Detection method for analyzing components and in-blood components of purpleflower holly fruit kidney-clearing granules

Abstract

The invention provides purpleflower holly seed kidney-clearing granules and a detection method for analyzing components entering blood, and the detection method for analyzing the components of the purpleflower holly seed kidney-clearing granules comprises the following steps: determining a test solution by adopting an ultra-high performance liquid chromatography-high resolution mass spectrometry method, and determining 103 components in the test solution, and reference is provided for quality control and clinical application guidance of the purpleflower holly seed kidney-clearing granules. The detection method comprises the following steps: preparing blank plasma and administration plasma into a test solution, determining the test solution by adopting an ultra-high performance liquid chromatography-high resolution mass spectrometry combined method, and identifying 77 compounds including 20 prototype components and 57 metabolites, the method can reflect the drug-derived component condition after the drug enters the blood, makes up the blank of qualitative detection of the blood-entering component of the Ilex chinensis kidney-clearing granules, and provides a basis for further research on the metabolic cycle and metabolic pathway of the drug.

Description

Technical Field

[0001] The present invention belongs to the technical field of research on the chemical composition of traditional Chinese medicine preparations, and relates to a detection method for analyzing the components and blood components of Dongqingzi Qingshen Granules. Background Art

[0002] Chronic nephritis generally refers to chronic glomerulonephritis, and its etiology has not been fully clarified. It has a slow onset, and its clinical manifestations can include edema, hypertension, proteinuria, hematuria, etc., which can present as one or several of them, and sometimes severe hypertension may be accompanied. The condition of this disease is protracted, sometimes mild and sometimes severe, and is often induced or aggravated by infection. If not treated promptly and effectively, the condition can gradually develop into chronic renal failure, and some even require dialysis treatment. In traditional Chinese medicine, it belongs to the categories of edema, hematuria, low back pain, consumptive fatigue, etc., and the syndromes of qi-yin deficiency, spleen-kidney yang deficiency, spleen-kidney qi deficiency, damp-heat accumulation, etc. are the most common. The commonly used treatment methods include strengthening the spleen and tonifying the kidney, clearing heat and promoting diuresis, replenishing qi and activating blood circulation, etc.

[0003] As an in-hospital preparation of our hospital, Dongqingzi Qingshen Granules have significant clinical efficacy in the treatment of chronic nephritis and have been approved by the Shanghai Food and Drug Administration. Dongqingzi Qingshen Granules are composed of medicinal herbs such as Ilex pubescens, Artemisia anomala, Viscum coloratum, Cortex Periplocae, etc., promoting blood circulation and inducing diuresis, dispelling wind and dredging collaterals. It contains a wide variety of components with complex structures. At present, there has been no reported relatively comprehensive qualitative and quantitative detection method for the compound components and blood components of Dongqingzi Qingshen Granules. Summary of the Invention

[0004] In view of the above-mentioned disadvantages of the prior art, the purpose of the present invention is to provide a detection method for analyzing the components and blood components of Dongqingzi Qingshen Granules, so as to solve the problem that there is a lack of a method for comprehensively qualitatively detecting the chemical components in Dongqingzi Qingshen Granules in the prior art.

[0005] To achieve the above object and other related objects, the first aspect of the present invention provides a detection method for analyzing the components of Dongqingzi Qingshen Granules, including the following steps:

[0006] 1) Add the Dongqingzi Qingshen sample to a solvent for dissolution, perform ultrasonic extraction, then centrifuge and take the supernatant to obtain a test solution;

[0007] 2) Determine 103 components in the test solution by using ultra-high performance liquid chromatography-high resolution mass spectrometry (UPLC-Q-TOF / MS).

[0008] Preferably, in step 1), the Dongqingzi Qingshen Granule sample should be ground into a powder.

[0009] Preferably, in step 1), the solvent is a 40 - 60 (v / v)% methanol solution. The specific concentration of the methanol solution can be, for example, 40 - 50 (v / v)%, 50 - 60 (v / v)%, 45 - 55 (v / v)%, and is preferably a 50 (v / v)% methanol solution. The 40 - 60 (v / v)% methanol solution is an aqueous methanol solution with a volume percentage of methanol being 40 - 60%, and the 50 (v / v)% methanol solution is an aqueous methanol solution with a volume percentage of methanol being 50%.

[0010] Preferably, in step 1), the ratio of the mass g of the prepared Ilex pubescens kidney - clearing granule sample to the volume mL of the solvent added is 0.5:5 - 15, specifically, for example, 0.5:5 - 7.5, 0.5:7.5 - 10, 0.5:10 - 12.5, 0.5:12.5 - 15, and is preferably 0.5:10.

[0011] Preferably, in step 1), the time for ultrasonic extraction is 20 - 40 min, specifically, for example, 20 - 30 min, 30 - 40 min, 25 - 35 min, and is preferably 30 min.

