CN114858959B - Detection method of bufadienolide component of musk heart-activating dripping pill in rat plasma and application thereof - Google Patents
Detection method of bufadienolide component of musk heart-activating dripping pill in rat plasma and application thereof Download PDFInfo
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- CN114858959B CN114858959B CN202110072178.7A CN202110072178A CN114858959B CN 114858959 B CN114858959 B CN 114858959B CN 202110072178 A CN202110072178 A CN 202110072178A CN 114858959 B CN114858959 B CN 114858959B
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- YBPMPRDOWHIVNA-XTBIJCDISA-N bufadienolide Chemical compound C=1([C@H]2CC[C@@H]3[C@H]4[C@@H]([C@]5(CCCCC5CC4)C)CC[C@@]32C)C=CC(=O)OC=1 YBPMPRDOWHIVNA-XTBIJCDISA-N 0.000 title claims abstract description 61
- 238000001514 detection method Methods 0.000 title claims abstract description 58
- 241000402754 Erythranthe moschata Species 0.000 title claims abstract description 44
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- 229950006858 bufogenin Drugs 0.000 claims abstract description 86
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
- G01N2030/8822—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving blood
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
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- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention provides a method for detecting bufadienolide components in musk heart-through dropping pills in rat plasma and application thereof, which specifically adopts ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to carry out qualitative and quantitative detection on bufogenin, far bufogenin, desacetylbufogenin, bufogenin and ester bufogenin in the rat plasma, and specifically comprises the steps of adding internal standard substances into the rat plasma of the stomach-filling musk heart-through dropping pills, and precipitating by methanol protein; detection was performed by UPLC-MS/MS. Wherein the detection limit of bufogenin is 1.00ng/mL, and the quantitative limit is 3.00ng/mL; the detection limit of the bufogenin, the cinobufagin and the ester bufogenin is 2.00ng/mL, and the quantitative limit is 6.00ng/mL; the limit of detection of the cinobufagin is 3.00ng/mL, and the limit of quantification is 9.00ng/mL. The method can complete detection within 15min, complete quantitative calculation by combining the retention time and peak area ratio of the MRM chromatogram and a standard curve, is more convenient to operate, reduces the interference of endogenous substances in blood plasma, and can be used for pharmacokinetics research of musk heart-penetrating dropping pills.
Description
Technical Field
The invention relates to a detection method of a compound traditional Chinese medicine component in rat plasma and application thereof, in particular to detection of bufadienolide components in musk heart-dredging dropping pills in the plasma.
Background
The musk heart-smoothing drop pill is a traditional Chinese medicine compound, and is used for treating coronary heart disease stable type exertional angina pectoris, and symptoms of chest pain and chest distress, palpitation and shortness of breath, lassitude and hypodynamia are seen in the syndrome of qi deficiency and blood stasis by the differentiation of the traditional Chinese medicine. The whole prescription comprises seven medicinal materials of venenum bufonis, artificial musk, red sage root, ginseng stem and leaf total saponin, artificial bezoar, bear gall powder and borneol. Studies show that musk heart-dredging dripping pills protect endothelial cells from atherosclerosis by reducing endothelin-1 (ET-1), C-reactive protein (CRP) and tumor necrosis factor alpha (TNF-alpha) in blood and simultaneously increasing nitric oxide content in blood. In addition, the musk heart-smoothing dripping pill can reduce harmful esters in human bodies and improve abnormal haemorheology indexes.
The toad venom is used as a monarch drug of musk heart-smoothing dropping pills, and the main medicinal component is bufadienolide compounds, and has the effects of resisting tumor, strengthening heart, anesthesia, pain relieving, anti-inflammatory and the like. In addition, the compounds have certain toxicity, and blood content detection has important significance for pharmacokinetic study and reasonable medication.
Currently, chemical component detection of traditional Chinese medicine compound preparations such as musk heart-smoothing dropping pills is mainly aimed at patent medicines. The musk heart-protecting pill contains artificial musk, ginseng, storax, toad venom, artificial bezoar, borneol and cinnamon, wherein the musk heart-protecting pill quality standard improvement research (second army medical university, 2018) adopts ultra-high performance synthetic phase chromatography-tandem mass spectrometry for analysis, adopts mobile phase B (methanol containing 10, 20 and 30mM ammonium acetate) and supercritical fluid for elution, and detects 28 active ingredients including bufogenin, ester bufogenin and bufogenin. In the research, the musk heart-smoothing dripping pills are different from musk heart-protecting pills in composition, and the effective components of the toad venom are limited in detection quantity and cannot be quantified. The quality basis and the effect of musk heart-activating dropping pills on myocardial ischemia are discussed based on PI3KAkt signal transduction pathway (university of Fujian traditional Chinese medicine, 2017) and are qualitatively analyzed by adopting an HPLC-Q-TOF-MS/MS method, wherein acetonitrile-formic acid water is adopted as a mobile phase in a liquid phase condition, and sarbufogenin, bufogenin and ester bufogenin are detected, but the peak and the separation degree are poor, and quantification cannot be performed. The patent CN201911410029.6 (a method for constructing a characteristic spectrum of a compound traditional Chinese medicine and measuring the content of the compound traditional Chinese medicine) (2019.12.31 of application date) adopts a high performance liquid chromatography to realize the quantitative detection of 10 medicinal components such as bufotalin, bufogenin, cinobufagin, ester bufogenin and the like in musk heart-dredging dropping pills synchronously and quantitatively, wherein the mobile phase is 0.005-0.05% acetonitrile (A) phosphate and 0.005-0.05% phosphoric acid water (B), the column temperature is 29-31 ℃, the detection wavelength is 198-206 nm, and the flow rate is 1.28-1.32 mL/min.
