Method for detecting in vivo prototype components in an antiviral traditional Chinese medicine composition

Abstract

The present application relates to the technical field of pharmaceutical analysis, and discloses a method for detecting in-vivo prototype components in an antiviral traditional Chinese medicine composition. The method uses an octadecyl-bonded silica gel chromatographic column as a detection chromatographic column, 0.08%-0.12% formic acid aqueous solution-acetonitrile as a mobile phase, adopts a gradient elution procedure, and cooperates with specific mass spectrometry conditions to achieve comprehensive identification of 46 blood-entering prototype components in the antiviral traditional Chinese medicine composition, thereby providing reliable experimental basis for identifying the pharmacodynamic material basis of the antiviral traditional Chinese medicine composition. Meanwhile, the method is further conducive to studying the pharmacokinetic process of the prototype components in the antiviral traditional Chinese medicine composition in the body, and can screen pharmacokinetic markers of the antiviral traditional Chinese medicine composition according to the absorption of the compounds in the body, thereby laying a data foundation for in-depth study of the action mechanism of the antiviral traditional Chinese medicine composition.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical analysis, in particular to a method for detecting in-vivo prototype components in an antiviral traditional Chinese medicine composition. BACKGROUND

[0002] Patent CN1483463A discloses an antiviral traditional Chinese medicine composition composed of forsythia suspense, honeysuckle, fried ephedra, fried bitter apricot kernel, gypsum, isatis root, mao kuding, houttuynia cordata, patchouli, rhubarb, rhodiola, menthol and licorice, etc. Each component plays a different role in the medicine, and together determines the overall efficacy of the traditional Chinese medicine composition. The traditional Chinese medicine composition has the effects of dispelling pathogenic wind and detoxifying, and promoting lung and discharging heat, and is mainly used for treating symptoms such as fever, nasal congestion, runny nose, cough, headache, dry throat, sore throat, etc. caused by influenza.

[0003] As can be seen from the composition of the prescription, the composition of the antiviral traditional Chinese medicine composition is complex, and exploring its material basis and mechanism of action will play an important role in the clinical application of the traditional Chinese medicine composition and the development of new antiviral traditional Chinese medicine based on the theory of traditional Chinese medicine. At present, the research on the pharmacokinetics of the traditional Chinese medicine composition has been published in many literatures, which only describes the simple kinetics of the medicinal pairs or single herbs, and does not systematically describe the overall PK behavior characteristics. At present, there is a lack of systematic pharmacokinetic research. Therefore, in order to better reveal the pharmacodynamic material basis of the traditional Chinese medicine composition, it is necessary to develop a content determination method for detecting in-vivo prototype components in the traditional Chinese medicine composition, which will play a very important role in the clinical application of the traditional Chinese medicine composition and the development of new antiviral traditional Chinese medicine in the future. SUMMARY

[0004] In order to better reveal the pharmacodynamic material basis of the traditional Chinese medicine composition composed of forsythia suspense, honeysuckle, fried ephedra, fried bitter apricot kernel, gypsum, isatis root, mao kuding, houttuynia cordata, patchouli, rhubarb, rhodiola, menthol and licorice, etc., the present application provides a method for detecting in-vivo prototype components in the traditional Chinese medicine composition. Through the detection method provided by the present application, the in-vivo prototype components and metabolites can be determined, and the components with high exposure can be determined, which is convenient for monitoring the pharmacokinetic process.

[0005] To solve the above technical problems, the technical scheme provided by the present application is:

[0006] A detection method of in-vivo prototype components in an antiviral traditional Chinese medicine composition, the in-vivo prototype components of the antiviral traditional Chinese medicine composition include ephedrine, pseudoephedrine, methylephedrine, quinine acid, gallic acid, (R, S)-yohimbine, rhodioloside, neochlorogenic acid, forsythoside E, caffeic acid, amygdalin, wild black cherry glycoside, p-coumaric acid, broken oxygenated loganin, ferulic acid, glycyrrhizin, isoglycyrrhizin, forsythoside I, quercetrin, luteoloside, forsythoside B, hyperoside, forsythoside A, centaureidol I, isoquercitrin, kaempferol-O-rutinoside, rhein-8-O-beta-D-glucoside, 3, 5-di-O-caffeoylquinic acid, sophoricoside, 4, 5-di-O-caffeoylquinic acid, glycyrrhizin, forsythoside, emodin-8-O-beta-D-glucoside, quercitrin, luteolin, sophoricoside, aloe-emodin, isoglycyrrhizin, glycyrrhizic acid, rheic acid, emodin, beta-glycyrrhetic acid, chlorogenic acid, cryptochlorogenic acid, rutin and kaempferol, the detection is performed by using high performance liquid chromatography and mass spectrometry, wherein the high performance liquid chromatography conditions are as follows:

[0007] A C18 chromatographic column is used, 0.08%-0.12% formic acid aqueous solution is used as mobile phase A, and acetonitrile is used as mobile phase B, and gradient elution is adopted;

[0008] The mass spectrometry adopts an ESI ion source, and the scanning mode is an MRM monitoring mode.

[0009] It should be noted that the antiviral traditional Chinese medicine composition in the present application includes forsythia, honeysuckle, isatis root, bitter almond, menthol, houttuynia cordata, rhubarb, pachystachys jutanca, marmoraceae, rhodiolae, ephedra, licorice and gypsum.

[0010] Compared with the prior art, the detection method of in-vivo prototype components in the antiviral traditional Chinese medicine composition provided by the present application uses an octadecyl-bonded silica gel chromatographic column as a detection chromatographic column, uses 0.08%-0.12% formic acid aqueous solution-acetonitrile as a mobile phase, adopts a gradient elution program, and cooperates with specific mass spectrometry conditions, so that the defects of too many traditional Chinese medicine flavors, complex components and mutual interference in the antiviral traditional Chinese medicine composition are effectively overcome, and the 46 in-vivo prototype components in the antiviral traditional Chinese medicine composition are comprehensively identified and identified. The method provided by the present application is verified through methodology researches such as specificity, precision and stability, and it is found that the method has good accuracy and reproducibility, which provides reliable experimental basis for identifying the pharmacodynamic material basis of the antiviral traditional Chinese medicine composition. Meanwhile, it is further beneficial to study the pharmacokinetic process of the prototype components in the above traditional Chinese medicine composition in the body, and the pharmacokinetic markers of the traditional Chinese medicine composition can also be screened according to the absorption of the compounds in the body, which lays a data foundation for in-depth study on the action mechanism of the traditional Chinese medicine composition.

[0011] As a specific embodiment of the present application, if the detected in vivo prototype components are neochlorogenic acid, p-coumaric acid, secooxyloganin, glycyrrhizin, isoglycyrrhizin, apigenin glycoside, jafficin, hyperoside, isoquercitrin, rhein-8-O-β-D-glucoside, 3,5-di-O-caffeoylquinic acid, sophoricoside, 4,5-di-O-caffeoylquinic acid, glycyrrhizin, forsythoside, emodin-8-O-β-D-glucoside, quercitrin, jafficin, sophoricoside, aloe-emodin, isorhamnetin, glycyrrhizic acid, rheic acid, emodin, β-glycyrrhetic acid, chlorogenic acid, cryptochlorogenic acid, rutin and kaempferol, the high performance liquid chromatography conditions are as follows:

[0012] Column: CAPCELL CORE C18, 2.1 mm x 75 mm, 2.7 μm;

[0013] Mobile phase A: 0.08%-0.12% formic acid aqueous solution;

[0014] Mobile phase B: acetonitrile;

[0015] 0-1 min, 95%→90% mobile phase A, 5%→10% mobile phase B;

[0016] 1-2.5 min, 90%→89% mobile phase A, 10%→11% mobile phase B;

[0017] 2.5-3 min, 89%→82.5% mobile phase A, 11%→17.5% mobile phase B;

[0018] 3-4.5 min, 82.5% mobile phase A, 17.5% mobile phase B;

[0019] 4.5-6 min, 82.5%→20% mobile phase A, 17.5%→80% mobile phase B;

[0020] 6-7 min, 20%→17% mobile phase A, 80%→83% mobile phase B;

[0021] 7-7.01 min, 17%→95% mobile phase A, 83%→5% mobile phase B;

[0022] 7.01-9 min, 95% mobile phase A, 5% mobile phase B.

[0023] Further, the column temperature is 35°C, the flow rate is 0.4 mL / min, and the injection volume is 10 μL.

[0024] The preferred high performance liquid chromatography conditions can improve the separation degree between each component peak and the detection sensitivity, so that the detection result is accurate and quantitative, and the precision is high.

[0025] As a specific embodiment of the present application, the mass spectrometer adopts negative ion detection mode, the capillary voltage is 5.5 kV, the nebulizing gas flow rate is 55 L / min, the dissolving gas flow rate is 20 L / min, the collision gas flow rate is 9 L / min, and the heating block temperature is 550 ℃.

