A method for detecting the original and metabolized components of a throat lozenge in vivo.

Abstract

This invention relates to the field of drug metabolism component analysis technology, specifically a method for detecting the original and metabolized components of Yanli Shuang oral drops in vivo. This application establishes a method for detecting the original and metabolized components in vivo based on GC-MS and UHPLC / Q Exactive Plus MS technology. The specific UHPLC-MS / MS method for detecting non-volatile original components and metabolites is as follows: Chromatographic conditions: LC system: Vanquishhorizon; Column: Hypersil gold, 2.1×100mm, 1.9μm; Column temperature: 40℃; Injection volume: 1μL; Flow rate: 0.3mL·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; Scanning mode: HESI+ & HESI-; Cone voltage: 3.5kV (HESI-), 2.5kV (HESI-); Desolvation gas temperature: 350℃; Nebulizer gas: 50arb; Desolvation gas: 10arb; Capillary: 320℃; Scan range (m / z): 100-1500; Resolution (MS): 70000; Resolution (MS / MS): 15000. This patent develops a method for identifying the in vivo prototype drug components and metabolites of Yanli Shuang oral drops. Analysis can clarify the drug's metabolic pathway and excretion patterns, thus providing a scientific basis for the quality control of the formulation and research on its pharmacological material basis.

Description

Technical Field

[0001] This invention relates to the field of drug metabolism component analysis technology, specifically a method for detecting the original components and metabolites of Yanlishuang oral drops in vivo. Background Technology

[0002] Yanlishuang Kouhan Diwan is a nationally exclusive Miao medicine product of Guizhou Huangguoshuli Shuang Pharmaceutical Co., Ltd. (National Medicine Approval Number Z20025286). It is a Miao medicine compound preparation composed of Artemisia argyi tablets, Artemisia argyi oil, peppermint oil, menthol, and glycyrrhizic acid monoammonium salt. It has the effects of dispelling wind and heat, reducing swelling and relieving pain, and clearing the throat (Miao medicine: Song Gongzheng, Taikai Taimeng). It is used to treat acute pharyngitis and acute exacerbations of chronic pharyngitis, with symptoms such as sore throat, dry throat, and halitosis.

[0003] Currently, research on the directly acting substances of Yanlishuang oral drops in vivo is lacking. The true active ingredients that exert the efficacy of traditional Chinese medicine (TCM) are the original components and their metabolites, which accurately reflect the changes of TCM components in vivo and represent the overall pharmacodynamic material basis of TCM. Furthermore, elucidating the metabolic pathways of drugs in vivo is one of the keys to optimizing formulation quality control and assessing safety. The use of multidimensional mass spectrometry techniques (such as GC-MS and UHPLC / Q Exactive PlusMS) provides highly sensitive and high-resolution analytical methods for the metabolic study 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 pathways and excretion patterns, providing a scientific basis for the quality control and material basis of this preparation. Summary of the Invention

[0005] To address the aforementioned technical problems in the prior art, this invention provides a method for detecting the original and metabolized components of Yanli Shuang oral drops in vivo, as detailed below:

[0006] A method for detecting the original components and metabolites of Yanli Shuang oral drops in vivo, using GC-MS to detect volatile original components and UHPLC-MS / MS to detect non-volatile original components and metabolites;

[0007] The specific methods for detecting non-volatile precursors and metabolites using UHPLC-MS / MS are as follows:

[0008] Chromatographic conditions: LC system: Vanquish Horizon; Column: Hypersil Gold, 2.1 × 100 mm, 1.9 μm; Column temperature: 40℃; 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 gas 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 detection using UHPLC-MS / MS have the following elution gradient:

[0011] Liquid chromatography conditions

[0012]

[0013] The specific steps for detecting volatile prototype components using GC-MS are as follows:

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

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

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

[0017]

[0018] Furthermore, the mass spectrometry conditions for detecting volatile prototype components using GC-MS, and the ion information for each index component in 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 specifically involves taking 100 μL of urine or bile, placing it in a 1.5 mL centrifuge tube, adding 100 μL of n-hexane:ethyl acetate (v / v, 1:2), vortexing for 2 min, and incubating at 4 °C and 14000 rpm. -1Centrifuge for 10 min, then transfer 100 μL into a vial containing the inner tube for GC-MS analysis.

[0022] Furthermore, the biological sample pretreatment for GC-MS detection specifically involves weighing 0.3 g of feces, adding 1.2 mL of n-hexane:ethyl acetate (v / v, 1:2) for extraction, vortexing for 5 min at 4 °C and 14000 r·min. -1 Centrifuge for 10 min. Use blank feces from rats that have not been given the oral drops as a blank control. Take 100 μL of rat fecal supernatant into a sample vial containing an inner tube and analyze by GC-MS.

[0023] Furthermore, the biological sample pretreatment for UHPLC-MS / MS detection involves taking 1 mL of urine or bile, adding 4 mL of methanol, vortexing for 2 min, sonicating (500 W, 40 kHz) for 10 min, and centrifuging (4 °C, 8000 r·min). -1 (10 min), collect the supernatant, blow dry with nitrogen at 37℃, add 1 mL of methanol to precipitate the protein again, vortex for 2 min, sonicate (500 W, 40 kHz) for 10 min, and centrifuge (4℃, 8000 r·min). -1 After 10 min, the supernatant was collected, dried under nitrogen at 37℃, and redissolved in 200 μL of 50% methanol. The supernatant was then injected for analysis.

