Method for detecting multiple components in Lianqing granules

Abstract

According to the application, liquid chromatography-mass spectrometry (LC-MS) is adopted to identify the chemical components of the Lianqing granules, and the contents of cowherb seed flavonoid glycoside, polydatin, naringin and hesperidin in the Lianqing granules are determined through high performance liquid chromatography (HPLC). And the liquid chromatography-mass spectrometry analysis adopts an electrospray ionization source and positive and negative ion scanning. An Agilent Poroshell 120EC-C18 chromatographic column (3.0 mm * 150 mm, 2.7 [mu] m) is adopted for quantitative analysis; a mobile phase is acetonitrile-water (containing 0.1% of formic acid); performing gradient elution; the volume flow rate is 0.4 mL / min; the column temperature is 45 DEG C; and the detection wavelengths are 280 nm and 365 nm. According to the application, 139 compounds are totally identified from Lianqing granules. The linear relation of the four index components in the respective ranges is good (R2gt; the average sample adding recovery rate is 97.29 percent to 105.08 percent, and the RSD (Relative Standard Deviation) is 2.07 percent to 4.60 percent. The LC-MS qualitative method established by the invention is simple, convenient and efficient, and can be used for rapidly identifying the chemical components in the Lianqing granules; the HPLC quantitative method is accurate and high in specificity, and can be used for determining the content of cowherb seed flavonoid glycoside, polydatin, naringin and hesperidin in the Lianqing granules.

Description

Technical Field

[0001] The present specification relates to the technical field of chemical composition research of traditional Chinese medicine preparations, and in particular to a method for detecting multiple components in Lianshuqing granules. Background Art

[0002] In traditional Chinese medicine, urinary stones belong to the category of "stone stranguria" and "blood stranguria" in "gonorrhea". In recent years, with the development of surgical equipment, minimally invasive surgery to treat urolithiasis has become more popular, but surgical treatment is prone to various complications and sequelae. At the same time, there are still some small stones that originally remained in the body or remained after lithotripsy that cannot be discharged. Traditional Chinese medicine has antispasmodic, diuretic, and anti-inflammatory effects, and has the effect of inhibiting and promoting the discharge of stones, and can relieve patients' symptoms such as urination discomfort. Therefore, timely use of traditional Chinese medicine treatment can not only effectively prevent and treat urinary stones, but also reduce the trauma caused by surgery.

[0003] Lianshuqingfang (formerly known as Xiaoshifang) is derived from the clinical experience of Professor Ye Jinghua, a famous old Chinese medicine doctor in Shanghai. It is composed of Herba Lysimachiae, Atractylodes, Polygonum cuspidatum, Citrus aurantium, Rhizoma Cynanchum, Trillium, Curcuma, Pyrus citrifolia, Vaccaria, and Gleditsia sinensis. It has the effects of regulating qi and activating blood circulation, dispersing nodules and eliminating stones, and has a significant effect on urinary tract stones. On the basis of clearing heat and removing dampness, this prescription focuses more on regulating qi and activating blood circulation, removing stasis and eliminating stones, and alleviating pain, achieving the effect of treating both the symptoms and the root causes. Herba Lysimachiae is the main drug in the prescription, which tastes sweet and slightly bitter, is cool in nature, and enters the liver, gallbladder, kidney, and bladder meridians. It has the effects of diuresis, relieving stranguria, clearing heat and detoxifying, and dissolving stones and eliminating stones. Atractylodes and Citrus aurantium are the assistant drugs. Atractylodes is pungent and bitter in taste, warm in nature, and enters the spleen, stomach, and liver meridians. It has the effects of drying dampness and strengthening the spleen, dispelling wind and dispersing cold; Citrus aurantium is bitter and pungent in taste, warm in nature, and enters the liver, gallbladder, spleen, and lung meridians, and has the effects of soothing the liver and regulating qi. Polygonum cuspidatum tastes slightly bitter and is slightly cold in nature. It enters the liver, gallbladder, and lung meridians and has the effects of promoting blood circulation, dispersing blood stasis, relieving pain, clearing away heat, promoting dampness, and detoxifying. Rhizoma Cynoglossi is sweet and slightly bitter in taste and is neutral in nature. It enters the liver and kidney meridians and has the effects of promoting blood circulation, dredging menstruation, promoting diuresis, relieving stranguria, relieving pain, and anti-inflammatory. Rhizoma Trigonosciae, Rhizoma Curcumae, Rhizoma Citri Reticulatae, Rhizoma Vaccariae, and Rhizoma Gleditsiae have the effects of promoting blood circulation and removing blood stasis. They are all adjuvant drugs. The whole formula is used together to play the role of regulating qi, promoting blood circulation, relieving stranguria, and eliminating stones.

[0004] Lianshuqing Granules (also known as Lianshuqing Granules) have been approved by the Shanghai Food and Drug Administration and obtained the "Registration Certificate for the Preparation of Traditional Chinese Medicine Preparations by Medical Institutions Using Traditional Crafts". This granule is a hospital preparation and its basic research is not yet in-depth. At present, there is no comprehensive qualitative and quantitative detection method for the ingredients in Lianshuqing Granules. Summary of the invention

[0005] In order to solve the above technical problems, this application uses liquid chromatography-mass spectrometry (LC-MS) technology to identify the chemical components of Lianshuqing Granules. On this basis, high performance liquid chromatography (HPLC) is used to establish a quantitative method for fenugreek flavonoid glycosides, polydatin, naringin, and hesperidin in Lianshuqing Granules, in order to provide a reference for the basic research on chemical substances and the establishment of quality standards for this compound preparation.

[0006] The present application provides a method for detecting multiple components in Lianshuqing granules, comprising the following steps: 1) adding a Lianshuqing granule sample to a solvent to dissolve, ultrasonically extracting, filtering, and taking a filtrate to obtain a test solution; 2) measuring the test solution by liquid chromatography-mass spectrometry to determine 139 components in the test solution.