However, the musk dripping pill with the function of dredging the heart has the difference between blood entering components and the patent medicine, and is mainly characterized by metabolic products formed after the original medicine components are metabolized. Taking Bufonis venenum as an example, in Analysis of the bioactive constituents of ChanSu in rat plasma by high performance liquid chromatography with mass spectrometric detection (high performance liquid chromatography-mass spectrometry analysis of bioactive components of Bufonis venenum in rat plasma) (Journal of Pharmaceutical and Biomedical Analysis,2010,53 (3): 646-654), chemical analysis is performed by high performance liquid chromatography-mass spectrometry on control plasma and Bufonis venenum extract to obtain 4 metabolites and 20 crude drug compounds. Because the structure of the metabolite has correlation with the original medicine, the metabolite has a certain influence on the detection of the original medicine components. In addition, after the compound traditional Chinese medicine preparation is added into blood, the metabolites are more complex than the single medicinal materials, and the detection result is more difficult to judge.
Currently, the research on blood entering components of musk heart-dredging dripping pills is less. The closest prior art is to explore the mechanism of musk heart-dredging dripping pills for treating coronary heart disease based on serum pharmaceutical chemistry and network pharmacology (Chinese patent medicine, 2020,42 (10): 2768-2777) discloses the liquid quality detection of blood-entering components of musk heart-dredging dripping pills. After the musk dripping pill suspension for treating heart disease is administrated by lavage of healthy rats, blood components including sabatorin, taurocholate, cinobufagin, glycocholic acid, deacetylated cinobufagin, bufogenin, ursodeoxycholic acid and ester bufogenin are added into serum samples. In Hydro-RP C 18 Chromatographic column (250 mm. Times.4.6 mm,4 μm); mobile phase 0.4% acetonitrile (A) -0.2% water (B), gradient elution (0-5 min,5% A, 5-13 min, 5-20% A, 13-40 min, 20-22% A, 40-60 min, 22-45% A, 60-75 min, 45-75A%); column temperature is 30 ℃; volume flow rate is 1.3mL/min; under the chromatographic condition of 10 mu L of sample injection quantity, ESI+ mode, dry gas temperature 320 ℃ and volume flow rate 10L/min; sheath gas temperature is 350 ℃, and volume flow is 12L/min; the fragmentation energy is 175V; capillary voltage 3kV; and detecting under the mass spectrum condition of the ion acquisition range m/z of 50-1500 to obtain the bufogenin, the cinobufagin, the desacetylcinobufagin, the bufogenin and the ester bufogenin. However, the retention time of all chromatographic peaks of 5 bufadienolide components is longer than 70min, and the detection time is long; bufogenin and ester bufogenin have poor separation degree and peak shape, a base line drifts, fragments can be extracted for quantification, and a quantitative value cannot be judged directly through the retention time and the peak area of an MRM (molecular resonance marker) chromatogram; the basal components of the blank serum are complex, and the detection result of bufadienolide components is greatly influenced.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a detection method of musk heart-dredging dripping pills bufadienolide components in rat plasma, which can rapidly, directly, qualitatively and quantitatively detect the effective components in the plasma, reduce the detection influence of plasma base components and primary drug secondary metabolites on the bufadienolide components, ensure that the detection result is true and reliable, and provide data support for subsequent pharmacokinetics research.
The invention discloses a method for detecting bufadienolide components in musk heart-dredging dripping pills in rat plasma, which adopts ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to detect the bufadienolide components in the rat plasma, wherein the bufadienolide components comprise bufogenin, telecinobufagin, deacetylcinobufagin, bufagin and ester bufogenin.
Further, the detection method comprises the following steps:
(1) Plasma test sample treatment: taking rat plasma of the stomach-filling musk heart-dredging dripping pill, adding an internal standard substance, uniformly mixing, adding methanol, swirling and centrifuging; taking supernatant, blow-drying with nitrogen, adding methanol for redissolution, swirling, centrifuging, and taking a supernatant filtering membrane to prepare a plasma test sample; the internal standard is tinidazole solution;
(2) Standard curve preparation: adding bufadienolide component standard substance into blank rat plasma to prepare a series of standard curve samples; adopting the method of the step (1), replacing the standard curve sample with the rat plasma of the stomach-filling musk heart-dredging dripping pill, and preparing to obtain a standard curve plasma sample; injecting a standard curve plasma sample into an ultra-high performance liquid chromatography-tandem mass spectrometer for analysis; regression is carried out by taking the concentration of bufadienolide components as an abscissa and the peak area ratio of the bufadienolide components to the internal standard substance as an ordinate to obtain a standard curve;
the liquid chromatography conditions are as follows: chromatographic column: acquisity (a Chinese character)BEH C18 (100 mm. Times.2.1 mm,1.7 μm); column temperature: 30-40 ℃; sample injection amount: 5-10 mu L; flow rate: 0.25-0.35 mL/min; the mobile phase A is 1-3 mmol/L ammonium acetate solution, the mobile phase B is acetonitrile, and the gradient elution procedure is as follows:
table 1 liquid chromatography elution procedure
| Time (min) | Mobile phase a (%) |
| 0→6 | 80→(78～72) |
| 6→(10～13) | (78～72)→(48～53) |
| (10～13)→13.1 | (48～53)→5 |
| 13.1→15 | 5 |
(3) And (3) content measurement: and (3) detecting the plasma test sample prepared in the step (1) by adopting a UPLC-MS/MS method in the step (2) to obtain the ratio of the bufadienolide component to the peak area of the internal standard substance, and substituting the ratio into the fitted standard curve in the step (2) to calculate so as to obtain the concentration of the bufadienolide component in the plasma test sample.
