Method for detecting and quantitatively analyzing multiple components in a deflating granule

Abstract

The present application relates to the technical field of traditional Chinese medicine analysis, in particular to a detection method and quantitative analysis method for multiple components in Xiaoshang granules. Liquid chromatography-mass spectrometry (LC-MS) is used to identify the chemical components of Xiaoshang granules, and high performance liquid chromatography (HPLC) is used to determine the contents of caffeic acid, wild scutellarein, aurantiamarin and hesperidin in Xiaoshang granules. A total of 70 compounds are identified from Xiaoshang granules. The linear relationships of the four index components are good within their respective ranges, the average sample recovery rates are 95.84-102.08%, and the RSDs are 0.84-2.27%. The established LC-MS qualitative method is simple and efficient, and can be used for rapid identification of the chemical components in Xiaoshang granules. The HPLC quantitative method is accurate and specific, and can be used for determination of the contents of caffeic acid, wild scutellarein, aurantiamarin and hesperidin in Xiaoshang granules.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine analysis technology, and in particular to a method for detecting and quantitatively analyzing multiple components in anti-inflammatory granules. Background Technology

[0002] Abdominal distension is a common symptom of digestive tract disorders, associated with functional gastrointestinal inflammation and organic diseases, with or without related symptoms. Studies show that approximately 15% to 30% of the global population experiences abdominal distension. Traditional Chinese medicine often attributes the pathogenesis of abdominal distension to impaired Qi flow and dysfunction of the digestive system. Clinical treatment primarily involves acupuncture, moxibustion, massage, and cupping. Herbal medicine treatment mainly focuses on strengthening the spleen and stomach, regulating Qi, and relieving distension.

[0003] The anti-bloating granules are composed of eight single herbs: areca nut, dried tangerine peel, germinated barley, patchouli, malt, costus root, citron, and perilla leaf. Areca nut is bitter and pungent, warm in nature, and enters the stomach and large intestine meridians. It has pharmacological effects such as anthelmintic, anti-inflammatory, improving gastrointestinal function, lowering blood lipids, preventing arteriosclerosis, and antidepressant effects. Dried tangerine peel is bitter and pungent, warm in nature, and enters the lung and spleen meridians. It has the effects of regulating qi and strengthening the spleen, drying dampness and resolving phlegm, and is mainly used to treat abdominal distension, poor appetite, vomiting and diarrhea, and cough with excessive phlegm. Germinated barley is sweet and warm in nature, and enters the spleen and stomach meridians. It has the effects of promoting digestion and strengthening the spleen and stomach, and is often used to treat indigestion and spleen and stomach weakness. Patchouli is pungent and slightly warm in nature, and enters the spleen, stomach, and lung meridians. It has the effects of stimulating appetite, stopping vomiting, releasing exterior pathogens, and relieving summer heat. Malt is sweet and neutral in nature, and enters the spleen and stomach meridians. It has the effects of strengthening the stomach, promoting digestion, and suppressing lactation. The aroma of *Citrus aurantium* is pungent, bitter, and warm in nature. It enters the spleen, stomach, large intestine, triple burner, and gallbladder meridians. It promotes gastrointestinal motility, stops diarrhea, protects the gastric mucosa, has anti-ulcer effects, and promotes bile secretion. Citron is pungent, bitter, and sour in taste, and warm in nature. It enters the liver, spleen, and lung meridians. It has the effects of soothing the liver and regulating qi, resolving phlegm, and anti-oxidation. Perilla leaves are pungent and warm in nature. They enter the lung and spleen meridians. They have the effects of regulating qi and stomach, anti-inflammatory effects, and anti-oxidation.

[0004] Xiaozhang Granules (also known as YaYa Xiaozhang Granules) have been approved by the Shanghai Municipal Drug Administration and have obtained the "Registration Certificate for Traditional Chinese Medicine Preparations Prepared by Medical Institutions Using Traditional Processes". This granule preparation is a hospital preparation, and its basic research is still in its early stages. Currently, there are no comprehensive qualitative and quantitative detection methods for the components of Xiaozhang Granules. Summary of the Invention

[0005] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a method for detecting and quantitatively analyzing multiple components in anti-swelling granules, in order to solve the problems in the prior art.

[0006] To achieve the above and other related objectives, the present invention first provides a method for detecting multiple components in anti-swelling granules, comprising the following steps:

[0007] 1) Dissolve the anti-swelling granule sample in a solvent, sonicate it, filter it, and collect the filtrate to obtain the test solution;

[0008] 2) The test solution was analyzed by liquid chromatography-mass spectrometry to determine the components in the test solution.

[0009] This invention also provides a method for detecting the content of caffeic acid, baicalin, rutin, or hesperidin in anti-swelling granules, comprising the following steps:

[0010] 1) Prepare the test solution according to step 1) of the above-mentioned detection method for multiple components in anti-swelling granules;

[0011] 2) Prepare reference solutions of caffeic acid, baicalin, rutin, and hesperidin;

[0012] 3) The test solution and the reference solution were determined by high performance liquid chromatography to determine the contents of caffeic acid, baicalin, rutin, and hesperidin in the test solution.

