CN113917031B - Method for determining blood concentration of lung-tonifying and blood-activating capsule in vivo by UHPLC-MS/MS method - Google Patents
Method for determining blood concentration of lung-tonifying and blood-activating capsule in vivo by UHPLC-MS/MS method Download PDFInfo
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Abstract
The invention provides a method for determining blood concentration of a lung-tonifying and blood-activating capsule in vivo by a UHPLC-MS/MS method, which comprises the following steps: dissolving psoralen glycoside, isopsoralen glycoside, calycosin glycoside, formononetin, psoralen, isopsoralen, 3-hydroxy-9, 10-dimethoxy rosewood alkane and neopsoralen isoflavone respectively, and fixing the volume; after constant volume, mixing and diluting the solution step by step into mixed contrast solution with serial concentrations; preparation of test samples: preparing a plasma sample from blood of a tester, and adding an internal standard solution to obtain a test sample; UHPLC-MS/MS measurement: and performing qualitative and quantitative determination on the mixed control solution and the test sample by using ultra performance liquid chromatography and secondary mass spectrometry. The invention screens the blood concentration of 45 active substances in the lung-tonifying and blood-activating capsule and screens 8 ingredients which are possibly closely related to the clinical treatment effect of the lung-tonifying and blood-activating capsule. The adopted UHPLC-MS/MS method can quickly detect the blood concentration of the lung tonifying and blood circulation promoting capsule with strong specificity and good repeatability, and is suitable for the pharmacokinetic research and clinical test of the lung tonifying and blood circulation promoting capsule.
Description
Technical Field
The invention belongs to the technical field of molecules, and particularly relates to a method for determining blood concentration of a lung-tonifying and blood-activating capsule in vivo by a UHPLC-MS/MS method.
Background
The capsule for tonifying lung and activating blood consists of three traditional Chinese medicines of astragalus, red paeony root and fructus psoraleae, has the effects of tonifying qi and activating blood, and tonifying lung and reinforcing kidney, is used for treating the deficiency of vital energy and blood stasis of pulmonary heart disease (remission stage), is also commonly used for treating Chronic Obstructive Pulmonary Disease (COPD) and novel coronavirus pneumonia (COVID-19) in the recovery stage in recent years, and is listed in a plurality of provincial and commercial COVID-19 recovery-stage treatment medicine recommendation catalogues. Modern pharmacological research shows that the lung tonifying and blood circulation invigorating capsule can regulate the level of inflammatory factors such as IL-6, IL-1 beta, IL-10 and the like of a body, reduce the deposition of collagen fibers in lung tissues, improve the ventilation function of the lung, and relieve the symptoms such as cough, shortness of breath, palpitation and the like caused by chronic respiratory diseases.
CN107505418A discloses a method for constructing HPLC fingerprint of a lung-tonifying and blood-activating capsule and a standard fingerprint, the invention determines the content of calycosin glucoside, calycosin, paeoniflorin, isopsoralen and psoralen in the lung-tonifying and blood-activating capsule and provides the fingerprint; provides scientific basis for the standardized research of the lung tonifying and blood circulation invigorating capsule. But the present invention measured only the content of 5 components therein.
CN107456479A discloses a preparation and detection method of fructus psoraleae formula granules, the invention can provide a detection method of fructus psoraleae formula granule products, establish a characteristic map of the fructus psoraleae formula granules, and determine the content and transfer rate of index components of the fructus psoraleae formula granules; however, the invention only measures the content of 4 components including psoralen, isopsoralen, psoralen and isopsoralen.
Both the two inventions only measure the contents of partial components in the capsule for tonifying lung and activating blood and establish a fingerprint spectrum, and do not measure the components comprehensively. Modern medicine believes that drug molecules must enter the blood before reaching the target of action, thereby exerting the drug effect. Therefore, the research on the blood-entering components and the metabolism rule thereof in the body has important significance for disclosing the drug effect substance basis of the traditional Chinese medicine compound. The research shows that the pharmacokinetics law of the Chinese medicine is changed when the Chinese medicine is used in a compound form clinically due to the multi-component interaction among the medicines. At present, the research on the blood-entering components and the pharmacokinetics of the single medicines of the astragalus, the psoralea fruit and the red paeony root is not lacked, but the research on the in vivo process of the main chemical components of the lung-tonifying and blood-activating capsule is rarely seen.