Preferably, in the treatment of the blood plasma test sample in the step (1), 100-300 mu L of blood plasma of the rat in the stomach-filling musk heart-dredging dripping pill is taken, 2.5-10 mu L of internal standard solution with the concentration of 1-5 mu g/mL is added, evenly mixed, 0.3-1.5 mL of methanol is added, vortex is carried out for 5-10 min, and centrifugation is carried out for 5-15 min at the temperature of 2-6 ℃ and at the speed of 8000-12000 r/min; taking supernatant, blowing nitrogen to near dryness at room temperature, adding 200-500 mu L of methanol for re-dissolution, swirling for 5-15 min, centrifuging for 5-10 min at 2-6 ℃ at 8000-12000 r/min, and taking a supernatant filtering membrane to obtain a plasma test sample.
Further, in the treatment of the blood plasma test sample in the step (1), 200 mu L of blood plasma of a rat with the stomach-filling musk heart-dredging dripping pill is taken, 5 mu L of internal standard substance solution with the concentration of 2 mu g/mL is added, the mixture is uniformly mixed, 1mL of methanol is added, vortex is carried out for 5min, and centrifugation is carried out for 10min at the temperature of 4 ℃ and 10000 r/min; taking supernatant, blowing nitrogen to near dryness at room temperature, adding 300 mu L of methanol for redissolution, swirling for 5min, centrifuging for 5min at 4 ℃ at 10000r/min, and taking supernatant to obtain a blood plasma test sample.
Preferably, the column temperature in the liquid chromatography condition is 35 ℃; the sample injection amount is 5 mu L; the flow rate is 0.3mL/min; mobile phase A was 2mmol/L ammonium acetate solution, B was acetonitrile, gradient elution procedure was as follows:
table 2 liquid chromatography elution procedure optimization results
| Time (min) | A(％) |
| 0→6 | 80→75 |
| 6→13 | 75→50 |
| 13→13.1 | 50→5 |
| 13.1→15 | 5 |
Further, the mass spectrum conditions in UPLC-MS/MS: an atmospheric electrospray ion source, positive ionization mode; the temperature of the ion source interface is 290-310 ℃; the temperature of the desolventizing pipe is 245-255 ℃; the temperature of the heating module is 390-410 ℃; the flow rate of heating gas is 8-12L/min; the flow rate of the atomized gas is 2-3L/min; the flow rate of the drying gas is 8-12L/min; monitoring mode MRM, monitoring scanning mode is as follows:
table 3 mass spectrometry condition monitoring scan mode
Preferably, the ion source interface temperature is 300 ℃; the temperature of the desolventizing pipe is 250 ℃; the temperature of the heating module is 400 ℃; heating air flow rate is 10L/min; the flow rate of the atomized gas is 3L/min; drying gas flow rate: 10L/min.
Further, in the MRM monitoring scan mode, Q1 Pre Bias, collision energy, and Q3 Pre Bias are as follows:
TABLE 4 optimization results of Q1, collision energy, and Q3 in a Mass Spectrometry Condition monitoring scan
| Sequence number | Compounds of formula (I) | Q1 Pre Bias(V) | Crash energy (V) | Q3 Pre Bias(V) |
| 1 | Sabafoxin essence | -21 | -28 | -28 |
| 2 | Cinobufagin extract | -22 | -28 | -26 |
| 3 | Deacetylated cinobufagin | -21 | -20 | -25 |
| 4 | Bufotalin | -16 | -25 | -27 |
| 5 | Ester bufogenin | -17 | -23 | -26 |
| 6 | Tinidazole | -13 | -17 | -12 |
The invention has the following advantages and technical effects:
1. according to the invention, a UPLC-MS/MS method is adopted to synchronously detect the sabin essence, the far bufalin essence, the deacetylated bufalin essence, the bufalin and the ester bufogenin of the musk heart-dredging dripping pill in the rat blood sample, and the peak can be generated within 15 minutes, so that compared with the retention time (more than 70 minutes) of chromatographic peaks of 5 bufadienolide components in the paper closest to the mechanism of researching musk heart-dredging dripping pill based on serum pharmaceutical chemistry and network pharmacology in the prior art, the detection time is greatly shortened.
2. In the UPLC-MS/MS method adopted in the technical proposal of the invention, the pretreatment of the blood sample is adopted, and then the acquisition is carried outBEH C18 (100 mm. Times.2.1 mm,1.7 μm) column; column temperature: 30-40 ℃; sample injection amount: 5-10 mu L; flow rate: 0.25-0.35 mL/min; the mobile phase A is 1-3 mmol/L ammonium acetate solution, the mobile phase B is acetonitrile, and the chromatographic base line of the chromatographic profile of the 5 bufadienolide components obtained by detection is stable, and the chromatographic peak separation degree R is the same as that of the chromatographic base line>1.5, the quantitative calculation can be completed by directly combining the retention time and the peak area ratio of the MRM chromatogram with a standard curve. The standard curve, detection limit and quantitative limit of 5 bufadienolide components are shown in Table 5. The bufogenin and bufogenin closest to the prior art are poor in separation degree and peak shape, and baseline shifts, and fragments are required to be extracted for quantification. The invention has more convenient operation and is beneficial to batch detection.