[0012] Preferably, in step 1), the rotation speed for centrifugation is 10000 - 15000 r·min -1 , specifically, for example, 10000 - 12000 r·min -1 , 12000 - 15000 r·min -1 , 11000 - 13000 r·min -1 , and is preferably 12000 r·min -1 .

[0013] Preferably, in step 1), the time for centrifugation is 1 - 10 min, specifically, for example, 1 - 5 min, 5 - 10 min, 4 - 6 min, and is preferably 5 min.

[0014] Preferably, after ultrasonic treatment in step 1), it needs to be cooled to room temperature and shaken well.

[0015] Preferably, in step 2), in the ultra - high performance liquid chromatography - high - resolution mass spectrometry method, the conditions for ultra - high performance liquid chromatography determination are as follows: The chromatographic column is a C 18 chromatographic column, preferably Welch Ultimate UHPLC AQ - C18 (2.1×100 mm, 1.8 μm); column temperature: 30 - 40 °C, preferably 35 °C; flow rate: 0.2 - 0.5 mL / min, preferably 0.3 mL / min; injection volume: 1 - 3 μL, preferably 2 μL; mobile phase A is acetonitrile; mobile phase B is a 0.05 - 0.15% formic acid aqueous solution, preferably 0.1% formic acid aqueous solution; analysis time: 40 min; gradient elution.

[0016] In the above mobile phase B, the 0.05-0.15% formic acid aqueous solution is an aqueous solution of formic acid with a volume percentage of 0.05-0.15%, and the 0.1% formic acid aqueous solution is an aqueous solution of formic acid with a volume percentage of 0.1%.

[0017] More preferably, the specific gradient elution program is as follows: 0-3 min, mobile phase A: mobile phase B volume ratio is 0-3:100-97; 3-7 min, mobile phase A: mobile phase B volume ratio is 3-8:97-92; 7-20 min, mobile phase A: mobile phase B volume ratio is 8-20:92-80; 20-33 min, mobile phase A: mobile phase B volume ratio is 20-40:80-60; 33-38 min, mobile phase A: mobile phase B volume ratio is 10-95:60-5; 38-41 min, 95:5; 41-41.1 min, 95-0:5-100; 41.1-44 min, 0:100.

[0018] Preferably, in step 2), in the ultra-high performance liquid chromatography-high resolution mass spectrometry method, the mass spectrometry determination conditions are: the ion source is an electrospray ionization source (ESI); the detection mode is the positive ion mode (ESI + ) and the negative ion mode (ESI - ); the spray gas pressure is 50-60 psi, preferably 50 psi; the ionization voltage is 4500-5500 V in the positive ion mode, preferably 5000 V; the ionization voltage is -4000 to -5000 V in the negative ion mode, preferably -4500 V; the ion source temperature is 450-550 °C, preferably 500 °C; the first-stage mass spectrometry selects the MS mode, and the mass scanning range is 50-1700 m / z; the second-stage mass spectrometry selects the MS / MS mode, and the mass scanning range is 50-1250 m / z; the collision voltage is 35-45 eV, preferably 40 eV.

[0019] In the above ultra-high performance liquid chromatography-high resolution mass spectrometry method, Analyst TF 1.7.1 software is used for data acquisition. The data processing software is Peakview 1.2. During identification, the mass spectrometry data is preferentially matched with the Natural Products HR-MS / MS Spectral Library 1.0 database. Compounds are preliminarily screened based on the score information of each chromatographic peak, and further confirmed based on the primary and secondary information of each chromatographic peak, etc. The Natural Products HR-MS / MS Spectral Library 1.0 database contains multi-stage mass spectrometry diagrams of standard products with confirmed structures, including different acquisition modes, different adduct ions, different collision energies, etc. The compound information is comprehensive, and all are diagrams of actual collected standard products without any simulated and speculated diagrams. Therefore, the matching result has high accuracy. For compounds not included in the database, identification is carried out according to literature reports, mass spectrometry fragmentation rules, etc.

[0020] Preferably, in step 2), 103 components in the test solution are qualitatively analyzed by mass spectrometry in the ultra-high performance liquid chromatography-high resolution mass spectrometry method.

[0021] As described above, a detection method for the component analysis of Dongqingzi Qingshen Granules provided by the present invention uses ultra-high performance liquid chromatography-high resolution mass spectrometry technology, combining the high-efficiency separation ability of UPLC and the high selectivity and high sensitivity of mass spectrometry. According to the multi-stage mass spectrometry information of the sample, 103 compounds are identified from the compound sample of Dongqingzi Qingshen Granules by combining the high-resolution mass spectrometry database of natural products and relevant literature, providing a reference for the quality control and clinical application guidance of Dongqingzi Qingshen Granules.

[0022] First of all, the present invention optimizes the test sample preparation conditions and chromatographic separation conditions through experiments, including the concentration of the extraction solvent methanol and the solid-liquid ratio. The results show that when preparing the test solution with 50% methanol and a liquid-solid ratio of 0.5:10 (v:w), it can better balance the detection of polar components and weakly polar components in the compound and will not cause chromatographic solvent effects.