[0026] The parent ion of neochlorogenic acid is 353 m / z, the daughter ion is 191 m / z, the declustering voltage is 76 V, and the fragmentation voltage is 31 eV; the parent ion of p-coumaric acid is 163 m / z, the daughter ion is 119 m / z, the declustering voltage is 66 V, and the fragmentation voltage is 23 eV; the parent ion of deoxyloganin is 403.2 m / z, the daughter ion is 179.2 m / z, the declustering voltage is 86 V, and the fragmentation voltage is 18 eV; the parent ion of glycyrrhizin is 417.3 m / z, the daughter ion is 135.1 m / z, the declustering voltage is 125 V, and the fragmentation voltage is 39 eV; the parent ion of isoglycyrrhizin is 417.2 m / z, the daughter ion is 148.1 m / z, the declustering voltage is 127 V, and the fragmentation voltage is 48 eV; the parent ion of trypsin is 549.1 m / z, the daughter ion is 255.3 m / z, the declustering voltage is 146 V, and the fragmentation voltage is 45 eV; the parent ion of luteoloside is 447.1 m / z, the daughter ion is 285.3 m / z, the declustering voltage is 154 V, and the fragmentation voltage is 45 eV; the parent ion of hyperoside is 463 m / z, the daughter ion is 300.7 m / z, the declustering voltage is 125 V, and the fragmentation voltage is 36 eV; the parent ion of isoquercitrin is 463.2 m / z, the daughter ion is 255 m / z, the declustering voltage is 113 V, and the fragmentation voltage is 58 eV; the parent ion of rhein-8-O-β-D-glucoside is 445 m / z, the daughter ion is 239 m / z, the declustering voltage is 85 V, and the fragmentation voltage is 35 eV; the parent ion of 3,5-di-O-caffeoylquinic acid is 515.3 m / z, the daughter ion is 191.2 m / z, the declustering voltage is 91 V, and the fragmentation voltage is 14 eV; the parent ion of ononin is 475.1 m / z, the daughter ion is 267 / z, the declustering voltage is 65 V, and the fragmentation voltage is 20 eV; the parent ion of 4,5-di-O-caffeoylquinic acid is 515.2 m / z, the daughter ion is 173 m / z, the declustering voltage is 81 V, and the fragmentation voltage is 39 eV; the parent ion of glycyrrhizin is 255 m / z, the daughter ion is 135.1 m / z, the declustering voltage is 83 V, and the fragmentation voltage is 20 eV; the parent ion of forsythoside is 579.2 m / z, the daughter ion is 371.4 m / z, the declustering voltage is 80 V, and the fragmentation voltage is 40 eV; the parent ion of emodin-8-O-β-D-glucoside is 431.2 m / z, the daughter ion is 269.2 m / z, the declustering voltage is 123 V, and the fragmentation voltage is 39 eV; the parent ion of quercitrin is 447.2 m / z, the daughter ion is 300.2 m / z, the declustering voltage is 99 V, and the fragmentation voltage is 38 eV; the parent ion of luteolin is 285.1 m / z, the daughter ion is 151.1 m / z, the declustering voltage is 103 V, and the fragmentation voltage is 35 eV; the parent ion of onocyanin is 267.1 m / z, the daughter ion is 252.2 m / z, cluster voltage 106 V, cracking voltage 30 eV; aloe-emodin parent ion 269 m / z, daughter ion 240 m / z, cluster voltage 104 V, cracking voltage 31 eV; isoliquiritigenin parent ion 255.1 m / z, daughter ion 119.1 m / z, cluster voltage 5 V, cracking voltage 42 eV; glycyrrhizic acid parent ion 821.5 m / z, daughter ion 350.9 m / z, cluster voltage 52 V, cracking voltage 41 eV; rhein parent ion 283.2 m / z, daughter ion 183.1 m / z, cluster voltage 52 V, cracking voltage 38 eV; emodin parent ion 269 m / z, daughter ion 241 m / z, cluster voltage 127 V, cracking voltage 38 eV; β-glycyrrhetic acid parent ion 469.3 m / z, daughter ion 355.3 m / z, cluster voltage 151 V, cracking voltage 67 eV; chlorogenic acid parent ion 353.3 m / z, daughter ion 191.1 m / z, cluster voltage 64 V, cracking voltage 22 eV; cryptochlorogenic acid parent ion 353.2 m / z, daughter ion 173.1 m / z, cluster voltage 76 V, cracking voltage 22 eV; rutin parent ion 609.1 m / z, daughter ion 300.2 m / z, cluster voltage 134 V, cracking voltage 50 eV; kaempferol parent ion 285 m / z, daughter ion 210.9 m / z, cluster voltage 85 V, cracking voltage 45 eV.

[0027] The preferred mass spectrometry detection condition can maximize the accuracy of determination of the multiple blood-entering prototype components in the anti-virus traditional Chinese medicine composition capsule.

[0028] As a specific embodiment of the present application, if the in-vivo prototype component to be detected is quinic acid, gallic acid, rhodioloside, caffeic acid, wild blackcherry glycoside, forsythoside E, forsythoside A, forsythoside B, forsythoside I and ferulic acid, the high performance liquid chromatography condition is as follows:

[0029] Chromatographic column: CAPCELL PAK C18, 2.0 mm x 50 mm, 2 μm;

[0030] Mobile phase A: 0.08%-0.12% formic acid aqueous solution;

[0031] Mobile phase B: acetonitrile;

[0032] 0-1 min, 95%→83% mobile phase A, 5%→17% mobile phase B;

[0033] 1-3.5 min, 83%→80% mobile phase A, 17%→20% mobile phase B;

[0034] 3.5-4min, 80%→35% mobile phase A, 20%→65% mobile phase B;

[0035] 4-6min, 35%→5% mobile phase A, 65%→95% mobile phase B;

[0036] 6-6.01min, 5%→95% mobile phase A, 95%→5% mobile phase B;

[0037] 6.01-7.5min, 95% mobile phase A, 5% mobile phase B.

[0038] Further, the column temperature is 35°C, the flow rate is 0.35mL / min, and the injection volume is 10μL.

[0039] Further, the mobile phase A is 0.1% formic acid aqueous solution.

[0040] The preferred high performance liquid chromatography conditions can improve the separation degree and detection sensitivity between the peaks of quinic acid, gallic acid, rhodioloside, caffeic acid, wild black cherry glycoside, forsythoside E, forsythoside A, forsythoside B, forsythoside I and ferulic acid components, so that the detection result is accurate and precise.

[0041] Further, the mass spectrometry adopts negative ion detection mode, the capillary voltage is 4kV, the atomizing gas flow rate is 10L / min, the dissolving gas flow rate is 30L / min, the desolvent gas line temperature is 325°C, and the heating block temperature is 325°C.

[0042] The parent ion of quinic acid is 191.1 m / z, the daughter ion is 85 m / z, the declustering voltage is 130 V, and the fragmentation voltage is 25 eV; the parent ion of gallic acid is 169.1 m / z, the daughter ion is 125.1 m / z, the declustering voltage is 90 V, and the fragmentation voltage is 10 eV; the parent ion of salidroside is 345.1 m / z, the daughter ion is 119 m / z, the declustering voltage is 75 V, and the fragmentation voltage is 15 eV; the parent ion of caffeic acid is 179 m / z, the daughter ion is 135.1 m / z, the declustering voltage is 85 V, and the fragmentation voltage is 5 eV; the parent ion of wild blackcherry glycoside is 340.1 m / z, the daughter ion is 161.1 m / z, the declustering voltage is 85 V, and the fragmentation voltage is 10 eV; the parent ion of forsythoside E is 461.1 m / z, the daughter ion is 135 m / z, the declustering voltage is 105 V, and the fragmentation voltage is 35 eV; the parent ion of forsythoside A is 623.2 m / z, the daughter ion is 161.1 m / z, the declustering voltage is 180 V, and the fragmentation voltage is 45 eV; the parent ion of forsythoside B is 755.2 m / z, the daughter ion is 161.1 m / z, the declustering voltage is 135 V, and the fragmentation voltage is 50 eV; the parent ion of forsythoside I is 623.2 m / z, the daughter ion is 161.1 m / z, the declustering voltage is 145 V, and the fragmentation voltage is 40 eV; the parent ion of ferulic acid is 193 m / z, the daughter ion is 133.9 m / z, the declustering voltage is 85 V, and the fragmentation voltage is 15 eV.

[0043] The preferred mass spectrometry detection condition can maximize the accuracy of determination of the multiple blood-entering prototype components in the antiviral traditional Chinese medicine composition.

[0044] As a specific embodiment of the present application, if the in vivo prototype component to be detected is (R,S)-emetine, pseudoephedrine, ephedrine, methylephedrine, amygdalin, centaureidin I and kaempferol-O-rutinoside, the high performance liquid chromatography condition is as follows:

[0045] Chromatographic column: CAPCELL PAK C18, 2.0 mm x 50 mm, 2 μm;

[0046] Mobile phase A: 0.08%-0.12% formic acid aqueous solution;

[0047] Mobile phase B: acetonitrile;

[0048] 0-3 min, 98%→95% mobile phase A, 2%→5% mobile phase B;

[0049] 3-3.5 min, 95%→75% mobile phase A, 5%→25% mobile phase B;

[0050] 3.5-6 min, 75%→60% mobile phase A, 25%→30% mobile phase B;

[0051] 6-6.01 min, 70%→98% mobile phase A, 30%→2% mobile phase B;

[0052] 6.01-7.5 min, 98% mobile phase A, 2% mobile phase B.

[0053] Further, the column temperature is 25°C, the flow rate is 0.35 mL / min, and the injection volume is 20 μL.

[0054] Further, the mobile phase A is 0.1% formic acid aqueous solution.

[0055] The preferred high performance liquid chromatography conditions can improve the separation degree and the sensitivity of detection between the peaks of (R,S)-ademetionine, pseudoephedrine, ephedrine, methylephedrine, amygdalin, centaureidin I and kaempferol-O-rutinoside components, so that the detection result is quantitatively accurate and has high precision.

[0056] Further, the mass spectrometry adopts a positive ion detection mode, the capillary voltage is 4 kV, the flow rate of atomization gas is 10 L / min, the flow rate of dissolving gas is 30 L / min, the temperature of desolvation gas line is 325°C, and the temperature of heating block is 325°C.

[0057] The parent ion of (R,S)-ademetionine is 130.1 m / z, the daughter ion is 70.2 m / z, the declustering voltage is 60 V, and the fragmentation voltage is 5 eV; the parent ion of pseudoephedrine is 166.2 m / z, the daughter ion is 133.2 m / z, the declustering voltage is 70 V, and the fragmentation voltage is 20 eV; the parent ion of ephedrine is 166.2 m / z, the daughter ion is 148.2 m / z, the declustering voltage is 70 V, and the fragmentation voltage is 10 eV; the parent ion of methylephedrine is 180.2 m / z, the daughter ion is 91.2 m / z, the declustering voltage is 85 V, and the fragmentation voltage is 40 eV; the parent ion of amygdalin is 475.1 m / z, the daughter ion is 163.1 m / z, the declustering voltage is 120 V, and the fragmentation voltage is 10 eV; the parent ion of centaureidin I is 595.2 m / z, the daughter ion is 286.9 m / z, the declustering voltage is 120 V, and the fragmentation voltage is 45 eV; the parent ion of kaempferol-O-rutinoside is 595.2 m / z, the daughter ion is 286.9 m / z, the declustering voltage is 120 V, and the fragmentation voltage is 40 eV.

[0058] The preferred mass spectrometry detection conditions can maximize the accuracy of the determination of the multiple blood-entering original components in the antiviral traditional Chinese medicine composition.