Disclosure of Invention
In view of the defects of the prior art in researching the pharmacokinetic characteristics of the components of the lung-tonifying and blood-activating capsule in blood plasma, the invention provides a method for determining the blood concentration of the lung-tonifying and blood-activating capsule in vivo by using a UHPLC-MS/MS method.
The invention provides a method for determining blood concentration of a lung-tonifying and blood-activating capsule in vivo by a UHPLC-MS/MS method, which comprises the following steps:
preparation of control solution: dissolving psoralen glycoside, isopsoralen glycoside, calycosin glycoside, formononetin, psoralen, isopsoralen, 3-hydroxy-9, 10-dimethoxy rosewood alkane and neopsoralen isoflavone respectively, and fixing the volume; after constant volume, mixing and diluting the solution step by step into mixed contrast solution with serial concentrations;
preparation of test samples: preparing a plasma sample from blood of a tester and adding an internal standard solution to obtain a test sample;
UHPLC-MS/MS determination: and performing qualitative and quantitative determination on the mixed control solution and the test sample by using ultra performance liquid chromatography and secondary mass spectrometry.
Specifically, the chromatographic conditions of the ultra-high performance liquid chromatography are as follows:
the chromatographic column is ACQUITYUPLCBEHC18A column;
the mobile phase A is 0.1 percent of formic acid water, and the mobile phase B is 0.1 percent of acetonitrile;
gradient elution procedure:
0-2min，95-88％A；
2-4min，88-85％A；
4-8min，85-65％A；
8-9min，65-60％A；
9-12min，60-40％A；
12-13min，40-15％A；
13-14min，15-95％A；
14-15min，95％A；
column temperature 35 deg.C, flow rate 0.4 mL/min-1The amount of sample was 4. mu.L.
Specifically, the mass spectrum conditions of the secondary mass spectrum are as follows:
ionization mode: ESI+/ESI-；
The detection mode is as follows: detecting multiple reactions;
electrospray voltage: 4500V;
atomizing gas pressure: 413.7 kPa;
air curtain pressure: 137.9 kPa;
flow rate of auxiliary gas: 413.7 kPa;
ion source temperature: 400 ℃;
collision gas: and Low.
The mass spectrometry conditions are determined based on the optimization of the monitoring substance aimed at by the invention, and the invention cannot be normally completed by adopting other parameters.
Preferably, the solvent of the control solution is methanol.
Preferably, the step of preparing a plasma sample from the blood of the subject is: blood of a tester is collected and placed in a test tube coated with heparin sodium solution in advance for centrifugation, and supernatant is taken after centrifugation.
Further preferably, the internal standard solution is a mixed methanol solution of clarithromycin and chloramphenicol.
Further preferably, the acetonitrile is added simultaneously with the addition of the internal standard solution.
Further preferably, the plasma sample is added with the internal standard solution and acetonitrile, centrifuged, the supernatant is removed and concentrated to be dry, methanol is added for redissolving, centrifuged, and the supernatant is taken as the test sample.
Preferably, the ultra performance liquid chromatography apparatus is a Waters acquisition UPLC system.
Preferably, the instrument for secondary mass spectrometry is a QTRAP 6500 triple quadrupole linear ion trap mass spectrometer system.
During sample pretreatment, this experiment compared methanol, acetonitrile and ethyl acetate: n-hexane (8:1)3 pretreatment methods.
The specific treatment method comprises the following steps: three identical drug-containing plasma samples were taken, and the plasma samples were treated with methanol, acetonitrile and ethyl acetate: after n-hexane (8:1) treatment, a verified liquid chromatography method is adopted for sample injection analysis. The areas of peak are 656697, 685118 and 541805 respectively according to the determination of psoralen glycoside. Therefore, acetonitrile was finally selected as the protein precipitation solvent for the experiment.
