Fingerprint spectrum and multi-component content determination method of gentian liver-fire purging pill

Abstract

The invention provides a radix gentianae liver-fire purging pill fingerprint spectrum and a multi-component content determination method. The invention also provides a detection method of the fingerprint spectrum of the gentian liver-fire purging pill. According to the fingerprint spectrum of the Gentian Xiegan pill and the multi-component content determination method provided by the invention, six components in the Gentian Xiegan pill can be quantitatively analyzed, the fingerprint spectrum of the Gentian Xiegan pill is established, and reference is provided for scientifically and comprehensively controlling the quality of the Gentian Xiegan pill.

Description

Technical Field

[0001] The present invention belongs to the technical field of detection of traditional Chinese medicine components, and relates to a fingerprint and multi-component content determination method for Longdan Xiegan Pills, specifically to a content determination method for 6 chemical components in Longdan Xiegan Pills: geniposidic acid, gardenoside, baicalin, wogonoside, glycyrrhizic acid, and ligustilide, and a fingerprint determined based on these 6 components. Background Art

[0002] Longdan Xiegan Pills are composed of ten herbs including gentiana, scutellaria, alisma, plantago asiatica L. var. major (Ting) Tsai & Tseng, rehmannia glutinosa, bupleurum chinense, gardenia jasminoides, akebia quinata, angelica sinensis, and roasted licorice root. It originated from "Collection of Medical Formulas" in the Qing Dynasty and can be clinically used for damp-heat in the liver and gallbladder, dizziness and red eyes, tinnitus and deafness, rib pain and bitter taste in the mouth, red urine, and damp-heat leukorrhea. In the formula, gentiana is extremely bitter and cold, which can not only purge the excessive fire of the liver and gallbladder but also remove damp-heat from the liver meridian, purging fire and removing dampness, and is used as the monarch drug; scutellaria and gardenia are bitter and cold, purging fire and removing dampness, strengthening the monarch drug's power of purging fire and removing dampness, and are used as the minister drugs; alisma, akebia quinata, and plantago asiatica L. var. major (Ting) Tsai & Tseng guide downward, promoting diuresis and clearing heat, and angelica sinensis and rehmannia glutinosa nourish blood and yin, so that pathogenic factors are removed without damaging yin and blood. All of the above are adjuvant drugs; bupleurum chinense soothes the qi of the liver and gallbladder and can lead all the drugs to the liver and gallbladder meridians, and licorice root harmonizes all the drugs and protects the stomach and soothes the middle, and the two drugs also serve as both adjuvant and guiding drugs. All the drugs work together to clear the liver and gallbladder and promote diuresis and remove dampness.

[0003] Currently, Longdan Xiegan Pills (water pills) are included in the first volume of the 2020 edition of the "Chinese Pharmacopoeia". Its quality control indicators are gentiopicroside, gardenoside, and baicalin. The number of indicator components is small and the quality control range is narrow. Therefore, it is necessary to further analyze the components of Longdan Xiegan Pills to comprehensively and objectively reflect the quality of Longdan Xiegan Pills and more effectively control the quality of Longdan Xiegan Pills. Summary of the Invention

[0004] In view of the above-mentioned disadvantages of the prior art, the purpose of the present invention is to provide a fingerprint and multi-component content determination method for Longdan Xiegan Pills. By using optimized pretreatment and high-performance liquid chromatography determination conditions, a content determination method for 6 components in Longdan Xiegan Pills: geniposidic acid, gardenoside, baicalin, wogonoside, glycyrrhizic acid, and ligustilide, and a fingerprint determined based on these 6 indicator components are established, providing reference for scientifically controlling the quality of Longdan Xiegan Pills.

[0005] To achieve the above object and other related objects, the first aspect of the present invention provides a multi-component content determination method for Longdan Xiegan Pills, including the following steps:

[0006] 1) Preparation of the test solution: Add a Longdan Xiegan Pills sample to a solvent for dissolution, perform ultrasonic extraction, cool it, filter, and take the subsequent filtrate to obtain the test solution;

[0007] 2) Preparation of the reference substance solution: Dissolve one or more reference substances of geniposide acid, geniposide, baicalin, wogonoside, glycyrrhizic acid, and ligustilide in a solvent and make up the volume to obtain the reference substance solution.

[0008] 3) Determination: Use high performance liquid chromatography (HPLC) to separately determine the test solution in step 1) and the reference substance solution in step 2), and calculate the content of one or more components of geniposide acid, geniposide, baicalin, wogonoside, glycyrrhizic acid, and ligustilide in the test solution by the external standard method.

[0009] Preferably, in step 1), the Gentian Liver-Clearing and Gallbladder-Draining Pills sample is a powdered sample that has been crushed and sieved.

[0010] More preferably, the sieve for sieving is sieve No. 3 - 5, preferably sieve No. 4.

[0011] Preferably, in step 1), the Gentian Liver-Clearing and Gallbladder-Draining Pills sample is accurately weighed before adding the solvent.

[0012] Preferably, in step 1), the weight is weighed after adding the solvent.

[0013] Preferably, in step 1), the ratio of the weight (g) of the Gentian Liver-Clearing and Gallbladder-Draining Pills sample added to the volume (mL) of the solvent added is 0.4 - 0.6:20 - 30, specifically such as 0.4 - 0.6:20 - 23, 0.4 - 0.6:23 - 27, 0.4 - 0.6:27 - 30, for example 0.4:23 - 27, 0.5:23 - 27, 0.6:23 - 27, preferably 0.5:25.

