UHPLC Q-Exactive Orbitrap MS / MS (Ultra High Performance Liquid Chromatography Q-Exactive Orbitrap Mass Spectrometry / Mass Spectrometry) rapid identification method for chemical components and serum migration components of compound radix scutellariae capsule for clearing heat from throat

Abstract

The invention discloses a UHPLC Q-Exactive Orbitrap MS / MS (Ultra High Performance Liquid Chromatography Q-Exactive Orbitrap Mass Spectrometry / Mass Spectrometry) rapid identification method for chemical components and serum migration components of a compound radix scutellariae capsule for clearing heat from throat, which is characterized in that the chemical components and the serum migration components of the compound radix scutellariae capsule for clearing heat from throat are rapidly qualitatively identified by adopting UHPLC Q-Exactive Orbitrap MS / MS; 21 in-vitro components (including flavonoids, phenolic acids, triterpenes and the like) and 21 in-blood migration components (including 9 in-blood prototype components and 12 metabolites) are identified. The chemical components and serum migration components of the compound radix scutellariae capsule for clearing heat from throat are defined, a foundation is laid for fundamental research of pharmacodynamic substances of the preparation, and the blank of chemical research of serum drugs of the compound radix scutellariae capsule for clearing heat from throat is filled up.

Description

Technical Field

[0001] This invention belongs to the field of compound traditional Chinese medicine component detection, specifically involving a rapid UHPLC Q-Exactive Orbitrap MS / MS method for the identification of chemical components and serum migrating components of Compound Scutellaria baicalensis throat-clearing capsules. Background Technology

[0002] Compound Scutellaria baicalensis throat-clearing capsules are a pure traditional Chinese medicine compound preparation made by scientific extraction and purification, with Scutellaria baicalensis as the principal ingredient, Scrophularia ningpoensis as the assistant ingredient, and Terminalia chebula and Platycodon grandiflorus as adjuvant ingredients. It has the effects of clearing the lungs and detoxifying, and relieving sore throat and improving voice. Currently, its clinical efficacy is certain, but the pharmacodynamic material basis is still unclear, hindering the further clinical application of this preparation.

[0003] The efficacy of traditional Chinese medicine (TCM) compound preparations stems from the holistic effect of their constituent chemical components. The "pharmacological basis" of TCM and its compound preparations refers to the chemical components that can be absorbed by the body into the bloodstream, exist in the blood in their original or metabolite form, and ultimately reach the site of action to produce biological activity. These are the key substances that enable TCM to exert its direct effects in the body.

[0004] UHPLC Q-Exactive Plus Orbitrap MS / MS technology integrates the high separation capability of ultra-high performance liquid chromatography with the precise qualitative advantages of high-resolution mass spectrometry. It features high sensitivity, high resolution, high scan speed, and powerful multi-stage fragment resolution capabilities, making it particularly suitable for the rapid screening, identification, and metabolite study of trace and ultra-low-level compounds in complex systems of traditional Chinese medicine compound preparations. This technology provides a powerful tool for in-depth research on the in vivo and in vitro chemical composition of traditional Chinese medicine compound preparations and their dynamic changes.

[0005] Currently, there are no reports on the qualitative detection of the in vivo and in vitro components of Compound Scutellaria baicalensis throat-clearing capsules. Therefore, this invention leverages the advantages of UHPLCQ-Exactive Plus Orbitrap MS / MS to rapidly analyze the chemical components of Compound Scutellaria baicalensis throat-clearing capsules and biological samples after administration, studying its chemical components and migrating components in serum, providing a basis for its pharmacodynamic material basis and subsequent pharmacokinetic and mechanism of action studies. Summary of the Invention

[0006] This invention aims to provide a rapid UHPLC Q-Exactive Orbitrap MS / MS method for the identification of chemical components and serum migrating components of Compound Scutellaria baicalensis throat-clearing capsules. This method provides a reference for in vivo pharmacodynamic material basis research by rapidly identifying the chemical components and serum migrating components of Compound Scutellaria baicalensis throat-clearing capsules.

[0007] The objective of this invention is achieved through the following technical solution: a rapid UHPLC Q-Exactive Orbitrap MS / MS method for identifying the chemical components and serum migrating components of Compound Scutellaria baicalensis Throat-Clearing Capsules. The Compound Scutellaria baicalensis Throat-Clearing Capsules, by weight, are mainly prepared from 25-30 parts of Scutellaria baicalensis, 15-20 parts of Scrophularia ningpoensis, 10-15 parts of Terminalia chebula, and 12-20 parts of Platycodon grandiflorus. The preparation method involves decocting Scutellaria baicalensis, Scrophularia ningpoensis, Terminalia chebula, and Platycodon grandiflorus with water three times. The first extraction is performed with 8-10 times the amount of water for 1.5-2 hours; the second extraction is performed with 6-8 times the amount of water for 1-1.5 hours; and the third extraction is performed with 4-6 times the amount of water for 1-1.5 hours. The extract is concentrated under reduced pressure to a relative density of 1.12-1.14 at 80°C, spray-dried, and then microcrystalline cellulose is added. After thorough mixing, the mixture is encapsulated, with a specification of 0.3g / capsule. The UHPLC Q-Exactive Orbitrap MS / MS rapid identification method includes the following steps: (1) Preparation of test solution: Accurately weigh the powder of compound Scutellaria baicalensis throat clearing capsule, place it in a volumetric flask, add 45-55% methanol and sonicate for 25-35 min to dissolve, make up to volume and mix well, centrifuge at 3.5-4.5℃ and 10000-20000r / min for 8-12 min, take the supernatant to obtain the test solution; (2) Preparation of reference solutions: Accurately weigh vanillin reference standard, baicalein reference standard, quercetin reference standard, baicalin reference standard, syringaldehyde reference standard, neochlorogenic acid reference standard, harpagoside reference standard, harpagoside reference standard, caffeic acid reference standard, apigenin-7-O-β-D-glucoside reference standard, apigenin reference standard, chlorogenic acid reference standard, and baicalin reference standard, respectively, and place them in volumetric flasks. Add 45-55% methanol, sonicate to dissolve, and dilute to volume to obtain 0.8-1.2 mg / mL vanillin reference solution, 0.8-1.2 mg / mL baicalein reference solution, 0.8-1.2 mg / mL quercetin reference solution, and 0.8-1.2 mg / mL vanillin reference solution, respectively. The following reference solutions were prepared: 0.8-1.2 mg / mL baicalin, 0.8-1.2 mg / mL syringaldehyde, 0.8-1.2 mg / mL neochlorogenic acid, 0.8-1.2 mg / mL harpagoside, 0.8-1.2 mg / mL harpagoside, 0.8-1.2 mg / mL caffeic acid, 0.8-1.2 mg / mL apigenin-7-O-β-D-glucoside, 0.8-1.2 mg / mL apigenin, 0.8-1.2 mg / mL jasmin, 0.8-1.2 mg / mL chlorogenic acid, and 0.8-1.2 mg / mL baicalin. These solutions were stored at -20°C for later use. (3) Preparation of drug-containing serum test solution: Rats were administered the test solution by gavage at a dose of 180-200 mg / kg each time, 2-3 times daily for 5 consecutive days. Blood was collected 1.8-2.2 h after the last administration, and 0.8-1.2 mL of plasma was separated. 3-5 times the volume of methanol was added to precipitate the protein. The mixture was allowed to stand at -20℃ for 25-35 min, then vortexed for 2.5-3.5 min. The mixture was then stirred at 3.5-4.5℃ and 10000-20000 r / min. Centrifuge for 8-12 minutes at 3.5-4.5℃ and 10000-20000 r / min, and collect the supernatant of the drug-treated serum sample. Take the supernatant of the drug-treated serum sample and blow it dry with nitrogen at 36-38℃. Redissolve the residue with 280-320 μL of 45-55% methanol, vortex for 2-4 minutes, and sonicate for 8-12 minutes. Centrifuge again at 3.5-4.5℃ and 10000-20000 r / min for 8-12 minutes, and collect the supernatant to obtain the drug-containing serum test solution. (4) The test solution and the drug-containing serum test solution were analyzed by UHPLC Q-Exactive Orbitrap MS / MS; UHPLC system chromatographic conditions: Vanquish Horizon, Hypersil Gold column (2.1 × 150 mm, 1.9 μm), column temperature 40℃, injection volume 2 µL, mobile phase A 0.1% formic acid-acetonitrile solution, mobile phase B 0.1% formic acid-water solution, elution gradient 0–7 min, 95%–83% B; 7–13 min, 83–81% B; 13–20 min, 81–77% B; 20–25 min, 77–75% B; 25–30 min, 75–71% B; 30–35 min, 75–10% B; 35–36 min, 10% B; 36–36.5 min, 10%–95% B; 36.5–40 min, 95% B. MS system chromatographic conditions: Q Exactive Plus Orbitrap; scan mode HESI+ & HESI-, cone voltage 3.5kV (+) / 2.5kV (-), desolvent gas temperature 350℃; nebulizer gas pressure 50arb; desolvent gas pressure 10arb; capillary temperature 300℃; scan range m / z 100-1500; MS resolution 120000; MS / MS resolution 15000. (5) Identification of the components of the test solution: The chromatographic peaks of the test solution were analyzed using Compound Discover 3.2 and Xcalibar 4.7 software. Combined with the retention time, precise molecular weight and secondary fragment information provided by high resolution mass spectrometry, and by comparing with the chromatographic peaks of the reference solution and multiple databases, a total of 13 compounds were finally identified from Compound Scutellaria baicalensis throat clearing capsules. (6) Identification of components in drug-containing serum test solution: Using rat blank serum as a reference, the retention time, quasi-molecular ion peak and characteristic secondary mass spectrometry fragment ions of the chromatographic peak of the drug-containing serum test solution were compared with those of the corresponding reference solution. A total of 9 original blood-entry components of Compound Scutellaria baicalensis throat-clearing capsule and 12 metabolites were identified in rat drug-containing serum.

