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

Abstract

The invention relates to the technical field of pharmaceutical analysis, and particularly discloses a method for detecting in-vivo prototype components in an antiviral traditional Chinese medicine composition. According to the method, an octadecyl bonded silica gel chromatographic column is used as a detection chromatographic column, 0.08%-0.12% formic acid aqueous solution-acetonitrile is used as a mobile phase, a gradient elution procedure is adopted, and specific mass spectrum conditions are matched, so that more comprehensive identification and identification of 46 blood-entering prototype components in the antiviral traditional Chinese medicine composition are realized; and a reliable experimental basis is provided for identifying the pharmacodynamic material basis of the antiviral traditional Chinese medicine composition. Meanwhile, research on the in-vivo pharmacokinetic process of prototype components in the antiviral traditional Chinese medicine composition is further facilitated, pharmacokinetic markers of the antiviral traditional Chinese medicine composition can be screened according to the in-vivo absorption condition of the compounds, and a data foundation is laid for deep research on the action mechanism of the antiviral traditional Chinese medicine composition.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a method for detecting in vivo prototype components in an antiviral traditional Chinese medicine composition. Background Art

[0002] Patent CN1483463A discloses an antiviral Chinese medicine composition, which is composed of a variety of Chinese herbal medicines such as forsythia, honeysuckle, roasted ephedra, stir-fried bitter almonds, gypsum, isatis root, cyperus rotundus, houttuynia, patchouli, rhubarb, rhodiola rosea, menthol and licorice. Each component plays a different role in the medicine, which together determine the overall efficacy of the Chinese medicine composition. The Chinese medicine composition has the effects of clearing away plague and detoxifying, ventilating the lungs and relieving heat, and is mainly used to treat fever, nasal congestion, runny nose, cough, headache, dry throat, sore throat and other symptoms caused by influenza.

[0003] It can be seen from the composition of the prescription that the composition of the antiviral Chinese medicine composition is complex. Exploring its material basis and mechanism of action will play an important role in the clinical application of the Chinese medicine composition and the development of new anti-epidemic Chinese medicines based on traditional Chinese medicine theory. At present, most of the published literature on the pharmacokinetics of the Chinese medicine composition only provides a simple kinetic description of the drug pairs or single drugs, and does not systematically explain its overall PK behavior characteristics. There is currently a lack of systematic pharmacokinetic research. Therefore, in order to better reveal the material basis of the efficacy of the Chinese medicine composition, it is necessary to develop a method for simultaneously detecting the content of the prototype components in the Chinese medicine composition, which will play a very important role in the future clinical application of the Chinese medicine composition and the development of new anti-epidemic Chinese medicines. Summary of the invention

[0004] In order to better reveal the pharmacodynamic material basis of a Chinese medicine composition composed of various Chinese herbal medicines such as Forsythia suspensa, honeysuckle, roasted ephedra, stir-fried bitter almonds, gypsum, isatis root, cyperus rotundus, houttuynia cordata, patchouli, rhubarb, rhodiola rosea, menthol and licorice, the present invention provides a method for detecting the prototype components in the above Chinese medicine composition. The detection method provided by the present invention can clarify the prototype components and metabolites in the body, and clarify the components with high exposure, so as to facilitate the monitoring of the pharmacokinetic process.

[0005] In order to solve the above technical problems, the technical solution provided by the present invention is:

[0006] A method for detecting in vivo prototype components in an antiviral Chinese medicine composition, wherein the in vivo prototype components of the antiviral Chinese medicine composition include ephedrine, pseudoephedrine, methylephedrine, quinic acid, gallic acid, (R, S)-goitrin, salidroside, neochlorogenic acid, forsythiaside E, caffeic acid, amygdalin, prunin, p-coumaric acid, strychnine, ferulic acid, liquiritin, isoliquiritigenin, forsythiaside I, apigenin, luteolin, forsythiaside B, hyperoside, forsythiaside A, scutellarin I, isoquercitrin, bergamot Naphthol-O-rutinoside, rhein-8-O-β-D-glucoside, 3,5-di-O-caffeoylquinic acid, formononetin, 4,5-di-O-caffeoylquinic acid, liquiritigenin, forsythin, rhein-8-O-β-D-glucoside, quercetin, luteolin, formononetin, aloe-emodin, isoliquiritigenin, glycyrrhizic acid, rhein, rhein, β-glycyrrhetinic acid, chlorogenic acid, cryptochlorogenic acid, rutin and kaempferol were detected by high performance liquid chromatography-mass spectrometry, wherein the high performance liquid chromatography conditions were as follows:

[0007] A C18 chromatographic column was used, with a 0.08% to 0.12% formic acid aqueous solution as mobile phase A and acetonitrile as mobile phase B, and gradient elution was performed;

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

[0009] It should be noted that the antiviral Chinese medicine composition described in the present invention includes Forsythia suspensa, honeysuckle, isatis root, bitter almond, menthol, houttuynia cordata, rhubarb, patchouli, Cynomorium mongolicum, rhodiola rosea, ephedra, licorice, and gypsum.

[0010] Compared with the prior art, the detection method of the prototype components in the antiviral Chinese medicine composition provided by the present invention uses an octadecyl bonded silica gel chromatographic column as the detection chromatographic column, 0.08%-0.12% formic acid aqueous solution-acetonitrile as the mobile phase, and a gradient elution program, in combination with specific mass spectrometry conditions, effectively overcomes the defects of the antiviral Chinese medicine composition having a variety of Chinese medicinal flavors, complex components and mutual interference, and realizes a more comprehensive identification and identification of 46 blood-entering prototype components in the antiviral Chinese medicine composition. And the method provided by the present invention has been studied and verified by methodological studies such as specificity, precision and stability, and it is found that the method of the present invention has good accuracy and reproducibility, which provides a reliable experimental basis for identifying the pharmacodynamic material basis of the antiviral Chinese medicine composition. At the same time, it is further conducive to studying the pharmacokinetic process of the prototype components in the above-mentioned Chinese medicine composition in vivo, and the pharmacokinetic markers of the Chinese medicine composition can also be screened according to the absorption of the compound in vivo, laying a data foundation for in-depth research on the mechanism of action of the Chinese medicine composition.

[0011] As a specific embodiment of the present invention, if the in vivo prototype components detected are neochlorogenic acid, p-coumaric acid, broken oxidized strychnine, liquiritin, isoliquiritigenin, liquiritigenin, hyperoside, isoquercetin, rhein-8-O-β-D-glucoside, 3,5-di-O-caffeoylquinic acid, formononetin, 4,5-di-O-caffeoylquinic acid, liquiritigenin, forsythin, rhein-8-O-β-D-glucoside, quercetin, luteolin, formononetin, aloe-rhein, isoliquiritigenin, glycyrrhizic acid, rhein, rhein, β-glycyrrhetinic acid, chlorogenic acid, cryptochlorogenic acid, rutin and kaempferol, the high performance liquid chromatography conditions are:

[0012] Chromatographic column: CAPCELL CORE C18, 2.1mm×75mm, 2.7μm;

[0013] Mobile phase A: 0.08%-0.12% formic acid in water;

[0014] Mobile phase B: acetonitrile;

[0015] 0-1min, 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] 7.01-9min, 95% mobile phase A, 5% mobile phase B.

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

[0024] The preferred HPLC conditions can improve the separation between component peaks and the detection sensitivity, making the detection results quantitatively accurate and highly precise.

