Method for determining contents of multiple components in compound radix scutellariae throat-clearing capsule based on UHPLC-MS / MS (ultra-high performance liquid chromatography-mass spectrometry / mass spectrometry)

Abstract

The invention discloses a UHPLC-MS / MS (Ultra High Performance Liquid Chromatography-Mass Spectrometry / Mass Spectrometry)-based multi-component content determination method for a compound radix scutellariae throat-clearing capsule, which is characterized in that a multi-component simultaneous quantitative analysis method is established by adopting a UHPLC-MS / MS technology, and the quality of the compound radix scutellariae throat-clearing capsule is evaluated from the quantitative aspect. 21 components including p-hydroxybenzaldehyde, vanillin, caffeic acid, syringaldehyde, citric acid, tanshinol, chrysin, baicalein, genistein, quercetin, chlorogenic acid, neochlorogenic acid, rosmarinic acid, harpagide, puerarin, apigenin-7-O-beta-D-glucoside, baicalin, oleanolic acid, scutellarin, glycyrrhetinic acid and harpagoside are selected as quantitative indexes; and determining the content of the compound radix scutellariae throat-clearing capsule. According to the method, the contents of 21 components in the compound radix scutellariae throat-clearing capsule can be accurately measured at the same time within 14 min, and the method has good precision and stability.

Description

Technical Field

[0001] This invention belongs to the field of compound traditional Chinese medicine component detection, specifically involving a method for determining the content of multiple components in compound Scutellaria baicalensis throat-clearing capsules based on UHPLC-MS / MS. 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 definite, but there is no method for determining the content of Compound Scutellaria baicalensis throat-clearing capsules.

[0003] Ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry (UHPLC-MS / MS) is a precision analytical instrument that combines ultra-high performance liquid chromatography with triple quadrupole mass spectrometry. It possesses high sensitivity and efficient separation capabilities, making it particularly suitable for the rapid screening and identification of trace and ultra-low-level compounds and the study of metabolites in complex systems of traditional Chinese medicine compound preparations. This technology provides a powerful tool for in-depth research into the in vivo and in vitro chemical composition of traditional Chinese medicine compound preparations and their dynamic changes.

[0004] The applicant previously conducted a study on the migration analysis of its chemical components in serum. Based on the component identification results, combined with the content and pharmacological activity of each component, a method for determining the content of Compound Scutellaria baicalensis and Phlegm-Clearing Capsules was further developed. A multi-component simultaneous quantitative analysis method was established using UHPLC-MS / MS technology to evaluate its quality quantitatively. This method is applicable to quality control research of Compound Scutellaria baicalensis and Phlegm-Clearing Capsules. The establishment of this method can provide experimental basis for improving and enhancing the quality standards of Compound Scutellaria baicalensis and Phlegm-Clearing Capsules, and has important guiding significance for the comprehensive quality control of this preparation. Summary of the Invention

[0005] This invention aims to provide a method for determining the content of multiple components in Compound Scutellaria baicalensis throat-clearing capsules based on UHPLC-MS / MS. A method for simultaneous quantitative analysis of multiple components was established using UHPLC-MS / MS technology to evaluate the quality from a quantitative perspective. This method can accurately determine the content of 21 components in Compound Scutellaria baicalensis throat-clearing capsules simultaneously within 14 minutes, and has good precision and stability.