[0059] Specifically, the preparation of the reference substance solution includes the following steps:

[0060] Accurately weigh a certain amount of ephedrine, pseudoephedrine, methylephedrine, quinic acid, gallic acid, (R, S)-yohimbine, rhodioloside, neochlorogenic acid, forsythoside E, caffeic acid, amygdalin, wild blackcherry glycoside, p-coumaric acid, seco-oxymatrine, ferulic acid, glycyrrhizin, isoglycyrrhizin, forsythoside I, sericoside, luteoloside, forsythoside B, hyperoside, forsythoside A, centaureidin I, isoquercitrin, kaempferol-O-rutinoside, rhein-8-O-β-D-glucoside, 3,5-di-O-caffeoylquinic acid, mutamoside, 4,5-di-O-caffeoylquinic acid, glycyrrhizin, forsythoside, emodin-8-O-β-D-glucoside, quercitrin, luteolin, mutamosine, aloeemodin, isoglycyrrhizin, glycyrrhizic acid, rheic acid, emodin, β-glycyrrhetic acid, chlorogenic acid, cryptochlorogenic acid, rutin and kaempferol reference substances, dissolve in methanol to prepare each reference substance mother liquor with a concentration of 2 mg / mL.

[0061] Mixing stock solution 1: accurately weigh the above prepared reference substance mother liquor, dilute with 50% methanol solution to prepare a mixing stock solution 1 containing 24 μg / mL (ephedrine, pseudoephedrine, methylephedrine, ferulic acid, glycyrrhizin, isoglycyrrhizin, sericoside, luteoloside, forsythoside B, rutin, hyperoside, forsythoside A, centaureidin I, isoquercitrin, kaempferol-O-rutinoside, rhein-8-O-β-D-glucoside, 3,5-di-O-caffeoylquinic acid, mutamoside, 4,5-di-O-caffeoylquinic acid, glycyrrhizin, emodin-8-O-β-D-glucoside, quercitrin, luteolin, mutamosine, aloeemodin, isoglycyrrhizin, emodin); 120 μg / mL ((R, S)-yohimbine, amygdalin, caffeic acid, forsythoside I, β-glycyrrhetic acid, seco-oxymatrine, neochlorogenic acid).

[0062] Mixing stock solution 2: accurately weigh the above prepared reference substance mother liquor, dilute with 50% methanol solution to prepare a mixing stock solution 2 containing 480 μg / mL (rheic acid, wild blackcherry glycoside); 72 μg / mL (gallic acid, forsythoside E, glycyrrhizic acid, kaempferol).

[0063] Mixing stock solution 3: accurately weigh the above prepared reference substance mother liquor, dilute with 50% methanol solution to prepare a mixing stock solution 3 containing 240 μg / mL (rhodioloside, quinic acid, cryptochlorogenic acid, chlorogenic acid, p-coumaric acid).

[0064] Take 50 μL of the above mixing stock solution 1, mixing stock solution 2 and mixing stock solution 3, add 450 μL of 50% methanol solution to obtain a mixed reference working solution.

[0065] The above mixed control working solution was precisely pipetted 0.5 μL, 1.5 μL, 5 μL, 15 μL, 40 μL, 60 μL, 80 μL, respectively, and diluted with 50% methanol solution to 100 μL, to obtain a series of concentration mixed control solutions, which were used to establish the standard curve for quantitative detection of the corresponding substances.

[0066] Further, the content of 46 components in the antiviral traditional Chinese medicine composition is calculated by using an internal standard method, and the internal standard substances are selected from gallic acid-d2 (IS-1), ferulic acid-d3 (IS-2) and acetaminophen (IS-3).

[0067] Specifically, the test sample in the present application is Lianhuaqingwen capsule.

[0068] Specifically, the preparation of the test sample solution comprises the following steps:

[0069] S1, an appropriate amount of Lianhuaqingwen capsule content is weighed, and a dosing solution with a concentration of 0.80 g / mL is prepared with ultrapure water, the dosing dose is 12 g / kg, and the dosing volume is 15 mL / kg;

[0070] S2, the mouse is given 12 g / kg of Lianhuaqingwen dosing solution at a time, and then 0.5 mL of blood is taken from the orbit at 5 min, 15 min, 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, 9 h, 12 h and 24 h after the last administration, and placed in a heparin sodium tube, centrifuged at a centrifugal force of 2348 g for 10 min, and 60 μL of supernatant is accurately absorbed into a 1.5 mL EP tube, and stored in a-80℃ refrigerator;

[0071] S3, 0.2% ice NaCl solution is used to prepare 100 U / 5 μL of β-glucuronidase and 1 U / 5 μL of sulfatase solution, and a mixed enzyme is obtained;

[0072] S4, 5 μL of internal standard working solution and 10 μL of mixed enzyme are added to 60 μL of plasma sample, and incubated at 37℃ water bath for 1 h, and the enzyme hydrolysis is quickly stopped in a-80℃ refrigerator, three times of methanol (180 μL) is added, vortexed for 1 min, centrifuged at a centrifugal force of 2348 g for 5 min, and the supernatant is taken, nitrogen blowing, and 100 μL of initial mobile phase (0.1% formic acid water:acetonitrile (v:v=95:5)) (100 μL) is redissolved, centrifuged at a centrifugal force of 15871 g for 10 min, and a test sample solution is obtained.

[0073] It has important significance to develop and research traditional Chinese medicine by modern method, and provides an objective and overall evaluation basis for the quality of traditional Chinese medicine. The HPLC-MS / MS method is used for establishing the blood plasma quantitative analysis method of related components in 46 antiviral traditional Chinese medicine compositions, including alkaloid components in ephedra herb, lignan, phenethyl alcohol, flavonoid glycoside components in forsythia suspense herb, triterpenoid saponin components in licorice herb, flavone, organic acid components in honeysuckle flower herb, anthraquinone components in rhubarb herb, cyanogenic glycoside components in fried bitter apricot kernel herb and the like. The detection method is verified in terms of selectivity, accuracy, precision and stability, and the method is simple and accurate, and meets the requirements of ICH M10 for biological analysis method. By the detection method, the pharmacokinetics of the blood entering original components in the antiviral traditional Chinese medicine composition in the body can be conveniently researched, and the pharmacokinetic markers of the antiviral traditional Chinese medicine composition can be screened according to the absorption of the compounds in the body, and the method has high practical value. BRIEF DESCRIPTION OF DRAWINGS

[0074] Figure 1 Figure (B) is a partial enlarged view of the green square frame in figure (A), and figure (C) is a partial enlarged view of the orange square frame in figure (A);

[0075] Figure 2 Figure (B) is a partial enlarged view of the green square frame in figure (A), and figure (C) is a partial enlarged view of the orange square frame in figure (A);

[0076] Figure 3 Figure (B) is a partial enlarged view of the green square frame in figure (A), and figure (C) is a partial enlarged view of the orange square frame in figure (A);

[0077] Figure 4 Figure (B) is a partial enlarged view of the green square frame in figure (A), and figure (C) is a partial enlarged view of the orange square frame in figure (A);

[0078] Figure 5 Figure (B) is a partial enlarged view of the green square frame in figure (A), and figure (C) is a partial enlarged view of the orange square frame in figure (A);

[0079] Figure 6 Figure (B) is a partial enlarged view of the green square frame in figure (A), and figure (C) is a partial enlarged view of the orange square frame in figure (A);

[0080] Figure 7 MRM chromatograms for amygdalin under item 2.4 in Example 2, where (A) blank plasma + internal standard, (B) lower limit of quantitation concentration sample LLOQ, (C) real rat plasma sample collected at 15 min post single dose of LHQW;

[0081] Figure 8 MRM chromatograms for hyperoside I and kaempferol-3-O-rutinoside under item 2.4 in Example 2, where (A) blank plasma + internal standard, (B) lower limit of quantitation concentration sample LLOQ, (C) real rat plasma sample collected at 15 min post single dose of LHQW;

[0082] Figure 9 MRM chromatograms for caffeic acid under item 2.4 in Example 2, where (A) blank plasma + internal standard, (B) lower limit of quantitation concentration sample LLOQ, (C) real rat plasma sample collected at 15 min post single dose of LHQW;

[0083] Figure 10 MRM chromatograms for quinic acid under item 2.4 in Example 2, where (A) blank plasma + internal standard, (B) lower limit of quantitation concentration sample LLOQ, (C) real rat plasma sample collected at 15 min post single dose of LHQW;

[0084] Figure 11 MRM chromatograms for gallic acid under item 2.4 in Example 2, where (A) blank plasma + internal standard, (B) lower limit of quantitation concentration sample LLOQ, (C) real rat plasma sample collected at 15 min post single dose of LHQW;

[0085] Figure 12 MRM chromatograms for ferulic acid under item 2.4 in Example 2, where (A) blank plasma + internal standard, (B) lower limit of quantitation concentration sample LLOQ, (C) real rat plasma sample collected at 15 min post single dose of LHQW;

[0086] Figure 13 MRM chromatograms for forsythoside A and forsythoside I under item 2.4 in Example 2, where (A) blank plasma + internal standard, (B) lower limit of quantitation concentration sample LLOQ, (C) real rat plasma sample collected at 15 min post single dose of LHQW;

[0087] Figure 14 MRM chromatograms for forsythoside B under item 2.4 in Example 2, where (A) blank plasma + internal standard, (B) lower limit of quantitation concentration sample LLOQ, (C) real rat plasma sample collected at 15 min post single dose of LHQW;

[0088] Figure 15 MRM chromatogram of forsythoside E under item 2.4 in Example 2, wherein (A) blank plasma + internal standard, (B) sample at lower limit of quantification LLOQ, (C) real rat plasma sample collected at 15 min after single-dose administration of LHQW;

[0089] Figure 16 MRM chromatogram of salidroside under item 2.4 in Example 2, wherein (A) blank plasma + internal standard, (B) sample at lower limit of quantification LLOQ, (C) real rat plasma sample collected at 15 min after single-dose administration of LHQW;

[0090] Figure 17 MRM chromatogram of prunusin under item 2.4 in Example 2, wherein (A) blank plasma + internal standard, (B) sample at lower limit of quantification LLOQ, (C) real rat plasma sample collected at 15 min after single-dose administration of LHQW;

[0091] Figure 18 MRM chromatogram of neochlorogenic acid under item 2.4 in Example 2, wherein (A) blank plasma + internal standard, (B) sample at lower limit of quantification LLOQ, (C) real rat plasma sample collected at 15 min after single-dose administration of LHQW;

[0092] Figure 19 MRM chromatogram of chlorogenic acid under item 2.4 in Example 2, wherein (A) blank plasma + internal standard, (B) sample at lower limit of quantification LLOQ, (C) real rat plasma sample collected at 15 min after single-dose administration of LHQW;

[0093] Figure 20 MRM chromatogram of cryptochlorogenic acid under item 2.4 in Example 2, wherein (A) blank plasma + internal standard, (B) sample at lower limit of quantification LLOQ, (C) real rat plasma sample collected at 15 min after single-dose administration of LHQW;

