A method for analyzing fingerprints of Huoxiangzhengqi soft capsules

Abstract

The invention provides a determination method for analyzing the fingerprint spectrum of Huoxiang Zhengqi soft capsules, and controls the quality of the Huoxiang Zhengqi soft capsules. The method comprises the following steps: a chromatographic condition and a system suitability test, wherein octadecylsilane bonded silica gel is used as a filler; acetonitrile is used as a mobile phase A, 0.1% phosphoric acid is used as a mobile phase B, and gradient elution is performed (the chromatographic conditions are 0-15 min, 10%→47% A, 15-20 min, 47% A, 20-30 min, 47%→60% A, 30-40 min, 60% A); the detection wavelength is 283 nm for 0-12 min and 254 nm for 12-50 min; and the column temperature is 30 DEG C. The number of theoretical plates calculated based on the imperatorin peak should be no less than 5000; preparation of reference solution; preparation of test solution; finally, determination by the assay method and extraction with methanol to ensure the stability and accuracy of the ingredients in Huoxiang Zhengqi soft capsules, and HPLC method is used to establish the fingerprint of Huoxiang Zhengqi soft capsules, which can reflect its overall chemical characteristics. The method is simple and fast, and provides a scientific basis for comprehensively improving its quality.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a method for determining a fingerprint spectrum of Huoxiangzhengqi soft capsules by combining gradient wavelength-high performance liquid chromatography. Background Art

[0002] Huoxiang Zhengqi Soft Capsule is a Chinese patent medicine, composed of Atractylodes, Tangerine Peel, Magnolia Bark, Huoxiang Zhengqi Soft Capsule and Licorice, which can dispel dampness, regulate Qi and harmonize the middle. It is used for colds caused by exogenous wind-cold, internal injury and dampness, or summer heat and dampness, with symptoms of headache, heaviness, chest tightness, abdominal distension and pain, vomiting and diarrhea.

[0003] At present, the fingerprint of Huoxiang Zhengqi soft capsule has not been reported, and its related research mainly focuses on content analysis. Therefore, in addition to establishing the content determination method of the active ingredient and setting the content determination limit, the quality control of Huoxiang Zhengqi soft capsule should establish the fingerprint analysis method of the active ingredient group, which is more conducive to fully grasping the quality of the medicinal materials. The fingerprint technology of traditional Chinese medicine has very important practical significance for increasing the quality control content of traditional Chinese medicine, improving the overall level of the traditional Chinese medicine industry, and realizing the globalization of traditional Chinese medicine.

[0004] The main function of fingerprint is to reflect the uniformity and stability of the internal quality of traditional Chinese medicines and their preparations with complex ingredients. With the application of fingerprint technology as the center, the modernization of traditional Chinese medicine quality standards is promoted. The State Food and Drug Administration has begun to require enterprises to gradually establish fingerprint quality standards from traditional Chinese medicine injections, and will gradually expand the scope of application of fingerprint technology. In combination with the "Pharmacopoeia of the People's Republic of China" (2005 edition), the national standards for traditional Chinese medicine will be further revised and improved, and the quality standards for traditional Chinese medicine pieces will be added to reflect the modernization of traditional Chinese medicine standards. Huoxiang Zhengqi Soft Capsule is a Chinese patent medicine, composed of a variety of ingredients such as Atractylodes, Tangerine Peel, Magnolia Bark, Huoxiang Zhengqi Soft Capsule and Licorice, which can relieve dampness, regulate qi and harmonize the middle. It is used for colds caused by exogenous wind-cold, internal injury and dampness, or summer injury and dampness, with symptoms of headache, heaviness, chest and diaphragm fullness, abdominal distension and pain, vomiting and diarrhea.

[0005] At present, the fingerprint of Huoxiang Zhengqi soft capsule has not been reported, and its related research mainly focuses on content analysis. Therefore, in addition to establishing the content determination method of the active ingredient and setting the content determination limit, the quality control of Huoxiang Zhengqi soft capsule should establish the fingerprint analysis method of the active ingredient group, which is more conducive to fully grasping the quality of the medicinal materials. The fingerprint technology of traditional Chinese medicine has very important practical significance for increasing the quality control content of traditional Chinese medicine, improving the overall level of the traditional Chinese medicine industry, and realizing the globalization of traditional Chinese medicine. Summary of the invention

[0006] The primary purpose of the present invention is to provide a method for determining the fingerprint spectrum of Huoxiang Zhengqi soft capsules by combining gradient wavelength and high performance liquid chromatography, so as to control the quality of Huoxiang Zhengqi soft capsules.