Table 55 Standard curve, detection Limit and quantitative Limit of bufadienolide Components
3. The UPLC-MS/MS method adopted by the invention adopts pretreatment of blood samples and mobile phase ammonium acetate (A) -acetonitrile (B), and combines a gradient elution flow, so that the detection influence of plasma base components and primary drug secondary metabolites on bufadienolide components can be better reduced, and the detection result is true and reliable. The method is closest to a blank plasma chromatogram in the prior art, and the chromatographic impurity peaks in the first 17min and 61-73 min are more, so that the detection of target components is greatly interfered; the chromatographic peak condition of the blank plasma detection result is shown in figures 1-6, and the impurity peak is less, so that conditions are provided for quantitative detection. The detection method is stable and reliable, and can be used for pharmacokinetics study of musk heart-dredging dropping pills.
Drawings
FIG. 1 MRM spectra of bufogenin in blank plasma, blank plasma label and positive plasma, FIG. 1-1 is blank plasma chromatogram, FIG. 1-2 is blank plasma label chromatogram, and FIG. 1-3 is positive plasma chromatogram. Wherein the retention time of bufogenin in FIGS. 1-2 is 10.545min and the retention time in FIGS. 1-3 is 10.495min.
FIG. 2 MRM spectra of bufogenin in blank plasma, blank plasma label and positive plasma, FIG. 2-1 is blank plasma chromatogram, FIG. 2-2 is blank plasma label chromatogram, and FIG. 2-3 is positive plasma chromatogram. Wherein the retention time of the cinobufagin in FIGS. 2-2 is 11.987min and the retention time in FIGS. 2-3 is 12.381min.
FIG. 3 MRM spectra of the blank plasma, the blank plasma label and the desacetylcinobufagin in the positive plasma, FIG. 3-1 is the blank plasma chromatogram, FIG. 3-2 is the blank plasma label chromatogram, and FIG. 3-3 is the positive plasma chromatogram. Wherein the retention time of the desacetylcinobufagin in FIG. 3-2 is 12.218min and the retention time in FIG. 3-3 is 12.602min.
FIG. 4 MRM spectra of bufalin in blank plasma, blank plasma label and positive plasma, FIG. 4-1 is blank plasma chromatogram, FIG. 4-2 is blank plasma label chromatogram, and FIG. 4-3 is positive plasma chromatogram. Wherein bufalin has a retention time of 13.309min in FIG. 4-2 and 13.656min in FIG. 4-3.
FIG. 5 MRM spectra of bufogenin in blank plasma, blank plasma label and positive plasma, FIG. 5-1 is blank plasma chromatogram, FIG. 5-2 is blank plasma label chromatogram, and FIG. 5-3 is positive plasma chromatogram. Wherein the retention time of the ester bufogenin in FIG. 5-2 is 14.204min and the retention time in FIG. 5-3 is 14.294min.
FIG. 6 MRM spectra of tinidazole in blank plasma, blank plasma addition label and positive plasma, FIG. 6-1 is blank plasma chromatogram, FIG. 6-2 is blank plasma addition label chromatogram, and FIG. 6-3 is positive plasma chromatogram. Wherein the retention time of tinidazole in FIG. 6-2 was 3.023min and the retention time in FIG. 6-3 was 3.015min.
FIG. 75A standard curve of bufadienolide components, wherein FIG. 7-1 is a bufogenin standard curve, FIG. 7-2 is a cinobufagin standard curve, FIG. 7-3 is a desacetylcinobufagin standard curve, FIG. 7-4 is a bufogenin standard curve, and FIG. 7-5 is an ester bufogenin standard curve.
Fig. 85 bufadienolide component content-time curves (mean ± standard deviation, n=6), wherein fig. 8-1 is a bufogenin content-time curve, fig. 8-2 is a telex bufogenin content-time curve, fig. 8-3 is a desacetylcinobufogenin content-time curve, fig. 8-4 is a bufogenin content-time curve, and fig. 8-5 is an ester bufogenin content-time curve.
Detailed Description
The present invention will be described in detail with reference to the following examples, but the present invention is not limited thereto.
Example 1: animal feeding, administration and standard solution preparation
1. Animal feeding: SPF SD rats, weighing 200 g-250 g, male. At the temperature of 20-26 ℃, the relative humidity of 40-70 percent, the illumination is carried out for 12 hours, and the brightness is alternated. Free drinking water, and feeding with standard block-shaped maintenance feed. Animals were acclimatized in the laboratory for a period of 5d prior to the experiment. Fasted for 12 hours before blood sampling, and water is not forbidden.
2. Administration: SD rats were weighed and the dose calculated, 6.4g/kg each was given by oral gavage, and the blank group was given the solvent control.
3. Blood collection: the anticoagulant is heparin sodium. The blank blood sample is collected by abdominal aorta, 14 blood samples, each of which is at least 5mL, and the blood is collected on the same day. The positive blood sample is taken by the venous sinus of the eye. The blood samples were centrifuged to separate the plasma, which was stored at-20℃in a frozen state.
4. Preparing bufadienolide component standard substance solution and tinidazole solution: dissolving standard substance and tinidazole with methanol to obtain bufogenin, cinobufagin, desacetylbufogenin, bufogenin, ester bufogenin and tinidazole solution.
Example 2: detection method 1 set up
1. Plasma test sample processing
Taking 200 mu L of rat plasma of the stomach-filling musk heart-smoothing dropping pill in the example 1, adding 5 mu L of tinidazole solution with the concentration of 2 mu g/mL, uniformly mixing, adding 1mL of methanol, swirling for 5min, and centrifuging for 10min at the temperature of 4 ℃ 10000 r/min; taking supernatant, blowing nitrogen to near dryness at room temperature, adding 300 mu L of methanol for redissolution, swirling for 5min, centrifuging for 5min at 4 ℃ at 10000r/min, and taking supernatant to obtain a filtering membrane to obtain a blood plasma test sample.
