Method for detecting in-vivo prototype components and metabolic components of dripping pills with refreshing throat

Abstract

The invention relates to the technical field of drug metabolic component analysis, in particular to a method for detecting in-vivo prototype components and metabolic components of a dripping pill with a refreshing and refreshing throat. According to the present invention, based on the GC-MS and the UHPLC / Q Exactive Plus MS technology, the detection method of the in vivo prototype component and the metabolic component is established; wherein the non-volatile prototypes and metabolites are detected by adopting UHPLC-MS / MS (Ultra High Performance Liquid Chromatography-Mass Spectrometry / Mass Spectrometry) according to the following specific conditions: chromatographic conditions: an LC (Liquid Chromatography) system: Vanquishhorizon; the chromatographic column is Hypersil gold, 2.1 * 100 mm, 1.9 [mu] m; the column temperature is 40 DEG C; the sample size is 1 [mu] L; the flow rate is 0.3 mL * min <-1 >; the composition and gradient of a mobile phase are as follows: A: 0.1% formic acid-acetonitrile, and B: 0.1% formic acid-water; the mass spectrum conditions are as follows: an MS system: Orbitrap Explores 240; the scanning mode is HESI + amp; hESI <->; the taper hole voltage is 3.5 kV (HESI-), and the taper hole voltage is 2.5 kV (HESI-); the temperature of the solvent removing gas is 350 DEG C; the atomization gas is 50 arb; solvent gas removal: 10 arb; the capillary tube is 320 DEG C; the scanning range (m / z) is 100-1500; the resolution ratio (MS) is 70000; and the resolution ratio (MS / MS) is 15000. The patent develops a method for identifying in-vivo prototype drug components and metabolites of the in-vivo prototype drug components, and the metabolic pathway and excretion rule of the drug can be determined through analysis, so that a scientific basis is provided for quality control of a preparation and fundamental research of pharmacological substances.

Description

Technical Field

[0001] The present invention relates to the technical field of drug metabolic component analysis, in particular to a method for detecting in vivo prototype components and metabolic components of Yanlishuang oral dropping pills. Background Art

[0002] Yan Lishuang oral drops are the exclusive Miao medicine product of Guizhou Huangguoshu Shushuang Pharmaceutical Co., Ltd. (National Medicine Approval Number Z20025286). It is a Miao medicine compound preparation composed of mugwort slices, mugwort sesame oil, peppermint oil, menthol and monoammonium glycyrrhizic acid. It has the effects of dispelling wind and heat, reducing swelling and relieving pain, and clearing the throat (Miao medicine: Song Gongzheng, Tai Kai Tai Meng). It is used to treat acute pharyngitis and acute attacks of chronic pharyngitis, with symptoms such as sore throat, dry throat, and bad breath.

[0003] Currently, research on the direct in vivo effects of Yanlishuang oral drops is lacking. The true active ingredients of traditional Chinese medicine (TCM) are the parent ingredients and their metabolites, which truly reflect the changes in the body and represent the overall material basis of the drug's efficacy. Furthermore, elucidating the metabolic pathways of drugs in the body is crucial for optimizing formulation quality control and assessing safety. The use of multidimensional mass spectrometry technologies (such as GC-MS and UHPLC / Q Exactive PlusMS) provides highly sensitive and high-resolution analytical tools for studying the metabolism of complex TCM components.

[0004] Therefore, finding a method to identify the in vivo prototype components and metabolites of Yanlishuang oral drops can clarify its metabolic pathway and excretion pattern, and provide a scientific basis for the quality control and material basis of the preparation. Summary of the Invention

[0005] In order to solve the above technical problems existing in the prior art, the present invention provides a method for detecting the prototype components and metabolic components of Yanlishuang oral dripping pills in vivo, which is as follows:

[0006] A method for detecting the parent component and metabolites of Yanlishuang oral dropping pills in vivo, using GC-MS to detect volatile parent components and UHPLC-MS / MS to detect non-volatile parent components and metabolites;

[0007] The specific method for detecting the non-volatile prototype and metabolites by UHPLC-MS / MS is as follows:

[0008] Chromatographic conditions: LC system: Vanquish horizon; chromatographic column: Hypersil gold, 2.1×100 mm, 1.9 μm; column temperature: 40°C; injection volume: 1 μL; flow rate: 0.3 mL min -1 ;Mobile phase composition and gradient: A: 0.1% formic acid-acetonitrile, B: 0.1% formic acid-water;

[0009] Mass spectrometry conditions: MS system: Orbitrap Exploris 240; scan mode: HESI+&HESI-; cone voltage: 3.5 kV (HESI-), 2.5 kV (HESI-); desolvation temperature: 350°C; nebulizer gas: 50 arb; desolvation gas: 10 arb; capillary: 320°C; scan range (m / z): 100-1500; resolution (MS): 70000; resolution (MS / MS): 15000.

[0010] Furthermore, the chromatographic conditions for the UHPLC-MS / MS detection are as follows:

[0011] Liquid chromatography conditions

[0012]

[0013] The volatile prototype components detected by GC-MS are as follows:

[0014] Chromatographic conditions: Column: SH-Stabilwax, 30 m × 0.32 mm, 0.25 μm film thickness capillary column; Injection port temperature: 220 °C; Splitless injection; Injection volume: 1 μL

[0015] Mass spectrometry conditions: SIM mode; ion source temperature: 200°C; interface temperature: 230°C; ionization mode: electron impact ionization (EI); detection voltage: 0.2 kV; solvent delay: 2 min; mass number scanning range: 30-400 amu.

[0016] Furthermore, the chromatographic conditions for detecting the volatile prototype components using GC-MS are programmed to rise in temperature as follows:

[0017]

[0018] Furthermore, the mass spectrometry conditions for detecting volatile prototype components using GC-MS, and the detection ion information of each index component in the SIM mode are as follows:

[0019]

[0020] Furthermore, the metabolic components are any one or more of urine, feces, and bile.