[0007] The main technical scheme is: a method for determining the fingerprint of Huoxiang Zhengqi soft capsules by gradient wavelength-high performance liquid chromatography, and controlling the quality of Huoxiang Zhengqi soft capsules, comprising the following steps:

[0008] (1) Preparation of reference solution;

[0009] (2) Preparation of test solution: Take 0.2 g of the contents of Huoxiang Zhengqi soft capsules, weigh it, place it in a stoppered conical flask, add 25 ml of methanol, weigh it, treat it with ultrasound, take it out, cool it, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain the solution;

[0010] Determination method: Accurately pipette 10 μl of reference solution and test solution, inject into liquid chromatograph, and determine;

[0011] Chromatographic conditions:

[0012] Filler: Octadecylsilane bonded silica gel,

[0013] Mobile phase A: acetonitrile,

[0014] Mobile phase B: 0.1% phosphoric acid,

[0015] Detection wavelength: 283nm for 0-12 minutes, 254nm for 12-50 minutes,

[0016] The column temperature was 30°C.

[0017] Gradient elution,

[0018]

[0019] Preferably, the number of theoretical plates calculated based on the imperatorin peak is not less than 5000.

[0020] Preferably, the specific steps in step (1) are to weigh the reference substance, weigh it, and add methanol to prepare a mixed solution containing 6 μg of liquiritin, 4 μg of naringin, 6 μg of hesperidin, 6 μg of neohesperidin, 2 μg of zanthoxylum toxicol, 5 μg of hydrated oxypeucedanum, 5 μg of white angelicae, 2 μg of zanthoxylum toxicol, 2 μg of bergamot lactone, 5 μg of white angelicae, 10 μg of imperatorin, 4 μg of magnolol, 5 μg of corallina, 4 μg of magnolol, and 10 μg of isoimperatorin as the reference substance solution.

[0021] Preferably, in step (2), the ultrasonic treatment is performed for 30 minutes, with a power of 250 W and a frequency of 33 kHz.

[0022] The beneficial effects of the present invention are:

[0023] This method uses methanol for extraction. In this fingerprint establishment method, 254nm and 283nm gradient wavelengths are selected as the measurement wavelengths; acetonitrile-0.1% phosphoric acid is used as the mobile phase and gradient elution is performed; methanol is preferably used as the extraction solvent and ultrasonic treatment is performed for 30 minutes. The established method can ensure the stability and accuracy of the ingredients in Huoxiang Zhengqi Soft Capsules, and the fingerprint of Huoxiang Zhengqi Soft Capsules is established by HPLC, which can reflect its overall chemical characteristics. The method is simple and fast, and provides a scientific basis for comprehensively improving its quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Other features, objects and advantages of the present application will become more apparent by reading the detailed description of non-limiting embodiments made with reference to the following drawings:

[0025] Figure 1 is the maximum absorption spectrum of the liquiritin component in the present invention;

[0026] Figure 2 is the maximum absorption spectrum of the naringin component in the present invention;

[0027] Figure 3 is the maximum absorption spectrum of the hesperidin component in the present invention;

[0028] Figure 4 is the maximum absorption spectrum of the neohesperidin component in the present invention;

[0029] Figure 5 is the maximum absorption spectrum of the Zanthoxylum bungeanum toxic phenol component in the present invention;

[0030] Figure 6 is the maximum absorption spectrum of the hydrated oxidized purpurogenol component in the present invention;

[0031] Figure 7 is the maximum absorption spectrum of the white angelica component in the present invention;

[0032] Figure 8 is the maximum absorption spectrum of the Zanthoxylum bungeanum toxin component in the present invention;

[0033] Fig. 9 is the maximum absorption spectrum of the bergamot lactone component in the present invention;

[0034] Fig.10 is the maximum absorption spectrum of the white angelica brain component in the present invention;

[0035] Fig.11 is the maximum absorption spectrum of the imperatorin component in the present invention;

[0036] Fig.12 is the maximum absorption spectrum of the magnolol component in the present invention;

[0037] Fig.13 is the maximum absorption spectrum of the anthocyanin component in the present invention;