2. Standard curve preparation
Adding bufadienolide component standard substance solution into blank plasma to prepare a series of plasma standard curve samples; adopting the method of the step (1), replacing the standard curve plasma sample with the rat plasma of the stomach-filling musk heart-dredging dripping pill, and preparing to obtain the standard curve plasma sample; injecting a standard curve plasma sample into an ultra-high performance liquid chromatography-tandem mass spectrometer for analysis; regression is carried out by taking the concentration of bufadienolide components as an abscissa and the peak area ratio of the bufadienolide components to tinidazole as an ordinate to obtain a standard curve;
instrument apparatus: LCMS-8045 ultra high performance liquid chromatography-triple quadrupole mass spectrometer.
The liquid chromatography conditions were: chromatographic column: acquisity (a Chinese character)BEH C18 (100 mm. Times.2.1 mm,1.7 μm); column temperature: 35 ℃; sample injection amount: 5. Mu.L; flow rate: 0.3mL/min; mobile phase A was 2mmol/L ammonium acetate solution, mobile phase B was acetonitrile, gradient elution procedure was as follows:
table 2 liquid chromatography elution procedure optimization results
| Time/min | Mobile phase a (%) |
| 0→6 | 80→75 |
| 6→13 | 75→50 |
| 13→13.1 | 50→5 |
| 13.1→15 | 5 |
Ionization mode esi+; the temperature of the ion source interface is 300 ℃; the temperature of the desolventizing pipe is 250 ℃; the temperature of the heating module is 400 ℃; heating air flow rate is 10L/min; the flow rate of the atomized gas is 3L/min; the flow rate of the drying gas is 10L/min; monitoring mode MRM, monitoring scanning mode is as follows:
table 6 monitoring scanning mode 1
3. Content determination
And (3) detecting the plasma test sample prepared in the step (1) by adopting a UPLC-MS/MS method in the step (2) to obtain the ratio of various bufadienolide components to tinidazole peak areas, and substituting the obtained ratio into the fitted standard curve in the step (2) to calculate, so that the concentration of 5 bufadienolide components in the plasma test sample can be obtained.
4. Experimental results
4.1 Selectivity and specificity
The blank plasma, the blank plasma added with the control and the positive plasma are detected, and each group is divided into 3 parallel samples, and the details are shown in figures 1 to 6. Wherein R values of chromatographic peaks of bufogenin, cinobufagin, desacetylcinobufagin, bufogenin, ester bufogenin and tinidazole in positive blood plasma are more than 1.5, retention times are 10.495, 12.381, 12.602, 13.656, 14.294 and 3.015min respectively, the retention times correspond to retention times of components in blank blood plasma added with a control, and endogenous interference substances are not contained in blood plasma.
TABLE 7 Positive plasma MRM figure chromatographic peak-to-peak conditions
| Sequence number | Names of Compounds | Tailing factor | Degree of separation (R) |
| 1 | Tinidazole | 0.807 | — |
| 2 | Sabafoxin essence | 1.207 | 13.273 |
| 3 | Cinobufagin extract | 0.932 | 10.571 |
| 4 | Deacetylated cinobufagin | 0.904 | 1.517 |
| 5 | Bufotalin | 0.852 | 7.328 |
| 6 | Ester bufogenin | 0.831 | 6.723 |
4.2 Standard Curve, detection Limit and quantitative Limit
The linearity of the test method 1 was carried out by analyzing blank plasma plus bufadienolide component standard, the curves each contained 8 non-0 concentration points (bufalin, far bufalin and ester bufalin standard curve concentrations are respectively 4, 20, 40, 100, 200, 400, 1000 and 2000ng/mL, the desacetylbufalin standard curve concentrations are respectively 6, 30, 60, 150, 300, 600, 1500 and 3000ng/mL, the bufalin standard curve concentrations are respectively 2, 10, 20, 50, 100, 200, 500 and 1000 ng/mL), the bufadienolide component concentrations are taken as the abscissa, and the peak area ratio of bufadienolide component to tinidazole is taken as the ordinate. The standard curve is shown in FIG. 7, and the information such as the detection limit and the quantitative limit is shown in Table 5.
Table 55 Standard curve, detection Limit and quantitative Limit of bufadienolide Components
As shown in the results, the peak shapes of 5 bufadienolide components were symmetrical and the linearity was good (R 2 >0.999). The quantitative limit of each bufadienolide component is the lowest concentration with signal to noise ratio above 10, and the quantitative limit of bufogenin, desbufogenin, bufogenin and ester bufogenin is 6, 9, 3 and 6ng/mL respectively. To verify that the quantitative limits of each compound meet a certain precision and accuracy, a blank matrix is taken, standard substance addition test is performed according to the quantitative limits of 5 compounds, 5 parallels are set, and the Relative Error (RE) and Relative Standard Deviation (RSD) of each compound are calculated3.58 to 9.77 percent and 1.21 to 3.34 percent, which are all within the range of +/-15 percent, and are shown in the table 8 in detail.
Table 8 precision and accuracy of quantitative limits (n=5)
Example 3: detection method 2 set up
1. Plasma test sample processing
Taking 100 mu L of rat plasma of the stomach-filling musk heart-smoothing drop pill in the example 1, adding 2.5 mu L of tinidazole solution with the concentration of 1 mu g/mL, uniformly mixing, adding 0.3mL of methanol, swirling for 7min, and centrifuging for 5min at the temperature of 8000r/min at the temperature of 2 ℃; taking supernatant, blowing nitrogen to near dryness at room temperature, adding 200 mu L of methanol for redissolution, swirling for 5min, centrifuging for 5min at the temperature of 8000r/min, and taking supernatant for filtering a membrane to obtain a plasma test sample.