[0025] As a specific embodiment of the present invention, the mass spectrometer adopts negative ion detection mode, the capillary voltage is 5.5 kV, the nebulizer gas flow rate is 55 L / min, the solution gas flow rate is 20 L / min, the collision gas flow rate is 9 L / min, and the heating block temperature is 550°C;

[0026] The parent ion of neochlorogenic acid is 353m / z, the daughter ion is 191m / z, the declustering voltage is 76V, and the fragmentation voltage is 31eV; the parent ion of p-coumaric acid is 163m / z, the daughter ion is 119m / z, the declustering voltage is 66V, and the fragmentation voltage is 23eV; the parent ion of strychnos nux vomica is 403.2m / z, the daughter ion is 179.2m / z, the declustering voltage is 86V, and the fragmentation voltage is 18eV; the parent ion of glycyrrhizin is 417.3m / z, the daughter ion is 135.1m / z, the declustering voltage is 125V, and the fragmentation voltage is 39eV; the parent ion of isoliquiritigenin is 417.2m / z, the daughter ion is 148.1m / z, the declustering voltage is 127V, and the fragmentation voltage is 23eV. The voltage was 48eV; the parent ion of liquiritin was 549.1m / z, the daughter ion was 255.3m / z, the declustering voltage was 146V, and the fragmentation voltage was 45eV; the parent ion of oligurin was 447.1m / z, the daughter ion was 285.3m / z, the declustering voltage was 154V, and the fragmentation voltage was 45eV; the parent ion of hyperoside was 463m / z, the daughter ion was 300.7m / z, the declustering voltage was 125V, and the fragmentation voltage was 36eV; the parent ion of isoquercetin was 463.2m / z, the daughter ion was 255m / z, the declustering voltage was 113V, and the fragmentation voltage was 58eV; the parent ion of rhein-8-O-β-D-glucoside was 445m / z, the daughter ion was 2 The parent ion of 3,5-di-O-caffeoylquinic acid is 515.3m / z, the daughter ion is 191.2m / z, the declustering voltage is 91V, and the fragmentation voltage is 14eV; the parent ion of formononetin is 475.1m / z, the daughter ion is 267 / z, the declustering voltage is 65V, and the fragmentation voltage is 20eV; the parent ion of 4,5-di-O-caffeoylquinic acid is 515.2m / z, the daughter ion is 173m / z, the declustering voltage is 81V, and the fragmentation voltage is 39eV; the parent ion of glycyrrhizin is 255m / z, the daughter ion is 135.1m / z, the declustering voltage is 83V, and the fragmentation voltage is 20eV ; The parent ion of forsythin was 579.2m / z, the daughter ion was 371.4m / z, the declustering voltage was 80V, and the fragmentation voltage was 40eV; the parent ion of rhubarb-8-O-β-D-glucoside was 431.2m / z, the daughter ion was 269.2m / z, the declustering voltage was 123V, and the fragmentation voltage was 39eV; the parent ion of quercetin was 447.2m / z, the daughter ion was 300.2m / z, the declustering voltage was 99V, and the fragmentation voltage was 38eV; the parent ion of luteolin was 285.1m / z, the daughter ion was 151.1m / z, the declustering voltage was 103V, and the fragmentation voltage was 35eV; the parent ion of formononetin was 267.1m / z, the daughter ion was 252.2m / z, declustering voltage was 106V, and fragmentation voltage was 30eV; the parent ion of aloe-emodin was 269m / z, the daughter ion was 240m / z, the declustering voltage was 104V, and the fragmentation voltage was 31eV; the parent ion of isoliquiritigenin was 255.1m / z, the daughter ion was 119.1m / z, the declustering voltage was 5V, and the fragmentation voltage was 42eV; the parent ion of glycyrrhizic acid was 821.5m / z, the daughter ion was 350.9m / z, the declustering voltage was 52V, and the fragmentation voltage was 41eV; the parent ion of rhein was 283.2m / z, the daughter ion was 183.1m / z, the declustering voltage was 52V, and the fragmentation voltage was 38eV; the parent ion of rhein was 269m / z, the daughter ion was 241m / z, the declustering voltage was 127V, and the fragmentation voltage was The decomposition voltage is 38eV; the parent ion of β-glycyrrhetinic acid is 469.3m / z, the daughter ion is 355.3m / z, the declustering voltage is 151V, and the fragmentation voltage is 67eV; the parent ion of chlorogenic acid is 353.3m / z, the daughter ion is 191.1m / z, the declustering voltage is 64V, and the fragmentation voltage is 22eV; the parent ion of cryptochlorogenic acid is 353.2m / z, the daughter ion is 173.1m / z, the declustering voltage is 76V, and the fragmentation voltage is 22eV; the parent ion of rutin is 609.1m / z, the daughter ion is 300.2m / z, the declustering voltage is 134V, and the fragmentation voltage is 50eV; the parent ion of kaempferol is 285m / z, the daughter ion is 210.9m / z, the declustering voltage is 85V, and the fragmentation voltage is 45eV. .

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

[0028] As a specific embodiment of the present invention, if the in vivo prototype components detected are quinic acid, gallic acid, salidroside, caffeic acid, prunin, forsythiaside E, forsythiaside A, forsythiaside B, forsythiaside I and ferulic acid, the HPLC conditions are:

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

[0030] Mobile phase A: 0.08%-0.12% formic acid in water;

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

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

[0036] 6-6.01 min, 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 was 35°C, the flow rate was 0.35 mL / min, and the injection volume was 10 μL.

[0039] Furthermore, the mobile phase A is a 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, salidroside, caffeic acid, prunin, forsythiaside E, forsythiaside A, forsythiaside B, forsythiaside I and ferulic acid components, so that the detection results are quantitatively accurate and have high precision.

[0041] Further, the mass spectrometer adopted negative ion detection mode, the capillary voltage was 4 kV, the nebulizer gas flow rate was 10 L / min, the solvent gas flow rate was 30 L / min, the desolvation gas line temperature was 325°C, and the heating block temperature was 325°C;

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

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

[0044] As a specific embodiment of the present invention, if the in vivo prototype components detected are (R, S)-gaitrin, pseudoephedrine, ephedrine, methylephedrine, amygdalin, limonene I and kaempferol-O-rutinoside, the HPLC conditions are:

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

[0046] Mobile phase A: 0.08%-0.12% formic acid in water;

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

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

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

[0055] The preferred high performance liquid chromatography conditions can improve the separation degree and detection sensitivity between the component peaks of (R, S)-gaoipine, pseudoephedrine, ephedrine, methylephedrine, amygdalin, scutellarin I and kaempferol-O-rutinoside, so that the detection results are quantitatively accurate and have high precision.

[0056] Further, the mass spectrometer adopted a positive ion detection mode, 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°C, and a heating block temperature of 325°C;

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

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

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

[0060] Accurately weigh a certain amount of ephedrine, pseudoephedrine, methylephedrine, quinic acid, gallic acid, (R, S)-goitrin, salidroside, neochlorogenic acid, forsythiaside E, caffeic acid, amygdalin, prunin, p-coumaric acid, strychnine, ferulic acid, liquiritin, isoliquiritigenin, forsythiaside I, licorice glycoside, luteolin, forsythiaside B, hyperoside, forsythiaside A, clematisin I, isoquercetin, kaempferol-O-rutinoside, rhein-8-O -β-D-glucoside, 3,5-di-O-caffeoylquinic acid, formononetin, 4,5-di-O-caffeoylquinic acid, liquiritigenin, forsythin, emodin-8-O-β-D-glucoside, quercetin, luteolin, formononetin, aloe-emodin, isoliquiritigenin, glycyrrhizic acid, rhein, emodin, β-glycyrrhetinic acid, chlorogenic acid, cryptochlorogenic acid, rutin and kaempferol reference substances were dissolved in methanol and prepared into a mother solution of each reference substance with a concentration of 2 mg / mL.