[0094] Figure 21 MRM chromatogram of seco-oxymatrine under item 2.4 in Example 2, wherein (A) blank plasma + internal standard, (B) sample at lower limit of quantification LLOQ, (C) real rat plasma sample collected at 15 min after single-dose administration of LHQW;

[0095] Figure 22 MRM chromatogram of p-coumaric acid under item 2.4 in Example 2, wherein (A) blank plasma + internal standard, (B) sample at lower limit of quantification LLOQ, (C) real rat plasma sample collected at 15 min after single-dose administration of LHQW;

[0096] Figure 23MRM chromatogram of rutin under item 2.4 in Example 2, wherein (A) blank plasma + internal standard, (B) lower limit of quantification concentration sample LLOQ, (C) real rat plasma sample collected at 15 min after single-dose administration of LHQW;

[0097] Figure 24 MRM chromatogram of sylvestroside A under item 2.4 in Example 2, wherein (A) blank plasma + internal standard, (B) lower limit of quantification concentration sample LLOQ, (C) real rat plasma sample collected at 15 min after single-dose administration of LHQW;

[0098] Figure 25 MRM chromatogram of glycyrrhizin under item 2.4 in Example 2, wherein (A) blank plasma + internal standard, (B) lower limit of quantification concentration sample LLOQ, (C) real rat plasma sample collected at 15 min after single-dose administration of LHQW;

[0099] Figure 26 MRM chromatogram of rhein-8-β-D-glucoside under item 2.4 in Example 2, wherein (A) blank plasma + internal standard, (B) lower limit of quantification concentration sample LLOQ, (C) real rat plasma sample collected at 15 min after single-dose administration of LHQW;

[0100] Figure 27 MRM chromatogram of hyperoside under item 2.4 in Example 2, wherein (A) blank plasma + internal standard, (B) lower limit of quantification concentration sample LLOQ, (C) real rat plasma sample collected at 15 min after single-dose administration of LHQW;

[0101] Figure 28 MRM chromatogram of isoquercitrin under item 2.4 in Example 2, wherein (A) blank plasma + internal standard, (B) lower limit of quantification concentration sample LLOQ, (C) real rat plasma sample collected at 15 min after single-dose administration of LHQW;

[0102] Figure 29 MRM chromatogram of jujuboside A under item 2.4 in Example 2, wherein (A) blank plasma + internal standard, (B) lower limit of quantification concentration sample LLOQ, (C) real rat plasma sample collected at 15 min after single-dose administration of LHQW;

[0103] Figure 30 MRM chromatogram of 3,5-O-caffeoylquinic acid under item 2.4 in Example 2, wherein (A) blank plasma + internal standard, (B) lower limit of quantification concentration sample LLOQ, (C) real rat plasma sample collected at 15 min after single-dose administration of LHQW;

[0104] Figure 31MRM chromatogram of 4,5-O-caffeoylquinic acid under item 2.4 in Example 2, where (A) blank plasma + internal standard, (B) lower limit of quantification concentration sample LLOQ, (C) real rat plasma sample collected at 15 min after single dose of LHQW;

[0105] Figure 32 MRM chromatogram of quercitrin under item 2.4 in Example 2, where (A) blank plasma + internal standard, (B) lower limit of quantification concentration sample LLOQ, (C) real rat plasma sample collected at 15 min after single dose of LHQW;

[0106] Figure 33 MRM chromatogram of isoliquiritigenin under item 2.4 in Example 2, where (A) blank plasma + internal standard, (B) lower limit of quantification concentration sample LLOQ, (C) real rat plasma sample collected at 15 min after single dose of LHQW;

[0107] Figure 34 MRM chromatogram of formononetin under item 2.4 in Example 2, where (A) blank plasma + internal standard, (B) lower limit of quantification concentration sample LLOQ, (C) real rat plasma sample collected at 15 min after single dose of LHQW;

[0108] Figure 35 MRM chromatogram of phillyrin under item 2.4 in Example 2, where (A) blank plasma + internal standard, (B) lower limit of quantification concentration sample LLOQ, (C) real rat plasma sample collected at 15 min after single dose of LHQW;

[0109] Figure 36 MRM chromatogram of glycyrrhizin under item 2.4 in Example 2, where (A) blank plasma + internal standard, (B) lower limit of quantification concentration sample LLOQ, (C) real rat plasma sample collected at 15 min after single dose of LHQW;

[0110] Figure 37 MRM chromatogram of emodin-8-β-D-glucoside under item 2.4 in Example 2, where (A) blank plasma + internal standard, (B) lower limit of quantification concentration sample LLOQ, (C) real rat plasma sample collected at 15 min after single dose of LHQW;

[0111] Figure 38 MRM chromatogram of luteolin under item 2.4 in Example 2, where (A) blank plasma + internal standard, (B) lower limit of quantification concentration sample LLOQ, (C) real rat plasma sample collected at 15 min after single dose of LHQW;

[0112] Figure 39MRM chromatograms for glycyrrhizic acid under item 2.4 in Example 2, where (A) blank plasma + internal standard, (B) lower limit of quantitation concentration sample LLOQ, (C) real rat plasma sample collected at 15 min post single dose of LHQW;

[0113] Figure 40 MRM chromatograms for kaempferol under item 2.4 in Example 2, where (A) blank plasma + internal standard, (B) lower limit of quantitation concentration sample LLOQ, (C) real rat plasma sample collected at 15 min post single dose of LHQW;

[0114] Figure 41 MRM chromatograms for isoliquiritigenin under item 2.4 in Example 2, where (A) blank plasma + internal standard, (B) lower limit of quantitation concentration sample LLOQ, (C) real rat plasma sample collected at 15 min post single dose of LHQW;

[0115] Figure 42 MRM chromatograms for formononetin under item 2.4 in Example 2, where (A) blank plasma + internal standard, (B) lower limit of quantitation concentration sample LLOQ, (C) real rat plasma sample collected at 15 min post single dose of LHQW;

[0116] Figure 43 MRM chromatograms for aloe-emodin under item 2.4 in Example 2, where (A) blank plasma + internal standard, (B) lower limit of quantitation concentration sample LLOQ, (C) real rat plasma sample collected at 15 min post single dose of LHQW;

[0117] Figure 44 MRM chromatograms for rhein under item 2.4 in Example 2, where (A) blank plasma + internal standard, (B) lower limit of quantitation concentration sample LLOQ, (C) real rat plasma sample collected at 15 min post single dose of LHQW;

[0118] Figure 45 MRM chromatograms for emodin under item 2.4 in Example 2, where (A) blank plasma + internal standard, (B) lower limit of quantitation concentration sample LLOQ, (C) real rat plasma sample collected at 15 min post single dose of LHQW;

[0119] Figure 46 MRM chromatograms for β-glycyrrhetic acid under item 2.4 in Example 2, where (A) blank plasma + internal standard, (B) lower limit of quantitation concentration sample LLOQ, (C) real rat plasma sample collected at 15 min post single dose of LHQW;

[0120] Figure 47 Pharmacokinetic profiles of quinic acid, ephedrine and pseudoephedrine in rats in Example 3;

[0121] Figure 48 Pharmacokinetic profiles of gallic acid, methylephedrine and (R,S)-ajmalicine in rats for Example 3;

[0122] Figure 49 Pharmacokinetic profiles of rhodiosin, neochlorogenic acid and forsythoside E in rats for Example 3;

[0123] Figure 50 Pharmacokinetic profiles of caffeic acid, amygdalin and prunasin in rats for Example 3;

[0124] Figure 51 Pharmacokinetic profiles of cryptochlorogenic acid, chlorogenic acid and p-coumaric acid in rats for Example 3;

[0125] Figure 52 Pharmacokinetic profiles of deoxyvomifoliol, ferulic acid and glycyrrhizin in rats for Example 3;

[0126] Figure 53 Pharmacokinetic profiles of isoglycyrrhizin, forsythoside I and apiosyl glycyrrhizin in rats for Example 3;

[0127] Figure 54 Pharmacokinetic profiles of jujuboside, forsythoside B and rutin in rats for Example 3;

[0128] Figure 55 Pharmacokinetic profiles of hyperoside, forsythoside A and centaureidin I in rats for Example 3;

[0129] Figure 56 Pharmacokinetic profiles of isoquercitrin, kaempferol-O-rutinoside, rhein-8-O-β-D-glucoside in rats for Example 3;

[0130] Figure 57 Pharmacokinetic profiles of 3,5-di-O-caffeoylquinic acid, mutamoside and 4,5-di-O-caffeoylquinic acid in rats for Example 3

[0131] Figure 58 Pharmacokinetic profiles of glycyrrhizin, forsythoside and emodin-8-O-β-D-glucoside in rats for Example 3;

[0132] Figure 59 Pharmacokinetic profiles of quercitrin, luteolin and kaempferol in rats for Example 3;

[0133] Figure 60 Pharmacokinetic profiles of mutamolin, aloeemodin and isoglycyrrhizin in rats for Example 3;

[0134] Figure 61Pharmacokinetic profile of glycyrrhizic acid, rhein and emodin in rats in Example 3.

[0135] Figure 62 Pharmacokinetic profile of β-glycyrrhetic acid in rats in Example 3. DETAILED DESCRIPTION

[0136] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and should not be used to limit the present application.

[0137] All solvents and reagents used in the following examples are chromatographically pure (HPLC) or analytically pure (AR), methanol, HPLC grade, purchased from TEDIA. Acetonitrile, HPLC grade, purchased from TEDIA. Formic acid HPLC grade purchased from Anaqua Chemical. The water used in the experiment is all deionized ultrapure water.

[0138] Example 1

[0139] 1. Reagents

[0140] β-glucuronidase (specification: 1918 U / mg, a total of 52.2 mg), sulfatase (specification: 30700 U / g, a total of 162.9 mg), acetaminophen (IS-1) (HPLC, purity > 98%) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0141] Lianhuaqingwen Capsule Dry Concentrate, provided by Shijiazhuang Yiling Pharmaceutical Co., Ltd., contains 8.41 g of raw medicinal materials per gram.