2. Standard curve preparation
Adding bufadienolide component standard substance solution into blank plasma to prepare a series of plasma standard curve samples; adopting the method of the step (1), replacing the standard curve plasma sample with the rat plasma of the stomach-filling musk heart-dredging dripping pill, and preparing to obtain the standard curve plasma sample; injecting a standard curve plasma sample into an ultra-high performance liquid chromatography-tandem mass spectrometer for analysis; and (3) carrying out regression by taking the concentration of the bufadienolide component as an abscissa and the peak area ratio of the bufadienolide component to the tinidazole as an ordinate to obtain a standard curve.
Instrument apparatus: LCMS-8045 ultra high performance liquid chromatography-triple quadrupole mass spectrometer.
The liquid chromatography conditions were: chromatographic column: acquisity (a Chinese character)BEH C18 (100 mm. Times.2.1 mm,1.7 μm); column temperature: 30 ℃; sample injection amount: 5. Mu.L; flow rate: 0.25mL/min; mobile phase a1mmol/L ammonium acetate solution, mobile phase B acetonitrile, gradient elution procedure as follows:
TABLE 9 liquid phase elution procedure 2
| Time/min | Mobile phase a/% |
| 0→6 | 80→78 |
| 6→10 | 78→53 |
| 10→13.1 | 53→5 |
| 13.1→15 | 5 |
Ionization mode esi+; the temperature of the ion source interface is 290 ℃; the temperature of the desolventizing pipe is 245 ℃; heating the module temperature to 390 ℃; heating air flow rate is 8L/min; the flow rate of the atomized gas is 2L/min; the flow rate of the drying gas is 8L/min; monitoring mode MRM, monitoring scanning mode is as follows:
table 10 monitoring scanning mode 2
3. Content determination
And (3) analyzing the plasma test sample prepared in the step (1) by adopting a UPLC-MS/MS method in the step (2) to obtain the ratio of various bufadienolide components to tinidazole peak areas, and substituting the ratio into the fitted standard curve in the step (2) to calculate, so that the concentration of 5 bufadienolide components in the plasma test sample can be obtained.
4. Experimental results
Blank plasma, control-added blank plasma and positive plasma were tested, 3 replicates each. Wherein, R value of the chromatographic peaks of the bufogenin, the cinobufagin, the desacetylcinobufagin, the bufogenin, the ester bufogenin and the tinidazole in positive plasma is more than 1.5, the chromatographic peak has symmetrical peak shape, the baseline is stable, and the quantitative calculation can be completed by directly passing through the peak outlet time and the peak area ratio of the MRM chromatogram and combining with a standard curve. Corresponding to the retention time of each component in the blank plasma added with the control, and no endogenous interference substances exist in the plasma.
Example 4: detection method 3 set up
1. Plasma test sample processing
Taking 300 mu L of rat plasma of the stomach-filling musk heart-smoothing drop pill in the example 1, adding 10 mu L of tinidazole solution with the concentration of 5 mu g/mL, uniformly mixing, adding 1.5mL of methanol, swirling for 10min, and centrifuging for 15min at the temperature of 12000r/min at the temperature of 6 ℃; taking supernatant, blowing nitrogen to near dryness at room temperature, adding 500 mu L of methanol for redissolution, swirling for 15min, centrifuging for 10min at 6 ℃ at 12000r/min, and taking supernatant to obtain a filtering membrane to obtain a plasma test sample.
2. Standard curve preparation
Adding bufadienolide component standard substance solution into blank plasma to prepare a series of plasma standard curve samples; adopting the method of the step (1), replacing the standard curve plasma sample with the rat plasma of the stomach-filling musk heart-dredging dripping pill, and preparing to obtain the standard curve plasma sample; injecting a standard curve plasma sample into an ultra-high performance liquid chromatography-tandem mass spectrometer for analysis; and (3) carrying out regression by taking the concentration of the bufadienolide component as an abscissa and the peak area ratio of the bufadienolide component to the tinidazole as an ordinate to obtain a standard curve.
Instrument apparatus: LCMS-8045 ultra high performance liquid chromatography-triple quadrupole mass spectrometer.
The liquid chromatography conditions were: chromatographic column: acquisity (a Chinese character)BEH C18 (100 mm. Times.2.1 mm,1.7 μm); column temperature: 40 ℃; sample injection amount: 10. Mu.L; flow rate: 0.35mL/min; mobile phase A is 3mmol/L ammonium acetate solution, mobile phase B is acetonitrile, gradient elution procedure is as follows:
TABLE 11 liquid phase elution procedure 3
| Time/min | Mobile phase a (%) |
| 0→6 | 80→72 |
| 6→13 | 72→48 |
| 13→13.1 | 48→5 |
| 13.1→15 | 5 |
Ionization mode esi+; the temperature of the ion source interface is 310 ℃; the desolventizing pipe temperature is 255 ℃; heating the module to a temperature of 410 ℃; heating air flow rate is 12L/min; the flow rate of the atomized gas is 3L/min; the flow rate of the drying gas is 12L/min; monitoring mode MRM, monitoring scanning mode is as follows:
table 12 monitoring scanning mode 3
3. Content determination
And (3) detecting the plasma test sample prepared in the step (1) by adopting UPLC-MS/MS to obtain the ratio of various bufadienolide components to the peak area of tinidazole, and substituting the obtained ratio into the fitted standard curve in the step (2) for calculation to obtain the concentration of 5 bufadienolide components in the plasma test sample.