[0021] Furthermore, the biological sample pretreatment for GC-MS detection is specifically to take 100 μL of urine or bile, place it in a 1.5 mL centrifuge tube, add 100 μL of n-hexane:ethyl acetate (v / v, 1:2), vortex mix for 2 minutes, and centrifuge at 4°C and 14000 r·min. -1After centrifugation for 10 min, 100 μL was taken into a sample injection bottle containing an inner tube and analyzed by GC-MS.

[0022] Furthermore, the biological sample pretreatment for GC-MS detection was as follows: 0.3 g of feces was weighed, extracted with 1.2 mL of n-hexane:ethyl acetate (v / v, 1:2), vortexed for 5 min, and heated at 4 °C and 14000 r / min. -1 Centrifuge for 10 minutes. Use blank feces from rats without the Yanlishuang oral pills as a blank control. Transfer 100 μL of the fecal supernatant to a vial containing an inner cannula for GC-MS analysis.

[0023] Furthermore, the biological sample pretreatment for UHPLC-MS / MS detection is to take 1 mL of urine or bile, add 4 mL of methanol, vortex for 2 minutes, ultrasonicate (500W, 40kHz) for 10 minutes, centrifuge (4℃, 8000r·min -1 , 10 min), the supernatant was taken, dried with nitrogen at 37 ° C, 1 mL of methanol was added, the protein was precipitated again, vortexed for 2 min, ultrasonicated (500 W, 40 kHz) for 10 min, and centrifuged (4 ° C, 8000 r·min -1 , 10 min), the supernatant was taken, dried under nitrogen at 37°C, 200 μL of 50% methanol was added for re-dissolution, and the supernatant was taken for sampling and analysis.

[0024] Furthermore, the biological sample pretreatment for UHPLC-MS / MS detection was as follows: 0.3 g of feces was weighed, 1.2 mL of physiological saline was added to homogenate, 1 mL of the supernatant homogenate was taken after centrifugation, 4 mL of methanol was added, vortexed for 2 min, ultrasonicated (500 W, 40 kHz) for 10 min, and centrifuged (4 ° C, 8000 r min -1 , 10 min), the supernatant was placed in an EP tube, blown dry with nitrogen at 37 ° C, and then 1 mL of methanol was added to the blown-dried sample, treated according to the above method, blown dry, and 200 μL of 50% methanol was added to re-dissolve, and the supernatant was taken for sampling and analysis.

[0025] Compared with the prior art, the technical effects created by the present invention are embodied in:

[0026] This application is based on GC-MS and UHPLC / Q Exactive Plus MS technology to establish a metabolic profile analysis method for Yanli Shuang oral drops in rats. 30 metabolites can be detected in bile, urine and feces, of which 18 metabolites are detected in bile, 13 metabolites are detected in feces, and 24 metabolites are detected in urine, which further indicates that the prototype components of the preparation are mainly excreted through urine after phase I (hydrolysis, oxidation) and phase II (mainly glucuronidation) metabolism, followed by bile and feces. The diversity of metabolites reflects the synergistic metabolic characteristics of the compound ingredients, among which glucuronide conjugates dominate. This application provides a theoretical basis for the formulation of quality control standards for Yanli Shuang oral drops, and lays a methodological foundation for subsequent basic research on pharmacological substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 These are the SRM chromatograms of three analytes and the internal standard: A. blank bile; B. blank bile + mixed reference solution; C. drug-containing bile.

[0028] Figure 2 These are the SRM chromatograms of three analytes and the internal standard: A. blank feces; B. blank feces + mixed reference solution; C. drug-containing feces.

[0029] Figure 3 These are the SRM chromatograms of three analytes and the internal standard: A. blank urine; B. blank urine + mixed reference solution; C. drug-containing urine.

[0030] Figure 4 The total ion currents of blank bile and rat bile after drug administration in positive and negative ionization modes.

[0031] Figure 5 The total ion currents of blank feces and rat feces after drug administration were obtained in positive and negative ionization modes.

[0032] Figure 6 The total ion currents of blank urine and rat urine after drug administration were obtained in positive and negative ionization modes.

[0033] Figure 7 This is the possible metabolic pathway of Yanlishuang oral drops in rats; Note: Glu is glucoside; GluA is glucuronide. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further defined below in conjunction with specific implementation methods, but the scope of protection required is not limited to the description.

[0035] Example:

[0036] 1 Experimental Materials

[0037] 1.1 Instrument

[0038] UHPLC / Q Exactive Plus Orbitrap HRMS (Thermo Fisher Scientific), vortex mixer (VX-III, Beijing Tajin Technology Co., Ltd.), nitrogen blowdown apparatus (MTN-2800D, Tianjin Aotesense Instrument Co., Ltd.), gas chromatography-mass spectrometer (GC-MS-TQ8050NX, Shimadzu Enterprise Management (China) Co., Ltd.), low-temperature high-speed centrifuge (Typ. Allegra 30R Centrifuge, Beckman Coulter Co., Ltd., USA), multi-tube vortex oscillator (Typ. VX-III, Beijing Tajin Technology Co., Ltd.), electronic balance (EL204, Mettler-Toledo Instrument Shanghai Co., Ltd.), and ultrasonic cleaner (Typ. CQ250A-TS, Shanghai Yuejin Medical Optical Instrument Factory).

[0039] 1.2 Drug testing

[0040] Yanlishuang oral dropping pills (YLS-R), formic acid, methanol, and acetonitrile (chromatographic grade) were purchased from Merck, Germany. Purified water (Guangzhou Watsons Food and Beverage Co., Ltd.) and coated pills (YLS-T) were provided by Guizhou Huangguoshu Shushuang Pharmaceutical Co., Ltd., batch number: 20230227. Borneol, camphor, and menthol (batch numbers: 110881-202410, 110747-202412, and 110728-201707, with purities of 97.2%, 99.6%, and 99.6%, respectively, were purchased from the China Food and Drug Inspection Institutes); naphthalene (batch number 20130702, purity 99.5% was purchased from Sinopharm Chemical Reagent Co., Ltd.); ethyl acetate (chromatographic grade, Tianjin Komiou Chemical Reagent Co., Ltd.); and n-hexane (analytical grade, Tianjin Fuyu Fine Chemical Co., Ltd.) were provided.