[0014] Preferably, in step 1), the ultrasonic extraction time is 25 - 35 min, specifically such as 25 - 28 min, 28 - 32 min, 32 - 35 min, for example 25 min, 28 min, 30 min, 32 min, 35 min, preferably 30 min.

[0015] Preferably, in step 1), the power of the ultrasonic extraction is 90 - 110 W, preferably 100 W; the frequency of the ultrasonic extraction is 30 - 50 kHz, preferably 40 kHz.

[0016] Preferably, in step 1), the cooling is to stand and cool to room temperature. The room temperature is 20 - 30 °C.

[0017] Preferably, in step 1), the weight is weighed again after cooling and then the weight is made up.

[0018] Preferably, in step 1), the solvent used for making up the weight is an aqueous solution containing methanol with a volume percentage concentration of 70 - 80%, preferably 72 - 78%, more preferably 75%. The weight should be made up to compensate for the lost mass.

[0019] Preferably, in step 1), the filtration is membrane filtration.

[0020] More preferably, the membrane is a 0.45 μm membrane.

[0021] The subsequent filtrate is the filtrate collected after pouring out the initial filtrate.

[0022] Preferably, in step 1) or 2), the solvent is an aqueous solution containing 70 - 80% methanol by volume percentage concentration, preferably 72 - 78%, and more preferably 75%.

[0023] Preferably, in step 2), the reference substance solution can be directly added to the solvent to prepare a mixed reference substance solution, or it can be prepared by first adding the solvent to prepare a reference substance stock solution and then adding the solvent for step - by - step dilution.

[0024] More preferably, in the reference substance stock solution, the content of geniposide acid is 100.90 μg / mL; the content of geniposide is 202.00 μg / mL; the content of baicalin is 579.60 μg / mL; the content of wogonoside is 204.80 μg / mL; the content of glycyrrhizic acid is 162.00 μg / mL; the content of ligustilide is 300.00 μg / mL.

[0025] The reference substance stock solution is stored in the dark at 4 °C for later use.

[0026] Preferably, in step 2), the CAS number of geniposide acid is 27741 - 01 - 1, the CAS number of geniposide is 24512 - 63 - 8, the CAS number of baicalin is 21967 - 41 - 9, the CAS number of wogonoside is 51059 - 44 - 0, the CAS number of glycyrrhizic acid is 1405 - 86 - 3, and the CAS number of ligustilide is 4431 - 01 - 0.

[0027] Preferably, in step 2), the content range of geniposide acid in the reference substance solution is 6.31 - 100.90 μg / mL; the content range of geniposide is 12.63 - 202.00 μg / mL; the content range of baicalin is 36.23 - 579.60 μg / mL; the content range of wogonoside is 12.80 - 204.80 μg / mL; the content range of glycyrrhizic acid is 10.13 - 162.00 μg / mL; the content range of ligustilide is 18.75 - 300.00 μg / mL.

[0028] Preferably, in step 3), the chromatographic column in the high - performance liquid chromatography is a C 18 chromatographic column. More preferably, the chromatographic column in the high - performance liquid chromatography is an Agilent TC - C 18Chromatographic column (4.6 mm × 250 mm, 5 μm), and the packing material is octadecylsilyl silica gel.

[0029] Preferably, in step 3), the detector in the high performance liquid chromatography is a photodiode array detector (DAD).

[0030] Preferably, in step 3), in the ultra high performance liquid chromatography, the column temperature is 30 - 40 °C, specifically such as 30 - 33 °C, 33 - 37 °C, 37 - 40 °C, and preferably 35 °C.

[0031] Preferably, in step 3), the injection volume in the high performance liquid chromatography is 8 - 12 μL. More preferably, the injection volume in the high performance liquid chromatography is, for example, 8 μL, 9 μL, 10 μL, 11 μL, 12 μL, and preferably 10 μL.

[0032] Preferably, in step 3), the flow rate in the high performance liquid chromatography is 0.5 - 1.5 mL / min, specifically such as 0.5 - 0.8 mL / min, 0.8 - 1.2 mL / min, 1.2 - 1.5 mL / min, for example 0.8 mL / min, 0.9 mL / min, 1.0 mL / min, 1.1 mL / min, 1.2 mL / min, and preferably 1.0 mL / min.

[0033] Preferably, in step 3), the detection wavelength in the high performance liquid chromatography is selected from one or more combinations of 236 - 240 nm, 252 - 256 nm, 278 - 282 nm, preferably 236 - 240 nm, 252 - 256 nm, and 278 - 282 nm, and more preferably 238 nm, 254 nm, and 280 nm.

[0034] More preferably, the detection wavelength changes with the time of the gradient elution program, and the specific change situation is: 0 - 10 min, the detection wavelength is 252 - 256 nm, preferably 254 nm; 10 - 27 min, the detection wavelength is 236 - 240 nm, preferably 238 nm; 27 - 33 min, the detection wavelength is 252 - 256 nm, preferably 254 nm; 33 - 50 min, the detection wavelength is 278 - 282 nm, preferably 280 nm; 50 - 54 min, the detection wavelength is 252 - 256 nm, preferably 254 nm; 54 - 65 min, the detection wavelength is 278 - 282 nm, preferably 280 nm.