[0006] The objective of this invention is achieved through the following technical solution: A method for determining the content of multiple components in Compound Scutellaria baicalensis Throat-Clearing Capsules based on UHPLC-MS / MS is disclosed. The 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 involves decocting the Scutellaria baicalensis, Scrophularia ningpoensis, Terminalia chebula, and Platycodon grandiflorus with water three times. For the first extraction, 8-10 times the amount of water is added, and extraction lasts 1.5-2 hours. For the second extraction, 6-8 times the amount of water is added, and extraction lasts 1-1.5 hours. For the third extraction, 4-6 times the amount of water is added, and extraction lasts 1-1.5 hours. The extract is concentrated under reduced pressure to a relative density of 1.12-1.14 at 80℃, spray-dried, and then microcrystalline cellulose is added. After thorough mixing, the mixture is encapsulated, with a specification of 0.3g / capsule. The method for determining the content of multiple components in Compound Scutellaria baicalensis Throat-Clearing Capsules includes the following steps: (1) Preparation of test solution: Remove the capsule shell of Compound Scutellaria baicalensis throat clearing capsule, weigh 0.09-0.11g of the powder inside the Compound Scutellaria baicalensis throat clearing capsule into a 100mL volumetric flask, add 50% methanol, sonicate for 25-35min, cool, add 50% methanol to make up to the mark, shake well, centrifuge at 13000-15000r / min for 8-12min at 3-5℃, separate the supernatant, filter membrane, take the filtrate to obtain the test solution, and use it for later use; (2) Preparation of single reference solutions: Accurately weigh p-hydroxybenzaldehyde reference, vanillin reference, caffeic acid reference, eugenol reference, citric acid reference, tanshinone reference, salicylic acid reference, baicalin reference, genistein reference, quercetin reference, chlorogenic acid reference, neochlorogenic acid reference, rosmarinic acid reference, harpagoside reference, puerarin reference, apigenin-7-O-β-D-glucoside reference, baicalin reference, oleanolic acid reference, baicalin reference, glycyrrhetinic acid reference, and harpagoside reference, dissolve them in methanol and make up to volume to prepare 21 single reference solutions with a concentration of 0.90-1.10 mg / mL for later use; (3) Preparation of mixed reference solution 1: Accurately measure an appropriate amount of the 21 single reference solutions in step (2), place them in a volumetric flask, and dilute with 50% methanol solution to prepare a solution containing 0.90-1.10 ng / mL p-hydroxybenzaldehyde reference, 1.90-2.10 ng / mL vanillin reference, 15.00-17.00 ng / mL caffeic acid reference, 0.90-1.10 ng / mL syringaldehyde reference, 6000.00-6500.00 ng / mL citric acid reference, 29.00-32.00 ng / mL tanshinone reference, 1.90-2.10 ng / mL salinomycin reference, 370.00-380.00 ng / mL baicalein reference, 0.90-1.10 ng / mL genistein reference, and 3.50-4.50 ng / mL quercetin. Reference standards: 7.50-8.50 ng / mL chlorogenic acid, 7.50-8.50 ng / mL neochlorogenic acid, 95.00-105.00 ng / mL rosmarinic acid, 195.00-205.00 ng / mL harbazoside, 0.23-0.26 ng / mL puerarin, 0.23-0.26 ng / mL apigenin-7-O-β-D. - A mixed reference solution 1 containing glucoside reference standard, 3800.00-4200.00 ng / mL baicalin reference standard, 7.50-8.50 ng / mL oleanolic acid reference standard, 120.00-130.00 ng / mL baicalin reference standard, 0.90-1.10 ng / mL glycyrrhetinic acid reference standard and 55.00-65.00 ng / mL harpagoside reference standard, for use; (4) Preparation of mixed reference solution 2: Accurately measure an appropriate amount of the 21 single reference solutions in step (2), place them in a volumetric flask, and dilute with 50% methanol solution to prepare a solution containing 1.80-2.20 ng / mL p-hydroxybenzaldehyde reference, 1.80-2.20 ng / mL vanillin reference, 30.00-34.00 ng / mL caffeic acid reference, 1.80-2.20 ng / mL syringaldehyde reference, 12000.00-13000.00 ng / mL citric acid reference, 58.00-64.00 ng / mL tanshinone reference, 3.80-4.20 ng / mL salinomycin reference, 740.00-760.00 ng / mL baicalein reference, 1.80-2.20 ng / mL genistein reference, and 7.00-9.00 ng / mL quercetin reference. 15.00-17.00 ng / mL chlorogenic acid reference standard, 15.0-17.00 ng / mL neochlorogenic acid reference standard, 190.00-210.00 ng / mL rosmarinic acid reference standard, 390.00-410.00 ng / mL harbazoside reference standard, 0.46-0.54 ng / mL puerarin reference standard, 0.46-0.54 ng / mL apigenin-7-O-β-D- Two mixed reference solutions of glucoside, baicalin (7600.00-8400.00 ng / mL), oleanolic acid (15.00-17.00 ng / mL), baicalin (240.00-260.00 ng / mL), glycyrrhetinic acid (1.80-2.20 ng / mL), and harpagoside (110.00-130.00 ng / mL) are prepared for use. (5) Preparation of mixed reference solution 3: Accurately measure an appropriate amount of the 21 single reference solutions from step (2), place them in a volumetric flask, and dilute with 50% methanol solution to prepare a solution containing 3.60-4.40 ng / mL p-hydroxybenzaldehyde reference, 3.60-4.40 ng / mL vanillin reference, 60.00-68.00 ng / mL caffeic acid reference, and 3.60-4.40 ng / mL syringaldehyde reference. Citric acid reference standard (24000.00-26000.00 ng / mL), tanshinone reference standard (116.00-128.00 ng / mL), salinomycin reference standard (7.60-8.40 ng / mL), baicalin reference standard (1480.00-1520.00 ng / mL), genistein reference standard (3.60-4.40 ng / mL), quercetin reference standard (15.00-18.00 ng / mL). 30.00-34.00 ng / mL chlorogenic acid reference standard, 30.00-34.00 ng / mL neochlorogenic acid reference standard, 380.00-420.00 ng / mL rosmarinic acid reference standard, 780.00-820.00 ng / mL harbazoside reference standard, 0.92-1.08 ng / mL puerarin reference standard, 0.92-1.08 ng / mL apigenin-7-O-β-D- 3 mixed reference solutions of glucoside reference standard, 15200.00-16800.00 ng / mL baicalin reference standard, 30.00-34.00 ng / mL oleanolic acid reference standard, 480.00-520.00 ng / mL baicalin reference standard, 3.60-4.40 ng / mL glycyrrhetinic acid reference standard and 220.00-260.00 ng / mL harpagoside reference standard, are prepared for use. (6) Preparation of mixed reference solution 4: Accurately measure an appropriate amount of the 21 single reference solutions from step (2), place them in a volumetric flask, and dilute with 50% methanol solution to prepare a solution containing 7.20-8.80 ng / mL p-hydroxybenzaldehyde reference, 7.20-8.80 ng / mL vanillin reference, 120.00-136.00 ng / mL caffeic acid reference, and 7.20-8.80 ng / mL syringaldehyde reference. Citric acid reference standard (48000.00-52000.00 ng / mL), tanshinone reference standard (235.00-256.00 ng / mL), salinomycin reference standard (15.20-16.80 ng / mL), baicalin reference standard (2960.00-3040.00 ng / mL), genistein reference standard (7.20-8.80 ng / mL), quercetin reference standard (30.00-36.00 ng / mL). Standard, 60.00-68.00 ng / mL chlorogenic acid reference standard, 60.00-68.00 ng / mL neochlorogenic acid reference standard, 760.00-840.00 ng / mL rosmarinic acid reference standard, 1560.00-1640.00 ng / mL harbazoside reference standard, 1.84-2.16 ng / mL puerarin reference standard, 1.84-2.16 ng / mL apigenin-7-O-β-D - A mixed reference solution 4 containing glucoside reference standard, 30400.00-33600.00 ng / mL baicalin reference standard, 60.00-68.00 ng / mL oleanolic acid reference standard, 960.00-1040.00 ng / mL baicalin reference standard, 7.20-8.80 ng / mL glycyrrhetinic acid reference standard and 440.00-520.00 ng / mL harpagoside reference standard, for use; (7) Preparation of mixed reference solution 5: Accurately measure an appropriate amount of the 21 single reference solutions from step (2), place them in a volumetric flask, and dilute with 50% methanol solution to prepare a solution containing 14.40-17.60 ng / mL p-hydroxybenzaldehyde reference, 14.40-17.60 ng / mL vanillin reference, 240.0-272.0 ng / mL caffeic acid reference, and 14.40-17.60 ng / mL syringaldehyde reference. 96000.00-104000.00 ng / mL citric acid reference standard, 470.00-512.00 ng / mL tanshinone reference standard, 30.40-33.60 ng / mL apigenin reference standard, 5920.00-6080.00 ng / mL baicalein reference standard, 14.40-17.60 ng / mL genistein reference standard, 60.00-72.00 ng / mL quercetin reference standard, 1 20.00-136.00 ng / mL chlorogenic acid reference standard, 120.00-136.00 ng / mL neochlorogenic acid reference standard, 1520.00-1680.00 ng / mL rosmarinic acid reference standard, 3120.00-3280.00 ng / mL harbazoside reference standard, 3.68-4.32 ng / mL puerarin reference standard, 3.68-4.32 ng / mL apigenin-7-O-β-D-glucan 5 mixed reference solutions of glucoside reference standard, 60800.00-67200.00 ng / mL baicalin reference standard, 120.00-136.00 ng / mL oleanolic acid reference standard, 1920.00-2080.00 ng / mL baicalin reference standard, 14.40-17.60 ng / mL glycyrrhetinic acid reference standard and 880.00-1040.00 ng / mL harpagoside reference standard, are prepared for use. (8) Preparation of mixed reference solution 6: Accurately measure an appropriate amount of the 21 single reference solutions from step (2), place them in a volumetric flask, and dilute with 50% methanol solution to prepare a solution containing 28.80-35.20 ng / mL p-hydroxybenzaldehyde reference, 28.80-35.20 ng / mL vanillin reference, 480.00-544.00 ng / mL caffeic acid reference, and 28.80-35.20 ng / mL syringaldehyde reference. Citric acid reference standard (192000.00-208000.00 ng / mL), tanshinone reference standard (940.00-1024.00 ng / mL), apigenin reference standard (60.80-67.20 ng / mL), baicalin reference standard (11840.00-12160.00 ng / mL), genistein reference standard (28.80-35.20 ng / mL), quercetin reference standard (120.00-144.00 ng / mL). 240.00-272.00 ng / mL chlorogenic acid reference standard, 240.00-272.00 ng / mL neochlorogenic acid reference standard, 3040.00-3340.00 ng / mL rosmarinic acid reference standard, 6240.00-6560.00 ng / mL harbazoside reference standard, 7.36-8.64 ng / mL puerarin reference standard, 7.36-8.64 ng / mL apigenin-7-O-β-D-glucan A mixed reference solution 6 containing glucoside reference standard, 121600.00-134400.00 ng / mL baicalin reference standard, 240.00-272.00 ng / mL oleanolic acid reference standard, 3840.00-4160.00 ng / mL baicalin reference standard, 28.80-35.20 ng / mL glycyrrhetinic acid reference standard and 1760.00-2080.00 ng / mL harpagoside reference standard, is prepared for use. (9) Chromatographic conditions: The chromatographic column was a Waters ACQUITY UPLC.  The chromatographic column was a BEH C18, with dimensions of 2.1 mm × 100 mm and a diameter of 1.7 µm; the guard column was a Waters Van Guard BEH C18, with dimensions of 2.1 mm × 5 mm and a diameter of 1.7 µm; the column temperature was 40 °C; mobile phase A was 0.1% formic acid acetonitrile, and mobile phase D was 0.1% formic acid water; the injection volume was 5 µL; the gradient elution conditions were as follows: Time (min) Flow rate (mL / min) A（%） D（%） 0.0 0.3 5 95 1.0 0.3 5 95 3.5 0.3 40 60 6.5 0.3 55 45 11.5 0.3 90 10 12.5 0.3 90 10 13.0 0.3 5 95 14.0 0.3 5 95 (10) Mass spectrometry conditions: Heated electrospray ionization source, positive ion mode; Electrospray voltage: 3500V, Sheath Gas: 20Arb; Aux Gas: 5Arb; Sweep Gas: 10Arb; IonTransfer Tube Temp: 325℃; Vaporizer Temp: 275℃; Auxiliary gas and sheath gas: N2; Q1 Resolution: 0.7; Q3 Resolution: 0.7; CID Gas: 2 mTorr; Scanning mode was selected ion monitoring; mass spectrometry data acquisition and processing software was Xcalibur workstation. The following are the monitoring ions used for the quantitative analysis of 21 components: p-hydroxybenzaldehyde, vanillin, caffeic acid, syringaldehyde, citric acid, tanshinone, baicalin, genistein, quercetin, chlorogenic acid, neochlorogenic acid, rosmarinic acid, harpagoside, puerarin, apigenin-7-O-β-D-glucoside, baicalin, oleanolic acid, baicalin, glycyrrhetinic acid, and harpagoside: Detected items Parent ion (m / z) Daughter ions (m / z) Collision energy (V) RF voltage (V) p-Hydroxybenzaldehyde 123.212 95.183 10.25 67 Vanillin 153.162 93.183 14.1 68 caffeic acid 181.112 163.071 10.25 67 Syringaldehyde 183.162 123.111 14.55 78 Citric acid 193.162 129.071 10.25 50 Danshensu 199.162 153.071 10.25 55 Poplar 255.112 153.014 27.9 169 Baicalein 271.088 123.054 31.64 144 Genistein 271.088 153 26.43 156 Quercetin 303.15 229 27.65 193 chlorogenic acid 355.138 163 10.25 103 Neochlorogenic acid 355.188 163.071 10.25 90 Rosemary acid 361.15 145.04 25.93 79 Harbaginoside 387.15 207 24.26 210 Puerarin 417.15 296.929 24.36 126 Apigenin-7-O-β-D-glucoside 433.138 271 17.84 133 baicalin 447.138 270.929 18.6 134 Oleanolic acid 457.388 203.169 22.19 105 Baicalin 463.15 287 19.71 132 glycyrrhetinic acid 471.388 189.111 31.64 216 harpagoside 495.238 167 11.97 116 (11) Determination of the content of multiple components: Mixed reference solution 1, mixed reference solution 2, mixed reference solution 3, mixed reference solution 4, mixed reference solution 5 and mixed reference solution 6 were taken and analyzed according to the chromatographic and mass spectrometric conditions. A standard curve was plotted with concentration as the abscissa and peak area as the ordinate to obtain the regression equation of 21 components. Then, the test sample solution was taken and analyzed according to the chromatographic and mass spectrometric conditions. The content of 21 components in the sample, namely p-hydroxybenzaldehyde, vanillin, caffeic acid, eugenol, citric acid, tanshinone, scutellarin, baicalin, genistein, quercetin, chlorogenic acid, neochlorogenic acid, rosmarinic acid, harpagoside, puerarin, apigenin-7-O-β-D-glucoside, baicalin, oleanolic acid, baicalin, glycyrrhetinic acid and harpagoside, was calculated according to the regression equation.

[0007] In step (1) above, the preparation of the test solution is as follows: Take the capsule shell of Compound Scutellaria baicalensis and remove it, weigh 0.095-0.105g of the powder inside the Compound Scutellaria baicalensis and put it into a 100mL volumetric flask, add 30-60mL of 50% methanol, sonicate for 28-32min, cool, add 50% methanol to make up to the mark, shake well, centrifuge at 14000r / min for 9-11min at 4℃, separate the supernatant, filter through a 0.22μm filter membrane, and take the filtrate to obtain the test solution for later use.

[0008] Specifically, in step (1) above, the preparation of the test solution is as follows: Take the capsule shell of Compound Scutellaria baicalensis throat-clearing capsule, weigh 0.10g of the powder inside the capsule into a 100mL volumetric flask, add 30-60mL of 50% methanol, sonicate for 30min, cool, add 50% methanol to make up to the mark, shake well, centrifuge at 14000r / min for 10min at 4℃, separate the supernatant, filter through a 0.22μm filter membrane, and take the filtrate to obtain the test solution for later use.

[0009] In step (2) above, the preparation of single reference solutions is as follows: accurately weigh p-hydroxybenzaldehyde reference standard, vanillin reference standard, caffeic acid reference standard, eugenol reference standard, citric acid reference standard, tanshinone reference standard, salicylic acid reference standard, baicalin reference standard, genistein reference standard, quercetin reference standard, chlorogenic acid reference standard, neochlorogenic acid reference standard, rosmarinic acid reference standard, harpagoside reference standard, puerarin reference standard, apigenin-7-O-β-D-glucoside reference standard, baicalin reference standard, oleanolic acid reference standard, baicalin reference standard, glycyrrhetinic acid reference standard, and harpagoside reference standard, dissolve them in methanol and make up to volume to prepare 21 single reference solution solutions with a concentration of 1.00 mg / mL for later use.

[0010] In step (3) above, the preparation of mixed reference solution 1 is as follows: Accurately measure an appropriate amount of the 21 single reference solutions from step (2), place them in a volumetric flask, and dilute with 50% methanol solution to prepare a solution containing 1.00 ng / mL p-hydroxybenzaldehyde reference, 1.00 ng / mL vanillin reference, 16.00 ng / mL caffeic acid reference, 1.00 ng / mL syringaldehyde reference, 6250.00 ng / mL citric acid reference, 30.00 ng / mL tanshinone reference, 2.00 ng / mL syringin reference, 375.00 ng / mL baicalein reference, 1.00 ng / mL genistein reference, and 4.00 ng / mL styracin reference. A mixed reference solution 1 containing quercetin (mL), chlorogenic acid (8.00 ng / mL), neochlorogenic acid (8.00 ng / mL), rosmarinic acid (100.00 ng / mL), harpagoside (200.00 ng / mL), puerarin (0.24 ng / mL), apigenin-7-O-β-D-glucoside (0.24 ng / mL), baicalin (4000.00 ng / mL), oleanolic acid (8.00 ng / mL), baicalin (125.00 ng / mL), glycyrrhetinic acid (1.00 ng / mL), and harpagoside (60.00 ng / mL) is prepared for use.