[0023] Secondly, the present invention compares the effects of different chromatographic columns on separation and finally selects Welch Ultimate UHPLC AQ-C18 (2.1×100 mm, 1.8 μm), and each component can obtain good retention and sharp peak shapes.

[0024] Furthermore, since the mobile phase not only affects the retention time, peak shape, and separation efficiency of compounds, but also affects the ionization of analytes in the mass spectrometer, thereby affecting the detection sensitivity of target compounds, the mobile phase and gradient were optimized through experiments. As a result, it was found that when acetonitrile and 0.1% formic acid were used as the mobile phase and separated according to a specific gradient elution program, the effect was better.

[0025] In addition, through precise molecular weight mass matching and MS / MS fragment information, 103 chemical components were identified in the present invention, and the medicinal material attribution was determined. The chemical profile of Dongqingzi Qingshen Granules was analyzed from two aspects: medicinal materials and compound categories. The results showed that the active ingredients in the formula mainly came from Ilex pubescens Hook. et Arn., Artemisia anomala S. Moore, Viscum coloratum (Kom.) Nakai, and Periploca sepium Bunge, and some chemical components were shared by multiple medicinal materials. The main components in the compound granules can be quickly identified by UPLC-Q-TOF-MS, which lays a foundation for the study of its quality attributes and provides a theoretical basis for the clinical use of this compound granule.

[0026] The second aspect of the present invention provides a detection method for analyzing the components entering the blood of Dongqingzi Qingshen Granules, including the following steps:

[0027] (3) Respectively take blank plasma and administered plasma, add a solvent to precipitate proteins, vortex and mix well, let stand, centrifuge, and take the supernatant. Centrifuge and concentrate to dryness for standby; before analysis, re-dissolve the residue blank plasma and administered plasma with a solvent, vortex and mix well, let stand, centrifuge, and take the supernatant to obtain the test solution;

[0028] (4) The test solution was determined by ultra-high performance liquid chromatography-high resolution mass spectrometry (UPLC-Q-TOF / MS), and 77 compounds were identified, including 20 prototype components and 57 metabolites.

[0029] Preferably, in step (1), the solvent is mass spectrometry grade methanol.

[0030] Preferably, in step (1), the volume of the added solvent is 2 to 4 times that of the blank plasma or administered plasma, which can be 2 to 3 times, or 3 to 4 times, preferably 3 times.

[0031] Preferably, in step (1), the time for vortex mixing is 3 to 8 min, which can be 3 to 4 min, 4 to 5 min, 5 to 6 min, 6 to 7 min, or 7 to 8 min, preferably 5 min.

[0032] Preferably, in step (1), the standing is at 4°C for 10 to 20 min, which can be 10 to 15 min, or 15 to 20 min, preferably 15 min.

[0033] Preferably, in step (1), the rotation speed of the centrifugation is 10,000 - 15,000 r·min -1 , specifically such as 10,000 - 12,000 r·min -1 , 12,000 - 15,000 r·min -1 , 11,000 - 13,000 r·min -1 , preferably 12,000 r·min -1 .

[0034] Preferably, in step (1), the centrifugation time is 10 - 20 min, specifically such as 10 - 12 min, 12 - 15 min, 15 - 18 min, 18 - 20 min, preferably 15 min.

[0035] Preferably, in step (1), the standby is storing and placing at -80 °C.

[0036] Preferably, in step (1), the solvent added for reconstitution is a 40 - 60 (v / v)% methanol solution, and the specific concentration of the methanol solution is such as 40 - 50 (v / v)%, 50 - 60 (v / v)%, 45 - 55 (v / v)%, preferably a 50 (v / v)% methanol solution. The 40 - 60 (v / v)% methanol solution is an aqueous methanol solution with a volume percentage of 40 - 60%, and the 50 (v / v)% methanol solution is an aqueous methanol solution with a volume percentage of 50%.

[0037] Preferably, in step (1), the time for vortex mixing during reconstitution is 1 - 5 min, it can be 1 - 2 min, 2 - 3 min, 3 - 4 min, or it can also be 4 - 5 min, preferably 3 min.

[0038] Preferably, in step (1), the rotation speed of the centrifugation during reconstitution is 10,000 - 15,000 r·min -1 , specifically such as 10,000 - 12,000 r·min -1 , 12,000 - 15,000 r·min -1 , 11,000 - 13,000 r·min -1 , preferably 12,000 r·min -1 .

[0039] Preferably, in step (1), the centrifugation time during reconstitution is 10 - 20 min, specifically such as 10 - 12 min, 12 - 15 min, 15 - 18 min, 18 - 20 min, preferably 15 min.