[0142] Ephedrine, pseudoephedrine and methylephedrine were purchased from China Food and Drug Control Institute (Beijing, China). Quinic acid, gallic acid, (R,S)-yohimbine, rhodiosin, neochlorogenic acid, forsythoside E, caffeic acid, amygdalin, prunol, p-coumaric acid, secoxyloganin, ferulic acid, glycyrrhizin, isoglycyrrhizin, forsythoside I, apigenin glycoside, luteoloside, forsythoside B, hyperoside, forsythoside A, centaureidin I, isoquercitrin, kaempferol-O-rutinoside, rhein-8-O-β-D-glucoside, 3,5-di-O-caffeoylquinic acid, mutamoside, 4,5-di-O-caffeoylquinic acid, glyasperin, forsythoside, emodin-8-O-β-D-glucoside, quercitrin, luteolin, mutamoside, aloeemodin, isoglycyrrhizin, glycyrrhizic acid, rheic acid, emodin, β-glycyrrhetic acid were purchased from Baoji Chen Guang Biological Co., Ltd (Baoji, China). Prunol was purchased from Yunnan Xili Biological Technology Co., Ltd (Yunnan, China). Chlorogenic acid, cryptochlorogenic acid, rutin and kaempferol were purchased from Chengdu Zhishang Hapure Biological Technology Co., Ltd (Chengdu, China). The purity of all control substances was greater than 98%.

[0143] Gallic acid-d2 (IS-2) and ferulic acid-d3 (IS-3) were purchased from Toronto Research Chemicals (Toronto, Canada).

[0144] 1.2 Drugs

[0145] Lianhuaqingwen Capsules content was provided by Shijiazhuang Yiling Pharmaceutical Co., Ltd (China); specifications: 8.39 g of crude drug / g dry paste, 0.240 g per unit of preparation.

[0146] 1.3 Experimental animals

[0147] SD rats, male; clean grade, age: 7-8 weeks, body weight 220 g-250 g, purchased from Shanghai Slek Experimental Animal Co., Ltd (purchased through Xiamen University Experimental Animal Center, No. XMULAC20200112).

[0148] Rats were raised in the animal observation room of the School of Pharmacy, Xiamen University (12 h light per day, temperature 25±2℃, relative humidity 55±5%), and experimental animals could freely drink water and eat, and experiments began after one week of environmental adaptation. Experimental animals were fasted for 12 h before the experiment, and could freely drink water. The study complied with the regulations established by the Xiamen University Animal Experiment Ethics Committee.

[0149] 2. Preparation of solutions

[0150] 2.1 Preparation of solutions

[0151] 2.1.1 Standard stock solution

[0152] A certain amount of ephedrine, pseudoephedrine, methylephedrine, quinic acid, gallic acid, (R, S)-yohimbine, rhodioloside, neochlorogenic acid, forsythoside E, caffeic acid, amygdalin, prunol, p-coumaric acid, seco-oxymatrine, ferulic acid, glycyrrhizin, isoglycyrrhizin, forsythoside I, sericoside, luteoloside, forsythoside B, hyperoside, forsythoside A, centaureidol I, isoquercitrin, kaempferol-O-rutinoside, rhein-8-O-β-D-glucoside, 3,5-di-O-caffeoylquinic acid, mutamoside, 4,5-di-O-caffeoylquinic acid, glycyrrhizin, forsythoside, emodin-8-O-β-D-glucoside, quercitrin, luteolin, mutamoside, aloeemodin, isoglycyrrhizin, glycyrrhizic acid, rheic acid, emodin, β-glycyrrhetic acid, chlorogenic acid, cryptochlorogenic acid, rutin, and kaempferol reference substances were precisely weighed, dissolved in methanol to prepare each reference substance stock solution at a concentration of 2 mg / mL, 1.5 mL brown glass vials were used for storage, and the vials were stored at -80°C.

[0153] Mixed stock solution 1: The above prepared reference substance stock solutions were precisely measured and diluted with 50% methanol to prepare a mixed stock solution 1 containing 24 μg / mL (ephedrine, pseudoephedrine, methylephedrine, ferulic acid, glycyrrhizin, isoglycyrrhizin, sericoside, luteoloside, forsythoside B, rutin, hyperoside, forsythoside A, centaureidol I, isoquercitrin, kaempferol-O-rutinoside, rhein-8-O-β-D-glucoside, 3,5-di-O-caffeoylquinic acid, mutamoside, 4,5-di-O-caffeoylquinic acid, glycyrrhizin, emodin-8-O-β-D-glucoside, quercitrin, luteolin, mutamoside, aloeemodin, isoglycyrrhizin, emodin); 120 μg / mL ((R, S)-yohimbine, amygdalin, caffeic acid, forsythoside I, β-glycyrrhetic acid, seco-oxymatrine, neochlorogenic acid).

[0154] Mixed stock solution 2: The above prepared reference substance stock solutions were precisely measured and diluted with 50% methanol to prepare a mixed stock solution 2 containing 480 μg / mL (rheic acid, prunol); 72 μg / mL (gallic acid, forsythoside E, glycyrrhizic acid, kaempferol).

[0155] Mixed stock solution 3: The above prepared reference substance stock solutions were precisely measured and diluted with 50% methanol to prepare a mixed stock solution 3 containing 240 μg / mL (rhodioloside, quinic acid, cryptochlorogenic acid, chlorogenic acid, p-coumaric acid).

[0156] 50 μL of each of the above mixed stock solutions 1, 2, and 3 was taken, 450 μL of 50% methanol was added, and a mixed reference substance working solution (Cal-8) was obtained.

[0157] The above mixed control working solution (Cal-8) was precisely transferred by 0.5 μL, 1.5 μL, 5 μL, 15 μL, 40 μL, 60 μL, 80 μL, respectively, and diluted with 50% methanol solution to 100 μL, respectively, to obtain a series of concentration of mixed control solution, which was used to establish the standard curve for quantitative detection of corresponding substances. The preparation method is shown in Table 1.

[0158] Table 1 Preparation and dilution process of mixed control solution and quality control sample solution

[0159]

[0160] 2.1.2 Internal standard working solution

[0161] Internal standard composition: gallic acid-d2 (IS-1), ferulic acid-d3 (IS-2), acetaminophen (IS-3).

[0162] Internal standard stock solution preparation: a certain amount of gallic acid-d2 (IS-1), ferulic acid-d3 (IS-2), and acetaminophen (IS-3) was precisely weighed in different 1.5 mL EP tubes, methanol was added, and ultrasonic dissolution was performed to prepare each internal standard stock solution with a concentration of 1 mg / mL.

[0163] Internal standard working solution (IS-mix) preparation: 20 μL of each of the above three internal standard stock solutions (1 mg / mL) was added to 950 μL of 50% methanol solution to obtain an internal standard stock solution (20 μg / mL), which was stored in a brown liquid phase vial at -20°C. 25 μL of the internal standard stock solution was diluted with 975 μL of 50% methanol solution to obtain the internal standard working solution (500 ng / mL).

[0164] 2.1.3 Preparation of enzyme solution

[0165] The β-glucuronidase solution of 100 U / 5 μL (10.4275 mg / mL) and the sulfatase solution of 1 U / 5 μL (6.5146 mg / mL) were prepared with 0.2% ice NaCl solution, and were used immediately after preparation.

[0166] 2.2 Preparation of animal administration solution

[0167] An appropriate amount of Lianhuaqingwen capsule content was weighed into a 50 mL centrifuge tube, and a concentration of 0.80 g / mL of administration solution (dose of 12 g / kg, 15 mL / kg) was prepared with ultrapure water. The addition process was continuously stirred, and ultrasonic mixing was performed until it became a uniform suspension liquid. This process was completed at room temperature.

[0168] 2.3 Administration and biological collection

[0169] Animals were acclimated for one week prior to the experiment, and were fasted for 12 h before the experiment. The blank blood (0.3 mL) was taken from the orbit before the administration. The single administration of 12 g / kg Lianhuaqingwen administration solution was given. The blood was taken from the orbit 5 min, 15 min, 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, 9 h, 12 h and 24 h after the last administration, 0.5 mL / time, and was placed in a commercial heparin sodium tube. The supernatant was accurately taken 60 μL to a 1.5 mL EP tube after centrifugation at 2348 g for 10 min, and was stored in a -80°C refrigerator.

[0170] 2.4 Plasma sample processing

[0171] Test plasma sample processing: 5 μL of internal standard working solution (under item 2.1.2) and 10 μL of mixed enzyme (under item 2.1.3) were added to 60 μL of plasma sample, which was incubated at 37°C water bath for 1 h, and then was quickly stopped for enzyme hydrolysis in a -80°C refrigerator. Three times of methanol (180 μL) was added, vortexed for 1 min, and centrifuged at 2348 g for 5 min. The supernatant was taken, nitrogen-blowing was performed, 100 μL of initial mobile phase (0.1% formic acid water: acetonitrile (v:v = 95:5)) was used for re-dissolution, and the supernatant was centrifuged at 15871 g for 10 min. The supernatant was taken to a 96-well plate, and was injected according to the specified injection amount.

[0172] Blank plasma sample processing: the remaining operation process was the same as that under the item “Test plasma sample processing”, except that the test plasma was replaced by blank plasma.

[0173] 3. Chromatography / mass spectrometry conditions of HPLC-MS / MS analysis method

[0174] (1) Analysis method 1

[0175] Instrument: AB SCIEX Triple Quad TM , analysis software: MultiQuant TM quantitative software;

[0176] Analytical compounds (29): neochlorogenic acid, p-coumaric acid, seco-oxymatrine, glycyrrhizin, isoglycyrrhizin, apigenin-8-C-β-D-glucoside, luteolin-8-C-β-D-glucoside, hyperoside, isoquercitrin, rhein-8-O-β-D-glucoside, 3,5-di-O-caffeoylquinic acid, ononin, 4,5-di-O-caffeoylquinic acid, glycyrrhizin, forsythoside, emodin-8-O-β-D-glucoside, quercitrin, luteolin, onocytin, aloe-emodin, isoglycyrrhizin, glycyrrhizic acid, rhein, emodin, β-glycyrrhetic acid, chlorogenic acid, cryptochlorogenic acid, rutin and kaempferol.