[0061] Mixed stock solution 1: Accurately measure the prepared reference substance mother solution above and dilute it with 50% methanol aqueous solution to prepare a mixed stock solution containing 24 μg / mL (ephedrine, pseudoephedrine, methylephedrine, ferulic acid, liquiritin, isoglycyrrhizin, apigenin, luteolin, forsythiaside B, rutin, hyperoside, forsythiaside A, celery glycoside I, isoquercetin, kaempferol-O-rutinoside, rhein-8-O-β-D-glucoside, 3-hydroxy-1-hydroxy-1-hydroxy-2-ol-glucoside, ... ,5-di-O-caffeoylquinic acid, formononetin, 4,5-di-O-caffeoylquinic acid, glycyrrhizin, rheum-8-O-β-D-glucoside, quercetin, luteolin, formononetin, aloe rhubarb, isoliquiritigenin, rheum); 120μg / mL ((R,S)-gaoichun, amygdalin, caffeic acid, forsythiaside I, β-glycyrrhetinic acid, strychnine-oxidized strychnine, neochlorogenic acid) mixed stock solution 1.

[0062] Mixed stock solution 2: Accurately measure the prepared reference substance mother solutions above and dilute them with 50% methanol aqueous solution to prepare mixed stock solution 2 containing 480 μg / mL (rhein, prunin); 72 μg / mL (gallic acid, forsythiaside E, glycyrrhizic acid, kaempferol).

[0063] Mixed stock solution 3: Accurately measure the prepared reference substance mother solutions above and dilute them with 50% methanol aqueous solution to prepare a mixed stock solution 3 of 240 μg / mL (salidroside, quinic acid, cryptochlorogenic acid, chlorogenic acid, p-coumaric acid).

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

[0065] Accurately pipette 0.5 μL, 1.5 μL, 5 μL, 15 μL, 40 μL, 60 μL, and 80 μL of the above-mentioned mixed reference working solution, respectively, and dilute them to 100 μL with 50% methanol aqueous solution to obtain mixed reference solution of a series of concentrations for establishing a standard curve for quantitative detection of the corresponding substances.

[0066] Furthermore, the present invention adopts the internal standard method when calculating the contents of 46 components in the antiviral traditional Chinese medicine composition, and the internal standards are gallic acid-d2 (IS-1), ferulic acid-d3 (IS-2), and acetaminophen (IS-3).

[0067] Specifically, the test sample described in the present invention is Lianhua Qingwen Capsule.

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

[0069] S1, weigh an appropriate amount of Lianhua Qingwen Capsules, and prepare a dosing solution with a concentration of 0.80 g / mL using ultrapure water. The dosing dose is 12 g / kg, and the dosing volume is 15 mL / kg;

[0070] S2, a single dose of 12 g / kg Lianhua Qingwen solution was given to mice, and then 0.5 mL of blood was collected through the eye socket 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 dose. The blood was placed in a sodium heparin tube, centrifuged at a centrifugal force of 2348 g for 10 min, and 60 μL of supernatant was accurately pipetted into a 1.5 mL EP tube and stored in a -80 °C refrigerator;

[0071] S3, preparing 100U / 5μL β-glucuronidase and 1U / 5μL sulfatase solution with 0.2% ice NaCl solution to obtain a mixed enzyme;

[0072] S4, add 5 μL of internal standard working solution and 10 μL of mixed enzyme to 60 μL of plasma sample, incubate in 37 ° C water bath for 1 h, quickly stop enzymatic hydrolysis in -80 ° C refrigerator, add three times methanol (180 μL), vortex for 1 min, centrifuge at a centrifugal force of 2348g for 5 min, take the supernatant, blow with nitrogen, re-dissolve with 100 μL of initial mobile phase (0.1% formic acid water: acetonitrile (v:v = 95:5)) (100 μL), centrifuge at a centrifugal force of 15871g for 10 min to obtain the test solution.

[0073] It is of great significance to develop and research Chinese patent medicines using modern methods, which provides an objective and overall evaluation basis for the quality of Chinese patent medicines. The present invention uses HPLC-MS / MS method to establish a plasma quantitative analysis method for 46 relevant components in antiviral Chinese medicine compositions, including alkaloid components in Ephedra medicinal materials; lignans, phenylethanol, and flavonoid glycoside components in Forsythia medicinal materials; triterpenoid saponins in Licorice medicinal materials; flavonoids and organic acid components in Honeysuckle medicinal materials; anthraquinone components in Rhubarb medicinal materials; cyanogenic glycoside components in stir-fried bitter almond medicinal materials, etc. The detection method is verified in terms of selectivity, accuracy, precision, stability, etc., indicating that the established method is simple and accurate, and meets the requirements of ICH M10 for bioanalysis methods. The detection method provided by the present invention is convenient for studying the pharmacokinetics of the blood-entering prototype components in the antiviral Chinese medicine composition in vivo, and the pharmacokinetic markers of the antiviral Chinese medicine composition can also be screened according to the absorption of the compound in vivo, which has high practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 The MRM chromatogram of the test solution detected by analytical method 1 under 2.2 of Example 2; (B) is a partial enlarged view of the green box in (A), and (C) is a partial enlarged view of the orange box in (A);

[0075] Figure 2 The MRM chromatogram of the test solution detected by analytical method 2 under item 2.2 of Example 2 in Example 2;

[0076] Figure 3 The MRM chromatogram of the test solution detected by analytical method 3 under item 2.2 of Example 2;

[0077] Figure 4 The MRM chromatogram of (R,S)-goitrin 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 15 minutes after a single dose of LHQW;

[0078] Figure 5 The MRM chromatograms of ephedrine and pseudoephedrine under Item 2.4 of Example 2, wherein (A) blank plasma + internal standard, (B) lower limit of quantification concentration sample LLOQ, (C) real rat plasma sample collected 15 minutes after a single dose of LHQW;

[0079] Figure 6 The MRM chromatogram of methylephedrine under Item 2.4 of Example 2, wherein (A) blank plasma + internal standard, (B) lower limit of quantification concentration sample LLOQ, (C) real rat plasma sample collected 15 minutes after a single dose of LHQW;

[0080] Figure 7 The MRM chromatogram of amygdalin 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 15 minutes after a single dose of LHQW;

[0081] Figure 8 The MRM chromatograms of calendula officinalis I and kaempferol-3-O-rutinoside 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 15 minutes after a single dose of LHQW;

[0082] Fig. 9 The MRM chromatogram of caffeic 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 15 minutes after a single dose of LHQW;

[0083] Fig.10 The MRM chromatogram of quinic 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 15 minutes after a single dose of LHQW;

[0084] Fig.11 The MRM chromatogram of gallic 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 15 minutes after a single dose of LHQW;

[0085] Fig.12 The MRM chromatogram of ferulic 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 15 minutes after a single dose of LHQW;

[0086] Fig.13 The MRM chromatograms of forsythiaside A and forsythiaside I under 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 15 minutes after a single dose of LHQW;

[0087] Fig.14 The MRM chromatogram of Forsythiaside B 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 15 minutes after a single dose of LHQW;

[0088] Fig.15 The MRM chromatogram of forsythiaside E 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 15 minutes after a single dose of LHQW;

[0089] Fig.16 The MRM chromatogram of salidroside 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 15 minutes after a single dose of LHQW;

[0090] Fig.17 This is the MRM chromatogram of prunin under Item 2.4 of Example 2, wherein (A) blank plasma + internal standard, (B) lower limit of quantification concentration sample LLOQ, (C) real rat plasma sample collected 15 minutes after a single dose of LHQW;

[0091] Fig.18 This is the MRM chromatogram of neochlorogenic 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 15 minutes after a single dose of LHQW;

[0092] Fig.19 The MRM chromatogram of chlorogenic 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 15 minutes after a single dose of LHQW;

[0093] Fig. 20 The MRM chromatogram of cryptochlorogenic 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 15 minutes after a single dose of LHQW;

[0094] Fig.21 The MRM chromatogram of oxidized strychnine under 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 15 minutes after a single dose of LHQW;

[0095] Fig. 22 The MRM chromatogram of p-coumaric 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 15 minutes after a single dose of LHQW;