[0040] Preferably, in step (2), in the ultra - performance liquid chromatography - high - resolution mass spectrometry method, the ultra - performance liquid chromatography determination conditions are: the chromatographic column is C 18Chromatographic column, preferably Welch Ultimate UHPLC AQ-C18 (2.1×100 mm, 1.8 μm); column temperature: 30 - 40 °C, preferably 35 °C; flow rate: 0.2 - 0.5 mL / min, preferably 0.3 mL / min; injection volume: 3 - 8 μL, preferably 5 μL; mobile phase A is acetonitrile; mobile phase B is 0.05 - 0.15% formic acid aqueous solution, preferably 0.1% formic acid aqueous solution; analysis time: 40 min; gradient elution.

[0041] In the above mobile phase B, the 0.05 - 0.15% formic acid aqueous solution is a formic acid aqueous solution with a volume percentage of 0.05 - 0.15%, and the 0.1% formic acid aqueous solution is a formic acid aqueous solution with a volume percentage of 0.1%.

[0042] More preferably, the specific gradient elution program is as follows: 0 - 3 min, volume ratio of phase A to phase B is 0 - 3:100 - 97; 3 - 7 min, volume ratio of phase A to phase B is 3 - 8:97 - 92; 7 - 20 min, volume ratio of phase A to phase B is 8 - 20:92 - 80; 20 - 33 min, volume ratio of phase A to phase B is 20 - 40:80 - 60; 33 - 38 min, volume ratio of phase A to phase B is 10 - 95:60 - 5; 38 - 41 min, 95:5; 41 - 41.1 min, 95 - 0:5 - 100; 41.1 - 44 min, 0:100.

[0043] Preferably, in step (2), in the ultra-high performance liquid chromatography - high resolution mass spectrometry method, the mass spectrometry determination conditions are: the ion source is an electrospray ionization source (ESI); the detection mode is the positive ion mode (ESI + ) and the negative ion mode (ESI - ); the spray gas pressure is 50 - 60 psi, preferably 50 psi; the ionization voltage is 4500 - 5500 V in the positive ion mode, preferably 5000 V; the ionization voltage is -4000 - -5000 V in the negative ion mode, preferably -4500 V; the ion source temperature is 450 - 550 °C, preferably 500 °C; the first-stage mass spectrometry selects the MS mode, and the mass scanning range is 50 - 1700 m / z; the second-stage mass spectrometry selects the MS / MS mode, and the mass scanning range is 50 - 1250 m / z; the collision voltage is 35 - 45 eV, preferably 40 eV.

[0044] In the above ultra-high performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) method, data acquisition was carried out using Analyst TF 1.7.1 software. The data processing software was Peakview 1.2. According to the multi-stage mass spectrometry information of the samples, combined with the original prescription identification results and relevant literature, 77 compounds were identified from the administered plasma samples, including 20 prototype components and 57 metabolites (Tables 7-10 below).

[0045] Compared with the prior art, the advantages and beneficial effects of the present invention include:

[0046] 1. The present invention has established an analytical method for detecting the components of Dongqingzi Qingshen Granules by UPLC-HRMS. By this method, 103 components in Dongqingzi Qingshen Granules can be effectively detected, providing a reference for the quality control and clinical application guidance of Dongqingzi Qingshen Granules, and also laying a foundation for the establishment of a quantitative detection method for Dongqingzi Qingshen Granules; an analytical method for detecting the blood components of Dongqingzi Qingshen Granules by UPLC-HRMS has been established. 77 compounds were identified, including 20 prototype components and 57 metabolites, which can reflect the drug-derived component situation after the drug enters the blood, filling the blank in the qualitative detection of the blood components of Dongqingzi Qingshen Granules and providing a basis for further studying the drug metabolism cycle and metabolic pathway.

[0047] 2. The detection method established by the present invention uses the UPLC-HRMS method for detection. By optimizing the chromatographic conditions and mass spectrometry parameters, the required time is short, and it can be completed in only 40 minutes. With the Natural Products HR-MS / MS Spectral Library 1.0 database, the detection speed is fast and the identification accuracy is high. It can accurately and reliably detect the complex compounds in the complex systems of the compound of Dongqingzi Qingshen Granules and plasma. Moreover, the pretreatment method is simple, and the overall method is easy to operate, suitable for popularization and application. Description of the Drawings

[0048] Figure 1 Shown is the base peak chromatogram (BPC) - negative ion mode of the compound sample of Dongqingzi Qingshen Granules by UPLC-HRMS.

[0049] Figure 2 Shown is the base peak chromatogram (BPC) - positive ion mode of the compound sample of Dongqingzi Qingshen Granules by UPLC-HRMS.

[0050] Figure 3 Shown is the ultraviolet chromatogram (UV-254nm) of the compound sample of Dongqingzi Qingshen Granules.

[0051] Figure 4 Shown is the base peak chromatogram (BPC) - negative ion mode of the plasma sample of Dongqingzi Qingshen Granules by UPLC-HRMS.

[0052] Figure 5 Shown as the base peak ion chromatogram (BPC) - positive ion mode of the plasma sample of Dongqingzi Qingshen Granules by UPLC-HRMS.