[0038] Fig.14 is the maximum absorption spectrum of the isoimperatorin component in the present invention;

[0039] Fig.15 is the maximum absorption spectrum of the magnolol component in the present invention;

[0040] Fig.16 This is the durability spectrum of the column temperature at 28°C in the present invention;

[0041] Fig.17 This is the durability spectrum of the column temperature at 30°C in the present invention;

[0042] Fig.18 This is the durability spectrum of the column temperature at 32°C in the present invention;

[0043] Fig.19 This is the durability spectrum of the flow rate of 0.9 ml / min in the present invention;

[0044] Fig. 20 This is the column temperature durability spectrum at a flow rate of 1.0 ml / min in the present invention;

[0045] Fig.21 This is the durability graph of the flow rate of 1.1 ml / min in the present invention;

[0046] Fig. 22 This is the durability spectrum of the COSMOSIL5C18-MS-Ⅱ (5μm, 4.6×250mm) chromatographic column in the present invention;

[0047] Fig.23 This is the durability spectrum of the Shiseido Capcellpak C18MGⅡ (5μm, 4.6×250mm) chromatographic column in the present invention;

[0048] Fig.24 This is the durability spectrum of the SHIMADZUVP-ODSC18 (5 μm, 4.6×250 mm) chromatographic column in the present invention;

[0049] Fig.25 The sample and reference atlas R of Huoxiangzhengqi soft capsule with batch number 22021711 in the present invention;

[0050] Fig.26 The test sample and reference atlas R of Huoxiangzhengqi soft capsules with batch numbers 2930160, 2930161 and 2930162 in the present invention;

[0051] Fig. 27The sample and reference atlas R of Huoxiangzhengqi soft capsules with batch numbers of 20042211, 20020411, 20070111, 20102211, 2011211121010911, 21031611, 21041611, 22022811, 22031912, and 22053111 in the present invention;

[0052] Fig.28 It is reference peak No. 17 in the present invention. DETAILED DESCRIPTION

[0053] The present application is further described in detail below in conjunction with the accompanying drawings and embodiments. It is to be understood that the specific embodiments described herein are only used to explain the relevant invention, rather than to limit the invention. It is also necessary to explain that, for ease of description, only the parts related to the invention are shown in the accompanying drawings.

[0054] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0055] Example:

[0056] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler; acetonitrile was used as mobile phase A, 0.1% phosphoric acid was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; the detection wavelengths were 254nm, 283nm; the column temperature was 30°C. The theoretical plate number calculated based on the imperatorin peak should be no less than 5000.

[0057]

[0058] Preparation of reference solution: Weigh an appropriate amount of reference substance, accurately weigh it, and add methanol to prepare a mixed solution containing 6 μg of liquiritin, 4 μg of naringin, 6 μg of hesperidin, 6 μg of neohesperidin, 2 μg of zanthoxylum toxicol, 5 μg of hydrated oxypeucedanum, 5 μg of white angelicae, 2 μg of zanthoxylum toxicol, 2 μg of bergamot lactone, 5 μg of white angelicae, 10 μg of imperatorin, 4 μg of magnolol, 5 μg of corallina, 4 μg of magnolol, and 10 μg of isoimperatorin as the reference solution.

[0059] Preparation of the test solution: Take about 0.2 g of the content of Huoxiang Zhengqi soft capsules, weigh it accurately, put it in a stoppered conical flask, add 25 ml of methanol accurately, weigh the weight, ultrasonically treat (power 250 W, frequency 33 kHz) for 30 minutes, take it out, cool, make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain the solution.

[0060] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0061] The above examples were methodologically validated according to the requirements of the Pharmacopoeia, and the results showed that the method was accurate, reliable, sensitive, and specific, and all indicators met the requirements of quality control. The fingerprint determination method can be applied to the quality control of Huoxiang Zhengqi soft capsules.

[0062] 1. Instruments and test drugs

[0063] 1.1 Instrument:

[0064] Ultimate3000 high performance liquid chromatograph (quaternary pump, diode array detector);

[0065] Waterse2695-2998 (quaternary pump, diode array detector);

[0066] Mettler XPE26 analytical balance (0.001 mg);

[0067] Mettler XS105DU analytical balance (0.01 mg);

[0068] Ultrapure water instrument from Millipore, USA.