[0007] The present application also provides a method for attributing multiple components in Lianshuqing granules, comprising the following steps: A) preparation of single medicinal material sample solutions: preparing any one or more of ten medicinal material samples of Herba Lysimachiae, Atractylodes, Polygonum cuspidatum, Citrus aurantium, Rhizoma Cynoglossi, Rhizoma Trillium, Rhizoma Curcumae, Citrus aurantium, Vaccaria segetalis, and Spina Gleditsiae according to step 1) of the above-mentioned method for detecting multiple components in Lianshuqing granules, and obtaining at least one single medicinal material sample solution respectively; B) preparation of negative sample solutions: preparing any one or more of ten medicinal material samples of Herba Lysimachiae, Atractylodes, Polygonum cuspidatum, Citrus aurantium, Rhizoma Cynoglossi, Rhizoma Trillium, Rhizoma Curcumae, Citrus aurantium, Vaccaria segetalis, and Spina Gleditsiae according to step 1) of the above-mentioned method for detecting multiple components in Lianshuqing granules, and obtaining at least one single medicinal material sample solution respectively; The samples of 10 medicinal materials, namely, Semen Ligusticum chuanxiong, and Gleditsiae Spina, are each lacking one medicinal material, and are prepared according to step 1) of the above-mentioned method for detecting multiple components in Lianshuqing granules to obtain negative sample solutions lacking Herba Lysimachiae, Herba Atractylodis Macrocephalae, Herba Polygoni Cuspidati, Herba Citri Reticulatae, Herba Cynoglossi, Herba Trillii, Herba Curcumae, Herba Citrus Aurantii Immaturus, Herba Vaccariae, and Herba Gleditsiae Spinae; C) determination: the single medicinal material sample solution in step a), the sample solution lacking Herba Lysimachiae, Herba Atractylodis Macrocephalae, Herba Polygoni Cuspidati, Herba Citri Reticulatae, and Herba Gleditsiae Spinae are respectively determined by liquid chromatography-mass spectrometry under the same conditions as step 2) of the above-mentioned method for detecting multiple components in Lianshuqing granules. The negative sample solution of Polygonum cuspidatum, lacking green peel, lacking Cyathula officinalis, lacking Trillium, lacking Curcuma zedoaria, lacking Citrus aurantium, lacking Vaccaria segetalis, and lacking Gleditsia sinensis is obtained, and the liquid chromatograms of the negative sample solutions of single medicinal material sample solution, lack of Lysimachia chinensis, lack of Atractylodes lancea, lacking Polygonum cuspidatum, lacking green peel, lacking Cyathula officinalis, lacking Trillium, lacking Curcuma zedoaria, lacking Citrus aurantium, lacking Vaccaria segetalis, and lacking Gleditsia sinensis are obtained respectively; D) obtaining the control liquid chromatogram: the test sample solution prepared according to step 1) of the above-mentioned method for detecting multiple components in Lianshuqing granules is used in the same manner as the method for detecting multiple components in Lianshuqing granules. Step 2) analyze to obtain a control liquid chromatogram of Lianshuqing Granules; E) attribution: compare the liquid chromatograms of single medicinal material sample solutions, negative sample solutions lacking Centella asiatica, lacking Atractylodes lancea, lacking Polygonum cuspidatum, lacking Citrus aurantium, lacking Rhizoma Cynoglossi, lacking Trillium gracile, lacking Curcuma zedoaria, lacking Citrus aurantium, lacking Vaccaria segetalis, and lacking Gleditsia sinensis with the control liquid chromatogram of Lianshuqing Granules, identify the corresponding characteristic peaks of the single medicinal material sample solutions in the control liquid chromatogram of Lianshuqing Granules through relative retention time, and thereby attribute and locate the components in the control liquid chromatogram of Lianshuqing Granules.

[0008] The present application also provides a method for detecting the content of vaccariae flavonoid glycosides, polydatin, naringin or hesperidin in Lianshuqing granules, comprising the following steps: 1) preparing according to step 1) of the above-mentioned method for detecting multiple components in Lianshuqing granules to obtain a test solution; 2) preparing vaccariae flavonoid glycosides, polydatin, naringin and hesperidin reference solution; 3) measuring the test solution and the reference solution by high performance liquid chromatography to determine the content of vaccariae flavonoid glycosides, polydatin, naringin and hesperidin in the test solution.

[0009] The beneficial effects brought by this application include but are not limited to: This application identified a total of 139 compounds from Lianshuqing Granules. The four index components have good linear relationships within their respective ranges (R2>0.9999), with an average sample recovery rate of 97.29% to 105.08%, and RSD 2.07% to 4.60%. The LC-MS qualitative method established in this application is simple and efficient, and can be used to quickly identify the chemical components in Lianshuqing Granules; the HPLC quantitative method is accurate and highly specific, and can be used to determine the content of fenugreek flavonoid glycosides, polydatin, naringin, and hesperidin in Lianshuqing Granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The present application will be further described in the form of exemplary embodiments, which will be described in detail with reference to the accompanying drawings. These embodiments are not restrictive, and include:

[0011] Figure 1 This is an overlay diagram of the extracted ion currents of Lian Shuqing particles in the positive and negative modes according to some embodiments of the present application.

[0012] Figure 2 HPLC chromatograms of blank solvent, mixed reference and test solution at wavelengths of 280 nm and 365 nm according to some embodiments of the present application. DETAILED DESCRIPTION

[0013] In order to more clearly illustrate the technical solutions of the embodiments of this specification, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some examples or embodiments of this specification. For ordinary technicians in this field, this specification can also be applied to other similar scenarios based on these drawings without creative work. Unless it is obvious from the language environment or otherwise explained, the same reference numerals in the figures represent the same structure or operation.

[0014] As shown in this specification and claims, unless the context clearly indicates an exception, the words "a", "an", "an" and / or "the" do not refer to the singular and may also include the plural. Generally speaking, the terms "comprise" and "include" only indicate the inclusion of the steps and elements that have been clearly identified, and these steps and elements do not constitute an exclusive list. The method or device may also include other steps or elements.

[0015] Flowcharts are used in this specification to illustrate the operations performed by the system according to the embodiments of this specification. It should be understood that the preceding or following operations are not necessarily performed precisely in order. Instead, the steps may be processed in reverse order or simultaneously. At the same time, other operations may also be added to these processes, or one or more operations may be removed from these processes.

[0016] The present application provides a method for detecting multiple components in Lianshuqing granules, comprising the following steps: 1) adding a Lianshuqing granule sample to a solvent to dissolve, ultrasonically extracting, filtering, and taking a filtrate to obtain a test solution; 2) measuring the test solution by liquid chromatography-mass spectrometry to determine 139 components in the test solution.

[0017] In some embodiments, in step 1), the solvent may be a 60-80% methanol solution. In some embodiments, in step 1), the solvent may be a 62-78% methanol solution. In some embodiments, in step 1), the solvent may be a 64-76% methanol solution. In some embodiments, in step 1), the solvent may be a 66-74% methanol solution. In some embodiments, in step 1), the solvent may be a 68-72% methanol solution. In some embodiments, in step 1), the solvent may be a 70-72% methanol solution. In some embodiments, preferably, in step 1), the solvent may be a 70% methanol solution.

[0018] In some embodiments, in step 1), the ratio of the mass of the Lianshuqing granules sample added to the volume of the solvent added can be 0.2: (9-11) g / mL. In some embodiments, preferably, in step 1), the ratio of the mass of the Lianshuqing granules sample added to the volume of the solvent added can be 0.2: 10 g / mL.

[0019] In some embodiments, in step 1), the ultrasonic extraction time may be 5 to 15 minutes. In some embodiments, in step 1), the ultrasonic extraction time may be 7.5 to 12.5 minutes. In some embodiments, in step 1), the ultrasonic extraction time may be 10 to 12.5 minutes. In some embodiments, preferably, in step 1), the ultrasonic extraction time may be 10 minutes.

[0020] In some embodiments, in step 2), in the liquid chromatography-mass spectrometry method, the chromatographic column can be a C18 chromatographic column. In some embodiments, preferably, in step 2), in the liquid chromatography-mass spectrometry method, the chromatographic column can be an Agilent Poroshell 120EC-C18 chromatographic column.

[0021] In some embodiments, in step 2), in the liquid chromatography-mass spectrometry, mobile phase A can be acetonitrile; mobile phase B can be 0.05-0.15% formic acid aqueous solution; analysis time: 50 min; gradient elution. In some embodiments, in step 2), in the liquid chromatography-mass spectrometry, mobile phase B can be 0.075-0.125% formic acid aqueous solution. In some embodiments, in step 2), in the liquid chromatography-mass spectrometry, mobile phase B can be 0.01-0.125% formic acid aqueous solution. In some embodiments, preferably, in step 2), in the liquid chromatography-mass spectrometry, mobile phase B can be 0.1% formic acid aqueous solution.

[0022] In some embodiments, in step 2), the column temperature in the liquid chromatography-mass spectrometry is 20 to 30°C. In some embodiments, in step 2), the column temperature in the liquid chromatography-mass spectrometry is 22 to 28°C. In some embodiments, in step 2), the column temperature in the liquid chromatography-mass spectrometry is 24 to 26°C. In some embodiments, preferably, in step 2), the column temperature in the liquid chromatography-mass spectrometry is 25°C.