[0011] In step (4) above, the preparation of mixed reference solution 2 is as follows: Accurately measure an appropriate amount of the 21 single reference solutions from step (2), place them in a volumetric flask, and dilute with 50% methanol solution to prepare a solution containing 2.00 ng / mL p-hydroxybenzaldehyde reference, 2.00 ng / mL vanillin reference, 32.00 ng / mL caffeic acid reference, 2.00 ng / mL syringaldehyde reference, 12500.00 ng / mL citric acid reference, 60.00 ng / mL tanshinone reference, 4.00 ng / mL salicylic acid reference, 750.00 ng / mL baicalein reference, 2.00 ng / mL genistein reference, and 8.00 ng / mL... A mixed reference solution 2 containing L-quercetin reference standard, 16.00 ng / mL chlorogenic acid reference standard, 16.00 ng / mL neochlorogenic acid reference standard, 200.00 ng / mL rosmarinic acid reference standard, 400.00 ng / mL harpagoside reference standard, 0.48 ng / mL puerarin reference standard, 0.48 ng / mL apigenin-7-O-β-D-glucoside reference standard, 8000.00 ng / mL baicalin reference standard, 16.00 ng / mL oleanolic acid reference standard, 250.00 ng / mL baicalin reference standard, 2.00 ng / mL glycyrrhetinic acid reference standard and 120.00 ng / mL harpagoside reference standard, is prepared for use.

[0012] In step (5) above, the preparation of mixed reference solution 3 is as follows: Accurately measure an appropriate amount of the 21 single reference solutions from step (2), place them in a volumetric flask, and dilute with 50% methanol solution to prepare a solution containing 4.00 ng / mL p-hydroxybenzaldehyde reference, 4.00 ng / mL vanillin reference, 64.00 ng / mL caffeic acid reference, 4.00 ng / mL syringaldehyde reference, 25000.00 ng / mL citric acid reference, 120.00 ng / mL tanshinone reference, 800 ng / mL salicylic acid reference, 1500.00 ng / mL baicalein reference, 4.00 ng / mL genistein reference, and 16.00 ng / mL... A mixed reference solution 3 containing L-quercetin reference standard, 32.00 ng / mL chlorogenic acid reference standard, 32.00 ng / mL neochlorogenic acid reference standard, 400.00 ng / mL rosmarinic acid reference standard, 800.00 ng / mL harpagoside reference standard, 0.96 ng / mL puerarin reference standard, 0.96 ng / mL apigenin-7-O-β-D-glucoside reference standard, 16000.00 ng / mL baicalin reference standard, 32.00 ng / mL oleanolic acid reference standard, 500.00 ng / mL baicalin reference standard, 4.00 ng / mL glycyrrhetinic acid reference standard, and 240.00 ng / mL harpagoside reference standard is prepared for use.

[0013] In step (6) above, the preparation of mixed reference solution 4 is as follows: Accurately measure an appropriate amount of the 21 single reference solutions from step (2), place them in a volumetric flask, and dilute with 50% methanol solution to prepare a solution containing 8.00 ng / mL p-hydroxybenzaldehyde reference, 8.00 ng / mL vanillin reference, 128.00 ng / mL caffeic acid reference, 8.00 ng / mL syringaldehyde reference, 50000.00 ng / mL citric acid reference, 240.00 ng / mL tanshinone reference, 16.00 ng / mL salicylic acid reference, 3000.00 ng / mL baicalein reference, 8.00 ng / mL genistein reference, and 32.00 ng / mL salicylic acid reference. A mixed reference solution of quercetin (mL), chlorogenic acid (64.00 ng / mL), neochlorogenic acid (64.00 ng / mL), rosmarinic acid (800.00 ng / mL), harpagoside (1600.00 ng / mL), puerarin (1.92 ng / mL), apigenin-7-O-β-D-glucoside (1.92 ng / mL), baicalin (32000.00 ng / mL), oleanolic acid (64.00 ng / mL), baicalin (1000.00 ng / mL), glycyrrhetinic acid (8.00 ng / mL), and harpagoside (480.00 ng / mL) is prepared for use.

[0014] In step (7) above, the preparation of mixed reference solution 5 is as follows: Accurately measure an appropriate amount of the 21 single reference solutions from step (2), place them in a volumetric flask, and dilute with 50% methanol solution to prepare a solution containing 16.00 ng / mL p-hydroxybenzaldehyde reference, 16.00 ng / mL vanillin reference, 256.00 ng / mL caffeic acid reference, 16.00 ng / mL syringaldehyde reference, 100000.00 ng / mL citric acid reference, 480.00 ng / mL tanshinone reference, 32.00 ng / mL salicylic acid reference, 6000.00 ng / mL baicalein reference, 16.00 ng / mL genistein reference, and 64.00 ng / mL salicylic acid reference. A mixed reference solution of quercetin (mL), chlorogenic acid (128.00 ng / mL), neochlorogenic acid (128.00 ng / mL), rosmarinic acid (1600.00 ng / mL), harpagoside (3200.00 ng / mL), puerarin (3.84 ng / mL), apigenin-7-O-β-D-glucoside (3.84 ng / mL), baicalin (64000.00 ng / mL), oleanolic acid (128.00 ng / mL), baicalin (2000.00 ng / mL), glycyrrhetinic acid (16.00 ng / mL), and harpagoside (960.00 ng / mL) is prepared for use.

[0015] In step (8) above, the preparation of mixed reference solution 6 is as follows: Accurately measure an appropriate amount of the 21 single reference solutions from step (2), place them in a volumetric flask, and dilute with 50% methanol solution to prepare a solution containing 32.00 ng / mL p-hydroxybenzaldehyde reference, 32.00 ng / mL vanillin reference, 512.00 ng / mL caffeic acid reference, 32.00 ng / mL syringaldehyde reference, 200,000.00 ng / mL citric acid reference, 960.00 ng / mL tanshinone reference, 64.00 ng / mL salicylic acid reference, 12,000.00 ng / mL baicalein reference, 32.00 ng / mL genistein reference, and 128.00 ng / mL salicylic acid reference. A mixed reference solution of quercetin (mL), chlorogenic acid (256.00 ng / mL), neochlorogenic acid (256.00 ng / mL), rosmarinic acid (3200.00 ng / mL), harpagoside (6400.00 ng / mL), puerarin (7.68 ng / mL), apigenin-7-O-β-D-glucoside (7.68 ng / mL), baicalin (128000.00 ng / mL), oleanolic acid (240.00 ng / mL), baicalin (4000.00 ng / mL), glycyrrhetinic acid (32.00 ng / mL), and harpagoside (1920.00 ng / mL) is prepared for use.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention establishes a multi-component simultaneous quantitative analysis method using UHPLC-MS / MS technology for the first time, evaluating the quality of Compound Scutellaria baicalensis throat-clearing capsules from a quantitative perspective. Twenty-one components were selected as quantitative indicators: p-hydroxybenzaldehyde, vanillin, caffeic acid, eugenol, citric acid, tanshinone, baicalin, genistein, quercetin, chlorogenic acid, neochlorogenic acid, rosmarinic acid, harpagoside, puerarin, apigenin-7-O-β-D-glucoside, baicalin, oleanolic acid, baicalin, glycyrrhetinic acid, and harpagoside. The content of these components in Compound Scutellaria baicalensis throat-clearing capsules was determined. This method can simultaneously and accurately determine the content of 21 components in Compound Scutellaria baicalensis Throat-Clearing Capsules within 14 minutes, exhibiting good precision and stability. It is suitable for quality control research of Compound Scutellaria baicalensis Throat-Clearing Capsules. The establishment of this method can provide experimental basis for improving and enhancing the quality standards of Compound Scutellaria baicalensis Throat-Clearing Capsules, and has important guiding significance for the comprehensive control of the quality of this preparation. Attached Figure Description

[0017] Figure 1 The total ion chromatogram of 21 components in mixed reference solution 3; Figure 2 Total ion chromatogram of 21 components in the test solution of Compound Scutellaria baicalensis throat-clearing capsules; Figure 3 Extraction ion chromatograms of 21 components in the test solution of Compound Scutellaria baicalensis throat-clearing capsules (1: p-hydroxybenzaldehyde; 2: vanillin; 3: caffeic acid; 4: syringaldehyde; 5: citric acid; 6: tanshinone; 7: guarin; 8: baicalin; 9: genistein; 10: quercetin; 11: chlorogenic acid; 12: neochlorogenic acid; 13: rosmarinic acid; 14: harpagoside; 15: puerarin; 16: apigenin-7-O-β-D-glucoside; 17: baicalin; 18: oleanolic acid; 19: baicalin; 20: glycyrrhetinic acid; 21: harpagoside). Figure 4 The extraction ion chromatograms of 21 components in mixed reference solution 3 are shown below (1: p-hydroxybenzaldehyde; 2: vanillin; 3: caffeic acid; 4: syringaldehyde; 5: citric acid; 6: tanshinone; 7: guarin; 8: baicalin; 9: genistein; 10: quercetin; 11: chlorogenic acid; 12: neochlorogenic acid; 13: rosmarinic acid; 14: harpagoside; 15: puerarin; 16: apigenin-7-O-β-D-glucoside; 17: baicalin; 18: oleanolic acid; 19: baicalin; 20: glycyrrhetinic acid; 21: harpagoside). Detailed Implementation

[0018] 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.