The results show that the elution amount of the components in 3 solvents has no obvious difference, and the peak area of the components is larger after the acetonitrile treatment under the same conditions according to the peak area comparison. But acetonitrile precipitates protein completely, the recovery and extraction rate of a sample is high, and the peak shape is good. In the initial stage of the experiment, when water (a) -acetonitrile (B) was used as the mobile phase, it was found that the peak shape of the target component was very trailing, and as shown in fig. 18 and 19, the peak shape was improved by adding 0.1% formic acid to the aqueous phase, so that the experiment finally selected acetonitrile as the protein precipitant and 0.1% formic acid (a) -acetonitrile (B) as the mobile phase.
The invention has the beneficial effects that:
1. aiming at 45 active substances in the lung-tonifying and blood-activating capsule, 8 ingredients which are closely related to the clinical treatment effect of the lung-tonifying and blood-activating capsule are screened out through experiments; active ingredients which do not actually exert efficacy are removed, and a foundation is laid for determining the lung-tonifying and blood-activating capsules by a UHPLC-MS/MS method; there is no precedent for screening in this manner.
2. Aiming at 8 active ingredients screened from the lung-tonifying and blood-activating capsules, the invention reselects various conditions such as mobile phase selection, control sample selection, mass spectrum monitoring mode and the like in order that the ultra-high performance liquid chromatography tandem mass spectrometry can rapidly and accurately detect the 8 active ingredients at one time, and the conditions cannot be directly used from other detection targets.
3. The UHPLC-MS/MS is carried out under the selected conditions and the screened control sample group, so that the blood concentration of the lung-tonifying and blood-activating capsule can be detected quickly, specifically and repeatedly, and the method is suitable for pharmacokinetic research and clinical test of the lung-tonifying and blood-activating capsule.
Drawings
FIGS. 1-8 show the chemical structures of psoralen glycoside, isopsoralen glycoside, calycosin glycoside, formononetin, psoralen, isopsoralen, 3-hydroxy-9, 10-dimethyl pterocarpan and neopsoralen isoflavone.
FIG. 9 is a MRM map of 8 major components and internal standards in rat plasma.
FIGS. 10-17 show the blood concentration-time curves of psoralen, isopsoralen, calycosin, formononetin, psoralen, isopsoralen, 3-hydroxy-9, 10-dimethylpterocarpan and neopsoralen isoflavone (respectively)
n＝8)。
Fig. 18 is a liquid mass diagram using pure water-acetonitrile as a mobile phase.
Fig. 19 is a liquid mass diagram using 0.1% formic acid water-acetonitrile as a mobile phase.
In fig. 9: A. blank plasma; B. blank plasma + mixed reference + internal standard solution; C. the capsule for tonifying lung and promoting blood circulation contains blood plasma; 1. psoralen glycosides; 2. isobavachin; 3. calycosin; 4. formononetin; 5. psoralen; 6. isopsoralen; 7.3-hydroxy-9, 10-dimethoxypterocarpan; 8. new psoralen isoflavones; IS1Chloramphenicol; IS2Clarithromycin.
Detailed Description
The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.
The instrument comprises the following steps:
waters ACQUITY UPLC system (including quaternary pump solvent, in-line degasser and autosampler; Waters Inc., Milford, USA).
QTRAP 6500 triple quadrupole rod linear ion trap mass spectrometer system (AB SCIEX, usa) equipped with an electrospray ion source and analysis 1.6.3 software.
MS105, ML204 electronic analytical balance (METTLER-TOLEDO, Inc.).
An Anke GL-16 GII type centrifuge (Shanghai' an pavilion scientific instruments factory); WH-micro vortex mixer (Shanghai province of analytical instruments Co., Ltd.).
Millipore Q pure water system (Millipore, USA).
Drugs and reagents:
psoralen (batch No. Y07A7S12664), isopsoralen (batch No. Y27A9S60238), calycosin (batch No. Y27F9H54731), formononetin (batch No. R28O8F46957), psoralen (batch No. C22A9Q68562), isopsoralen (batch No. C05M10Q82078), 3-hydroxy-9, 10-dimethoxy rosewood alkane (batch No. Y28M10H84302), neopsoralen isoflavone (batch No. ZN1112 13), and clarithromycin (batch No. Y31M7C12157) were all obtained from Shanghai-sourced leaf Biotech Limited;
chloramphenicol (batch: A1608010) was purchased from Shanghai Allantin Biotechnology, Inc.