[0013] As described above, the method for detecting and quantitatively analyzing multiple components in anti-swelling granules of the present invention has the following beneficial effects:

[0014] 1) This invention identified 70 compounds from the anti-swelling particles;

[0015] 2) The four index components exhibit good linear relationships within their respective ranges (R0). 2 The average recovery rate was 95.84–102.08%, and the RSD was 0.84–2.27%. (>0.9996).

[0016] 3) The LC-MS qualitative method established in this invention is simple and efficient, and can be used to quickly identify the chemical components in the anti-swelling granules; the HPLC quantitative method is accurate and specific, and can be used to determine the content of caffeic acid, baicalin, rutin, and hesperidin in the anti-swelling granules. Attached Figure Description

[0017] Figure 1 The image shown is a superimposed diagram of the extracted ion flow of the deswelling particles in positive and negative modes in Embodiment 1 of the present invention.

[0018] Figures 2-4 The results shown are the identification results obtained from identifying the main compounds in the swelling-reducing granules.

[0019] Figure 5 The image shows the HPLC chromatograms of the blank solvent, mixed reference standard, and test solution in Example 2 of this invention at a wavelength of 280 nm.

[0020] Figure 6The linear relationship of each component is shown in Embodiment 2 of the present invention.

[0021] Figure 7 The results are shown in the sample recovery experiment of Example 2 of this invention.

[0022] Figure 8 The results show the content determination of each component in the anti-swelling granules in Example 2 of this invention. Detailed Implementation

[0023] This study used liquid chromatography-mass spectrometry (LC-MS) to identify the main chemical components in the anti-swelling granules; at the same time, high performance liquid chromatography (HPLC) was used to establish a quantitative method for four indicator components in the anti-swelling granules: caffeic acid, baicalin, rutin, and hesperidin, in order to provide theoretical and experimental basis for the basic chemical research and quality standard establishment of this compound preparation.

[0024] The present invention first provides a method for detecting multiple components in anti-swelling granules, comprising the following steps:

[0025] 1) Dissolve the anti-swelling granule sample in a solvent, sonicate it, filter it, and collect the filtrate to obtain the test solution;

[0026] 2) The test solution was analyzed by liquid chromatography-mass spectrometry to determine the components in the test solution.

[0027] In some embodiments of the present invention, in step 1), the solvent is an aqueous methanol solution. The volume fraction of the aqueous methanol solution is 60-80%. The volume fraction of the aqueous methanol solution is selected from any of the following ranges: 60-64%, 64-68%, 68-72%, 72-76%, 76-80%. In a preferred embodiment of the present invention, the volume fraction of the aqueous methanol solution is 68-72%. In a specific embodiment of the present invention, the volume fraction of the aqueous methanol solution is 70%.

[0028] In some embodiments of the present invention, in step 1), the mass ratio of the deswelling granule sample to the volume of the solvent is 0.2 g : (8~12) mL. The mass ratio of the deswelling granule sample to the volume of the solvent is selected from any of the following: 0.2 g : (8~9) mL, 0.2 g : (9~10) mL, 0.2 g : (10~11) mL, 0.2 g : (11~12) mL. In a specific embodiment of the present invention, the mass ratio of the deswelling granule sample to the volume of the solvent is 0.2 g : 10 mL.

[0029] In some embodiments of the present invention, in step 1), the ultrasonic treatment time is 20-40 min. The ultrasonic treatment time is selected from any of the following ranges: 20-25 min, 25-30 min, 30-35 min, 35-40 min. In a specific embodiment of the present invention, the ultrasonic treatment time is 30 min.

[0030] In some embodiments of the present invention, in step 2), the chromatographic column used in the liquid chromatography-mass spectrometry (LC-MS) method is a C18 column. In a preferred embodiment of the present invention, the C18 column is an Agilent Poroshell 120 EC-C18 column.

[0031] In some embodiments of the present invention, in step 2), the mobile phase A in the liquid chromatography-mass spectrometry method is acetonitrile.

[0032] In some embodiments of the present invention, in step 2), the mobile phase B in the liquid chromatography-mass spectrometry (LC-MS) method is a 0.05-0.20% (v / v) formic acid aqueous solution. The mobile phase B is selected from any of the following: 0.05-0.10% (v / v) formic acid aqueous solution, 0.10-0.15% (v / v) formic acid aqueous solution, or 0.15-0.20% (v / v) formic acid aqueous solution. In a specific embodiment of the present invention, the mobile phase B is a 0.1% (v / v) formic acid aqueous solution.

[0033] In some embodiments of the present invention, in step 2), the analysis time in the liquid chromatography-mass spectrometry method is 50 min.

[0034] In some embodiments of the present invention, in step 2), the elution method in the liquid chromatography-mass spectrometry is gradient elution.

[0035] In some embodiments of the present invention, the gradient elution program is as follows: 0-8 min, A phase:B phase volume ratio is 5:95-10:90; 8-14 min, A phase:B phase volume ratio is 10:90-12:88; 14-28 min, A phase:B phase volume ratio is 12:88-18:82; 28-38 min, A phase:B phase volume ratio is 18:82-30:70; 38-50 min, A phase:B phase volume ratio is 30:70-100:0.