[0069] Chromatographic column:

[0070] WatersSymmtry, C18(5μm,4.6×250mm)

[0071] COSMOSIL5C18-MS-Ⅱ(5μm,4.6×250mm)

[0072] Shiseido Capcellpak C18MGⅡ(5μm,4.6×250mm)

[0073] 1.2 Reagents and test drugs:

[0074] Reagents: methanol (Sinopharm Chemical Reagent Co., Ltd., batch number: 10014118, analytical grade);

[0075] Acetonitrile (Thermo Fisher Scientific (China) Co., Ltd., batch number: F22M2A201, chromatographic grade);

[0076] The water was ultrapure water.

[0077] Reference substances: Liquorice glycoside reference substance (batch number 111610-201106, calculated as 93.7%), Hesperidin reference substance (batch number 110721-201316, calculated as 95.3%), Neohesperidin reference substance (batch number 111857-201102, calculated as 99.6%), Honokiol reference substance (batch number 110730-201313, calculated as 99.1%), Honokiol reference substance (Batch No. 110729-201714, calculated as 100%), imperatorin reference substance (Batch No. 110826-201918, calculated as 99.0%), isoimperatorin reference substance (Batch No. 110827-202113), Magnolia officinalis reference substance (Batch No. 121285-200301), Tangerine peel reference substance (Batch No. 120969-200507), Atractylodes macrocephala reference substance (Batch No. 12 0983-201004), Atractylodes macrocephala reference substance (batch number 120932-201006) were purchased from China Food and Drug Inspection Institute; Zanthoxylum toxicity phenol reference substance (batch number 2009-24-7, calculated as 98%), white angelicain reference substance (482-25-7, calculated as 98%), hydrated oxydepescens reference substance (batch number 2643-85-8, calculated as 98%), white angelica brain reference substance (batch number 26091-79-2, calculated as 98%), and coral acanthin reference substance (batch number 2543-94-4, calculated as 98%) were purchased from Shanghai Shidande Standard Technology Service Co., Ltd.; Zanthoxylum toxicity phenol reference substance (batch number DST210407-062, calculated as 98%), bergamot lactone reference substance (batch number DST210409-012, calculated as 98%) were purchased from Chengdu Desit Biotechnology Co., Ltd.

[0078] 2. Method determination

[0079] 2.1 Selection of chromatographic conditions

[0080] 2.1.1 Wavelength selection

[0081] In the literature, the wavelengths of 254nm and 300nm were mostly selected for the determination of Huoxiang Zhengqi soft capsules. Ten reference solutions were taken and the spectrum was scanned at 200nm to 800nm. The maximum absorption of 15 reference solutions was Figure 1 The test showed that liquiritin, naringin, hesperidin and neohesperidin had good peak shape and good separation at 283nm wavelength, while the remaining 11 reference substances had good peak shape and good separation at 254nm wavelength, so the gradient wavelengths of 254nm and 283nm were selected as the measurement wavelengths of the fingerprint.

[0082] 2.1.2 Selection of extraction solvent

[0083] The extraction conditions of samples by methanol, ethanol and acidic methanol were investigated respectively. The results showed that the components in Huoxiang Zhengqi soft capsules can be well reflected in the methanol extract. Therefore, methanol was selected as the extraction condition.

[0084] 2.1.2 Selection of mobile phase

[0085] The effects of different mobile phase systems acetonitrile-water and acetonitrile-0.1% phosphoric acid on the chromatographic peaks were investigated. The results showed that when the mobile phase system was acetonitrile-0.1% phosphoric acid solution (gradient elution), the baseline of the chromatogram was stable and the separation effect of the chromatographic peaks was better.

[0086] 3. Investigation of fingerprint methodology

[0087] 3.1 Precision investigation Weigh 0.2g of the content of the same batch of Huoxiangzhengqi soft capsules (batch number 20042211) accurately, prepare the test solution according to the method of "Preparation of test solution", inject 6 times continuously, record the chromatogram, conduct precision investigation, take the imperatorin peak as the reference peak S, calculate the relative retention time and relative peak area of ​​the other common peaks and the S peak, and calculate the RSD value. The results are shown in Table 1 and Table 2, and the results are all less than 1.0%, indicating that the instrument has good precision.