The purity of the above reference substances is more than or equal to 98%.
Sodium carboxymethylcellulose (lot # 20181019) was purchased from national pharmaceutical group chemical reagents, Inc.;
the lung-tonifying and blood-activating capsule (batch: 022004) was supplied by Guangdong Leyun pharmaceutical industry Co., Ltd;
methanol and acetonitrile as chromatographies (Merck, Germany);
formic acid is chromatographically pure (ACS, usa);
ultrapure water is self-made in laboratories.
Example 1 animal preliminary preparation
Experimental animals: the male Wistar rat is SPF-grade, the weight is 250-280 g, and the qualification number SCXK (Zhe) 2019-containing 0001 is provided by Nanjing university of traditional Chinese medicine laboratory animal center. The breeding temperature is 20-25 deg.C, and the ambient humidity is (45 + -10)%.
Chromatographic conditions are as follows: the chromatographic column is ACQUITYUPLCBEHC18Columns (100 mm. times.2.1 mm, 1.7 μm); mobile phase 0.1% formic acid (a) -acetonitrile (B), gradient elution procedure: 0-2min, 95-88% A; 2-4min, 88-85% A; 4-8min, 85-65% of A; 8-9min, 65-60% A; 9-12min, 60-40% A; 12-13min, 40-15% A; 13-14min, 15-95% A; 14-15min, 95% A. Column temperature 35 deg.C, flow rate 0.4 mL/min-1The sample size was 4. mu.L.
Mass spectrum conditions: ionization mode: ESI+/ESI-(ii) a The detection mode comprises the following steps: multiple reaction detection (MRM); electrospray voltage: 4500V; atomizing gas pressure: 413.7 kPa; air curtain pressure: 137.9 kPa; flow rate of auxiliary gas: 413.7 kPa; ion source temperature: 400 ℃; collision gas: low; the MRM parameters for the 8 test substances and the 2 internal standard substances are shown in Table 1.
TABLE 1 Mass Spectrometry parameters of 8 analytes and 2 internal standards in rat plasma
Preparation of control solutions:
1. preparation of mixed reference solution A proper amount of psoralen glycoside, isopsoralen glycoside, calycosin glycoside, formononetin, psoralen, isopsoralen, 3-hydroxy-9, 10-dimethoxy rosewood alkane and neopsoralen isoflavone reference is weighed, dissolved in methanol, and subjected to constant volume to prepare a reference stock solution with a certain concentration. Precisely measuring appropriate amount of control stock solution, and mixing to obtain a mixture containing psoralen glycoside (3250 ng. mL)-1) Isopsoralen glycoside (2792 ng/mL)-1) Calycosin (27.73 ng/mL)-1) Formononetin (34.93 ng. mL)-1) Psoralen (2295 ng/mL)-1) Isopsoralen (3767 ng/mL)-1) 3-hydroxy-9, 10-dimethoxypterocarpan (32.13 ng. mL)-1) Psoralea fruit isoflavone (15.83 ng. mL)-1) The mixed reference stock solution is gradually diluted by methanol to obtainMixed control solutions of series concentrations. Storing in a refrigerator at 4 deg.C.