4. Experimental results
Blank plasma, control-added blank plasma and positive plasma were tested, 3 replicates each. Wherein, R value of the chromatographic peaks of the bufogenin, the cinobufagin, the desacetylcinobufagin, the bufogenin, the ester bufogenin and the tinidazole in positive plasma is more than 1.5, the chromatographic peak has symmetrical peak shape, the baseline is stable, and the quantitative calculation can be completed by directly passing through the peak outlet time and the peak area ratio of the MRM chromatogram and combining with a standard curve. Corresponding to the retention time of each component in the blank plasma added with the control, and no endogenous interference substances exist in the plasma.
Example 5 methodological verification of detection method 1
1. Precision and accuracy
Taking 3 groups of blank plasma samples, 6 parts of each group, respectively adding control substances with low, medium and high concentration gradients (the adding standard amounts of bufogenin, far bufogenin and ester bufogenin are 1.2ng, 2.0ng and 4.0ng, the adding scalar of desacetylbufogenin is 1.8ng, 3.0ng and 6.0ng, and the adding scalar of bufogenin is 0.6ng, 1.0ng and 2.0 ng), and completing detection on the same day according to the detection method in the example 2 to calculate the daily precision and accuracy; three consecutive days of detection were performed to calculate the daytime precision and accuracy.
The results of the daily and daytime precision and accuracy analysis of detection method 1 are detailed in Table 13. For different concentration levels, the relative daily error (RE) and the Relative Standard Deviation (RSD) of the measurement results of 5 bufadienolide components are respectively-5.03-7.08% and 0.23-2.57%, and the relative daily error (RE) and the Relative Standard Deviation (RSD) are respectively-1.13-6.97% and 0.64-8.14%, and are respectively within 15%, which indicates that the method has better precision and accuracy for detecting 5 bufadienolide components in blood plasma.
Table 13 results of intra-day and inter-day precision and accuracy analysis (n=6)
2. Extraction recovery and matrix effects
Three sets of samples were prepared: sample A after pretreatment after the blank plasma is added with the reference substance; the blank plasma is pretreated and then added with a sample B of a reference substance; control solution C without any matrix added. The above groups were supplemented with controls of three concentration gradients (1.2 ng, 2.0ng and 4.0ng for bufogenin, 1.8ng, 3.0ng and 6.0ng for desacetylbufogenin, 0.6ng, 1.0ng and 2.0ng for bufogenin) at low, medium and high concentrations, 6 replicates were set for each concentration, and the detection method of example 2 was used. The extraction recovery rate is the comparison of the corresponding average value of each compound in the group A and the corresponding average value of each compound in the group B; the matrix effect is the ratio of the corresponding average value of each compound in group B to the corresponding average value of each compound in group C.
The extraction recovery and the results of the matrix effect analysis are detailed in Table 14. Extraction recovery = group a/group B measurements 100%; matrix effect = group B/group C assay 100%.
Table 14 extraction recovery and matrix effect analysis results (n=6)
The average extraction recovery rate of 5 bufadienolide components with different concentration levels is 100.32-114.31%, the RSD is between 0.73-5.02%, the average matrix effect ratio is between 93.37-119.37%, and the RSD is between 1.98-6.49%, which indicates that the method has higher extraction recovery rate and no obvious matrix effect.
3. Stability of
Blank plasma samples were taken and three controls of different concentrations (1.2 ng, 2.0ng and 4.0ng of bufogenin, 1.8ng, 3.0ng and 6.0ng of bufogenin and 0.6ng of bufogenin and 2.0ng of bufogenin) were added at low, medium and high concentrations, respectively, with 6 replicates for each concentration. The sample was left at room temperature for 8 hours, at-20℃for 1 month, and after storage at-20℃for 12 hours after thawing, it was measured by the method of example 2.
The stability analysis results are detailed in Table 15. From the experimental results, the sample is placed at room temperature for 8 hours before being treated, the extraction recovery rate of 5 bufadienolide components with different concentration levels is between 101.2 and 116.7 percent, and the RSD is between 1.24 and 6.37 percent; the sample treatment is carried out at the temperature of minus 20 ℃ for 1 month, the extraction rate of 5 bufadienolide components with different concentration levels is between 98.0 and 112.7 percent, and the RSD percent is between 0.91 and 4.62 percent; the sample is kept at the temperature of minus 20 ℃ before being treated, and is placed for 12 hours after being thawed, the extraction recovery rate of 5 bufadienolide components with different concentration levels is between 101.6 and 113.0 percent, and the RSD is between 1.29 and 8.50 percent, which shows that the method has better stability for detecting the 5 bufadienolide components in the blood plasma.
Table 15 stability analysis results (n=6)
EXAMPLE 6 pharmacokinetic uses
The rats were given 6.4g/kg each, and the vehicle control was given in the blank group. The blank blood sample is collected by abdominal aorta, each of which is at least 5mL, and the blood is collected on the same day. The positive blood sample is taken from the venous sinus of the eye, 14 groups of 6 groups of blood are arranged, and the blood is taken from each group at 0.5min, 5min, 15min, 30min, 1h, 1.5h, 2h, 3h, 4h, 6h, 8h, 10h, 12h and 24h of administration respectively. Blood samples were centrifuged to separate plasma, which was processed as in example 2 and subjected to ultra performance liquid chromatography-tandem mass spectrometry. The results of the sample measurement are shown in tables 16 to 20, and the time-dependent changes in the content are shown in FIG. 8.
Table 16 results of sarbufogenin detection in plasma (n=6)
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Note that: N.D. indicates no detection, 30min and no detection after; -representing no data.