[0023] In some embodiments, in step 2), the flow rate of the liquid chromatography-mass spectrometry is 0.2 to 0.4 mL / min. In some embodiments, preferably, in step 2), the flow rate of the liquid chromatography-mass spectrometry is 0.2 to 0.4 mL / min.

[0024] In some embodiments, in step 2), the injection volume in the liquid chromatography-mass spectrometry method is 4 to 6 μL. In some embodiments, preferably, in step 2), the injection volume in the liquid chromatography-mass spectrometry method is 5 μL.

[0025] In some embodiments, the gradient elution procedure can be specifically: 0-10 min, the volume ratio of phase A:phase B is 5:95-25:75; 10-30 min, the volume ratio of phase A:phase B is 25:75-40:60; 30-50 min, the volume ratio of phase A:phase B is 40:60-100:0.

[0026] In some embodiments, in step 2), in the liquid chromatography-mass spectrometry method, the ion source can be an electrospray ion source ESI.

[0027] In some embodiments, in step 2), the detection mode of the liquid chromatography-mass spectrometry method can be positive ion electrospray ionization mode ESI + and negative ion electrospray ionization mode ESI - .

[0028] In some embodiments, in step 2), in the liquid chromatography-mass spectrometry method, the drying gas can be nitrogen.

[0029] In some embodiments, in step 2), in the liquid chromatography-mass spectrometry, the temperature of the drying gas may be 340-360°C. For example, in step 2), in the liquid chromatography-mass spectrometry, the temperature of the drying gas may be about 340, 342, 344, 346, 348, 350, 352, 354, 356, 358 or 360°C. It also includes any range characterized by a combination of the above end values, which will not be repeated here. In some embodiments, preferably, in step 2), in the liquid chromatography-mass spectrometry, the temperature of the drying gas may be 350°C.

[0030] In some embodiments, in step 2), the flow rate of the drying gas in the liquid chromatography-mass spectrometry method can be 9-11 L / min. In some embodiments, preferably, in step 2), the flow rate of the drying gas in the liquid chromatography-mass spectrometry method can be 10 L / min.

[0031] In some embodiments, in step 2), in the liquid chromatography-mass spectrometry, the atomizing gas pressure can be 25-35psi. For example, in step 2), in the liquid chromatography-mass spectrometry, the atomizing gas pressure can be 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35psi. It also includes any range characterized by a combination of the above end values, which will not be repeated here. In some embodiments, preferably, in step 2), in the liquid chromatography-mass spectrometry, the atomizing gas pressure can be 30psi.

[0032] In some embodiments, in step 2), in the liquid chromatography-mass spectrometry, the capillary voltage can be 3400-3600V. For example, in step 2), in the liquid chromatography-mass spectrometry, the capillary voltage can be 3400, 3410, 3420, 3430, 3440, 3450, 3460, 3470, 3480, 3490, 3500, 3510, 3520, 3530, 3540, 3550, 3560, 3570, 3580, 3590 or 3600V. It also includes any range characterized by a combination of the above end values, which will not be repeated here. In some embodiments, preferably, in step 2), in the liquid chromatography-mass spectrometry, the capillary voltage can be 3500V.

[0033] In some embodiments, in step 2), in the liquid chromatography-mass spectrometry, the capillary outlet voltage can be 115-135V. For example, in step 2), in the liquid chromatography-mass spectrometry, the capillary outlet voltage can be 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134 or 135V. It also includes any range characterized by a combination of the above end values, which will not be repeated here. In some embodiments, preferably, in step 2), in the liquid chromatography-mass spectrometry, the capillary outlet voltage can be 125V.

[0034] In some embodiments, in step 2), in the liquid chromatography-mass spectrometry, the Skimmer voltage can be 60-70V. For example, in step 2), in the liquid chromatography-mass spectrometry, the Skimmer voltage can be 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70V. It also includes any range characterized by a combination of the above end values, which will not be repeated here. In some embodiments, preferably, in step 2), in the liquid chromatography-mass spectrometry, the Skimmer voltage can be 65V.

[0035] In some embodiments, in step 2), in the liquid chromatography-mass spectrometry, the octopole radio frequency voltage can be 700-800V. For example, in step 2), in the liquid chromatography-mass spectrometry, the octopole radio frequency voltage can be 700, 710, 720, 730, 740, 750, 760, 770, 780, 790 or 800V. It also includes any range characterized by a combination of the above end values, which will not be repeated here. In some embodiments, preferably, in step 2), in the liquid chromatography-mass spectrometry, the octopole radio frequency voltage can be 750V.

[0036] In some embodiments, in step 2), in the liquid chromatography-mass spectrometry, the primary mass spectrometry uses the MS1 mode, and the mass-to-charge ratio scanning range can be 50-1200m / z. For example, in step 2), in the liquid chromatography-mass spectrometry, the primary mass spectrometry uses the MS1 mode, and 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 1200m / z. It also includes any range characterized by a combination of the above end values, which will not be repeated here.

[0037] In some embodiments, in step 2), in the liquid chromatography-mass spectrometry, the secondary mass spectrometry uses the Auto-MS / MS mode, and the collision voltage can be 25-35eV. For example, in step 2), in the liquid chromatography-mass spectrometry, the secondary mass spectrometry uses the Auto-MS / MS mode, and the collision voltage can be 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35eV. It also includes any range characterized by a combination of the above end values, which will not be repeated here. In some embodiments, preferably, in step 2), in the liquid chromatography-mass spectrometry, the secondary mass spectrometry uses the Auto-MS / MS mode, and the collision voltage can be 30eV.

[0038] In some embodiments, in step 2), the components in the test solution can be selected from lysine, serine, glutamic acid, gluconic acid, valine, cytidine, malic acid, adenine, 2-furancarboxylic acid, adenosine * , pyroglutamic acid, uracil, fumaric acid, tyrosine, citric acid, phenylalanine, tyramine, isoleucine, leucine, succinic acid, N-methyltyramine, gallic acid, hordenine, 5-hydroxymethylfurfural, vanillin, synephrine, vanillic acid, 5-hydroxy-2-(hydroxymethyl)pyridine, neochlorogenic acid, chlorogenic acid, protocatechuic acid, n-butyl-O-β-D-pyranofructose, catechol, syringin, scopoletin, cryptochlorogenic acid, cyclotide G, erythrine, epicatechin * , catechins, p-hydroxybenzoic acid, kaempferol-3-O-β-acacia disaccharide, chrysophanol-8-O-β-glucoside, hesperidin, vaccariae cyclopeptide H, naringin, 2-methoxy flat orange alkaloids, vaccariae flavonoid glycosides * , Caffeic acid * , ferulic acid, caffeoylmalic acid, sinapinic aldehyde, saponin, isorientin, resveratrol-4'-O-glucoside, isovitexin-2"-O-arabinoside, orientin, p-hydroxybenzaldehyde, epicatechin-5-O-β-D-pyranoglucopyranoside, isoschafta glycoside, vaccaria cyclopeptide F, isorhamnetin-3-O-rutinoside, eriocitrin, rutin, 4-hydroxyacetophenone, vitexin, atractylodesin * , phlorizin, p-coumaric acid, p-hydroxycinnamic acid,