[0035] Preferably, in step 3), in the high performance liquid chromatography, the mobile phase is acetonitrile - 0.09 - 0.11% phosphoric acid aqueous solution; in the acetonitrile - 0.09 - 0.11% phosphoric acid aqueous solution, phase A is acetonitrile and phase B is 0.09 - 0.11% phosphoric acid aqueous solution.

[0036] More preferably, in the high performance liquid chromatography, the mobile phase is acetonitrile - 0.1% phosphoric acid aqueous solution; in the acetonitrile - 0.1% phosphoric acid aqueous solution, phase A is acetonitrile and phase B is 0.1% phosphoric acid aqueous solution.

[0037] The 0.09 - 0.11% phosphoric acid aqueous solution is a phosphoric acid aqueous solution with a volume percentage of 0.09 - 0.11%. The 0.1% phosphoric acid aqueous solution is a phosphoric acid aqueous solution with a volume percentage of 0.1%.

[0038] Preferably, in step 3), in the high performance liquid chromatography, gradient elution is adopted and the analysis time is 65 min.

[0039] More preferably, the specific program of the gradient elution is as follows:

[0040] 0 - 5 min, volume ratio of phase A to phase B is 4 - 6:94 - 96 - 9 - 11:89 - 91;

[0041] 5 - 10 min, volume ratio of phase A to phase B is 9 - 11:89 - 91 - 14 - 16:84 - 86;

[0042] 10 - 15 min, volume ratio of phase A to phase B is 14 - 16:84 - 86 - 19 - 21:79 - 81;

[0043] 15 - 25 min, volume ratio of phase A to phase B is 19 - 21:79 - 81 - 22 - 23:77 - 78;

[0044] 25 - 40 min, volume ratio of phase A to phase B is 22 - 23:77 - 78 - 29 - 31:69 - 71;

[0045] 40 - 55 min, volume ratio of phase A to phase B is 29 - 31:69 - 71 - 49 - 51:49 - 51;

[0046] 55 - 65 min, volume ratio of phase A to phase B is 49 - 51:49 - 51 - 67 - 69:31 - 33.

[0047] Even more preferably, the specific program of the gradient elution is shown in Table 1 and is as follows:

[0048] 0 - 5 min, volume ratio of phase A to phase B is 5:95 - 10:90;

[0049] 5 - 10 min, volume ratio of phase A to phase B is 10:90 - 15:85;

[0050] 10 - 15 min, volume ratio of phase A to phase B is 15:85 - 20:80;

[0051] 15 - 25 min, the volume ratio of phase A to phase B is 20:80 - 22:78;

[0052] 25 - 40 min, the volume ratio of phase A to phase B is 22:78 - 30:70;

[0053] 40 - 55 min, the volume ratio of phase A to phase B is 30:70 - 50:50;

[0054] 55 - 65 min, the volume ratio of phase A to phase B is 50:50 - 68:32.

[0055] Table 1

[0056]

[0057] Preferably, in step 3), the external standard method refers to: respectively pipetting a series of reference substance solutions with different volumes in step 2), respectively preparing a series of solutions with different concentrations, injecting and analyzing them using a high - performance liquid chromatograph, obtaining the linear relationship between the concentrations of 6 components in the reference substance solution and the peak areas, using the peak area of each component chromatogram corresponding to its corresponding concentration, plotting the corresponding standard working curve, and calculating the regression equation of each standard working curve. Then, detecting the test sample solution using a high - performance liquid chromatograph, substituting the peak areas of the 6 components in the obtained test sample solution into the regression equations of the respective standard working curves, and calculating to obtain the contents of the corresponding components.

[0058] More preferably, in the standard working curve, the peak area of each component is used as the ordinate, and the concentration of each component in the reference substance solution is used as the abscissa.

[0059] The second aspect of the present invention provides the use of a method for determining the contents of multiple components in Longdan Xiegan Pills in the detection of the fingerprint of Longdan Xiegan Pills.

[0060] The third aspect of the present invention provides a method for detecting the fingerprint of Longdan Xiegan Pills, comprising the following steps:

[0061] A) Preparation of the test sample solution: the same as step 1) of the method for determining the contents of multiple components in Longdan Xiegan Pills;

[0062] B) Preparation of the reference substance solution: the same as step 2) of the method for determining the contents of multiple components in Longdan Xiegan Pills;

[0063] C) Determination: Using high performance liquid chromatography (HPLC) under the same chromatographic conditions as in step 3) of the multi-component content determination method for Longdan Xiegan Pills, the test solution in step A) and the reference solution in step B) were respectively determined to obtain the fingerprint of the test solution and the fingerprint of the reference solution. The fingerprint of the test solution was compared with the fingerprint of the reference solution to assign and locate the target components in the fingerprint of the test solution, thereby obtaining the fingerprint of Longdan Xiegan Pills.

[0064] Preferably, in step C), when comparing the fingerprint of the test solution with the fingerprint of the reference solution, based on the known characteristic peaks in the fingerprint of the reference solution, the corresponding characteristic peaks in the fingerprint of the test solution were identified through relative retention time, so as to assign and locate the target components in the fingerprint of the test solution.

[0065] All water used in the present invention is pure water.

[0066] As described above, the present invention provides a fingerprint and a multi-component content determination method for Longdan Xiegan Pills, optimized pretreatment and high performance liquid chromatography determination conditions, using a detection method with segmented wavelengths to detect the contents of 6 components in Longdan Xiegan Pills: geniposide acid, gardenoside, baicalin, wogonoside, glycyrrhizic acid, and ligustilide, and establishing the fingerprint of Longdan Xiegan Pills.