[0036] In some embodiments of the present invention, in step 2), the column temperature in the liquid chromatography-mass spectrometry (LC-MS) method is 30-40°C. The column temperature is selected from any of the following ranges: 30-35°C, 35-40°C. In a specific embodiment of the present invention, the column temperature is 35°C.

[0037] In some embodiments of the present invention, in step 2), the flow rate of the liquid chromatography-mass spectrometry (LC-MS) is 0.2–0.6 mL / min. The flow rate is selected from any of the following ranges: 0.2–0.3 mL / min, 0.3–0.4 mL / min, 0.4–0.5 mL / min, or 0.5–0.6 mL / min. In a specific embodiment of the present invention, the flow rate is 0.4 mL / min.

[0038] In some embodiments of the present invention, in step 2), the injection volume in the liquid chromatography-mass spectrometry (LC-MS) method is 15-25 μL. The injection volume is selected from any of the following ranges: 15-17 μL, 17-19 μL, 19-21 μL, 21-23 μL, or 23-25 ​​μL. In a specific embodiment of the present invention, the injection volume is 20 μL.

[0039] In some embodiments of the present invention, in step 2), the ion source in the liquid chromatography-mass spectrometry method is an electrospray ionization (ESI) source.

[0040] In some embodiments of the present invention, in step 2), the detection mode of the liquid chromatography-mass spectrometry method is positive ion electrospray ionization mode (ESI). + and negative ion electrospray ionization mode ESI - .

[0041] In some embodiments of the present invention, in step 2), the drying gas in the liquid chromatography-mass spectrometry method is nitrogen.

[0042] In some embodiments of the present invention, in step 2), the flow rate of the drying gas in the liquid chromatography-mass spectrometry (LC-MS) method is 8-12 L / min. In a specific embodiment of the present invention, the flow rate of the drying gas is 10 L / min.

[0043] In some embodiments of the present invention, in step 2), the temperature of the drying gas in the liquid chromatography-mass spectrometry (LC-MS) method is 320~380°C. The temperature of the drying gas is selected from any of the following ranges: 320~330°C, 330~340°C, 340~350°C, 350~360°C, 360~370°C, or 370~380°C. In a specific embodiment of the present invention, the temperature of the drying gas is 350°C.

[0044] In some embodiments of the present invention, in step 2), the nebulizer pressure of the drying gas in the liquid chromatography-mass spectrometry (LC-MS) method is 25-35 psi. The nebulizer pressure of the drying gas is selected from any of the following ranges: 25-27 psi, 27-29 psi, 29-31 psi, 31-33 psi, or 33-35 psi. In a specific embodiment of the present invention, the nebulizer pressure of the drying gas is 30 psi.

[0045] In some embodiments of the present invention, in step 2), the capillary voltage in the liquid chromatography-mass spectrometry (LC-MS) method is 3400~3600V. The capillary voltage is selected from any of the following ranges: 3400~3450V, 3450~3500V, 3500~3550V, or 3550~3600V. In a preferred embodiment of the present invention, the capillary voltage is 3450~3550V. In a specific embodiment of the present invention, the capillary voltage is 3500V.

[0046] In some embodiments of the present invention, in step 2), the Skimmer voltage in the liquid chromatography-mass spectrometry (LC-MS) method is 60-70V. The Skimmer voltage is selected from any of the following ranges: 60-62V, 62-64V, 64-66V, 66-68V, 68-70V. For example, it can be 60V, 61V, 62V, 63V, 64V, 65V, 66V, 67V, 68V, 69V, or 70V. In a specific embodiment of the present invention, the Skimmer voltage is 65V.

[0047] In some embodiments of the present invention, in step 2), the octapole radio frequency voltage in the liquid chromatography-mass spectrometry (LC-MS) method is 700-800V. The octapole radio frequency voltage is selected from any of the following ranges: 700-720V, 720-740V, 740-760V, 760-780V, or 780-800V. For example, the octapole radio frequency voltage is 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, or 800V. In a specific embodiment of the present invention, the octapole radio frequency voltage can be 750V.

[0048] In some embodiments of the present invention, in step 2), the capillary outlet voltage in the liquid chromatography-mass spectrometry (LC-MS) method is 120-130V. The capillary outlet voltage is selected from any of the following ranges: 120-122V, 122-124V, 124-126V, 126-128V, or 128-130V. For example, the capillary outlet voltage is 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, or 130V. In a specific embodiment of the present invention, the capillary outlet voltage can be 125V.

[0049] In some embodiments of the present invention, in step 2), the liquid chromatography-mass spectrometry method is described in which the primary mass spectrometry data is acquired in full scan mode (Full MS), and the secondary fragment data of the compound is acquired in automatic secondary ion mode (Auto MS / MS).

[0050] In some embodiments of the present invention, in step 2), the mass-to-charge ratio scanning range of the liquid chromatography-mass spectrometry (LC-MS) method is 50–1200 m / z. The mass-to-charge ratio scanning range is selected from any of the following: 50–100, 100–200, 200–300, 300–400, 400–500, 500–600, 600–700, 700–800, 800–900, 900–1000, 1000–1100, or 1100–1200. For example, the mass-to-charge ratio scanning range can be 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150 or 1200 m / z.

