Fingerprint spectral construction and detection method of Jiawei Huoxiang Zhengqi Pill

Abstract

This invention relates to a method for constructing a fingerprint spectrum of Jiawei Huoxiang Zhengqi Pills, comprising the following steps: extracting multiple batches of Jiawei Huoxiang Zhengqi Pills samples with extraction solvent A and collecting the extract A to prepare multiple sample solutions; detecting the multiple sample solutions using ultra-high performance liquid chromatography (UHPLC), and importing the obtained multiple sample chromatograms into a traditional Chinese medicine chromatographic fingerprint spectrum similarity evaluation system to formulate a fingerprint spectrum of Jiawei Huoxiang Zhengqi Pills. The fingerprint spectrum of Jiawei Huoxiang Zhengqi Pills constructed in this application includes 49 characteristic peaks, which can more comprehensively reflect the overall chemical composition of Jiawei Huoxiang Zhengqi Pills, improve the accuracy and comprehensiveness of quality control of Jiawei Huoxiang Zhengqi Pills, and thus better evaluate the overall quality of Jiawei Huoxiang Zhengqi Pills.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine detection, and particularly relates to a method for constructing a fingerprint spectrum and a detection method for Huoxiang Zhengqi Pills with Added Flavors. Background Art

[0002] The prescription of Huoxiang Zhengqi Pills with Added Flavors consists of Pogostemonis Herba, Folium Perillae, Radix Angelicae Dahuricae, Atractylodis Macrocephalae Rhizoma Praeparatum, Pericarpium Citri Reticulatae, Pinelliae Rhizoma Praeparatum Cum Zingibere, Magnoliae Officinalis Cortex Praeparatus Cum Zingibere, Poria, Platycodonis Radix, Glycyrrhizae Radix et Rhizoma, and Semen Arecae, and has the effects of dispelling pathogenic wind and dampness, regulating qi and harmonizing the middle, and can be used for treating exogenous wind-cold, internal injury of dampness retention, headache and dizziness, stuffiness and oppression in the chest and diaphragm, abdominal distension and pain, vomiting, and diarrhea. Huoxiang Zhengqi Pills with Added Flavors (concentrated water pills) are included in the fourteenth volume of the Drug Standards of Traditional Chinese Medicine Prepared Formula of the Ministry of Health. The standards only include the routine inspection and microscopic identification of pills. In addition, there are few literatures on the quality research of Huoxiang Zhengqi Pills with Added Flavors, mainly focusing on the thin-layer identification methods of Radix Angelicae Dahuricae, Pericarpium Citri Reticulatae, Pogostemonis Herba, and Atractylodis Macrocephalae Rhizoma in Huoxiang Zhengqi Pills with Added Flavors and the high-performance liquid chromatography content determination of components such as patchouli alcohol, magnolol, honokiol, and glycyrrhizic acid. There is no research report on the overall quality control of Huoxiang Zhengqi Pills with Added Flavors.

[0003] Traditional detection methods related to Huoxiang Zhengqi Pills with Added Flavors are as follows:

[0004] In the invention patent application named "A Quality Control Method for Huoxiang Zhengqi Pills with Added Flavors" applied by Cheng Yanyang in 2009, a thin-layer identification method for Magnoliae Officinalis Cortex, Pogostemonis Herba, and Radix Angelicae Dahuricae and a method for determining the content of hesperidin in Pericarpium Citri Reticulatae by high-performance liquid chromatography are recorded. The applicant of this invention patent application is Guangzhou Baiyunshan Zhongyi Pharmaceutical Co., Ltd., the application number is CN200910193643.1, the publication number is CN101698034A, and the authorized publication number is CN101698034B.

[0005] In the article titled "Determination of the Content of Patchouli Alcohol in Huoxiang Zhengqi Pills with Added Flavors by Gas Chromatography" published by Huo Jiayin in 2010, a GC content determination method for patchouli alcohol in Huoxiang Zhengqi Pills with Added Flavors is recorded.

[0006] In the article titled "Determination of the Contents of Patchouli Alcohol, Magnolol, and Honokiol in Huoxiang Zhengqi Pills with Added Flavors by Gas Chromatography" published by Zhang Xiaolong in 2008, a GC content determination method for patchouli alcohol, magnolol, and honokiol in Huoxiang Zhengqi Pills with Added Flavors is reported.

[0007] In the article titled "Extraction and Content Determination of Magnolol and Honokiol in Huoxiang Zhengqi Pills with Added Flavors" published by Jiang Ning in 2008, a method for rapidly extracting magnolol and honokiol with an ASE300 type accelerated solvent extraction system and determining the contents of magnolol and honokiol by high-performance liquid chromatography is recorded.

[0008] In their 2011 article titled "Determination of the content of baiqiuli alcohol in Jiawei Huoxiang Zhengqi Pills", Song Weizhong et al. described a thin-layer chromatography method for identifying and determining the GC content of baiqiuli alcohol in Jiawei Huoxiang Zhengqi Pills.

[0009] In their 2007 article titled "Research on the Quality Standard of Jiawei Huoxiang Zhengqi Pills," Zhang Xiaobing et al. described a thin-layer chromatography method for identifying Angelica dahurica, Pogostemon cablin, Citrus reticulata peel, and Atractylodes macrocephala in Jiawei Huoxiang Zhengqi Pills, as well as a method for determining the GC content of baicalein.

[0010] In their 2014 article titled "Determination of the Contents of Magnolol and Honokiol in Jiawei Huoxiang Zhengqi Pills by High Performance Liquid Chromatography", Zhao Xia et al. reported a method for determining the contents of magnolol and honokiol in Jiawei Huoxiang Zhengqi Pills using high performance liquid chromatography (HPLC).

[0011] In their 2006 article titled "Determination of Hesperidin Content in Jiawei Huoxiang Zhengqi Pills by High Performance Liquid Chromatography," Liu Yingmin et al. reported a method for determining the hesperidin content in Jiawei Huoxiang Zhengqi Pills using high performance liquid chromatography (HPLC).