[0067] The methodological verification results of this content determination method showed that the extraction conditions, linear range, repeatability, precision, stability, and sample addition recovery of this method were investigated. The results showed that this method was stable and reliable, the separation degree of 6 active components in Longdan Xiegan Pills was good, the peak shape was symmetrical, the method was simple and reliable, easy to operate, and had good reproducibility, providing reference for scientifically and comprehensively controlling the quality of Longdan Xiegan Pills. Description of the Drawings

[0068] Figure 1 Shown are the reference chromatograms of the reference substances and test samples of 6 chemical components in the Longdan Xiegan Pills of the present invention. Among them, S1: the chromatogram of the reference substance; S2: the chromatogram of the test sample; 1: geniposide acid; 2: geniposide acid; 3: baicalin; 4: wogonoside; 5: glycyrrhizic acid; 6: ligustilide. Detailed Embodiments

[0069] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the protection scope of the present invention.

[0070] The following specific examples illustrate the implementation modes of the present invention. Those skilled in the art can easily understand the other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation modes. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0071] The reagents and instruments used in the following examples are as follows:

[0072] 1. Reagents

[0073] Reference substances: Geniposidic acid (batch number: 111828 - 201805), Gardenoside (batch number: 22102223), Baicalin (batch number: 110715 - 201821), Wogonoside (batch number: 112002 - 202303), Glycyrrhizic acid (batch number: 17032205), Ligustilide (batch number: 8155). The above reference substances are all purchased from the National Institutes for Food and Drug Control.

[0074] Samples: Longdan Xiegan Pills (batch number: 20240701), provided by Shanghai Hutchison Pharmaceuticals Co., Ltd.

[0075] Medicinal materials: Gentiana scabra Bunge, Scutellaria baicalensis Georgi, Alisma orientale (Sam.) Juz., Plantago asiatica L. var. major (Nakai) Kitag., Rehmannia glutinosa Libosch., Bupleurum chinense DC., Gardenia jasminoides Ellis, Akebia quinata (Thunb.) Decne., Angelica sinensis (Oliv.) Diels, and Glycyrrhiza uralensis Fisch. are all commonly used medicinal materials, provided by Shanghai Hutchison Pharmaceuticals Co., Ltd.

[0076] Reagents: Methanol (analytical pure AR, Sinopharm Chemical Reagent Co., Ltd.), Acetonitrile, Phosphoric acid (chromatographic pure, Fisher Scientific, USA), and ultrapure water is prepared by a Milli - Q ultrapure water treatment system.

[0077] 2. Instruments

[0078] Agilent 1260 high performance liquid chromatograph (Agilent Technologies, Inc., USA, including Agilent OpenLAB CDS ChemStation workstation; G1311C quaternary pump system; G1329B standard autosampler; G1316A column oven; G4212B diode array detector); AL204 one in ten thousand electronic balance and X205BDU one in one hundred thousand electronic balance (METTLER TOLEDO Instruments Shanghai Co., Ltd.); DFY-500 high-speed traditional Chinese medicine grinder (Dade Traditional Chinese Medicine Machinery Co., Ltd., Wenling, Zhejiang); TDL-40B tabletop centrifuge (Shanghai Anting Scientific Instrument Factory); DHG-9123A electrothermal blast drying oven (Shanghai Yiheng Scientific Instrument Co., Ltd.); SB-5200 ultrasonic cleaner (Ningbo Xinzhi Biotechnology Co., Ltd.); Mill-Q Advantage A10 ultrapure water preparation system (MILLIPORE Shanghai Trading Co., Ltd.).

[0079] Example 1

[0080] 1. Sample pretreatment

[0081] Preparation of test solution: The Gentian Liver-Clearing and Gallbladder-Draining Pills sample of batch 20240701 was pulverized and passed through a No. 4 sieve. The obtained sample powder was accurately weighed 0.5 g and placed in a stoppered conical flask. 25 mL of 75% methanol was accurately added, the flask was stoppered, weighed, ultrasonically extracted (power 100 W, frequency 40 kHz) for 30 minutes, allowed to stand and cool to room temperature, weighed again, and the lost weight was made up with 75% methanol. It was filtered through a 0.45 μm microporous membrane, and the subsequent filtrate was taken to obtain test solution 1#.

[0082] Preparation of reference solution: The reference substances of geniposide acid, geniposide, baicalin, wogonoside, glycyrrhizic acid, and ligustilide were accurately weighed respectively and placed in the same volumetric flask. They were dissolved and diluted to the scale with 75% methanol and shaken well to prepare a reference stock solution. In the reference stock solution, the content of geniposide acid was 100.90 μg / mL; the content of geniposide was 202.00 μg / mL; the content of baicalin was 579.60 μg / mL; the content of wogonoside was 204.80 μg / mL; the content of glycyrrhizic acid was 162.00 μg / mL; the content of ligustilide was 300.00 μg / mL. It was refrigerated in the dark at 4 °C for standby.

[0083] The reference substance stock solution was further diluted with 75% methanol to prepare a series of reference substance solutions with different concentrations. In the series of reference substance solutions with different concentrations, the content range of geniposidic acid was 6.31 - 100.90 μg / mL; the content range of gardenoside was 12.63 - 202.00 μg / mL; the content range of baicalin was 36.23 - 579.60 μg / mL; the content range of wogonoside was 12.80 - 204.80 μg / mL; the content range of glycyrrhizic acid was 10.13 - 162.00 μg / mL; the content range of ligustilide was 18.75 - 300.00 μg / mL.