The specific series of concentrations are:
psoralen glycosides (3250, 1625, 812.5, 406.2, 203.1, 101.6, 50.78, 25.39, 12.70, 6.348, 3.174, 1.587, 0.7934 ng/mL)-1)；
Isopsoralen glycoside (2792, 1396, 697.9, 349.0, 174.5, 87.24, 43.62, 21.81, 10.90, 5.452, 2.726, 1.363, 0.6816 ng/mL)-1)；
Calycosin (27.73, 13.87, 6.933, 3.467, 1.733, 0.8667, 0.4333, 0.2167, 0.1083, 0.05417, 0.0271, 0.01354, 0.006836 ng/mL)-1)；
Formononoside (34.93, 17.46, 8.732, 4.367, 2.183, 1.092, 0.5458, 0.2729, 0.1364, 0.06823, 0.03411, 0.01706, 0.008529ng & mL)-1)；
Psoralen (2295, 1148, 573.8, 286.9, 143.4, 71.72, 35.86, 17.93, 8.965, 4.482, 2.241, 1.121, 0.5603ng mL)-1)；
Isopsoralen (3766, 1883, 941.7, 470.8, 235.4, 117.7, 58.85, 29.43, 14.71, 7.357, 3.678, 1.839, 0.9196ng mL)-1)；
3-hydroxy-9, 10-dimethoxy-pterocarpan (32.13, 16.07, 8.033, 4.017, 2.008, 1.004, 0.5021, 0.2510, 0.1255, 0.06276, 0.03138, 0.01569, 0.007845 ng. mL)-1)；
Neopsoralen isoflavone (15.83, 7.915, 3.9575, 1.97875, 0.9894, 0.4947, 0.2473, 0.1237, 0.06184, 0.0309, 0.01546, 0.007729, 0.003865ng mL)-1)。
In the invention, the preparation of the mixed standard solution can be realized by using the single standard solution with any concentration.
2. Preparation of internal standard solution A proper amount of clarithromycin and chloramphenicol reference substances are weighed and placed in a 10mL volumetric flask, and methanol is dissolved and the volume is determined to prepare internal standard solution stock solution with a certain concentration. Sucking an appropriate amount of internal standard solution stock solution, and diluting with methanol to obtain concentrations of 197.6 ng/mL-1And 58.80 ng/mL-1Mixed internal standard solution of (4). Storing in a refrigerator at 4 deg.C.
3. Administration and plasma sample Collection
Male SD rats, 16, were acclimatized for one week and then randomized into blank and dosed groups. After fasting (free drinking water) for 12h, the administration group drenches the capsule suspension for tonifying the stomach, the lung and the blood according to the clinical equivalent dose (0.38g/kg) (the capsule powder for tonifying the lung and the blood is weighed and is added with 0.5 percent sodium carboxymethylcellulose solution to prepare the suspension with the concentration of 0.038 g/mL), and the blank group drenches the physiological saline with the same volume. Collecting blood at 400 μ L via orbital venous plexus at 5min, 10min, 15min, 30min, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 10h, 12h, and 24h after administration, respectively, placing the plasma sample in EP tube pre-coated with 10 μ L0.1% heparin sodium solution, and collecting blood at 4000 r.min-1Centrifuging for 10min under the condition, collecting supernatant as plasma sample, and storing in a refrigerator at-80 deg.C for use.
4. Plasma sample processing
Thawing rat plasma sample in refrigerator at 4 deg.C, vortexing for 2min, precisely sucking 200 μ L, adding mixed internal standard solution 10 μ L and acetonitrile 590 μ L, vortexing for 2min, standing for 5min, and centrifuging for 10min (4 deg.C, 13500 r.min)-1) Centrifuging the supernatant at 37 deg.C, concentrating to dry, adding 50% methanol 50 μ L for redissolving, and centrifuging for 10min (4 deg.C, 13500r min)-1) And taking the supernatant as the plasma sample test solution.
Example 2 animal Pre-Experimental preparation
1. The specificity is as follows: and respectively taking the blank plasma, the blank plasma plus a reference substance and an internal standard of the rat, adding the internal standard of the dosed plasma sample of the rat, processing according to a plasma sample processing method, carrying out UHPLC-MS/MS analysis under the conditions to obtain blank plasma, the blank plasma plus the reference substance and a plasma sample chromatogram after the dosing, wherein the result is shown in figure 9. The result shows that the components to be detected and the internal standard are not interfered by impurities and endogenous substances in plasma, and the components to be detected and the internal standard can be completely separated, which indicates that the established method has good specificity.
2. Standard curve and linear range: precisely measuring 200 μ L of rat blank plasma, adding 50 μ L of serial concentration reference solution, and 10 μ L of internal standard solutionAnd 540. mu.L acetonitrile, plasma samples were processed and prepared in a series of concentrations according to the "plasma sample treatment" method and subjected to UHPLC-MS/MS analysis as described above. The mass concentration of each component (ng. mL)-1) Taking the ratio of the peak area of the corresponding sample to the peak area of the internal standard as the abscissa (X), taking the ordinate (Y), and performing regression operation by adopting a weighted least square method with the weight of 1/X2And drawing a standard curve. The results show that the compounds are in good linear relationship within the corresponding ranges (R)2Greater than 0.99) meets the requirements of the analysis method of the biological sample. The standard curve equation, correlation coefficient and linear range of each component to be measured are shown in table 2.