[0012] In their 2015 article titled "Study on the Determination of Hesperidin Content in Jiawei Huoxiang Zhengqi Pills by Reversed-Phase High Performance Liquid Chromatography", Qian Fang et al. described a method for determining the hesperidin content in Jiawei Huoxiang Zhengqi Pills using reversed-phase high performance liquid chromatography.

[0013] In their 2007 article titled "Determination of Glycyrrhizic Acid Content in Jiawei Huoxiang Zhengqi Pills by HPLC", Li Li et al. described a method for determining the glycyrrhizic acid content in Jiawei Huoxiang Zhengqi Pills using high performance liquid chromatography.

[0014] Traditional testing methods for Jiawei Huoxiang Zhengqi Pills mainly involve using high-performance liquid chromatography or gas chromatography to determine the content of one or a few components (such as hesperidin, magnolol, honokiol, glycyrrhizic acid, and baicalein) in the pills. These methods cannot provide a comprehensive and holistic quality control and evaluation of the Jiawei Huoxiang Zhengqi Pills product.

[0015] In view of the above, this application is hereby submitted. Summary of the Invention

[0016] The purpose of this application is to provide a method for constructing a fingerprint spectrum of Jiawei Huoxiang Zhengqi Pills. The fingerprint spectrum constructed using this method can more comprehensively reflect the overall chemical composition of Jiawei Huoxiang Zhengqi Pills, improve the accuracy and comprehensiveness of the quality control of Jiawei Huoxiang Zhengqi Pills, and is more conducive to the comprehensive quality control and evaluation of Jiawei Huoxiang Zhengqi Pills.

[0017] In a first aspect of this application, a method for constructing the fingerprint spectrum of Jiawei Huoxiang Zhengqi Pills is provided, the method comprising the following steps:

[0018] Multiple batches of Jiawei Huoxiang Zhengqi Pills were extracted using extraction solvent A, and extract A was collected to prepare multiple test solutions accordingly.

[0019] Ultra-high performance liquid chromatography was used to detect multiple test sample solutions, and the obtained chromatograms of multiple test samples were imported into the traditional Chinese medicine chromatographic fingerprint identification evaluation system to formulate the fingerprint spectrum of Jiawei Huoxiang Zhengqi Pill.

[0020] The conditions for the ultra-high performance liquid chromatography method include:

[0021] The stationary phase is a C18 column;

[0022] The mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution of formic acid with a volume percentage of 0.1%-0.4%, and mobile phase B is acetonitrile;

[0023] A gradient elution method is used, and the gradient elution procedure includes:

[0024] From 0 min to 30 min, the volume percentage of the mobile phase B increased from 5% to 35%.

[0025] Between 30 and 52 minutes, the volume percentage of the mobile phase B increased from 35% to 46%.

[0026] Between 52 and 85 minutes, the volume percentage of mobile phase B increased from 46% to 100%.

[0027] In some of these embodiments, the ultra-high performance liquid chromatography method further satisfies one or more of the following conditions:

[0028] (1) Multi-wavelength ultraviolet detection is adopted; optionally, the detection wavelength of multi-wavelength ultraviolet detection is 254nm-281nm.

[0029] (2) The column temperature is 25℃-45℃; optionally, the column temperature is 35℃-45℃; and,

[0030] (3) The specifications of the C18 chromatographic column are: column length of 80mm-250mm, inner diameter of 1.5mm-3mm, and packing particle size of 1.5μm-2.5μm; optionally, the specifications of the C18 chromatographic column are: column length of 100mm-200mm, inner diameter of 1.8mm-2.4mm, and packing particle size of 1.8μm-2.4μm.

[0031] In some of these embodiments, the ultra-high performance liquid chromatography method further satisfies one or more of the following conditions:

[0032] (A) Flow rate is 0.1 mL / min - 0.6 mL / min;

[0033] (B) The injection volume is 1 μL–6 μL; optionally, the injection volume is 3.5 μL–4.5 μL; and,

[0034] (C) The sampling frequency is 4Hz-10Hz; optionally, the sampling frequency is 4.5Hz-5.5Hz.

[0035] In some embodiments, the preparation step of the test solution satisfies one or more of the following conditions:

[0036] (a) The extraction solvent A comprises an aqueous methanol solution with a methanol volume percentage of 10%-95%; optionally, the extraction solvent A comprises an aqueous methanol solution with a methanol volume percentage of 40%-95%.

[0037] (b) The extraction method includes ultrasonic extraction; optionally, the ultrasonic extraction power is 125W-350W, the frequency is 35kHz-45kHz, and the extraction time is 10min-90min; further optionally, the ultrasonic extraction power is 130W-140W, the ultrasonic extraction frequency is 40kHz-45kHz, and the ultrasonic extraction time is 15min-90min; and,

[0038] (c) Extract A can be collected by centrifugation or filtration; optionally, the centrifugation speed is 12,000 rpm to 18,000 rpm and the centrifugation time is 8 min to 15 min.

[0039] In some embodiments, the construction method further includes:

[0040] A reference solution is provided, and the reference solution is detected by the ultra-high performance liquid chromatography method. The common chromatographic peaks on the fingerprint spectrum are identified by referring to the obtained reference chromatogram.

[0041] The reference solution contains one or more of the following: hesperidin, glycyrrhizin, glycyrrhizin, isoglycyrrhizin, norihesperidin, glycyrrhizic acid, citrulline, imperatorin, glycyrrhizin chalcone A, isoimperatorin, magnolol, honokiol, and glycyrrhetinic acid.

[0042] In some of these embodiments, the solvent for the reference solution comprises methanol.