[0008] In step (1) above, the preparation of the test solution is as follows: accurately weigh 0.45-0.55g of compound Scutellaria baicalensis throat-clearing capsule powder, place it in a volumetric flask, add 6-8.5mL of 48-52% methanol, sonicate for 28-32min, dissolve, make up to volume, mix well, centrifuge at 3.8-4.2℃ and 12000-18000r / min for 9-11min, take the supernatant, and the test solution is obtained.

[0009] Specifically, in the aforementioned step (1), the preparation of the test solution is as follows: accurately weigh 0.5g of compound Scutellaria baicalensis throat-clearing capsule powder, place it in a 10mL volumetric flask, add 7-8mL of 50% methanol, sonicate for 30min, dissolve, make up to volume, mix well, centrifuge at 4℃ and 14000r / min for 10min, take the supernatant, and the test solution is obtained.

[0010] In step (2) above, the preparation of the reference solutions is as follows: Accurately weigh vanillin reference standard, baicalin reference standard, quercetin reference standard, baicalin reference standard, syringaldehyde reference standard, neochlorogenic acid reference standard, harpagoside reference standard, harpagoside reference standard, caffeic acid reference standard, apigenin-7-O-β-D-glucoside reference standard, apigenin reference standard, chlorogenic acid reference standard, and baicalin reference standard, respectively, and place them in volumetric flasks. Add 6-8.5 mL of 48-52% methanol to each flask, sonicate to dissolve, and dilute to volume to obtain 0.9-1.1 mg / mL vanillin reference solution, 0.9-1.1 mg / mL baicalin reference solution, 0.9-1.1 mg / mL quercetin reference solution, and 0... 0.9-1.1 mg / mL baicalin reference solution, 0.9-1.1 mg / mL syringaldehyde reference solution, 0.9-1.1 mg / mL neochlorogenic acid reference solution, 0.9-1.1 mg / mL harpagoside reference solution, 0.9-1.1 mg / mL harpagoside reference solution, 0.9-1.1 mg / mL caffeic acid reference solution, 0.9-1.1 mg / mL apigenin-7-O-β-D-glucoside reference solution, 0.9-1.1 mg / mL apigenin reference solution, 0.9-1.1 mg / mL apigenin reference solution, 0.9-1.1 mg / mL chlorogenic acid reference solution, and 0.9-1.1 mg / mL baicalin reference solution should be stored at -20℃ for later use.

[0011] Specifically, in step (2) above, the preparation of the reference solutions is as follows: Vanillin reference, baicalin reference, quercetin reference, baicalin reference, syringaldehyde reference, neochlorogenic acid reference, harpagoside reference, harpagoside reference, caffeic acid reference, apigenin-7-O-β-D-glucoside reference, apigenin reference, chlorogenic acid reference, and baicalin reference are accurately weighed and placed in 10 mL volumetric flasks respectively. 7-8 mL of 50% methanol is added to each flask. Dissolved by ultrasonication in mL and diluted to volume, 1 mg / mL vanillin reference solution, 1 mg / mL baicalein reference solution, 1 mg / mL quercetin reference solution, 1 mg / mL baicalin reference solution, 1 mg / mL syringaldehyde reference solution, 1 mg / mL neochlorogenic acid reference solution, 1 mg / mL harpagoside reference solution, 1 mg / mL harpagoside reference solution, 1 mg / mL caffeic acid reference solution, 1 mg / mL apigenin-7-O-β-D-glucoside reference solution, 1 mg / mL apigenin reference solution, 1 mg / mL juglans regia reference solution, 1 mg / mL chlorogenic acid reference solution and 1 mg / mL baicalin reference solution were obtained, and stored at -20℃ for later use.

[0012] In step (3) above, the preparation of the drug-containing serum test solution is as follows: Rats are administered the drug-containing serum test solution by gavage at a dose of 185-195 mg / kg twice daily for 5 consecutive days. Blood is collected 1.9-2.1 h after the last administration. 0.9-1.1 mL of plasma is separated, and 3.5-4.5 times the volume of methanol is added to precipitate the protein. The mixture is allowed to stand at -20℃ for 28-32 min, then vortexed for 2.8-3.2 min. The mixture is then vortexed at 3.8-4.2℃ and 12000-18000 r / min. Centrifuge for 9-11 min at 36.5-37.5℃, collect the upper serum layer to obtain the supernatant of the drug-treated serum sample; take the supernatant of the drug-treated serum sample and blow it dry with nitrogen at 36.5-37.5℃, reconstitute the residue with 290-310 μL of 48-52% methanol, vortex for 2.5-3.5 min, sonicate for 9-11 min, and centrifuge again at 3.8-4.2℃ and 12000-18000 r / min for 9-11 min, collect the supernatant to obtain the drug-containing serum test solution.

[0013] Specifically, in step (3) above, the preparation of the drug-containing serum test solution is as follows: rats are given a dose of 189 mg / kg by gavage twice a day for 5 consecutive days. Blood is collected 2 hours after the last administration, 1 mL of plasma is separated, 4 times the volume of methanol is added to precipitate the protein, and the mixture is allowed to stand at -20℃ for 30 min, vortexed for 3 min, and centrifuged at 4℃ and 14000 r / min for 10 min. The upper serum layer is collected to obtain the supernatant of the drug-treated serum sample. The supernatant of the drug-treated serum sample is dried by nitrogen blowing at 37℃. The residue is reconstituted with 300 μL of 50% methanol, vortexed for 3 min, sonicated for 10 min, and centrifuged again at 4℃ and 14000 r / min for 10 min. The supernatant is collected to obtain the drug-containing serum test solution.

[0014] In step (5) above, the 13 compounds are harpagoside, neochlorogenic acid, chlorogenic acid, caffeic acid, syringaldehyde, vanillin, cosmos glycoside, baicalin, quercetin, baicalin, baicalin, harpagoside and strophin.

[0015] In step (5) above, the multiple databases include mzCloud, mzVault and Chemispider.