[0051] In some embodiments of the present invention, in step 2), the collision voltage in the liquid chromatography-mass spectrometry (LC-MS) method is 25-35 eV. The collision voltage is selected from any of the following ranges: 25-30 eV, 30-35 eV. For example, the collision voltage can be 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 eV. In a specific embodiment of the present invention, the collision voltage can be 30 eV.

[0052] In some embodiments of the present invention, in step 2), the components of the test solution are selected from arginine, lysine, asparagine, aspartic acid, alanine, γ-aminobutyric acid, threonine, glutamine, glutamic acid, glucose, maltose, proline, adenine, valine, arecoline, arecoline*, adenosine, tyrosine, synephrine, leucine, N-methyltyramine, phenylalanine, syringic acid, protocatechuic acid, neochlorogenic acid*, tryptophan, proanthocyanidin B2, p-hydroxybenzoic acid, chlorogenic acid*, catechin, cryptochlorogenic acid, caffeic acid*, epicatechin*, luteolin-7-O-rutin, p-coumaric acid, citric acid, etc. The following are one or more of the following: naringin, ferulic acid*, luteolin*, sennain, rutin, luteolin, baicalin*, naringenin-7-O-glucoside, rutin, apigenin-7-glucuronic acid, hesperidin*, isochromine, rosmarinic acid hydrated oxypenicillin, quercetin*, limonin, isochromine, sennain, robinin, sennain, costunolide*, kaempferol*, hesperidin, patchouli, 5,7-dimethoxycoumarin, sennain, limonene, nomiline, isohesperidin, citrulline, berberine, demethylnodohesperidin, or dehydrocostunolide*.

[0053] This invention also provides a method for detecting the content of caffeic acid, baicalin, rutin, or hesperidin in anti-swelling granules, comprising the following steps:

[0054] 1) Prepare the test solution according to step 1) of the above-mentioned detection method for multiple components in anti-swelling granules;

[0055] 2) Prepare reference solutions of caffeic acid, baicalin, rutin, and hesperidin;

[0056] 3) The test solution and the reference solution were determined by high performance liquid chromatography to determine the contents of caffeic acid, baicalin, rutin, and hesperidin in the test solution.

[0057] In some embodiments of the present invention, in step 3), the chromatographic column used in the high-performance liquid chromatography is a C18 column. In a preferred embodiment of the present invention, the C18 column is an Agilent Poroshell 120EC-C18 column.

[0058] In some embodiments of the present invention, in step 3), the mobile phase A in the high performance liquid chromatography is acetonitrile.

[0059] In some embodiments of the present invention, in step 3), the mobile phase B in the high-performance liquid chromatography is a 0.05-0.15% (v / v) aqueous solution of phosphoric acid. In a preferred embodiment of the present invention, the mobile phase B is a 0.1% (v / v) aqueous solution of phosphoric acid.

[0060] In some embodiments of the present invention, in step 3), the analysis time in the high performance liquid chromatography is 50 min.

[0061] In some embodiments of the present invention, in step 3), the elution method in the high performance liquid chromatography is gradient elution.

[0062] Furthermore, the gradient elution program is as follows: 0-8 min, A phase:B phase volume ratio is 5:95-10:90; 8-14 min, A phase:B phase volume ratio is 10:90-12:88; 14-28 min, A phase:B phase volume ratio is 12:88-18:82; 28-38 min, A phase:B phase volume ratio is 18:82-30:70; 38-50 min, A phase:B phase volume ratio is 30:70-100:0.

[0063] In some embodiments of the present invention, in step 3), the flow rate of the high-performance liquid chromatography is 0.3 to 0.5 mL / min. The flow rate is selected from any of the following ranges: 0.3 to 0.4 mL / min, 0.4 to 0.5 mL / min. In a specific embodiment of the present invention, the flow rate is 0.4 mL / min.

[0064] In some embodiments of the present invention, in step 3), the column temperature in the high-performance liquid chromatography is 30-40°C. The column temperature is selected from any of the following temperatures: 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40°C. In a specific embodiment of the present invention, the column temperature is 35°C.

[0065] In some embodiments of the present invention, in step 3), the injection volume in the high-performance liquid chromatography is 4-6 μL. In a specific embodiment of the present invention, the injection volume is 5 μL.

[0066] In some embodiments of the present invention, in step 3), the ultraviolet detection wavelength in the high performance liquid chromatography is 280 nm.

[0067] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand 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 embodiments, and 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.

[0068] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0069] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0070] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent companies. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0071] The instruments, equipment, and reagents used in the embodiments of this application are as follows:

[0072] 1. Instruments:

[0073] AL104 analytical balance (Mettler-Toledo, Switzerland); EPED-E2-10TF laboratory ultrapure water system (Nanjing Yipuyida Technology Development Co., Ltd.); KS-500DE CNC ultrasonic cleaner (Kunshan Jielimei Ultrasonic Instrument Co., Ltd.); Agilent 1290 UPLC-6530 QTOF-MS liquid chromatography-mass spectrometry system (Agilent Technologies, USA); ThermoFisher Scientific Vanquish high performance liquid chromatograph (Thermo Fisher Scientific, USA) equipped with a diode array detector.