[0024] Furthermore, the biological sample pretreatment for UHPLC-MS / MS detection involves weighing 0.3g of feces, adding 1.2mL of physiological saline to homogenize, centrifuging, taking 1mL of the supernatant homogenate, adding 4mL of methanol, vortexing for 2min, sonicating (500W, 40kHz) for 10min, and centrifuging (4℃, 8000r·min). -1 (10 min), take the supernatant and place it in an EP tube, blow it dry with nitrogen at 37℃, then add 1 mL of methanol to the dried sample, process it according to the above method, blow it dry, add 200 μL of 50% methanol to redissolve, and take the supernatant for analysis.

[0025] Compared with the prior art, the technical effects of this invention are reflected in:

[0026] This application establishes a metabolic profile analysis method for Yanlishuang oral drops in rats based on GC-MS and UHPLC / Q Exactive Plus MS techniques. Thirty metabolites were detected in bile, urine, and feces, with 18 metabolites detected in bile, 13 in feces, and 24 in urine. This indicates that the original components of the formulation, after phase I (hydrolysis and oxidation) and phase II (mainly glucuronidation) metabolism, are primarily excreted in urine, followed by bile and feces. The diversity of metabolites reflects the synergistic metabolic characteristics of the compound components, with glucuronide conjugates being dominant. This application provides a theoretical basis for the establishment of quality control standards for Yanlishuang oral drops and lays a methodological foundation for subsequent pharmacodynamic material basis research. Attached Figure Description

[0027] Figure 1 The following are SRM chromatograms of three analytes and an internal standard: A. Blank bile; B. Blank bile + mixed reference solution; C. Drug-containing bile.

[0028] Figure 2 The following are SRM chromatograms of three analytes and an internal standard: A. Blank stool sample; B. Blank stool sample + mixed reference solution; C. Stool sample containing the drug.

[0029] Figure 3 The following are SRM chromatograms of three analytes and an internal standard: A. Blank urine; B. Blank urine + mixed reference solution; C. Drug-containing urine.

[0030] Figure 4 This is a total ion chromatogram of blank bile and rat bile after drug administration under positive and negative ionization modes.

[0031] Figure 5 This is a total ion chromatogram of blank feces and feces of rats after drug administration under positive and negative ionization modes.

[0032] Figure 6 This is a total ion flow diagram of blank urine and rat urine after drug administration under positive and negative ionization modes.

[0033] Figure 7 This describes the possible metabolic pathway of Yanlishuang oral drops in rats; Note: Glu is a glucosinolate; GluA is a glucuronide. Detailed Implementation

[0034] The technical solution of the present invention will be further defined below with reference to specific embodiments, but the scope of protection is not limited to the description made.

[0035] Example:

[0036] 1. Experimental Materials

[0037] 1.1 Instruments

[0038] UHPLC / Q Exactive Plus Orbitrap HRMS (Thermo Fisher Scientific), vortex mixer (Model VX-III, Beijing Taji Technology Co., Ltd.), nitrogen evaporator (MTN-2800D, Tianjin Outsen Instruments Co., Ltd.), gas chromatography-mass spectrometry (GC-MS-TQ8050NX, Shimadzu (China) Co., Ltd.), low-temperature high-speed centrifuge (Type Allegra 30R Centrifuge, Beckman Coulter, Inc., USA), multi-tube vortex oscillator (Type VX-III, Beijing Taji Technology Co., Ltd.), electronic balance (EL204, Mettler Toledo Instruments Shanghai Co., Ltd.), ultrasonic cleaner (Type CQ250A-TS, Shanghai Yuejin Medical Optical Instruments Factory).

[0039] 1.2 Reagents

[0040] Pharyngeal Lishuang Kouhan Dropping Pills (ordinary pills, abbreviated as YLS-R), formic acid, methanol, and acetonitrile (chromatographic grade) were all purchased from Merck KGaA, Germany. Purified water (Guangzhou Watson's Food & Beverage Co., Ltd.) and coated pills (abbreviated as YLS-T) were provided by Guizhou Huangguoshu Lishuang Pharmaceutical Co., with the batch number: 20230227; borneol, camphor, menthol (batch numbers were 110881-202410, 110747-202412, 110728-201707 respectively, and the purities were 97.2%, 99.6%, 99.6% respectively, purchased from National Institutes for Food and Drug Control); naphthalene (batch number 20130702, purity 99.5% purchased from Sinopharm Chemical Reagent Co., Ltd.) ethyl acetate (chromatographic grade, Tianjin Kemiou Chemical Reagent Co., Ltd.); n-hexane (analytical grade, Tianjin Fuyu Fine Chemical Co., Ltd.).

[0041] 1.3 Experimental animals

[0042] SD rats, weighing 180 - 220 g, male. Provided by the Animal Management Center of Guizhou Medical University, purchased from Changsha Tianqin Biotechnology Co., Ltd., license number (SCKK(Xiang)2022-0011). The laboratory had sufficient light, good ventilation, room temperature of 18 - 25 °C, and relative humidity of 50 - 70%. All animal experiments were approved by the Animal Ethics Committee of Guizhou Medical University, and all experimental procedures strictly complied with the regulations of the "Regulations on the Administration of Laboratory Animals (Revised in 2017)" and the "General Requirements for Laboratory Animal Experiments (GB / T35823-2018)" of the state.