[0177] High performance liquid chromatography conditions:

[0178] Column: CAPCELL CORE C18, 2.1 mm x 75 mm, 2.7 μm;

[0179] Mobile phase A: 0.1% formic acid in water;

[0180] Mobile phase B: 100% acetonitrile;

[0181] Column temperature: 35 °C;

[0182] Flow rate: 0.4 mL / min;

[0183] Injection volume: 10 μL;

[0184] Gradient elution sequence:

[0185] 0-1 min, 95%→90% mobile phase A, 5%→10% mobile phase B;

[0186] 1-2.5 min, 90%→89% mobile phase A, 10%→11% mobile phase B;

[0187] 2.5-3 min, 89%→82.5% mobile phase A, 11%→17.5% mobile phase B;

[0188] 3-4.5 min, 82.5% mobile phase A, 17.5% mobile phase B;

[0189] 4.5-6 min, 82.5%→20% mobile phase A, 17.5%→80% mobile phase B;

[0190] 6-7 min, 20%→17% mobile phase A, 80%→83% mobile phase B;

[0191] 7-7.01 min, 17%→95% mobile phase A, 83%→5% mobile phase B;

[0192] 7.01-9 min, 95% mobile phase A, 5% mobile phase B;

[0193] Mass spectrometry conditions:

[0194] The ion source was an electrospray ion source (ESI source) in negative ion detection mode, with a capillary voltage of 5.5 KV, nebulizer gas of 55.0 L / min, solvent gas of 20.0 L / min, and collision gas of 9.0 L / min; the heating block temperature was 550 °C; the scan mode was multiple reaction monitoring (MRM) mode, and the quantitative parent ions, daughter ions, fragmentation voltages, and declustering voltages are shown in Table 2. The experimental data were collected and processed using MultiQuant TM quantification software.

[0195] (2) Analysis Method 2

[0196] The analysis was performed using Agilent 1260 series liquid chromatograph coupled with Agilent 6460 triple quadrupole mass spectrometer (QQQ) for detection.

[0197] Analytical compounds (10): quinic acid, gallic acid, rhodioloside, caffeic acid, wild black cherry glycoside, forsythoside E, forsythoside A, forsythoside B, forsythoside I and ferulic acid.

[0198] High performance liquid chromatography conditions:

[0199] Chromatographic column: CAPCELL PAK C18, 2.0 mm x 50 mm, 2 μm;

[0200] Mobile phase A: 0.1% formic acid aqueous solution;

[0201] Mobile phase B: 100% acetonitrile;

[0202] Column temperature: 35°C;

[0203] Flow rate: 0.35 mL / min;

[0204] Injection volume: 10 μL

[0205] Gradient elution sequence:

[0206] 0-1 min, 95%→83% mobile phase A, 5%→17% mobile phase B;

[0207] 1-3.5 min, 83%→80% mobile phase A, 17%→20% mobile phase B;

[0208] 3.5-4 min, 80%→35% mobile phase A, 20%→65% mobile phase B;

[0209] 4-6 min, 35%→5% mobile phase A, 65%→95% mobile phase B;

[0210] 6-6.01 min, 5%→95% mobile phase A, 95%→5% mobile phase B;

[0211] 6.01-7.5 min, 95% mobile phase A, 5% mobile phase B.

[0212] Mass spectrometry conditions:

[0213] The ion source was an electrospray ion source (ESI source), negative ion detection mode, capillary voltage: 4.0 kV, nebulizer gas: 10.0 L / min, dissolving gas: 30.0 L / min, desolvation gas line temperature was 325℃, heating block temperature: 325℃, and the scanning mode was MRM monitoring mode. The quantitative parent ions, daughter ions, fragmentation voltages and declustering voltages are shown in Table 2. The experimental data were collected and processed using Agilent MassHunter data processing workstation and Agilent Qualitative Analysis B.07.00 Tools software.

[0214] (3) Analysis Method 3

[0215] An Agilent 1260 series liquid chromatograph coupled with an Agilent 6460 triple quadrupole mass spectrometer (QQQ) was used for detection and analysis.

[0216] Seven compounds were analyzed: (R,S)-codeine, pseudoephedrine, ephedrine, methylephedrine, amygdalin, centaureidin I and kaempferol-O-rutinoside.

[0217] The high-performance liquid chromatography conditions were as follows:

[0218] Chromatographic column: CAPCELL PAK C18, 2.0 mm x 50 mm, 2 μm;

[0219] Mobile phase A: 0.1% formic acid aqueous solution;

[0220] Mobile phase B: 100% acetonitrile;

[0221] Column temperature: 25℃;

[0222] Flow rate: 0.35 mL / min;

[0223] Injection volume: 20 μL;

[0224] Gradient elution sequence:

[0225] 0-3 min, 98%→95% mobile phase A, 2%→5% mobile phase B;

[0226] 3-3.5 min, 95%→75% mobile phase A, 5%→25% mobile phase B;

[0227] 3.5-6 min, 75%→60% mobile phase A, 25%→30% mobile phase B;

[0228] 6-6.01 min, 70%→98% mobile phase A, 30%→2% mobile phase B;

[0229] 6.01-7.5 min, 98% mobile phase A, 2% mobile phase B.

[0230] Mass spectrometry conditions:

[0231] The ion source was an electrospray ion source (ESI source) in positive ion detection mode, with a capillary voltage of 4.0 KV, nebulizer gas of 10.0 L / min, and a solvent gas temperature of 325 °C. The heated block temperature was 325 °C. The scan mode was MRM monitoring mode, and the quantitative parent ions, daughter ions, fragmentation voltages, and de-clustering voltages are shown in Table 2. Experimental data were collected and processed using Agilent MassHunter data processing workstations and Agilent Qualitative Analysis B.07.00 Tools software.

[0232] Table 2 MRM mass spectrometry parameters for 46 compounds to be tested in Lianhua Qingwen capsules

[0233]

[0234]

[0235]

[0236] Example 2

[0237] Method validation:

[0238] 2.1 Linear range

[0239] Prepare a mixed control solution and a quality control sample solution according to the preparation of each compound in item 2.1.1 in Example 1, and the concentrations of each compound in the mixed control solution and the quality control sample solution are shown in Table 3.

[0240] Add 5 μL of each concentration of the mixed control solution (cal-1 to cal-8) or the quality control working solution in Table 3, 5 μL of the internal standard working solution (item 2.1.2 in Example 1), and 10 μL of the mixed enzyme (item 2.1.3 in Example 1) to 55 μL of blank plasma, respectively, and incubate at 37 °C for 1 h. Stop the enzyme reaction quickly in a -80 °C refrigerator, add three times the amount of methanol (180 μL), vortex for 1 min, centrifuge at 2348 g for 5 min, take the supernatant, nitrogen blow, and redissolve with 100 μL of the initial mobile phase (0.1% formic acid water:acetonitrile (v:v = 95:5)) 100 μL, centrifuge at 15871 g for 10 min, take the supernatant to a 96-well plate, and inject according to the specified injection amount.

[0241] Table 3 Standard curve and quality control concentration concentration settings for compounds to be tested

[0242]

[0243]

[0244]

[0245] Using the ratio of the peak area of ​​the 46 target compounds to the internal standard as the ordinate and the concentration of the target compounds as the abscissa, linear regression was performed using the least squares method (the weight coefficient of each compound is W = 1 / x) to obtain the linear equation of the matrix standard curve and the linear correlation coefficient r, as shown in Table 4.

[0246] Table 4. Linear equations and linear ranges for 46 test compounds.

[0247]

[0248]

[0249]

[0250]

[0251] The results show that, within the linear range, the ratio of peak area to internal standard peak area of ​​the 46 target compounds has a good linear relationship with concentration, and the correlation coefficient r is higher than 0.99.

[0252] 2.2 Detection of the test solution

[0253] Add 5 μL of the cal-8 mixed control solution from Table 3 above, 5 μL of internal standard working solution (under section 2.1.2 in Example 1), and 10 μL of mixed enzyme (under section 2.1.3 in Example 1) to 55 μL of blank plasma. The subsequent treatment is the same as under section 2.1 to prepare the test solution.

[0254] The prepared test solution was analyzed according to the HPLC-MS / MS method in Example 1, and the results were... Figures 1-3 As shown.

[0255] As can be seen from the figure, the peak shapes of the 46 test compounds in Lianhua Qingwen are good, and the separation between the test components is good, proving that the detection method provided by the present invention can accurately detect the 46 blood-entering original components in Lianhua Qingwen capsules.

[0256] 2.3 Accuracy and Precision

[0257] The low concentration plasma quality control sample (LLOQ), the medium concentration plasma quality control sample (MQC) and the high concentration plasma quality control sample (HQC) under item 2.1 were taken to investigate the precision and accuracy of the plasma. Six samples of each concentration were determined in parallel, the ratio of the peak area of the target determination compound and the internal standard was substituted into the standard curve to calculate the concentration. The precision was represented by the intra-day and inter-day precision RSD of the quality control sample, and the RSD should generally be less than 15%, and the accuracy requirement was within the range of 85%-115%. The intra-day and inter-day precision and accuracy of the measured concentration of the quality control sample at three different concentration levels are shown in Table 5.

[0258] Table 5 Intra-day and inter-day precision and accuracy of 46 compounds in rat plasma

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266] It can be seen from the results that the RSD of the intra-day precision and the inter-day precision is less than 14.64% and 14.93% respectively, and the accuracy range is within 85-115%, which can meet the requirements of the determination accuracy.

[0267] 2.4 Selectivity and specificity

[0268] The blank plasma sample of the rat + internal standard, the blank plasma sample added with the mixed control solution, the internal standard and the quality control sample of the mixed enzyme (preparation method same as the LLOQ under item 2.1 in Example 2), and the plasma sample of the rat after administration (preparation method same as items 2.2-2.4 in Example 1) were respectively subjected to protein precipitation pretreatment, and then injected into the three HPLC-MS / MS analysis methods of Example 1 for detection, and the chromatograms were recorded. The chromatograms of each compound in the chromatograms were analyzed and compared, and the results are shown in Table 6. Figures 4-46

[0269] It can be seen from the results that the blank biological matrix and the potential related interfering substances have no interference on the determination of the 46 target determination compounds and the internal standard, and the isomers have good separation degree, which indicates that the established method is suitable for the determination of the 46 target determination compounds. ​

[0270] 2.5 Stability

[0271] 2.5.1 Stability after processing

[0272] Take the low concentration plasma quality control sample (LQC), medium concentration plasma quality control sample (MQC) and high concentration plasma quality control sample (HQC) under item 2.1, according to the plasma sample processing under item 2.4 in Example 1, after processing, sample analysis after 24h at room temperature (25℃), substitute into the standard curve, calculate the concentration and SD value, the results are shown in Table 6.

[0273] 2.5.2 Freeze-thaw stability

[0274] Take the low concentration plasma quality control sample (LQC), medium concentration plasma quality control sample (MQC) and high concentration plasma quality control sample (HQC) under item 2.1, respectively, store in -80℃ refrigerator for 24h, all taken out and naturally thawed at room temperature (25℃), then put back into -80℃ refrigerator, repeat freeze-thaw twice at intervals of 24h, according to the plasma sample processing under item 2.4 in Example 1, after processing, sample analysis, substitute into the standard curve, calculate the concentration and SD value, the results are shown in Table 6.

