram of the test product is compared with the characteristic spectrum obtained by the method for establishing the HPLC-ELSD characteristic spectrum of the standard decoction of the Jijizi decoction pieces;

[0034] The chromatographic conditions of the high performance liquid chromatography-evaporative light scattering detection method include: acetonitrile as mobile phase A, water as mobile phase B, and gradient elution;

[0035] The gradient elution procedure includes:

[0036] From 0 to 25 minutes, the volume percentage of the mobile phase A increased from 16% to 32%, and the volume percentage of the mobile phase B decreased from 84% to 68%;

[0037] 25min~30min, the volume percentage of the mobile phase A is 32%, and the volume percentage of the mobile phase B is 68%;

[0038] The detection conditions of the high performance liquid chromatography-evaporative light scattering detection method include: a gain of 1 to 3; a carrier gas flow rate of 2.5 L / min to 3.5 L / min; and a temperature of 110° C. to 120° C.

[0039] In some embodiments of the identification method of the present invention, the chromatographic conditions of the high performance liquid chromatography-evaporative light scattering detection method further include: using an Agilent Poroshell 120EC-C18 chromatographic column.

[0040] In some embodiments of the identification method of the present invention, the chromatographic conditions of the high performance liquid chromatography-evaporative light scattering detection method further include: a flow rate of 0.8 mL / min to 1.2 mL / min.

[0041] In some embodiments of the identification method of the present invention, the chromatographic conditions of the high performance liquid chromatography-evaporative light scattering detection method further include: a column temperature of 33°C to 37°C.

[0042] In some embodiments of the identification method of the present invention, the step of preparing the test solution includes:

[0043] The sample to be tested is mixed with a second alcohol-containing aqueous solution having a volume fraction of 50% to 80% for extraction, concentrated, the residue is dissolved, filtered, and the filtrate is collected;

[0044] The alcohol in the second alcohol-containing aqueous solution is selected from methanol or ethanol;

[0045] The extraction method is ultrasonic extraction or heating reflux extraction, and the conditions of ultrasonic extraction include: power of 200W to 300W, frequency of 35kHz to 45kHz, and time of 30min to 60min.

[0046] In some embodiments of the identification method of the present invention, in the step of preparing the test sample solution, the amount of ethanol solution used for every 0.1 g of the test sample is 15 mL to 25 mL.

[0047] In some embodiments of the identification method of the present invention, the test product includes at least one of Perilla seed, Chrysanthemum daisy seed, Amomum villosum seed, Radish seed, Complanatus complanatus seed and Malus domestica seed.

[0048] Compared with traditional technologies, the present invention has the following beneficial effects: the present invention utilizes high performance liquid chromatography-evaporative light scattering detection to detect, and provides a method for establishing an HPLC-ELSD characteristic spectrum of a standard decoction of Jijizi medicinal slices by selecting appropriate conditions. The method is fast, stable and highly specific, and the established HPLC-ELSD characteristic spectrum can be used to identify the standard decoction of confused products that have lost their medicinal properties, providing a more comprehensive and effective evaluation method for the quality control of the standard decoction of Jijizi medicinal slices. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is the chromatogram of the different chromatographic conditions of the standard decoction of Jijizi;

[0050] Figure 2 This is the chromatogram of different chromatographic columns of the standard decoction of Jijizi;

[0051] Figure 3 This is a graph showing the characteristic spectrum of the standard decoction of Jijizi, obtained by investigating different extraction solvents;

[0052] Figure 4 This is a graph showing the investigation of different extraction methods for the characteristic spectrum of the standard decoction of Jijizi;

[0053] Figure 5 This is the characteristic spectrum of the standard decoction of Jijizi at different extraction times;

[0054] Figure 6 This is a graph showing the specificity of the characteristic spectrum of the standard decoction of Jijizi;

[0055] Figure 7 This is an overlay of characteristic spectra of 21 batches of Jijizi standard decoctions;

[0056] Figure 8 This is the characteristic spectrum of the control medicinal material of Jijizi; among them, Peak 1: Quercetin-3-O-[α-L-rhamnosyl-(1→2)-β-D-glucosyl]-5-O-β-D-glucoside; Peak 2: Impatiens tetraol saponin B; Peak 4: Impatiens tetraol saponin K; Peak 8: Impatiens tetraol saponin G; Peak 9: Impatiens tetraol saponin A; Peak 10: Impatiens tetraol saponin L;

[0057] Figure 9This is the reference characteristic spectrum of the standard decoction of Jijizi; among them, Peak 1: Quercetin-3-O-[α-L-rhamnosyl-(1→2)-β-D-glucosyl]-5-O-β-D-glucoside; Peak 2: Impatiens tetraol saponin B; Peak 4: Impatiens tetraol saponin K; Peak 8: Impatiens tetraol saponin G; Peak 9: Impatiens tetraol saponin A; Peak 10: Impatiens tetraol saponin L;

[0058] Figure 10 This is the total ion current graph and ultraviolet absorption chromatogram of the test solution of the standard decoction of Jijizi decoction pieces;

[0059] Figure 11 The results were confirmed by the reference substance for the characteristic spectrum of the standard decoction of Jijizi;

[0060] Figure 12 This is a comparison chart of the characteristic spectra of the standard decoctions of Perilla seeds, Chrysanthemum seeds and Jijizi;

[0061] Figure 13 This is a comparison chart of the characteristic spectra of the standard decoctions of Radish Seed and Jijizi;

[0062] Figure 14 This is a comparison chart of the characteristic spectra of standard decoctions of Fructus Lycoris and Fructus Aconitum;

[0063] Figure 15 This is a comparison chart of the characteristic spectra of the standard decoctions of Astragalus and Aconitum;

[0064] Figure 16 This is a comparison chart of the characteristic spectra of the standard decoctions of Wangjiangnan and Jijizi. DETAILED DESCRIPTION

[0065] To facilitate understanding of the present invention, the present invention will be described more fully below with reference to the accompanying drawings. The accompanying drawings illustrate preferred embodiments of the present invention. However, the present invention may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the present disclosure.