[0043] 2 Methods and results

[0044] 2.1 Preparation of administration solutions

[0045] The dosage of Yanlishuang oral drops is 2-4 drops (25mg per drop) taken sublingually, 4 times a day, which is 0.4g per day. Based on a human body weight of 60kg, this equates to approximately 6.67mg / kg per day. The single-dose dosage calculation for SD rats is approximately 6.3 times the clinically equivalent dose in humans, meaning the clinically equivalent dose for rats is 0.042g / kg. This metabolic experiment used a dose 10 times the clinically equivalent dose of 0.42g / kg.

[0046] After grinding and thoroughly mixing the Yanli Shuang oral drops, weigh an appropriate amount of powder, add 0.5% sodium carboxymethyl cellulose solution, sonicate for 10 minutes, and shake to 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 (blank group and treatment group), with 4 rats in the blank group and 6 rats in the treatment group. The rats were placed in metabolic cages for acclimatization feeding for 3 days. They were fasted for 12 hours before administration, but had free access to water. Blank urine and feces were collected from the blank group from 0 to 48 hours. The treatment group was given a single oral administration of 0.5% CMC-Na-prepared throat lozenges. Urine and feces were collected from 0 to 48 hours in segments, and the urine volume and the weight of the freeze-dried feces at each time point were recorded. Equal amounts of feces and urine were mixed at each time point.

[0049] Ten additional SD rats underwent bile duct cannulation and were administered a single oral gavage solution of 0.5% CMC-Na. Bile was collected in fractions from 0 to 48 hours, and equal volumes of bile from each time point were mixed. The samples were then stored at -80°C for later use.

[0050] 2.3 Processing of Metabolic Samples

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

[0052] Take 100 μL of well-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), vortex for 2 min, and incubate at 4 °C and 14000 rpm. -1 Centrifuge for 10 min, then transfer 100 μL into a vial containing the inner tube for GC-MS analysis.

[0053] Weigh 0.3g of finely ground rat feces from each time period, add 1.2mL of n-hexane:ethyl acetate (v / v, 1:2) for extraction, vortex for 5min, 4℃, 14000r·min. -1Centrifuge for 10 min. Use blank feces from rats that have not been given the oral drops as a blank control. Take 100 μL of rat fecal supernatant into a sample vial containing an inner tube and analyze by GC-MS.

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

[0055] Take 1 mL of well-mixed urine or bile from each time period, add 4 mL of methanol, vortex for 2 min, sonicate (500 W, 40 kHz) for 10 min, and centrifuge (4 °C, 8000 r·min). -1 (10 min), collect the supernatant, blow dry with nitrogen at 37℃, add 1 mL of methanol to precipitate the protein again, vortex for 2 min, sonicate (500 W, 40 kHz) for 10 min, and centrifuge (4℃, 8000 r·min). -1 After 10 min, the supernatant was collected, dried under nitrogen at 37℃, and redissolved in 200 μL of 50% methanol. The supernatant was then injected for analysis.

[0056] Weigh 0.3g of dried rat feces from each time point and add 1.2mL of physiological saline to homogenize. Use the corresponding fecal homogenate from rats not receiving the drug as a blank control. After centrifugation, take 1mL of the supernatant homogenate, add 4mL of methanol, vortex for 2min, sonicate (500W, 40kHz) for 10min, and centrifuge (4℃, 8000r·min). -1 (10 min), take the supernatant and place it in an EP tube, blow it dry with nitrogen at 37℃, then add 1 mL of methanol to the dried sample, process it according to the above method, blow it dry, add 200 μL of 50% methanol to redissolve, and take the supernatant for 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 (30m×0.32mm, film thickness 0.25μm) capillary column; injection port temperature: 220℃; splitless injection; injection volume 1μL; temperature program details are in Table 1.

[0061] Table 1. Column Oven Temperature Program

[0062]

[0063]

[0064] (2) Mass spectrometry conditions

[0065] SIM mode; ion source temperature 200℃; interface temperature 230℃; ionization method: electron impact ion (EI); detection voltage: 0.2KV; solvent delay 2min; mass number scan range: 30~400amu; detailed ion information for each index component in SIM mode is shown in Table 2.

[0066] Table 23 Mass Spectrometry Conditions for Indicator Components and Internal Standards

[0067]

[0068] 2.4.2 Results of GC-MS Detection of Volatile Prototype Components

[0069] Under the same GC-MS conditions, the chromatograms of the sample and the reference standard are as follows: Figures 1-3 As shown in the results, the endogenous components in the blank matrix did not interfere with the determination of each component, and the conditions were stable and feasible. This instrumental method can detect the prototype components of menthol, camphor, and borneol in Yanli Shuang oral drops.

[0070] 2.5 Detection of Non-volatile Progenitors and Metabolites Based on UHPLC-MS / MS

[0071] 2.5.1 Instrument Conditions

[0072] (1) Chromatographic conditions

[0073] LC system: Vanquish Horizon; Column: Hypersil Gold (2.1 × 100 mm, 1.9 μm); Column temperature: 40℃; 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 is shown in Table 3.