[0016] In the aforementioned step (6), the original blood-entry components of the nine compound Scutellaria baicalensis throat-clearing capsules are harbacoside, neochlorogenic acid, chlorogenic acid, vanillin, quercetin, baicalin, harbacoside, salicylic acid and oleanolic acid.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention enables rapid qualitative analysis of the chemical components and serum-transferred components of Compound Scutellaria baicalensis Throat-Clearing Capsules. A total of 21 compounds were detected in Compound Scutellaria baicalensis Throat-Clearing Capsules, including 12 flavonoids, 3 phenolic acids, 1 triterpenoid, and 5 other components. A total of 21 substances were detected in rat serum containing the drug, including 9 original components and 12 metabolites. This clarifies the chemical components and serum-transferred components of Compound Scutellaria baicalensis Throat-Clearing Capsules, laying the foundation for the study of the pharmacodynamic material basis of this preparation.

[0018] 2. This invention studies the compound traditional Chinese medicine as a whole. The blood-entering components and metabolites discovered in the research results may be related to the efficacy. The absorbed blood-entering components may be the effective component group for the compound Scutellaria baicalensis throat-clearing capsule to exert its efficacy in vivo. This can provide a reference for elucidating the serum pharmacochemical pharmacochemical material basis and mechanism of action of the compound Scutellaria baicalensis throat-clearing capsule, and fill the gap in the serum pharmacochemical research of the compound Scutellaria baicalensis throat-clearing capsule. Attached Figure Description

[0019] Figure 1 The high-resolution mass spectrometry (HMS) total ion chromatograms of Compound Scutellaria baicalensis throat-clearing capsules in positive and negative ion modes are shown (A is the total ion chromatogram in positive ion mode; B is the total ion chromatogram in negative ion mode). Figure 2 The following are the total ion chromatograms of the serum containing Compound Scutellaria baicalensis and Phlegm-Clearing Capsules (A is the total ion chromatogram of the drug-containing serum in positive ion mode; B is the total ion chromatogram of the drug-containing serum in negative ion mode; C is the total ion chromatogram of the blank serum in positive ion mode; D is the total ion chromatogram of the blank serum in negative ion mode). Figure 3 This refers to the metabolic pathway of harbazoside; Figure 4 The metabolic pathway for chlorogenic acid / neochlorogenic acid; Figure 5 This is the cleavage pathway of vanillin; Figure 6 This represents the cleavage pathway of baicalin; Figure 7 This represents the cleavage pathway of harpagoside; Figure 8 This represents the cleavage pathway of salicylic acid. Detailed Implementation

[0020] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0021] Example 1: UHPLC Q-Exactive Orbitrap MS / MS Rapid Identification Method Preparation of the test solution: Accurately weigh 0.5g of compound Scutellaria baicalensis throat-clearing capsule powder, place it in a 10mL volumetric flask, add 7-8mL of 50% methanol, sonicate for 30min, dissolve, make up to volume, mix well, centrifuge at 4℃ and 14000r / min for 10min, take the supernatant, and the test solution is obtained.

[0022] Preparation of reference solutions: Accurately weigh vanillin reference standard, baicalin reference standard, quercetin reference standard, baicalin reference standard, syringaldehyde reference standard, neochlorogenic acid reference standard, harpagoside reference standard, harpagoside reference standard, caffeic acid reference standard, apigenin-7-O-β-D-glucoside reference standard, apigenin reference standard, chlorogenic acid reference standard, and baicalin reference standard, and place them in separate 10 mL volumetric flasks. Add 7-8 mL of 50% methanol to each flask. Dissolved by ultrasonication in mL and diluted to volume, 1 mg / mL vanillin reference solution, 1 mg / mL baicalein reference solution, 1 mg / mL quercetin reference solution, 1 mg / mL baicalin reference solution, 1 mg / mL syringaldehyde reference solution, 1 mg / mL neochlorogenic acid reference solution, 1 mg / mL harpagoside reference solution, 1 mg / mL harpagoside reference solution, 1 mg / mL caffeic acid reference solution, 1 mg / mL apigenin-7-O-β-D-glucoside reference solution, 1 mg / mL apigenin reference solution, 1 mg / mL juglans regia reference solution, 1 mg / mL chlorogenic acid reference solution and 1 mg / mL baicalin reference solution were obtained, and stored at -20℃ for later use.

[0023] Preparation of drug-containing serum test solution: Rats were administered the drug-containing serum test solution by gavage at a dose of 189 mg / kg twice daily for 5 consecutive days. Blood was collected 2 hours after the last administration, 1 mL of plasma was separated, and 4 times the volume of methanol was added to precipitate the protein. The mixture was allowed to stand at -20℃ for 30 min, vortexed for 3 min, and centrifuged at 4℃ and 14000 r / min for 10 min. The supernatant of the drug-containing serum sample was collected. The supernatant of the drug-containing serum sample was dried by nitrogen blowing at 37℃. The residue was reconstituted with 300 μL of 50% methanol, vortexed for 3 min, sonicated for 10 min, and centrifuged again at 4℃ and 14000 r / min for 10 min. The supernatant was collected to obtain the drug-containing serum test solution.

[0024] The test solution and the drug-containing serum test solution were analyzed by UHPLC Q-Exactive Orbitrap MS / MS; UHPLC system chromatographic conditions: Vanquish Horizon, Hypersil Gold column (2.1 × 150 mm, 1.9 μm), column temperature 40℃, injection volume 2 µL, mobile phase A 0.1% formic acid-acetonitrile solution, mobile phase B 0.1% formic acid-water solution, elution gradient 0–7 min, 95%–83% B; 7–13 min, 83–81% B; 13–20 min, 81–77% B; 20–25 min, 77–75% B; 25–30 min, 75–71% B; 30–35 min, 75–10% B; 35–36 min, 10% B; 36–36.5 min, 10%–95% B; 36.5–40 min, 95% B. MS system chromatographic conditions: Q Exactive Plus Orbitrap; scan mode HESI+ & HESI-, cone voltage 3.5kV (+) / 2.5kV (-), desolvent gas temperature 350℃; nebulizer gas pressure 50arb; desolvent gas pressure 10arb; capillary temperature 300℃; scan range m / z 100-1500; MS resolution 120000; MS / MS resolution 15000.

[0025] Identification of the components of the test solution: The chromatographic peaks of the test solution were analyzed using Compound Discover 3.2 and Xcalibar 4.7 software, and combined with high-resolution mass spectrometry, the chromatographic peaks were compared with those of the reference solution and multiple databases; Identification of components in drug-containing serum test solution: Using rat blank serum as a reference, the chromatographic peaks of the drug-containing serum test solution were compared with the chromatographic peak retention times, quasi-molecular ion peaks, and characteristic secondary mass spectrometry fragment ions of the corresponding reference solution.