[0066] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0067] The complexity and diversity of Chinese herbal medicine's chemical composition underpins its therapeutic efficacy, but also presents both a challenge and a key point in evaluating its quality. Fingerprints / characteristic profiles can comprehensively and systematically characterize the similarities between the primary chemical components within a herbal sample; chemical pattern recognition can identify differences between herbal samples for comprehensive quality assessment. Research indicates that due to limited research on the material basis of schisandra, limited studies on its fingerprints / characteristic profiles are required, making their study challenging. At present, the research on the fingerprint / characteristic spectrum of Jinzi mainly focuses on medicinal materials. For example, Xu Yan et al. established the fingerprint spectrum of Jinzi medicinal materials in the study of HPLC fingerprint spectrum of Jinzi. Moreover, the separation effect of each chromatographic peak of the fingerprint / characteristic spectrum of Jinzi established in the report is poor. Most studies only involve one type of compound (flavonoids or saponins), such as Tan Lina, Study on saponin components of Jinzi [D]; Pei Hui et al., Determination of the contents of four balsamina tetraol glycosides in Jinzi by HPLC-ELSD method [J]; Wang Wei et al., Preliminary study on HPLC fingerprint spectrum of flavonoid components in Jinzi [J]. It is difficult to fully reflect the intrinsic quality of Jinzi.

[0068] As a reference for the quality control of related Chinese medicine products, the standard decoction of Chinese herbal medicine slices is a key hub connecting Chinese herbal medicine slices and modern Chinese medicine preparations. Due to the loss of medicinal material properties, the standard decoction of Jinjizi slices cannot be identified by traditional medicinal material identification methods such as properties and smell, and then different confusing products can be distinguished, which seriously affects the safety of its use based on the difference in efficacy. Therefore, this study established a characteristic spectrum of the standard decoction of Jinjizi slices, and simultaneously characterized as many of the main chemical components (flavonoids and saponins) in the standard decoction of Jinjizi as possible, which can effectively qualitatively identify related confusing products. In addition, the establishment of this characteristic spectrum not only provides a basis for the establishment of the quality standard of the standard decoction of Jinjizi slices, but also provides an important reference for the establishment of the quality standard of Jinjizi formula granules and related Chinese medicine preparations.

[0069] In the present invention, the standard decoction of Jijizi slices can be prepared by the following method:

[0070] 1. Preparation of Jijizi decoction pieces

[0071] Since the Chinese Pharmacopoeia (2020 edition) does not include a section on the preparation of decoction pieces for Jijizi, the preparation method, referring to the medicinal material regulations, is as follows: Take the Jijizi raw material and remove impurities and spoiled products. Based on the principle of "pounding all shells and breaking all seeds" under "Preparation of decoctions for research and characterization of standard decoctions" in the "Technical Requirements for Quality Control and Standardization of Traditional Chinese Medicine Formula Granules," Jijizi decoction pieces are crushed.

[0072] 2. Preparation of standard decoction of Jijizi slices

[0073] Get Fructus Aconitum decoction pieces 100g, add water and decoct twice, decoct for the first time and add 8 times of water gagings, soak 30 minutes, after high fire (500W) boils, slow fire (200W) keeps slightly and boils 30 minutes, decocting solution with 350 mesh screens filtered while hot, and filtrate cools off with cold water rapidly.Decoction for the second time and add 6 times of water gagings, after high fire (500W) boils, slow fire (200W) keeps slightly and boils 25 minutes, decocting solution with 350 mesh screens filtered while hot, and filtrate cools off with cold water rapidly, merges twice filtrate.Filtrate is transferred in 2000ml round-bottomed flask, reduced pressure low temperature is concentrated to 100ml, vacuum freeze drying, obtains standard decoction lyophilized powder.

[0074] The standard decoction of the Jijizi medicinal slices in the embodiment of the present invention can be prepared by the above preparation method. It can be understood that it is not limited to this and one or more steps can be adjusted on the basis of the above preparation method.

[0075] The first aspect of the present invention provides a method for establishing an HPLC-ELSD characteristic spectrum of a standard decoction of Jijizi decoction pieces, comprising the following steps:

[0076] Prepare a reference solution by taking one or more of the reference substances impatiens tetraol saponin K, impatiens tetraol saponin A, impatiens tetraol saponin G, impatiens tetraol saponin L, impatiens tetraol saponin B, and quercetin-3-O-[α-L-rhamnosyl-(1→2)-β-D-glucosyl]-5-O-β-D-glucoside;

[0077] Prepare the test solution from the standard decoction of Jijizi slices;

[0078] The reference solution and the test solution were respectively detected by high performance liquid chromatography-evaporative light scattering detection;

[0079] The chromatographic conditions of the high performance liquid chromatography-evaporative light scattering detection method include: acetonitrile as mobile phase A, water as mobile phase B, and gradient elution;

[0080] The gradient elution procedure includes:

[0081] From 0 to 25 minutes, the volume percentage of the mobile phase A increased from 16% to 32%, and the volume percentage of the mobile phase B decreased from 84% to 68%;

[0082] 25min~30min, the volume percentage of the mobile phase A is 32%, and the volume percentage of the mobile phase B is 68%;

[0083] The detection conditions of the high performance liquid chromatography-evaporative light scattering detection method include: a gain of 1 to 3; a carrier gas flow rate of 2.5 L / min to 3.5 L / min; and a temperature of 110° C. to 120° C.

[0084] Preferably, the detection conditions of the high performance liquid chromatography-evaporative light scattering detection method include: a gain of 2; a carrier gas flow rate of 3.0 L / min; and a temperature of 110°C.

[0085] In some examples of the present invention, the chromatographic conditions of the high performance liquid chromatography-evaporative light scattering detection method further include: using an Agilent Poroshell 120EC-C18 chromatographic column.

[0086] Preferably, the specifications of the chromatographic column are: column length 150 mm, inner diameter 4.6 mm, and filler particle size 2.7 μm.

[0087] In some examples of the present invention, the chromatographic conditions of the high performance liquid chromatography-evaporative light scattering detection method further include: a flow rate of 0.8 mL / min to 1.2 mL / min.

[0088] It can be understood that in the present invention, the flow rate is, for example, 0.8 mL / min, 0.9 mL / min, 1.0 mL / min, 1.1 mL / min, 1.2 mL / min, and preferably, the flow rate is 1.0 mL / min.

[0089] In some examples of the present invention, the chromatographic conditions of the high performance liquid chromatography-evaporative light scattering detection method further include: a column temperature of 30°C to 37°C.