[0088] Table 1 Precision relative to retention time

[0089]

[0090]

[0091] Table 2 Precision relative peak area

[0092]

[0093]

[0094] 3.2 Repeatability test Weigh 0.2g of the content of the same batch of Huoxiangzhengqi soft capsules (batch number 20042211) accurately, prepare 6 test solutions in parallel according to the method of "Preparation of test solution", record the peak area, take the imperatorin peak as the reference peak S, calculate the relative retention time and relative peak area of ​​the remaining common peaks and the S peak, and calculate the RSD value. The results are shown in Tables 3 and 4, and the results are all less than 1.0%, indicating that the method has good repeatability.

[0095] Table 3 Reproducibility relative retention time

[0096]

[0097]

[0098] Table 4 Repeatability relative peak area

[0099]

[0100]

[0101] 3.3 Stability test Accurately weigh the contents of Huoxiang Zhengqi Soft Capsules (Batch No. 20042211), prepare the test solution according to the method of "Preparation of test solution", and inject and measure at 0, 2, 4, 6, 8, 12, and 24 hours respectively. Take the imperatorin peak as the reference peak S, calculate the relative retention time and relative peak area of ​​the remaining common peaks and the S peak, and calculate the RSD value. The results are shown in Tables 5 and 6. The results are all less than 2.0%, indicating that the test solution has good stability within 24 hours.

[0102] Table 5 Stability relative retention time

[0103]

[0104]

[0105] Table 6 Stability relative peak area

[0106]

[0107] 3.4 Durability test

[0108] 3.4.1 Column temperature investigation The same batch of test samples were measured at different column temperatures. The results are shown in Table 7. Figure 2 , the result was less than 1.0%, indicating that the chromatographic system had good durability to column temperature.

[0109] Table 7 Relative retention time at different column temperatures

[0110]

[0111]

[0112] 3.4.2 Flow rate investigation The same batch of test products were measured at different flow rates. The results are shown in Table 8. Figure 3 , the result was less than 2.0%, indicating that the chromatographic system has good durability to the flow rate.

[0113] Table 8 Relative retention time at different flow rates

[0114]

[0115]

[0116] 3.4.3 Chromatographic column investigation Three different chromatographic columns were used to measure the same batch of Huoxiang Zhengqi soft capsules. The results are shown in Table 9 and the chromatograms are shown in Figure 4The peak time of each main component in the chromatogram of the test sample was appropriate and the separation was good, and the result was less than 3.0%, indicating that the chromatographic system had good durability for the chromatographic column.

[0117] Table 9 Relative retention time of different chromatographic columns

[0118]

[0119] Note: ⅠCOSMOSIL5C 18 -MS-Ⅱ(5μm,4.6×250mm);ⅡShiseido CapcellpakC18MGⅡ(5μm,4.6×250mm);ⅢSHIMADZUVP-ODSC 18 (5μm,4.6×250mm)

[0120] 3.4.4 Instrument investigation The same chromatographic column (SHIMADZUVP-ODS, C18, 5μm, 4.6×250mm) was used to measure the same batch of test samples using high performance liquid chromatographs of different brands. The measurement results are shown in Table 10. The results are greater than 3.0%, indicating that the chromatographic system has good durability for the instrument.

[0121] Table 10 Relative retention time of different instruments

[0122]

[0123] Note: ⅠUltimate3000 HPLC; ⅡWaterse2695-2998

[0124] 4. Fingerprint establishment and similarity evaluation

[0125] Take 15 batches of Huoxiangzhengqi soft capsule test solution (S1S15) and record the chromatogram. Use the "Chinese medicine fingerprint similarity evaluation system 2012 version", use the S1 test sample chromatogram as the reference spectrum, the median method as the reference spectrum generation method, the time window width is 0.1, and the multi-point correction is automatically matched to generate the superimposed spectrum of the test sample and the reference spectrum R (see Figure 5 There are 22 common peaks in 15 batches of samples. Since peak 17 is well separated from adjacent chromatographic peaks, has a large peak area, and a relatively stable retention time, the peak 17 of imperatorin in the chromatographic peak of the test sample is selected as the reference peak (see Figure 6 ).

[0126] Table 1115 batches of samples with common peaks relative retention time

[0127]

[0128] Table 1215 batches of samples have common peak relative peak areas

[0129]

[0130] Table 13 Similarity of 15 batches of samples

[0131]

[0132] The results in Table 13 show that the similarities of the 15 batches of samples are all greater than 0.8, indicating that their quality is relatively stable and their chemical compositions have good consistency.