[0041] 1.3 Experimental animals

[0042] Male SD rats, weighing 180–220 g, were provided by the Animal Care Center of Guizhou Medical University and purchased from Changsha Tianqin Biotechnology Co., Ltd. under license number (SCKK(Xiang)2022-0011). The laboratory was well-lit and well-ventilated, with a room temperature of 18–25°C and a relative humidity of 50–70%. All animal experiments were approved by the Animal Ethics Committee of Guizhou Medical University and strictly adhered to the "Regulations on the Administration of Laboratory Animals (2017 Revision)" and the "General Requirements for Laboratory Animal Experiments (GB / T35823-2018)."

[0043] 2 Methods and Results

[0044] 2.1 Preparation of dosing solution

[0045] Yanlishuang Oral Drops: Take 2-4 pills (25mg) four times daily, equivalent to 0.4g per day. Based on a 60kg human body weight, this translates to approximately 6.67mg / kg per day. The single-dose dosage in SD rats is approximately 6.3 times the clinically equivalent human dose, or 0.042g / kg. For this metabolism study, the dosage was 10 times the clinically equivalent dose of 0.42g / kg.

[0046] Grind the Yanlishuang oral dropping pills and mix them thoroughly, then weigh an appropriate amount of powder, add 0.5% sodium carboxymethyl cellulose solution, ultrasonicate for 10 minutes, and shake and mix for 2 minutes to keep it uniform. Prepare and use immediately.

[0047] 2.2 Collection of metabolic samples

[0048] Ten SD rats were randomly divided into two groups (a blank group with 4 rats and a drug group with 6 rats). The rats were placed in metabolic cages for adaptive feeding for 3 days. Before drug administration, they were fasted for 12 hours and had free access to water. Urine and feces were collected from the blank group for 0 to 48 hours. The drug group was given a single oral gavage of a 0.5% CMC-Na-containing Yanlishuang oral dropper solution. Urine and feces were collected in sections for 0 to 48 hours. The urine volume and freeze-dried feces weight were recorded at each time point. Equal amounts of feces and urine were mixed at each time point.

[0049] Ten SD rats underwent bile duct cannulation and received a single oral gavage of a 0.5% CMC-Na solution of Yanlishuang oral drops. Bile was collected at intervals from 0 to 48 hours, and equal amounts of bile from each time point were combined. After processing, the samples were stored at -80°C until further use.

[0050] 2.3 Processing of metabolic samples

[0051] 2.3.1 Biological sample pretreatment for GC-MS analysis

[0052] Take 100 μL of the mixed rat urine or bile and place it in a 1.5 mL centrifuge tube. Then add 100 μL of n-hexane:ethyl acetate (v / v, 1:2) and vortex mix for 2 min at 4 °C and 14000 r / min. -1 After centrifugation for 10 min, 100 μL was taken into a sample injection bottle containing an inner tube and analyzed by GC-MS.

[0053] Weigh 0.3 g of rat feces mixed and ground evenly at each time point, add 1.2 mL of n-hexane:ethyl acetate (v / v, 1:2) for extraction, vortex mix for 5 min, and rotate at 4 °C and 14000 r / min. -1Centrifuge for 10 minutes. Use blank feces from rats without the Yanlishuang oral pills as a blank control. Transfer 100 μL of the fecal supernatant to a vial containing an inner cannula for GC-MS analysis.

[0054] 2.3.2 Biological Sample Pretreatment for UHPLC-MS / MS Detection

[0055] 1 mL of urine or bile was mixed evenly at each time point, added with 4 mL of methanol, vortexed for 2 min, ultrasonicated (500 W, 40 kHz) for 10 min, and centrifuged (4 °C, 8000 r·min). -1 , 10 min), the supernatant was taken, dried with nitrogen at 37 ° C, 1 mL of methanol was added, the protein was precipitated again, vortexed for 2 min, ultrasonicated (500 W, 40 kHz) for 10 min, and centrifuged (4 ° C, 8000 r·min -1 , 10 min), the supernatant was taken, dried under nitrogen at 37°C, 200 μL of 50% methanol was added for re-dissolution, and the supernatant was taken for sampling and analysis.

[0056] Weigh 0.3 g of dried rat feces at each time period and add 1.2 mL of normal saline to homogenize. The corresponding fecal homogenate without drug administration was used as a blank control. After centrifugation, take 1 mL of the supernatant homogenate, add 4 mL of methanol, vortex mix for 2 minutes, ultrasonicate (500 W, 40 kHz) for 10 minutes, and centrifuge (4°C, 8000 r·min). -1 , 10 min), the supernatant was placed in an EP tube, blown dry with nitrogen at 37 ° C, and then 1 mL of methanol was added to the blown-dried sample, treated according to the above method, blown dry, and 200 μL of 50% methanol was added to re-dissolve, and the supernatant was taken for sampling and analysis.

[0057] 2.4 Detection of volatile prototype components based on GC-MS

[0058] 2.4.1 Instrument conditions

[0059] (1) Chromatographic conditions

[0060] Chromatographic column: SH-Stabilwax (30 m × 0.32 mm, film thickness 0.25 μm) capillary column; injection port temperature: 220°C; splitless injection; injection volume: 1 μL; temperature program details are shown in Table 1.

[0061] Table 1 Column oven temperature program

[0062]

[0063]

[0064] (2) Mass spectrometry conditions

[0065] SIM mode; ion source temperature 200°C; interface temperature 230°C; ionization mode: electron impact ionization (EI); detection voltage: 0.2 kV; solvent delay 2 min; mass number scanning range: 30-400 amu; detailed information on the detection ions of each indicator component in SIM mode is shown in Table 2.