[0090] It can be understood that in the present invention, the column temperature is, for example, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, or 37°C. Preferably, the column temperature is 35°C.

[0091] In some examples of the present invention, methanol is used as a dissolving reagent to prepare the reference solution; the concentration of the impatiens tetraol saponin K reference solution is 280 μg / mL to 320 μg / mL; the concentration of the impatiens tetraol saponin A reference solution is 260 μg / mL to 300 μg / mL; the concentration of the impatiens tetraol saponin G reference solution is 270 μg / mL to 310 μg / mL; the concentration of the impatiens tetraol saponin L reference solution is 290 μg / mL to 330 μg / mL; the concentration of the impatiens tetraol saponin B reference solution is 270 μg / mL to 310 μg / mL; and the concentration of the quercetin-3-O-[α-L-rhamnosyl-(1→2)-β-D-glucosyl]-5-O-β-D-glucoside reference solution is 140 μg / mL to 180 μg / mL.

[0092] In some examples of the present invention, the steps of preparing the test solution include: mixing the standard decoction of Jijizi slices with a first alcohol-containing aqueous solution having a volume fraction of 50% to 80% for extraction, concentrating, dissolving the residue, filtering, and obtaining a filtrate;

[0093] The alcohol in the first alcohol-containing aqueous solution is selected from methanol or ethanol;

[0094] The extraction method is ultrasonic extraction or heating reflux extraction, and the conditions of ultrasonic extraction include: power of 200W to 300W, frequency of 35kHz to 45kHz, and time of 30min to 60min.

[0095] It can be understood that the volume percentage of the first alcohol-containing aqueous solution includes but is not limited to 50%, 60%, 70%, and 80%, and is preferably a 50% ethanol aqueous solution.

[0096] It can be understood that the dissolving agent for dissolving the residue is consistent with the extraction solvent used in the extraction process.

[0097] It can be understood that in the present invention, the power of the ultrasonic extraction is, for example, 200W, 220W, 250W, 280W, and 300W, the frequency of the ultrasonic extraction is, for example, 35kHz, 36kHz, 38kHz, 40kHz, 42kHz, and 45kHz, and the time of the ultrasonic extraction is, for example, 30min, 40min, 45min, 48min, 50min, 55min, and 60min. Preferably, the conditions of the ultrasonic extraction include: a power of 250W, a frequency of 40kHz, and a time of 45min.

[0098] In some examples of the present invention, in the step of preparing the test solution, the amount of ethanol solution corresponding to each 0.1g of the standard decoction of Jijizi medicinal pieces test sample is 15mL~25mL.

[0099] It can be understood that the amount of ethanol solution corresponding to each 0.1g of the standard decoction of the Jijizi medicinal slices is 15mL, 18mL, 20mL, 22mL, or 25mL. Preferably, the amount of ethanol solution corresponding to each 0.1g of the standard decoction of the Jijizi medicinal slices is 20mL.

[0100] In some examples of the present invention, 10 characteristic peaks are presented in the characteristic spectrum, and the peak corresponding to the peak of the reference substance of impatiens tetraol saponin K is the S peak. The relative retention times of peaks 1 to 3, peaks 5 to 8, and peak 10 and the S peak are calculated, and the relative retention times are within ±10% of the specified values, and the specified values ​​are: peak 1 is 0.21, peak 2 is 0.78, peak 3 is 0.89, peak 5 is 1.04, peak 6 is 1.07, peak 7 is 1.14, peak 8 is 1.24, and peak 10 is 1.59.

[0101] In some examples of the present invention, the establishment method further comprises the step of performing high-resolution mass spectrometric identification of characteristic peaks in the characteristic spectrum using a combined mass spectrometry method;

[0102] The mass spectrometry conditions include:

[0103] HESI ion source parameters include: sheath gas flow rate of 30 arb to 40 arb, auxiliary gas flow rate of 8 arb to 12 arb, spray voltage of 3.5 kV to 4.0 kV, S-lens voltage of 45 V to 55 V, heating temperature of 320°C to 380°C, and capillary temperature of 320°C to 380°C;

[0104] The mass spectrometry scanning parameters include: scanning mode is positive ion mode and / or negative ion mode, scanning range is 120m / z to 1200m / z, normalized collision energy is 40V, and mass spectrum type is peak shape diagram.

[0105] It can be understood that in the present invention, the sheath gas flow rate is, for example, 30arb, 32arb, 35arb, 38arb, and 40arb, the auxiliary gas flow rate is, for example, 8arb, 9arb, 10arb, 11arb, and 12arb, the spray voltage is, for example, 3.5kV, 3.6kV, 3.8kV, and 4.0kV, the S-lens voltage is, for example, 45V, 48V, 50V, 52V, and 55V, the heating temperature is, for example, 320°C, 350°C, 360°C, and 380°C, and the capillary temperature is, for example, 320°C, 350°C, 360°C, and 380°C.

[0106] Preferably, the sheath gas flow rate is 35 arb, the auxiliary gas flow rate is 10 arb, the spray voltage is 3.8 kV, the S-lens voltage is 50 V, the heating temperature is 350°C, and the capillary temperature is 350°C.

[0107] A second aspect of the present invention further provides a method for identifying a standard decoction of Jijizi decoction pieces, comprising the following steps:

[0108] Prepare a test solution by taking the test product;

[0109] The test solution is detected by high performance liquid chromatography-evaporative light scattering detection, and the obtained chromatogram of the test product is compared with the characteristic spectrum obtained by the method for establishing the HPLC-ELSD characteristic spectrum of the standard decoction of the Jijizi decoction pieces;

[0110] The chromatographic conditions of the high performance liquid chromatography-evaporative light scattering detection method include: acetonitrile as mobile phase A, water as mobile phase B, and gradient elution;

[0111] The gradient elution procedure includes:

[0112] From 0 to 25 minutes, the volume percentage of the mobile phase A increased from 16% to 32%, and the volume percentage of the mobile phase B decreased from 84% to 68%;

[0113] 25min~30min, the volume percentage of the mobile phase A is 32%, and the volume percentage of the mobile phase B is 68%;

[0114] The detection conditions of the high performance liquid chromatography-evaporative light scattering detection method include: a gain of 1 to 3; a carrier gas flow rate of 2.5 L / min to 3.5 L / min; and a temperature of 110° C. to 120° C.