[0043] In some embodiments, the fingerprint spectrum includes 49 chromatographic peaks, wherein peak 21 is hesperidin, peak 23 is glycyrrhizin, peak 24 is glycyrrhizin, peak 30 is isoglycyrrhizin, peak 33 is norihesperidin, peak 34 is glycyrrhizic acid, peak 38 is citrulline, peak 41 is imperatorin, peak 42 is glycyrrhizin chalcone A, peak 44 is isoimperatorin, peak 45 is magnolol, peak 47 is magnolol, and peak 49 is glycyrrhetinic acid.

[0044] In a second aspect of this application, a method for detecting Jiawei Huoxiang Zhengqi Pills is provided, the method comprising the following steps:

[0045] Extract the sample to be tested using extraction solvent B and collect the extract B to prepare the sample solution to be tested;

[0046] The sample solution to be tested is detected by ultra-high performance liquid chromatography, and the obtained sample spectrum is compared with the fingerprint spectrum constructed by the construction method described in the first aspect. The mass of the sample to be tested is determined based on the comparison result.

[0047] The ultra-high performance liquid chromatography method is as defined in the first aspect.

[0048] In some embodiments, the extraction solvent B comprises an aqueous methanol solution with a methanol volume percentage of 10%-95%; alternatively, the extraction solvent B comprises an aqueous methanol solution with a methanol volume percentage of 40%-95%.

[0049] In some embodiments, the preparation steps of the test sample solution satisfy one or more of the following conditions:

[0050] (I) The extraction method includes ultrasonic extraction; optionally, the ultrasonic extraction power is 125W-350W, the frequency is 35kHz-45kHz, and the extraction time is 10min-90min; further optionally, the ultrasonic extraction power is 130W-140W, the ultrasonic extraction frequency is 40kHz-45kHz, and the ultrasonic extraction time is 15min-90min; and,

[0051] (II) Extraction B can be collected by centrifugation or filtration; optionally, the centrifugation speed is 12000rpm-18000rpm and the centrifugation time is 8min-15min.

[0052] Compared with traditional technologies, this application has the following advantages:

[0053] This application utilizes ultra-high performance liquid chromatography (UHPLC) to construct a fingerprint spectrum of Jiawei Huoxiang Zhengqi Pills under appropriate UHPLC conditions. This fingerprint spectrum includes 49 characteristic peaks (peak 21 is hesperidin, peak 23 is glycyrrhizin, peak 24 is glycyrrhizin, peak 30 is isoglycyrrhizin, peak 33 is norihesperidin, peak 34 is glycyrrhizic acid, peak 38 is citrulline, peak 41 is imperatorin, peak 42 is glycyrrhizin chalcone A, peak 44 is isoimperatorin, peak 45 is magnolol, peak 47 is magnolol, and peak 49 is glycyrrhetinic acid). This fingerprint spectrum more comprehensively reflects the overall chemical composition of Jiawei Huoxiang Zhengqi Pills, improving the accuracy and comprehensiveness of quality control and enabling better evaluation of the overall quality of Jiawei Huoxiang Zhengqi Pills. Attached Figure Description

[0054] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0055] Figure 1 The UPLC spectra of 15 batches of Jiawei Huoxiang Zhengqi Pills samples from Example 1 are shown below.

[0056] Figure 2 The standard fingerprint spectrum of the modified Huoxiang Zhengqi Pill in Example 1;

[0057] Figure 3 Chromatograms of samples of Huoxiang Zhengqi Pills extracted with different solvents in Example 2;

[0058] Figure 4 Chromatograms of Huoxiang Zhengqi Pills extracted at different times in Example 2;

[0059] Figure 5 The chromatograms of the Jiawei Huoxiang Zhengqi Pill samples at different detection wavelengths in Example 2 are shown.

[0060] Figure 6 The following are separation chromatograms of different mobile phase systems in Example 2;

[0061] Figure 7 The following are chromatograms showing the separation of different mobile phase additives in Example 2;

[0062] Figure 8 Chromatograms of separation using different chromatographic columns in Example 2;

[0063] Figure 9 The following are separation chromatograms at different column temperatures in Example 2;

[0064] Figure 10 This is the chromatogram of the gradient elution sample from Example 2;

[0065] Figure 11 This is the mass spectrometry spectrum of the flavored Huoxiang Zhengqi Pill sample from Example 3. Detailed Implementation

[0066] The present invention will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the invention. The purpose of providing these embodiments and examples is to enable a more thorough and complete understanding of the disclosure of the present invention. It should also be understood that the present invention can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present invention, and the equivalent forms obtained also fall within the protection scope of this application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present invention. It should be understood that the present invention can be implemented without one or more of these details.

[0067] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for descriptive purposes only and is not intended to be limiting of the invention.

[0068] the term

[0069] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0070] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0071] In this invention, terms such as "multiple", "various", "multiple times", and "multi-source" are used, and unless otherwise specified, they refer to a quantity greater than or equal to 2. For example, "one or more" means one or more types.

[0072] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0073] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this invention, solve the technical problem of this invention, and achieve the expected technical effect of this invention.

[0074] In this article, terms such as "preferred," "better," "more suitable," and "ideal" are merely used to describe implementation methods or examples that achieve better results, and should be understood not to limit the scope of protection of this invention.

[0075] In this invention, terms such as "further," "even more," and "particularly" are used for descriptive purposes and to indicate differences in content, but should not be construed as limiting the scope of protection of this invention.

[0076] In this invention, "optionally," "optionally," and "optional" mean that they are optional, that is, they are selected from either "with" or "without." If multiple "options" appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "option" is independent.

[0077] In this invention, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on the quantity.

[0078] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0079] In this invention, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0080] Unless otherwise specified, the temperature parameters in this invention can be either constant temperature treatment or variations within a certain temperature range. It should be understood that the constant temperature treatment allows temperature fluctuations within the precision range controlled by the instrument. Fluctuations are permitted within ranges such as ±5℃, ±4℃, ±3℃, ±2℃, and ±1℃.