[0039] Polydatin * , paeonol, luteolin * , Vaccaria flavonoid glycoside E, isoscopolamine, umbelliferone, cyperasterone, naringin * , naringenin-7-O-glucoside, dihydroquercetin, wild sumac glycoside, 24-hydroxy calixasterone, phenyl-β-glucopyranoside, neohesperidin, hesperidin, naringenin, 5-demethylnobiletin, hesperidin *, isoprunin, 6,7-dihydroxycoumarin, rosmarinic acid, azelaic acid, isovitexin, atractylodes lactone II, coumarin, epigallocatechin, apigenin, 6,7-dimethoxycoumarin, triangular acid, angelica lactone, resveratrol, hesperidin, 5,4'-dihydroxy-6,7,8,3'-tetramethoxyflavonoid isomers, kaempferol-3-O-rutinoside, 6-hydroxyaloe-emodin-8-methyl ether, vaccariae cyclopeptide B, quercetin * , citrus glycosides, kaempferol, rhamnolide-8-O-β-D-glucoside, cassia pine, costus lactone, n-butyl-O-β-D-pyranose, vaccaria cyclopeptide C, vaccaria cyclopeptide A, rhamnolide methyl ether-8-O-β-D-glucoside, 7-hydroxy-4-methoxy-5-methylcoumarin isomers, vaccaria cyclopeptide D, baicalin, bergamot lactone, 4'-hydroxy-5,6,7-trimethoxyflavone, deacetylnomirin, limonin, atractylolactone III, isorhodoptilometrin, nobiletin, atractylol acetate, nomilin, dehydrocurdione, atractylone, hibiscus lactone, tangeretin, isoimperatorin, curcuma epoxide, rhamnolide * , curcumadione, echinocystic acid, physaccharin methyl ether or β-eudesmol.

[0040] The present application also provides a method for attributing multiple components in Lianshuqing granules, comprising the following steps: A) preparation of single medicinal material sample solutions: preparing any one or more of ten medicinal material samples of Herba Lysimachiae, Atractylodes, Polygonum cuspidatum, Citrus aurantium, Rhizoma Cynoglossi, Rhizoma Trillium, Rhizoma Curcumae, Citrus aurantium, Vaccaria segetalis, and Spina Gleditsiae according to step 1) of the above-mentioned method for detecting multiple components in Lianshuqing granules, and obtaining at least one single medicinal material sample solution respectively; B) preparation of negative sample solutions: preparing any one or more of ten medicinal material samples of Herba Lysimachiae, Atractylodes, Polygonum cuspidatum, Citrus aurantium, Rhizoma Cynoglossi, Rhizoma Trillium, Rhizoma Curcumae, Citrus aurantium, Vaccaria segetalis, and Spina Gleditsiae according to step 1) of the above-mentioned method for detecting multiple components in Lianshuqing granules, and obtaining at least one single medicinal material sample solution respectively; The samples of 10 medicinal materials, namely, Semen Ligusticum chuanxiong, and Gleditsiae Spina, are each lacking one medicinal material, and are prepared according to step 1) of the above-mentioned method for detecting multiple components in Lianshuqing granules to obtain negative sample solutions lacking Herba Lysimachiae, Herba Atractylodis Macrocephalae, Herba Polygoni Cuspidati, Herba Citri Reticulatae, Herba Cynoglossi, Herba Trillii, Herba Curcumae, Herba Citrus Aurantii Immaturus, Herba Vaccariae, and Herba Gleditsiae Spinae; C) determination: the single medicinal material sample solution in step a), the sample solution lacking Herba Lysimachiae, Herba Atractylodis Macrocephalae, Herba Polygoni Cuspidati, Herba Citri Reticulatae, and Herba Gleditsiae Spinae are respectively determined by liquid chromatography-mass spectrometry under the same conditions as step 2) of the above-mentioned method for detecting multiple components in Lianshuqing granules. The negative sample solution of Polygonum cuspidatum, lacking green peel, lacking Cyathula officinalis, lacking Trillium, lacking Curcuma zedoaria, lacking Citrus aurantium, lacking Vaccaria segetalis, and lacking Gleditsia sinensis is obtained, and the liquid chromatograms of the negative sample solutions of single medicinal material sample solution, lack of Lysimachia chinensis, lack of Atractylodes lancea, lacking Polygonum cuspidatum, lacking green peel, lacking Cyathula officinalis, lacking Trillium, lacking Curcuma zedoaria, lacking Citrus aurantium, lacking Vaccaria segetalis, and lacking Gleditsia sinensis are obtained respectively; D) obtaining the control liquid chromatogram: the test sample solution prepared according to step 1) of the above-mentioned method for detecting multiple components in Lianshuqing granules is used in the same manner as the method for detecting multiple components in Lianshuqing granules. Step 2) Analyze to obtain the control liquid chromatogram of Lianshuqing Granules; E) Attribution: Compare the liquid chromatograms of the single medicinal material sample solution, the negative sample solution lacking Herba Lysimachiae, lacking Atractylodes, lacking Polygonum cuspidatum, lacking Citrus aurantium, lacking Tripterygium wilfordii, lacking Curcuma zedoaria, lacking Citrus citrifolia, lacking Vaccaria segetalis, and lacking Gleditsia sinensis with the control liquid chromatogram of Lianshuqing Granules, and identify the corresponding characteristic peaks of the single medicinal material sample solution in the control liquid chromatogram of Lianshuqing Granules by relative retention time, so as to attribute and locate the components in the control liquid chromatogram of Lianshuqing Granules. The attribution of the corresponding components in the medicinal materials is shown in Table 1.

[0041] The present application also provides a method for detecting the content of vaccariae flavonoid glycosides, polydatin, naringin or hesperidin in Lianshuqing granules, comprising the following steps: 1) preparing according to step 1) of the above-mentioned method for detecting multiple components in Lianshuqing granules to obtain a test solution; 2) preparing vaccariae flavonoid glycosides, polydatin, naringin and hesperidin reference solution; 3) measuring the test solution and the reference solution by high performance liquid chromatography to determine the content of vaccariae flavonoid glycosides, polydatin, naringin and hesperidin in the test solution.

[0042] In some embodiments, in the HPLC determination conditions, the chromatographic column can be a C18 chromatographic column. In some embodiments, preferably, in the HPLC determination conditions, the chromatographic column can be an Agilent Poroshell 120EC-C18 chromatographic column.

[0043] In some embodiments, in the HPLC determination conditions, mobile phase A can be acetonitrile; mobile phase B can be 0.05-0.15% phosphoric acid aqueous solution; analysis time: 30 min; gradient elution. In some embodiments, preferably, the gradient elution program can be specifically: 0-10 min, the volume ratio of phase A: phase B is 5:95-25:75; 10-20 min, the volume ratio of phase A: phase B is 25:75; 20-30 min, the volume ratio of phase A: phase B is 25:75-100:0. In some embodiments, preferably, in the HPLC determination conditions, mobile phase A can be acetonitrile; mobile phase B can be 0.1% phosphoric acid aqueous solution.

[0044] In some embodiments, the flow rate of the HPLC assay conditions may be 0.3-0.5 mL / min. In some embodiments, preferably, the flow rate of the HPLC assay conditions may be 0.4 mL / min.

[0045] In some embodiments, in the HPLC assay conditions, the column temperature may be 40-50°C. For example, in the HPLC assay conditions, the column temperature may be 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50°C. Any range characterized by a combination of the above end values ​​is also included, which will not be repeated here. In some embodiments, preferably, in the HPLC assay conditions, the column temperature may be 45°C.

[0046] In some embodiments, in the HPLC assay conditions, the injection volume can be 4 to 6 μL. In some embodiments, preferably, in the HPLC assay conditions, the injection volume can be 5 μL.

[0047] In some embodiments, in the HPLC assay conditions, the UV detection wavelengths may be 280 nm and 365 nm.

[0048] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified. The quantitative tests in the following examples are repeated three times, and the results are averaged.