[0074] Table 3 Liquid Chromatography Conditions

[0075]

[0076] (2) Mass spectrometry conditions

[0077] MS System: Orbitrap Exploris 240; Scan Mode: HESI + &HESI - Tapered hole voltage: 3.5kV (HESI) - ), 2.5kV (HESI) - Desolvent gas temperature: 350℃; Nebulizer gas: 50arb; Desolvent gas: 10arb; Capillary: 320℃; Scan range (m / z): 100-1500; Resolution (MS): 70000; Resolution (MS / MS): 15000.

[0078] 2.5.2 Analysis of Non-volatile Progenitors and Metabolites Based on UHPLC-MS / MS UHPLC / Q ExactivePlus Orbitrap HRMS technology was used to analyze samples of Yanlishuang oral drops, drug-containing bile, feces, urine, and empty matrix. Total ion chromatograms of each sample were obtained in both positive and negative ion modes. The total ion chromatograms of blank samples and bile, feces, and urine after drug administration are shown in [Figure 1]. Figures 4-6 This experiment used Compound Discover 3.2 and Xcalibar software to analyze data in positive mode. Chromatographic peaks in the samples were extracted using preprocessing methods such as peak extraction, peak matching, and background subtraction. Using a blank matrix as a reference, components present in the administered group but absent in the blank matrix were identified. Furthermore, using the chromatogram of Yanlishuang oral drops as a reference, components present in the administered group but absent in the Yanlishuang oral drops were identified as metabolites, while other components were identified as parent compounds. Based on the retention time, precise molecular weight, and secondary fragmentation information provided by UHPLC / Q Exactive Plus Orbitrap HRMS technology, and by comparing with the reference standard and combining with the database, the metabolites were identified. Four parent compounds of Yanlishuang oral drops were found, with 30 metabolites in bile, feces, and urine, 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 original form and metabolites after administration of Yanlishuang oral drops.

[0080]

[0081]

[0082]

[0083]

[0084] Note: Camphor, menthol, and borneol are volatile components and were identified by GC-MS; "*" indicates standard samples for matching and confirmation; 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's t R The time was 23.75 min, and the quasi-molecular ion produced in negative mode had a m / z of 821.3928 [MH]. - Its molecular formula is predicted to be C 42 H 62 O 16The secondary major fragment ions are m / z 425.3098, 200.9992, 162.9437, 134.5173, and 113.0228, which are consistent with the retention time and fragmentation mode of glycyrrhizic acid reference standard. It is speculated that P4 is the original component of glycyrrhizic acid in Yanlishuang oral drops.

[0087] M1's t R The time was 19.00 min, and the quasi-molecular ion produced in positive mode had a m / z of 155.1426 [M+H]. + Its molecular formula is predicted to be C 10 H 18 O, compared to the menthol quasi-molecular ion peak m / z 157.1587 [M+H] + The difference of 2 is consistent with the reduction of two H molecules in menthol. 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 that it is a fragment ion formed by the removal of two H molecules. Therefore, it is speculated that it is a metabolite obtained by the oxidation of the hydroxyl group on the six-membered ring of menthol to a ketone group.

[0088] M2's t R The time was 4.83 min, and the quasi-molecular ion produced in negative mode had an m / z of 251.0956 [MH]. - Its molecular formula is predicted to be C 10 H 20 O5S, compared to the menthol quasi-molecular ion peak, m / z 155.1441 [MH] - The value of 96 is consistent with the addition of one molecule of sulfate (SO3) after menthol is oxidized to M1. The main secondary fragment ions are m / z 159.8588, 150.5030, 134.5023, and 79.9563. Among them, m / z 134.5023 is 16 less than m / z 150.5030, which is a fragment ion formed by the removal of one molecule of O. m / z 79.9563 is 80 less than m / z 159.8588, both of which are fragment ions formed by the removal of one molecule of sulfate (SO3). Therefore, it is speculated that these are metabolites obtained from the oxidation and sulfation of menthol.

[0089] M3's t R The time was 24.41 min, and the quasi-molecular ion produced in negative mode had an m / z of 195.1382 [MH]. - Its molecular formula is predicted to be C 12 H 20 O2, compared to the menthol quasi-molecular ion peak, has a m / z of 155.1441 [MH]. -The value of 40 is consistent with the addition of an acetyl group (C2H2O) after menthol is oxidized to M1. The major secondary fragment ions are m / z 138.0673, 125.0961, 109.0645, and 96.3625. Among them, m / z 125.0961 is 16 less than m / z 109.0645, which is a fragment ion formed by the removal of an O molecule. m / z 96.3625 is 42 less than m / z 138.0673, which is a fragment ion formed by the removal of a C2H2O molecule. Therefore, it is speculated that these are metabolites obtained from the oxidation and acetylation of menthol.

[0090] M4's t R The time was 23.24 min, and the quasi-molecular ions produced in negative mode were m / z 331.1762 [MH]. - Its molecular formula is predicted to be C 16 H 28 O7, compared to the menthol quasi-molecular ion peak, has a m / z of 155.1441 [MH]. - The difference of 176 is consistent with the addition of one molecule of glucuronide (C6H8O6) to menthol. The major secondary fragment ions are m / z 329.1163, 153.1272, and 75.0074, with m / z 153.1272 being 176 less than m / z 329.1163, indicating the removal of one molecule of C6H8O6. 10 The fragment ions formed by O5 suggest that it is a metabolite obtained from the glucuronidation of menthol. Furthermore, M4 shows consistent fragmentation patterns and retention times with the borneol-2-O-glucuronide standard, suggesting that they are the same substance.