[0081] In this invention, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0082] All references to this invention are incorporated herein by reference as if each document were individually incorporated by reference. Unless they conflict with the inventive purpose and / or technical solution of this application, the referenced documents involved in this invention are incorporated in their entirety and for all purposes. When references are made in this invention, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When references are made in this invention, examples and preferred embodiments of the relevant technical features cited may also be incorporated herein by reference, but only to the extent that they enable the implementation of this invention. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptively based on the description in this application.

[0083] HPLC: High Performance Liquid Chromatography.

[0084] UPLC: Ultra Performance Liquid Chromatography.

[0085] RSD: Relative Standard Deviation.

[0086] CAD detector: Electrospray detector.

[0087] VWD detector: Ultraviolet detector.

[0088] With the development and widespread application of traditional Chinese medicine (TCM) fingerprinting technology, based on the fact that TCM fingerprinting can systematically and comprehensively reflect the common characteristic peaks contained in TCM materials or prepared TCM products, it has become an important means of controlling the quality of TCM materials and prepared TCM products, and has been widely used in the overall quality control of various TCM materials and prepared TCM products. However, no fingerprinting method for Jiawei Huoxiang Zhengqi Wan (a TCM formula) has been reported to date.

[0089] The first aspect of this application

[0090] This application provides a method for constructing the fingerprint spectrum of Jiawei Huoxiang Zhengqi Pill, the method comprising the following steps:

[0091] Multiple batches of Jiawei Huoxiang Zhengqi Pills were extracted using extraction solvent A, and extract A was collected to prepare multiple test solutions accordingly.

[0092] Ultra-high performance liquid chromatography was used to detect multiple test sample solutions, and the obtained chromatograms of multiple test samples were imported into the traditional Chinese medicine chromatographic fingerprint identification evaluation system to formulate the fingerprint spectrum of Jiawei Huoxiang Zhengqi Pill.

[0093] The conditions for the ultra-high performance liquid chromatography method include:

[0094] The stationary phase is a C18 column;

[0095] The mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution of formic acid with a volume percentage of 0.1%-0.4%, and mobile phase B is acetonitrile;

[0096] A gradient elution method is used, and the gradient elution procedure includes:

[0097] From 0 min to 30 min, the volume percentage of the mobile phase B increased from 5% to 35%.

[0098] Between 30 and 52 minutes, the volume percentage of the mobile phase B increased from 35% to 46%.

[0099] Between 52 and 85 minutes, the volume percentage of mobile phase B increased from 46% to 100%.

[0100] Optionally, the ultra-high performance liquid chromatography method further satisfies one or more of the following conditions:

[0101] (1) Multi-wavelength ultraviolet detection is adopted; optionally, the detection wavelength of multi-wavelength ultraviolet detection is 254nm-281nm (e.g., 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 281nm);

[0102] (2) The column temperature is 25℃-45℃ (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45℃); optionally, the column temperature is 25℃-45℃;

[0103] and,

[0104] (3) The specifications of the C18 chromatographic column are as follows: column length 80mm-250mm (e.g., 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250mm), inner diameter 1.5mm-3mm (e.g., 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9mm). The C18 column has a particle size of 1.5 μm-2.5 μm (e.g., 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5 μm). Optionally, the C18 column has the following specifications: column length of 100 mm-200 mm, inner diameter of 1.8 mm-2.4 mm, and particle size of 1.8 μm-2.4 μm; for example, a 150 mm × 2.1 mm column with a particle size of 1.9 μm, which can be ThermoFisher Hypersil gold C18 (150 mm × 2.1 mm, 1.9 μm).

[0105] Optionally, the ultra-high performance liquid chromatography method further satisfies one or more of the following conditions:

[0106] (A) Flow rate is 0.1 mL / min to 0.6 mL / min (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 mL / min);

[0107] (B) The injection volume is 1 μL–6 μL (e.g., 1, 1.5, 2, 2.5, 3, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 5, 5.5, 6 μL); optionally, the injection volume is 3.5 μL–4.5 μL; and,

[0108] (C) The sampling frequency is 4Hz-10Hz (e.g., 4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10Hz); optionally, the frequency is 4.5Hz-5.5Hz.

[0109] In some embodiments, the preparation step of the test solution satisfies one or more of the following conditions:

[0110] (a) The extraction solvent A comprises an aqueous methanol solution with a volume percentage of 10%-95% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%).

[0111] (b) The extraction method includes ultrasonic extraction; optionally, the ultrasonic extraction power is 125W-350W (e.g., 120, 125, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350W), frequency... The extraction frequency is 35kHz-45kHz (e.g., 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45kHz), and the extraction time is 10min-90min (e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90min); further optionally, the ultrasonic extraction power is 130W-140W, the ultrasonic extraction frequency is 40kHz-45kHz, and the ultrasonic extraction time is 15min-90min; and,

[0112] (c) The extraction liquid A can be collected by centrifugation or filtration; optionally, the centrifugation speed is 12,000 rpm to 18,000 rpm (e.g., 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000 rpm), and the centrifugation time is 8 min to 15 min (e.g., 8, 9, 10, 11, 12, 13, 14, 15 min).

[0113] Optionally, the construction method further includes:

[0114] A reference solution is provided, and the reference solution is detected by the ultra-high performance liquid chromatography method. The common chromatographic peaks on the fingerprint spectrum are identified by referring to the obtained reference chromatogram.

[0115] The reference solution contains one or more of the following: hesperidin, glycyrrhizin, glycyrrhizin, isoglycyrrhizin, norihesperidin, glycyrrhizic acid, citrulline, imperatorin, glycyrrhizin chalcone A, isoimperatorin, magnolol, honokiol, and glycyrrhetinic acid.

[0116] Optionally, the solvent of the reference solution includes methanol.

[0117] Optionally, the fingerprint spectrum includes 49 chromatographic peaks, wherein peak 21 is hesperidin, peak 23 is glycyrrhizin, peak 24 is glycyrrhizin, peak 30 is isoglycyrrhizin, peak 33 is norihesperidin, peak 34 is glycyrrhizic acid, peak 38 is citrulline, peak 41 is imperatorin, peak 42 is glycyrrhizin chalcone A, peak 44 is isoimperatorin, peak 45 is magnolol, peak 47 is magnolol, and peak 49 is glycyrrhetinic acid.