[0096] Fig.23The MRM 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 15 minutes after a single dose of LHQW;

[0097] Fig.24 The MRM chromatogram of apigenin 2.4 in Example 2, wherein (A) blank plasma + internal standard, (B) lower limit of quantification sample LLOQ, (C) real rat plasma sample collected 15 minutes after a single dose of LHQW;

[0098] Fig.25 The MRM chromatogram of liquiritin 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 15 minutes after a single dose of LHQW;

[0099] Fig.26 The 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 15 minutes after a single dose of LHQW;

[0100] Fig. 27 The 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 15 minutes after a single dose of LHQW;

[0101] Fig.28 The MRM chromatogram of isoquercetin 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 15 minutes after a single dose of LHQW;

[0102] Fig.29 The MRM chromatogram of luteolin 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 15 minutes after a single dose of LHQW;

[0103] Fig.30 The 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 15 minutes after a single dose of LHQW;

[0104] Fig.31The MRM chromatogram of 4,5-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 15 minutes after a single dose of LHQW;

[0105] Fig.32 The MRM chromatogram of quercetin under Item 2.4 of Example 2, wherein (A) blank plasma + internal standard, (B) lower limit of quantification concentration sample LLOQ, (C) real rat plasma sample collected 15 minutes after a single dose of LHQW;

[0106] Fig.33 The MRM chromatogram of isoliquiritigenin 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 15 minutes after a single dose of LHQW;

[0107] Fig.34 The MRM chromatogram of formononetin 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 15 minutes after a single dose of LHQW;

[0108] Fig.35 The MRM chromatogram of forsythiaside 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 15 minutes after a single administration of LHQW;

[0109] Fig.36 The MRM chromatogram of licorice in item 2.4 of Example 2, wherein (A) blank plasma + internal standard, (B) lower limit of quantification concentration sample LLOQ, (C) real rat plasma sample collected 15 minutes after a single dose of LHQW;

[0110] Fig.37 The MRM chromatogram of emodin-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 15 minutes after a single dose of LHQW;

[0111] Fig.38 The MRM chromatogram of luteolin 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 15 minutes after a single dose of LHQW;

[0112] Fig.39The MRM chromatogram of glycyrrhizic 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 15 minutes after a single dose of LHQW;

[0113] Fig.40 The MRM chromatogram of kaempferol under Item 2.4 of Example 2, wherein (A) blank plasma + internal standard, (B) lower limit of quantification concentration sample LLOQ, (C) real rat plasma sample collected 15 minutes after a single dose of LHQW;

[0114] Fig.41 The MRM chromatogram of isoliquiritigenin 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 15 minutes after a single dose of LHQW;

[0115] Fig.42 The MRM chromatogram of formononetin 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 15 minutes after a single dose of LHQW;

[0116] Fig.43 The MRM chromatogram of aloe-emodin 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 15 minutes after a single dose of LHQW;

[0117] Fig.44 The MRM chromatogram of rhein 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 15 minutes after a single dose of LHQW;

[0118] Fig.45 The MRM chromatogram of emodin 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 15 minutes after a single dose of LHQW;

[0119] Fig.46 The MRM chromatogram of β-glycyrrhetinic 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 15 minutes after a single dose of LHQW;

[0120] Fig.47 1 is the drug-time curve of quinic acid, ephedrine and pseudoephedrine in rats in Example 3;

[0121] Fig.48 1 is the drug-time curve of gallic acid, methylephedrine and (R,S)-gaitrin in rats in Example 3;

[0122] Fig.49 The drug-time curves of salidroside, neochlorogenic acid and forsythiaside E in rats in Example 3;

[0123] Fig.50 The drug-time curves of caffeic acid, amygdalin and prunin in rats in Example 3;

[0124] Fig.51 1 is the drug-time curve of cryptochlorogenic acid, chlorogenic acid and p-coumaric acid in rats in Example 3;

[0125] Fig.52 The drug-time curves of interrupted oxidation of strychnine, ferulic acid and liquiritin in rats in Example 3;

[0126] Fig.53 The drug-time curves of isoliquiritigenin, forsythiaside I and liquiritigenin in rats in Example 3;

[0127] Fig.54 The drug-time curves of luteolin, forsythiaside B and rutin in rats in Example 3;

[0128] Fig.55 The drug-time curves of hyperoside, forsythiaside A and cyperus foetida I in rats in Example 3;

[0129] Fig.56 The drug-time curves of isoquercetin, kaempferol-O-rutinoside, and rhein-8-O-β-D-glucoside in rats in Example 3;

[0130] Fig.57 The drug-time curves of 3,5-di-O-caffeoylquinic acid, formononetin and 4,5-di-O-caffeoylquinic acid in rats in Example 3 are shown in FIG.

[0131] Fig.58 The drug-time curves of liquiritigenin, forsythiaside and rhamnolide-8-O-β-D-glucoside in rats in Example 3;

[0132] Fig.59 The drug-time curves of quercetin, luteolin and kaempferol in rats in Example 3;

[0133] Fig.60 The drug-time curves of formononetin, aloe-emodin and isoliquiritigenin in rats in Example 3;

[0134] Fig.61The drug-time curves of glycyrrhizic acid, rhein and rhein in rats in Example 3;

[0135] Fig.62 This is the drug-time curve of β-glycyrrhetinic acid in rats in Example 3. DETAILED DESCRIPTION

[0136] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

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

[0138] Example 1

[0139] 1. Reagents

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

[0141] Lianhua Qingwen Capsule dry paste is provided by Shijiazhuang Yiling Pharmaceutical Co., Ltd. and contains 8.41g of raw materials per gram.

[0142] Ephedrine, pseudoephedrine, and methylephedrine were purchased from the National Institutes for Food and Drug Control (Beijing, China). Quinic acid, gallic acid, (R,S)-gallin, salidroside, neochlorogenic acid, forsythiaside E, caffeic acid, amygdalin, prunin, p-coumaric acid, strychnoside, ferulic acid, liquiritin, isoliquiritigenin, forsythiaside I, licorice glycoside, luteolin, forsythiaside B, hyperoside, forsythiaside A, scutellarin I, isoquercitrin, kaempferol-O-rutinoside, rhein-8 -O-β-D-glucoside, 3,5-di-O-caffeoylquinic acid, formononetin, 4,5-di-O-caffeoylquinic acid, liquiritigenin, forsythin, rhein-8-O-β-D-glucoside, quercetin, luteolin, formononetin, aloe-rhein, isoliquiritigenin, glycyrrhizic acid, rhein, rhein, β-glycyrrhetinic acid were purchased from Baoji Chenguang Biological Co., Ltd. (Baoji, China). Prunin was purchased from Yunnan Xili Biotechnology Co., Ltd. (Yunnan, China). Chlorogenic acid, cryptochlorogenic acid, rutin, and kaempferol were purchased from Chengdu Zhibiao Huachun Biotechnology Co., Ltd. (Chengdu, China). The purity of all reference 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 Medicines

[0145] Contents of the traditional Chinese medicine Lianhua Qingwen Capsules, provided by Shijiazhuang Yiling Pharmaceutical Co., Ltd. (China); specifications: 8.39 g crude drug / g dry paste, 0.240 g / preparation unit.

[0146] 1.3 Experimental animals

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

[0148] Rats were raised in the animal observation room of the School of Pharmacy of Xiamen University (12 h light per day, temperature of 25 ± 2 ° C, relative humidity of 55 ± 5%). The experimental animals were free to drink water and eat. The experiment began after one week of adaptation to the environment. The experimental animals were fasted for 12 h before the experiment and were free to drink water. The study followed the procedures established by the Animal Experiment Ethics Committee of Xiamen University.