[0091] M5's t R The time was 18.92 min, and the quasi-molecular ion produced in negative mode had an m / z of 347.1715 [MH]. - Its molecular formula is predicted to be C 16 H 28 O8, compared to the menthol quasi-molecular ion peak, has a m / z of 155.1441 [MH]. - The value of 192 is consistent with the oxidation of menthol to M1 followed by the addition of one molecule of gluconate (C6H8O6). The major secondary fragment ions are m / z 272.2710, 171.1387, 155.1061, and 96.3220. Among them, m / z 171.1387 is 16 less than m / z 155.1061, which is a fragment ion formed by the removal of one molecule of O. m / z 272.2710 is 176 less than m / z 96.3220, both of which are fragment ions formed by the removal of one molecule of gluconate (C6H8O6). Therefore, it is speculated that this is a metabolite obtained from the oxidation and glucuronidation of menthol.

[0092] M6's tR The time was 0.96 min, and the quasi-molecular ions produced in negative mode had an m / z of 193.0346 [MH]. - Its molecular formula is predicted to be C6H. 10 O7 is identical to a molecule of glucuronic acid. Its major secondary fragment ions are m / z 162.7277, 134.4170, and 113.0231. The fragmentation pattern is similar to that of glycyrrhizic acid, therefore it is speculated that M6 is a glucuronide formed from the hydrolysis of glycyrrhizic acid.

[0093] M7's t R The time was 25.65 min, and the quasi-molecular ions generated in negative mode had m / z 469.3303 [MH]. - Its molecular formula is predicted to be C 30 H 46 O4. The peak value of the glycyrrhizic acid quasi-molecular ion is 821.3929 m / z [MH]. - The value of 352 is consistent with the reduction of two molecules of glucuronide (C6H8O6) in glycyrrhizic acid. The major secondary fragment ions are m / z 425.3406, 162.8264, 134.5991, and 113.0333, exhibiting a fragmentation pattern similar to that of glycyrrhizic acid. Therefore, it is presumed to be a metabolite obtained from the hydrolysis of glycyrrhizic acid by the removal of two molecules of glucuronide. Comparison with glycyrrhetinic acid standards shows that its retention time matches the fragmentation pattern, suggesting that M7 represents glycyrrhetinic acid.

[0094] M8's t R The time was 24.36 min, and the quasi-molecular ions produced in negative mode had m / z values ​​of 483.3119 [MH]. - Its molecular formula is predicted to be C 30 H 44 O5. The peak value of the quasi-molecular ion of glycyrrhizic acid is 821.3929 m / z [M+H]. + The value of 338 is consistent with the oxidation of the methyl group on the benzene ring to a ketone carbonyl group after the hydrolysis of glycyrrhizic acid to form M7. The main secondary fragment ions of M8 are m / z 453.3097, 439.3221, 409.2742, and 353.2643. Among them, the fragment ions at m / z 453.3097 and 409.2742 are generated by the successive loss of CH2O and CO2, while the fragment ions at m / z 439.3221 and 353.2643 are generated by the loss of CO and the fragmentation of the benzene ring. Therefore, it is speculated that M8 is a metabolite obtained from the hydrolysis and oxidation of glycyrrhizic acid.

[0095] M9-M11's t R At time intervals of 24.25, 24.35, and 24.50 min, the quasi-molecular ions generated in negative mode had m / z values ​​of 499.3064 [MH]. -m / z 499.3068 [MH] - m / z499.3069[MH] - Its molecular formula is predicted to be C 30 H 44 O6. The peak value of the glycyrrhizic acid quasi-molecular ion is 821.3929 m / z [MH]. - The value of 322 is consistent with the oxidation of the methyl group on the benzene ring to a carboxylic acid after glycyrrhizic acid loses two molecules of glucuronide (C6H8O6). The major secondary fragment ions for M9 are m / z 437.3080, 393.3166, 162.8852, and 134.5000; for M10, m / z 437.3056, 393.3452, and 162.8852; and for M11, m / z 437.3069, 393.3162, and 134.4669. The peak at m / z 437 is a quasi-molecular ion, while m / z 499 [MH] is a quasi-molecular ion peak. - The fragment ions formed by the removal of one molecule of CO2 and one molecule of H2O, with m / z 393 representing fragment ions formed by the shedding of CO2 and CH2, and m / z 162 and 134 exhibiting similar fragmentation patterns to glycyrrhizic acid, suggest that M9-M11 are obtained by the oxidation of the methyl group on the benzene ring after the hydrolysis of glycyrrhizic acid and the removal of two molecules of glucuronide.

[0096] M12's t R The time was 24.59 min, and the quasi-molecular ions produced in negative mode were m / z 485.3272 [MH]. - Its molecular formula is predicted to be C 30 H 46 O5. The peak value of the quasi-molecular ion of glycyrrhizic acid is 821.3929 m / z [M+H]. + The value of 336 is consistent with the reduction of two glucuronide molecules (C6H8O6) in glycyrrhizic acid followed by the addition of one O molecule. The major secondary fragment ions are m / z 441.3399, 200.9798, 162.8195, and 134.5529. Among them, m / z 441.3399 is the fragment ion formed by adding one O molecule to glycyrrhizic acid (m / z 425.3098), and the fragmentation patterns of m / z 200.9798, 162.8195, and 134.5529 are similar to those of glycyrrhizic acid. Therefore, it is speculated that M12 glycyrrhizic acid is obtained by oxidizing after hydrolysis to remove two glucuronide molecules.