[0019] Example 1: Chromatographic and mass spectrometric conditions for the determination of multiple components Chromatographic conditions: Waters ACQUITY UPLC column. The chromatographic column was a BEH C18, with dimensions of 2.1 mm × 100 mm and a diameter of 1.7 µm; the guard column was a Waters Van Guard BEH C18, with dimensions of 2.1 mm × 5 mm and a diameter of 1.7 µm; the column temperature was 40 °C; mobile phase A was 0.1% formic acid acetonitrile, and mobile phase D was 0.1% formic acid water; the injection volume was 5 µL; the gradient elution conditions were as follows: Time (min) Flow rate (mL / min) A（%） D（%） 0.0 0.3 5 95 1.0 0.3 5 95 3.5 0.3 40 60 6.5 0.3 55 45 11.5 0.3 90 10 12.5 0.3 90 10 13.0 0.3 5 95 14.0 0.3 5 95 Mass spectrometry conditions: Heated electrospray ionization source, positive ion mode; Electrospray voltage: 3500V; Sheath Gas: 20 Arb; Aux Gas: 5 Arb; Sweep Gas: 10 Arb; IonTransfer Tube Temp: 325℃; Vaporizer Temp: 275℃; Auxiliary gas and sheath gas: N2; Q1 Resolution: 0.7; Q3 Resolution: 0.7; CID Gas: 2 mTorr; Scanning mode was selected ion monitoring; mass spectrometry data acquisition and processing software was Xcalibur workstation. The following are the monitoring ions used for the quantitative analysis of 21 components: p-hydroxybenzaldehyde, vanillin, caffeic acid, syringaldehyde, citric acid, tanshinone, baicalin, genistein, quercetin, chlorogenic acid, neochlorogenic acid, rosmarinic acid, harpagoside, puerarin, apigenin-7-O-β-D-glucoside, baicalin, oleanolic acid, baicalin, glycyrrhetinic acid, and harpagoside: Detected items Parent ion (m / z) Daughter ions (m / z) Collision energy (V) RF voltage (V) p-Hydroxybenzaldehyde 123.212 95.183 10.25 67 Vanillin 153.162 93.183 14.1 68 caffeic acid 181.112 163.071 10.25 67 Syringaldehyde 183.162 123.111 14.55 78 Citric acid 193.162 129.071 10.25 50 Danshensu 199.162 153.071 10.25 55 Poplar 255.112 153.014 27.9 169 Baicalein 271.088 123.054 31.64 144 Genistein 271.088 153 26.43 156 Quercetin 303.15 229 27.65 193 chlorogenic acid 355.138 163 10.25 103 Neochlorogenic acid 355.188 163.071 10.25 90 Rosemary acid 361.15 145.04 25.93 79 Harbaginoside 387.15 207 24.26 210 Puerarin 417.15 296.929 24.36 126 Apigenin-7-O-β-D-glucoside 433.138 271 17.84 133 baicalin 447.138 270.929 18.6 134 Oleanolic acid 457.388 203.169 22.19 105 Baicalin 463.15 287 19.71 132 glycyrrhetinic acid 471.388 189.111 31.64 216 harpagoside 495.238 167 11.97 116 Example 2: Determination of the content of multiple components Preparation of the test solution: Remove the capsule shell from Compound Scutellaria baicalensis throat-clearing capsules, weigh 0.10 g of the powder from the capsules into a 100 mL volumetric flask, add 30-60 mL of 50% methanol, sonicate for 30 min, cool, then add 50% methanol to the mark, shake well, centrifuge at 14000 r / min for 10 min at 4℃, separate the supernatant, filter through a 0.22 μm filter membrane, and collect the filtrate to obtain the test solution for later use; (2) Preparation of single reference solutions: Accurately weigh p-hydroxybenzaldehyde reference, vanillin reference, caffeic acid reference, eugenol reference, citric acid reference, tanshinone reference, salicylic acid reference, baicalin reference, genistein reference, quercetin reference, chlorogenic acid reference, neochlorogenic acid reference, rosmarinic acid reference, harpagoside reference, puerarin reference, apigenin-7-O-β-D-glucoside reference, baicalin reference, oleanolic acid reference, baicalin reference, glycyrrhetinic acid reference, and harpagoside reference, dissolve them in methanol and make up to volume to prepare 21 single reference solutions with a concentration of 1.00 mg / mL for later use.

[0020] (3) Accurately measure an appropriate amount of the 21 single reference solutions from step (2), place them in a volumetric flask, and dilute with 50% methanol solution to prepare the following 6 groups of mixed reference solutions of different concentrations: (4) Determination of the content of multiple components: Mixed reference solution 1, mixed reference solution 2, mixed reference solution 3, mixed reference solution 4, mixed reference solution 5 and mixed reference solution 6 were taken and analyzed according to the chromatographic and mass spectrometric conditions of Example 1. A standard curve was plotted with concentration as the abscissa and peak area as the ordinate to obtain the regression equation of 21 components. Then, the test sample solution was taken and analyzed according to the chromatographic and mass spectrometric conditions. The content of 21 components in the sample, namely p-hydroxybenzaldehyde, vanillin, caffeic acid, eugenol, citric acid, tanshinone, scutellarin, baicalin, genistein, quercetin, chlorogenic acid, neochlorogenic acid, rosmarinic acid, harpagoside, puerarin, apigenin-7-O-β-D-glucoside, baicalin, oleanolic acid, baicalin, glycyrrhetinic acid and harpagoside, was calculated according to the regression equation.

[0021] Example 3: Determination of the content of multiple components (1) Preparation of test solution: Take the capsule shell of Compound Scutellaria baicalensis throat clearing capsule, weigh 0.09g of the powder inside the capsule into a 100mL volumetric flask, add 50% methanol, sonicate for 25min, cool, add 50% methanol to make up to the mark, shake well, centrifuge at 13000r / min for 8-12min at 3℃, separate the supernatant, filter the membrane, take the filtrate, and the test solution is obtained for use; (2) Preparation of single reference solutions: Accurately weigh p-hydroxybenzaldehyde reference, vanillin reference, caffeic acid reference, eugenol reference, citric acid reference, tanshinone reference, salicylic acid reference, baicalin reference, genistein reference, quercetin reference, chlorogenic acid reference, neochlorogenic acid reference, rosmarinic acid reference, harpagoside reference, puerarin reference, apigenin-7-O-β-D-glucoside reference, baicalin reference, oleanolic acid reference, baicalin reference, glycyrrhetinic acid reference, and harpagoside reference, dissolve them in methanol and dilute to volume to prepare 21 single reference solutions with a concentration of 0.90 mg / mL for later use; (3) Accurately measure an appropriate amount of the 21 single reference solutions from step (2), place them in a volumetric flask, and dilute with 50% methanol solution to prepare the following 6 groups of mixed reference solutions of different concentrations: (4) Determination of the content of multiple components: Mixed reference solution 1, mixed reference solution 2, mixed reference solution 3, mixed reference solution 4, mixed reference solution 5 and mixed reference solution 6 were taken and analyzed according to the chromatographic and mass spectrometric conditions of Example 1. A standard curve was plotted with concentration as the abscissa and peak area as the ordinate to obtain the regression equation of 21 components. Then, the test sample solution was taken and analyzed according to the chromatographic and mass spectrometric conditions. The content of 21 components in the sample, namely p-hydroxybenzaldehyde, vanillin, caffeic acid, eugenol, citric acid, tanshinone, scutellarin, baicalin, genistein, quercetin, chlorogenic acid, neochlorogenic acid, rosmarinic acid, harpagoside, puerarin, apigenin-7-O-β-D-glucoside, baicalin, oleanolic acid, baicalin, glycyrrhetinic acid and harpagoside, was calculated according to the regression equation.

[0022] Example 4: Determination of the content of multiple components (1) Preparation of test solution: Remove the capsule shell of Compound Scutellaria baicalensis throat clearing capsule, weigh 0.11g of the powder inside the Compound Scutellaria baicalensis throat clearing capsule into a 100mL volumetric flask, add 50% methanol, sonicate for 35min, cool, add 50% methanol to make up to the mark, shake well, centrifuge at 15000r / min for 12min at 5℃, separate the supernatant, filter membrane, take the filtrate to obtain the test solution, and use it for later use; (2) Preparation of single reference solutions: Accurately weigh p-hydroxybenzaldehyde reference, vanillin reference, caffeic acid reference, eugenol reference, citric acid reference, tanshinone reference, salicylic acid reference, baicalin reference, genistein reference, quercetin reference, chlorogenic acid reference, neochlorogenic acid reference, rosmarinic acid reference, harpagoside reference, puerarin reference, apigenin-7-O-β-D-glucoside reference, baicalin reference, oleanolic acid reference, baicalin reference, glycyrrhetinic acid reference, and harpagoside reference, dissolve them in methanol and dilute to volume to prepare 21 single reference solutions with a concentration of 1.10 mg / mL for later use; (3) Accurately measure an appropriate amount of the 21 single reference solutions from step (2), place them in a volumetric flask, and dilute with 50% methanol solution to prepare the following 6 groups of mixed reference solutions of different concentrations: (4) Determination of the content of multiple components: Mixed reference solution 1, mixed reference solution 2, mixed reference solution 3, mixed reference solution 4, mixed reference solution 5 and mixed reference solution 6 were taken and analyzed according to the chromatographic and mass spectrometric conditions of Example 1. A standard curve was plotted with concentration as the abscissa and peak area as the ordinate to obtain the regression equation of 21 components. Then, the test sample solution was taken and analyzed according to the chromatographic and mass spectrometric conditions. The content of 21 components in the sample, namely p-hydroxybenzaldehyde, vanillin, caffeic acid, eugenol, citric acid, tanshinone, scutellarin, baicalin, genistein, quercetin, chlorogenic acid, neochlorogenic acid, rosmarinic acid, harpagoside, puerarin, apigenin-7-O-β-D-glucoside, baicalin, oleanolic acid, baicalin, glycyrrhetinic acid and harpagoside, was calculated according to the regression equation.

[0023] 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 and Methods 1.1 Drugs and Reagents Compound Scutellaria baicalensis throat-clearing capsules were provided by Guizhou Ruihe Pharmaceutical Co., Ltd. (batch numbers: 20231201, 20231202, 20231203); oleanolic acid, vanillin, baicalein, quercetin, puerarin, baicalin, citric acid, and rosmarinic acid were purchased from the National Institutes for Food and Drug Control (batch numbers: 110709-201808, 100491-202203, 111595-201808, 100081-201610, 110752-202217, 110842-202010, 111679-202403, 111871-202408); p-hydroxybenzaldehyde, genistein, eugenol, sodium tanshinone, and neochlorogenic acid were also included. Acetic acid, harpagoside, harpagoside, and caffeic acid were purchased from Chengdu Efa Biotechnology Co., Ltd. (batch numbers AFCC0754, AFDA0354, AFCJ2454, AB0388, AF20030804, AFBG1391, AFCA2953, and AF9051082, respectively); apigenin-7-O-β-D-glucoside, apigenin, and chlorogenic acid were purchased from Sichuan Weikeqi Biotechnology Co., Ltd. (batch numbers WP24060306, WP24090610, and WP24012303, respectively); baicalin (Shanghai Shidander, batch number: RS01861120) and glycyrrhetinic acid (Chengdu Pusi Biotechnology Co., Ltd., batch number: PS010486).

[0024] 1.2 Methods 1.2.1 Preparation of reference solution Accurately weigh appropriate amounts of each reference standard under section "1.1", dissolve them separately in methanol, and dilute to 10 mL to prepare single reference standard stock solutions with a mass concentration of approximately 1 mg / mL. Accurately pipette appropriate amounts of the above single reference standard solutions into 10 mL volumetric flasks, dilute with 50% methanol (v / v), and dilute to volume to prepare mixed reference standard solutions containing 21 components including p-hydroxybenzaldehyde. Then, dilute to the series concentrations shown in Table 1 according to the specified ratios.