[0026] Example 2: UHPLC Q-Exactive Orbitrap MS / MS Rapid Identification Method (1) Preparation of test solution: Accurately weigh 0.55g of compound Scutellaria baicalensis throat clearing capsule powder, place it in a volumetric flask, add 55% methanol and sonicate for 35min. After dissolving, make up to volume and mix well. Centrifuge at 4.5℃ and 10000-20000r / min for 12min. Take the supernatant to obtain the test solution. (2) Preparation of reference solutions: Accurately weigh vanillin reference standard, baicalein reference standard, quercetin reference standard, baicalin reference standard, syringaldehyde reference standard, neochlorogenic acid reference standard, harpagoside reference standard, harpagoside reference standard, caffeic acid reference standard, apigenin-7-O-β-D-glucoside reference standard, apigenin reference standard, chlorogenic acid reference standard and baicalin reference standard, respectively, and place them in volumetric flasks. Add 7-8 mL of 55% methanol to each flask, sonicate to dissolve and dilute to volume, to obtain 1.2 mg / mL vanillin reference solution, 1.2 mg / mL baicalein reference solution, and 1.2 mg / mL quercetin reference solution, respectively. The following reference solutions were prepared: 1.2 mg / mL baicalin, 1.2 mg / mL syringaldehyde, 1.2 mg / mL neochlorogenic acid, 1.2 mg / mL harpagoside, 1.2 mg / mL caffeic acid, 1.2 mg / mL apigenin-7-O-β-D-glucoside, 1.2 mg / mL apigenin, 1.2 mg / mL chrysogenin, 1.2 mg / mL chlorogenic acid, and 1.2 mg / mL baicalin. These solutions were stored at -20°C for later use. (3) Preparation of drug-containing serum test solution: Rats were administered the drug-containing serum test solution by gavage at a dose of 180 mg / kg each time, 3 times a day for 5 consecutive days. Blood was collected 2.2 h after the last administration, 1.2 mL of plasma was separated, 5 times the volume of methanol was added to precipitate the protein, and the solution was allowed to stand at -20℃ for 35 min, vortexed for 2.5 min, and centrifuged at 3.5℃ and 10000 r / min for 12 min. The upper serum layer was collected to obtain the supernatant of the drug-containing serum sample. The supernatant of the drug-containing serum sample was dried by nitrogen blowing at 36℃. The residue was reconstituted with 320 μL of 55% methanol, vortexed for 2 min, sonicated for 8 min, and centrifuged again at 3.5℃ and 10000 r / min for 12 min. The supernatant was collected to obtain the drug-containing serum test solution. (4) The test solution and the drug-containing serum test solution were analyzed by UHPLC Q-Exactive Orbitrap MS / MS; UHPLC system chromatographic conditions: Vanquish Horizon, Hypersil Gold column (2.1 × 150 mm, 1.9 μm), column temperature 40℃, injection volume 2 µL, mobile phase A 0.1% formic acid-acetonitrile solution, mobile phase B 0.1% formic acid-water solution, elution gradient 0–7 min, 95%–83% B; 7–13 min, 83–81% B; 13–20 min, 81–77% B; 20–25 min, 77–75% B; 25–30 min, 75–71% B; 30–35 min, 75–10% B; 35–36 min, 10% B; 36–36.5 min, 10%–95% B; 36.5–40 min, 95% B. MS system chromatographic conditions: Q Exactive Plus Orbitrap; scan mode HESI+ & HESI-, cone voltage 3.5kV (+) / 2.5kV (-), desolvent gas temperature 350℃; nebulizer gas pressure 50arb; desolvent gas pressure 10arb; capillary temperature 300℃; scan range m / z 100-1500; MS resolution 120000; MS / MS resolution 15000. (5) Identification of the components of the test solution: The chromatographic peaks of the test solution were analyzed using Compound Discover 3.2 and Xcalibar 4.7 software. The retention time, precise molecular weight and secondary fragment information provided by high-resolution mass spectrometry were compared with the chromatographic peaks of the reference solution and multiple databases. (6) Identification of components in drug-containing serum test solution: Using rat blank serum as a reference, the chromatographic peaks of the drug-containing serum test solution were compared with the chromatographic peak retention time, quasi-molecular ion peaks and characteristic secondary mass spectrometry fragment ions of the corresponding reference solution.

[0027] Example 3: UHPLC Q-Exactive Orbitrap MS / MS Rapid Identification Method (1) Preparation of test solution: Accurately weigh 0.45g of compound Scutellaria baicalensis throat clearing capsule powder, place it in a volumetric flask, add 45% methanol and sonicate for 25min. After dissolving, make up to volume and mix well. Centrifuge at 3.5℃ and 10000r / min for 8min, and take the supernatant to obtain the test solution. (2) Preparation of reference solutions: Accurately weigh vanillin reference, baicalin reference, quercetin reference, baicalin reference, syringaldehyde reference, neochlorogenic acid reference, harpagoside reference, harpagoside reference, caffeic acid reference, apigenin-7-O-β-D-glucoside reference, apigenin reference, chlorogenic acid reference, and baicalin reference, and place them in volumetric flasks respectively. Add 45% methanol and sonicate for 7-8 minutes. The solutions were diluted to a final volume of mL to obtain 0.8 mg / mL vanillin reference solution, 0.8 mg / mL baicalein reference solution, 0.8 mg / mL quercetin reference solution, 0.8 mg / mL baicalin reference solution, 0.8 mg / mL syringaldehyde reference solution, 0.8 mg / mL neochlorogenic acid reference solution, 0.8 mg / mL harpagoside reference solution, 0.8 mg / mL harpagoside reference solution, 0.8 mg / mL caffeic acid reference solution, 0.8 mg / mL apigenin-7-O-β-D-glucoside reference solution, 0.8 mg / mL apigenin reference solution, 0.8 mg / mL juglansine reference solution, 0.8 mg / mL chlorogenic acid reference solution, and 0.8 mg / mL baicalin reference solution. These solutions were stored at -20℃ for later use. (3) Preparation of drug-containing serum test solution: Rats were administered the drug-containing serum test solution by gavage at a dose of 200 mg / kg twice a day for 5 consecutive days. Blood was collected 1.8 h after the last administration, 0.8 mL of plasma was separated, and 3 times the volume of methanol was added to precipitate the protein. The solution was allowed to stand at -20℃ for 25 min, vortexed for 3.5 min, and centrifuged at 4.5℃ and 20000 r / min for 8 min. The upper serum layer was collected to obtain the supernatant of the drug-containing serum sample. The supernatant of the drug-containing serum sample was dried by nitrogen blowing at 38℃. The residue was reconstituted with 280 μL of 45% methanol, vortexed for 4 min, sonicated for 12 min, and centrifuged again at 4.5℃ and 20000 r / min for 8 min. The supernatant was collected to obtain the drug-containing serum test solution. (4) The test solution and the drug-containing serum test solution were analyzed by UHPLC Q-Exactive Orbitrap MS / MS; UHPLC system chromatographic conditions: Vanquish Horizon, Hypersil Gold column (2.1 × 150 mm, 1.9 μm), column temperature 40℃, injection volume 2 µL, mobile phase A 0.1% formic acid-acetonitrile solution, mobile phase B 0.1% formic acid-water solution, elution gradient 0–7 min, 95%–83% B; 7–13 min, 83–81% B; 13–20 min, 81–77% B; 20–25 min, 77–75% B; 25–30 min, 75–71% B; 30–35 min, 75–10% B; 35–36 min, 10% B; 36–36.5 min, 10%–95% B; 36.5–40 min, 95% B. MS system chromatographic conditions: Q Exactive Plus Orbitrap; scan mode HESI+ & HESI-, cone voltage 3.5kV (+) / 2.5kV (-), desolvent gas temperature 350℃; nebulizer gas pressure 50arb; desolvent gas pressure 10arb; capillary temperature 300℃; scan range m / z 100-1500; MS resolution 120000; MS / MS resolution 15000. (5) Identification of the components of the test solution: The chromatographic peaks of the test solution were analyzed using Compound Discover 3.2 and Xcalibar 4.7 software. The retention time, precise molecular weight and secondary fragment information provided by high-resolution mass spectrometry were compared with the chromatographic peaks of the reference solution and multiple databases. (6) Identification of components in drug-containing serum test solution: Using rat blank serum as a reference, the chromatographic peaks of the drug-containing serum test solution were compared with the chromatographic peak retention time, quasi-molecular ion peaks and characteristic secondary mass spectrometry fragment ions of the corresponding reference solution.

[0028] To obtain the solution of this invention and verify its technical effects, the inventors conducted extensive experimental research, some of which are recorded below: 1. Materials 1.1 Instruments UHPLC Q-Exactive Plus Orbitrap MS / MS (Thermo Fisher Scientific), vortex mixer (Model VX-III, Beijing Taji Technology Co., Ltd.), nitrogen evaporator (Model MTN-2800D, Tianjin Outsen Instruments Co., Ltd.), low-temperature high-speed centrifuge (Allegra 30R Centrifuge, Beckman Coulter, Inc., USA), electronic balance (EL204, Mettler Toledo Instruments Shanghai Co., Ltd.), ultrasonic cleaner (CQ250A-TS, Shanghai Yuejin Medical Optical Instruments Factory).