[0074] 2. Reagents and Drugs

[0075] Six batches of anti-swelling granules were collected, with batch numbers 20230301, 20230302, 20230303, 20230401, 20230402, and 20230403.

[0076] Areca nut, dried tangerine peel, germinated barley, patchouli, malt, costus root, citron, and perilla leaves are sourced from Shanghai Wanshicheng Traditional Chinese Medicine Products Co., Ltd.

[0077] Hesperidin (batch number PCS0167, purity ≥98%) was purchased from Chengdu Zhibiao Chemical Pure Biotechnology Co., Ltd.; epicatechin (batch number 013375460, purity ≥98%), chlorogenic acid (batch number 01061375, purity ≥98%), HBr arecoline (batch number 014215479, purity ≥98%), and baicalin (batch number 013232291, purity ≥98%) were all purchased from Shanghai Titan Technology Co., Ltd.; luteolin (batch number HS21053B1, purity ≥98%) was purchased from Baoji Chenguang Biotechnology Co., Ltd.; caffeic acid (batch number PRF7102121, purity ≥98%) was purchased from Chengdu Purifa Technology Development Co., Ltd.; quercetin (batch number H-009-170516, purity >98%), kaempferol ( The following products were purchased from Chengdu Ruifensi Biotechnology Co., Ltd.: batch number S-014-171216 (purity ≥98%); tangeretin (batch number YRC008-220101, purity ≥98%) and neochlorogenic acid (batch number WKQ21010802, purity ≥98%); dehydroausyl lactone (batch number 111525-200907, purity 100%) and ferulic acid (batch number JMRRZRSB, purity ≥99.7%); China National Institutes for Food and Drug Control; aucubin (batch number DSTD002201, purity ≥98%); and rutin (batch number PS011583, purity ≥98%). Formic acid and acetonitrile for mass spectrometry were of mass spectrometry grade and purchased from Merck AG, Germany; acetonitrile was of chromatographic grade and purchased from Beijing Bailingwei Technology Co., Ltd.; methanol was of analytical grade and purchased from Shanghai Wohua Chemical Co., Ltd.; phosphoric acid was of analytical grade and purchased from Shanghai Titan Technology Co., Ltd.; and water was ultrapure water.

[0078] Example 1

[0079] 1. Method

[0080] 1.1 Solution Preparation

[0081] 1.1.1 Preparation of reference solution

[0082] Accurately weigh appropriate amounts of dehydroauric acid lactone, auric acid lactone, hesperidin, chlorogenic acid, neochlorogenic acid, novocheriin, kaempferol, ferulic acid, arecoline, epicatechin, luteolin, caffeic acid, and quercetin. Dissolve them in methanol and then dilute to prepare a mixed reference solution with a mass concentration of approximately 50 µg / mL for LC-MS identification.

[0083] Accurately weigh appropriate amounts of caffeic acid, baicalin, naringin, and hesperidin reference standards, place them in the same volumetric flask, dissolve them in methanol, and dilute to volume to prepare mixed reference standard stock solutions with mass concentrations of 0.0870, 0.2025, 1.103, and 3.562 mg / mL, respectively. Then, serially dilute with methanol to obtain a series of mixed reference standard solutions, in which the mass concentrations of caffeic acid were 87.0, 43.5, 17.4, 8.70, and 4.35 μg / mL, the mass concentrations of baicalin were 202.5, 101.2, 40.50, 20.25, and 10.12 μg / mL, respectively, and the mass concentrations of naringin were 1103, 551.5, 220.6, 110.3, and 55.15 μg / mL, respectively. The mass concentrations of hesperidin were 3562, 1781, 712.4, 356.2, and 178.1 μg / mL, respectively.

[0084] 1.1.2 Test solution

[0085] Accurately weigh 0.2 g of this product and place it in a stoppered conical flask. Accurately add 10 mL of 70% methanol, weigh the sample, sonicate for 30 min, cool, and replenish the lost mass with 70% methanol. Shake well and filter through a 0.22 µm microporous membrane. Use the filtrate for subsequent qualitative and quantitative analysis.

[0086] Accurately weigh 0.2g each of areca nut, dried tangerine peel, germinated rice, patchouli, malt, costus root, citron, and perilla leaf, and prepare a test solution using the method described above. Take the filtrate for single-herb component attribution analysis.

[0087] 1.2 LC-MS Analysis Method

[0088] 1.2.1 Chromatographic conditions

[0089] The chromatographic column was an Agilent Poroshell 120 EC-C. 18 (3.0 mm × 150 mm, 2.7 µm); mobile phase was acetonitrile (A) - 0.1% formic acid aqueous solution (B), gradient elution (0–8 min, 5%–10% A; 8–14 min, 10%–12% A; 14–28 min, 12%–18% A; 28–38 min, 18%–30% A; 38–50 min, 30%–100% A); flow rate 0.4 mL·min -1 Column temperature: 35 ℃; Injection volume: 20 µL.