[0025] Table 1 Concentration of a series of standard solutions 1.2.2 Preparation of the test solution Weigh 0.1 g of Compound Scutellaria Phlegm-Clearing Capsule Powder into a 100 mL volumetric flask, add a certain amount of 50% methanol (v / v), sonicate for 30 min, cool, add 50% methanol to the mark, shake well, centrifuge at 14000 r / min for 10 min at 4 °C, separate the supernatant, filter through a 0.22 μm filter membrane, and take the filtrate for analysis.

[0026] 1.2.3 Chromatographic and Mass Spectrometry Conditions Chromatographic conditions: Column: Waters ACQUITY UPLC BEH C18 (2.1 mm × 100 mm, 1.7 µm) column; Guard column: Waters Van Guard BEH C18 (2.1 mm × 5 mm, 1.7 µm); Column temperature: 40 °C; Mobile phase: 0.1% formic acid acetonitrile (A) - 0.1% formic acid water (D); Injection volume: 5 µL. Gradient elution conditions are shown in Table 2.

[0027] Table 2 Chromatographic conditions for 21 components including p-hydroxybenzaldehyde Time (min) Flow rate (mL / min) A（%） D（%） 0.0 0.3 5 95 1.0 0.3 5 95 3.5 0.3 40 60 6.5 0.3 55 45 11.5 0.3 90 10 12.5 0.3 90 10 13.0 0.3 5 95 14.0 0.3 5 95 Mass spectrometry conditions: Heated electrospray ionization (H-ESI) source, positive ion mode; electrospray voltage: 3500 V; Sheath Gas: 20 Arb; Aux Gas: 5 Arb; Sweep Gas: 10 Arb; Ion Transfer Tube Temp: 325℃; Vaporizer Temp: 275℃; Auxiliary gas and sheath gas: N2; Q1 Resolution: 0.7; Q3 Resolution: 0.7; CID Gas: 2 mTorr; Selected ion monitoring (SRM) mode; Xcalibur workstation was used for mass spectrometry data acquisition and processing. The monitoring ions used for the quantitative analysis of 21 components, including p-hydroxybenzaldehyde, are shown in Table 3.

[0028] Table 3 Mass spectrometry conditions for 21 components including p-hydroxybenzaldehyde Detected items Parent ion (m / z) Daughter ions (m / z) Collision energy (V) RF voltage (V) p-Hydroxybenzaldehyde 123.212 95.183 10.25 67 Vanillin 153.162 93.183 14.1 68 caffeic acid 181.112 163.071 10.25 67 Syringaldehyde 183.162 123.111 14.55 78 Citric acid 193.162 129.071 10.25 50 Danshensu 199.162 153.071 10.25 55 Poplar 255.112 153.014 27.9 169 Baicalein 271.088 123.054 31.64 144 Genistein 271.088 153 26.43 156 Quercetin 303.15 229 27.65 193 chlorogenic acid 355.138 163 10.25 103 Neochlorogenic acid 355.188 163.071 10.25 90 Rosemary acid 361.15 145.04 25.93 79 Harbaginoside 387.15 207 24.26 210 Puerarin 417.15 296.929 24.36 126 Apigenin-7-O-β-D-glucoside 433.138 271 17.84 133 baicalin 447.138 270.929 18.6 134 Oleanolic acid 457.388 203.169 22.19 105 Baicalin 463.15 287 19.71 132 glycyrrhetinic acid 471.388 189.111 31.64 216 harpagoside 495.238 167 11.97 116 1.2.4 Validation of Analytical Methods 1.2.4.1 Specificity Examination Accurately pipette 5 μL of the mixed reference standard 3 and the compound Scutellaria baicalensis throat-clearing capsule sample solution, and inject them for analysis according to the chromatographic and mass spectrometric conditions in section "1.2.3". Record the chromatograms.

[0029] See results Figure 1 , Figure 2 , Figure 3 and Figure 4 The test solution and the mixed reference solution showed the same chromatographic peaks at the corresponding positions in their chromatograms, and the separation of each component was good, indicating that the method has good specificity and meets the requirements for quantitative analysis.

[0030] 1.2.4.2 Linearity Examination Accurately pipette the series of mixed reference solutions 1, 2, 3, 4, 5, and 6 under section "1.2.1", and analyze them according to the chromatographic and mass spectrometric conditions under section "1.2.3". Plot a standard curve with concentration (x) as the abscissa and peak area (y) as the ordinate to obtain the regression equation for each component.

[0031] The results are shown in Table 4. The 21 components, including p-hydroxybenzaldehyde, showed good linearity within their respective concentration ranges, with r ≥ 0.9902.

[0032] Table 4. Results of linearity studies for 21 components including p-hydroxybenzaldehyde compound Regression equation Linear range r p-Hydroxybenzaldehyde y = 792.64x - 493.08 0.99~31.62 0.9923 Vanillin y = 1187x + 2974 1.94~62.15 0.9969 caffeic acid y = 585.05x - 11362 15.72~502.90 0.9937 Syringaldehyde y = 654.29x - 17.791 0.98~31.25 0.9962 Citric acid y = 1.3174x - 11773 6250.00~200000.00 0.9972 Danshensu y = 6.0823x + 531.38 31.23~999.38 0.9902 Poplar y = 1145.2x - 320.09 1.95~62.40 0.9991 Baicalein y = 1078.7x + 275770 375.39~12012.40 0.9905 Genistein y = 490.6x - 238.71 0.99~31.72 0.9968 Quercetin y = 153.98x - 712.58 3.88~124.32 0.9904 chlorogenic acid y = 509.9x - 2853.2 7.78~249.11 0.9968 Neochlorogenic acid y = 358.4x - 1806.5 7.81~249.78 0.9939 Rosemary acid y = 39.238x + 476.82 96.84~3099.00 0.9961 Harbaginoside y = 4.8213x + 259.6 200.63~6420.00 0.9958 Puerarin y = 844.04x - 129.16 0.24~7.81 0.9973 Apigenin-7-O-β-D-glucoside y = 2829x - 151.7 0.24~7.83 0.9963 baicalin y = 564.1x + 10000000 4123.97~131967.00 0.9925 Oleanolic acid y = 47.714x - 13.035 7.81~249.85 0.9997 Baicalin y = 1252.7x + 153759 125.31~4009.87 0.994 glycyrrhetinic acid y = 185.36x - 190.10 0.98~31.51 0.9942 harpagoside y = 1.4691x - 90.483 62.18~1989.68 0.9966 1.2.4.3 Precision Test Take an appropriate amount of mixed reference standard 3, and continuously inject and determine it 6 times according to the chromatographic and mass spectrometric conditions under section "1.2.3". Record the peak area of ​​21 components such as p-hydroxybenzaldehyde, and calculate the RSD value of the peak area of ​​each component to evaluate the precision of the instrument.

[0033] The results (see Table 5) show that the RSDs of the peak areas of p-hydroxybenzaldehyde, vanillin, caffeic acid, eugenol, citric acid, tanshinone, scutellarin, baicalin, genistein, quercetin, chlorogenic acid, neochlorogenic acid, rosmarinic acid, harpagoside, puerarin, apigenin-7-O-β-D-glucoside, baicalin, oleanolic acid, baicalin, glycyrrhetinic acid, and harpagoside were 0.50%, 0.91%, 0.40%, 3.19%, 0.94%, 1.59%, 1.36%, 1.87%, 0.37%, 0.79%, 0.54%, 0.76%, 2.33%, 1.47%, 0.56%, 1.12%, 0.20%, 0.91%, 0.09%, 0.71%, and 1.47%, respectively, indicating good instrument precision.

[0034] Table 5. Precision test results of 21 components including p-hydroxybenzaldehyde (n = 6) compound Average peak area RSD / % p-Hydroxybenzaldehyde 90131 0.50 Vanillin 155405 0.91 caffeic acid 130682 0.40 Syringaldehyde 83464 3.19 Citric acid 17889 0.94 Danshensu 2282 1.59 Poplar 148563 1.36 Baicalein 1062224 1.87 Genistein 61844 0.37 Quercetin 18266 0.79 chlorogenic acid 56071 0.54 Neochlorogenic acid 40390 0.76 Rosemary acid 29520 2.33 Harbaginoside 2129 1.47 Puerarin 122075 0.56 Apigenin-7-O-β-D-glucoside 394201 1.12 baicalin 12673815 0.20 Oleanolic acid 19579 0.91 Baicalin 813105 0.09 glycyrrhetinic acid 28709 0.71 harpagoside 223 1.47 1.2.4.4 Repeatability Test Take 6 portions of Compound Scutellaria baicalensis and Phlegm-Clearing Capsules, prepare test solutions according to the method in section "1.2.2", analyze them according to the chromatographic and mass spectrometric conditions in section "1.2.3", record the peak areas of 21 components including p-hydroxybenzaldehyde, and calculate the content of each component and the RSD value of the content based on the regression equation obtained from the linear relationship investigation, and evaluate the repeatability of the method.

[0035] The results (see Table 6) showed that the contents of p-hydroxybenzaldehyde, vanillin, caffeic acid, eugenol, citric acid, tanshinone, salinomycin, baicalin, genistein, quercetin, chlorogenic acid, neochlorogenic acid, rosmarinic acid, harpagoside, puerarin, apigenin-7-O-β-D-glucoside, baicalin, oleanolic acid, baicalin, glycyrrhetinic acid, and harpagoside were 0.006, 0.009, 0.114, 0.005, 64.693, 0.378, 0.024, 0.358, 0.005, 0.008, 0.037, 0.041, 0.318, 1.243, 0.001, 0.005, 110.565, 0.171, 0.492, 0.008, and 0.242, respectively. The RSDs for mg / g were 1.27%, 0.67%, 0.11%, 0.57%, 0.39%, 2.91%, 0.67%, 0.20%, 1.07%, 1.23%, 1.48%, 2.14%, 1.33%, 2.39%, 1.61%, 0.74%, 0.38%, 1.52%, 0.43%, 1.43%, and 1.74%, respectively, indicating that the method has good repeatability.