[0049] The instruments and reagents used in the examples of this application are as follows:

[0050] instrument:

[0051] AL104 analytical balance (Mettler Toledo, Switzerland); EPED-E2-10TF laboratory ultrapure water machine (Nanjing Yipu Yida Technology Development Co., Ltd.); KS-500DE CNC ultrasonic cleaner (Kunshan Jielimei Ultrasonic Instrument Co., Ltd.); Agilent 1290UPLC-6530QTOF-MS liquid spectrometer (Agilent Technologies, USA); ThermoFisher Scientific Vanquish high performance liquid chromatograph (Thermo Fisher Scientific, USA) equipped with a diode array detector.

[0052] Reagents and drugs:

[0053] There are 6 batches of Lianshuqing granules samples, batch numbers are 20231001-20231006.

[0054] Vaccariae flavonoid glycosides (batch number PCS0888, purity ≥98%), hesperidin (batch number PCS0167, purity ≥98%), and rhamnosin (batch number PCS0249, purity ≥98%) were purchased from Chengdu Zhibiao Huachun Biotechnology Co., Ltd.; polydatin (batch number T109800, purity ≥98%) was purchased from Jiangsu Anhuai Chemical Technology Co., Ltd., naringin (batch number K200680, purity ≥98%), adenosine (batch number P328025, purity ≥99%) were purchased from Shanghai Dibai Chemical Technology Co., Ltd.; atractylodesin (batch number ZA05 47, purity ≥99.97%), purchased from Chengdu Mansite Biotechnology Co., Ltd.; epicatechin (batch number 013375460, purity 98%), purchased from Shanghai Titan Technology Co., Ltd.; luteolin (batch number HS21053B1, purity ≥98%), purchased from Baoji Chenguang Biotechnology Co., Ltd.; caffeic acid (batch number PRF7102121, purity ≥98%), purchased from Chengdu Purifa Technology Development Co., Ltd.; quercetin (batch number H-009-170516, purity >98%), purchased from Chengdu Ruifensi Biotechnology Co., Ltd. Formic acid and acetonitrile for mass spectrometry were of mass spectrometry grade and purchased from Merck Co., Ltd., 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.; water was ultrapure water.

[0055] Example 1

[0056] 1. Methods

[0057] 1.1 Solution preparation

[0058] 1.1.1 Preparation of reference solution

[0059] Accurately weigh appropriate amounts of fenugreek flavonoid glycosides, hesperidin, rhamnolide, polydatin, naringin, adenosine, atractylodesin, epicatechin, luteolin, caffeic acid, and quercetin, dissolve and dilute with methanol to prepare a mixture reference solution with a mass concentration of each component of about 20 μg / mL for LC-MS identification.

[0060] Accurately weigh appropriate amounts of fenugreek flavonoid glycosides, polydatin, naringin, and hesperidin reference substances, place them in the same volumetric flask, dissolve and dilute to volume with methanol to prepare mixed reference substance stock solutions with a mass concentration of 1 mg / mL, and then add methanol to dilute them step by step to obtain reference substance solutions with mass concentrations of 1, 2, 5, 10, 20, 50, 100, and 150 μg / mL of each component, respectively.

[0061] 1.1.2 Test solution

[0062] Accurately weigh 0.2 g of this product and place it in a stoppered conical flask. Accurately add 10 mL of 70% methanol and weigh the mass. Ultrasonicate for 10 min, make up the lost mass with 70% methanol, shake well, filter through a 0.22 μm microporous membrane, and take the filtrate for qualitative and quantitative analysis.

[0063] Accurately weigh 0.2 g each of the powders of Herba Lysimachiae, Atractylodes, Polygonum cuspidatum, Citrus aurantium, Rhizoma Cyathulae, Rhizoma Trillium, Rhizoma Curcumae, Pyrus Lycii, Vaccariae Segetalis, and Spina Gleditsiae, and prepare the test solution in the same way. Take the filtrate for the analysis of the attribution of the individual medicinal components.

[0064] 1.2 LC-MS analysis method

[0065] 1.2.1 Chromatographic conditions

[0066] The chromatographic column is Agilent Poroshell 120EC-C 18 (3.0 mm × 150 mm, 2.7 μm); mobile phase: acetonitrile (A)-0.1% formic acid aqueous solution (B), gradient elution (0-10 min, 5%-25% A; 10-30 min, 25%-40% A; 30-50 min, 40%-100% A); flow rate: 0.3 mL min -1 ; Column temperature: 25℃; Injection volume: 5μL.

[0067] 1.2.2 Mass spectrometry conditions

[0068] Electrospray ion source (ESI) was used to scan in positive and negative ion modes, full scan mode (full MS) was used to collect primary mass spectrometry data, and automatic secondary ion mode (Auto MS / MS) was used to collect secondary fragmentation data of compounds. The optimized mass spectrometry parameters are as follows: drying gas flow rate 10L / min; drying gas temperature 350℃; nebulizer gas pressure 30psi; capillary voltage 3500V; skimmer voltage 65V; octopole radio frequency voltage 750V; capillary outlet voltage 125V; collision voltage 30eV; mass-to-charge ratio scanning range: m / z 50-1200.

[0069] 1.3 HPLC quantitative method

[0070] 1.3.1 Chromatographic conditions

[0071] The chromatographic column is the same as that in 2.2.1; the mobile phase is acetonitrile (A)-0.1% phosphoric acid aqueous solution (B), gradient elution (0-10 min, 5%-25% A; 10-20 min, 25% A; 20-30 min, 25%-100% A); the flow rate is 0.4 mL min -1 ; Column temperature: 45℃; Injection volume: 5μL; UV detection wavelength: 280nm and 365nm.

[0072] 2. Results

[0073] 2.1 UPLC-Q-TOF-MS / MS component identification

[0074] Take appropriate amount of each test solution and inject it into LC-MS for analysis. The superposition of extracted ion current of Lianshuqing granules in positive and negative modes is shown in Figure 1 By consulting databases such as CNKI, PubMed, SciFinder, TCMID (Traditional Chinese medicine integrated database) and related literature, a database of the main chemical components of Lianshuqing Granules was established. The main compounds in Lianshuqing Granules were identified by combining the retention time of the reference substance, the accurate mass-to-charge ratio of the quasi-molecular ion peak, secondary fragments and other related information. The results are shown in Table 1.

[0075] The structural formula of 5,4'-dihydroxy-6,7,8,3'-tetramethoxyflavone isomers is shown in Formula 1 below:

[0076]

[0077] The structural formula of 7-hydroxy-4-methoxy-5-methylcoumarin isomers is shown in Formula 2 below:

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085] * Marked as a reference substance for determination

[0086] **Centella asiatica JQC, Atractylodes macrocephala CZ, Polygonum cuspidatum HZ, Citrus aurantium QP, Cyathula officinalis CNX, Trillium sclerotium SL, Curcuma zedoaria EZ, Citrus aurantium GJL, Vaccaria segetalis WBLX, Gleditsia sinensis ZJC

[0087] Example 2 - Attribution of single herbal ingredients

[0088] Ten kinds of medicinal materials, including Herba Lysimachiae, Atractylodes, Polygonum cuspidatum, Citrus aurantium, Rhizoma Cyathulae, Rhizoma Trillium, Rhizoma Curcumae, Pyrus Lycii, Vaccariae Segetalis, and Spina Gleditsiae, were ground and passed through a 50-mesh sieve. The sample pretreatment steps in the above Example 1 were used to prepare 10 single medicinal material sample solutions.

[0089] The 10 medicinal materials of Herba Lysimachiae, Atractylodes, Polygonum cuspidatum, Citrus aurantium, Rhizoma Cynoglossi, Sparganium, Rhizoma Curcumae, Pyrus erythrorhizon, Vaccaria segetalis, and Spina Gleditsiae were each lacking one medicinal material, and the sample pretreatment steps in the above Example 1 were used for preparation, and negative sample solutions lacking Herba Lysimachiae, Atractylodes, Polygonum cuspidatum, Citrus aurantium, Rhizoma Cynoglossi, Sparganium, Rhizoma Curcumae, Pyrus erythrorhizon, Vaccaria segetalis, and Spina Gleditsiae were obtained respectively.