TABLE 2 Linear equation, limit of quantitation and Linear Range of the Components to be measured
3. Precision and accuracy: taking 200 mu L of rat blank plasma, adding reference substance solutions with low, medium and high mass concentrations to obtain quality control samples (QC) containing psoralen glycoside, isobavarin glycoside, calycosin glycoside, formononetin, psoralen, isobavarin, 3-hydroxy-9, 10-dimethoxy rosewood alkane and neopsoralen isoflavone with low, medium and high mass concentrations. Plasma samples were processed according to the "plasma sample processing" method and subjected to UHPLC-MS/MS analysis as described above. Continuously sampling for 6 times in the same day, and calculating the precision in the same day; the measurement was continued for 3d, and the daytime precision was calculated. Accuracy is expressed in Relative Error (RE) and precision in RSD. The result shows (table 3), the accuracy of 8 components to be measured in the plasma is 88.63% -111.3% in the day and the daytime, and the precision RSD is lower than 12%, which shows that the method meets the requirement of biological sample measurement.
Table 3 precision and accuracy analysis of 8 components in rat plasma (n ═ 6)
4. Extraction recovery and matrix effect: taking the QC samples with the low, medium and high mass concentrations, preparing 6 parts of QC samples with each mass concentration in parallel, processing the plasma samples according to a plasma sample processing method, carrying out UHPLC-MS/MS analysis according to the conditions, and recording the ratio (A) of component peak area to internal standard peak area; taking blank plasma of a rat, processing the blank plasma by the same method, adding a QC sample, carrying out sample injection analysis, recording the ratio (B) of the peak area of the component to the peak area of the internal standard, and extracting the recovery rate (%) < A/B x 100%; taking methanol aqueous solution to replace blank plasma, and performing the other operations to obtain the ratio (C) of the peak area of the component to the peak area of the internal standard, wherein the matrix effect (%) is A/C multiplied by 100%. The result shows that the extraction recovery rate of psoralen glycoside, isopsoralen glycoside, calycosin glycoside, formononetin, psoralen, isopsoralen, 3-hydroxy-9, 10-dimethoxy rosewood alkane and neopsoralen isoflavone in the plasma is 85.65-106.65%, the matrix effect is 88.17-103.70%, the detection influence of endogenous substances in rat plasma on the components to be detected is small, the extraction recovery rate of samples in the plasma meets the detection requirement, and the result is shown in table 4.
Table 4 extraction recovery and matrix effect of 8 components (n ═ 6)
5. Stability: precisely measuring blank plasma 200 mu L to prepare QC samples with low, medium and high 3 mass concentrations, preparing 6 parts of QC samples with each mass concentration in parallel, respectively investigating the stability of the QC samples after being placed for 2h at room temperature for reprocessing, the plasma samples after being processed being placed in an automatic sample injector for 12h, repeated freeze-thaw cycles being performed for 3 times, and the plasma samples being placed for 21 days at-80 ℃. The results show that the RSD of psoralen glycoside, isopsoralen glycoside, calycosin glycoside, formononetin, psoralen, isopsoralen, 3-hydroxy-9, 10-dimethoxy rosewood alkane and neopsoralen isoflavone in the plasma under the 4 storage conditions is less than 11 percent, which indicates that the plasma samples are stable when placed under the conditions, and the results are shown in Table 5.