[0036] Table 6. Results of repeatability testing for 21 components including p-hydroxybenzaldehyde (n = 6) compound Content (mg / g) RSD / % p-Hydroxybenzaldehyde 0.006 1.27 Vanillin 0.009 0.67 caffeic acid 0.114 0.11 Syringaldehyde 0.005 0.57 Citric acid 64.693 0.39 Danshensu 0.378 2.91 Poplar 0.024 0.67 Baicalein 0.358 0.20 Genistein 0.005 1.07 Quercetin 0.008 1.23 chlorogenic acid 0.037 1.48 Neochlorogenic acid 0.041 2.14 Rosemary acid 0.318 1.33 Harbaginoside 1.243 2.39 Puerarin 0.001 1.61 Apigenin-7-O-β-D-glucoside 0.005 0.74 baicalin 110.565 0.38 Oleanolic acid 0.171 1.52 Baicalin 0.492 0.43 glycyrrhetinic acid 0.008 1.43 harpagoside 0.242 1.74 1.2.4.5 Stability Test Take the test solution under section “1.2.4.4” and measure it at 0, 4, 6, 12 and 24 h respectively. Record the peak area of ​​21 components such as p-hydroxybenzaldehyde, and calculate the RSD value of the peak area of ​​each component to evaluate the stability of the test solution.

[0037] The results (see Table 7) showed that the RSDs of the peak areas of p-hydroxybenzaldehyde, vanillin, caffeic acid, eugenol, citric acid, tanshinone, salicylic acid, baicalin, genistein, quercetin, chlorogenic acid, neochlorogenic acid, rosmarinic acid, harpagoside, puerarin, apigenin-7-O-β-D-glucoside, baicalin, oleanolic acid, baicalin, glycyrrhetinic acid, and harpagoside were 1.18%, 0.52%, 0.14%, 0.67%, 0.50%, 1.44%, 0.75%, 0.13%, 1.20%, 2.99%, 1.77%, 2.52%, 1.23%, 1.65%, 2.27%, 0.84%, 0.46%, 1.51%, 0.38%, 1.82%, and 2.43%, respectively, indicating that the test solution had good stability within 24 h.

[0038] Table 7. Stability test results of 21 components including p-hydroxybenzaldehyde (n = 5) compound Average peak area RSD / % p-Hydroxybenzaldehyde 4432 1.18 Vanillin 14008 0.52 caffeic acid 56981 0.14 Syringaldehyde 3656 0.67 Citric acid 75554 0.50 Danshensu 3000 1.44 Poplar 27322 0.75 Baicalein 671774 0.13 Genistein 2070 1.20 Quercetin 547 2.99 chlorogenic acid 16768 1.77 Neochlorogenic acid 13471 2.52 Rosemary acid 13330 1.23 Harbaginoside 6446 1.65 Puerarin 453 2.27 Apigenin-7-O-β-D-glucoside 13835 0.84 baicalin 53891624 0.46 Oleanolic acid 8392 1.51 Baicalin 785519 0.38 glycyrrhetinic acid 1362 1.82 harpagoside 273 2.43 1.2.4.6 Recovery rate Accurately weigh 6 portions of Compound Scutellaria baicalensis throat-clearing capsules with known content, each approximately 0.05 g. Add a certain amount of each reference solution to each portion. Prepare samples according to the method in section "1.2.2". Inject and analyze according to the chromatographic and mass spectrometric conditions in section "1.2.3". Record the peak areas of 21 components, including p-hydroxybenzaldehyde. Calculate the recovery rate and RSD value of each component based on the regression equation obtained from the linear relationship study. Evaluate the recovery rate of the method.

[0039] The results (see Table 8) showed that the average recoveries of p-hydroxybenzaldehyde, vanillin, caffeic acid, eugenol, citric acid, tanshinone, salinomycin, baicalin, genistein, quercetin, chlorogenic acid, neochlorogenic acid, rosmarinic acid, harpagoside, puerarin, apigenin-7-O-β-D-glucoside, baicalin, oleanolic acid, baicalin, glycyrrhetinic acid, and harpagoside were 99.70%, 102.03%, 99.64%, 103.62%, 100.19%, 102.21%, 103.12%, 100.17%, 101.23%, 107.89%, 105.97%, and 101.35%, respectively. The recoveries were 102.98%, 100.59%, 103.27%, 102.16%, 99.99%, 98.88%, 100.11%, 97.65%, and 100.52%, with corresponding RSDs of 4.79%, 2.23%, 1.86%, 3.16%, 2.42%, 3.79%, 3.24%, 1.30%, 1.25%, 0.96%, 2.21%, 2.55%, 1.02%, 2.95%, 3.27%, 3.06%, 0.02%, 3.10%, 0.92%, 4.25%, and 4.41%, respectively, indicating that the recovery rates of each component in this method are good.

[0040] Table 8 Results of recovery of 21 components including p-hydroxybenzaldehyde (n = 6) compound Sample content / μg Amount added / μg Measured amount / μg Average recovery rate / % RSD / % p-Hydroxybenzaldehyde 0.306 0.309 0.614 99.70 4.79 Vanillin 2.073 2.073 0.682 102.03 2.23 caffeic acid 5.43 5.457 10.867 99.64 1.86 Syringaldehyde 0.302 0.303 0.616 103.62 3.16 Citric acid 3163.605 3163.52 6333.283 100.19 2.42 Danshensu 20.905 20.91 42.278 102.21 3.79 Poplar 0.843 0.845 1.714 103.12 3.24 Baicalein 19.253 19.261 38.547 100.17 1.30 Genistein 0.159 0.159 0.320 101.23 1.25 Quercetin 0.254 0.255 0.529 107.89 0.96 chlorogenic acid 1.803 1.804 3.715 105.97 2.21 Neochlorogenic acid 2.004 2.008 4.039 101.35 2.55 Rosemary acid 15.393 15.401 31.253 102.98 1.02 Harbaginoside 48.646 48.792 97.724 100.59 2.95 Puerarin 0.034 0.034 0.069 103.27 3.27 Apigenin-7-O-β-D-glucoside 0.233 0.233 0.471 102.16 3.06 baicalin 4836.18 4836.744 9672.573 99.99 0.02 Oleanolic acid 6.666 6.671 13.262 98.88 3.10 Baicalin 22.102 22.178 44.305 100.11 0.92 glycyrrhetinic acid 0.353 0.354 0.699 97.65 4.25 harpagoside 12.961 12.995 26.024 100.52 4.41 1.2.5 Determination of Multiple Component Content Take each batch of Compound Scutellaria baicalensis and Phlegm-Clearing Capsules, prepare test solutions according to the method in section "1.2.2", and analyze them according to the chromatographic and mass spectrometric conditions in section "1.2.3". Calculate the content of 21 components, including p-hydroxybenzaldehyde, in the samples based on the regression equation obtained from the linear relationship in section "1.2.4.2".

[0041] The content determination results of 21 components in three batches of Compound Scutellaria baicalensis throat-clearing capsules are shown in Table 9.

[0042] Table 9. Results of content determination of 21 components in 3 batches of samples (n = 3) 2. Discussion and Conclusion Based on the component identification results, combined with the content and pharmacological activity of each component, 21 components were selected as quantitative indicators for the determination of the content of Compound Scutellaria baicalensis throat-clearing capsules. These components include p-hydroxybenzaldehyde, vanillin, caffeic acid, eugenol, citric acid, tanshinone, baicalin, gentiolic acid, genistein, quercetin, chlorogenic acid, neochlorogenic acid, rosmarinic acid, harpaquinone, puerarin, apigenin-7-O-β-D-glucoside, baicalin, oleanolic acid, baicalin, glycyrrhetinic acid, and harpaquinone. The results showed that baicalin and citric acid had the highest contents, followed by harpaquinone, baicalin, tanshinone, baicalin, and harpaquinone, while p-hydroxybenzaldehyde and other components had lower contents. The contents of each component in different batches of Compound Scutellaria baicalensis throat-clearing capsules varied to some extent, which may be related to factors such as the quality of the raw materials, the harvest period, and the processing and storage of the samples. Improper sample storage can lead to capsules absorbing moisture and the contents clumping, thus affecting the content determination results of Compound Scutellaria baicalensis throat-clearing capsules.

[0043] Based on the identification results of blood-entering components, this study established a multi-component simultaneous quantitative analysis method using UHPLC-MS / MS technology to evaluate its quality from a quantitative perspective. This method can accurately determine the content of 21 components in Compound Scutellaria baicalensis Throat-Clearing Capsules simultaneously within 14 minutes, exhibiting good precision and stability. It is suitable for quality control research of Compound Scutellaria baicalensis Throat-Clearing Capsules. The establishment of this method can provide experimental basis for improving and enhancing the quality standards of Compound Scutellaria baicalensis Throat-Clearing Capsules, and has important guiding significance for the comprehensive quality control of this preparation.