[0097] M13's t R The time was 24.33 min, and the quasi-molecular ions produced in negative mode were m / z 645.3616 [MH]. - Its molecular formula is predicted to be C 36 H 54 O 10The peak value of the quasi-molecular ion of glycyrrhizic acid is 821.3929 m / z [MH]. - The decrease of 176 is consistent with the reduction of one molecule of glycyrrhizic acid glucuronide (C6H8O6). The major secondary fragment ions are m / z 469.3315, 332.3896, and 156.0127, with m / z 469.3315 being significantly lower than m / z 645.3616 [MH]. - The difference of 176 from m / z 156.0127 compared to m / z 332.3896 indicates that both are fragment ions formed by the removal of one molecule of gluconate ester (C6H8O6), suggesting that it is a metabolite obtained from the hydrolysis of glycyrrhizic acid by removing one molecule of glucuronide. Furthermore, comparison of M13 with the standard glycyrrhetinic acid monoglucuronide shows consistent fragmentation patterns and retention times, suggesting that they are the same substance.

[0098] M14, M15 t R At 19.00 and 22.30 min, the quasi-molecular ions generated in positive mode had m / z values ​​of 137.1321 [M+H]. + 137.1321[M+H] + Its molecular formula is predicted to be C 10 H 16 The peak value of the borneol quasi-molecular ion is 155.1426 m / z [M+H]. + The difference of 18 is consistent with the reduction of one molecule of water (H2O) in borneol. The major secondary fragment ions of M14 are m / z 113.9737, 109.1011, 95.0857, and 91.0545. Among them, m / z 95.0857 is 18 less than m / z 113.9737, and m / z 91.0545 is 18 less than m / z 109.1011. Both are fragment ions formed by the removal of one molecule of H2O. The major secondary fragment ions of M15 are m / z 109.10121 and 91.05436. Among them, m / z 91.05436 is 18 less than m / z 109.10121. It is also a fragment ion formed by the removal of one molecule of H2O. Therefore, it is speculated that M14 and M15 are metabolites obtained from the dehydration of borneol.

[0099] M16's t R The time was 16.30 min, and the quasi-molecular ions generated in positive mode were m / z 171.1374 [M+H]. + Its molecular formula is predicted to be C 10 H 18 O2, compared to the borneol quasi-molecular ion peak m / z 155.1426 [M+H] +The difference of 16 is consistent with the addition of one molecule of O to borneol. The main secondary fragment ions are m / z 170.0955, 154.1304, 146.9607, and 130.9660. Among them, m / z 154.1304 is 16 less than m / z 170.0955, and m / z 130.9660 is 16 less than m / z 146.9607. These are all fragment ions formed by the removal of one molecule of O. It is speculated that these are metabolites obtained from the oxidation of borneol.

[0100] M17's t R The time was 21.46 min, and the quasi-molecular ion produced in negative mode had a m / z of 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 difference of 80 is consistent with the addition of one molecule of sulfate (SO3) to borneol. The main secondary fragment ions are m / z 214.4837 and 134.4867, with m / z 134.4867 being 80 less than m / z 214.4837. This difference represents fragment ions formed by the removal of one molecule of sulfate (SO3), suggesting that it is a metabolite from the sulfation of borneol.

[0101] M18, M19 t R The time intervals were 22.19 and 22.39 min, and the quasi-molecular ions generated in positive mode had m / z values ​​of 329.1604 [MH]. - 329.1606 [MH] - Its molecular formula is predicted to be C 16 H 26 O7, with a peak m / z of 153.1284 [MH] compared to the borneol quasi-molecular ion peak. - The difference of 176 is consistent with the addition of one molecule of glucuronide (C6H8O6) to borneol. The major secondary fragment ions of M18 are m / z 295.1529, 119.0852, 111.1168, and 85.0282, with m / z 119.08517 being 176 fewer 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 being 176 fewer than m / z 313.1616. It is speculated that these are fragment ions formed by the removal of one molecule of glucuronide. It is presumed that M18 and M19 are metabolites obtained from the glucuronidation of borneol. Among them, M18 and borneol-2-O-glucuronide showed consistent cleavage patterns and retention times, suggesting that they are the same substance.

[0102] M20, M21's t R The time intervals were 15.41 and 15.75 min, and the quasi-molecular ions generated in positive mode had m / z values ​​of 347.1690 [M+H]. + 347.1692 [M+H] + Its molecular formula is predicted to be C 16 H 26 O8, compared to the borneol quasi-molecular ion peak, m / z 155.1426 [M+H]. + More than 192, with borneol, add one molecule of O, and then add one molecule of glucuronide (C6H) 10 This is consistent with O5. The major secondary fragment ions of M20 are m / z 171.1373, 153.1269, and 109.1011, while those 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 obtained by the combination of borneol oxidation and glucuronide.