[0066] Table 2 Mass spectrometry conditions of 3 index components and internal standards

[0067]

[0068] 2.4.2 Results of volatile prototype components detected by GC-MS

[0069] Under the same GC-MS conditions, the spectra of the sample and the reference are as follows Figures 1 to 3 The results show that the endogenous components in the blank matrix do not interfere with the determination of the components, and the conditions are stable and feasible. This instrumental method can detect the original components of menthol, camphor, and borneol in Yanlishuang oral dripping pills.

[0070] 2.5 Detection of non-volatile prototypes and metabolites based on UHPLC-MS / MS

[0071] 2.5.1 Instrument conditions

[0072] (1) Chromatographic conditions

[0073] LC system: Vanquish horizon; chromatographic column: Hypersil gold (2.1 × 100 mm, 1.9 μm); column temperature: 40°C; injection volume: 1 μL; flow rate: 0.3 mL min -1 ; Mobile phase composition and gradient: A: 0.1% formic acid-acetonitrile, B: 0.1% formic acid-water; elution gradient see Table 3:

[0074] Table 3 Liquid chromatography conditions

[0075]

[0076] (2) Mass spectrometry conditions

[0077] MS system: Orbitrap Exploris 240; Scan mode: HESI + &HESI - ; Cone voltage: 3.5kV (HESI - ), 2.5kV(HESI - ); desolvation temperature: 350°C; nebulizer gas: 50 arb; desolvation gas: 10 arb; capillary: 320°C; scan range (m / z): 100-1500; resolution (MS): 70000; resolution (MS / MS): 15000.

[0078] 2.5.2 Analysis of non-volatile prototypes and metabolites based on UHPLC-MS / MS Results UHPLC / Q ExactivePlus Orbitrap HRMS technology was used to analyze Yanlishuang oral dripping pills, bile containing drug, feces, urine and blank matrix samples. The total ion current of each sample in positive and negative ion modes was obtained. The total ion current of the blank and post-dose bile, feces and urine is shown in Figure 2. Figures 4 to 6 . In this experiment, Compound Discover 3.2 and Xcalibar software were used to analyze the data in the positive mode, and the chromatographic peaks in the samples were extracted by pre-treatment methods such as peak extraction, peak matching, and background subtraction. Using the blank matrix as a reference, the components contained in the drug-treated group but not in the blank matrix were found. In addition, using the atlas of Yanli Shuang oral drops as a reference, the components contained in the drug-treated group but not in the Yanli Shuang oral drops samples were found to be metabolites, and the other components were prototype components. According to the retention time, accurate molecular weight, secondary fragmentation and other information provided by UHPLC / Q Exactive PlusOrbitrap HRMS technology, by comparing with the reference substance and combining with the database to identify its metabolites, 4 prototype components of Yanli Shuang oral drops were obtained, and its metabolites in bile, feces, and urine were 30, including 18 in bile, 13 in feces, and 24 in urine. The results are shown in Table 4. Figure 7 .

[0079] Table 4 Identification of the prototype and metabolites of Yanlishuang oral dropping pills after administration

[0080]

[0081]

[0082]

[0083]

[0084] Note: Camphor, menthol, and borneol are volatile components identified by GC-MS; “*” indicates matching confirmation with standard products; B, F, and U represent bile, feces, and urine, respectively.

[0085] 2.5.3 Identification and analysis of non-volatile prototype components and metabolites

[0086] P4 t R The quasi-molecular ion generated in negative mode was m / z 821.3928 [MH] - , its molecular formula is predicted to be C 42 H 62 O 16The secondary main fragment ions were m / z 425.3098, 200.9992, 162.9437, 134.5173, and 113.0228, which were consistent with the retention time and fragmentation pattern of glycyrrhizic acid reference substance. It was speculated that P4 was the prototype component glycyrrhizic acid in Yanlishuang oral drops.

[0087] M1's t R The quasi-molecular ion generated in the positive mode was m / z 155.1426 [M+H] + , its molecular formula is predicted to be C 10 H 18 O, menthol quasi-molecular ion peak m / z 157.1587 [M+H] + The number of H molecules is 2 less, consistent with menthol losing two H molecules. The major secondary fragment ions are m / z 111.1170 and 109.1011, with m / z 109.1011 being 2 less than m / z 111.11703, indicating the fragment ion formed by the removal of two H molecules. Therefore, it is speculated that this is a metabolite formed by the oxidation of the hydroxyl group on the six-membered ring of menthol to a keto group.

[0088] M2 t R The quasi-molecular ion generated in negative mode was m / z 251.0956 [MH] - , its molecular formula is predicted to be C 10 H 20 O5S, menthol quasi-molecular ion peak m / z 155.1441 [MH] - The excess of 96 is consistent with the addition of a sulfate (SO3) molecule after menthol is oxidized to M1. The main secondary fragment ions are m / z 159.8588, 150.5030, 134.5023, and 79.9563. m / z 134.5023 has a 16-fold difference from m / z 150.5030, indicating the fragment ion is formed by the removal of a single oxygen molecule. m / z 79.9563 has a 80-fold difference from m / z 159.8588, indicating the fragment ion is formed by the removal of a single sulfate (SO3) molecule. Therefore, it is speculated to be a metabolite of menthol derived from oxidation and sulfation.

[0089] M3's t R The quasi-molecular ion generated in negative mode was m / z 195.1382 [MH] - , its molecular formula is predicted to be C 12 H 20 O2, menthol quasi-molecular ion peak m / z 155.1441 [MH] -The 40-fold increase in the number of fragments is consistent with the addition of an acetyl group (C₂H₂O) after menthol is oxidized to M1. The main secondary fragment ions are m / z 138.0673, 125.0961, 109.0645, and 96.3625. m / z 125.0961 has a 16-fold decrease compared to m / z 109.0645, indicating the fragment ion formed by the removal of an oxygen molecule. m / z 96.3625 has a 42-fold decrease compared to m / z 138.0673, indicating the fragment ion formed by the removal of a C₂H₂O molecule. Therefore, it is speculated to be a metabolite of menthol derived from oxidation and acetylation.