[0090] The instrumental analysis conditions in the above Example 1 were used to respectively measure the test solution, 10 single medicinal material sample solutions, and negative sample solutions lacking Herba Lysimachiae, lacking Atractylodes, lacking Polygonum cuspidatum, lacking Citrus aurantium, lacking Rhizoma Cynoglossi, lacking Trillium gracile, lacking Curcuma zedoaria, lacking Citrus aurantium, lacking Vaccaria segetalis, and lacking Spina Gleditsiae, and obtain the control liquid chromatograms of the test solution, the liquid chromatograms of the single medicinal material sample solution, and the liquid chromatograms of the negative sample solutions lacking Herba Lysimachiae, lacking Atractylodes, lacking Polygonum cuspidatum, lacking Citrus aurantium, lacking Rhizoma Cynoglossi, lacking Trillium gracile, lacking Curcuma zedoaria, lacking Citrus aurantium, lacking Vaccaria segetalis, and lacking Spina Gleditsiae, respectively. The liquid chromatograms of the single medicinal material sample solution, the negative sample solution lacking Herba Lysimachiae, lacking Atractylodes, lacking Polygonum cuspidatum, lacking Citrus aurantium, lacking Rhizoma Cynoglossi, lacking Trillium, lacking Curcuma, lacking Citrus aurantium, lacking Vaccaria segetalis, and lacking Gleditsia sinensis were compared with the control liquid chromatogram of Lianshuqing Granules, and the corresponding characteristic peaks of the single medicinal material sample solution in the control liquid chromatogram of Lianshuqing Granules were identified by relative retention time, so as to attribute and locate the components in the control liquid chromatogram of Lianshuqing Granules. The attribution information of the components in Lianshuqing Granules in the medicinal materials is shown in Table 1 above.

[0091] The compound results obtained by liquid-mass spectrometry analysis included 41 flavonoids, 15 phenolic acids, 10 terpenes, 16 amino acids and cyclic peptides, 13 phenylpropanoids, 9 alkaloids, and 35 other compounds. The 139 compounds identified were classified as single herbal ingredients, including 43 in Lysimachia chinensis, 11 in Atractylodes macrocephala, 29 in Polygonum cuspidatum, 27 in Citrus aurantium, 8 in Cyathula officinalis, 21 in Trigonella ternata, 33 in Citrus citrifolia, 3 in Curcuma zedoaria, 14 in Vaccaria segetalis, and 16 in Gleditsia sinensis.

[0092] Example 3 - HPLC quantitative methodology study

[0093] 1. Specificity test

[0094] Take appropriate amount of reference substance and test substance solution, and perform the test under the conditions of "1.2" and "1.3". The results are shown in Figure 2 The separation of each index component is ideal, indicating that the method has good specificity.

[0095] 2. Linear relationship investigation

[0096] Accurately pipette an appropriate amount of the reference stock solution under item "1.1", dilute it with 70% methanol to form a series of standard solutions of different mass concentrations, and inject and measure under the conditions of item "1.3". Regression is performed with the mass concentration of the reference as the horizontal axis (X) and the peak area as the vertical axis (Y). The results are shown in Table 2. Each component has a good linear relationship within its respective range. Dilute the mixed reference solution step by step and measure it under the conditions under item "1.3". The solution concentration when the signal-to-noise ratio (S / N) is 3:1 is used as the LOD, and the solution concentration when the S / N is 10:1 is used as the LOQ. The results are shown in Table 2.

[0097] Table 2 Linear relationship of each component

[0098]

[0099] 3. Precision test

[0100] Take an appropriate amount of the reference solution with a concentration of 50 μg / mL under item "1.1", and make six injections under the conditions of item "1.3". The RSDs of fenugreek flavonoid glycosides, polydatin, naringin and hesperidin were 0.57%, 0.29%, 0.30% and 0.23%, respectively, indicating that the instrument has good precision.

[0101] 4. Stability test

[0102] Take appropriate amount of the test solution under "1.1", and inject samples for determination at 0, 2, 4, 8, 12, and 24 h. The RSDs of fenugreek flavonoid glycosides, polydatin, naringin, and hesperidin were 0.62%, 1.02%, 0.70%, and 0.42%, respectively, indicating that the solution had good stability within 24 h.

[0103] 5. Repeatability test

[0104] Take 6 portions of this product (batch number 20231002), prepare the test solution according to the method under "1.1", and measure the sample under the conditions of "1.3". The RSDs of the contents of fenugreek flavonoid glycosides, polydatin, naringin and hesperidin were 0.01%, 1.00%, 0.83% and 1.62%, respectively, indicating that the method has good repeatability.

[0105] 6. Sample recovery test

[0106] Take 6 portions of this product (batch number 20231002) with known content of each component, 0.1g each, add 1mL of mixed reference solution under "1.1" respectively, prepare the test solution according to the method under "1.1", inject and measure under the conditions of "1.3", calculate the recovery rate, and the results are shown in Table 3. The average recovery rates of fenugreek flavonoid glycosides, polydatin, naringin, and hesperidin were calculated to be 98.64%, 101.04%, 97.29%, and 105.08%, respectively, and the RSDs were 4.60%, 3.31%, 3.02%, and 2.07%, respectively.

[0107] Table 3. Experimental results of sample recovery

[0108]

[0109] 7. Sample content determination

[0110] Six batches of samples were taken, with 3 portions in each batch. The test solutions were prepared according to the method under "1.1". The samples were injected and measured under the conditions of "1.3". The results are shown in Table 4. The contents of fenugreek flavonoid glycosides, polydatin, naringin and hesperidin in the six batches of samples were 0.65-0.71 mg / g, 2.31-2.80 mg / g, 2.02-2.17 mg / g and 4.76-5.11 mg / g, respectively. The results showed that the contents of the four active ingredients in different batches of Lianshuqing granules were slightly different, and the production process was stable.

[0111] Table 4 Determination results of the contents of each component in Lianshuqing granules (mean ± SD, mg / g, n = 3)

[0112]

[0113] The experiment of this application used LC-MS to identify the main chemical components in Lianshuqing Granules, and used HPLC to determine the contents of its four index components.

[0114] First, the chromatographic analysis conditions were optimized. The results of chromatographic column screening showed that Agilent Poroshell 120EC-C18 filled with core-shell filler particles had low mass transfer resistance, fast peak elution, high analytical throughput, and ideal chromatographic peak shape and separation efficiency. The results of mobile phase screening showed that adding an appropriate amount of phosphoric acid to the aqueous phase could inhibit the ionization of weakly acidic compounds and improve the peak shape of the chromatographic peak. The effects of column temperature (25, 35, 45°C) and flow rate (0.3, 0.4, 0.5 mL / min) on the separation were investigated. It was found that when the column temperature was 45°C, the peak shape and separation of flavonoid glycosides from Vaccaria were better; the flow rate had little effect on the peak shape and separation of each index component. Polydatin, naringin, and hesperidin had the highest detection sensitivity at a detection wavelength of 280 nm, while flavonoid glycosides from Vaccaria had high detection sensitivity and low interference at a detection wavelength of 365 nm. Therefore, the multi-wavelength simultaneous detection function of the diode array detector was used to simultaneously determine the four index components at two wavelengths.