[0103] M22-M24's t R The time intervals were 6.62, 11.76, and 15.76 min, and the quasi-molecular ions generated in positive mode had m / z values ​​of 135.1166 [M+H]. + 135.1167[M+H] + 135.1166[M+H] + Its molecular formula is predicted to be C 10 H 14 The camphor quasi-molecular ion peak m / z is 153.1267 [M+H]. + The difference of 18 m / z is consistent with the reduction of one molecule of water (H2O) in camphor. The major secondary fragment ions of M22 are m / z 117.0694, 107.0856, and 93.0701; those of M23 are m / z 118.0651, 107.0856, and 93.0701; and those of M24 are m / z 117.0691, 107.0855, and 93.0701. Among these, m / z 117 and 93 differ from camphor fragment ions m / z 135 and 111 by 18 m / z, respectively, and are presumably fragment ions formed by the reduction of one molecule of H2O. The fragmentation pattern of m / z 118, 117, 107, and 93 is similar to that of camphor. Therefore, it is speculated that M22-M24 are metabolites obtained from the dehydration of camphor.

[0104] M25, M26 t R The time intervals were 12.36 and 14.61 min, and the quasi-molecular ions generated in positive mode had m / z values ​​of 169.1219 [M+H]. +169.1217[M+H] + Its molecular formula is predicted to be C 10 H 16 O2, camphor quasi-molecular ion peak m / z 153.1267 [M+H] + The increase of 16 is consistent with the addition of one molecule of O to camphor. The major secondary fragment ions of M25 are m / z 152.1145, 151.1114, and 123.0801, while those of M26 are m / z 152.1150, 151.1112, and 123.0799. The m / z values ​​of 152, 151, and 123 are 16 higher than those of camphor fragment ions (m / z 136, 135, and 107), respectively, suggesting that they are fragment ions formed by the addition of one molecule of O. The fragmentation patterns of m / z 152, 151, and 123 are similar to those of camphor, suggesting that they are metabolites obtained from the oxidation of camphor.

[0105] M27's t R The time was 20.77 min, and the quasi-molecular ions generated in 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 difference of 42 is consistent with the addition of one acetyl group (C2H2O) to camphor. The major secondary fragment ions have m / z values ​​of 177.1270, 153.0912, 149.0950, 136.1199, and 135.1164. Among them, m / z values ​​of 177.1270, 153.0912, and 149.0950 differ from camphor fragment ions m / z values ​​of 135.11635, 111.08028, and 107.08541 by 42, respectively, suggesting that they are fragment ions formed by the addition of one acetyl group. The m / z values ​​of 136.1199 and 135.1164 are similar to those of camphor fragment ions m / z values ​​of 136.11942 and 135.11635. Therefore, it is speculated that they are metabolites obtained from the acetylation of camphor.

[0106] M28, M29 t R The time intervals were 13.84 and 14.59 min, and the quasi-molecular ions generated in positive mode had m / z values ​​of 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 / z 153.1267 [M+H] + More than 192, with camphor plus one molecule of O and then one molecule of glucuronide (C6H) 10This is consistent with O5. The major secondary fragment ions of M28 are m / z 151.1111, 123.0802, and 107.0854, while those of 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 presumably metabolites obtained by the combination of camphor oxidation and glucuronide.

[0107] M30's t R The time was 19.01 min, and the quasi-molecular ions produced in 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 than 178, with camphor plus one molecule of O and then one molecule of glucoside (C6H) 10 This matches the O5) pattern. The major secondary fragment ions are m / z 285.1676, 135.1160, and 107.0854. The difference of 178 between m / z 285.1676 and 107.0854 is presumably due to the successive addition of one molecule of O and one molecule of glucoside (C6H). 10 The fragment ions obtained from O5, with m / z values ​​of 135.1160 and 107.0854, exhibited fragmentation patterns similar to those of camphor. Therefore, it is speculated that these fragments are metabolites obtained from the combination of camphor oxidation and glucosides.

[0108] 3 Discussion

[0109] The active substances of traditional Chinese medicine (TCM) include the original bioactive compounds and metabolites within the herb, which are crucial for ensuring the safety and efficacy of TCM. The active substance basis of TCM compound prescriptions refers to the group of chemical components that act on multiple targets and produce overall efficacy after entering the human body. These chemical components may originate from inherent components in the medicinal materials, products formed during preparation, or metabolites generated by the interaction between the drug and the human body. This study identified numerous pharmacological activities of the original components of the Yanli Shuang oral drops in bile, feces, and urine for acute pharyngitis, such as menthol, borneol, and camphor, which have anti-inflammatory and analgesic effects. Furthermore, the metabolites glycyrrhetinic acid, monoglucuronic acid glycyrrhetinic acid, borneol-2-O-glucuronide, and menthol-1-O-glucuronide were detected in rat bile, feces, and urine, indicating that these metabolites may be some of the active substances contributing to the efficacy of Yanli Shuang oral drops. It is suggested that, in addition to the original components, quantitative detection of the main metabolites of this compound preparation can be added to the quality control standards to ensure the efficacy stability of the preparation.

[0110] Experimental results showed that after oral administration to rats, the metabolism of Yanlishuang oral drops mainly involved hydrolysis, oxidation, and glucuronic acid conjugation, with small amounts of glucose conjugates and sulfation products. This primarily involved phase I and phase II metabolism, and the hypothesized metabolic pathways were largely consistent with literature reports. Furthermore, the urine sample contained the highest number of metabolites, suggesting that renal excretion is the main elimination pathway, a result consistent with the mechanism of glomerular filtration of water-soluble metabolites. Eighteen metabolites were detected in bile, possibly related to enterohepatic circulation or secondary metabolism mediated by gut microbiota.