[0090] M4's t R The quasi-molecular ions generated in the negative mode were m / z 331.1762[MH] - , its molecular formula is predicted to be C 16 H 28 O7, menthol quasi-molecular ion peak m / z 155.1441 [MH] - The number of ions is 176, which is consistent with the addition of one molecule of glucuronide (C6H8O6) to menthol. The secondary main fragment ions are m / z 329.1163, 153.1272, and 75.0074, and m / z 153.1272 has 176 less than m / z 329.1163, which is consistent with the removal of one molecule of C6H 10 O5 formed a fragment ion, so it was speculated to be a metabolite of menthol glucuronidation. In addition, M4 was compared with a standard of borneol-2-O-glucuronide, and the fragmentation pattern and retention time were consistent, suggesting that the two are the same substance.

[0091] M5's t R The quasi-molecular ion generated in negative mode was m / z 347.1715 [MH] - , its molecular formula is predicted to be C 16 H 28 O8, menthol quasi-molecular ion peak m / z 155.1441 [MH] - The excess of 192 is consistent with menthol oxidation to M1 followed by the addition of a glucuronide molecule (C6H8O6). The major secondary fragment ions are m / z 272.2710, 171.1387, 155.1061, and 96.3220. m / z 171.1387 has a 16-fold difference from m / z 155.1061, indicating the removal of an oxygen molecule. m / z 272.2710 has a 176-fold difference from m / z 96.3220, indicating the removal of a glucuronide molecule (C6H8O6). Therefore, it is hypothesized to be a metabolite of menthol oxidation and glucuronidation.

[0092] M6 tR The quasi-molecular ions generated in the negative mode were m / z 193.0346 [MH] - , its molecular formula is predicted to be C6H 10 O7 is identical to a single glucuronic acid molecule. The primary secondary fragment ions are m / z 162.7277, 134.4170, and 113.0231. The fragmentation patterns are similar to those of glycyrrhizic acid, leading us to speculate that M6 is a glucuronide generated by the hydrolysis of glycyrrhizic acid.

[0093] M7's t R The quasi-molecular ions generated in the negative mode were m / z 469.3303 [MH] - , its molecular formula is predicted to be C 30 H 46 O4. Quasi-molecular ion peak of glycyrrhizic acid m / z 821.3929 [MH] - The number of residues decreased by 352, which is consistent with the reduction of two glucuronides (C6H8O6) by glycyrrhizic acid. The secondary major fragment ions are m / z 425.3406, 162.8264, 134.5991, and 113.0333, and the fragmentation pattern is similar to that of glycyrrhizic acid. Therefore, it is speculated that it is a metabolite obtained by the hydrolysis of glycyrrhizic acid to remove two glucuronides. Compared with the glycyrrhetinic acid standard, its retention time is consistent with the fragmentation pattern, and it is speculated that M7 is glycyrrhetinic acid.

[0094] M8's t R The quasi-molecular ions generated in the negative mode were m / z 483.3119 [MH] - , its molecular formula is predicted to be C 30 H 44 O5. Quasi-molecular ion peak of glycyrrhizic acid m / z 821.3929 [M+H] + The number of ions is 338, consistent with the oxidation of the methyl group on the benzene ring to a keto carbonyl group after glycyrrhizic acid hydrolysis to produce M7. The major secondary fragment ions of M8 are m / z 453.3097, 439.3221, 409.2742, and 353.2643. The fragment ions at m / z 453.3097 and 409.2742 are produced by the sequential loss of CH2O and CO2, while the fragment ions at m / z 439.3221 and 353.2643 are produced by the loss of CO and the fragmentation of the benzene ring. Therefore, M8 is presumed to be a metabolite resulting from the hydrolysis and oxidation of glycyrrhizic acid.

[0095] M9-M11 t R The quasi-molecular ions generated in the negative mode were m / z 499.3064 [MH] at 24.25, 24.35, and 24.50 min, respectively. -、m / z 499.3068[MH] - 、m / z499.3069[MH] - Its molecular formula is predicted to be C 30 H 44 O6. Quasi-molecular ion peak of glycyrrhizic acid m / z 821.3929 [MH] - The number of fragments is 322, which is consistent with the oxidation of the methyl group on the benzene ring to carboxylic acid after glycyrrhizic acid reduces two molecules of glucuronide (C6H8O6). The secondary major fragment ions of M9 are m / z 437.3080, 393.3166, 162.8852, and 134.5000, the secondary major fragment ions of M10 are m / z 437.3056, 393.3452, and 162.8852, and the secondary major fragment ions of M11 are m / z 437.3069, 393.3162, and 134.4669. Among them, m / z 437 is the quasi-molecular ion peak m / z 499 [MH] - The fragment ions formed by the reduction of one CO2 molecule and one H2O molecule are: m / z 393 is formed by the shedding of CO2 and CH2, and the fragmentation patterns of m / z 162 and 134 are similar to those of glycyrrhizic acid. Therefore, it is speculated that M9-M11 are formed by the oxidation of the methyl group on the phenyl ring after the hydrolysis of glycyrrhizic acid to remove two glucuronides.

[0096] M12 t R The quasi-molecular ions generated in the negative mode were m / z 485.3272 [MH] - , its molecular formula is predicted to be C 30 H 46 O5. Quasi-molecular ion peak of glycyrrhizic acid m / z 821.3929 [M+H] + The number of m / z 1200 m / s was 336, consistent with glycyrrhizic acid reducing two glucuronides (C6H8O6) and then adding one oxygen molecule. The main secondary fragment ions were m / z 441.3399, 200.9798, 162.8195, and 134.5529. m / z 441.3399 was formed by the addition of one oxygen molecule to glycyrrhizic acid (m / z 425.3098). The fragmentation patterns of m / z 200.9798, 162.8195, and 134.5529 were similar to those of glycyrrhizic acid. Therefore, it is speculated that M12 was formed by the hydrolysis of glycyrrhizic acid, which removed two glucuronides, followed by oxidation.