[0149] 2. Solution Preparation

[0150] 2.1 Solution preparation

[0151] 2.1.1 Standard stock solution

[0152] Accurately weigh a certain amount of ephedrine, pseudoephedrine, methylephedrine, quinic acid, gallic acid, (R, S)-goitrin, salidroside, neochlorogenic acid, forsythiaside E, caffeic acid, amygdalin, prunin, p-coumaric acid, strychnoside, ferulic acid, liquiritin, isoliquiritigenin, forsythiaside I, licorice glycoside, luteolin, forsythiaside B, hyperoside, forsythiaside A, clematisin I, isoquercetin, kaempferol-O-rutinoside, rhein-8-O-β-D-glucoside, 3,5-di-O-caffeoylquinic acid, formononetin, 4,5-di-O-caffeoylquinic acid, glycyrrhizin, forsythin, emodin-8-O-β-D-glucoside, quercetin, luteolin, formononetin, aloe-emodin, isoliquiritigenin, glycyrrhizic acid, rhein, emodin, β-glycyrrhetinic acid, chlorogenic acid, cryptochlorogenic acid, rutin and kaempferol reference substances were dissolved in methanol and prepared into 2 mg / mL mother solutions of each reference substance, respectively, and packaged in 1.5 mL brown glass bottles and stored at -80°C.

[0153] Mixed stock solution 1: Accurately measure the prepared reference substance mother solution above and dilute it with 50% methanol aqueous solution to prepare a mixed stock solution containing 24 μg / mL (ephedrine, pseudoephedrine, methylephedrine, ferulic acid, liquiritin, isoglycyrrhizin, apigenin, luteolin, forsythiaside B, rutin, hyperoside, forsythiaside A, celery glycoside I, isoquercetin, kaempferol-O-rutinoside, rhein-8-O-β-D-glucoside, 3-hydroxy-1-hydroxy-1-hydroxy-2-ol-glucoside, ... ,5-di-O-caffeoylquinic acid, formononetin, 4,5-di-O-caffeoylquinic acid, glycyrrhizin, rheum-8-O-β-D-glucoside, quercetin, luteolin, formononetin, aloe rhubarb, isoliquiritigenin, rheum); 120μg / mL ((R,S)-gaoichun, amygdalin, caffeic acid, forsythiaside I, β-glycyrrhetinic acid, strychnine-oxidized strychnine, neochlorogenic acid) mixed stock solution 1.

[0154] Mixed stock solution 2: Accurately measure the prepared reference substance mother solutions above and dilute them with 50% methanol aqueous solution to prepare mixed stock solution 2 containing 480 μg / mL (rhein, prunin); 72 μg / mL (gallic acid, forsythiaside E, glycyrrhizic acid, kaempferol).

[0155] Mixed stock solution 3: Accurately measure the prepared reference substance mother solutions above and dilute them with 50% methanol aqueous solution to prepare a mixed stock solution 3 of 240 μg / mL (salidroside, quinic acid, cryptochlorogenic acid, chlorogenic acid, p-coumaric acid).

[0156] Take 50 μL of each of the mixed stock solution 1, mixed stock solution 2 and mixed stock solution 3, add 450 μL of 50% methanol aqueous solution to obtain a mixed reference substance working solution (Cal-8).

[0157] Accurately pipette 0.5 μL, 1.5 μL, 5 μL, 15 μL, 40 μL, 60 μL, and 80 μL of the mixed reference substance working solution (Cal-8) and dilute them to 100 μL with 50% methanol aqueous solution to obtain mixed reference substance solutions of a series of concentrations for establishing a standard curve for quantitative detection of the corresponding substances. The preparation method is shown in Table 1.

[0158] Table 1 Preparation and dilution process of mixed reference 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] Preparation of internal standard stock solutions: accurately weigh a certain amount of gallic acid-d2 (IS-1), ferulic acid-d3 (IS-2), and acetaminophen (IS-3) into different 1.5 mL EP tubes, add methanol, and ultrasonically dissolve them to prepare internal standard stock solutions with a concentration of 1 mg / mL.

[0163] Preparation of internal standard working solution (IS-mix): Take 20 μL of each of the three internal standard mother solutions (1 mg / mL) and add 950 μL of 50% methanol aqueous solution to obtain internal standard stock solution (20 μg / mL), put it in a brown liquid phase vial and store at -20°C. Take 25 μL of the internal standard stock solution and add 975 μL of 50% methanol aqueous solution to dilute it to obtain the internal standard working solution (500 ng / mL).

[0164] 2.1.3 Preparation of enzyme solution

[0165] Use 0.2% ice NaCl solution to prepare 100 U / 5 μL (10.4275 mg / mL) of β-glucuronidase and 1 U / 5 μL (6.5146 mg / mL) of sulfatase solution, and use them immediately after preparation.

[0166] 2.2 Preparation of drug solution for animals

[0167] Weigh an appropriate amount of the contents of Lianhua Qingwen Capsules into a 50 mL centrifuge tube, and use ultrapure water to prepare a dosing solution with a concentration of 0.80 g / mL (dose is 12 g / kg, 15 mL / kg). Stir continuously during the addition process and mix by ultrasound until it becomes a completely suspended uniform liquid. This process is completed at room temperature.

[0168] 2.3 Drug administration and biological collection

[0169] Animals were adaptively fed for one week before the animal experiment, and fasted but not watered for 12 hours before the experiment. Blank blood (0.3 mL) was collected from the eye sockets before administration, and a single dose of 12 g / kg Lianhua Qingwen solution was given. The administration group collected blood 0.5 mL / time through the eye sockets 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, respectively, and placed in a commercial sodium heparin tube, centrifuged at a centrifugal force of 2348 g for 10 min, and 60 μL of supernatant was accurately drawn into a 1.5 mL EP tube and stored in a -80 °C refrigerator.

[0170] 2.4 Plasma sample processing

[0171] Treatment of plasma samples to be tested: add 5 μL of internal standard working solution (under 2.1.2) and 10 μL of mixed enzyme (under 2.1.3) to 60 μL of plasma sample, incubate in a 37°C water bath for 1 h, then quickly stop enzymatic hydrolysis in a -80°C refrigerator, add three times methanol (180 μL), vortex for 1 min, centrifuge at a centrifugal force of 2348 g for 5 min, take the supernatant, blow with nitrogen, reconstitute with 100 μL of 100L initial mobile phase (0.1% formic acid water: acetonitrile (v:v=95:5)), centrifuge at a centrifugal force of 15871 g for 10 min, take the supernatant into a 96-well plate, and inject according to the injection volume specified in the method.

[0172] Blank plasma sample processing: Except for replacing the plasma to be tested with blank plasma, the rest of the operation process is the same as under “Plasma sample processing to be tested”.

[0173] 3. Chromatographic / mass spectrometric conditions for HPLC-MS / MS analysis

[0174] (1) Analysis method 1

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

[0176] Analyzed compounds (29): neochlorogenic acid, p-coumaric acid, strychnine, liquiritigenin, isoliquiritigenin, liquiritigenin, hyperoside, isoquercetin, rhein-8-O-β-D-glucoside, 3,5-di-O-caffeoylquinic acid, formononetin, 4,5-di-O-caffeoylquinic acid, liquiritigenin, forsythin, rhein-8-O-β-D-glucoside, quercetin, luteolin, formononetin, aloe-emodin, isoliquiritigenin, glycyrrhizic acid, rhein, rhein, β-glycyrrhetinic acid, chlorogenic acid, cryptochlorogenic acid, rutin and kaempferol.

[0177] HPLC conditions:

[0178] Chromatographic column: CAPCELL CORE C18, 2.1mm×75mm, 2.7μm;

[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] 0-1min, 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] 7.01-9min, 95% mobile phase A, 5% mobile phase B;

[0193] Mass spectrometry conditions:

[0194] The ion source was an electrospray ion source (ESI source), negative ion detection mode, capillary voltage: 5.5 KV, nebulizer gas: 55.0 L / min, dissolved gas: 20.0 L / min, collision gas: 9.0 L / min; heating block temperature: 550 °C; scanning mode was multiple reaction monitoring (MRM) mode, and the quantitative parent ion, daughter ion, fragmentation voltage and declustering voltage are shown in Table 2. MultiQuant TM Quantitative software collects and processes experimental data.