[0053] Figure 6 Shown as the extracted ion chromatogram (EIC) - negative ion mode of the administered plasma sample by UPLC-HRMS.

[0054] Figure 7 Shown as the extracted ion chromatogram (EIC) - positive ion mode of the administered plasma sample by UPLC-HRMS. Detailed implementation manners

[0055] The present invention will be further elaborated below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the protection scope of the present invention.

[0056] The following illustrates the implementation manners of the present invention through specific examples. 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 implementation manners. 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.

[0057] Example 1

[0058] 1. Reagents

[0059] Dongqingzi Qingshen Granules, hospital preparation, batch number: Shanghai Medicine Preparation No. Z20240012000; acetonitrile, mass spectrometry grade, D618978, CNW Company; methanol, mass spectrometry grade, D618975, CNW Company; water, purified water, 20240717C, Guangzhou Watson's Food and Beverage Co., Ltd.; formic acid, mass spectrometry grade, B2307771, Shanghai Aladdin Biochemical Technology Co., Ltd.

[0060] 2. Instruments

[0061] Waters H-Class ultra-high performance liquid chromatograph, Waters Technology Co., Ltd. 4600 high-resolution mass spectrometer, SCIEX Company; electronic balance, ME104, Mettler-Toledo International Trade (Shanghai) Co., Ltd.; ultrasonic cleaner, KQ-300BD, Kunshan Ultrasonic Instruments Co., Ltd.; high-speed centrifuge, H1650-W, Hunan Xiangyi Laboratory Instrument Development Co., Ltd.

[0062] 3. Pretreatment

[0063] Accurately weigh 0.500 g of the powdered Dongqingzi Qingshen Granules sample, place it in a 50-ml stoppered conical flask, add 10.0 mL of 50 (v / v)% methanol solution, extract it by ultrasound (power 300 W, frequency 40 kHz) for 30 min, take it out, let it cool, shake well, take 2 ml and put it into a centrifuge tube, and centrifuge at 12000 r·min -1 Centrifuge for 5 min, take the supernatant, and thus obtain the test solution.

[0064] 4. Qualitative analysis by ultra-high performance liquid chromatography-high resolution mass spectrometry

[0065] 4.1 Chromatographic conditions

[0066] Chromatographic column: Welch Ultimate UHPLC AQ-C18 (2.1×100 mm, 1.8 μm)

[0067] Column temperature: 35 °C

[0068] Flow rate: 0.3 ml / min

[0069] Injection volume: 2 μl

[0070] Detection wavelength: 254 nm, 190 - 400 nm

[0071] Mobile phase ratio: Phase A is acetonitrile, Phase B is 0.1% formic acid aqueous solution, and the gradient is shown in Table 1.

[0072] Table 1 Mobile phase gradient

[0073]

[0074] 4.2 Mass spectrometry conditions

[0075] Mass spectrometry detection mode: ESI-Negative / Positive ion mode

[0076] Mass spectrometry parameters: See Table 2.

[0077] Table 2 Mass parameters (Sciex TripleTOF 4600 LC-MS)

[0078]

[0079] The relevant spectra are as Figures 1 to 3 shown.

[0080] The compound sample of Dongqingzi Qingshen Granules was analyzed by ultra-high performance liquid chromatography-high resolution mass spectrometry (UPLC-Q-TOF / MS). According to the multi-stage mass spectrometry information of the sample, combined with the high-resolution mass spectrometry database of natural products and relevant literature, 103 compounds were identified from the compound sample of Dongqingzi Qingshen Granules (see Tables 3 and 4).

[0081] Table 3 Information Table of Chemical Constituents in Dongqingzi Qingshen Granules

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088] Table 4 Attribution of Chemical Constituents in Dongqingzi Qingshen Granules

[0089]

[0090]

[0091]

[0092]

[0093]

[0094] Note: All means all.

[0095] As can be seen from Tables 3 and 4, a total of 103 components were detected from Dongqingzi Qingshen Granules, and the specific type attribution can be seen in Table 4 above.

[0096] The data acquisition software is Analyst TF 1.7.1, and the data processing software is Peakiew 1.2. During the identification, the mass spectrometry data is preferentially matched with the Natural Products HR-MS / MS Spectral Library 1.0 database. The compounds are preliminarily screened according to the score information of each chromatographic peak, and further confirmed according to the primary and secondary information of each chromatographic peak, etc. The Natural Products HR-MS / MS Spectral Library 1.0 database contains multi-stage mass spectrometry diagrams of standard substances with confirmed structures, including different acquisition modes, different adduct ions, different collision energies, etc. The compound information is comprehensive, and all are actual spectra of the collected standard substances without any simulated or speculative spectra. Therefore, the matching results are highly accurate. For compounds not included in the database, they are identified according to literature reports, mass spectrometry fragmentation rules, etc. It can be detected by the above method that a total of 103 compounds have been identified from the Dongqingzi Qingshen Granules.