[0097] M13 t R The quasi-molecular ions generated in the negative mode were m / z 645.3616[MH] - , its molecular formula is predicted to be C 36 H 54 O 10. Quasi-molecular ion peak of glycyrrhizic acid m / z 821.3929[MH] - The number of glycyrrhizic acid molecules decreased by 176, which is consistent with the reduction of one molecule of glucuronide (C6H8O6) from glycyrrhizic acid. The secondary major fragment ions are m / z 469.3315, 332.3896, and 156.0127, among which m / z 469.3315 is significantly higher than m / z 645.3616 [MH] - The m / z 156.0127 is 176 less than the m / z 332.3896, both of which are fragment ions formed by the removal of a single molecule of glucuronide (C6H8O6). Therefore, it is speculated to be a metabolite derived from the hydrolysis of glycyrrhizic acid by removing a single molecule of glucuronide. Furthermore, when M13 was compared with a monoglucuronic acid glycyrrhetinic acid standard, the fragmentation pattern and retention time were consistent, suggesting that the two are the same substance.

[0098] M14, M15 t R The quasi-molecular ions generated in the positive mode were m / z 137.1321 [M+H] + 、137.1321[M+H] + , its molecular formula is predicted to be C 10 H 16 , borneol quasi-molecular ion peak m / z155.1426[M+H] + The number of fragments of M14 is 18 less than that of m / z 113.9737, consistent with the removal of one molecule of water (H2O). The main secondary fragment ions of M14 are m / z 113.9737, 109.1011, 95.0857, and 91.0545. Among them, m / z 95.0857 has 18 less fragments than m / z 113.9737, and m / z 91.0545 has 18 less fragments than m / z 109.1011, both of which are formed by the removal of one molecule of H2O. The main secondary fragment ions of M15 are m / z 109.10121 and 91.05436. Among them, m / z 91.05436 has 18 less fragments than m / z 109.10121, which is formed by the removal of one molecule of H2O. Therefore, M14 and M15 are speculated to be metabolites of borneol dehydration.

[0099] M16 t R The quasi-molecular ions generated in the positive mode were m / z 171.1374 [M+H] + , its molecular formula is predicted to be C 10 H 18 O2, borneol quasi-molecular ion peak m / z 155.1426[M+H] +The excess of 16 is consistent with the addition of one molecule of oxygen to borneol. The main secondary fragment ions are m / z 170.0955, 154.1304, 146.9607, and 130.9660. m / z 154.1304 has 16 fewer atoms than m / z 170.0955, and m / z 130.9660 has 16 fewer atoms than m / z 146.9607. These fragments are all formed by the removal of one molecule of oxygen. These are presumed to be metabolites obtained after oxidation of borneol.

[0100] M17's t R The quasi-molecular ion generated in negative mode was m / z 233.0854 [MH] - , its molecular formula is predicted to be C 10 H 18 O4S, borneol quasi-molecular ion peak m / z 153.1285[MH] - The excess of 80 is consistent with the addition of one sulfate (SO3) molecule to borneol. The secondary major fragment ions are m / z 214.4837 and 134.4867. m / z 134.4867 has 80 fewer fragments than m / z 214.4837, indicating the fragment ion formed by the removal of one sulfate (SO3) molecule. Therefore, it is speculated to be a metabolite of borneol sulfation.

[0101] M18, M19 t R The quasi-molecular ions generated in the positive mode were m / z 329.1604 [MH] and - 、329.1606[MH] - , its molecular formula is predicted to be C 16 H 26 O7, borneol quasi-molecular ion peak m / z153.1284[MH] - The excess of 176 is consistent with borneol plus one molecule of glucosylcarboxylate (C6H8O6). The major secondary fragment ions of M18 are m / z 295.1529, 119.0852, 111.1168, and 85.0282, with m / z 119.08517 having 176 fewer fragments than m / z 295.15292. The major secondary fragment ions of M19 are m / z 313.1616, 137.1323, and 111.1168, with m / z 137.13225 having 176 fewer fragments than m / z 313.1616. This is presumably the fragment ion formed by the removal of one molecule of glucosylcarboxylate. It is speculated that M18 and M19 are metabolites derived from the glucuronidation of borneol. Among them, M18 was compared with borneol-2-O-glucuronide, and the fragmentation pattern and retention time were consistent, so it was speculated that the two were the same substance.

[0102] M20, M21 t R The quasi-molecular ions generated in the positive mode were m / z 347.1690 [M+H] and m / z 347.1690 [M+H], respectively. + 、347.1692[M+H] + , its molecular formula is predicted to be C 16 H 26 O8, borneol quasi-molecular ion peak m / z155.1426[M+H] + More 192, with borneol plus one molecule of O and then one molecule of glucuronide (C6H 10 O5). The major secondary fragment ions of M20 are m / z 171.1373, 153.1269, and 109.1011, and the major secondary fragment ions of M21 are m / z 153.1269, 135.1164, and 109.1012. The fragmentation patterns of m / z 153, 135, and 109 are similar to those of borneol, suggesting that M20 and M21 are metabolites of borneol oxidized and conjugated with glucuronides.