Claims

1. A method for determining the content of multiple components in Compound Scutellaria baicalensis Throat-Clearing Capsules based on UHPLC-MS / MS, wherein 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 mixing evenly, the mixture is encapsulated, with a specification of 0.3g / capsule. The method is characterized by: The method for determining the content of multiple components in Compound Scutellaria baicalensis Throat-Clearing Capsules includes the following steps: (1) Preparation of test solution: Remove the capsule shell of Compound Scutellaria baicalensis throat clearing capsule, weigh 0.09-0.11g of the powder inside the Compound Scutellaria baicalensis throat clearing capsule into a 100mL volumetric flask, add 50% methanol, sonicate for 25-35min, cool, add 50% methanol to make up to the mark, shake well, centrifuge at 13000-15000r / min for 8-12min at 3-5℃, separate the supernatant, filter membrane, take the filtrate to obtain the test solution, and use it for later use; (2) Preparation of single reference solutions: Accurately weigh the following reference standards: p-hydroxybenzaldehyde, vanillin, caffeic acid, eugenol, citric acid, tanshinone, salicylic acid, baicalin, genistein, quercetin, chlorogenic acid, neochlorogenic acid, rosmarinic acid, harpagoside, puerarin, apigenin-7-O-β-D-glucoside, baicalin, oleanolic acid, baicalin, glycyrrhetinic acid, and harpagoside. Dissolve each in methanol and bring the volume to a concentration of 0.90-1.10 mg / mL for later use. (3) Preparation of mixed reference solution 1: Accurately measure an appropriate amount of the 21 single reference solutions in step (2), place them in a volumetric flask, and dilute with 50% methanol solution to prepare a solution containing 0.90-1.10 ng / mL p-hydroxybenzaldehyde reference, 1.90-2.10 ng / mL vanillin reference, 15.00-17.00 ng / mL caffeic acid reference, 0.90-1.10 ng / mL syringaldehyde reference, 6000.00-6500.00 ng / mL citric acid reference, 29.00-32.00 ng / mL tanshinone reference, 1.90-2.10 ng / mL salinomycin reference, 370.00-380.00 ng / mL baicalein reference, 0.90-1.10 ng / mL genistein reference, and 3.50-4.50 ng / mL quercetin. Reference standards: 7.50-8.50 ng / mL chlorogenic acid, 7.50-8.50 ng / mL neochlorogenic acid, 95.00-105.00 ng / mL rosmarinic acid, 195.00-205.00 ng / mL harbazoside, 0.23-0.26 ng / mL puerarin, 0.23-0.26 ng / mL apigenin-7-O-β-D. - A mixed reference solution 1 containing glucoside reference standard, 3800.00-4200.00 ng / mL baicalin reference standard, 7.50-8.50 ng / mL oleanolic acid reference standard, 120.00-130.00 ng / mL baicalin reference standard, 0.90-1.10 ng / mL glycyrrhetinic acid reference standard and 55.00-65.00 ng / mL harpagoside reference standard, for use; (4) Preparation of mixed reference solution 2: Accurately measure an appropriate amount of the 21 single reference solutions in step (2), place them in a volumetric flask, and dilute with 50% methanol solution to prepare a solution containing 1.80-2.20 ng / mL p-hydroxybenzaldehyde reference, 3.80-4.20 ng / mL vanillin reference, 30.00-34.00 ng / mL caffeic acid reference, 1.80-2.20 ng / mL syringaldehyde reference, 12000.00-13000.00 ng / mL citric acid reference, 58.00-64.00 ng / mL tanshinone reference, 3.80-4.20 ng / mL syringin reference, 740.00-760.00 ng / mL baicalein reference, 1.80-2.20 ng / mL genistein reference, and 7.00-9.00 ng / mL quercetin reference. 15.00-17.00 ng / mL chlorogenic acid reference standard, 15.0-17.00 ng / mL neochlorogenic acid reference standard, 190.00-210.00 ng / mL rosmarinic acid reference standard, 390.00-410.00 ng / mL harbazoside reference standard, 0.46-0.52 ng / mL puerarin reference standard, 0.46-0.52 ng / mL apigenin-7-O-β-D- Two mixed reference solutions of glucoside, baicalin (7600.00-8400.00 ng / mL), oleanolic acid (15.00-17.00 ng / mL), baicalin (240.00-260.00 ng / mL), glycyrrhetinic acid (1.80-2.20 ng / mL), and harpagoside (110.00-130.00 ng / mL) are prepared for use. (5) Preparation of mixed reference solution 3: Accurately measure an appropriate amount of the 21 single reference solutions from step (2), place them in a volumetric flask, and dilute with 50% methanol solution to prepare a solution containing 3.60-4.40 ng / mL p-hydroxybenzaldehyde reference, 7.60-8.40 ng / mL vanillin reference, 60.00-68.00 ng / mL caffeic acid reference, and 3.60-4.40 ng / mL syringaldehyde reference. Citric acid reference standard (24000.00-26000.00 ng / mL), tanshinone reference standard (116.00-128.00 ng / mL), salinomycin reference standard (7.60-8.40 ng / mL), baicalin reference standard (1480.00-1520.00 ng / mL), genistein reference standard (3.60-4.40 ng / mL), quercetin reference standard (14.00-18.00 ng / mL). 30.00-34.00 ng / mL chlorogenic acid reference standard, 30.00-34.00 ng / mL neochlorogenic acid reference standard, 380.00-420.00 ng / mL rosmarinic acid reference standard, 780.00-820.00 ng / mL harbazoside reference standard, 0.92-1.04 ng / mL puerarin reference standard, 0.92-1.04 ng / mL apigenin-7-O-β-D- 3 mixed reference solutions of glucoside reference standard, 15200.00-16800.00 ng / mL baicalin reference standard, 30.00-34.00 ng / mL oleanolic acid reference standard, 480.00-520.00 ng / mL baicalin reference standard, 3.60-4.40 ng / mL glycyrrhetinic acid reference standard and 220.00-260.00 ng / mL harpagoside reference standard, are prepared for use. (6) Preparation of mixed reference solution 4: Accurately measure an appropriate amount of the 21 single reference solutions from step (2), place them in a volumetric flask, and dilute with 50% methanol solution to prepare a solution containing 7.20-8.80 ng / mL p-hydroxybenzaldehyde reference, 15.20-16.80 ng / mL vanillin reference, 120.00-136.00 ng / mL caffeic acid reference, and 7.20-8.80 ng / mL syringaldehyde. Reference standards: Citric acid (48000.00-52000.00 ng / mL), Tanshinone (232.00-256.00 ng / mL), Pueraria lobata (15.20-16.80 ng / mL), Baicalein (2960.00-3040.00 ng / mL), Gentianone (7.20-8.80 ng / mL), Quercetin (28.00-36.00 ng / mL). Reference standards: 60.00-68.00 ng / mL chlorogenic acid, 60.00-68.00 ng / mL neochlorogenic acid, 760.00-840.00 ng / mL rosmarinic acid, 1560.00-1640.00 ng / mL harbazoside, 1.84-2.08 ng / mL puerarin, and 1.84-2.08 ng / mL apigenin-7-O-β-D. - A mixed reference solution 4 containing glucoside reference standard, 30400.00-33600.00 ng / mL baicalin reference standard, 60.00-68.00 ng / mL oleanolic acid reference standard, 960.00-1040.00 ng / mL baicalin reference standard, 7.20-8.80 ng / mL glycyrrhetinic acid reference standard and 440.00-520.00 ng / mL harpagoside reference standard, for use; (7) Preparation of mixed reference solution 5: Accurately measure an appropriate amount of the 21 single reference solutions from step (2), place them in a volumetric flask, and dilute with 50% methanol solution to prepare a solution containing 14.40-17.60 ng / mL p-hydroxybenzaldehyde reference, 30.40-33.60 ng / mL vanillin reference, 240.0-272.0 ng / mL caffeic acid reference, and 14.40-17.60 ng / mL syringaldehyde reference. 96000.00-104000.00 ng / mL citric acid reference standard, 464.00-512.00 ng / mL tanshinone reference standard, 30.40-33.60 ng / mL apigenin reference standard, 5920.00-6080.00 ng / mL baicalein reference standard, 14.40-17.60 ng / mL genistein reference standard, 56.00-72.00 ng / mL quercetin reference standard, 1 20.00-136.00 ng / mL chlorogenic acid reference standard, 120.00-136.00 ng / mL neochlorogenic acid reference standard, 1520.00-1680.00 ng / mL rosmarinic acid reference standard, 3120.00-3280.00 ng / mL harbazoside reference standard, 3.68-4.16 ng / mL puerarin reference standard, 3.68-4.16 ng / mL apigenin-7-O-β-D-glucan 5 mixed reference solutions of glucoside reference standard, 60800.00-67200.00 ng / mL baicalin reference standard, 120.00-136.00 ng / mL oleanolic acid reference standard, 1920.00-2080.00 ng / mL baicalin reference standard, 14.40-17.60 ng / mL glycyrrhetinic acid reference standard and 880.00-1040.00 ng / mL harpagoside reference standard, are prepared for use. (8) Preparation of mixed reference solution 6: Accurately measure an appropriate amount of the 21 single reference solutions from step (2), place them in a volumetric flask, and dilute with 50% methanol solution to prepare a solution containing 28.80-35.20 ng / mL p-hydroxybenzaldehyde reference, 60.80-67.20 ng / mL vanillin reference, 480.00-544.00 ng / mL caffeic acid reference, and 28.80-35.20 ng / mL syringaldehyde reference. Citric acid reference standard (192000.00-208000.00 ng / mL), tanshinone reference standard (928.00-1024.00 ng / mL), apigenin reference standard (60.80-67.20 ng / mL), baicalin reference standard (11840.00-12160.00 ng / mL), genistein reference standard (28.80-35.20 ng / mL), quercetin reference standard (112.00-144.00 ng / mL). 240.00-272.00 ng / mL chlorogenic acid reference standard, 240.00-272.00 ng / mL neochlorogenic acid reference standard, 3040.00-3340.00 ng / mL rosmarinic acid reference standard, 6240.00-6560.00 ng / mL harbazoside reference standard, 7.36-8.32 ng / mL puerarin reference standard, 7.36-8.32 ng / mL apigenin-7-O-β-D-glucan A mixed reference solution 6 containing glucoside reference standard, 121600.00-134400.00 ng / mL baicalin reference standard, 240.00-272.00 ng / mL oleanolic acid reference standard, 3840.00-4160.00 ng / mL baicalin reference standard, 28.80-35.20 ng / mL glycyrrhetinic acid reference standard and 1760.00-2080.00 ng / mL harpagoside reference standard, is prepared for use. (9) Chromatographic conditions: The chromatographic column was a Waters ACQUITY UPLCBEH C18 with dimensions of 2.1 mm × 100 mm and a diameter of 1.7 µm; the guard column was a Waters Van Guard BEH C18 with dimensions of 2.1 mm × 5 mm and a diameter of 1.7 µm; the column temperature was 40 °C; the mobile phase A was 0.1% formic acid acetonitrile, and the mobile phase D was 0.1% formic acid water; the injection volume was 5 µL; the gradient elution conditions were as follows: (10) Mass spectrometry conditions: Heated electrospray ionization source, positive ion mode; Electrospray voltage: 3500V, Sheath Gas: 20Arb; Aux Gas: 5Arb; Sweep Gas: 10Arb; IonTransfer Tube Temp: 325℃; Vaporizer Temp: 275℃; Auxiliary gas and sheath gas: N2; Q1 Resolution: 0.7; Q3 Resolution: 0.7; CID Gas: 2 mTorr; Scanning mode was selected ion monitoring; mass spectrometry data acquisition and processing software was Xcalibur workstation. The following are the monitoring ions used for the quantitative analysis of 21 components: p-hydroxybenzaldehyde, vanillin, caffeic acid, syringaldehyde, citric acid, tanshinone, baicalin, genistein, quercetin, chlorogenic acid, neochlorogenic acid, rosmarinic acid, harpagoside, puerarin, apigenin-7-O-β-D-glucoside, baicalin, oleanolic acid, baicalin, glycyrrhetinic acid, and harpagoside: (11) Determination of the content of multiple components: Mixed reference solution 1, mixed reference solution 2, mixed reference solution 3, mixed reference solution 4, mixed reference solution 5 and mixed reference solution 6 were taken and analyzed according to the chromatographic and mass spectrometric conditions. A standard curve was plotted with concentration as the abscissa and peak area as the ordinate to obtain the regression equation of 21 components. Then, the test sample solution was taken and analyzed according to the chromatographic and mass spectrometric conditions. The content of 21 components in the sample, namely p-hydroxybenzaldehyde, vanillin, caffeic acid, eugenol, citric acid, tanshinone, scutellarin, baicalin, genistein, quercetin, chlorogenic acid, neochlorogenic acid, rosmarinic acid, harpagoside, puerarin, apigenin-7-O-β-D-glucoside, baicalin, oleanolic acid, baicalin, glycyrrhetinic acid and harpagoside, was calculated according to the regression equation.