[0115] The preparation conditions of the test solution, such as ultrasonic time (10, 20, 30 min), extraction solvent (0, 30, 50, 70, 100% methanol), and solid-solution ratio (v:w) (1:10, 1:25, 1:50, 1:100), were investigated respectively, and it was found that the ultrasonic time had little effect on the extraction rate of each component, so the ultrasonic time was selected as 10 min; the extraction rate was higher when 70% methanol was used as the extraction solvent; when the solid-solution ratio was 1:50, both the extraction rate and the peak intensity were higher.

[0116] Under the optimized conditions, the chromatographic peaks of flavonoid glycosides of Vaccariae, Polydatin, Naringin, and Hesperidin in Lianshuqing Granules were high in height, symmetrical in shape, and well separated, and were selected as quantitative index components. The method validation results showed that the linear relationship of the four index components was good, and the precision, repeatability, stability, and recovery rate all met the determination requirements. The quantitative method was fast and accurate, and could be used for the content determination of Lianshuqing Granules.

[0117] The results of LC-MS qualitative analysis show that Lianshuqing Granules are rich in chemical components, among which there are many types of flavonoids with high content. Modern pharmacological studies have shown that the important active ingredients such as flavonoids, phenolic acids, and volatile substances contained in the main drug of this prescription, the herb Centella asiatica, have an inhibitory effect on the formation of calcium oxalate monohydrate crystals, the main component of urinary stones, and can be used in the clinical treatment of diseases such as kidney stones, gallstones, and urinary tract stones. The flavonoids contained in the compound, such as quercetin, can significantly reduce CaO x The formation of kidney stones plays an effective role in preventing kidney stones; kaempferol, rutin, etc. play an important role in anti-inflammatory response; Vaccaria flavonoid glycoside is one of the main active ingredients of Vaccaria, which has multiple effects such as anti-inflammatory, antioxidant and vasodilation; Polydatin is one of the main active ingredients of Polygonum cuspidatum, which has multiple effects such as anti-inflammatory, antitussive and antioxidant; Naringin has multiple biological activities, including analgesia, anti-allergic reaction, antioxidant and other effects; Hesperidin has the effects of improving immunity, anti-inflammatory and antibacterial. Phenylpropanoid components such as umbelliferone and isoscopolamine have significant antioxidant and anti-inflammatory activities; terpenoid components such as atractylodesin and β-eudesmol have anti-inflammatory and antioxidant effects. Therefore, Lianshuqing granules can reduce the formation of stones through various ways, improve immunity, reduce inflammation of patients, and achieve the effects of regulating qi and activating blood circulation, dispersing knots and eliminating stones.

[0118] The basic concepts have been described above. Obviously, for those skilled in the art, the above detailed disclosure is only for example and does not constitute a limitation of this specification. Although not explicitly stated here, those skilled in the art may make various modifications, improvements and corrections to this specification. Such modifications, improvements and corrections are suggested in this specification, so such modifications, improvements and corrections still belong to the spirit and scope of the exemplary embodiments of this specification.

[0119] At the same time, this specification uses specific words to describe the embodiments of this specification. For example, "one embodiment", "an embodiment", and / or "some embodiments" refer to a certain feature, structure or characteristic related to at least one embodiment of this specification. Therefore, it should be emphasized and noted that "one embodiment" or "an embodiment" or "an alternative embodiment" mentioned twice or more in different positions in this specification does not necessarily refer to the same embodiment. In addition, certain features, structures or characteristics in one or more embodiments of this specification can be appropriately combined.

[0120] In some embodiments, numbers describing the number of components and attributes are used. It should be understood that such numbers used in the description of the embodiments are modified by the modifiers "about", "approximately" or "substantially" in some examples. Unless otherwise specified, "about", "approximately" or "substantially" indicate that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may change according to the required features of individual embodiments. In some embodiments, the numerical parameters should take into account the specified significant digits and adopt the general method of retaining digits. Although the numerical domains and parameters used to confirm the breadth of their range in some embodiments of this specification are approximate values, in specific embodiments, the setting of such numerical values ​​is as accurate as possible within the feasible range.

[0121] Finally, it should be understood that the embodiments described in this specification are only used to illustrate the principles of the embodiments of this specification. Other variations may also fall within the scope of this specification. Therefore, as an example and not a limitation, alternative configurations of the embodiments of this specification may be considered consistent with the teachings of this specification. Accordingly, the embodiments of this specification are not limited to the embodiments explicitly introduced and described in this specification.

Claims

1. A method for detecting multiple components in Lianshuqing granules, comprising the following steps: 1) Add the Lianshuqing granule sample to the solvent to dissolve, perform ultrasonic extraction, filter, and take the filtrate to obtain the test solution; 2) The test solution was measured by liquid chromatography-mass spectrometry to determine 139 components in the test solution.

2. The detection method according to claim 1, characterized in that In step 1), the solvent is a 60-80% methanol solution; and / or the ratio of the mass of the Lianshuqing granules sample added to the volume of the solvent added is 0.2: (9-11) g / mL.

3. The detection method according to claim 1, characterized in that In step 1), the ultrasonic extraction time is 5 to 15 minutes.

4. The detection method according to claim 1, characterized in that In step 2), in the liquid chromatography-mass spectrometry method, the liquid chromatography determination conditions include any one or more of the following: A1) the chromatographic column is a C18 chromatographic column, preferably, the chromatographic column is an Agilent Poroshell 120EC-C18 chromatographic column; A2) Mobile phase A is acetonitrile; mobile phase B is 0.05-0.15% formic acid aqueous solution; analysis time: 50 min; Gradient elution; A3) Column temperature: 20-30°C; A4) Flow rate: 0.2-0.4 mL / min; A5) Injection volume: 4-6 μL.

5. The detection method according to claim 4, characterized in that: The gradient elution procedure is specifically as follows: 0-10 min, the volume ratio of phase A: phase B is 5:95-25:75; 10-30 min, the volume ratio of phase A: phase B is 25:75-40:60; 30-50 min, the volume ratio of phase A: phase B is 40:60-100:

0.

6. The detection method according to claim 1, characterized in that In step 2), in the liquid chromatography-mass spectrometry method, the mass spectrometry conditions further include any one or more of the following: C1) The ion source is an electrospray ion source ESI; C2) Detection mode is positive ion electrospray ionization mode ESI + and negative ion electrospray ionization mode ESI - ; C3) Drying gas is nitrogen; C4) The temperature of the drying gas is 340-360°C; C5) The flow rate of drying gas is 9-11 L / min; C6) Atomizing gas pressure is 25-35 psi; C7) capillary voltage is 3400-3600V; C8) The capillary outlet voltage is 115-135V; C9) Skimmer voltage 60-70V; C10) Octopole RF voltage 700-800V; C11) The primary mass spectrometer was in MS1 ​​mode with a mass-to-charge ratio scanning range of 50-1200 m / z; C12) The secondary mass spectrometer was in Auto-MS / MS mode with a collision voltage of 25-35 eV.