[0103] M22-M24 t R The quasi-molecular ions generated in the positive mode were m / z 135.1166 [M+H], + 、135.1167[M+H] + 、135.1166[M+H] + Its molecular formula is predicted to be C 10 H 14 , camphor quasi-molecular ion peak m / z 153.1267[M+H] + The main secondary fragment ions of M22 were m / z 117.0694, 107.0856, and 93.0701, those of M23 were m / z 118.0651, 107.0856, and 93.0701, and those of M24 were m / z 117.0691, 107.0855, and 93.0701. m / z 117 and 93 differed by 18 from the camphor fragment ions m / z 135 and 111, respectively, suggesting that they were formed by the reduction of one molecule of H2O. The fragmentation patterns of m / z 118, 117, 107, and 93 were similar to those of camphor, suggesting that M22-M24 are metabolites derived from the dehydration of camphor.

[0104] M25, M26 t R The quasi-molecular ions generated in the positive mode were m / z 169.1219 [M+H] and 12.36 and 14.61 min, respectively. +、169.1217[M+H] + Its molecular formula is predicted to be C 10 H 16 O2, camphor quasi-molecular ion peak m / z153.1267[M+H] + The addition of 16 ions is consistent with the addition of one molecule of oxygen to camphor. The major secondary fragment ions of M25 are m / z 152.1145, 151.1114, and 123.0801, and the major secondary fragment ions of M26 are m / z 152.1150, 151.1112, and 123.0799. m / z 152, 151, and 123 have an addition of 16 ions compared to the camphor fragment ions m / z 136, 135, and 107, respectively, presumably resulting from the addition of one molecule of oxygen. The fragmentation patterns of m / z 152, 151, and 123 are similar to those of camphor, suggesting that they are metabolites derived from the oxidation of camphor.

[0105] M27's t R The quasi-molecular ions generated in the positive mode were m / z 195.1375 [M+H] + , its molecular formula is predicted to be C 12 H 18 O2, camphor quasi-molecular ion peak m / z 153.1267 [M+H] + The addition of 42 molecule is consistent with the addition of an acetyl group (C2H2O) to camphor. The main secondary fragment ions are m / z 177.1270, 153.0912, 149.0950, 136.1199, and 135.1164. m / z 177.1270, 153.0912, and 149.0950 differ by 42 molecule from the camphor fragment ions m / z 135.11635, 111.08028, and 107.08541, respectively, suggesting that they are fragment ions formed by the addition of an acetyl group. m / z 136.1199 and 135.1164 are similar to the camphor fragment ions m / z 136.11942 and 135.11635, respectively. Therefore, these are presumed to be metabolites derived from the acetylation of camphor.

[0106] M28, M29 t R The quasi-molecular ions generated in the positive mode were m / z 345.1531 [M+H] + 、345.1530[M+H] + Its molecular formula is predicted to be C 16 H 24 O8, camphor quasi-molecular ion peak m / z153.1267[M+H] + More 192, add one molecule of O to camphor and then add one molecule of glucuronide (C6H 10O5). The major secondary fragment ions for M28 are m / z 151.1111, 123.0802, and 107.0854, and those for M29 are m / z 123.0803, 111.0804, and 107.0855. The fragmentation patterns of m / z 123, 111, and 107 are similar to those of camphor. Therefore, M28 and M29 are hypothesized to be metabolites of camphor oxidized and conjugated to glucuronides.

[0107] M30 t R The quasi-molecular ions generated in the positive mode were m / z 331.1740 [M+H] + , its molecular formula is predicted to be C 16 H 26 O7, camphor quasi-molecular ion peak m / z 153.1267 [M+H] + More 178, add one molecule of O to camphor and then add one molecule of glucoside (C6H 10 The secondary main fragment ions are m / z 285.1676, 135.1160, and 107.0854, among which m / z 285.1676 and 107.0854 differ by 178, which is speculated to be the result of the successive addition of one molecule of O and one molecule of glucoside (C6H 10 The fragment ions m / z 135.1160 and 107.0854 obtained by PCR (O5) have similar fragmentation patterns to camphor. Therefore, it is speculated that they are metabolites of camphor after oxidation and binding to glucoside.

[0108] 3 Discussions

[0109] The pharmacological substances of traditional Chinese medicine (TCM) include the biologically active compounds and their metabolites, which are crucial for ensuring the safety and efficacy of TCM. The pharmacological basis of TCM compounds is the cluster of chemical components that, upon entry into the human body, act on multiple targets and produce their overall efficacy. This cluster of chemical components may originate from inherent components of the medicinal materials, products formed during the preparation process, or metabolites produced by drug interactions with the body. The prototype components of Yanlishuang oral drops identified in this study, expressed in bile, feces, and urine, exhibit numerous pharmacological activities against acute pharyngitis. For example, menthol, borneol, and camphor exhibit anti-inflammatory and analgesic effects. Furthermore, the metabolites glycyrrhetinic acid, glycyrrhetinic acid monoglucuronide, borneol-2-O-glucuronide, and menthol-1-O-glucuronide were detected in rat bile, feces, and urine, suggesting that these metabolites may be part of the active substances responsible for the efficacy of Yanlishuang oral drops. It is suggested that in addition to the prototype components, quantitative detection of its main metabolites can be added to the quality control standards of this compound preparation to ensure the pharmacological stability of the preparation.

[0110] Experimental results indicate that after oral administration to rats, the metabolism of Yanlishuang oral drops is primarily through hydrolysis, oxidation, and glucuronic acid conjugation, with the presence of minor glucose conjugates and sulfation products. These products primarily involve phase I and phase II metabolism, and the proposed metabolic pathways are generally consistent with those reported in the literature. Furthermore, the highest number of metabolites was found in urine, suggesting that renal excretion is the primary elimination pathway, a finding consistent with glomerular filtration of water-soluble metabolites. Eighteen metabolites were detected in bile, potentially related to enterohepatic circulation or secondary metabolism mediated by intestinal microbiota.