2. The method for determining the content of multiple components of Compound Scutellaria baicalensis throat-clearing capsules based on UHPLC-MS / MS according to claim 1, characterized in that: In step (1), the preparation of the test solution is as follows: Take the capsule shell of Compound Scutellaria baicalensis throat-clearing capsule, weigh 0.095-0.105g of the powder inside the capsule into a 100mL volumetric flask, add 30-60mL of 50% methanol, sonicate for 28-32min, cool, add 50% methanol to the mark, shake well, centrifuge at 14000r / min for 9-11min at 4℃, separate the supernatant, filter through a 0.22μm filter membrane, and take the filtrate to obtain the test solution for later use.

3. The method for determining the content of multiple components in Compound Scutellaria baicalensis throat-clearing capsules based on UHPLC-MS / MS according to claim 2, characterized in that: In step (1), the preparation of the test solution is as follows: Take the capsule shell of Compound Scutellaria baicalensis throat-clearing capsule, weigh 0.10 g of the powder inside the capsule into a 100 mL volumetric flask, add 30-60 mL of 50% methanol, sonicate for 30 min, cool, add 50% methanol to make up to the mark, shake well, centrifuge at 14000 r / min for 10 min at 4℃, separate the supernatant, filter through a 0.22 μm filter membrane, and take the filtrate to obtain the test solution for later use.

4. The method for determining the content of multiple components of Compound Scutellaria baicalensis throat-clearing capsules based on UHPLC-MS / MS according to claim 1, characterized in that: In step (2), the preparation of single reference solutions is as follows: accurately weigh p-hydroxybenzaldehyde reference standard, vanillin reference standard, caffeic acid reference standard, eugenol reference standard, citric acid reference standard, tanshinone reference standard, salicylic acid reference standard, baicalin reference standard, genistein reference standard, quercetin reference standard, chlorogenic acid reference standard, neochlorogenic acid reference standard, rosmarinic acid reference standard, harpagoside reference standard, puerarin reference standard, apigenin-7-O-β-D-glucoside reference standard, baicalin reference standard, oleanolic acid reference standard, baicalin reference standard, glycyrrhetinic acid reference standard, and harpagoside reference standard, dissolve them in methanol and make up to volume to prepare 21 single reference solution solutions with a concentration of 1.00 mg / mL for later use.

5. The method for determining the content of multiple components of Compound Scutellaria baicalensis throat-clearing capsules based on UHPLC-MS / MS according to claim 1, characterized in that: In step (3), the preparation of mixed reference solution 1 is as follows: Accurately measure an appropriate amount of the 21 single reference solutions from step (2), place them in a volumetric flask, and dilute with 50% methanol solution to prepare a solution containing 1.00 ng / mL p-hydroxybenzaldehyde reference, 2.00 ng / mL vanillin reference, 16.00 ng / mL caffeic acid reference, 1.00 ng / mL syringaldehyde reference, 6250.00 ng / mL citric acid reference, 30.00 ng / mL tanshinone reference, 2.00 ng / mL salinomycin reference, 375.00 ng / mL baicalein reference, 1.00 ng / mL genistein reference, and 4.00 ng / mL salinomycin reference. A mixed reference solution 1 containing quercetin (mL), chlorogenic acid (8.00 ng / mL), neochlorogenic acid (8.00 ng / mL), rosmarinic acid (100.00 ng / mL), harpagoside (200.00 ng / mL), puerarin (0.24 ng / mL), apigenin-7-O-β-D-glucoside (0.24 ng / mL), baicalin (4000.00 ng / mL), oleanolic acid (8.00 ng / mL), baicalin (125.00 ng / mL), glycyrrhetinic acid (1.00 ng / mL), and harpagoside (60.00 ng / mL) is prepared for use.

6. The method for determining the content of multiple components of Compound Scutellaria baicalensis throat-clearing capsules based on UHPLC-MS / MS according to claim 1, characterized in that: In step (4), the preparation of mixed reference solution 2 is as follows: Accurately measure an appropriate amount of the 21 single reference solutions from step (2), place them in a volumetric flask, and dilute with 50% methanol solution to prepare a solution containing 2.00 ng / mL p-hydroxybenzaldehyde reference, 4.00 ng / mL vanillin reference, 32.00 ng / mL caffeic acid reference, 2.00 ng / mL syringaldehyde reference, 12500.00 ng / mL citric acid reference, 60.00 ng / mL tanshinone reference, 4.00 ng / mL salinomycin reference, 750.00 ng / mL baicalein reference, 2.00 ng / mL genistein reference, and 8.00 ng / mL salinomycin reference. A mixed reference solution 2 containing L-quercetin reference standard, 16.00 ng / mL chlorogenic acid reference standard, 16.00 ng / mL neochlorogenic acid reference standard, 200.00 ng / mL rosmarinic acid reference standard, 400.00 ng / mL harpagoside reference standard, 0.48 ng / mL puerarin reference standard, 0.48 ng / mL apigenin-7-O-β-D-glucoside reference standard, 8000.00 ng / mL baicalin reference standard, 16.00 ng / mL oleanolic acid reference standard, 250.00 ng / mL baicalin reference standard, 2.00 ng / mL glycyrrhetinic acid reference standard and 120.00 ng / mL harpagoside reference standard, is prepared for use.

7. The method for determining the content of multiple components of Compound Scutellaria baicalensis throat-clearing capsules based on UHPLC-MS / MS according to claim 1, characterized in that: In step (5), the preparation of mixed reference solution 3 is as follows: Accurately measure an appropriate amount of the 21 single reference solutions from step (2), place them in a volumetric flask, and dilute with 50% methanol solution to prepare a solution containing 4.00 ng / mL p-hydroxybenzaldehyde reference, 8.00 ng / mL vanillin reference, 64.00 ng / mL caffeic acid reference, 4.00 ng / mL syringaldehyde reference, 25000.00 ng / mL citric acid reference, 120.00 ng / mL tanshinone reference, 8.00 ng / mL salinomycin reference, 1500.00 ng / mL baicalein reference, 4.00 ng / mL genistein reference, and 16.00 ng / mL salinomycin reference. A mixed reference solution of quercetin (mL), chlorogenic acid (32.00 ng / mL), neochlorogenic acid (32.00 ng / mL), rosmarinic acid (400.00 ng / mL), harpagoside (800.00 ng / mL), puerarin (0.96 ng / mL), apigenin-7-O-β-D-glucoside (0.96 ng / mL), baicalin (16000.00 ng / mL), oleanolic acid (32.00 ng / mL), baicalin (500.00 ng / mL), glycyrrhetinic acid (4.00 ng / mL), and harpagoside (240.00 ng / mL) is prepared for use.

8. The method for determining the content of multiple components of Compound Scutellaria baicalensis throat-clearing capsules based on UHPLC-MS / MS according to claim 1, characterized in that: In step (6), the preparation of mixed reference solution 4 is as follows: Accurately measure an appropriate amount of the 21 single reference solutions from step (2), place them in a volumetric flask, and dilute with 50% methanol solution to prepare a solution containing 8.00 ng / mL p-hydroxybenzaldehyde reference, 16.00 ng / mL vanillin reference, 128.00 ng / mL caffeic acid reference, 8.00 ng / mL syringaldehyde reference, 50000.00 ng / mL citric acid reference, 240.00 ng / mL tanshinone reference, 16.00 ng / mL salinomycin reference, 3000.00 ng / mL baicalein reference, 8.00 ng / mL genistein reference, and 32.00 ng / mL salinomycin reference. A mixed reference solution of quercetin (mL), chlorogenic acid (64.00 ng / mL), neochlorogenic acid (64.00 ng / mL), rosmarinic acid (800.00 ng / mL), harpagoside (1600.00 ng / mL), puerarin (1.92 ng / mL), apigenin-7-O-β-D-glucoside (1.92 ng / mL), baicalin (32000.00 ng / mL), oleanolic acid (64.00 ng / mL), baicalin (1000.00 ng / mL), glycyrrhetinic acid (8.00 ng / mL), and harpagoside (480.00 ng / mL) is prepared for use.

9. The method for determining the content of multiple components of Compound Scutellaria baicalensis throat-clearing capsules based on UHPLC-MS / MS according to claim 1, characterized in that: In step (7), the preparation of mixed reference solution 5 is as follows: Accurately measure an appropriate amount of the 21 single reference solutions from step (2), place them in a volumetric flask, and dilute with 50% methanol solution to prepare a solution containing 16.00 ng / mL p-hydroxybenzaldehyde reference, 32.00 ng / mL vanillin reference, 256.00 ng / mL caffeic acid reference, 16.00 ng / mL syringaldehyde reference, 100,000.00 ng / mL citric acid reference, 480.00 ng / mL tanshinone reference, 32.00 ng / mL salinomycin reference, 6000.00 ng / mL baicalein reference, 16.00 ng / mL genistein reference, and 64.00 ng / mL salinomycin. A mixed reference solution of quercetin (mL), chlorogenic acid (128.00 ng / mL), neochlorogenic acid (128.00 ng / mL), rosmarinic acid (1600.00 ng / mL), harpagoside (3200.00 ng / mL), puerarin (3.84 ng / mL), apigenin-7-O-β-D-glucoside (3.84 ng / mL), baicalin (64000.00 ng / mL), oleanolic acid (128.00 ng / mL), baicalin (2000.00 ng / mL), glycyrrhetinic acid (16.00 ng / mL), and harpagoside (960.00 ng / mL) is prepared for use.

10. The method for determining the content of multiple components of Compound Scutellaria baicalensis throat-clearing capsules based on UHPLC-MS / MS according to claim 1, characterized in that: In step (8), the preparation of mixed reference solution 6 is as follows: Accurately measure an appropriate amount of the 21 single reference solutions from step (2), place them in a volumetric flask, and dilute with 50% methanol solution to prepare a solution containing 32.00 ng / mL p-hydroxybenzaldehyde reference, 64.00 ng / mL vanillin reference, 512.00 ng / mL caffeic acid reference, 32.00 ng / mL syringaldehyde reference, 200,000.00 ng / mL citric acid reference, 960.00 ng / mL tanshinone reference, 64.00 ng / mL salinomycin reference, 12,000.00 ng / mL baicalein reference, 32.00 ng / mL genistein reference, and 128.00 ng / mL salinomycin reference. A mixed reference solution of quercetin (mL), chlorogenic acid (256.00 ng / mL), neochlorogenic acid (256.00 ng / mL), rosmarinic acid (3200.00 ng / mL), harpagoside (6400.00 ng / mL), puerarin (7.68 ng / mL), apigenin-7-O-β-D-glucoside (7.68 ng / mL), baicalin (128000.00 ng / mL), oleanolic acid (240.00 ng / mL), baicalin (4000.00 ng / mL), glycyrrhetinic acid (32.00 ng / mL), and harpagoside (1920.00 ng / mL) is prepared for use.

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