[0090] 1.2.2 Mass Spectrometry Conditions

[0091] Electrospray ionization (ESI) was used for scanning in both positive and negative ion modes. Primary mass spectrometry data were acquired in full scan mode (Full MS), while secondary fragment data of the compounds were acquired in automatic secondary ionization mode (Auto MS / MS). The optimized mass spectrometry conditions were as follows: nitrogen as the drying gas; nitrogen flow rate 10 L / min; nitrogen temperature 350 °C; nebulizer gas pressure 30 psi; capillary voltage 3500 V; Skimmer voltage 65 V; octapole RF voltage 750 V; capillary outlet voltage 125 V; collision voltage 30 eV; mass-to-charge ratio scan range: m / z 50–1200.

[0092] 1.3 HPLC Quantitative Method

[0093] 1.3.1 Chromatographic conditions

[0094] The chromatographic column was the same as described in section 1.2.1; the mobile phase was acetonitrile (A)-0.1% phosphoric acid aqueous solution (B), with gradient elution (0–8 min, 5%–10% A; 8–14 min, 10%–12% A; 14–28 min, 12%–18% A; 28–38 min, 18%–30% A; 38–50 min, 30%–100% A); the flow rate was 0.4 mL / min. -1 Column temperature: 35 ℃; injection volume: 5 µL; UV detection wavelength: 280 nm.

[0095] 2. Results

[0096] 2.1 UPLC-Q-TOF-MS / MS component identification

[0097] Take an appropriate amount of each test solution from section 1.1.2 and inject it into the LC-MS for analysis to obtain the following results: Figure 1 The image shows a superimposed plot of extracted ion currents at different intensities under positive and negative modes for the anti-swelling granules. By consulting databases such as CNKI, PubMed, SciFinder, and TCMID (Traditional Chinese Medicine Integrated Database) and relevant literature, the main chemical components that may be present in the anti-swelling granules were identified, and a database was established. The main compounds in the anti-swelling granules were identified by combining information such as retention time of reference standards, precise mass-to-charge ratio of quasi-molecular ion peaks, and secondary fragments. The identification results are shown below. Figures 2-4 .

[0098] 2.2 Classification of Components of Single Herbs

[0099] The compounds identified by liquid chromatography-mass spectrometry (LC-MS) analysis contained 30 flavonoids, 11 phenolic acids, 15 amino acids, 5 alkaloids, 3 coumarins, 2 sugars, and 4 other compounds. Furthermore, the 70 identified compounds were assigned as single-herb components: 19 from areca nut, 27 from malt, 19 from dried tangerine peel, 19 from germinated barley, 2 from costus root, 12 from patchouli, 17 from citron, and 17 from perilla leaf.

[0100] Example 2: Investigation of HPLC Quantitative Methodology

[0101] 2.1 Specificity test

[0102] Take appropriate amounts of the reference standard and test solution, and inject them for determination under the conditions specified in sections "1.2" and "1.3". The results are shown in the table below. Figure 5 The good separation of the components indicates that the method has good specificity.

[0103] 2.2 Examination of Linear Relationships

[0104] Accurately pipette an appropriate amount of the reference standard stock solution from section "1.1", add 70% methanol, and dilute sequentially to prepare a series of standard solutions with different mass concentrations. Inject and determine the solutions under the conditions described in section "1.3". After linear fitting with the reference standard mass concentration as the abscissa (X) and peak area as the ordinate (Y), standard curves for the corresponding index components are obtained. The results are shown in Table 2. Each index component exhibits good linearity within its respective mass concentration range. The mixed reference standard solution is serially diluted and injected and determined under the conditions described in section "1.3". The solution concentration with a signal-to-noise ratio (S / N) of 3:1 is taken as the limit of quantitation (LOD), and the solution concentration with an S / N of 10:1 is taken as the limit of detection (LOQ). The results are shown in Table 2. Figure 6 .

[0105] 2.3 Precision Test

[0106] Take an appropriate amount of the test solution under section "1.1" and inject it under the conditions in section "1.3" for 6 determinations. The RSDs of caffeic acid, baicalin, rutin, and hesperidin were 0.33%, 0.06%, 0.16%, and 0.14%, respectively, indicating that the instrument used has good precision.

[0107] 2.4 Stability Test

[0108] Take an appropriate amount of the test solution under section "1.1" and inject it at 0, 2, 4, 8, 12 and 24 h respectively for determination. The RSD of caffeic acid, baicalin, rutin, and hesperidin were 0.59%, 0.38%, 0.44% and 0.45% respectively, indicating that the solution has good stability within 24 h.

[0109] 2.5 Repeatability Test

[0110] Take 6 portions of this product (batch number 20230301), prepare the test solution according to the method in section "1.1", and inject it under the conditions in section "1.3" for determination. The RSDs of caffeic acid, baicalin, rutin, and hesperidin were 0.82%, 2.72%, 1.00%, and 1.08%, respectively, indicating that the method has good repeatability.