[0118] Second aspect of this application

[0119] This application provides a method for detecting modified Huoxiang Zhengqi Pills, the method comprising the following steps:

[0120] Extract the sample to be tested using extraction solvent B and collect the extract B to prepare the sample solution to be tested;

[0121] The sample solution to be tested is detected by ultra-high performance liquid chromatography, and the obtained sample spectrum is compared with the fingerprint spectrum constructed by the construction method described in the first aspect. The mass of the sample to be tested is determined based on the comparison result.

[0122] The ultra-high performance liquid chromatography method is as defined in the first aspect.

[0123] Optionally, the extraction solvent B comprises an aqueous methanol solution with a methanol volume percentage of 10%-95% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%); or, alternatively, the extraction solvent B comprises an aqueous methanol solution with a methanol volume percentage of 40%-95%.

[0124] Optionally, the preparation steps of the sample solution to be tested satisfy one or more of the following conditions:

[0125] (I) The extraction method includes ultrasonic extraction; optionally, the ultrasonic extraction power is 125W-350W (e.g., 125, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350W), and the frequency is 35kHz-45kHz (e.g., The extraction frequencies are 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, and 45 kHz, with extraction times ranging from 10 to 90 minutes (e.g., 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, and 90 minutes). Further optionally, the ultrasonic extraction power is 130 W-140 W, the ultrasonic extraction frequency is 40 kHz-45 kHz, and the ultrasonic extraction time is 10 to 90 minutes.

[0126] (II) The extraction liquid B can be collected by centrifugation or filtration; optionally, the centrifugation speed is 12,000 rpm to 18,000 rpm (e.g., 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000 rpm), and the centrifugation time is 8 min to 15 min (e.g., 8, 9, 10, 11, 12, 13, 14, 15 min).

[0127] The embodiments of the present invention will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this invention, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0128] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0129] Example 1

[0130] This embodiment provides a method for constructing the fingerprint spectrum of Jiawei Huoxiang Zhengqi Pill, including the following steps:

[0131] 1. Experimental instruments and reagents

[0132] 1.1 Instruments: Ultimate 3000 Rapid Separation Liquid Chromatography System (Dionex Corporation, USA), including: HPG-3400RS binary high-pressure gradient pump, WPS-3000TRS autosampler, TCC-3000RS column oven, RS Variable wavelength UV detector, and Chameleon chromatography management software (SR7.2); Milli-Q ultrapure water treatment system (Millipore Corporation, USA); high-speed cryogenic centrifuge (Thermo Fisher Scientific, USA); LTQ-Orbitrap XL combined high-resolution mass spectrometer.

[0133] 1.2 Reagents: Acetonitrile, methanol, and formic acid were all chromatographically pure reagents. The ultrapure water (18.2 Mohm·cm) used in the experiment was prepared by the Milli-Q ultrapure water system.

[0134] Standard substances: hesperidin, neohesperidin, limonene, hesperidin, geraniol, tartrazine, norihesperidin, citrulline, magnolol, honokiol, platycodon D, deapigenin platycodon D, deapigenin platycodon D2, deapigenin platycodon D3, platycodon D2, platycodon D3, glycyrrhizic acid, glycyrrhetinic acid, glycyrrhizin, isoglycyrrhizin, glycyrrhizin, glycyrrhizin chalcone B, isoglycyrrhizin, spicated glycyrrhizin chalcone, glycyrrhizin chalcone A, glycyrrhizin, imperatorin, and isoimperatorin were all purchased from Chengdu Manster Biotechnology Co., Ltd. and all had a purity ≥95%. All standard substances were confirmed by ultraviolet absorption spectroscopy, high-resolution mass spectrometry, and multi-stage fragment data before use.

[0135] Experimental Samples: All batches of the finished product samples of Jiawei Huoxiang Zhengqi Pills in the experiment were provided by Guangzhou Baiyunshan Zhongyi Pharmaceutical Co., Ltd., as detailed in Table 1.

[0136] Table 1. Sample Information of Huoxiang Zhengqi Pills

[0137]

[0138]

[0139] 2. Methods

[0140] 2.1 Chromatographic conditions

[0141] The chromatographic column was a ThermoFisher Hypersil gold C18 (150 mm × 2.1 mm, 1.9 μm).

[0142] Mobile phase A was 0.1% formic acid in water, and mobile phase B was acetonitrile. Gradient elution was used: 0–30 min, 5%–35% B; 30–52 min, 35%–46% B; 52–85 min, 46%–100% B. Flow rate: 0.4 mL / min; column temperature: 35℃; injection volume: 4 μL. Multi-wavelength ultraviolet detector (VWD) detection conditions: detection wavelength: 254 nm; sampling frequency: 5 Hz.

[0143] Ionization method: electrospray ionization; spray voltage: 4.5kV; ion transmission tube temperature: 380℃; maximum ion implantation time: 300ms for single-stage full scan, 150ms for multi-stage scan.

[0144] Scanning method: positive ion scan; scan range: m / z 100~1500; scan resolution: 30,000; secondary data-dependent scans were performed on the two strongest ions with the strongest response in the full scan; fragmentation method: collision-induced fragmentation (CID), energy 35%.

[0145] 2.2 Preparation of test solution

[0146] Accurately weigh approximately 2g of Jiawei Huoxiang Zhengqi Pill powder, place it in a stoppered conical flask, accurately add 20mL of 70% methanol, seal tightly, weigh, and sonicate (BRANSON 5510; power / frequency, 135W / 42kHz) for 20min. Cool, weigh, and make up the weight loss with 70% methanol. Shake well, let stand, and centrifuge the supernatant at 14000rpm for 10min. Place the supernatant in a liquid chromatography injection bottle to obtain the final product.