[0115] Preferably, the detection conditions of the high performance liquid chromatography-evaporative light scattering detection method include: a gain of 2; a carrier gas flow rate of 3.0 L / min; and a temperature of 110°C.

[0116] In some examples of the identification method of the present invention, the chromatographic conditions of the high performance liquid chromatography-evaporative light scattering detection method further include: using an Agilent Poroshell 120EC-C18 chromatographic column.

[0117] Preferably, the specifications of the chromatographic column are: column length 150 mm, inner diameter 4.6 mm, and filler particle size 2.7 μm.

[0118] In some examples of the identification method of the present invention, the chromatographic conditions of the high performance liquid chromatography-evaporative light scattering detection method further include: a flow rate of 0.8 mL / min to 1.2 mL / min.

[0119] It can be understood that in the present invention, the flow rate is, for example, 0.8 mL / min, 0.9 mL / min, 1.0 mL / min, 1.1 mL / min, 1.2 mL / min, and preferably, the flow rate is 1.0 mL / min.

[0120] In some examples of the identification method of the present invention, the chromatographic conditions of the high performance liquid chromatography-evaporative light scattering detection method further include: a column temperature of 33°C to 37°C.

[0121] It can be understood that in the present invention, the column temperature is, for example, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, or 37°C. Preferably, the column temperature is 35°C.

[0122] In some examples of the identification method of the present invention, the step of preparing the test solution includes:

[0123] The sample to be tested is mixed with a second alcohol-containing aqueous solution having a volume fraction of 50% to 80% for extraction, concentrated, the residue is dissolved, filtered, and the filtrate is collected;

[0124] The alcohol in the second alcohol-containing aqueous solution is selected from methanol or ethanol;

[0125] The extraction method is ultrasonic extraction or heating reflux extraction, and the conditions of ultrasonic extraction include: power of 200W to 300W, frequency of 35kHz to 45kHz, and time of 30min to 60min.

[0126] It can be understood that the volume percentage of the second alcohol-containing aqueous solution includes but is not limited to 50%, 60%, 70%, and 80%, and is preferably a 50% ethanol aqueous solution.

[0127] It can be understood that in the present invention, the power of the ultrasonic extraction is, for example, 200W, 220W, 250W, 280W, and 300W, the frequency of the ultrasonic extraction is, for example, 35kHz, 36kHz, 38kHz, 40kHz, 42kHz, and 45kHz, and the time of the ultrasonic extraction is, for example, 30min, 40min, 45min, 48min, 50min, 55min, and 60min. Preferably, the conditions of the ultrasonic extraction include: a power of 250W, a frequency of 40kHz, and a time of 45min.

[0128] In some embodiments of the identification method of the present invention, in the step of preparing the test sample solution, the amount of the extraction solvent used for every 0.1 g of the test sample is 15 mL to 25 mL.

[0129] It can be understood that the amount of ethanol solution corresponding to each 0.1g of the test product is 15mL, 18mL, 20mL, 22mL, or 25mL. Preferably, the amount of ethanol solution corresponding to each 0.1g of the test product is 20mL.

[0130] In some embodiments of the identification method of the present invention, the test product includes at least one of Perilla seed, Chrysanthemum daisy seed, Amomum villosum seed, Radish seed, Complanatus complanatus seed and Malus domestica seed.

[0131] The embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. For experimental methods in the following examples where specific conditions are not specified, reference is made to the guidance provided in the present invention, and may also be made according to experimental manuals or conventional conditions in the art, or according to conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0132] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0133] Example 1

[0134] A method for establishing HPLC-ELSD characteristic spectrum of standard decoction of Jijizi medicinal pieces

[0135] 1. Instruments, reagents and test drugs

[0136] Instruments: Waters ultra-high performance liquid chromatograph (H-Class PLus, Waters Technologies Inc.), Agilent ultra-high performance liquid chromatograph (1290, Agilent Technologies Inc.), Poroshell 120EC-C18 (4.6 mm × 150 mm, 2.7 μm, No. JS-320, JS-319, JS-321) columns, Waters Xbridge C18 (4.6 mm × 150 mm, 5 μm) columns, Waters Xselect HSS T3 (4.6 mm × 150 mm, 5 μm) columns, evaporative light scattering detector (ELSD6000, Alltech Chrom), 1 / 100 electronic analytical balance (JJ600, Changshu Shuangjie Testing Instrument Factory), 1 / 10,000 electronic analytical balance (ME204E, Mettler-Toledo), 1 / 100,000 electronic analytical balance (ABT220-5DM, Guangzhou Dexiang Technology Co., Ltd.), 1 / 1,000,000 electronic analytical balance (XP26, Mettler-Toledo), CNC ultrasonic cleaner (KQ-500DE, Kunshan Ultrasonic Instrument Co., Ltd.), electric constant temperature water bath (HWS28, Shanghai Yiheng Technology Co., Ltd.), and ultrapure water system (Milli-Q Direct, Merck AG).

[0137] Reagents: Methanol (Xilong Scientific Co., Ltd.) and ethanol (Xilong Scientific Co., Ltd.) were of analytical grade; acetonitrile (Merck AG) was of chromatographic grade; and water was ultrapure water (prepared in the laboratory).

[0138] Test drugs: Impatiens tetraol saponin K (batch number: 112005-201902, content: 91.2%, China Food and Drug Inspection Institute), Impatiens tetraol saponin A (batch number: 112009-202102, content: 95.4%, China Food and Drug Inspection Institute), Impatiens tetraol saponin G (batch number: 18020704, content: 99.47%, Chengdu Pufeide Biotechnology Co., Ltd.), Impatiens tetraol saponin B (batch number: 060032-201906, content: ≥98%, Guangzhou Langou Biotechnology Co., Ltd. Technology Co., Ltd.), impatiens tetraol saponin L (batch number: 060033-201906, content: ≥98%, Guangzhou Langou Biotechnology Co., Ltd.), quercetin-3-O-[α-L-rhamnosyl-(1→2)-β-D-glucosyl]-5-O-β-D-glucoside (content: ≥98%, Sichuan Weikeqi Biotechnology Co., Ltd.), acne-resistant radix syringae reference medicinal material (batch number: 121606-201602, China Food and Drug Inspection Institutes); 21 batches of lyophilized powders of standard decoctions prepared from acne-resistant radix syringae slices from different origins.