[0195] (2) Analysis method 2

[0196] An Agilent 1260 series liquid chromatograph coupled with an Agilent 6460 triple quadrupole mass spectrometer (QQQ) was used for detection and analysis.

[0197] Analyzed compounds (10): quinic acid, gallic acid, salidroside, caffeic acid, prunin, forsythiaside E, forsythiaside A, forsythiaside B, forsythiaside I and ferulic acid.

[0198] HPLC conditions:

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

[0200] Mobile phase A: 0.1% formic acid in water;

[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] 6.01-7.5min, 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, dissolved gas: 30.0 L / min, desolvation gas line temperature was 325 °C, heating block temperature: 325 °C, scanning mode was MRM monitoring mode, and the quantitative parent ion, daughter ion, fragmentation voltage and declustering voltage are shown in Table 2. Agilent MassHunter data processing workstation and Agilent Qualitative Analysis B.07.00 Tools software were used to collect and process the experimental data.

[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)-gaitrin, pseudoephedrine, ephedrine, methylephedrine, amygdalin, calendulaside I and kaempferol-O-rutinoside.

[0217] The HPLC conditions were:

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

[0219] Mobile phase A: 0.1% formic acid in water;

[0220] Mobile phase B: 100% acetonitrile;

[0221] Column temperature: 25°C;

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

[0230] Mass spectrometry conditions:

[0231] The ion source was an electrospray ion source (ESI source), positive ion detection mode, capillary voltage: 4.0 KV, nebulizer gas: 10.0 L / min, dissolved gas: 30.0 L / min, desolvation gas line temperature was 325 ° C, heating block temperature: 325 ° C, scanning mode was MRM monitoring mode, and the quantitative parent ion, daughter ion, fragmentation voltage and declustering voltage are shown in Table 2. Agilent MassHunter data processing workstation and Agilent Qualitative Analysis B.07.00 Tools software were used to collect and process the experimental data.

[0232] Table 2 MRM mass spectrometry parameters of 46 test compounds in Lianhua Qingwen Capsules

[0233]

[0234]

[0235]

[0236] Example 2

[0237] Methodological validation:

[0238] 2.1 Linear range

[0239] The mixed reference solution and the quality control sample solution were prepared according to the series of concentrations of each compound prepared under 2.1.1 in Example 1. The concentrations of each compound in the mixed reference solution and the quality control sample solution are shown in Table 3.

[0240] 5 μL of the mixed reference solution (cal-1 to cal-8) or quality control working solution of each concentration in Table 3, 5 μL of the internal standard working solution (under 2.1.2 in Example 1) and 10 μL of the mixed enzyme (under 2.1.3 in Example 1) were added to 55 μL of blank plasma, incubated in a 37°C water bath for 1 h, and the enzymatic hydrolysis was quickly stopped in a -80°C refrigerator. Three times methanol (180 μL) was added, vortexed for 1 min, centrifuged at a centrifugal force of 2348 g for 5 min, the supernatant was taken, nitrogen blown, and re-dissolved with 100 μL of the initial mobile phase (0.1% formic acid water: acetonitrile (v:v=95:5)), centrifuged at a centrifugal force of 15871 g for 10 min, the supernatant was taken into a 96-well plate, and the sample was injected according to the injection volume specified in the method.

[0241] Table 3 Standard curve and quality control concentration setting of the tested compounds

[0242]

[0243]

[0244]

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

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

[0247]

[0248]

[0249]

[0250]

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

[0252] 2.2 Test solution testing

[0253] To 55 μL of blank plasma, add 5 μL of the cal-8 mixed reference solution in Table 3, 5 μL of the internal standard working solution (under 2.1.2 in Example 1) and 10 μL of the mixed enzyme (under 2.1.3 in Example 1), and perform subsequent treatment as in 2.1 to prepare the test solution.

[0254] The test solution prepared above was tested according to the HPLC-MS / MS analysis method in Example 1. Figure 1-Figure 3 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, which proves that the detection method provided by the present invention can accurately detect the 46 blood-entering prototype components in Lianhua Qingwen Capsules.

[0256] 2.3 Accuracy and precision

[0257] Take low-concentration plasma quality control samples (LLOQ), medium-concentration plasma quality control samples (MQC) and high-concentration plasma quality control samples (HQC) under Item 2.1 to investigate the precision and accuracy of plasma. Six samples were measured in parallel for each concentration, and the ratio of the peak area of ​​the target compound and the internal standard was substituted into the standard curve to calculate the concentration. The precision is expressed by the intra-day and inter-day precision RSD of the quality control samples. The RSD should generally be less than 15%, and the accuracy requirement is within the range of 85%-115%. The intra-day and inter-day precision and accuracy results of the measured concentrations of quality control samples 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] The results showed that the RSDs of intra-day precision and inter-day precision were less than 14.64% and 14.93% respectively, and the accuracy range was within 85-115%, which could meet the requirements of measurement accuracy.

[0267] 2.4 Selectivity and specificity

[0268] The blank plasma samples of rats + internal standard, the blank plasma samples added with mixed reference solution, the quality control samples of internal standard and mixed enzyme (prepared by the same method as LLOQ under 2.1 in Example 2), and the plasma samples of rats after administration (prepared by the same method as 2.2-2.4 in Example 1) were respectively subjected to protein precipitation pretreatment, and then sampled and tested using the three HPLC-MS / MS analysis methods of Example 1, and the chromatograms were recorded. The chromatograms of the individual compounds in each chromatogram were analyzed and compared. The results are as follows: Figure 4-Figure 46 shown.

[0269] The results showed that the blank biological matrix and potential related interfering substances had no interference with the determination of 46 test compounds and internal standards in Lianhua Qingwen, and the isomers had good separation, indicating that the established method is suitable for the determination of these 46 target compounds.

[0270] 2.5 Stability

[0271] 2.5.1 Stability after treatment

[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, and process them according to the plasma sample under Item 2.4 in Example 1. After processing, place them at room temperature (25°C) for 24 hours and then analyze them. Substitute them into the standard curve, calculate the concentration and SD value, and 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, and place them in a -80°C refrigerator for 24 hours respectively. After all of them are taken out and thawed naturally at room temperature (25°C), they are put back into the -80°C refrigerator, and the freeze-thaw cycle is repeated twice at an interval of 24 hours. The plasma samples under Item 2.4 in Example 1 are processed. After processing, the samples are injected and analyzed, substituted into the standard curve, and the concentration and SD values ​​are calculated. 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, place them at 4°C for 24 hours, and then process them according to the plasma samples under Item 2.4 in Example 1. After processing, inject and analyze, substitute them into the standard curve, calculate the concentration and SD value, and the results are shown in Table 6.

[0277] 2.5.4 Long-term stability

[0278] 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 were placed at -80°C for 10 days, and then processed according to the plasma samples under Item 2.4 in Example 1. After processing, the samples were injected and analyzed, substituted into the standard curve, and the concentration and SD values ​​were calculated. 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 showed that the short-term stability RSD value of the 46 compounds to be tested was less than 14.82% when placed at 4°C for 24 hours; the long-term stability RSD value when placed at -80°C for 10 days was less than 14.74%; the RSD value of the repeated freeze-thaw stability was less than 14.93%; and the RSD value of the stability after being placed at room temperature (25°C) for 24 hours was less than 14.88%. The accuracy range was within 85-115%, which could meet the requirements of the stability of the determination.