[0147] 2.3 Preparation of reference solution

[0148] Accurately weigh appropriate amounts of the standard substances, dissolve them in chromatographic methanol, and dilute to 2 mL to prepare standard stock solutions. Store them in a refrigerator at 4°C for later use.

[0149] 2.4 Data Processing

[0150] In the experiment, all chromatograms were processed and exported using Chameleon Chromatography Management Software (SR7.2); the exported sample chromatograms were analyzed using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System V2.0" (National Pharmacopoeia Commission) to calculate sample similarity.

[0151] 3. Results and Discussion

[0152] 3.1 Methodological Examination

[0153] 3.1.1 Precision

[0154] The test solution was prepared according to the preparation method in section "2.2", and injected five times consecutively for UPLC analysis. The results showed that the RSD values ​​of the relative peak area and relative retention time of each common peak were less than 3.21% and 0.29%, respectively, indicating that the instrument precision was good and met the requirements of fingerprint spectrum.

[0155] 3.1.2 Stability

[0156] The test solution was prepared according to the preparation method in section "2.2" and UPLC analysis was performed at 0, 3, 6, 9, 12 and 24 h. The results showed that the RSD values ​​of the relative peak area and relative retention time of each common peak were less than 2.35% and 0.04%, respectively, indicating that the sample solution had good stability within 24 h.

[0157] 3.1.3 Repeatability

[0158] Five samples of the same batch of Jiawei Huoxiang Zhengqi Pills were taken and prepared into test solutions according to the preparation method in section "2.2". UPLC analysis was performed, and the results showed that the RSD values ​​of the relative peak area and relative retention time of each common peak were less than 4.89% and 0.28%, respectively, indicating that the method has good repeatability.

[0159] 3.2 Establishment of Standard Fingerprint Pattern

[0160] 3.2.1 Fingerprint Spectrum Detection

[0161] Take 15 batches of Jiawei Huoxiang Zhengqi Pills samples, prepare sample test solutions according to the method in section "2.2", perform UPLC-UV analysis according to the chromatographic conditions in section "2.1", and record the chromatogram for 90 min.

[0162] 3.2.2 Identification of common peaks by comparing the spectrum

[0163] The HPLC fingerprints of 15 batches of samples were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2004A". Peak 34 was used as the reference peak, and the median method was used for automatic peak matching. Figure 1 ), generate comparative fingerprint profiles, and establish common patterns ( Figure 2 Chromatographic peaks with good stability, strong absorption, and distinct characteristics were selected as common peaks, and 49 common peaks were determined based on the determination results of 15 batches of samples. Based on the selected glycyrrhizic acid reference peak (peak 34), its retention time and peak area were both set to 1.0, and the relative retention time and relative peak area of ​​each common peak were calculated and listed in Tables 2 and 3, respectively.

[0164] Table 2. Relative retention times of common peaks in fingerprint chromatograms of 15 batches of Jiawei Huoxiang Zhengqi Pills

[0165]

[0166]

[0167] Table 3. Relative peak area of ​​total peaks in fingerprint chromatograms of 15 batches of Jiawei Huoxiang Zhengqi Pills

[0168]

[0169] 3.2.3 Similarity Evaluation

[0170] The HPLC fingerprint chromatograms of 15 batches of samples were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2004A" to generate a reference fingerprint chromatogram of Jiawei Huoxiang Zhengqi Pill. In the analytical testing mode, the similarity of the fingerprint chromatograms of the 15 batches of samples was calculated. The results showed that the similarity between the 15 batches of samples and the reference fingerprint chromatogram was very high, all greater than >0.905 (Table 4).

[0171] Table 4. Fingerprint Similarity

[0172]

[0173] 3.2.4 Common Peak Attribution Analysis

[0174] The chromatograms of each reference standard and the whole formula sample were determined under the same chromatographic conditions. After comparison with the chromatograms of the reference standard solutions and confirmation by mass spectrometry, it was found that among the characteristic peaks of the fingerprint chromatogram, peak 21 was hesperidin, peak 23 was glycyrrhizin, peak 24 was glycyrrhizin, peak 30 was isoglycyrrhizin, peak 33 was norihesperidin, peak 34 was glycyrrhizic acid, peak 38 was citrulline, peak 41 was imperatorin, peak 42 was glycyrrhizin chalcone A, peak 44 was isoimperatorin, peak 45 was magnolol, peak 47 was magnolol, and peak 49 was glycyrrhetinic acid.

[0175] Example 2

[0176] This embodiment relates to the conditional investigation of the fingerprint spectrum construction method of Jiawei Huoxiang Zhengqi Pill.

[0177] 1. Optimization of extraction process

[0178] 1.1 Selection of extraction solvent

[0179] This experiment investigated the extraction solvent systems of the fingerprint spectrum test sample solution of Jiawei Huoxiang Zhengqi Pill. The ultrasonic extraction efficiencies of five solvent systems (water, 10%, 40%, 70%, and 100% methanol) were compared. The results are shown in [Figure number missing]. Figure 3When water and 10% methanol were used as extraction solvents, the chromatogram showed abundant peaks within 40 minutes, but almost no peaks appeared after 40 minutes, indicating that there were few weakly polar components in the sample. As the concentration of methanol in the extraction solvent increased, the number of detected peaks gradually increased, especially the peaks of weakly polar components after 40 minutes, and the peak intensities also gradually increased, indicating improved extraction efficiency. However, when 100% methanol was used as the extraction solvent, the intensity of the main peaks was significantly weakened, and the peaks of some weakly polar compounds even disappeared. Therefore, 70% methanol, which had the highest extraction efficiency, was ultimately selected as the extraction solvent for the fingerprint chromatogram sample of Jiawei Huoxiang Zhengqi Pill.