Table 5 stability test results for 8 ingredients (n ═ 6)
Example 3 pharmacokinetic Studies
After a rat is subjected to single-time intragastric administration, lung tonifying and blood circulation activating capsules, the blood concentration of each time point is determined by adopting a verified UHPLC-MS/MS method, and the time is plotted as an abscissa (X) and the blood concentration of the rat is plotted as an ordinate (Y), so that an average blood concentration-time curve graph of 8 components is obtained, and is shown in figures 10-17. Fitting of a non-compartmental model using DAS 3.2.8 software to calculate time to peak (T)max) Maximum blood concentration (C)max) Area under plasma concentration-time curve (AUC), elimination half-life (t)1/2z) Pharmacokinetic parameters such as Mean Residence Time (MRT) and the like, the results are shown in table 6. The result shows that the peak time (T) of the 8 components in the capsule for tonifying lung and activating bloodmax) Arranged from fast to slow, sequentially: calycosin < mangiferin < neopsoralen < psoralen < isopsoralen < 3-hydroxy-9, 10-dimethoxy rosewood alkane < isopsoralen < psoralen; by elimination of half-life (t)1/2z) Arranging, in sequence: psoralen > 3-hydroxy-9, 10-dimethoxy rosepan > isopsoralen > mangiferin > mangostin > neopsoralen > calycosin > psoralen > isopsoralen. 8 components researched by the experiment can reach the peak blood concentration of 1.02-1509.33 mug.L within 0.21-1.56 h-1The absorption of each component is faster; the exposure amount of the psoralen glycoside, isopsoralen, psoralen and isopsoralen in the rat body is far larger than that of other components; in addition, the time curve of psoralen, isopsoralen and 3-hydroxy-9, 10-dimethoxy pterocarpan has a 'double peak' phenomenon.
TABLE 6 pharmacokinetic parameters of 8 components of rat after gavage, lung tonifying and blood circulation promoting Capsule ((
n＝8)
Pharmacological research shows that the lung-tonifying and blood-activating capsule can obviously reduce the level of inflammatory factors in the body of patients with chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis, enhance the immunity of the body, improve the ventilation function of the lung and promote the restoration of the lung function. The radix astragali in the formula contains chemical components such as flavonoid, saponins and polysaccharides, and has effects of regulating immunity, inhibiting expression of lung tumor tissue protein pathway, and relieving lung injury. The fructus Psoraleae contains coumarins, flavonoids, monoterpene phenols, etc., and has effects of reducing active oxygen generation in myofibroblast, and inhibiting inflammatory cell infiltration in lung. The paeoniflorin contained in radix Paeoniae Rubra has effects of improving capillary microcirculation, promoting blood circulation, increasing coronary blood flow, and resisting inflammation, bacteria and oxidation. The components can be potentially associated with the exertion of the effect of the lung-tonifying and blood-activating capsule on treating pulmonary heart disease and COPD.
Accordingly, in the earlier stage of the invention, based on the UHPLC-MS/MS technology, under the condition of clinical equivalent dose, the blood concentrations of 45 components in the 3 medicinal materials in the drug-containing plasma of a rat after being infused with the stomach, lung and blood circulation invigorating capsule are tried to be measured, but except for the main active ingredients of psoralea corylifolia glycoside, isopsoralen, neopsoraleisoflavone in the psoralea corylifolia and the main active ingredients of calycosin glycoside, formononetin and 3-hydroxy-9, 10-dimethoxy rosewood alkane in astragalus root, the content of each component in the drug-containing plasma of the rat is lower than the detection limit, and the 8 components are presumed to be closely related to the exertion of the clinical therapeutic effect of the lung and blood circulation invigorating capsule.
The specific 45 components are respectively as follows:
paeoniflorin, albiflorin, oxypaeoniflorin, benzoylpaeoniflorin, galloyl paeoniflorin, gallic acid, paeonol, methyl gallate, 9-hydroxybenzoic acid, astragaloside I, astragaloside II, astragaloside III, astragaloside I, isoastragaloside II, isoastragaloside IV, calycosin, formononetin, calycosin, cycloastragenol, daidzein B, kaempferol, quercetin, 3-hydroxy-9, 10-dimethoxy santaline, 7,2 '-dihydroxy-3, 4' -dimethoxy isoflavan, isorhamnetin, oleanolic acid, psoralen glycoside, isobavalin, psoralen, isopsoralen, psoralen A (dihydropsoralen), psoralen, isopsoralen, isochalcone, etc, Bavachinin methyl ether, Corylifol A, bakuchiol, 8-methoxypsoralen (zanthoxylum piperitum toxin), bavachalcone, (S) -isopsoralen flavanone, 4' -O-methyl bavachalcone B, and bavachalcone.