[0139] 2. Preparation of Jijizi decoction pieces

[0140] Since the Chinese Pharmacopoeia (2020 edition) does not include a section on the preparation of decoction pieces under the section on Jijizi (Acute Chinese Medicine), the preparation method, referring to the medicinal material regulations, is as follows: Take the Jijizi raw material and remove impurities and spoiled products. Based on the principle of "pounding all shells and breaking all seeds" under the "Preparation of Decoctions for Research and Characterization of Standardized Decoctions" section in the "Technical Requirements for Quality Control and Standardization of Traditional Chinese Medicine Formula Granules," Jijizi decoction pieces are crushed.

[0141] 3. Preparation of standard decoction of Jijizi slices

[0142] Get Fructus Aconitum decoction pieces 100g, add water and decoct twice, decoct for the first time and add 8 times of water gagings, soak 30 minutes, high fire (500W) boils rear slow fire (200W) keeps slightly boiling 30 minutes, decoction is filtered while hot with 350 mesh sieves, and filtrate is cooled with cold water rapidly.Decoction for the second time and add 6 times of water gagings, high fire (500W) boils rear slow fire (200W) keeps slightly boiling 25 minutes, decoction is filtered while hot with 350 mesh sieves, and filtrate is cooled with cold water rapidly, merges twice filtrate.Filtrate is transferred in 2000ml round-bottom flask, is concentrated to 100ml under reduced pressure and freeze-dried in vacuo, obtains standard decoction freeze-dried powder.21 batches of standard decoction freeze-dried powders prepared by Fructus Aconitum decoction pieces from different places of origin are shown in Table 1.

[0143] Table 1 Origin information of 21 batches of freeze-dried powder and medicinal materials of Jijizi standard decoction

[0144]

[0145] 4. Chromatographic conditions and test solution preparation

[0146] 4.1 Chromatographic conditions

[0147] An Agilent Poroshell 120EC-C18 (4.6×150 mm, 2.7 μm) column was used with acetonitrile as mobile phase A and water as mobile phase B, and gradient elution was performed as specified in Table 2. The flow rate was 1.0 mL / min, the column temperature was 35°C, and the detection was performed with an evaporative light scattering detector with a gain of 2, a carrier gas flow rate of 3.0 L / min, and a temperature of 110°C.

[0148] Table 2 Gradient elution table

[0149]

[0150] 4.2 Preparation of reference solution

[0151] Accurately weigh 7.860 mg of impatiens tetraol saponin K reference substance, place it in a 25 ml volumetric flask, and add methanol to make a solution containing 299.963 μg of impatiens tetraol saponin K per 1 ml to obtain the impatiens tetraol saponin K reference substance solution.

[0152] Accurately weigh 7.755 mg of impatiens tetraol saponin A reference substance, place it in a 25 ml volumetric flask, and add methanol to make a solution containing 286.733 μg of impatiens tetraol saponin A per 1 ml to obtain the impatiens tetraol saponin A reference substance solution.

[0153] Accurately weigh 7.280 mg of impatiens tetraol saponin G reference substance, place it in a 25 ml volumetric flask, and add methanol to make a solution containing 289.657 μg of impatiens tetraol saponin G per 1 ml to obtain the impatiens tetraol saponin G reference substance solution.

[0154] Accurately weigh 7.674 mg of the Impatiens tetraol saponin L reference substance, place it in a 25 ml volumetric flask, and add methanol to make a solution containing 310.841 μg of Impatiens tetraol saponin L per 1 ml to obtain the Impatiens tetraol saponin L reference substance solution.

[0155] Accurately weigh 7.463 mg of impatiens tetraol saponin B reference substance, place it in a 25 ml volumetric flask, and add methanol to make a solution containing 292.574 μg of impatiens tetraol saponin B per 1 ml to obtain the impatiens tetraol saponin B reference substance solution.

[0156] Accurately weigh 1.628 mg of quercetin-3-O-[α-L-rhamnose-(1→2)-β-D-glucosyl]-5-O-β-D-glucoside reference substance and place it in a 10 ml volumetric flask. Add methanol to make a solution containing 159.544 μg of quercetin-3-O-[α-L-rhamnose-(1→2)-β-D-glucosyl]-5-O-β-D-glucoside per 1 ml to obtain the quercetin-3-O-[α-L-rhamnose-(1→2)-β-D-glucosyl]-5-O-β-D-glucoside reference substance solution.

[0157] 4.3 Preparation of control medicinal material solution

[0158] Take about 1g of the control medicinal material of Aconitum carmichaelii, accurately weigh it, put it in a stoppered conical flask, add 50mL of water, heat and reflux for 30 minutes, let it cool, filter, take the filtrate, evaporate to dryness, add 50ml of 50% ethanol to the residue, weigh the weight, heat and reflux for 45 minutes, let it cool, weigh it again, make up the lost weight with 50% ethanol, centrifuge, accurately measure 25ml of the supernatant, evaporate to dryness in a water bath, add 50% ethanol to dissolve the residue, transfer it to a 5ml volumetric flask, add 50% ethanol to the scale, shake well, filter, and take the filtrate as the reference solution of the control medicinal material.

[0159] 4.4 Preparation of test solution

[0160] Take an appropriate amount of this product, grind it into powder, take about 0.1g, accurately weigh it, place it in a stoppered conical flask, accurately add 20mL of 50% ethanol, weigh it, and treat it with ultrasound (power 250W, frequency 40kHz) for 45 minutes. Let it cool, weigh it again, make up the lost weight with 50% ethanol, centrifuge it, accurately measure 10ml of the supernatant, evaporate it to dryness in a water bath, dissolve the residue in 50% ethanol, transfer it to a 5mL volumetric flask, add 50% ethanol to the scale, shake it well, filter it, and take the filtrate to obtain it.

[0161] 4.5 Determination method

[0162] Accurately pipette 5 μL of the test solution, reference solution, and reference medicinal material solution respectively, inject them into the liquid chromatograph for determination.

[0163] 5. Optimization of chromatographic conditions

[0164] 5.1 Optimization of elution gradient

[0165] Take an appropriate amount of Jizi standard decoction (No. BT1), grind it into powder, take about 0.1g, and prepare the test solution according to the method under "4.4". Except for the different elution gradients, all other analyses were carried out according to the provisions under "4.1". The results showed that by comparing the chromatograms of different gradient elutions ( Figure 1), it can be found that when gradient 3 is selected, the baseline of the chromatogram is stable and the separation effect of each chromatographic peak is better, so gradient 3 is selected as the final elution gradient.