[0084] 2. Chromatographic conditions

[0085] The chromatographic conditions for high performance liquid chromatography were as follows: the chromatographic column was an Agilent TC-C 18 chromatographic column (4.6 mm × 250 mm, 5 μm); the detector was a photodiode array detector (DAD); the column temperature was 35 °C; the injection volume was 10 μL; the flow rate was 1.0 mL / min.

[0086] Analysis was carried out using a multi-wavelength method, and the detection wavelengths were 238 nm, 254 nm, and 280 nm. Specifically: from 0 - 10 min, the detection wavelength was 254 nm; from 10 - 27 min, the detection wavelength was 238 nm; from 27 - 33 min, the detection wavelength was 254 nm; from 33 - 50 min, the detection wavelength was 280 nm; from 50 - 54 min, the detection wavelength was 254 nm; from 54 - 65 min, the detection wavelength was 280 nm.

[0087] The mobile phase was acetonitrile - 0.1% phosphoric acid aqueous solution. Among them, phase A was acetonitrile and phase B was 0.1% phosphoric acid aqueous solution; the analysis time was 65 min; gradient elution was used.

[0088] The specific program for gradient elution was as follows:

[0089] From 0 - 5 min, the volume ratio of phase A to phase B was 5:95 - 10:90;

[0090] From 5 - 10 min, the volume ratio of phase A to phase B was 10:90 - 15:85;

[0091] From 10 - 15 min, the volume ratio of phase A to phase B was 15:85 - 20:80;

[0092] From 15 - 25 min, the volume ratio of phase A to phase B was 20:80 - 22:78;

[0093] From 25 - 40 min, the volume ratio of phase A to phase B was 22:78 - 30:70;

[0094] 40 - 55 min, volume ratio of phase A to phase B is 30:70 - 50:50;

[0095] 55 - 65 min, volume ratio of phase A to phase B is 50:50 - 68:32.

[0096] 3. Determination

[0097] Using the external standard method, a series of reference solutions with different volumes were respectively pipetted to prepare a series of solutions with different concentrations. The samples were analyzed by injecting them into a high-performance liquid chromatograph to plot the standard working curve. Then, the test solution obtained was analyzed by injecting it into the high-performance liquid chromatograph, and the analysis results were substituted into the standard working curve to obtain the contents of the 6 components in the test solution.

[0098] Specifically, a series of reference solutions with different volumes were respectively pipetted to prepare a series of solutions with different concentrations. The samples were analyzed by injecting them into a high-performance liquid chromatograph to obtain the linear relationship between the concentrations of the 6 components and the peak areas in the reference solutions. With the chromatographic peak area of each component corresponding to its respective concentration, the corresponding standard working curves were plotted, and the regression equations of each standard working curve were calculated. Then, the test solution was detected by a high-performance liquid chromatograph. The chromatographic peak areas of the 6 components in the obtained test solution were respectively substituted into the regression equations of the respective standard working curves to calculate the contents of the corresponding components. The specific chromatograms are shown in Figure 1 .

[0099] Example 2

[0100] 1. Sample pretreatment

[0101] Preparation of the test solution: The Gentian Liver-Clearing and Gallbladder-Draining Pills sample of batch 20240701 was crushed and passed through a No. 4 sieve. 0.5 g of the obtained sample powder was precisely weighed and placed in a stoppered conical flask. 24 mL of 76% methanol was precisely added, the stopper was tightly closed, and the weight was measured. It was ultrasonically extracted (power 100 W, frequency 40 kHz) for 32 minutes, allowed to stand and cool to room temperature, and then weighed again. The lost weight was made up with 76% methanol. It was filtered through a 0.45 μm microporous membrane, and the subsequent filtrate was taken to obtain the test solution 2#.

[0102] Preparation of the reference solution: The reference substances of geniposide acid, geniposide, baicalin, wogonoside, glycyrrhizic acid, and ligustilide were precisely weighed respectively and placed in the same volumetric flask. They were dissolved and diluted to the scale with 76% methanol and shaken well to prepare the reference stock solution. Then, the reference stock solution was diluted with 76% methanol respectively to prepare a series of reference solutions with different concentrations. The concentration ranges of the reference stock solution and the series of reference solutions with different concentrations were the same as those in step 1 of Example 1.

[0103] 2. Chromatographic conditions

[0104] The chromatographic conditions for high performance liquid chromatography are as follows: the chromatographic column is an Agilent TC-C 18 chromatographic column (4.6 mm × 250 mm, 5 μm); the detector is a photodiode array detector (DAD); the column temperature is 34 °C; the injection volume is 11 μL; the flow rate is 1.1 mL / min.

[0105] Analysis is carried out in a multi-wavelength manner, and the detection wavelengths are 237 nm, 253 nm and 279 nm. The specific conditions are as follows: from 0 to 10 min, the detection wavelength is 253 nm; from 10 to 27 min, the detection wavelength is 237 nm; from 27 to 33 min, the detection wavelength is 253 nm; from 33 to 50 min, the detection wavelength is 279 nm; from 50 to 54 min, the detection wavelength is 253 nm; from 54 to 65 min, the detection wavelength is 279 nm.