[0275] 2.5.3 Short-term stability

[0276] Take the low concentration plasma quality control sample (LQC), medium concentration plasma quality control sample (MQC) and high concentration plasma quality control sample (HQC) under item 2.1, store at 4℃ for 24h, then according to the plasma sample processing under item 2.4 in Example 1, after processing, sample analysis, substitute into the standard curve, calculate the concentration and SD value, the results are shown in Table 6.

[0277] 2.5.4 Long-term stability

[0278] Take the low concentration plasma quality control sample (LQC), medium concentration plasma quality control sample (MQC) and high concentration plasma quality control sample (HQC) under item 2.1, store at -80℃ for 10d, then according to the plasma sample processing under item 2.4 in Example 1, after processing, sample analysis, substitute into the standard curve, calculate the concentration and SD value, the results are shown in Table 6.

[0279] Table 6 Stability of 46 test compounds in rat plasma

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291] The results show that the short-term stability RSD value of the 46 compounds to be detected is less than 14.82% at 4°C for 24h; the long-term stability RSD value is less than 14.74% at -80°C for 10 days; the repeated freeze-thaw stability RSD value is less than 14.93%; and the stability RSD value after being placed at room temperature (25°C) for 24h is less than 14.88%. The accuracy range is within 85-115%, which meets the requirements of the determination stability.

[0292] Example 3

[0293] Drug determination concentration in rat plasma

[0294] The pretreatment method of enzyme hydrolysis followed by methanol protein precipitation was combined with the established HPLC-MS / MS method to quantitatively analyze the 46 main components in the plasma of rats after intragastrical administration. According to the blood drug concentration calculated by the calibration curve, the DAS2.0 software was introduced, and the non-compartment model was used to fit the pharmacokinetic parameters (AUC 0-t , T max , C max , t 1 / 2z , V z / F and CL z / F ) of the 46 compounds, and the specific results are shown in Table 7.

[0295] Table 7 Pharmacokinetic parameters of 46 compounds to be detected in rats (n=6)

[0296]

[0297]

[0298]

[0299] The average plasma concentration-time curve of the 46 compounds is shown in Figure 1. Figures 47-62From the average plasma concentration-time curves and pharmacokinetic analysis results, it can be seen that the plasma concentrations and metabolic characteristics of each group are quite different. Organic acids, anthraquinones, caffeic acid, phenylethanoid glycosides and alkaloids (except pseudoephedrine) compounds have the characteristics of fast absorption, short peak time and slow elimination, while rhodioside, flavonoids and triterpenoids have the characteristics of slow absorption and long peak time. AUC 0-t The top 10 compounds in terms of AUC values are: rhodioside (mainly derived from the medicinal material Rhodiolae Radix), caffeic acid (mainly derived from the medicinal material Lonicerae Japonicae Flos), rhein (mainly derived from the medicinal material Radix et Rhizoma Rhei), glycyrrhetinic acid (mainly derived from the medicinal material Radix Glycyrrhizae), kaempferol (mainly derived from the medicinal materials Rhodiolae Radix and Radix et Rhizoma Rhei), quinic acid (mainly derived from the medicinal material Lonicerae Japonicae Flos), wild black cherry glycoside (mainly derived from the medicinal material Semen Armeniacae Amarum), forsythia ester glycoside E (mainly derived from the medicinal material Forsythiae Fructus), gallic acid (mainly derived from the medicinal materials Rhodiolae Radix and Radix et Rhizoma Rhei), and ferulic acid (mainly derived from the medicinal materials Radix et Rhizoma Bupleuri and Herba Gentianae Macrophyllae). Among them, rhodioside and rhein are the quality control components of Rhodiolae Radix and Radix et Rhizoma Rhei, respectively, with AUC 0-t values of 132211.18±121869.67 μg / L*h and 32865.26±12913.65 μg / L*h in rat plasma, respectively, which are the main prototype components exposed in rat plasma after oral administration of Lianhua Qingwen Capsules, and can be used as potential pharmacokinetic markers of Lianhua Qingwen Capsules. Among them, glycosides include flavonoid glycosides such as formononetin, isoquercitrin, chrysosplenium glycoside, hyperoside, kaempferol-3-O-rutinoside and other compounds, which have low content in vivo. The possible reasons are: 1) the compounds undergo hydrolysis reaction in vivo to remove the glycoside bond, and exist in the form of metabolites; for example, chrysosplenium glycoside is rapidly absorbed (T max 0.11±0.07h; t 1 / 2 2.65±1.63h) in vivo and metabolized into chrysosplenium, which is the main form of chrysosplenium glycoside in rats; 2) β-glucuronidase is added in the pretreatment of plasma samples to hydrolyze the β-D-glucose bond combined to the non-reducing end, releasing β-D-glucose and the corresponding prototype ligand, which also hydrolyzes part of the glycoside prototype components, resulting in low content in vivo.

[0300] The plasma concentrations of compounds with small molecular weights such as caffeic acid, kaempferol, quinic acid, gallic acid, ferulic acid, and p-coumaric acid are high, and the main reasons are speculated to be the original content of the medicinal materials and the conversion from other components. For example, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, cryptogreen acid, chlorogenic acid, and neochlorogenic acid are all condensed phenolic acids generated from caffeic acid and quinic acid.

[0301] Pseudoephedrine, forsythoside B and kaempferol-3-O-rhamnoside have elimination half-lives less than 2 h, and rutin, glycyrrhizin, luteolin, isoliquiritigenin and glycyrrhetic acid have elimination half-lives greater than 25 h. The plasma concentration-time curves of multiple compounds exhibit double peaks. For example, glycyrrhizin appears at 0.08 h-1 h and 6 h-12 h after taking Lianhuaqingwen, respectively Figure 58 ), and prunusin appears at 0.5 h-1 h and 6 h-12 h after administration, respectively Figure 50 ), wherein the first peak is produced by the absorption of the prototype components in Lianhuaqingwen through the intestinal tract. The second peak of glycyrrhizin is produced by the de-glycosylation of glycyrrhizin in the body after the absorption of glycyrrhizin / glycyrrhizin apioside in the intestinal tract through microbial metabolism. The second peak of prunusin is mainly produced by the de-glycosylation of amygdalin.

[0302] In addition, by comparing the plasma concentrations and the detection of compounds in medicinal materials, it is found that the concentrations of prunusin and glycyrrhetic acid in medicinal materials are low, and the plasma concentrations gradually increase during the absorption and distribution in the body. In our previous studies, it is known that prunusin and glycyrrhetic acid are the main metabolites of amygdalin and glycyrrhizic acid in the body, respectively, and play a major pharmacodynamic effect. In the pharmacokinetic study this time, as the plasma concentrations of amygdalin and glycyrrhizic acid gradually decrease, the contents of prunusin and glycyrrhetic acid in the plasma gradually increase, and reach the peak of drug concentration at 6.17±4.40 h and 14.17±8.16 h, respectively. This data again confirms this conclusion.

[0303] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement or improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for detecting in vivo effective components in an antiviral traditional Chinese medicine composition, wherein the in vivo effective components of the antiviral traditional Chinese medicine composition include ephedrine, pseudoephedrine, methylephedrine, quinic acid, gallic acid, (R,S)-yohimbine, rhodioside, neochlorogenic acid, forsythoside E, caffeic acid, amygdalin, prunol, p-coumaric acid, secooxyloganin, ferulic acid, glycyrrhizin, isoglycyrrhizin, forsythoside I, sericoside, juncuside, forsythoside B, hyperoside, forsythoside A, centaureidol I, isoquercitrin, kaempferol-O-rutinoside, rhein-8-O-β-D-glucoside, 3,5-di-O-caffeoylquinic acid, ononin, 4,5-di-O-caffeoylquinic acid, liquiritin, forsythoside, emodin-8-O-β-D-glucoside, quercitrin, luteolin, formononetin, aloe-emodin, isoglycyrrhizin, glycyrrhizic acid, rheic acid, emodin, β-glycyrrhetic acid, chlorogenic acid, cryptochlorogenic acid, rutin, and kaempferol, characterized in that, The high performance liquid chromatography and mass spectrometry are used for detection, wherein the high performance liquid chromatography conditions are as follows: The volume concentration of 0.08%-0.12% formic acid aqueous solution is used as mobile phase A, and acetonitrile is used as mobile phase B, and gradient elution is used; The mass spectrometry uses ESI ion source, and the scanning mode is MRM monitoring mode; If the in-vivo effective components to be detected are neochlorogenic acid, p-coumaric acid, seco-oxymatridin, glycyrrhizin, isoglycyrrhizin, apigenin glycoside, jafficin, hyperoside, isoquercitrin, rhein-8-O-beta-D-glucoside, 3,5-di-O-caffeoylquinic acid, ononin, 4,5-di-O-caffeoylquinic acid, glycyrrhizin, forsythoside, emodin-8-O-beta-D-glucoside, quercitrin, luteolin, formononetin, aloe-emodin, glycyrrhetic acid, rhein, beta-glycyrrhetic acid, chlorogenic acid, cryptochlorogenic acid, rutin and kaempferol, the high performance liquid chromatography conditions are as follows: CAPCELL CORE C18, 2.1mm*75mm, 2.7um is used; The gradient elution sequence is as follows: 0-1min, 95%→90% mobile phase A, 5%→10% mobile phase B; 1-2.5min, 90%→89% mobile phase A, 10%→11% mobile phase B; 2.5-3min, 89%→82.5% mobile phase A, 11%→17.5% mobile phase B; 3-4.5min, 82.5% mobile phase A, 17.5% mobile phase B; 4.5-6min, 82.5%→20% mobile phase A, 17.5%→80% mobile phase B; 6-7min, 20%→17% mobile phase A, 80%→83% mobile phase B; 7-7.01min, 17%→95% mobile phase A, 83%→5% mobile phase B; 7.01-9min, 95% mobile phase A, 5% mobile phase B; The mass spectrometry uses negative ion detection mode, the capillary voltage is 5.5kV, the atomization gas flow rate is 55L / min, the dissolving gas flow rate is 20L / min, the collision gas flow rate is 9L / min, and the heating block temperature is 550℃.

2. The method for detecting the in vivo effective component of the antiviral traditional Chinese medicine composition according to claim 1, characterized in that, The column temperature is 35℃, the flow rate is 0.4mL / min, and the injection volume is 10uL.