7. The detection method according to claim 1, characterized in that In step 2), the components in the test solution are selected from lysine, serine, glutamic acid, gluconic acid, valine, cytidine, malic acid, adenine, 2-furancarboxylic acid, Adenosine * , pyroglutamic acid, uracil, fumaric acid, tyrosine, citric acid, phenylalanine, tyramine, isoleucine, leucine, succinic acid, N-methyltyramine, gallic acid, hordenine, 5-hydroxymethylfurfural, vanillin, synephrine, vanillic acid, 5-hydroxy-2-(hydroxymethyl)pyridine, neochlorogenic acid, chlorogenic acid, protocatechuic acid, n-butyl-O-β-D-pyranofructose, catechol, syringin, scopoletin, cryptochlorogenic acid, cyclotide G, erythrine, epicatechin * , catechins, p-hydroxybenzoic acid, kaempferol-3-O-β-acacia disaccharide, chrysophanol-8-O-β-glucoside, hesperidin, vaccariae cyclopeptide H, naringin, 2-methoxy flat orange alkaloids, vaccariae flavonoid glycosides * , Caffeic acid * , ferulic acid, caffeoylmalic acid, sinapinic aldehyde, saponin, isorientin, resveratrol-4'-O-glucoside, isovitexin-2"-O-arabinoside, orientin, p-hydroxybenzaldehyde, epicatechin-5-O-β-D-pyranoglucopyranoside, isoschafta glycoside, vaccaria cyclopeptide F, isorhamnetin-3-O-rutinoside, eriocitrin, rutin, 4-hydroxyacetophenone, vitexin, atractylodesin * , phlorizin, p-coumaric acid, p-hydroxycinnamic acid, polydatin * , paeonol, luteolin * , Vaccaria flavonoid glycoside E, isoscopolamine, umbelliferone, cyperasterone, naringin * , naringenin-7-O-glucoside, dihydroquercetin, wild sumac glycoside, 24-hydroxy calixasterone, phenyl-β-glucopyranoside, neohesperidin, hesperidin, naringenin, 5-demethylnobiletin, hesperidin * , isoprunin, 6,7-dihydroxycoumarin, rosmarinic acid, azelaic acid, isovitexin, atractylodes lactone II, coumarin, epigallocatechin, apigenin, 6,7-dimethoxycoumarin, triangular acid, angelica lactone, resveratrol, hesperidin, 5,4'-dihydroxy-6,7,8,3'-tetramethoxyflavonoid isomers, kaempferol-3-O-rutinoside, 6-hydroxyaloe-emodin-8-methyl ether, vaccariae cyclopeptide B, quercetin * , citrus glycosides, kaempferol, rhamnolide-8-O-β-D-glucoside, cassia pine, costus lactone, n-butyl-O-β-D-pyranose, vaccaria cyclopeptide C, vaccaria cyclopeptide A, rhamnolide methyl ether-8-O-β-D-glucoside, 7-hydroxy-4-methoxy-5-methylcoumarin isomers, vaccaria cyclopeptide D, baicalin, bergamot lactone, 4'-hydroxy-5,6,7-trimethoxyflavone, deacetylnomirin, limonin, atractylolactone III, isorhodoptilometrin, nobiletin, atractylol acetate, nomilin, dehydrocurdione, atractylone, hibiscus lactone, tangeretin, isoimperatorin, curcuma epoxide, rhamnolide * , curcumadione, echinocystic acid, physaccharin methyl ether or β-eudesmol.

8. A method for attributing the medicinal materials of multiple components in Lianshuqing granules, comprising the following steps: A) Preparation of single medicinal material sample solutions: prepare any one or more of the 10 medicinal material samples of Herba Lysimachiae, Rhizoma Atractylodis Macrocephalae, Rhizoma Polygoni Cuspidati, Rhizoma Citri Reticulatae, Rhizoma Cynoglossi, Rhizoma Trillium, Rhizoma Curcumae, Rhizoma Citri Reticulatae, Rhizoma Vaccariae, and Rhizoma Gleditsiae according to step 1) of the method for detecting multiple components in Lianshuqing Granules according to any one of claims 1 to 7, and obtain at least one single medicinal material sample solution respectively; B) Preparation of negative sample solution: 10 medicinal material samples of Herba Lysimachiae, Atractylodes, Polygonum cuspidatum, Citrus aurantium, Cyathula officinalis, Sparganium, Curcuma zedoaria, Citrus aurantium, Vaccaria segetalis, and Gleditsia sinensis are respectively lacking 1 medicinal material, and are prepared according to step 1) of the method for detecting multiple components in Lianshuqing granules according to any one of claims 1 to 7, to obtain negative sample solutions lacking Herba Lysimachiae, Atractylodes, Polygonum cuspidatum, Citrus aurantium, Cyathula officinalis, Sparganium, Curcuma zedoaria, Citrus aurantium, Vaccaria segetalis, and Gleditsia sinensis; C) Determination: using liquid chromatography-mass spectrometry under the same conditions as step 2) of the method for detecting multiple components in Lianshuqing granules according to any one of claims 1 to 7 to respectively determine the single medicinal material sample solution in step a), and the negative sample solution lacking Herba Lysimachiae, lacking Atractylodes, lacking Polygonum cuspidatum, lacking Citrus aurantium, lacking Rhizoma Cynoglossum, lacking Tripterygium wilfordii, lacking Curcuma zedoaria, lacking Citrus aurantium, lacking Vaccaria segetalis, and lacking Spina Gleditsiae in step b), and obtain liquid chromatograms of the single medicinal material sample solution, the negative sample solution lacking Herba Lysimachiae, lacking Atractylodes, lacking Polygonum cuspidatum, lacking Citrus aurantium, lacking Tripterygium wilfordii, lacking Tripterygium wilfordii, lacking Vaccaria segetalis, and lacking Spina Gleditsiae, respectively; D) Obtaining a reference liquid chromatogram: analyzing the test solution prepared in step 1) of the method for detecting multiple components in Lianshuqing granules according to any one of claims 1 to 7 using step 2) of the method for detecting multiple components in Lianshuqing granules according to any one of claims 1 to 7 to obtain a reference liquid chromatogram of Lianshuqing granules; E) Attribution: The liquid chromatograms of the single medicinal material sample solution, the negative sample solution lacking Centella asiatica, lacking Atractylodes lancea, lacking Polygonum cuspidatum, lacking Citrus aurantium, lacking Rhizoma Cynoglossi, lacking Trillium gracile, lacking Curcuma zedoaria, lacking Citrus aurantium, lacking Vaccaria segetalis, and lacking Gleditsia sinensis are compared with the control liquid chromatogram of Lianshuqing Granules, and the corresponding characteristic peaks of the single medicinal material sample solution in the control liquid chromatogram of Lianshuqing Granules are identified by relative retention time, thereby attributing and locating the components in the control liquid chromatogram of Lianshuqing Granules.

9. A method for detecting the content of fenugreek flavonoid glycosides, polydatin, naringin or hesperidin in Lianshuqing granules, comprising the following steps: 1) preparing according to step 1) of the method for detecting multiple components in Lianshuqing granules according to any one of claims 1 to 7 to obtain a test solution; 2) Prepare reference solutions of fenugreek flavonoid glycosides, polydatin, naringin, and hesperidin; 3) The test solution and the reference solution were measured by high performance liquid chromatography to determine the contents of fenugreek flavonoid glycosides, polydatin, naringin and hesperidin in the test solution.

10. The method for detecting the content of vaccariae flavonoid glycosides, polydatin, naringin or hesperidin in Lianshuqing granules according to claim 9, characterized in that: The HPLC assay conditions include any one or more of the following: A1) the chromatographic column is a C18 chromatographic column, preferably, the chromatographic column is an Agilent Poroshell 120EC-C18 chromatographic column; A2) mobile phase A is acetonitrile; mobile phase B is 0.05-0.15% phosphoric acid aqueous solution; analysis time: 30 min; gradient elution; preferably, the gradient elution program is specifically as follows: 0-10 min, the volume ratio of phase A: phase B is 5:95-25:75; 10-20 min, the volume ratio of phase A: phase B is 25:75; 20-30 min, the volume ratio of phase A: phase B is 25:75-100:0; A3) flow rate 0.3-0.5 mL / min; A4) Column temperature: 40-50°C; A5) Injection volume: 4-6 μL; A6) UV detection wavelengths: 280nm and 365nm.

Images