[0111] 2.6 Recovery Test

[0112] Take 6 portions of this product (batch number 20230301) with known content of each component, 0.1 g of each portion, and add 1 mL of the mixed reference solution under section "1.1" (containing 17.4 µg caffeic acid, 40.5 µg baicalin, 220.6 µg rutin, and 712.4 µg hesperidin). Prepare the test solution according to the method under section "1.1", and inject and determine under the conditions in section "1.3". Calculate the recovery rate. The results are shown in [Figure 1]. Figure 7 The average recoveries of caffeic acid, baicalin, naringin, and hesperidin were calculated to be 98.19%, 95.84%, 102.08%, and 98.59%, respectively, with RSDs of 2.27%, 1.82%, 0.84%, and 1.31%, respectively. The results indicate that the recoveries of the four components were good.

[0113] 2.7 Determination of Sample Content

[0114] Take 6 batches of samples, prepare 3 replicates for each batch, prepare the test solution according to the method in section "1.1", and inject and determine under the conditions in section "1.3". The results are shown in [Figure 1]. Figure 8 The contents of caffeic acid, baicalin, rutin, and hesperidin in the six batches of samples were 0.14~0.16 mg / g, 0.48~0.57 mg / g, 2.19~2.27 mg / g, and 7.12~7.36 mg / g, respectively. The results showed that the contents of the four active ingredients in the different batches of anti-swelling granules were relatively similar, and the production process was relatively stable.

[0115] This application uses LC-MS to identify the main chemical components in the anti-swelling granules and HPLC to determine the content of four indicator components.

[0116] First, chromatographic analysis conditions were optimized. After screening different chromatographic columns, the Agilent Poroshell 120 EC-C18 column, with its core-shell particle packing material, was found to have advantages such as low mass transfer resistance, fast peak elution rate, and high analytical throughput. Furthermore, the peak shapes and separation obtained using this column were relatively ideal. After screening different mobile phases, it was found that adding an appropriate amount of phosphoric acid to the aqueous phase could inhibit the ionization of weakly acidic compounds, effectively improving the peak shapes. Considering that phosphoric acid cannot be used in LC-MS / MS analysis, formic acid was substituted. The effects of column temperature (25, 35, 45 ℃) and flow rate (0.3, 0.4, 0.5 mL / min) on separation were also investigated. The results showed that a column temperature of 35 ℃ and a flow rate of 0.4 mL / min resulted in better peak shapes and separation for each indicator component. Caffeic acid, baicalin, rutin, and hesperidin showed the highest detection sensitivity at a wavelength of 280 nm. Therefore, a diode array detector was selected to simultaneously measure the four components at a wavelength of 280 nm.

[0117] The preparation conditions of the test sample solution, such as ultrasonic time (10, 20, 30 min), extraction solvent (0, 30, 50, 70, 100% methanol), and solid-solid ratio (v:w) (1:10, 1:25, 1:50, 1:100), were investigated. It was found that the extraction rate and peak intensity were higher when the ultrasonic time was 30 min, 70% methanol was used as the extraction solvent, and the solid-solid ratio was 1:50.

[0118] Under the optimized conditions obtained in the above experiments, the chromatographic peaks of caffeic acid, baicalin, rutin, and hesperidin in the anti-swelling granules showed good separation, symmetrical peak shape, and high peak height, and were therefore selected as quantitative indicator components. Method validation results showed that the four indicator components exhibited good linearity, and the precision, repeatability, stability, and spiked recovery all met the determination requirements. The quantitative method is rapid and accurate and can be used for the content determination of anti-swelling granules.

[0119] Qualitative analysis by LC-MS showed that the swelling-reducing granules contained a rich variety of chemical components, including 30 flavonoids, 11 phenolic acids, 15 amino acids, 5 alkaloids, 3 coumarins, 2 sugars, and 4 other compounds. Flavonoids were the most abundant and present in high concentrations. Modern pharmacological studies have shown that caffeic acid is one of the main active ingredients in perilla leaves, possessing anti-inflammatory, antioxidant, and antibacterial effects. Studies have also shown that the DPPH free radical scavenging capacity of fresh perilla leaves is significantly enhanced after in vitro digestion in gastric and intestinal fluids. Baicalin has protective effects on the cardiovascular and cerebrovascular systems, inhibits platelet aggregation, has antithrombotic, anti-inflammatory, and antitumor effects. The flavonoids in dried tangerine peel, such as rutin and hesperidin, have antitumor, anti-inflammatory, antioxidant, antiviral, and lipid-lowering effects. Arecoline is the main active ingredient in areca nut. It can increase the relative abundance of lactobacilli and bifidobacteria in the intestine, as well as the content of acetic acid and propionic acid in the intestine, thereby promoting intestinal peristalsis and digestion and playing a role in relieving bloating.

[0120] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A method for detecting multiple components in anti-swelling granules, characterized in that, Includes the following steps: 1) Dissolve the anti-swelling granule sample in a solvent, sonicate it, filter it, and collect the filtrate to obtain the test solution; 2) The test solution was analyzed by liquid chromatography-mass spectrometry to determine the components in the test solution.

2. The detection method according to claim 1, characterized in that, Step 1) includes one or more of the following features: 1a) The solvent is an aqueous methanol solution; preferably, the volume fraction of the aqueous methanol solution is 60-80%; 1b) The mass ratio of the deswelling granule sample to the volume ratio of the solvent is 0.2 g : (8~12) mL; 1c) The ultrasonic treatment time is 20~40 min.