[0166] Table 3 Gradient 1

[0167]

[0168] Table 4 Gradient 2

[0169]

[0170] Table 5 Gradient 3

[0171]

[0172] 5.2 Column selection

[0173] Take an appropriate amount of Jijizi standard decoction (No. BT1), grind it into powder, take about 0.1g, and prepare the test solution according to the method under "4.4". Except for using different chromatographic columns, all other analyses were carried out according to the provisions under "4.1". The results showed that when the Agilent Poroshell 120EC-C18 (4.6×150mm, 2.7μm) chromatographic column was selected for elution, the response values ​​of each characteristic peak were large, the baseline was stable, the interference was small, and the peak shape and separation effect of each chromatographic peak were good. Therefore, the Agilent Poroshell 120EC-C18 (4.6×150mm, 2.7μm) chromatographic column was selected as the best chromatographic column ( Figure 2 ).

[0174] 6 Investigation of test solution preparation method

[0175] 6.1 Extraction solvent investigation

[0176] Take appropriate amount of the same batch of Jijizi standard decoction (No.: BT1), grind it into powder, take about 0.1g, weigh it accurately, put it in a stoppered conical flask, accurately add 20ml of methanol, 80% methanol, 50% methanol, ethanol, 80% ethanol, and 50% ethanol respectively, weigh the weight, ultrasonically treat (power 250W, frequency 40kHz) for 45 minutes, let it cool, weigh it again, make up the lost weight with the corresponding solvent, centrifuge, accurately measure 10ml of the supernatant, evaporate it to dryness in a water bath, dissolve the residue in the corresponding solvent, transfer it to a 5ml volumetric flask, add the corresponding solvent to the scale, shake it well, filter it, and take the filtrate. The determination was carried out according to the chromatographic conditions specified under "4.1". The results showed that the characteristic spectra had obvious differences when different extraction solvents were used. When 50% ethanol was used as the extraction solvent, the chromatographic peak shape was better and the extraction rate was higher. Therefore, 50% ethanol was used as the extraction solvent ( Figure 3 ).

[0177] 6.2 Investigation of extraction methods

[0178] Take appropriate amount of the same batch of Jijizi standard decoction (No.: BT1), grind it into powder, take about 0.1g, weigh it accurately, put it in a stoppered conical flask, accurately add 20ml of 50% ethanol, weigh it, and treat it with ultrasound (power 250W, frequency 40kHz) for 45 minutes and reflux for 45 minutes respectively. Let it cool and weigh it again. Make up the lost weight with 50% ethanol, centrifuge it, accurately measure 10ml of the supernatant, evaporate it to dryness in a water bath, dissolve the residue in 50% ethanol, transfer it to a 5ml volumetric flask, add 50% ethanol to the scale, shake it well, filter it, and take the filtrate. Determine according to the chromatographic conditions specified under "4.1". The results show that ( Figure 4 ), using different extraction methods, there was no significant difference in the peak shape and separation effect of each characteristic peak. Considering the simplicity of operation, ultrasonic extraction was determined to be the extraction method.

[0179] 6.3 Extraction time investigation

[0180] Take appropriate amount of the same batch of Jizi standard decoction (No.: BT1), grind it into powder, take about 0.1g, weigh it accurately, put it in a stoppered conical flask, accurately add 20ml of 50% ethanol, weigh it, and ultrasonically treat it (power 250W, frequency 40kHz) for 30 minutes, 45 minutes, and 60 minutes respectively. Let it cool, weigh it again, make up the lost weight with 50% ethanol, centrifuge it, accurately measure 10ml of the supernatant, evaporate it in a water bath, dissolve the residue in 50% ethanol, transfer it to a 5ml volumetric flask, add 50% ethanol to the scale, shake it well, filter it, and take the filtrate. Determine it according to the chromatographic conditions specified under "4.1". The results show that different extraction times have little effect on the characteristic spectrum of Jizi standard decoction. In order to ensure complete extraction, the ultrasonic extraction time is selected as 45 minutes ( Figure 5 ).

[0181] 7 Methodological Review

[0182] 7.1 Specificity Investigation

[0183] Take an appropriate amount of Jijizi standard decoction (No. BT1), grind it into powder, take about 0.1g, prepare the test solution according to the method under "4.4", accurately aspirate the test solution, the reference solution under "4.2" and the blank solvent 5μl each, and inject and analyze according to the chromatographic conditions under "4.1". The results show that the chromatogram of the test sample has the same chromatographic peak at the corresponding retention time of the reference solution chromatogram, and there is no interference from the blank solvent, indicating that the method has good specificity ( Figure 6 ).

[0184] 7.2 Precision Investigation

[0185] Take an appropriate amount of the standard decoction of Jijizi (No. BT1), grind it into powder, and take approximately 0.1 g. Prepare the test solution according to the method under "4.4". Repeat the injection six times according to the chromatographic conditions under "4.1". Using impatiens tetraol saponin K as the reference peak S, calculate the relative retention time and relative peak area of ​​each characteristic peak relative to the S peak, and calculate the RSD value. The relative retention time RSD values ​​of the nine common peaks are within the range of 0.01% to 0.21%, and the relative peak area RSD values ​​are within the range of 0.41% to 2.73%, indicating good instrument precision.

[0186] 7.3 Repeatability Study

[0187] Take an appropriate amount of the standard decoction of Jijizi (No. BT1), grind it into powder, and take approximately 0.1 g. Prepare six test solutions according to the method in "4.4". Inject and measure the solution according to the chromatographic conditions in "4.1". Using impatiens tetraol saponin K as the reference peak S, calculate the relative retention time and relative peak area of ​​each characteristic peak relative to the S peak, and calculate the RSD values. The relative retention time RSD values ​​of the nine common peaks ranged from 0.02% to 0.30%, and the relative peak area RSD values ​​ranged from 1.29% to 2.88%, indicating that the method has good reproducibility.