[0097] Example 2

[0098] 1. Provide blank plasma and administered plasma

[0099] 1.1 Experimental animals

[0100] (1) Experimental environment: Wuchuang Biotechnology (Shanghai) Co., Ltd.; (2) Animal information: WT-SD-160-180g-♂-Jihui-2024.11.22; (3) Animal certificate number: SCXK (Shanghai) 2022-0009 (20220009031105); (4) Fast overnight before the last administration, drink water freely, and drink water and eat freely at other times.

[0101] 1.2 Animal grouping and administration

[0102] G1: CA24A-155 (Dongqingzi Qingshen Granules), oral gavage, 4.5 g / kg, administration volume 15 ml / kg;

[0103] 1.3 Administration and blood sampling routes

[0104] (1) Administration route: PO: by oral gavage

[0105] (2) Blood sampling route: jugular vein puncture

[0106] 1.4 Experimental instruments and consumables

[0107] Anticoagulant sample tube: EDTA-K2; Balance: JA103P; Centrifuge: Thermo TRESCO 17; Syringe: Shanghai Kantele; Pipette: eppendorf.

[0108] 1.5、Preparation of Administration Solvent

[0109] (1) Preparation Process

[0110] D1: G1 - PO: Accurately weigh 5.000 g of Dongqingzi Qingshen Granules, add 16.67 mL of pure water, vortex for 5 minutes, sonicate for 0 minutes, and heat - stir for 20 minutes; obtain a brown - colored suspension homogeneous administration solution with a concentration of 0.300 g / mL.

[0111] D2: G1 - PO: Accurately weigh 2.500 g of Dongqingzi Qingshen Granules, add 8.33 mL of pure water, vortex for 5 minutes, sonicate for 0 minutes, and heat - stir for 20 minutes; obtain a brown - colored suspension homogeneous administration solution with a concentration of 0.300 g / mL.

[0112] D3: G1 - PO: Accurately weigh 5.000 g of Dongqingzi Qingshen Granules, add 16.67 mL of pure water, vortex for 5 minutes, sonicate for 0 minutes, and heat - stir for 20 minutes; obtain a brown - colored suspension homogeneous administration solution with a concentration of 0.300 g / mL.

[0113] (2) Preparation Date and Storage Conditions

[0114] D1: Preparation Date: December 2, 2024; Store at room temperature and administer within 0.5 hour after preparation.

[0115] D2: Preparation Date: December 3, 2024; Store at room temperature and administer within 0.5 hour after preparation.

[0116] D3: Preparation Date: December 4, 2024; Store at room temperature and administer within 0.5 hour after preparation.

[0117] 1.6、Animal Body Weight and Administration Volume

[0118] Table 5 Animal Body Weight and Administration Volume

[0119]

[0120] 1.7、Blood Sampling Schedule after Administration

[0121] Table 6 Blood Sampling Schedule after Administration

[0122]

[0123] *BD: before dosing, collect blank plasma before administration.

[0124] 1.7、Clinical Observation and Sample Processing Record

[0125] (1) Clinical Observation: No obvious macroscopic abnormalities were observed

[0126] (2) Sample Processing:

[0127] Plasma samples: 1000 uL of whole blood was collected from the jugular vein at each time point, placed on wet ice after collection, centrifuged within 30 minutes by a centrifuge, with centrifugation conditions of 6000 revolutions per minute for 5 minutes at 4 °C. Approximately 500 uL of plasma was taken and placed in a 1.5 ML sample tube, and stored under dry ice conditions.

[0128] 2. Analytical detection method for components in blood

[0129] 2.1. Samples and reagents

[0130] Blank plasma samples and plasma samples after drug administration were sourced as above. All blank plasma samples were mixed to obtain blank plasma, and all plasma samples after drug administration were mixed to obtain plasma after drug administration; Acetonitrile, mass spectrometry grade, D618978, CNW Corporation; Methanol, mass spectrometry grade, D618975, CNW Corporation; Water, purified water, 20240831C, Guangzhou Watson's Food and Beverage Co., Ltd.; Formic acid, mass spectrometry grade, D821049, CNW Corporation.

[0131] 2.2. Instruments and equipment

[0132] Waters H-Class ultra-high performance liquid chromatograph, Waters Technology Co., Ltd.; 4600 high-resolution mass spectrometer, SCIEX Corporation; Electronic balance, ME104, Mettler Toledo International Trade (Shanghai) Co., Ltd.; Ultrasonic cleaner, KO-300BD, Kunshan Ultrasonic Instruments Co., Ltd.; High-speed centrifuge, H1650-W, Hunan Xiangyi Laboratory Instrument Development Co., Ltd.