[0111] 4 Conclusion

[0112] In this study, a metabolic profiling method for Yanlishuang oral drops in rats was established based on GC-MS and UHPLC / Q Exactive Plus MS techniques. A total of 30 metabolites were detected in bile, urine, and feces by comparing the metabolites and metabolic reaction types in rats. Of these, 18 metabolites were detected in bile, 13 in feces, and 24 in urine. These results indicate that the parent ingredient undergoes phase I (hydrolysis and oxidation) and phase II (primarily glucuronidation) metabolism, with the primary excretion being in urine, followed by bile and feces. The diverse metabolite profiles reflect the synergistic metabolism of the compound components, with glucuronidation conjugates predominating. This study provides a theoretical basis for the development of quality control standards for Yanlishuang oral drops and lays a methodological foundation for subsequent research on the basis of pharmacodynamic substances.

[0113] Finally, it should be noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solutions of the present invention are not limited to the above embodiments and are subject to numerous variations. All variations that can be directly derived or conceived by a person of ordinary skill in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A method for detecting the prototype components and metabolic components of Yanlishuang oral dripping pills in vivo, characterized in that: Volatile prototype components were detected by GC-MS, and non-volatile prototype and metabolites were detected by UHPLC-MS / MS; The specific method for detecting the non-volatile prototype and metabolites by UHPLC-MS / MS is as follows: Chromatographic conditions: LC system: Vanquish horizon; Column: Hypersil gold, 2.1 × 100 mm, 1.9 μm; Column temperature: 40°C; injection volume: 1 μL; flow rate: 0.3 mL min -1 ;Mobile phase composition and gradient: A: 0.1% formic acid-acetonitrile, B: 0.1% formic acid-water; Mass spectrometry conditions: MS system: Orbitrap Exploris 240; scan mode: HESI+ & HESI-; cone voltage: 3.5 kV (HESI-), 2.5 kV (HESI-); desolvation temperature: 350°C; nebulizer gas: 50 arb; desolvation gas: 10 arb; Capillary: 320 °C; scanning range (m / z): 100-1500; Resolution (MS): 70,000; Resolution (MS / MS): 15,000.

2. The method for detecting the prototype components and metabolic components of Yanlishuang oral dripping pills according to claim 1, characterized in that: The chromatographic conditions for UHPLC-MS / MS detection are as follows: Liquid chromatography conditions 3. The method for detecting the prototype components and metabolic components of Yanlishuang oral dropping pills according to claim 1, characterized in that: The volatile prototype components detected by GC-MS are as follows: Chromatographic conditions: Column: SH-Stabilwax, 30 m × 0.32 mm, 0.25 μm film thickness capillary column; Injection port temperature: 220 °C; Splitless injection; Injection volume: 1 μL Mass spectrometry conditions: SIM mode; ion source temperature: 200°C; interface temperature: 230°C; ionization mode: electron impact ionization (EI); detection voltage: 0.2 kV; solvent delay: 2 min; mass number scanning range: 30-400 amu.

4. The method for detecting the prototype components and metabolic components of Yanlishuang oral dropping pills according to claim 3, characterized in that: The chromatographic conditions for detecting the volatile prototype components using GC-MS are as follows:

5. The method for detecting the prototype components and metabolic components of Yanlishuang oral dropping pills according to claim 3, characterized in that: The mass spectrometry conditions for detecting volatile prototype components using GC-MS and the detection ion information of each index component in SIM mode are as follows:

6. The method for detecting the prototype components and metabolic components of Yanlishuang oral dropping pills according to claim 5, characterized in that: The metabolic components are any one or more of urine, feces, and bile.

7. The method for detecting the prototype components and metabolic components of Yanlishuang oral dropping pills according to claim 1, characterized in that: The biological sample pretreatment for GC-MS detection is as follows: 100 μL of urine or bile is taken and placed in a 1.5 mL centrifuge tube, 100 μL of n-hexane:ethyl acetate (v / v, 1:2) is added, vortexed for 2 minutes, and centrifuged at 4°C and 14000 r·min. -1 After centrifugation for 10 min, 100 μL was taken into a sample injection bottle containing an inner tube and analyzed by GC-MS.

8. The method for detecting the prototype components and metabolic components of Yanlishuang oral dripping pills according to claim 1, characterized in that: The biological sample pretreatment for GC-MS detection was as follows: 0.3 g of feces was weighed, extracted with 1.2 mL of n-hexane:ethyl acetate (v / v, 1:2), vortexed for 5 min, and heated at 4 °C and 14000 r / min. -1 Centrifuge for 10 minutes. Use blank feces from rats without the Yanlishuang oral pills as a blank control. Transfer 100 μL of the fecal supernatant to a vial containing an inner cannula for GC-MS analysis.

9. The method for detecting the prototype components and metabolic components of Yanlishuang oral dripping pills according to claim 1, characterized in that: The biological sample pretreatment for UHPLC-MS / MS detection is as follows: 1 mL of urine or bile is taken, 4 mL of methanol is added, the mixture is vortexed for 2 minutes, sonicated for 10 minutes, centrifuged, the supernatant is taken, and the mixture is blown dry with nitrogen at 37°C. 1 mL of methanol is added to precipitate the protein again, the mixture is vortexed for 2 minutes, sonicated for 10 minutes, centrifuged, the supernatant is taken, the mixture is blown dry with nitrogen at 37°C, 200 μL of 50% methanol is added for re-dissolution, and the supernatant is taken for sampling and analysis.

10. The method for detecting the prototype components and metabolic components of Yanlishuang oral dripping pills in vivo according to claim 1, characterized in that: The biological sample pretreatment for UHPLC-MS / MS detection is as follows: 0.3 g of feces is weighed, 1.2 mL of physiological saline is added for homogenization, 1 mL of the supernatant homogenate is taken after centrifugation, 4 mL of methanol is added, vortex mixing is performed for 2 minutes, sonication is performed for 10 minutes, centrifugation is performed, the supernatant is placed in an EP tube, and blown dry with nitrogen at 37°C. Then, 1 mL of methanol is added to the blown-dried sample, and the sample is treated according to the above method, blown dry, and 200 μL of 50% methanol is added for re-dissolution. The supernatant is taken for sampling and analysis.

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