[0188] 7.4 Stability investigation

[0189] Take an appropriate amount of the standard decoction of Jijizi (No. BT1), grind it into powder, and take about 0.1 g. Prepare the test solution according to the method under "4.4". Samples were injected and measured at 0, 2, 4, 6, 8, 10, 12, and 24 hours according to the chromatographic conditions under "4.1". Using impatiens tetraol saponin K as the reference peak S, the relative retention time and relative peak area of ​​each characteristic peak and the S peak were calculated, and the RSD values ​​were calculated. The relative retention time RSD values ​​of the 9 common peaks were in the range of 0.02% to 0.53%, and the relative peak area RSD values ​​were in the range of 2.03% to 2.90%, indicating that the test solution was relatively stable within 24 hours.

[0190] 8 Establishment of characteristic spectrum of standard decoction of Jijizi

[0191] 8.1 Results of Characteristic Spectrum Determination of Jijizi Standard Decoction

[0192] Take 21 batches of Jijizi standard decoction, according to the chromatographic conditions under "4.1" and the test solution preparation method determined under "4.4", with impatiens tetraol saponin K as the reference peak S, calculate the relative retention time of each characteristic peak and the S peak, and calculate the average relative retention time of each characteristic peak to be 0.21 (peak 1), 0.78 (peak 2), 0.89 (peak 3), 1.04 (peak 5), 1.07 (peak 6), 1.14 (peak 7), 1.24 (peak 8), 1.59 (peak 10). The characteristic spectra of 21 batches of Jijizi standard decoction are shown in Figure 7 shown.

[0193] 8.2 Establishment of the common pattern of characteristic spectra of standard decoctions of Jijizi

[0194] Take the reference medicinal material of Jijizi (batch number: 121606-201602, China Food and Drug Inspection Institute), prepare the reference medicinal material reference solution according to the method under "4.3", and prepare the test sample solution of 21 batches of Jijizi standard decoction according to the method under "4.4". The samples were injected and measured according to the chromatographic conditions under "4.1" to obtain the characteristic spectrum of the reference medicinal material of Jijizi ( Figure 8 ); using the "Chinese Herbal Chromatographic Fingerprint Similarity Evaluation Software" to generate a reference spectrum by the average method, and establish a reference characteristic spectrum for the standard decoction of Jijizi ( Figure 9 ).

[0195] Determine that the test sample should present 10 characteristic peaks in the chromatogram, and the retention times should correspond to the 10 characteristic peaks in the chromatogram of the reference medicinal material. Peak 4 should correspond to the retention time of the corresponding reference material peak. The peak corresponding to the peak of the reference material of impatiens tetraol saponin K is the S peak. Calculate the relative retention times of peaks 1 to 3, 5 to 8, and 10 to the S peak. The relative retention times should be within ±10% of the specified values, which are: 0.21 (peak 1), 0.78 (peak 2), 0.89 (peak 3), 1.04 (peak 5), 1.07 (peak 6), 1.14 (peak 7), 1.24 (peak 8), and 1.59 (peak 10).

[0196] High-resolution mass spectrometric identification of 9 characteristic peaks

[0197] 9.1 HPLC-MS Conditions

[0198] Liquid chromatography conditions: A Poroshell 120EC-C18 column (150 mm × 4.6 mm, 2.7 μm) was used; acetonitrile was used as mobile phase A, water was used as mobile phase B, and gradient elution was performed as specified in Table 6; the flow rate was 1.0 ml / min; the column temperature was 35°C; and detection was performed using an evaporative light scattering detector.

[0199] Table 6 Gradient elution table

[0200]

[0201] The mass spectrometry conditions are shown in Table 7:

[0202] Table 7 Mass spectrometry parameters

[0203]

[0204] 9.2 Preparation of test solution

[0205] The same as the preparation of test solution under "4.4".

[0206] 9.3 Determination

[0207] Accurately draw 5 μL of the test solution and inject it into the liquid chromatography-mass spectrometer. The test solution is detected using the above liquid chromatography conditions and mass spectrometry conditions. The total ion current and ultraviolet absorption chromatogram of the test solution are shown in Figure 10 .

[0208] 9.4 Results Analysis

[0209] By mass spectrometry accurate molecular weight, fragment ion comparison analysis, and matching with the Thermo Fisher Scientific mzVault standard database, identification and reference substance confirmation, the six characteristic peaks in the characteristic spectrum of the Jijizi decoction were determined to be: quercetin-3-O-[α-L-rhamnosyl-(1→2)-β-D-glucosyl]-5-O-β-D-glucoside (peak 1), impatiens tetraol saponin B (peak 2), impatiens tetraol saponin K (peak 4), impatiens tetraol saponin G (peak 8), impatiens tetraol saponin A (peak 9) and impatiens tetraol saponin A (peak 10). The compound information is shown in Table 8, and the reference substance confirmation results are shown in Figure 11 .

[0210] Table 8 Mass spectrometry identification results of compounds in the standard decoction of Jijizi decoction pieces

[0211]

[0212] Example 2

[0213] Identification of the standard decoction of Jijizi slices and different adulterants

[0214] Relevant literature reports and a catalog of 130 commonly used counterfeit and mixed products of traditional Chinese medicines mention that Perilla seed, Chrysanthemum chrysanthemum seed, Amomum villosum seed, Radish seed, Astragalus seed, and Plantago seed are similar to Atractylodes lancea in properties and are mixed products of Atractylodes lancea.

[0215] The standard decoctions of Perilla Seed, Chrysanthemum Grosvenori Seed, Amomum Vulgare Seed, Radish Seed, Astragalus Seed and Malus Vulgare Seed were prepared by the method for preparing the standard decoction of Jinjizi. The characteristic spectra of the standard decoctions of Perilla Seed, Chrysanthemum Grosvenori Seed, Amomum Vulgare Seed, Radish Seed, Astragalus Seed and Malus Vulgare Seed were determined by the method for preparing the standard decoction of Jinjizi and the characteristic spectrum detection method. The characteristic spectra were compared with those of the standard decoction of Jinjizi. The results are as follows: Figures 12 to 16 shown.

[0216] The results showed that the characteristic spectra of the standard decoctions of Perilla Seed, Chrysanthemum Grosbeckia Seed, Radish Seed, Astragalus Seed, and Winter Mellow Seed were significantly different from those of the standard decoction of Jinjizi. The characteristic spectra of the standard decoctions of Perilla Seed, Chrysanthemum Grosbeckia Seed, Chrysanthemum Grosbeckia Seed, and Winter Mellow Seed did not contain the 10 characteristic peaks found in the standard decoction of Jinjizi. The characteristic spectra of the standard decoction of Radish Seed only contained characteristic peak 2 with a very low response, and peak 11 was unique to Radish Seed. The characteristic spectra of the standard decoction of Astragalus Seed contained characteristic peaks 1, 2, and 8, but the response was lower than that of Jinjizi. Peaks 11 to 13 were unique to Jinjizi. In summary, the established characteristic spectrum method can effectively distinguish various adulterants of Jinjizi that have lost their medicinal properties.