[0133] 2.3. Experimental procedure

[0134] (1) Preparation of test solution

[0135] Respectively take blank plasma (2 ml) and plasma after drug administration (6 ml), and each add 3 times the amount of methanol (mass spectrometry grade) to precipitate proteins. Vortex and mix well for 5 minutes, let stand for 15 minutes (4 °C), centrifuge for 15 minutes (12000 revolutions per minute), take the supernatant, centrifuge and concentrate to dryness, and store at -80 °C. Before analysis, the residue of blank plasma was re-dissolved with 0.2 ml of 50% methanol, and the plasma after drug administration was re-dissolved with 0.4 ml of 50% methanol. Vortex and mix well for 3 minutes, centrifuge for 15 minutes (12000 revolutions per minute), and take the supernatant to obtain.

[0136] (2) Chromatographic conditions

[0137] Chromatographic column: Welch Ultimate UHPLC AQ-C18 (2.1 x 100 mm, 1.8 μm)

[0138] Column temperature: 35 °C

[0139] Flow rate: 0.3 ml / min

[0140] Sample injection volume: 5 μl

[0141] Detection wavelength: 190 - 400 nm

[0142] Mobile phase ratio: Phase A is acetonitrile, Phase B is 0.1% formic acid aqueous solution, and the gradient is shown in Table 1 above.

[0143] (3) Mass spectrometry conditions

[0144] Mass spectrometry detection mode: ESI - Negative / Positive ion mode

[0145] Mass spectrometry parameters: See Table 2 above.

[0146] 2.3. Experimental results

[0147] The relevant spectra are as Figures 4 to 7 shown.

[0148] The blank plasma and administered plasma samples of Dongqingzi Qingshen Granules were analyzed by ultra - performance liquid chromatography - high - resolution mass spectrometry (UPLC - O - TOF / MS). According to the multi - level mass spectrometry information of the samples, combined with the identification results of the original formula and relevant literature, 77 compounds were identified from the blank plasma and administered plasma samples, including 20 prototype components and 57 metabolites (see Tables 7 - 10).

[0149] The data acquisition software is Analyst TF 1.7.1, and the data processing software is Peakview 1.2.

[0150] Table 7 Information table of components in the administered plasma samples of Dongqingzi Qingshen Granules

[0151]

[0152] Table 8 Identification results of prototype components in the administered plasma of Dongqingzi Qingshen Granules

[0153]

[0154]

[0155] Table 9 Information table of metabolite components in the administered plasma samples of Dongqingzi Qingshen Granules

[0156]

[0157]

[0158]

[0159] Table 10 Identification Results of Metabolites in Plasma Samples of Dongqingzi Qingshen Granules

[0160]

[0161]

[0162]

[0163]

[0164] As mentioned above, it is only the preferred embodiment of the present invention, and it is not a limitation to the present invention in any form or substance. It should be pointed out that for those of ordinary skill in the art of this technology, without departing from the premise of the method of the present invention, several improvements and supplements can still be made, and these improvements and supplements should also be regarded as the protection scope of the present invention. Those who are familiar with this professional technology, without departing from the spirit and scope of the present invention, when making some minor changes, modifications and equivalent changes of evolution using the technical content disclosed above, are all equivalent embodiments of the present invention; at the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the substantial technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for analyzing the components of Holly Seed Qingshen Granules, characterized in that: The steps include: 1) Adding the holly seed clear kidney sample to a solvent to dissolve, ultrasonically extracting and centrifuging, taking the supernatant to obtain a test solution; 2) The test solution was measured by ultra performance liquid chromatography-high resolution mass spectrometry to determine the 103 components in the test solution.

2. The method for analyzing the components of Holly Seed Qingshen Granule according to claim 1, characterized in that: In step 1), one or more of the following features are included: (1) The Holly Seed Qingshen Granule sample is ground into powder; (2) The solvent is a 40-60 (v / v)% methanol solution; (3) The ratio of the mass g of the Dongqingzi Qingshen Granule sample added to the volume mL of the solvent added is 0.5:5-15; (4) The ultrasonic extraction time is 20-40 min; (5) The centrifugal speed is 10000-15000 r / min -1 ; (6) The centrifugation time is 1-10 min.

3. The method for analyzing the components of Holly Seed Qingshen Granule according to claim 1, characterized in that: In step 2), in the ultra-high performance liquid chromatography-high resolution mass spectrometry method, the ultra-high performance liquid chromatography determination conditions are: the chromatographic column is C 18 The chromatographic column is preferably Welch Ultimate UHPLCAQ-C18 (2.1×100mm, 1.8μm); column temperature: 30-40°C, preferably 35°C; flow rate: 0.2-0.5mL / min, preferably 0.3mL / min; injection volume: 1-3μL, preferably 2μL; mobile phase A is acetonitrile; mobile phase B is 0.05-0.15% formic acid aqueous solution, preferably 0.1% formic acid aqueous solution; analysis time: 40min; gradient elution.

4. The method for analyzing the components of Holly Seed Qingshen Granule according to claim 3, characterized in that: The specific gradient elution program is: 0-3min, the volume ratio of phase A: phase B is 0-3:100-97; 3-7min, the volume ratio of phase A: phase B is 3-8:97-92; 7-20min, the volume ratio of phase A: phase B is 8-20:92-80; 20-33min, the volume ratio of phase A: phase B is 20-40:80-60; 33-38min, the volume ratio of phase A: phase B is 10-95:60-5; 38-41min, 95:5; 41-41.1min, 95-0:5-100; 41.1-44min, 0:

100.