[0292] Example 3

[0293] Drug concentration in rat plasma

[0294] The pretreatment method of protein precipitation with methanol after enzymatic hydrolysis was combined with the established HPLC-MS / MS method to quantitatively analyze the 46 main components of Lianhua Qingwen in rat plasma after oral administration. The blood drug concentration calculated by the standard curve was imported into DAS2.0 software, and the pharmacokinetic parameters (AUC 0-t , T max , C max , t 1 / 2z , V z / F and CL z / F The specific results are shown in Table 7.

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

[0296]

[0297]

[0298]

[0299] The average plasma concentration-time curves of 46 compounds are shown in Figure 47-Figure 62As shown. From the average blood drug concentration-time curve and pharmacokinetic analysis results, it can be seen that the plasma concentrations of each component and its metabolic characteristics are quite different. Organic acids, anthraquinones, caffeoylquinic acid, phenylethanoid glycosides and alkaloids (except pseudoephedrine) have the characteristics of faster absorption, shorter peak time and slower elimination, while salidroside, flavonoids and triterpenoids have the characteristics of slower absorption and longer peak time. AUC 0-t The top 10 compounds with the highest AUC values ​​are: salidroside (mainly derived from the medicinal material Rhodiola), caffeic acid (mainly derived from the medicinal material honeysuckle), rhein (mainly derived from the medicinal material rhubarb), glycyrrhetinic acid (mainly derived from the medicinal material licorice), kaempferol (mainly derived from Rhodiola and rhubarb), quinic acid (mainly derived from honeysuckle), prunin (mainly derived from bitter almond), forsythiaside E (mainly derived from Forsythia), gallic acid (mainly derived from Rhodiola and rhubarb), ferulic acid (mainly derived from Isatis and Cyperus rotundus). Among them, salidroside and rhein are the quality control components of Rhodiola and rhubarb, respectively, and their AUC values ​​in rat plasma are 2.34 and 2.37, respectively. 0-t They were 132211.18±121869.67μg / L*h and 32865.26±12913.65μg / L*h, respectively. They are the main prototype components exposed in rat plasma after oral administration of Lianhua Qingwen Capsules and can be used as potential pharmacokinetic markers for Lianhua Qingwen Capsules. Among them, glycoside components include flavonoid glycosides, such as formononetin, isoquercitrin, luteolin, hyperoside, kaempferol-3-O-rutinoside and other compounds, which have low contents in the body. The possible reasons are speculated to be: 1) The compound undergoes hydrolysis reaction in the body to remove the glycosidic bond and exists in the form of metabolites; for example, luteolin is quickly absorbed after entering the body (T max 0.11±0.07h;t 1 / 2 2.65±1.63h) and metabolized to luteolin, which is the main form of luteolin in rats; 2) β-glucuronidase was added in the pretreatment of plasma samples, which hydrolyzed the β-D-glucose bond bound to the terminal non-reducing bond, released β-D-glucose and the corresponding prototype ligand, and also hydrolyzed some glycoside prototype components, resulting in low content in the body.

[0300] The plasma concentration of compounds with smaller molecular weights, such as caffeic acid, kaempferol, quinic acid, gallic acid, ferulic acid, and p-coumaric acid, is high. It is speculated that the main reason is that in addition to the original content of the medicinal materials, they can also be converted from other components by breaking bonds. For example, compounds such as 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, cryptochlorogenic acid, chlorogenic acid, and neochlorogenic acid in the medicinal materials are all deoxyphenolic acids generated from caffeic acid and quinic acid.

[0301] The elimination half-lives of pseudoephedrine, forsythiaside B and kaempferol-3-O-rhamnoside were all less than 2 hours, and the half-lives of rutin, liquiritigenin, luteolin, isoliquiritigenin and glycyrrhetinic acid were all greater than 25 hours. The blood concentration-time curves of multiple compounds showed double peaks. For example, liquiritigenin appeared at 0.08h-1h and 6h-12h after taking Lianhua Qingwen. Fig.58 ) and prunin appeared at peak values ​​at 0.5h-1h and 6h-12h after administration, respectively. Fig.50 ), the first peak of which is produced by the intestinal absorption of the prototype component in Lianhua Qingwen. The second peak of licorice is produced by the deglycosylation of unabsorbed licorice glycoside / licorice apigenin after it is metabolized into licorice glycoside by microorganisms in the intestine and then absorbed by the body. The second peak of wild black cherry glycoside is mainly produced by the deglycosylation of amygdalin.

[0302] In addition, by comparing the blood drug concentration and the detection of compounds in medicinal materials, it was found that the concentrations of prunus glycoside and glycyrrhizic acid in medicinal materials were low, while the absorption and distribution process in the body showed a gradual increase in blood drug concentration. In previous studies, we learned that prunus glycoside and glycyrrhizic acid are the main metabolites of amygdalin and glycyrrhizic acid in the body, respectively, and play a major pharmacodynamic role. In this pharmacokinetic study, as the plasma concentrations of amygdalin and glycyrrhizic acid gradually decreased, the levels of prunus glycoside and glycyrrhizic acid in plasma gradually increased, reaching peak drug concentrations at 6.17±4.40h and 14.17±8.16h, respectively. This data once again confirmed this conclusion.

[0303] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting in vivo prototype components in an antiviral Chinese medicine composition, wherein the in vivo prototype components of the antiviral Chinese medicine composition include ephedrine, pseudoephedrine, methylephedrine, quinic acid, gallic acid, (R, S)-goitrin, salidroside, neochlorogenic acid, forsythiaside E, caffeic acid, amygdalin, prunin, p-coumaric acid, strychnine, ferulic acid, liquiritin, isoliquiritigenin, forsythiaside I, apigenin, luteolin, forsythiaside B, hyperoside, forsythiaside A, scutellarin I, isoquercetin, kaempferol-O-rutinoside, rhein-8-O-β-D-glucoside, 3,5-di-O-caffeoylquinic acid, formononetin, 4,5-di-O-caffeoylquinic acid, liquiritigenin, forsythin, rhein-8-O-β-D-glucoside, quercetin, luteolin, formononetin, aloe-rhein, isoliquiritigenin, glycyrrhizic acid, rhein, rhein, β-glycyrrhetinic acid, chlorogenic acid, cryptochlorogenic acid, rutin and kaempferol, characterized in that, High performance liquid chromatography-mass spectrometry was used for detection, wherein the high performance liquid chromatography conditions were: A C18 chromatographic column was used, with a 0.08% to 0.12% formic acid aqueous solution as mobile phase A and acetonitrile as mobile phase B, and gradient elution was performed; The mass spectrometer adopts ESI ion source, and the scanning mode is MRM monitoring mode.

2. The method for detecting the in vivo prototype component in the antiviral Chinese medicine composition according to claim 1, characterized in that: If the prototype components detected in vivo are neochlorogenic acid, p-coumaric acid, strychnine, liquiritigenin, isoliquiritigenin, liquiritigenin, hyperoside, isoquercetin, rhein-8-O-β-D-glucoside, 3,5-di-O-caffeoylquinic acid, formononetin, 4,5-di-O-caffeoylquinic acid, liquiritigenin, forsythin, rhein-8-O-β-D-glucoside, quercetin, luteolin, formononetin, aloe-rhein, isoliquiritigenin, glycyrrhizic acid, rhein, rhein, β-glycyrrhetinic acid, chlorogenic acid, cryptochlorogenic acid, rutin and kaempferol, the HPLC conditions are: Chromatographic column: CAPCELL CORE C18, 2.1mm×75mm, 2.7μm; Mobile phase A: 0.08%-0.12% formic acid in water; Mobile phase B: acetonitrile; 0-1min, 95%→90% mobile phase A, 5%→10% mobile phase B; 1-2.5 min, 90% → 89% mobile phase A, 10% → 11% mobile phase B; 2.5-3 min, 89% → 82.5% mobile phase A, 11% → 17.5% mobile phase B; 3-4.5 min, 82.5% mobile phase A, 17.5% mobile phase B; 4.5-6 min, 82.5% → 20% mobile phase A, 17.5% → 80% mobile phase B; 6-7 min, 20% → 17% mobile phase A, 80% → 83% mobile phase B; 7-7.01 min, 17% → 95% mobile phase A, 83% → 5% mobile phase B; 7.01-9min, 95% mobile phase A, 5% mobile phase B.