[0029] 1.2 Reagents Vanillin, baicalein, quercetin, and scutellarin were all purchased from the National Institutes for Food and Drug Control (batch numbers were 100491-202203, 111595-201808, 100081-201610, 110752-202217, 110842-202010, 111679-202403, 111871-202408 respectively). Syringaldehyde, neochlorogenic acid, harpagide, harpagoside, and caffeic acid were all purchased from Chengdu Efa Biotechnology Co., Ltd. (batch numbers were AFCJ2454, AB0388, AF20030804, AFBG1391, AFCA2953, AF9051082 respectively). Apigenin-7-O-β-D-glucoside, chrysin, and chlorogenic acid were all purchased from Sichuan Weikeqi Biotechnology Co., Ltd. (batch numbers were WP24060306, WP24090610, WP24012303 respectively). Baicalin was purchased from Shanghai Standard & Dade (batch number: RS01861120); formic acid (mass spectrometry grade, product number: 28905, Thermo Fisher Scientific); acetonitrile (mass spectrometry grade, product number: 047138.K2, Thermo Fisher Scientific); other reagents were all of analytical grade.

[0030] 1.3 Experimental animals Healthy SD rats, with half males and half females, weighing 200 ± 20 g, were provided by Beijing Huafukang Biotechnology Co., Ltd. The feeding conditions were SPF level, and the animal license number was SCXK (Beijing) 2019-0008. After the rats were transported to the laboratory animal room, female rats and male rats were randomly separated and housed in cages of 8 rats each. The breeding room had sufficient light, good air conditioning and ventilation equipment systems, a breeding temperature of 20-25 °C, and a relative humidity of 40-70%.

[0031] 2 Methods 2.1 Solution preparation Test solution: Accurately weigh 0.5g of compound Scutellaria baicalensis throat-clearing capsule powder, place it in a 10mL volumetric flask, add an appropriate amount of 50% methanol, sonicate for 30min, dissolve, make up to volume, mix well, and centrifuge at 4℃ and 14000r / min for 10min to obtain the supernatant of the test solution.

[0032] Reference solution: Accurately weigh appropriate amounts of vanillin, baicalin, quercetin, baicalin, syringaldehyde, neochlorogenic acid, harpagoside, harpagoside, caffeic acid, apigenin-7-O-β-D-glucoside, apigenin, chlorogenic acid, and baicalin reference standards, place them in a 10 mL amber volumetric flask, add a small amount of 50% methanol, sonicate to dissolve, and dilute to volume. Obtain a 1 mg / mL stock solution. Store at -20°C for later use.

[0033] 2.2 Serum Pharmaceutical Chemistry Following the adult clinical usage guidelines for Compound Scutellaria baicalensis and Phlegm-Clearing Capsules, rats were administered a dose of 189 mg / kg / time via gavage, twice daily for 5 consecutive days. Two hours after the last administration, blood was collected, 1 mL of plasma was separated, and 4 times the volume of methanol was added to precipitate the protein. The mixture was allowed to stand at -20°C for 30 min, vortexed for 3 min, and then centrifuged at 4°C and 14000 r / min for 10 min. The supernatant serum was collected as the administered serum sample.

[0034] The supernatant of the drug-treated serum sample was dried by nitrogen blowing at 37℃. The residue was reconstituted with 300 μL of 50% methanol, vortexed for 3 min, and then sonicated for 10 min. It was then centrifuged again at 4℃ and 14000 r / min for 10 min. The supernatant was then injected for analysis.

[0035] 2.3 Instrument Conditions LC system: Vanquish horizon; Column: Hypersil gold (2.1×150mm, 1.9μm); Column temperature: 40℃; Injection volume: 2µL; Mobile phase composition and gradient: A: 0.1% formic acid-acetonitrile solution; B: 0.1% formic acid-aqueous solution; Elution gradient: 0~7min, 95%~83%B; 7~13min, 83~81%B; 13~20min, 81~77%B; 20~25min, 77~75%B; 25~30min, 75~71%B; 30~35min, 75~10%B; 35~36min, 10%B; 36~36.5min, 10%~95%B; 36.5~40min, 95%B.

[0036] MS system: Q Exactive Plus Orbitrap; Scan mode: HESI + & HESI -Cone voltage: 3.5kV (+) / 2.5kV (-); Desolvation gas temperature: 350°C; Nebulizer gas: 50arb; Desolvation gas: 10arb; Capillary: 300°C; Scan range (m / z): 100-1500; Resolution (MS): 120000; Resolution (MS / MS): 15000.

[0037] 3 Results 3.1 Identification of in vitro chemical components of Compound Scutellaria baicalensis throat-clearing capsules The compound Scutellaria baicalensis throat-clearing capsule sample was analyzed using UHPLC Q-Exactive Plus Orbitrap MS / MS technology. Total ion chromatograms were obtained under both positive and negative ion modes. (See attached image.) Figure 1 Further analysis was performed using Compound Discover 3.2 and Xcalibar 4.7 software. Chromatographic peaks in the samples were extracted using pretreatment methods such as peak extraction, peak matching, and background subtraction. Combined with information such as retention time, precise molecular weight, and secondary fragmentation provided by high-resolution mass spectrometry, and comparisons with reference standards and various databases (mzCloud, mzVault, Chemspider, etc.), a total of 21 compounds were identified from Compound Scutellaria baicalensis throat-clearing capsules, including flavonoids, phenolic acids, and triterpenoids. Thirteen of these compounds were confirmed using reference standards: harpagoside, neochlorogenic acid, chlorogenic acid, caffeic acid, syringaldehyde, vanillin, cosmososide, baicalin, baicalein, quercetin, baicalin, harpagoside, and baicalein. Detailed results are shown in Table 1.

[0038] Table 1. Identification and analysis of chemical components in Compound Scutellaria baicalensis throat-clearing capsules Note: "*" indicates comparison with a reference standard.

[0039] 3.2 Identification of migrating components in rat serum Based on the clarified chemical composition of Compound Scutellaria baicalensis and Phlegm-Clearing Capsules, serum samples after drug administration were further analyzed using rat blank serum as a reference. Total ion chromatograms under positive and negative ion modes are shown below. Figure 2 As shown, by comparing the retention time, quasi-molecular ion peak, and characteristic secondary mass spectrometry fragment ions with the corresponding reference standards, nine original blood-entry components of Compound Scutellaria baicalensis throat-clearing capsules were identified in rat serum containing the drug. These included harbagoside (P1), neochlorogenic acid (P2), chlorogenic acid (P3), vanillin (P4), quercetin (P5), baicalin (P6), harbagoside (P7), juglansin (P8), and oleanolic acid (P9). Furthermore, based on high-resolution mass spectrometry data and fragment analysis, a total of 12 metabolites (M1-M12) were deduced; detailed information is shown in Table 2.

[0040] Table 2. Identification of original components and metabolites in serum Two harbazoside (P1) metabolites (M1, M2) were identified, potentially involving methylation and desaturation pathways. Figure 3 M1's t R The wavelength was 4.87 min, and a quasi-molecular ion peak was generated in positive ion mode at m / z 399.12518 [M+Na]. + Based on high-resolution data, software predicts its molecular formula to be C10. 16 H 24 O 10 M1 has 12 more carbon atoms than harbazoside, consistent with the addition of one carbon molecule (C) to harbazoside. Further secondary mass spectrometry analysis confirmed the metabolite structure. The main fragment ions at m / z 201.44064 and 237.07178 are both one carbon molecule more than the main fragment ions at m / z 189.05194 and 225.07353 of harbazoside, suggesting they are ion fragments formed during methylation of harbazoside. Therefore, M1 is presumably a product of methylation of harbazoside. M2's t R The wavelength was 4.86 min, and a quasi-molecular ion peak was generated in negative ion mode at m / z 362.13538 [MH]. - Based on high-resolution data, software predicts its molecular formula to be C10. 15 H 22 O 10 The number of hydrogen molecules (H) in M2 is 2 fewer than that of harbazoside, consistent with the reduction of two hydrogen molecules (H) in harbazoside. Further secondary mass spectrometry analysis confirmed the metabolite structure. The main fragment ions (m / z 137.02350, 163.03931, 181.04967) are two fewer than the main fragment ions (m / z 139.03894, 165.05467, 183.06548) of harbazoside. These are presumed to be ion fragments formed from the dehydrogenation of harbazoside. Therefore, M2 is inferred to be a product obtained from the desaturation of harbazoside.