[0180] 1.2 Selection of extraction time

[0181] After selecting 70% methanol as the extraction solvent, in order to obtain higher extraction efficiency, the experiment further investigated the ultrasonic extraction time of the sample solution of Jiawei Huoxiang Zhengqi Pill fingerprint spectrum, including 10 min, 20 min, 30 min, 60 min, and 90 min. The results are as follows: Figure 4 As shown, the response intensity of each chromatographic peak gradually increases with the increase of extraction time. When the extraction time is 20 min, the chromatographic peak intensity reaches the highest. Moreover, after the extraction time is greater than 20 min, the chromatographic peak intensity does not increase significantly. Therefore, based on the principle of high extraction efficiency and saving time, 20 min is selected as the ultrasonic extraction time for the fingerprint chromatogram sample of Jiawei Huoxiang Zhengqi Pill.

[0182] 2. Optimization of chromatographic conditions

[0183] UPLC is used to separate and detect the test solution. The separation conditions are optimized, including detection wavelength, column type, column temperature, mobile phase composition, and mobile phase additives, so that the analyzed sample can obtain as many baseline separated chromatographic peaks as possible in the shortest time.

[0184] 2.1 Selection of detection wavelength

[0185] Jiawei Huoxiang Zhengqi Pills are composed of 13 traditional Chinese medicines, with a large number, properties, and types of chemical components. Therefore, the fingerprint chromatogram established in this experiment should obtain as much information as possible about these chemical components. Thus, this experiment selected a VWD ultraviolet detector (with multi-wavelength detection) to compare the chromatograms at commonly used ultraviolet detection wavelengths (254 nm and 281 nm). The results are as follows: Figure 5As shown, the sample components exhibit strong signal responses and high sensitivity at different wavelengths using the VWD (Vacuum-Driven Diode) detector. Therefore, the VWD detector was used for fingerprint chromatographic studies of Jiawei Huoxiang Zhengqi Pills. At a detection wavelength of 281 nm, the polar components showed high signal response intensity, while the moderately polar components showed low signal intensity and fewer visible peaks. After changing the detection wavelength to 254 nm, although the intensity of some chromatographic peaks of polar components decreased to some extent, the number of chromatographic peaks of moderately polar components increased significantly, and the signal response intensity was high. The UV absorption intensity range of all components showed strong consistency. Therefore, this experiment selected 254 nm as the UV detection wavelength.

[0186] 2.2 Selection of Mobile Phase

[0187] The experiment first investigated methanol-water and acetonitrile-water as mobile phases. Results showed that using acetonitrile-water as the mobile phase for gradient elution resulted in a stable baseline, more chromatographic peaks, better peak shapes, and higher sensitivity. Therefore, the acetonitrile-water system was chosen for this study. Figure 6 Meanwhile, to further improve peak shape, enhance chromatographic peak resolution, and improve detector signal response, different concentrations of formic acid as a mobile phase additive were investigated, including acetonitrile-0.1% formic acid solution, acetonitrile-0.2% formic acid solution, and acetonitrile-0.4% formic acid solution. Figure 7 It can be seen that there is no significant difference in the chromatogram under different additive concentrations. However, in the acetonitrile-0.1% formic acid water mobile phase system, the chromatographic peak signal response is enhanced within 50-70 min. Considering the column's tolerance to acidic mobile phase and the reproducibility of the experiment, this experiment uses acetonitrile-0.1% formic acid water as the mobile phase for gradient elution analysis.

[0188] 2.3 Selection of Chromatographic Column

[0189] This experiment investigated three different types of chromatographic columns under the same chromatographic conditions: Thermo Fisher Hypersil gold C18 (150 mm × 2.1 mm, 1.9 μm), Waters Xselect C18 (150 mm × 3 mm, 2.5 μm), and Waters BEH C18 (100 mm × 2.1 mm, 1.7 μm). The results are as follows: Figure 8 As shown, the Hypersil Gold C18 column exhibits stronger selectivity, better resolution and peak shape, and a moderate separation time. Therefore, the Hypersil Gold C18 column was chosen for subsequent fingerprint studies.

[0190] 2.4 Column Temperature Selection

[0191] The temperature of the chromatographic column has a significant impact on the separation of compounds with similar structures. This experiment investigated the effects of different column temperatures (35℃, 40℃, and 45℃) on sample separation. Figure 9 It can be seen that as the column temperature increases, the sample separation time gradually shortens and the separation efficiency improves, but the resolution between two adjacent chromatographic peaks decreases within 45-55 minutes. To ensure peak resolution and column life, a column temperature of 35℃ was selected for this experiment.

[0192] 2.5 Selection of Gradient Procedure

[0193] Jiawei Huoxiang Zhengqi Pills are composed of 13 traditional Chinese medicines, containing a large number and variety of chemical components. Furthermore, fingerprinting requires the separation and characterization of these chemical components as comprehensively as possible. Therefore, this study used UPLC-UV technology with a 1.9 μm particle size ultra-high performance liquid chromatography column for chromatographic analysis of the sample. Simultaneously, due to the varying properties of the chemical components, we optimized the gradient elution program to separate the maximum number of chromatographic peaks in the shortest possible time. Finally, the sample analysis was completed within 85 minutes. Figure 10 The preferred gradient procedure is shown in 2.1.

[0194] Example 3

[0195] This embodiment relates to a mass spectrometry method for identifying the chemical components of Jiawei Huoxiang Zhengqi Pills, as detailed below:

[0196] The fingerprint chromatographic method established was tandem with LTQ-Orbitrap combined high-resolution mass spectrometry to detect the effects of Jiawei Huoxiang Zhengqi Pills. Figure 11 Based on the obtained precise mass numbers and multi-level fragment information, and after comparison with relevant literature and compound standard materials, a total of 109 compounds were identified, as detailed in Table 5.

[0197] Table 5. Mass Spectrometry Identification Results of Compounds

[0198]

[0199]

[0200]

[0201]

[0202]

[0203] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples 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.