The invention adopts a UHPLC-MS/MS method for the first time to establish a method for measuring the blood concentration of 8 main active components in blood plasma of a rat after being infused with stomach, tonifying lung and activating blood capsules, meets the measurement requirement of a biological sample through methodology investigation, researches the pharmacokinetic characteristics in vivo of the rat with the 8 components, provides support for explaining the pharmacodynamic material basis of the lung-tonifying and activating blood capsules, and provides basis for the design of the clinical administration scheme of the lung-tonifying and activating blood capsules.
Claims (4)
1. A method for measuring the blood concentration of a lung-tonifying and blood-activating capsule in a body by a UHPLC-MS/MS method is characterized by comprising the following steps:
preparation of control solution: dissolving psoralen glycoside, isopsoralen glycoside, calycosin glycoside, formononetin, psoralen, isopsoralen, 3-hydroxy-9, 10-dimethoxy rosewood alkane and neopsoralen isoflavone respectively, and fixing the volume; after constant volume, mixing and diluting the solution step by step into mixed contrast solution with serial concentrations;
preparation of test samples: preparing a plasma sample from blood of a tester and adding an internal standard solution to obtain a test sample;
UHPLC-MS/MS determination: performing qualitative and quantitative determination on the mixed control solution and the test sample by using ultra-high performance liquid chromatography and secondary mass spectrometry;
the chromatographic conditions of the ultra-high performance liquid chromatography are as follows:
the chromatographic column is ACQUITYUPLCBEHC18A column;
the mobile phase A is 0.1 percent of formic acid water, and the mobile phase B is 0.1 percent of acetonitrile;
gradient elution procedure:
0-2min，95-88%A；
2-4min，88-85%A；
4-8min，85-65%A；
8-9min，65-60%A；
9-12min，60-40%A；
12-13min，40-15%A；
13-14min，15-95%A；
14-15min，95%A；
column temperature 35 deg.C, flow rate 0.4 mL/min-1The sampling amount is 4 mu L;
the mass spectrum conditions of the secondary mass spectrum are as follows:
ionization mode: ESI+/ESI−；
The detection mode is as follows: detecting multiple reactions;
electrospray voltage: 4500V;
atomizing gas pressure: 413.7 kPa;
air curtain pressure: 137.9 kPa;
flow rate of auxiliary gas: 413.7 kPa;
ion source temperature: 400 ℃;
collision gas: low;
the solvent of the control solution is methanol;
the steps of taking blood of a tester to prepare a plasma sample are as follows: collecting blood of a tester, placing the blood in a test tube coated with heparin sodium solution in advance, centrifuging, and taking supernate;
the internal standard solution is a mixed methanol solution of clarithromycin and chloramphenicol;
acetonitrile was added simultaneously with the addition of the internal standard solution.
2. The method for measuring the blood concentration of the lung-tonifying and blood-activating capsule in the body by the UHPLC-MS/MS method according to claim 1, characterized in that an internal standard solution and acetonitrile are added into a plasma sample, centrifugation is carried out, supernatant is removed and concentrated to be dry, methanol is added for redissolution, centrifugation is carried out, and the supernatant is taken as a test sample.
3. The method for measuring the blood concentration of the lung-tonifying and blood-activating capsule in vivo by the UHPLC-MS/MS method according to claim 1, wherein the ultra-high performance liquid chromatography is a Waters ACQUITY UPLC system.
4. The method for measuring the blood concentration of the lung-tonifying and blood-activating capsule in the body by the UHPLC-MS/MS method according to claim 1, wherein an instrument of a secondary mass spectrum is a QTRAP 6500 triple quadrupole linear ion trap mass spectrometer system.
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Denomination of invention: Method for Determining the Blood Concentration of Bufei Huoxue Capsules in vivo by UHPLC-MS/MS Effective date of registration: 20230329 Granted publication date: 20220607 Pledgee: Industrial and Commercial Bank of China Limited Yunfu Yun'an Branch Pledgor: GUANGDONG LEIYUNSHANG PHARMACEUTICAL Co.,Ltd. Registration number: Y2023980036720 |