[0041] Two chlorogenic acid (P3) / neochlorogenic acid (P2) metabolites (M3, M4) were identified, possibly involving hydration and oxidation pathways. Figure 4 M3 metabolite t R The wavelength was 2.31 min, and a quasi-molecular ion peak was generated in negative ion mode at m / z 371.09851 [MH]. - Based on high-resolution data, software predicts its molecular formula to be C10. 16 H 20 O 10The amount of chlorogenic acid (M3) is 18 more than that of neochlorogenic acid, consistent with the fact that chlorogenic acid / neochlorogenic acid contains one more water molecule (H2O). Further secondary mass spectrometry analysis confirmed the metabolite structure. The main fragment ions (m / z 135.044070, 151.07555, 219.06604) all contain one more water molecule (H2O) than the main fragment ions (m / z 117.03345, 133.02821, 201.24734) of chlorogenic acid / neochlorogenic acid. This is presumed to be ionic fragments formed after the hydration of chlorogenic acid / neochlorogenic acid. Therefore, it is inferred that M3 is a product of the hydration of chlorogenic acid / neochlorogenic acid; metabolite t of M4... R The wavelength is 8.08 min, and a quasi-molecular ion peak is generated in positive ion mode at m / z 388.12350 [M+NH4]. + Based on high-resolution data, software predicts its molecular formula to be C10. 16 H 18 O 10 The number of fragment ions is 16 more than that of chlorogenic acid / neochlorogenic acid, consistent with the presence of one oxygen molecule (O) in chlorogenic acid / neochlorogenic acid. Further secondary mass spectrometry analysis confirmed the metabolite structure. The main fragment ions (m / z 133.08586, 162.65863, 371.22696) all contain one more oxygen molecule (O) than the main fragment ions (m / z 117.03365, 146.03171, 355.19980) of chlorogenic acid / neochlorogenic acid. It is speculated that these are ionic fragments formed from the oxidation of chlorogenic acid / neochlorogenic acid. Therefore, it is presumed that M4 is a product of the oxidation of chlorogenic acid / neochlorogenic acid.

[0042] Two vanillin (P4) metabolites (M5, M6) were identified, potentially involving methylation and dehydration pathways. Figure 5 M5 metabolite t R The wavelength was 3.52 min, and a quasi-molecular ion peak was generated in positive ion mode at m / z 167.07013 [M+H]. + Based on high-resolution data analysis software, its molecular formula is predicted to be C9H. 10 O3, 14 more than vanillin, is consistent with the addition of one methyl molecule (CH2) to vanillin. Further secondary mass spectrometry analysis confirmed the metabolite structure. The main fragment ions (m / z 121.05693, 125.05987, 147.83771) all contain one more methyl molecule (CH2) than the main fragment ions (m / z 107.04936, 111.04429, 133.97757) of vanillin. This is presumed to be ion fragments formed during the methylation of vanillin. Therefore, M5 is presumed to be a product of vanillin methylation. Metabolite M6 t R The wavelength was 8.89 min, and a quasi-molecular ion peak was generated in positive ion mode at m / z 135.04396 [M+H]. +According to high-resolution data analysis software, its molecular formula is predicted to be C8H6O2, which is 18 less than that of vanillin, consistent with vanillin having one less water molecule (H2O). Further secondary mass spectrometry analysis confirmed the metabolite structure. The main fragment ions (m / z 93.07035, 95.04953, 108.0444) are all one less water molecule (H2O) than the main fragment ions (m / z 111.04429, 113.04892, 126.06313) of vanillin. It is speculated that these are ion fragments formed by the dehydration reaction of vanillin. Therefore, it is presumed that M6 is a product obtained from the dehydration of vanillin.

[0043] Two baicalin (P6) metabolites (M7, M8) were identified, potentially involving methylation and hydration pathways. Figure 6 M7 metabolite t R The wavelength was 23.47 min, and a quasi-molecular ion peak was generated in positive ion mode at m / z 461.10626 [M+H]. + Based on high-resolution data, software predicts its molecular formula to be C10. 22 H 20 O 11 The amount of methyl groups is 14 more than that of baicalin, consistent with the addition of a methyl group (CH2) to baicalin. Further secondary mass spectrometry analysis confirmed the metabolite structure. The main fragment ions (m / z 284.03168, 285.07507, 286.07837) all contain one more methyl group (CH2) than the main fragment ions (m / z 270.05118, 271.05939, 272.06400) of baicalin. It is speculated that M7 is an ion fragment formed by the addition of a methyl group to baicalin. Therefore, it is inferred that M7 is a product obtained from the methylation reaction of baicalin; metabolite M8 t... R The wavelength was 11.32 min, and a quasi-molecular ion peak was generated in negative ion mode at m / z 477.10422 [MH]. - Based on high-resolution data, software predicts its molecular formula to be C10. 22 H 22 O 12 The concentration of 32 ions is higher than that of baicalin, consistent with the presence of one additional water molecule (H2O) and one additional methyl molecule (CH2) in baicalin. Further secondary mass spectrometry analysis confirmed the metabolite structure. The main fragment ions (m / z 117.01811, 165.01843, 301.07178) are all higher than the main fragment ions of baicalin (m / z 85.02820, 133.02841, 269.04556) by one additional water molecule (H2O) and one additional methyl molecule (CH2). It is speculated that M8 is an ion fragment formed by the addition of one water molecule and one methyl molecule to baicalin. Therefore, it is inferred that M8 is a product obtained from the hydration and methylation of baicalin.

[0044] Two harpagosine (P7) metabolites (M9, M10) were identified, which may involve oxidation and desaturation methylation pathways. Figure 7 M9 metabolite t R The wavelength was 11.24 min, and a quasi-molecular ion peak was generated in positive ion mode at m / z 549.15918 [M+Na]. + Based on high-resolution data, software predicts its molecular formula to be C10. 24 H 30 O 13 The amount of ions with m / z 32 more than that of harpagosine is consistent with the fact that harpagosine contains two more oxygen molecules (O). Further secondary mass spectrometry analysis confirmed the metabolite structure. The main fragment ions at m / z 216.03951, 251.07114, and 281.07990 all contain two more oxygen molecules (O) than the main fragment ions of harpagosine at m / z 184.07315, 219.03896, and 249.04936. These are presumed to be ion fragments formed from the oxidation of harpagosine. Therefore, it is speculated that M9 is a product of the oxidation of harpagosine; metabolite t of M10... R The wavelength was 11.56 min, and a quasi-molecular ion peak was generated in negative ion mode at m / z 549.16217 [M+FA-H]. - Based on high-resolution data, software predicts its molecular formula to be C10. 25 H 28 O 11 The number of fragment ions is 10 more than that of harpagosine, consistent with the fact that harpagosine has two fewer hydrogen (H) molecules and one more carbon (C) molecule. Further secondary mass spectrometry analysis confirmed the metabolite structure. The main fragment ions were m / z 109.02821, 149.02365, and 211.07433, all of which have two fewer hydrogen (H) molecules and one more carbon (C) molecule than the main fragment ions of harpagosine (m / z 99.00745, 139.03896, and 201.07693). These are presumed to be ion fragments formed by the desaturation and methylation of harpagosine. Therefore, it is inferred that M10 is a product obtained from the desaturation and methylation of harpagosine.

[0045] Three salicylic acid (P8) metabolites (M11, M12) were identified, potentially involving oxidation and methylation pathways. Figure 8 M11 metabolite t R The wavelength was 15.39 min, and a quasi-molecular ion peak was generated in positive ion mode at m / z 271.05923 [M+H]. + Based on high-resolution data, software predicts its molecular formula to be C10. 15 H 10O5, 16 more than apigenin, consistent with the addition of one oxygen molecule (O) to apigenin. Further secondary mass spectrometry analysis confirmed the metabolite structure. The main fragment ions (m / z 229.04875, 247.86945, 270.05151) all contained one more oxygen molecule (O) than the main fragment ions (m / z 213.05470, 231.06544, 254.63176) of apigenin. These are presumed to be ionic fragments formed from the oxidation of apigenin. Therefore, M11 is presumed to be an oxidation product of apigenin; metabolite t of M12... R The wavelength is 23.47 min, and a quasi-molecular ion peak is generated in positive ion mode at m / z 285.07486 [M+H]. + Based on high-resolution data, software predicts its molecular formula to be C10. 16 H 12 O5, with 30 more ions than guarnin, is consistent with the fact that guarnin contains one molecule of oxygen (O) and one molecule of methyl (CH2). Further mass spectrometry analysis confirmed the metabolite structure. The main fragment ions (m / z 155.04906, 243.05969, 284.03104) all contain one more molecule of oxygen (O) and one more molecule of methyl (CH2) than the main fragment ions (m / z 125.02337, 213.05470, 254.63176) of guarnin. It is speculated that these are ion fragments formed by further methylation reactions following the oxidation of guarnin. Therefore, M12 is presumably a methylation product of guarnin.