[0106] The mobile phase is acetonitrile - 0.11% phosphoric acid aqueous solution. Among them, phase A is acetonitrile and phase B is 0.11% phosphoric acid aqueous solution; the analysis time is 65 min; gradient elution is used.

[0107] The specific program for gradient elution is as follows:

[0108] From 0 to 5 min, the volume ratio of phase A to phase B is 4:96 - 11:89;

[0109] From 5 to 10 min, the volume ratio of phase A to phase B is 11:89 - 14:86;

[0110] From 10 to 15 min, the volume ratio of phase A to phase B is 14:86 - 21:79;

[0111] From 15 to 25 min, the volume ratio of phase A to phase B is 21:79 - 23:77;

[0112] From 25 to 40 min, the volume ratio of phase A to phase B is 23:77 - 31:69;

[0113] From 40 to 55 min, the volume ratio of phase A to phase B is 31:69 - 51:49;

[0114] From 55 to 65 min, the volume ratio of phase A to phase B is 51:49 - 69:31.

[0115] 3. Determination

[0116] The specific determination process is the same as step 3 in Example 1.

[0117] Example 3

[0118] 1. Sample pretreatment

[0119] Preparation of the test solution: The Gentian Liver-Clearing and Gallbladder-Draining Pills sample from batch 20240701 was crushed and passed through a No. 4 sieve. The obtained sample powder was accurately weighed at 0.5 g, placed in a stoppered conical flask, accurately added with 26 mL of 74% methanol, stoppered, weighed, ultrasonically extracted (power 100 W, frequency 40 kHz) for 28 minutes, allowed to stand and cool to room temperature, weighed again, and the lost weight was made up with 74% methanol. It was filtered through a 0.45 μm microporous membrane, and the subsequent filtrate was taken to obtain the test solution 3#.

[0120] Preparation of the reference solution: The reference substances of geniposidic acid, gardenoside, baicalin, wogonoside, glycyrrhizic acid, and ligustilide were accurately weighed respectively, placed in the same volumetric flask, dissolved with 74% methanol and diluted to the scale and shaken well to prepare the reference stock solution. Then the reference stock solution was diluted with 74% methanol respectively to prepare a series of reference solutions with different concentrations. The concentration ranges of the reference stock solution and the series of reference solutions with different concentrations were the same as those in step 1 of Example 1.

[0121] 2. Chromatographic conditions

[0122] The chromatographic conditions for high performance liquid chromatography were as follows: The chromatographic column was an Agilent TC-C 18 chromatographic column (4.6 mm × 250 mm, 5 μm); the detector was a photodiode array detector (DAD); the column temperature was 36°C; the injection volume was 9 μL; the flow rate was 0.9 mL / min.

[0123] Analysis was carried out using a multi-wavelength method, and the detection wavelengths were 239 nm, 255 nm, and 281 nm. Specifically: from 0 - 10 min, the detection wavelength was 255 nm; from 10 - 27 min, the detection wavelength was 239 nm; from 27 - 33 min, the detection wavelength was 255 nm; from 33 - 50 min, the detection wavelength was 281 nm; from 50 - 54 min, the detection wavelength was 255 nm; from 54 - 65 min, the detection wavelength was 281 nm.

[0124] The mobile phase was acetonitrile - 0.09% phosphoric acid aqueous solution, where phase A was acetonitrile and phase B was 0.09% phosphoric acid aqueous solution; the analysis time was 65 min; gradient elution was performed.

[0125] The specific program for gradient elution was as follows:

[0126] From 0 - 5 min, the volume ratio of phase A:phase B was 6:94 - 9:91;

[0127] From 5 - 10 min, the volume ratio of phase A:phase B was 9:91 - 16:84;

[0128] From 10 - 15 min, the volume ratio of phase A:phase B was 16:84 - 19:81;

[0129] 15 - 25 min, the volume ratio of phase A to phase B is 19:81 - 22:78;

[0130] 25 - 40 min, the volume ratio of phase A to phase B is 22:78 - 29:71;

[0131] 40 - 55 min, the volume ratio of phase A to phase B is 29:71 - 49:51;

[0132] 55 - 65 min, the volume ratio of phase A to phase B is 49:51 - 67:33.

[0133] 3. Determination

[0134] The specific determination process is the same as step 3 in Example 1.

[0135] Example 4

[0136] Accurately weigh the reference substances of geniposidic acid, gardenoside, baicalin, wogonoside, glycyrrhizic acid, and ligustilide respectively, place them in the same volumetric flask, dissolve with 75% methanol and dilute to the scale, shake well, and prepare a reference substance stock solution. In the reference substance stock solution, the content of geniposidic acid is 100.90 μg / mL; the content of gardenoside is 202.00 μg / mL; the content of baicalin is 579.60 μg / mL; the content of wogonoside is 204.80 μg / mL; the content of glycyrrhizic acid is 162.00 μg / mL; the content of ligustilide is 300.00 μg / mL.

[0137] Then dilute the reference substance stock solution with 75% methanol respectively to prepare a series of reference substance solutions with different concentrations. In a series of reference substance solutions with different concentrations, the content range of geniposidic acid is 6.31 - 100.90 μg / mL; the content range of gardenoside is 12.63 - 202.00 μg / mL; the content range of baicalin is 36.23 - 579.60 μg / mL; the content range of wogonoside is 12.80 - 204.80 μg / mL; the content range of glycyrrhizic acid is 10.13 - 162.00 μg / mL; the content range of ligustilide is 18.75 - 300.00 μg / mL.