[0133] 5. Chromatographic peak identification and chemical component attribution

[0134] Depend on Figure 3 It can be seen that based on the consistency of retention time and UV spectrum of the test sample chromatogram and the mixed reference sample chromatogram, a total of 16 characteristic fingerprint peaks were identified, of which peak 1 was liquiritin, peak 2 was naringin, peak 3 was hesperidin, peak 4 was neohesperidin, peak 5 was zanthoxylum phenol, peak 6 was hydrated oxypeucedanin, peak 7 was white angelicaein, peak 9 was zanthoxylum toxicum, peak 10 was bergamot lactone, peak 11 was white angelicaein, peak 17 was imperatorin, peak 18 was honokiol, peak 19 was corallin, peak 20 was isoimperatorin, peak 21 was magnolol, and peak 22 was atractylodesin.

[0135] In summary, this method used gradient wavelength-HPLC to establish the fingerprint of Huoxiang Zhengqi soft capsules, and can reflect its overall chemical characteristics. This method is simple and fast, and provides a scientific basis for comprehensively improving the product quality of Huoxiang Zhengqi soft capsules.

[0136] The above description is only a preferred embodiment of the present application and an explanation of the technical principles used. Those skilled in the art should understand that the scope of the invention involved in the present application is not limited to the technical solution formed by a specific combination of the above technical features, but should also cover other technical solutions formed by any combination of the above technical features or their equivalent features without departing from the inventive concept. For example, the above features are replaced with (but not limited to) technical features with similar functions disclosed in the present application.

Claims

1. A method for determining the fingerprint of Huoxiang Zhengqi soft capsules by gradient wavelength-high performance liquid chromatography to control the quality of Huoxiang Zhengqi soft capsules, characterized in that: The following steps are involved: (1) Preparation of reference solution; (2) Preparation of test solution: Take 0.2 g of the contents of Huoxiang Zhengqi soft capsules, weigh it, place it in a stoppered conical flask, add 25 ml of methanol, weigh it, treat it with ultrasound, take it out, cool it, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain the solution; Determination method: Accurately pipette 10 μl of reference solution and test solution, inject into liquid chromatograph, and determine; Chromatographic conditions: Filler: Octadecylsilane bonded silica gel, Mobile phase A: acetonitrile, Mobile phase B: 0.1% phosphoric acid, Detection wavelength: 283nm for 0-12 minutes, 254nm for 12-40 minutes, The column temperature was 30°C. Gradient elution, After the determination, 16 characteristic fingerprint peaks were identified, among which peak 1 was liquiritigenin, peak 2 was naringin, peak 3 was hesperidin, peak 4 was neohesperidin, peak 5 was zanthoxylum toxicol, peak 6 was hydrated oxypeucedanum, peak 7 was white angelicaein, peak 9 was zanthoxylum toxin, peak 10 was bergamot lactone, peak 11 was white angelicaein, peak 17 was imperatorin, peak 18 was honokiol, peak 19 was corallin, peak 20 was isoimperatorin, peak 21 was magnolol, and peak 22 was atractylodesin.

2. The method for determining the fingerprint of Huoxiang Zhengqi soft capsules by gradient wavelength-high performance liquid chromatography according to claim 1, characterized in that: The number of theoretical plates calculated based on the imperatorin peak is not less than 5000.

3. The method for determining the fingerprint of Huoxiang Zhengqi soft capsules by gradient wavelength-high performance liquid chromatography according to claim 1, characterized in that: The specific steps in step (1) are to weigh the reference substance, weigh it, and add methanol to prepare a mixed solution containing 6 μg of liquiritin, 4 μg of naringin, 6 μg of hesperidin, 6 μg of neohesperidin, 2 μg of zanthoxylum toxicol, 5 μg of hydrated oxypeucedanum, 5 μg of white angelicae, 2 μg of zanthoxylum toxicol, 2 μg of bergamot lactone, 5 μg of white angelicae, 10 μg of imperatorin, 4 μg of honokiol, 5 μg of corallina, 4 μg of magnolol, and 10 μg of isoimperatorin as the reference substance solution.

4. The method for determining the fingerprint of Huoxiang Zhengqi soft capsules by gradient wavelength-high performance liquid chromatography according to claim 1, characterized in that: In the step (2), ultrasonic treatment is performed for 30 minutes, with a power of 250 W and a frequency of 33 kHz.

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