5. The method for analyzing the components of Holly Seed Qingshen Granule according to claim 1, characterized in that: In step 2), in the ultra-high performance liquid chromatography-high resolution mass spectrometry method, the mass spectrometry conditions are as follows: the ion source is an electrospray ion source; the detection modes are positive ion mode and negative ion mode; the spray gas pressure is 50-60psi, preferably 50psi; the ionization voltage is 4500-5500V in positive ion mode, preferably 5000V; the ionization voltage is -4000-5000V in negative ion mode, preferably -4500V; the ion source temperature is 450-550°C, preferably 500°C; the primary mass spectrometry uses MS mode, and the mass scanning range is 50-1700m / z; the secondary mass spectrometry uses MS / MS mode, and the mass scanning range is 50-1250m / z; the collision voltage is 35-45eV, preferably 40eV.

6. A method for analyzing the blood components of Holly Seed Qingshen Granule, characterized in that: The steps include: (1) Blank plasma and administration plasma were taken separately, solvent was added to precipitate protein, vortexed, allowed to stand, centrifuged, supernatant was taken, centrifuged and concentrated to dryness for later use; before analysis, the residual blank plasma and administration plasma were reconstituted with solvent, vortexed, allowed to stand, centrifuged, supernatant was taken, and the test solution was obtained; (2) The sample solution was measured by ultra-performance liquid chromatography-high-resolution mass spectrometry, and 77 compounds were identified, including 20 prototype components and 57 metabolites.

7. The method for analyzing blood components of Holly Seed Qingshen Granule according to claim 6, characterized in that: In step (1), one or more of the following features are included: 1) The solvent is mass spectrometry grade methanol; 2) The volume of the added solvent is 2 to 4 times that of the blank plasma or the administered plasma; 3) The vortex mixing time is 3 to 8 minutes; 4) the standing is standing at 4° C. for 10 to 20 minutes; 5) The centrifugal speed is 10000-15000 r·min -1 ; 6) The centrifugation time is 10-20 min; 7) The standby is stored at -80°C; 8) The solvent added for re-dissolving is 40-60 (v / v)% methanol solution; 9) Vortex and mix the solution for 1 to 5 minutes. 10) The speed of the reconstitution centrifugation is 10000-15000 r·min -1 ; 11) The time for reconstitution and centrifugation is 10-20 minutes.

8. The method for analyzing blood components of Holly Seed Qingshen Granule according to claim 6, characterized in that: In step (2), in the ultra-high performance liquid chromatography-high resolution mass spectrometry method, the ultra-high performance liquid chromatography determination conditions are: the chromatographic column is C 18 The chromatographic column is preferably Welch Ultimate UHPLC AQ-C18 (2.1×100 mm, 1.8 μm); column temperature: 30-40° C., preferably 35° C.; flow rate: 0.2-0.5 mL / min, preferably 0.3 mL / min; injection volume: 3-8 μL, preferably 5 μL; mobile phase A is acetonitrile; mobile phase B is 0.05-0.15% formic acid aqueous solution, preferably 0.1% formic acid aqueous solution; analysis time: 40 min; gradient elution.

9. The method for analyzing blood components of Holly Seed Qingshen Granule according to claim 8, characterized in that: In step (2), in the ultra-high performance liquid chromatography-high resolution mass spectrometry method, the gradient elution program is specifically as follows: 0-3 min, the volume ratio of phase A: phase B is 0-3:100-97; 3-7 min, the volume ratio of phase A: phase B is 3-8:97-92; 7-20 min, the volume ratio of phase A: phase B is 8-20:92-80; 20-33 min, the volume ratio of phase A: phase B is 20-40:80-60; 33-38 min, the volume ratio of phase A: phase B is 10-95:60-5; 38-41 min, 95:5; 41-41.1 min, 95-0:5-100; 41.1-44 min, 0:

100.

10. The method for analyzing blood components of Holly Seed Qingshen Granule according to claim 6, characterized in that: In step (2), in the ultra-high performance liquid chromatography-high resolution mass spectrometry method, the mass spectrometry conditions are as follows: the ion source is an electrospray ion source; the detection modes are positive ion mode and negative ion mode; the spray gas pressure is 50-60psi, preferably 50psi; the ionization voltage is 4500-5500V in positive ion mode, preferably 5000V; the ionization voltage is -4000-5000V in negative ion mode, preferably -4500V; the ion source temperature is 450-550°C, preferably 500°C; the primary mass spectrometry uses the MS mode, and the mass scanning range is 50-1700m / z; the secondary mass spectrometry uses the MS / MS mode, and the mass scanning range is 50-1250m / z; The collision voltage is 35-45 eV, preferably 40 eV.

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