3. The method for detecting the in vivo effective component of the antiviral traditional Chinese medicine composition according to claim 1 or 2, characterized in that, The parent ion of neochlorogenic acid is 353 m / z, the daughter ion is 191 m / z, the declustering voltage is 76 V, and the fragmentation voltage is 31 eV; the parent ion of p-coumaric acid is 163 m / z, the daughter ion is 119 m / z, the declustering voltage is 66 V, and the fragmentation voltage is 23 eV; the parent ion of deoxyloganin is 403.2 m / z, the daughter ion is 179.2 m / z, the declustering voltage is 86 V, and the fragmentation voltage is 18 eV; the parent ion of glycyrrhizin is 417.3 m / z, the daughter ion is 135.1 m / z, the declustering voltage is 125 V, and the fragmentation voltage is 39 eV; the parent ion of isoglycyrrhizin is 417.2 m / z, the daughter ion is 148.1 m / z, the declustering voltage is 127 V, and the fragmentation voltage is 48 eV; the parent ion of trypsin is 549.1 m / z, the daughter ion is 255.3 m / z, the declustering voltage is 146 V, and the fragmentation voltage is 45 eV; the parent ion of luteoloside is 447.1 m / z, the daughter ion is 285.3 m / z, the declustering voltage is 154 V, and the fragmentation voltage is 45 eV; the parent ion of hyperoside is 463 m / z, the daughter ion is 300.7 m / z, the declustering voltage is 125 V, and the fragmentation voltage is 36 eV; the parent ion of isoquercitrin is 463.2 m / z, the daughter ion is 255 m / z, the declustering voltage is 113 V, and the fragmentation voltage is 58 eV; the parent ion of rhein-8-O-β-D-glucoside is 445 m / z, the daughter ion is 239 m / z, the declustering voltage is 85 V, and the fragmentation voltage is 35 eV; the parent ion of 3,5-di-O-caffeoylquinic acid is 515.3 m / z, the daughter ion is 191.2 m / z, the declustering voltage is 91 V, and the fragmentation voltage is 14 eV; the parent ion of ononin is 475.1 m / z, the daughter ion is 267 / z, the declustering voltage is 65 V, and the fragmentation voltage is 20 eV; the parent ion of 4,5-di-O-caffeoylquinic acid is 515.2 m / z, the daughter ion is 173 m / z, the declustering voltage is 81 V, and the fragmentation voltage is 39 eV; the parent ion of glycyrrhizin is 255 m / z, the daughter ion is 135.1 m / z, the declustering voltage is 83 V, and the fragmentation voltage is 20 eV; the parent ion of forsythoside is 579.2 m / z, the daughter ion is 371.4 m / z, the declustering voltage is 80 V, and the fragmentation voltage is 40 eV; the parent ion of emodin-8-O-β-D-glucoside is 431.2 m / z, the daughter ion is 269.2 m / z, the declustering voltage is 123 V, and the fragmentation voltage is 39 eV; the parent ion of quercitrin is 447.2 m / z, the daughter ion is 300.2 m / z, the declustering voltage is 99 V, and the fragmentation voltage is 38 eV; the parent ion of luteolin is 285.1 m / z, the daughter ion is 151.1 m / z, the declustering voltage is 103 V, and the fragmentation voltage is 35 eV; the parent ion of onocyanin is 267.1 m / z, the daughter ion is 252.2 m / z, cluster voltage 106 V, cleavage voltage 30 eV; aloe-emodin parent ion 269 m / z, daughter ion 240 m / z, cluster voltage 104 V, cleavage voltage 31 eV; isoliquiritigenin parent ion 255.1 m / z, daughter ion 119.1 m / z, cluster voltage 5 V, cleavage voltage 42 eV; glycyrrhizic acid parent ion 821.5 m / z, daughter ion 350.9 m / z, cluster voltage 52 V, cleavage voltage 41 eV; rhein parent ion 283.2 m / z, daughter ion 183.1 m / z, cluster voltage 52 V, cleavage voltage 38 eV; emodin parent ion 269 m / z, daughter ion 241 m / z, cluster voltage 127 V, cleavage voltage 38 eV; β-glycyrrhetic acid parent ion 469.3 m / z, daughter ion 355.3 m / z, cluster voltage 151 V, cleavage voltage 67 eV; chlorogenic acid parent ion 353.3 m / z, daughter ion 191.1 m / z, cluster voltage 64 V, cleavage voltage 22 eV; cryptochlorogenic acid parent ion 353.2 m / z, daughter ion 173.1 m / z, cluster voltage 76 V, cleavage voltage 22 eV; rutin parent ion 609.1 m / z, daughter ion 300.2 m / z, cluster voltage 134 V, cleavage voltage 50 eV; kaempferol parent ion 285 m / z, daughter ion 210.9 m / z, cluster voltage 85 V, cleavage voltage 45 eV.

4. The method for detecting the in-vivo effective component of the antiviral traditional Chinese medicine composition according to claim 1, characterized in that, If the in-vivo effective components to be detected are quinic acid, gallic acid, salidroside, caffeic acid, wild black cherry glycoside, forsythoside E, forsythoside A, forsythoside B, forsythoside I and ferulic acid, the high performance liquid chromatography conditions are as follows: The chromatographic column is CAPCELL PAK C18, 2.0mm*50mm, 2um; The gradient elution sequence is as follows: 0-1min, 95%→83% mobile phase A, 5%→17% mobile phase B; 1-3.5min, 83%→80% mobile phase A, 17%→20% mobile phase B; 3.5-4min, 80%→35% mobile phase A, 20%→65% mobile phase B; 4-6min, 35%→5% mobile phase A, 65%→95% mobile phase B; 6-6.01min, 5%→95% mobile phase A, 95%→5% mobile phase B; 6.01-7.5 min, 95% mobile phase A, 5% mobile phase B.

5. The method for detecting the in-vivo effective component of the antiviral traditional Chinese medicine composition according to claim 4, characterized in that, The column temperature was 35℃, the flow rate was 0.35 mL / min, and the injection volume was 10 μL.

6. The method for detecting the in-vivo effective component of the antiviral traditional Chinese medicine composition according to claim 4 or 5, characterized in that, The mass spectrometer was operated in negative ion detection mode, with a capillary voltage of 4 kV, a nebulizer gas flow rate of 10 L / min, a solvent gas flow rate of 30 L / min, a desolvation gas line temperature of 325℃, and a heating block temperature of 325℃; The parent ion of quinic acid was 191.1 m / z, the daughter ion was 85 m / z, the declustering voltage was 130 V, and the fragmentation voltage was 25 eV; the parent ion of gallic acid was 169.1 m / z, the daughter ion was 125.1 m / z, the declustering voltage was 90 V, and the fragmentation voltage was 10 eV; the parent ion of rhodioside was 345.1 m / z, the daughter ion was 119 m / z, the declustering voltage was 75 V, and the fragmentation voltage was 15 eV; the parent ion of caffeic acid was 179 m / z, the daughter ion was 135.1 m / z, the declustering voltage was 85 V, and the fragmentation voltage was 5 eV; the parent ion of wild blackcherry glycoside was 340.1 m / z, the daughter ion was 161.1 m / z, the declustering voltage was 85 V, and the fragmentation voltage was 10 eV; the parent ion of forsythoside E was 461.1 m / z, the daughter ion was 135 m / z, the declustering voltage was 105 V, and the fragmentation voltage was 35 eV; the parent ion of forsythoside A was 623.2 m / z, the daughter ion was 161.1 m / z, the declustering voltage was 180 V, and the fragmentation voltage was 45 eV; the parent ion of forsythoside B was 755.2 m / z, the daughter ion was 161.1 m / z, the declustering voltage was 135 V, and the fragmentation voltage was 50 eV; the parent ion of forsythoside I was 623.2 m / z, the daughter ion was 161.1 m / z, the declustering voltage was 145 V, and the fragmentation voltage was 40 eV; the parent ion of ferulic acid was 193 m / z, the daughter ion was 133.9 m / z, the declustering voltage was 85 V, and the fragmentation voltage was 15 eV.

7. The method for detecting the in vivo effective component of the antiviral traditional Chinese medicine composition according to claim 1, characterized in that, If the detected active ingredients in vivo are (R,S)-diamorphine, pseudoephedrine, ephedrine, methylephedrine, amygdalin, centaureidin I, and kaempferol-O-rutinoside, the high performance liquid chromatography conditions are as follows: The column was CAPCELL PAK C18, 2.0 mm x 50 mm, 2 μm; The gradient elution sequence was as follows: 0-3 min, 98%→95% mobile phase A, 2%→5% mobile phase B; 3-3.5 min, 95%→75% mobile phase A, 5%→25% mobile phase B; 3.5-6 min, 75%→60% mobile phase A, 25%→30% mobile phase B; 6-6.01 min, 70%→98% mobile phase A, 30%→2% mobile phase B; 6.01-7.5 min, 98% mobile phase A, 2% mobile phase B.

8. The method for detecting the in-vivo effective component of the antiviral traditional Chinese medicine composition according to claim 7, characterized in that, The column temperature was 25℃, the flow rate was 0.35 mL / min, and the injection volume was 20 μL.

9. The method for detecting the in vivo effective component of the antiviral traditional Chinese medicine composition according to claim 7 or 8, characterized in that, The mass spectrum adopts positive ion detection mode, the capillary voltage is 4 kV, the atomizing gas flow rate is 10 L / min, the dissolving gas flow rate is 30 L / min, the desolvation gas line temperature is 325℃, and the heating block temperature is 325℃; The parent ion of (R,S)-diamorphine is 130.1 m / z, the daughter ion is 70.2 m / z, the declustering voltage is 60 V, and the fragmentation voltage is 5 eV; the parent ion of pseudoephedrine is 166.2 m / z, the daughter ion is 133.2 m / z, the declustering voltage is 70 V, and the fragmentation voltage is 20 eV; the parent ion of ephedrine is 166.2 m / z, the daughter ion is 148.2 m / z, the declustering voltage is 70 V, and the fragmentation voltage is 10 eV; the parent ion of methylephedrine is 180.2 m / z, the daughter ion is 91.2 m / z, the declustering voltage is 85 V, and the fragmentation voltage is 40 eV; the parent ion of amygdalin is 475.1 m / z, the daughter ion is 163.1 m / z, the declustering voltage is 120 V, and the fragmentation voltage is 10 eV; the parent ion of centaureidin I is 595.2 m / z, the daughter ion is 286.9 m / z, the declustering voltage is 120 V, and the fragmentation voltage is 45 eV; the parent ion of kaempferol-O-rutinoside is 595.2 m / z, the daughter ion is 286.9 m / z, the declustering voltage is 120 V, and the fragmentation voltage is 40 eV.

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