[0138] Inject 10 μL for determination according to the chromatographic conditions in step 2 of Example 1. Take the peak area (Y) as the ordinate and the concentration of each reference substance (X, μg / ml) as the abscissa, plot the standard curve and perform regression calculation, and the linear relationship of each component is good. The specific results are shown in Table 2.

[0139] Table 2

[0140]

[0141] Example 5

[0142] 1. Precision

[0143] Take the reference substance stock solution of Example 4, and perform the detection according to Step 2 in Example 1 above. Inject samples continuously for 6 times respectively for analysis, record the peak areas of the chromatographic peaks of each component, and calculate the RSD% of the 6 components. The results show that the RSD% of the peak areas of the 6 components are all less than 1.7%, indicating that the precision of the instrument is good.

[0144] 2. Stability

[0145] Take the Gentian Liver-Clearing and Gallbladder-Draining Pills sample of Batch 20240701, prepare 1 portion of the test solution according to Step 1 in Example 1, perform the detection according to Step 2 in Example 1 above, inject samples at 0, 2, 4, 8, 12, 24, 36, and 48 h respectively, record the chromatogram, measure the peak area, and calculate the RSD of the peak areas of the contents of the 6 components. The results show that the RSD% of the peak areas of each component are all less than 2.4%, indicating that the test solution is stable within 48 h.

[0146] 3. Repeatability

[0147] Take the Gentian Liver-Clearing and Gallbladder-Draining Pills sample of Batch 20240701, prepare 6 portions of the test solution in parallel according to Step 1 in Example 1, perform the detection according to Step 2 in Example 1 above, and record the peak areas of the chromatographic peaks of each component. The results show that the RSD% of the peak areas of the contents of each component are all less than 2.4%, indicating that the method has good repeatability.

[0148] 4. Sample recovery

[0149] Take 9 portions of the Gentian Liver-Clearing and Gallbladder-Draining Pills sample of Batch 20240701, weigh each portion accurately about 0.25 g, add reference substances of geniposide acid, geniposide, baicalin, wogonoside, glycyrrhizic acid, and ligustilide at three different concentration levels respectively, prepare the test solution according to Step 1 in Example 1, perform the detection according to Step 2 in Example 1 above, record the chromatogram, and calculate the sample recovery and RSD% of each component according to the measured amount and the added amount. The results are shown in Table 3. It can be seen from Table 3 that the average recoveries of geniposide acid, geniposide, baicalin, wogonoside, glycyrrhizic acid, and ligustilide are all between 97% and 102%, and the RSD% are all less than 3%, indicating that the accuracy of the method is good.

[0150] Table 3

[0151]

[0152]

[0153] Example 6

[0154] 1. Sample pretreatment

[0155] Preparation of the test solution: The preparation process of the test solution is the same as that in Step 1 of Example 1.

[0156] Preparation of the reference solution: The preparation process of the reference solution is the same as that in Step 1 of Example 1.

[0157] 2. Chromatographic conditions

[0158] The chromatographic conditions of the high performance liquid chromatography are the same as those in Step 2 of Example 1.

[0159] 3. Determination

[0160] Using the high performance liquid chromatography under the chromatographic conditions in Step 2 above, the test solution and the reference solution in Step 1 above are respectively determined to obtain the fingerprint of the test solution and the fingerprint of the reference solution. Among them, the fingerprint of the test solution should be compared with the fingerprint of the reference solution. According to the known characteristic peaks in the fingerprint of the reference solution, the corresponding characteristic peaks in the fingerprint of the test solution are identified through the relative retention time, so as to locate the target components in the fingerprint of the test solution and obtain the fingerprint of Longdan Xiegan Pills.

[0161] In summary, a method for fingerprint and multi-component content determination of Longdan Xiegan Pills provided by the present invention can quantitatively analyze 6 components in Longdan Xiegan Pills and establish the fingerprint of Longdan Xiegan Pills, providing reference for scientifically and comprehensively controlling the quality of Longdan Xiegan Pills. Therefore, the present invention overcomes various shortcomings in the prior art and has high industrial utilization value.

[0162] The above embodiments are only illustrative of the principles and effects of the present invention, and are not used to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes completed by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A method for determining the contents of multiple components in Longdan Xiegan Pills, comprising the following steps: 1) Preparation of the test solution: Add the Longdan Xiegan Pills sample to a solvent for dissolution, perform ultrasonic extraction, cool it down, filter and take the subsequent filtrate to obtain the test solution; 2) Preparation of the reference solution: Dissolve one or more reference substances of geniposide acid, geniposide, baicalin, wogonoside, glycyrrhizic acid, and ligustilide in a solvent and make up the volume to obtain the reference solution; 3) Determination: Use high-performance liquid chromatography to separately determine the test solution in step 1) and the reference solution in step 2), and calculate the contents of one or more components of geniposide acid, geniposide, baicalin, wogonoside, glycyrrhizic acid, and ligustilide in the test solution by the external standard method.