[0204] The above-described embodiments are merely illustrative of several implementation methods of the present invention, facilitating a detailed and specific understanding of the technical solutions of the present invention. However, they should not be construed as limiting the scope of protection of the invention patent. 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 all fall within the scope of protection of the present invention. Furthermore, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various alterations or modifications to the present invention, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided by the present invention through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this invention patent should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for constructing a fingerprint spectrum of Jiawei Huoxiang Zhengqi Pill, characterized in that, The construction method comprises the following steps: extracting multiple batches of flavored Huoxiang Zhengqi Pills test samples with extraction solvent A respectively and collecting the extraction liquid A to prepare multiple test sample solutions; detecting the multiple test sample solutions by using an ultra-high performance liquid chromatography method, and importing the obtained multiple test sample chromatograms into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to formulate a fingerprint of the flavored Huoxiang Zhengqi Pills; The conditions of the ultra-high performance liquid chromatography method comprise: The mobile phase comprises mobile phase A and mobile phase B, the mobile phase A is a formic acid aqueous solution with a formic acid volume percentage of 0.1%-0.4%, and the mobile phase B is acetonitrile; The gradient elution program comprises: 0min-30min, the volume percentage of the mobile phase B is increased from 5% to 35%; 30min-52min, the volume percentage of the mobile phase B is increased from 35% to 46%; 52min-85min, the volume percentage of the mobile phase B is increased from 46% to 100%; The chromatographic column is ThermoFisher Hypersil gold C18, 150 mm×2.1mm, 1.9μm; The fingerprint comprises 49 chromatographic peaks, wherein, peak No. 21 is hesperidin, peak No. 23 is glycyrrhizin, peak No. 24 is glycyrrhizin, peak No. 30 is isoglycyrrhizin, peak No. 33 is citropten, peak No. 34 is glycyrrhizic acid, peak No. 38 is aurantiamarin, peak No. 41 is imperatorin, peak No. 42 is glychalcone A, peak No. 44 is isoeuropin, peak No. 45 is honokiol, peak No. 47 is magnolol, and peak No. 49 is glycyrrhetic acid; The detection wavelength is 254nm-281nm; The extraction solvent A comprises a methanol aqueous solution with a methanol volume percentage of 40%-95%.

2. The method according to claim 1, characterized in that, The ultra-high performance liquid chromatography method also satisfies the following conditions: the column temperature is 25℃-45℃.

3. The method according to claim 2, characterized in that, The column temperature is 35℃-45℃.

4. The method according to claim 2, characterized in that, The ultra-high performance liquid chromatography method also satisfies one or more of the following conditions: (A) the flow rate is 0.1mL / min-0.6mL / min; (B) the injection volume is 1μL-6μL; and, (C) the sampling frequency is 4Hz-10Hz.

5. The method according to claim 4, characterized in that, The injection volume is 3.5μL-4.5μL.

6. The method according to claim 4, characterized in that, The sampling frequency is 4.5Hz-5.5Hz.

7. The method according to any one of claims 1 to 6, characterized in that, The preparation steps of the test sample solution satisfy one or more of the following conditions: (a) the extraction method comprises ultrasonic extraction; and, (b) the collection of the extraction liquid A comprises centrifugation or filtration.

8. The method according to claim 7, characterized in that, The ultrasonic extraction power is 125W-350W, the frequency is 35kHz-45kHz, and the extraction time is 10min-90min.

9. The method according to claim 8, characterized in that, The ultrasonic extraction power is 130W-140W, the ultrasonic extraction frequency is 40kHz-45kHz, and the ultrasonic extraction time is 15min-90min.

10. The method according to claim 7, characterized in that, The centrifugation speed is 12000rpm-18000rpm, and the centrifugation time is 8min-15min.

11. The method according to any one of claims 1 to 6, characterized in that, The construction method further comprises: A control solution is provided, the control solution is detected by the ultra-high performance liquid chromatography, and the common chromatographic peaks on the fingerprint spectrum are calibrated by referring to a control chromatogram obtained. The control in the control solution comprises one or more of hesperidin, glycyrrhizin, glycyrrhizin, isoglycyrrhizin, naringin, glycyrrhizic acid, citrin, imperatorin, glychalcone A, isoimperatorin, honokiol, magnolol, and glycyrrhetinic acid.

12. The method according to claim 11, wherein the Jia Wei Huo Xiang Zheng Qi Pill fingerprint spectrum is characterized by, The solvent of the control solution comprises methanol.

13. A method for detecting Jiawei Huoxiang Zhengqi Pills, characterized in that, The detection method comprises the following steps: The sample solution is prepared by extracting the sample to be detected with an extraction solvent B and collecting the extraction liquid B; The sample solution is detected by the ultra-high performance liquid chromatography, and the sample spectrum obtained is compared with the fingerprint spectrum constructed by the construction method of any one of claims 1 to 12, and the quality of the sample to be detected is determined according to the comparison result; The ultra-high performance liquid chromatography is defined in any one of claims 1 to 6. The extraction solvent B comprises a methanol aqueous solution with a methanol volume percentage of 40%-95%.

14. The method of claim 13, wherein the Jia Wei Huo Xiang Zheng Qi Wan is detected by, The preparation step of the sample solution to be detected satisfies one or more of the following conditions: (I) the extraction method comprises ultrasonic extraction; and (II) the collection of the extraction liquid B comprises centrifugation or filtration.

15. The method of claim 14, wherein the Jia Wei Huo Xiang Zheng Qi Wan is detected by, The power of ultrasonic extraction is 125W-350W, the frequency is 35kHz-45kHz, and the extraction time is 10min-90min.

16. The method of claim 15, wherein the Jia Wei Huo Xiang Zheng Qi Wan is detected by, The power of ultrasonic extraction is 130W-140W, the frequency of ultrasonic extraction is 40kHz-45kHz, and the time of ultrasonic extraction is 15min-90min.

17. The method of claim 14, wherein the Jia Wei Huo Xiang Zheng Qi Wan is detected by, The centrifugal speed is 12000rpm-18000rpm, and the centrifugal time is 8min-15min.

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