3. The method for detecting the in vivo prototype component in the antiviral Chinese medicine composition according to claim 2, characterized in that: The column temperature was 35 °C, the flow rate was 0.4 mL / min, and the injection volume was 10 μL.

4. The method for detecting the in vivo prototype component in the antiviral Chinese medicine composition according to claim 2 or 3, characterized in that: The mass spectrometer adopted negative ion detection mode, the capillary voltage was 5.5 kV, the nebulizer gas flow rate was 55 L / min, the solvent gas flow rate was 20 L / min, the collision gas flow rate was 9 L / min, and the heating block temperature was 550°C; The parent ion of neochlorogenic acid is 353m / z, the daughter ion is 191m / z, the declustering voltage is 76V, and the fragmentation voltage is 31eV; the parent ion of p-coumaric acid is 163m / z, the daughter ion is 119m / z, the declustering voltage is 66V, and the fragmentation voltage is 23eV; the parent ion of strychnos nux vomica is 403.2m / z, the daughter ion is 179.2m / z, the declustering voltage is 86V, and the fragmentation voltage is 18eV; the parent ion of glycyrrhizin is 417.3m / z, the daughter ion is 135.1m / z, the declustering voltage is 125V, and the fragmentation voltage is 39eV; the parent ion of isoliquiritigenin is 417.2m / z, the daughter ion is 148.1m / z, the declustering voltage is 127V, and the fragmentation voltage is 23eV. The voltage was 48eV; the parent ion of liquiritin was 549.1m / z, the daughter ion was 255.3m / z, the declustering voltage was 146V, and the fragmentation voltage was 45eV; the parent ion of oligurin was 447.1m / z, the daughter ion was 285.3m / z, the declustering voltage was 154V, and the fragmentation voltage was 45eV; the parent ion of hyperoside was 463m / z, the daughter ion was 300.7m / z, the declustering voltage was 125V, and the fragmentation voltage was 36eV; the parent ion of isoquercetin was 463.2m / z, the daughter ion was 255m / z, the declustering voltage was 113V, and the fragmentation voltage was 58eV; the parent ion of rhein-8-O-β-D-glucoside was 445m / z, the daughter ion was 2 The parent ion of 3,5-di-O-caffeoylquinic acid is 515.3m / z, the daughter ion is 191.2m / z, the declustering voltage is 91V, and the fragmentation voltage is 14eV; the parent ion of formononetin is 475.1m / z, the daughter ion is 267 / z, the declustering voltage is 65V, and the fragmentation voltage is 20eV; the parent ion of 4,5-di-O-caffeoylquinic acid is 515.2m / z, the daughter ion is 173m / z, the declustering voltage is 81V, and the fragmentation voltage is 39eV; the parent ion of glycyrrhizin is 255m / z, the daughter ion is 135.1m / z, the declustering voltage is 83V, and the fragmentation voltage is 20eV ; The parent ion of forsythin was 579.2m / z, the daughter ion was 371.4m / z, the declustering voltage was 80V, and the fragmentation voltage was 40eV; the parent ion of rhubarb-8-O-β-D-glucoside was 431.2m / z, the daughter ion was 269.2m / z, the declustering voltage was 123V, and the fragmentation voltage was 39eV; the parent ion of quercetin was 447.2m / z, the daughter ion was 300.2m / z, the declustering voltage was 99V, and the fragmentation voltage was 38eV; the parent ion of luteolin was 285.1m / z, the daughter ion was 151.1m / z, the declustering voltage was 103V, and the fragmentation voltage was 35eV; the parent ion of formononetin was 267.1m / z, the daughter ion was 252.2m / z, declustering voltage was 106V, and fragmentation voltage was 30eV; the parent ion of aloe-emodin was 269m / z, the daughter ion was 240m / z, the declustering voltage was 104V, and the fragmentation voltage was 31eV; the parent ion of isoliquiritigenin was 255.1m / z, the daughter ion was 119.1m / z, the declustering voltage was 5V, and the fragmentation voltage was 42eV; the parent ion of glycyrrhizic acid was 821.5m / z, the daughter ion was 350.9m / z, the declustering voltage was 52V, and the fragmentation voltage was 41eV; the parent ion of rhein was 283.2m / z, the daughter ion was 183.1m / z, the declustering voltage was 52V, and the fragmentation voltage was 38eV; the parent ion of rhein was 269m / z, the daughter ion was 241m / z, the declustering voltage was 127V, and the fragmentation voltage was The decomposition voltage is 38eV; the parent ion of β-glycyrrhetinic acid is 469.3m / z, the daughter ion is 355.3m / z, the declustering voltage is 151V, and the fragmentation voltage is 67eV; the parent ion of chlorogenic acid is 353.3m / z, the daughter ion is 191.1m / z, the declustering voltage is 64V, and the fragmentation voltage is 22eV; the parent ion of cryptochlorogenic acid is 353.2m / z, the daughter ion is 173.1m / z, the declustering voltage is 76V, and the fragmentation voltage is 22eV; the parent ion of rutin is 609.1m / z, the daughter ion is 300.2m / z, the declustering voltage is 134V, and the fragmentation voltage is 50eV; the parent ion of kaempferol is 285m / z, the daughter ion is 210.9m / z, the declustering voltage is 85V, and the fragmentation voltage is 45eV. .

5. The method for detecting the in vivo prototype component in the antiviral Chinese medicine composition according to claim 1, characterized in that: If the in vivo prototype components detected are quinic acid, gallic acid, salidroside, caffeic acid, prunin, forsythiaside E, forsythiaside A, forsythiaside B, forsythiaside I and ferulic acid, the HPLC conditions are: Chromatographic column: CAPCELL PAK C18, 2.0 mm × 50 mm, 2 μm; Mobile phase A: 0.08%-0.12% formic acid in water; Mobile phase B: acetonitrile; 0-1 min, 95% → 83% mobile phase A, 5% → 17% mobile phase B; 1-3.5 min, 83% → 80% mobile phase A, 17% → 20% mobile phase B; 3.5-4 min, 80% → 35% mobile phase A, 20% → 65% mobile phase B; 4-6 min, 35% → 5% mobile phase A, 65% → 95% mobile phase B; 6-6.01 min, 5% → 95% mobile phase A, 95% → 5% mobile phase B; 6.01-7.5min, 95% mobile phase A, 5% mobile phase B.

6. The method for detecting the in vivo prototype component in the antiviral Chinese medicine composition according to claim 5, characterized in that: The column temperature was 35 °C, the flow rate was 0.35 mL / min, and the injection volume was 10 μL.

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

8. The method for detecting the in vivo prototype component in the antiviral Chinese medicine composition according to claim 1, characterized in that: If the prototype components detected in vivo are (R, S)-gaitrin, pseudoephedrine, ephedrine, methylephedrine, amygdalin, calendulaside I and kaempferol-O-rutinoside, the HPLC conditions are: Chromatographic column: CAPCELL PAK C18, 2.0 mm × 50 mm, 2 μm; Mobile phase A: 0.08%-0.12% formic acid in water; Mobile phase B: acetonitrile; 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; 6.01-7.5min, 98% mobile phase A, 2% mobile phase B.

9. The method for detecting the in vivo prototype component in the antiviral Chinese medicine composition according to claim 8, characterized in that: The column temperature was 25 °C, the flow rate was 0.35 mL / min, and the injection volume was 20 μL.

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

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