[0217] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0218] The embodiments described above only express several implementation methods of the present invention, which are convenient for understanding the technical solutions of the present invention in a specific and detailed manner, but they cannot be understood as limiting the scope of protection of the invention patent. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present invention, several variations and improvements can be made, which all fall within the scope of protection of the present invention. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided by the present invention are all within the scope of protection of the claims attached to the present invention. Therefore, the scope of protection of the patent of the present invention shall be based on the content of the attached claims, and the description and drawings can be used to interpret the content of the claims.

Claims

1. A method for establishing a HPLC-ELSD characteristic spectrum of a standard decoction of Jijizi decoction pieces, characterized in that: The steps include: Prepare a reference solution by taking impatiens tetraol saponin K, impatiens tetraol saponin A, impatiens tetraol saponin G, impatiens tetraol saponin L, impatiens tetraol saponin B and quercetin-3-O-[α-L-rhamnosyl-(1→2)-β-D-glucosyl]-5-O-β-D-glucoside reference substances; Prepare the test solution from the standard decoction of Jijizi slices; The steps of preparing the test solution include: mixing the standard decoction of Jijizi slices with a first alcohol-containing aqueous solution with a volume fraction of 50% to 80% for extraction, concentrating, dissolving the residue, filtering, and obtaining a filtrate; the alcohol in the first alcohol-containing aqueous solution is selected from methanol or ethanol; The reference solution and the test solution were respectively detected by high performance liquid chromatography-evaporative light scattering detection; The chromatographic conditions of the high performance liquid chromatography-evaporative light scattering detection method include: using an Agilent Poroshell 120EC-C18 chromatographic column with the following specifications: column length 150 mm, inner diameter 4.6 mm, and filler particle size 2.7 μm; acetonitrile as mobile phase A and water as mobile phase B, with gradient elution; The gradient elution procedure includes: From 0 to 25 minutes, the volume percentage of the mobile phase A increased from 16% to 32%, and the volume percentage of the mobile phase B decreased from 84% to 68%; 25min~30min, the volume percentage of the mobile phase A is 32%, and the volume percentage of the mobile phase B is 68%; The detection conditions of the high performance liquid chromatography-evaporative light scattering detection method include: a gain of 1 to 3; a carrier gas flow rate of 2.5 L / min to 3.5 L / min; and a temperature of 110° C. to 120° C.

2. The method for establishing the HPLC-ELSD characteristic pattern of the standard decoction of the Jijizi decoction pieces according to claim 1, wherein The chromatographic conditions of the high performance liquid chromatography-evaporative light scattering detection method also include one or more of the following technical features: (1) Flow rate: 0.8 mL / min to 1.0 mL / min; (2) The column temperature is 33℃~37℃.

3. The method for establishing the HPLC-ELSD characteristic pattern of the standard decoction of the Jijizi decoction pieces according to claim 1, wherein Methanol is used as a dissolving agent to prepare the reference solution; the concentration of the impatiens tetraol saponin K reference solution is 280 μg / mL to 320 μg / mL; The concentration of the impatiens tetraol saponin A reference solution is 260 μg / mL to 300 μg / mL; The concentration of the impatiens tetraol saponin G reference solution is 270 μg / mL to 310 μg / mL; The concentration of the Impatiens tetraol saponin L reference solution is 290 μg / mL to 330 μg / mL; The concentration of the impatiens tetraol saponin B reference solution is 270 μg / mL to 310 μg / mL; The concentration of the quercetin-3-O-[α-L-rhamnosyl-(1→2)-β-D-glucosyl]-5-O-β-D-glucoside reference solution is 140 μg / mL to 180 μg / mL.

4. The method for establishing the HPLC-ELSD characteristic pattern of the standard decoction of the Jijizi decoction pieces according to claim 1, wherein The extraction method is ultrasonic extraction or heating reflux extraction, and the conditions of ultrasonic extraction include: power of 200W to 300W, frequency of 35kHz to 45kHz, and time of 30min to 60min.

5. The method for establishing the HPLC-ELSD characteristic pattern of the standard decoction of the Jijizi decoction pieces according to claim 4, wherein In the step of preparing the test solution, the amount of ethanol solution corresponding to every 0.1g of the standard decoction of the Jijizi medicinal slices is 15mL to 25mL.

6. The method for establishing the HPLC-ELSD characteristic spectrum of the standard decoction of Jijizi decoction pieces according to any one of claims 1 to 5, characterized in that: The characteristic spectrum presents 10 characteristic peaks, the peak corresponding to the peak of the reference substance of impatiens tetraol saponin K is the S peak, and the relative retention times of peaks 1 to 3, peaks 5 to 8, and peak 10 and the S peak are calculated. The relative retention times are within ±10% of the specified values, and the specified values ​​are: peak 1 is 0.21, peak 2 is 0.78, peak 3 is 0.89, peak 5 is 1.04, peak 6 is 1.07, peak 7 is 1.14, peak 8 is 1.24, and peak 10 is 1.

59.

7. The method for establishing the HPLC-ELSD characteristic spectrum of the standard decoction of Jijizi decoction pieces according to any one of claims 1 to 5, characterized in that: The establishment method further comprises the step of performing high-resolution mass spectrometry identification on the characteristic peaks in the characteristic spectrum by combined mass spectrometry; The mass spectrometry conditions include: HESI ion source parameters include: sheath gas flow rate of 30 arb to 40 arb, auxiliary gas flow rate of 8 arb to 12 arb, spray voltage of 3.5 kV to 4.0 kV, S-lens voltage of 45 V to 55 V, heating temperature of 320°C to 380°C, and capillary temperature of 320°C to 380°C; The mass spectrometry scanning parameters include: scanning mode is positive ion mode and / or negative ion mode, scanning range is 120m / z to 1200m / z, normalized collision energy is 40V, and mass spectrum type is peak shape diagram.

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