[0046] 4. Summary Traditional Chinese medicine (TCM) compound formulas, based on TCM theory, are composed of multiple medicinal herbs and are characterized by reduced toxicity and enhanced efficacy. The material basis for their efficacy is highly complex, essentially resulting from the interaction between the various chemical components contained in the formula and the human body. Traditional research often focuses on the main components with higher content, but these main components do not necessarily represent the key active ingredients. Modern pharmacological theory emphasizes that the fundamental prerequisite for the therapeutic effect of TCM compound formulas is the effective absorption of their active ingredients into the bloodstream, which constitutes a prerequisite for exploring their pharmacodynamic material basis. These active ingredients come from diverse sources; they may be directly absorbed into the bloodstream from the original compounds in the medicinal materials, or they may be secondary products formed after the original components undergo metabolism, transformation, or recombination in the body. Therefore, to deeply explore the pharmacodynamic material basis of TCM compound formulas, a comprehensive analysis of their in vitro and blood-borne components is necessary. Based on this, this study not only focuses on the original components but also deeply analyzes their in vivo biotransformation products, identifying a total of 12 metabolites. The study of metabolites is an indispensable part of elucidating the material basis of the efficacy of traditional Chinese medicine, because many components need to be activated by metabolism to exert their medicinal effects, or the metabolites themselves have unique activities. The original components and their metabolites together form a synergistic therapeutic network, which can, to a certain extent, explain the traditional medicine wisdom of "reducing toxicity and increasing efficacy" of Compound Scutellaria Phlegm-Clearing Capsules.

[0047] 5. Conclusion This invention utilizes UHPLC-Q-Exactive Orbitrap MS / MS technology to efficiently and accurately resolve the complex chemical composition of Compound Scutellaria baicalensis and its migrating components in rat serum. Twenty-one in vitro components (including flavonoids, phenolic acids, and triterpenoids) and 21 blood-migrating components (including nine blood-entering original components and 12 metabolites) were identified. The blood-entering components and metabolites discovered in this study may be related to the drug's efficacy, providing a scientific reference for its pharmacodynamic material basis, quality control research, and rational clinical use. This further confirms the application value of serum-based medicinal chemistry research strategies combined with high-resolution mass spectrometry in elucidating the in vivo and in vitro material basis of traditional Chinese medicine compound formulas.

Claims

1. A rapid UHPLC Q-Exactive Orbitrap MS / MS method for the identification of chemical components and serum-transmitted components of Compound Scutellaria baicalensis throat-clearing capsules, characterized in that: Compound Scutellaria baicalensis throat-clearing capsules, by weight, are mainly composed of 25-30 parts of Scutellaria baicalensis, 15-20 parts of Scrophularia ningpoensis, 10-15 parts of Terminalia chebula, and 12-20 parts of Platycodon grandiflorus. The preparation method is as follows: Scutellaria baicalensis, Scrophularia ningpoensis, Terminalia chebula, and Platycodon grandiflorus are decocted with water three times. For the first decoction, 8-10 times the amount of water is added, and the extraction time is 1.5-2 hours. For the second decoction, 6-8 times the amount of water is added, and the extraction time is 1-1.5 hours. For the third decoction, 4-6 times the amount of water is added, and the extraction time is 1-1.5 hours. The extract is concentrated under reduced pressure to a relative density of 1.12-1.14 at 80℃, spray-dried, microcrystalline cellulose is added, mixed evenly, and then encapsulated. The specification is 0.3g / capsule. The UHPLC Q-Exactive Orbitrap MS / MS rapid identification method includes the following steps: (1) Preparation of test solution: Accurately weigh the powder of compound Scutellaria baicalensis throat clearing capsule, place it in a volumetric flask, add 45-55% methanol and sonicate for 25-35 min to dissolve, make up to volume and mix well, centrifuge at 3.5-4.5℃ and 10000-20000r / min for 8-12 min, take the supernatant to obtain the test solution; (2) Preparation of reference solutions: Accurately weigh vanillin reference standard, baicalein reference standard, quercetin reference standard, baicalin reference standard, syringaldehyde reference standard, neochlorogenic acid reference standard, harpagoside reference standard, harpagoside reference standard, caffeic acid reference standard, apigenin-7-O-β-D-glucoside reference standard, apigenin reference standard, chlorogenic acid reference standard, and baicalin reference standard, respectively, and place them in volumetric flasks. Add 45-55% methanol, sonicate to dissolve, and dilute to volume to obtain 0.8-1.2 mg / mL vanillin reference solution, 0.8-1.2 mg / mL baicalein reference solution, 0.8-1.2 mg / mL quercetin reference solution, and 0.8-1.2 mg / mL vanillin reference solution, respectively. The following reference solutions were prepared: 0.8-1.2 mg / mL baicalin, 0.8-1.2 mg / mL syringaldehyde, 0.8-1.2 mg / mL neochlorogenic acid, 0.8-1.2 mg / mL harpagoside, 0.8-1.2 mg / mL harpagoside, 0.8-1.2 mg / mL caffeic acid, 0.8-1.2 mg / mL apigenin-7-O-β-D-glucoside, 0.8-1.2 mg / mL apigenin, 0.8-1.2 mg / mL jasmin, 0.8-1.2 mg / mL chlorogenic acid, and 0.8-1.2 mg / mL baicalin. These solutions were stored at -20°C for later use. (3) Preparation of drug-containing serum test solution: Rats were administered the test solution by gavage at a dose of 180-200 mg / kg each time, 2-3 times daily for 5 consecutive days. Blood was collected 1.8-2.2 h after the last administration, and 0.8-1.2 mL of plasma was separated. 3-5 times the volume of methanol was added to precipitate the protein. The mixture was allowed to stand at -20℃ for 25-35 min, then vortexed for 2.5-3.5 min. The mixture was then stirred at 3.5-4.5℃ and 10000-20000 r / min. Centrifuge for 8-12 minutes at 3.5-4.5℃ and 10000-20000 r / min, and collect the supernatant of the drug-treated serum sample. Take the supernatant of the drug-treated serum sample and blow it dry with nitrogen at 36-38℃. Redissolve the residue with 280-320 μL of 45-55% methanol, vortex for 2-4 minutes, and sonicate for 8-12 minutes. Centrifuge again at 3.5-4.5℃ and 10000-20000 r / min for 8-12 minutes, and collect the supernatant to obtain the drug-containing serum test solution. (4) The test solution and the drug-containing serum test solution were analyzed by UHPLC Q-Exactive Orbitrap MS / MS; UHPLC system chromatographic conditions: Vanquish Horizon, Hypersil Gold column (2.1 × 150 mm, 1.9 μm), column temperature 40℃, injection volume 2 µL, mobile phase A 0.1% formic acid-acetonitrile solution, mobile phase B 0.1% formic acid-water solution, elution gradient 0–7 min, 95%–83% B; 7–13 min, 83–81% B; 13–20 min, 81–77% B; 20–25 min, 77–75% B; 25–30 min, 75–71% B; 30–35 min, 75–10% B; 35–36 min, 10% B; 36–36.5 min, 10%–95% B; 36.5–40 min, 95% B. MS system chromatographic conditions: Q Exactive Plus Orbitrap; scan mode HESI+ & HESI-, cone voltage 3.5kV (+) / 2.5kV (-), desolvent gas temperature 350℃; nebulizer gas pressure 50arb; desolvent gas pressure 10arb; capillary temperature 300℃; scan range m / z 100-1500; MS resolution 120000; MS / MS resolution 15000. (5) Identification of the components of the test solution: The chromatographic peaks of the test solution were analyzed using Compound Discover 3.2 and Xcalibar 4.7 software. Combined with the retention time, precise molecular weight and secondary fragment information provided by high resolution mass spectrometry, and by comparing with the chromatographic peaks of the reference solution and multiple databases, a total of 13 compounds were finally identified from Compound Scutellaria baicalensis throat clearing capsules. (6) Identification of components in drug-containing serum test solution: Using rat blank serum as a reference, the retention time, quasi-molecular ion peak and characteristic secondary mass spectrometry fragment ions of the chromatographic peak of the drug-containing serum test solution were compared with those of the corresponding reference solution. A total of 9 original blood-entry components of Compound Scutellaria baicalensis throat-clearing capsule and 12 metabolites were identified in rat drug-containing serum.