2. The method for determining the contents of multiple components in Longdan Xiegan Pills according to claim 1, characterized in that, In step 1), any one or more of the following conditions are included: 11) The Longdan Xiegan Pills sample is a powdered sample after being crushed and sieved; preferably, the sieve for sieving is a No. 3 - 5 sieve; 12) The ratio of the weight of the added Longdan Xiegan Pills sample to the volume of the added solvent is 0.4 - 0.6: 20 - 30, g / mL; 13) The ultrasonic extraction time is 25 - 35 min; 14) The power of the ultrasonic extraction is 90 - 110 W; the frequency of the ultrasonic extraction is 30 - 50 kHz; 15) The cooling is to let it stand and cool to room temperature; 16) After cooling, weigh it and then make up the weight. The solvent used for making up the weight is an aqueous solution containing 70 - 80% methanol by volume percentage; 17) The filtration is by membrane filtration; preferably, the membrane is a 0.45 μm membrane; 18) The solvent is an aqueous solution containing 70 - 80% methanol by volume percentage.

3. The method for determining the contents of multiple components in Longdan Xiegan Pills according to claim 1, characterized in that, In step 2), any one or more of the following conditions are included: 21) The solvent is an aqueous solution containing 70 - 80% methanol by volume percentage; 22) The content range of geniposide acid in the reference solution is 6.31 - 100.90 μg / mL; the content range of geniposide is 12.63 - 202.00 μg / mL; the content range of baicalin is 36.23 - 579.60 μg / mL; the content range of wogonoside is 12.80 - 204.80 μg / mL; the content range of glycyrrhizic acid is 10.13 - 162.00 μg / mL; the content range of ligustilide is 18.75 - 300.00 μg / mL.

4. The method for determining the contents of multiple components in Longdan Xiegan Pills according to claim 1, characterized in that, In step 3), the high-performance liquid chromatography includes any one or more of the following conditions: 31) The chromatographic column is C 18 Chromatographic column; 32) The detector is a photodiode array detector; 33) The column temperature is 30 - 40 °C; 34) The injection volume is 8 - 12 μL; 35) The flow rate is 0.5 - 1.5 mL / min; 36) The detection wavelength is selected from one or more combinations of 236 - 240 nm, 252 - 256 nm, and 278 - 282 nm; 37) The mobile phase is acetonitrile - 0.09 - 0.11% phosphoric acid aqueous solution; in the acetonitrile - 0.09 - 0.11% phosphoric acid aqueous solution, phase A is acetonitrile and phase B is 0.09 - 0.11% phosphoric acid aqueous solution; 38) Gradient elution is adopted and the analysis time is 65 min.

5. The method for determining the contents of multiple components in Longdan Xiegan Pills according to claim 4, characterized in that, In item (31), the chromatographic column is an Agilent TC-C 18 chromatographic column, 4.6 mm × 250 mm, 5 μm.

6. The method for determining the contents of multiple components in Longdan Xiegan Pills according to claim 4, wherein, In item 36), the detection wavelengths are 236 - 240 nm, 252 - 256 nm, and 278 - 282 nm.

7. The method for determining the contents of multiple components in Longdan Xiegan Pills according to claim 6, wherein The detection wavelengths vary with the time of the gradient elution program. Specifically, the variations are as follows: from 0 - 10 min, the detection wavelength is 252 - 256 nm; from 10 - 27 min, the detection wavelength is 236 - 240 nm; from 27 - 33 min, the detection wavelength is 252 - 256 nm; from 33 - 50 min, the detection wavelength is 278 - 282 nm; from 50 - 54 min, the detection wavelength is 252 - 256 nm; from 54 - 65 min, the detection wavelength is 278 - 282 nm.

8. The method for determining the contents of multiple components in Longdan Xiegan Pills according to claim 4, characterized in that, In item 38), the specific program of the gradient elution is as follows: From 0 - 5 min, the volume ratio of phase A to phase B is 4 - 6:94 - 96 - 9 - 11:89 - 91; From 5 - 10 min, the volume ratio of phase A to phase B is 9 - 11:89 - 91 - 14 - 16:84 - 86; From 10 - 15 min, the volume ratio of phase A to phase B is 14 - 16:84 - 86 - 19 - 21:79 - 81; From 15 - 25 min, the volume ratio of phase A to phase B is 19 - 21:79 - 81 - 22 - 23:77 - 78; From 25 - 40 min, the volume ratio of phase A to phase B is 22 - 23:77 - 78 - 29 - 31:69 - 71; From 40 - 55 min, the volume ratio of phase A to phase B is 29 - 31:69 - 71 - 49 - 51:49 - 51; From 55 - 65 min, the volume ratio of phase A to phase B is 49 - 51:49 - 51 - 67 - 69:31 - 33.

9. Use of the method for determining the contents of multiple components in Longdan Xiegan Pills according to any one of claims 1 - 8 in the detection of the fingerprint of Longdan Xiegan Pills.

10. A method for detecting the fingerprint of Longdan Xiegan Pills, comprising the following steps: A) Preparation of the test solution: The same as step 1) of the method for determining the contents of multiple components in Longdan Xiegan Pills according to any one of claims 1 - 8; B) Preparation of the reference solution: The same as step 2) of the method for determining the contents of multiple components in Longdan Xiegan Pills according to any one of claims 1 - 8; C) Determination: Using high - performance liquid chromatography under the same chromatographic conditions as step 3) of the method for determining the contents of multiple components in Longdan Xiegan Pills according to any one of claims 1 - 8, respectively determine the test solution in step A) and the reference solution in step B), obtain the fingerprint of the test solution and the fingerprint of the reference solution, compare the fingerprint of the test solution with the fingerprint of the reference solution, and conduct attribution and localization of the target components in the fingerprint of the test solution, thereby obtaining the fingerprint of Longdan Xiegan Pills.

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