[0111] 4. Conclusion

[0112] This study established a metabolic profile analysis method for Yanlishuang oral drops in rats based on GC-MS and UHPLC / Q Exactive Plus MS techniques. By comparing the metabolites and metabolic reaction types in rats, a total of 30 metabolites were detected in bile, urine, and feces. Specifically, 18 metabolites were detected in bile, 13 in feces, and 24 in urine. The results indicate that the original components of this preparation, after phase I (hydrolysis and oxidation) and phase II (mainly glucuronidation) metabolism, are primarily excreted in urine, followed by bile and feces. The diversity of metabolites reflects the synergistic metabolic characteristics of the compound components, with glucuronide conjugates being the dominant metabolites. This study provides a theoretical basis for the formulation of quality control standards for Yanlishuang oral drops and lays a methodological foundation for subsequent pharmacodynamic material basis research.

[0113] Finally, it should be noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the content disclosed in this invention should be considered within the scope of protection of this invention.

Claims

1. A method for detecting the original and metabolized components of a throat lozenge in vivo, characterized in that, Volatile precursor components were detected by GC-MS, and non-volatile precursor components and metabolites were detected by UHPLC-MS / MS. The specific methods for detecting non-volatile precursors and metabolites using UHPLC-MS / MS are as follows: Chromatographic conditions: LC system: Vanquishhorizon; Column: Hypersilgold, 2.1×100mm, 1.9μm; Column temperature: 40℃; Injection volume: 1µL; Flow rate: 0.3mL·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 Explorer 240; Scan mode: HESI+ & HESI-; Cone voltage: 3.5 kV for HESI+, 2.5 kV for HESI-; Desolvent gas temperature: 350 °C; Nebulizer gas: 50 alb; Desolvent gas: 10 alb; Capillary: 320℃; Scanning range m / z: 100-1500; resolution MS: 70000; Resolution MS / MS: 15000; The chromatographic conditions for detection by UHPLC-MS / MS, with the following elution gradient: Liquid chromatography conditions The biological sample pretreatment for UHPLC-MS / MS detection is as follows: take 1 mL of urine or bile, add 4 mL of methanol, vortex for 2 min, sonicate for 10 min, centrifuge, take the supernatant, dry it under nitrogen at 37℃, add 1 mL of methanol, precipitate the protein again, vortex for 2 min, sonicate for 10 min, centrifuge, take the supernatant, dry it under nitrogen at 37℃, add 200 µL of 50% methanol to redissolve, and inject the supernatant for analysis. The pretreatment of biological samples for UHPLC-MS / MS detection involves weighing 0.3g of feces, adding 1.2mL of physiological saline to homogenize, centrifuging, taking 1mL of the supernatant homogenate, adding 4mL of methanol, vortexing for 2min, sonicating for 10min, centrifuging, taking the supernatant and placing it in an EP tube, drying it under nitrogen at 37℃, then adding 1mL of methanol to the dried sample, treating it according to the above method, drying it, adding 200µL of 50% methanol to reconstitute it, and taking the supernatant for analysis. The specific original and metabolized components of the Throat-Soothing Oral Droplets are as follows: Table 4. Identification of the original form and metabolites after administration of Yanlishuang oral drops. 。 2. The method for detecting the original and metabolized components of the throat-soothing oral drops in vivo according to claim 1, characterized in that, The specific steps for detecting volatile prototype components using GC-MS are as follows: Chromatographic conditions: Column: SH-Stabilwax, 30 m × 0.32 mm, 0.25 μm capillary column; Injector temperature: 220℃; Splitless injection; Injection volume: 1 μL Mass spectrometry conditions: SIM mode; ion source temperature 200 ℃; interface temperature 230 ℃; ionization method: electron impact ion (EI). Detection voltage: 0.2 kV; solvent delay: 2 min; mass number scan range: 30 ~ 400 amu.

3. The method for detecting the original and metabolized components of the throat-soothing oral drops in vivo according to claim 2, characterized in that, The chromatographic conditions for detecting volatile precursor components using GC-MS, with the following temperature program: 。 4. The method for detecting the original and metabolized components of the throat-soothing oral drops according to claim 2, characterized in that, The mass spectrometry conditions for detecting volatile prototype components using GC-MS, and the ion information for each index component in SIM mode are as follows: 。 5. The method for detecting the original and metabolized components of the throat-soothing oral drops in vivo according to claim 4, characterized in that, The metabolic components are any one or more of urine, feces, and bile.

6. The method for detecting the original and metabolized components of the throat-soothing oral drops in vivo according to claim 1, characterized in that, The biological sample pretreatment for GC-MS detection specifically involves taking 100 µL of urine or bile, placing it in a 1.5 mL centrifuge tube, then adding 100 µL of n-hexane:ethyl acetate (volume ratio 1:2), vortexing for 2 min at 4 °C and 14000 r·min. -1 Centrifuge for 10 min, then transfer 100 µL into a vial containing the inner tube for GC-MS analysis.

7. The method for detecting the original and metabolized components of the throat-soothing oral drops according to claim 1, characterized in that, The biological sample pretreatment for GC-MS detection specifically involves weighing 0.3 g of feces, adding 1.2 mL of n-hexane:ethyl acetate (volume ratio 1:2), vortexing for 5 min at 4 ℃ and 14000 r·min. -1 Centrifuge for 10 min; use blank feces without administering the oral drops as a blank control; take 100 μL of rat fecal supernatant into a sample bottle containing an inner tube, and analyze by GC-MS.

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