3. The detection method according to claim 1, characterized in that, In step 2), the liquid chromatography-mass spectrometry (LC-MS) method includes one or more of the following characteristics in the liquid chromatography determination conditions: 2a) The chromatographic column is a C18 column; preferably, the C18 column is an Agilent Poroshell 120 EC-C18 column; 2b) Mobile phase A is acetonitrile; mobile phase B is 0.05~0.20% (v / v) formic acid aqueous solution; 2c) The analysis time is 50 min; 2d) Column temperature is 30~40℃; 2e) The flow rate is 0.2~0.6 mL / min; 2f) The injection volume is 15~25 μL.

4. The detection method according to claim 1, characterized in that, In step 2), the elution method in the liquid chromatography-mass spectrometry is gradient elution.

5. The detection method according to claim 4, characterized in that, The gradient elution program was as follows: 0-8 min, A phase:B phase volume ratio 5:95-10:90; 8-14 min, A phase:B phase volume ratio 10:90-12:88; 14-28 min, A phase:B phase volume ratio 12:88-18:82; 28-38 min, A phase:B phase volume ratio 18:82-30:70; 38-50 min, A phase:B phase volume ratio 30:70-100:

0.

6. The detection method according to claim 1, characterized in that, In step 2), the mass spectrometry determination conditions in the liquid chromatography-mass spectrometry method include any one or more of the following characteristics: 2A) The ion source is an electrospray ionization (ESI) source; 2B) Detection mode is positive ion electrospray ionization mode (ESI). + and negative ion electrospray ionization mode ESI - ; 2C) The drying gas is nitrogen; 2D) The temperature of the drying gas is 320~380℃; 2E) The atomizing gas pressure of the drying gas is 25~35psi; 2F) The capillary voltage is 3400~3600V; 2G) Skimmer voltage is 60~70V; 2H) Octapole RF voltage is 700~800V; 2I) The capillary outlet voltage is 120~130V; 2J) Primary mass spectrometry acquisition uses full scan mode; the mass-to-charge ratio scan range is 50~1200 m / z; 2K) secondary mass spectrometry acquisition was performed in Auto-MS / MS mode with a collision voltage of 25~35eV.

7. The detection method according to claim 1, characterized in that, In step 2), the components in the test solution are selected from arginine, lysine, asparagine, aspartic acid, alanine, γ-aminobutyric acid, threonine, glutamine, glutamic acid, glucose, maltose, proline, adenine, valine, arecoline, arecoline, adenosine, tyrosine, synephrine, leucine, N-methyltyramine, phenylalanine, syringic acid, protocatechuic acid, neochlorogenic acid, tryptophan, proanthocyanidin B2, p-hydroxybenzoic acid, chlorogenic acid, catechin, cryptochlorogenic acid, caffeic acid, epicatechin, luteolin-7-O-rutin, p-coumaric acid, naringin, and other components. The following are one or more of the following: rutin, luteolin, senna, rutin, luteolin, baicalin, naringenin-7-O-glucoside, rutin, apigenin-7-glucuronic acid, hesperidin, isochromine, rosmarinic acid hydrated oxypenicillin, quercetin, limonin, isochromine, robinin, robinin, citric acid, costunolide, kaempferol, hesperidin, patchouli, 5,7-dimethoxycoumarin, succinate, limonene, nomiline, isohesperidin, citronellol, demethylnodosperidin, or dehydrocostunolide.

8. A method for detecting the content of caffeic acid, baicalin, rutin, or hesperidin in anti-swelling granules, characterized in that, Includes the following steps: 1) Prepare the test solution according to step 1) of the method for detecting multiple components in the anti-swelling granules according to any one of claims 1 to 7; 2) Prepare reference solutions of caffeic acid, baicalin, rutin, and hesperidin; 3) The test solution and the reference solution were determined by high performance liquid chromatography to determine the contents of caffeic acid, baicalin, rutin, and hesperidin in the test solution.

9. The detection method according to claim 8, characterized in that, In step 3), the determination conditions of the high-performance liquid chromatography include any one or more of the following: 3a) The chromatographic column is a C18 column; preferably, the C18 column is an Agilent Poroshell 120EC-C18 column; 3b) Mobile phase A is acetonitrile, and mobile phase B is a 0.05~0.15% (v / v) aqueous solution of phosphoric acid; 3c) The analysis time is 50 minutes; (3d) The flow rate is 0.3~0.5 mL / min; 3e) Column temperature is 30~40℃; 3f) The elution method is gradient elution; (3g) Injection volume is 4~6 μL; (3h) The ultraviolet detection wavelength is 280 nm.

10. The detection method according to claim 9, characterized in that, The gradient elution program is as follows: 0-8 min, A phase:B phase volume ratio is 5:95-10:90; 8-14 min, A phase:B phase volume ratio is 10:90-12:88; 14-28 min, A phase:B phase volume ratio is 12:88-18:82; 28-38 min, A phase:B phase volume ratio is 18:82-30:70; 38-50 min, A phase:B phase volume ratio is 30:70-100:0.

Images