2. The rapid identification method of UHPLC Q-Exactive Orbitrap MS / MS according to claim 1, characterized in that: In step (1), the preparation of the test solution is as follows: accurately weigh 0.45-0.55g of compound Scutellaria baicalensis throat-clearing capsule powder, place it in a volumetric flask, add 6-8.5mL of 48-52% methanol, sonicate for 28-32min, dissolve, make up to volume, mix well, centrifuge at 3.8-4.2℃ and 12000-18000r / min for 9-11min, take the supernatant, and the test solution is obtained.

3. The UHPLC Q-Exactive Orbitrap MS / MS rapid identification method according to claim 1 or 2, characterized in that: In step (1), the preparation of the test solution is as follows: accurately weigh 0.5g of compound Scutellaria baicalensis throat-clearing capsule powder, place it in a 10mL volumetric flask, add 7-8mL of 50% methanol, sonicate for 30min, dissolve, make up to volume, mix well, centrifuge at 4℃ and 14000r / min for 10min, take the supernatant, and the test solution is obtained.

4. The rapid identification method of UHPLC Q-Exactive Orbitrap MS / MS according to claim 1, characterized in that: In step (2), the preparation of the reference solutions is as follows: Vanillin reference standard, baicalein reference standard, quercetin reference standard, baicalin reference standard, syringaldehyde reference standard, neochlorogenic acid reference standard, harpagoside reference standard, harpagoside reference standard, caffeic acid reference standard, apigenin-7-O-β-D-glucoside reference standard, apigenin reference standard, chlorogenic acid reference standard, and baicalin reference standard are accurately weighed and placed in volumetric flasks respectively. 6-8.5 mL of 48-52% methanol is added to each flask, and the solutions are dissolved by sonication and diluted to volume to obtain 0.9-1.1 mg / mL vanillin reference solution, 0.9-1.1 mg / mL baicalein reference solution, 0.9-1.1 mg / mL quercetin reference solution, and 0... 0.9-1.1 mg / mL baicalin reference solution, 0.9-1.1 mg / mL syringaldehyde reference solution, 0.9-1.1 mg / mL neochlorogenic acid reference solution, 0.9-1.1 mg / mL harpagoside reference solution, 0.9-1.1 mg / mL harpagoside reference solution, 0.9-1.1 mg / mL caffeic acid reference solution, 0.9-1.1 mg / mL apigenin-7-O-β-D-glucoside reference solution, 0.9-1.1 mg / mL apigenin reference solution, 0.9-1.1 mg / mL apigenin reference solution, 0.9-1.1 mg / mL chlorogenic acid reference solution, and 0.9-1.1 mg / mL baicalin reference solution should be stored at -20℃ for later use.

5. The UHPLC Q-Exactive Orbitrap MS / MS rapid identification method according to claim 1 or 4, characterized in that: In step (2), the preparation of the reference solutions is as follows: Accurately weigh vanillin reference standard, baicalin reference standard, quercetin reference standard, baicalin reference standard, syringaldehyde reference standard, neochlorogenic acid reference standard, harpagoside reference standard, harpagoside reference standard, caffeic acid reference standard, apigenin-7-O-β-D-glucoside reference standard, apigenin reference standard, chlorogenic acid reference standard, and baicalin reference standard, and place them in 10 mL volumetric flasks respectively. Add 7-8 mL of 50% methanol to each flask. Dissolved by ultrasonication in mL and diluted to volume, 1 mg / mL vanillin reference solution, 1 mg / mL baicalein reference solution, 1 mg / mL quercetin reference solution, 1 mg / mL baicalin reference solution, 1 mg / mL syringaldehyde reference solution, 1 mg / mL neochlorogenic acid reference solution, 1 mg / mL harpagoside reference solution, 1 mg / mL harpagoside reference solution, 1 mg / mL caffeic acid reference solution, 1 mg / mL apigenin-7-O-β-D-glucoside reference solution, 1 mg / mL apigenin reference solution, 1 mg / mL juglans regia reference solution, 1 mg / mL chlorogenic acid reference solution and 1 mg / mL baicalin reference solution were obtained, and stored at -20℃ for later use.

6. The rapid identification method of UHPLC Q-Exactive Orbitrap MS / MS according to claim 1, characterized in that: In step (3), the preparation of the drug-containing serum test solution is as follows: Rats are administered the drug-containing serum test solution by gavage at a dose of 185-195 mg / kg twice daily for 5 consecutive days. Blood is collected 1.9-2.1 h after the last administration. 0.9-1.1 mL of plasma is separated, and 3.5-4.5 times the volume of methanol is added to precipitate the protein. The mixture is allowed to stand at -20℃ for 28-32 min, then vortexed for 2.8-3.2 min at 3.8-4.2℃ and 12000-18000 r / min. Centrifuge for 9-11 min at 36.5-37.5℃, collect the upper serum layer to obtain the supernatant of the drug-treated serum sample; take the supernatant of the drug-treated serum sample and blow it dry with nitrogen at 36.5-37.5℃, reconstitute the residue with 290-310 μL of 48-52% methanol, vortex for 2.5-3.5 min, sonicate for 9-11 min, and centrifuge again at 3.8-4.2℃ and 12000-18000 r / min for 9-11 min, collect the supernatant to obtain the drug-containing serum test solution.

7. The UHPLC Q-Exactive Orbitrap MS / MS rapid identification method according to claim 1 or 6, characterized in that: In step (3), the preparation of the drug-containing serum test solution is as follows: rats are given a dose of 189 mg / kg by gavage twice a day for 5 consecutive days. Blood is collected 2 hours after the last administration, 1 mL of plasma is separated, 4 times the volume of methanol is added to precipitate the protein, and the mixture is allowed to stand at -20℃ for 30 min, vortexed for 3 min, and centrifuged at 4℃ and 14000 r / min for 10 min. The upper serum layer is collected to obtain the supernatant of the drug-treated serum sample. The supernatant of the drug-treated serum sample is dried by nitrogen blowing at 37℃. The residue is reconstituted with 300 μL of 50% methanol, vortexed for 3 min, sonicated for 10 min, and centrifuged again at 4℃ and 14000 r / min for 10 min. The supernatant is collected to obtain the drug-containing serum test solution.

8. The rapid identification method of UHPLC Q-Exactive Orbitrap MS / MS according to claim 1, characterized in that: In step (5), the 13 compounds are harpagoside, neochlorogenic acid, chlorogenic acid, caffeic acid, syringaldehyde, vanillin, cosmos glycoside, baicalin, baicalein, quercetin, baicalin, harpagoside and succinic acid.

9. The rapid identification method of UHPLC Q-Exactive Orbitrap MS / MS according to claim 1, characterized in that: In step (5), the multiple databases include mzCloud, mzVault, and Chemspider.

10. The UHPLC Q-Exactive Orbitrap MS / MS rapid identification method according to claim 1, characterized in that: In step (6), the original blood-entry components of the nine compound Scutellaria baicalensis throat-clearing capsules are harbacoside, neochlorogenic acid, chlorogenic acid, vanillin, quercetin, baicalin, harbacoside, salicylic acid and oleanolic acid.

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