Table 17 results of the far cinobufagin assay in plasma (n=6)
Note that: N.D. indicates no detection, 30min and no detection after; -representing no data.
Table 18 determination of desacetylcinobufagin in plasma (n=6)
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Note that: N.D. indicates undetected, 12h and thereafter; -representing no data.
Table 19 bufogenin assay results in plasma (n=6)
Note that: N.D. indicates no detection, 30min and no detection after; -representing no data.
Table 20 results of the ester bufogenin assay in plasma (n=6)
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Note that: N.D. indicates no detection, 30min and no detection after; -representing no data.
As can be seen from tables 16 to 20 and FIG. 8, the total content of 5 bufadienolide components tended to decrease with time. Wherein the bufogenin, bufogenin and bufogenin reach a maximum value in 0.5min (the maximum average value of bufogenin is 127.8ng/mL, the maximum average value of bufogenin is 96.7ng/mL, the maximum average value of bufogenin is 85.8 ng/mL), and the maximum values of the far bufogenin and the desacetylcinolone reach a maximum value in 5min (the maximum average value of far bufogenin is 105.4ng/mL, and the maximum average value of desacetylcinolone is 152.7 ng/mL). Sabatigenin, bufogenin and bufogenin were not detected in the plasma after 15min, and no target compound was detected in the plasma after 10 h.
As can be seen, bufogenin and bufogenin are metabolized more rapidly in rats and acetobufogenin is metabolized more slowly in rats. The experimental result can provide reference for the pharmacokinetics research of musk heart-dredging dropping pills.
Claims (7)
1. A method for detecting bufadienolide components of musk heart-activating dropping pills in rat plasma, which is characterized by detecting bufadienolide components in the rat plasma by adopting ultra-high performance liquid chromatography-tandem mass spectrometry, wherein the bufadienolide components comprise bufogenin, telex-ray bufogenin, deacetylated bufogenin, bufogenin and ester bufogenin, and the method comprises the following steps:
(1) Plasma test sample treatment: taking rat plasma of the stomach-filling musk heart-dredging dripping pill, adding an internal standard substance, uniformly mixing, adding methanol, swirling and centrifuging; taking supernatant, blow-drying with nitrogen, adding methanol for redissolution, swirling, centrifuging, and taking a supernatant filtering membrane to prepare a plasma test sample; the internal standard is tinidazole solution;
(2) Standard curve preparation: adding bufadienolide component standard substance into blank rat plasma to prepare a series of standard curve samples; adopting the method of the step (1), replacing the standard curve sample with the rat plasma of the stomach-filling musk heart-dredging dripping pill, and preparing to obtain a standard curve plasma sample; injecting a standard curve plasma sample into an ultra-high performance liquid chromatography-tandem mass spectrometer for analysis; regression is carried out by taking the concentration of bufadienolide components as an abscissa and the peak area ratio of the bufadienolide components to the internal standard substance as an ordinate to obtain a standard curve;
the liquid chromatography conditions were: chromatographic column: acquisity (a Chinese character)BEH C18, 100mm by 2.1mm, filler particle size 1.7 μm; column temperature: 30-40 ℃; sample injection amount: 5-10 mu L; flow rate: 0.25-0.35 mL/min; the mobile phase A is 1-3 mmol/L ammonium acetate solution, the mobile phase B is acetonitrile, and the gradient elution procedure is as follows:
Or (b)
Mass spectrometry conditions: an atmospheric electrospray ion source, positive ionization mode; the temperature of the ion source interface is 290-310 ℃; the temperature of the desolventizing pipe is 245-255 ℃; the temperature of the heating module is 390-410 ℃; the flow rate of heating gas is 8-12L/min; the flow rate of the atomized gas is 2-3L/min; the flow rate of the drying gas is 8-12L/min; monitoring mode MRM, monitoring scanning mode is as follows:
(3) And (3) content measurement: and (3) detecting the plasma test sample prepared in the step (1) by adopting an ultra-high performance liquid chromatography-tandem mass spectrometry method in the step (2) to obtain the ratio of the bufadienolide component to the peak area of the internal standard substance, and substituting the ratio into the fitted standard curve in the step (2) to calculate so as to obtain the concentration of the bufadienolide component in the plasma test sample.
2. The method according to claim 1, wherein in the step (1) of treating the blood plasma test sample, 100-300 μl of blood plasma of the stomach-perfusing musk heart-penetrating dripping pill rat is taken, 2.5-10 μl of 1-5 μg/mL of internal standard solution is added, the mixture is uniformly mixed, 0.3-1.5 mL of methanol is added, vortexing is carried out for 5-10 min, and centrifugation is carried out for 5-15 min at 8000-12000 r/min at 2-6 ℃; taking supernatant, blowing nitrogen to near dryness at room temperature, adding 200-500 mu L of methanol for re-dissolution, swirling for 5-15 min, centrifuging for 5-10 min at 2-6 ℃ at 8000-12000 r/min, and taking a supernatant filtering membrane to obtain a plasma test sample.
3. The method according to claim 2, wherein in the step (1) of treating the blood plasma test sample, 200 μl of blood plasma of the rat in the form of the stomach-perfusing musk heart-penetrating dripping pill is taken, 5 μl of the internal standard solution with the concentration of 2 μg/mL is added, the mixture is uniformly mixed, 1mL of methanol is added, vortexing is carried out for 5min, and centrifugation is carried out for 10min at 4 ℃ and 10000 r/min; taking supernatant, blowing nitrogen to near dryness at room temperature, adding 300 mu L of methanol for redissolution, swirling for 5min, and centrifuging for 5min at 4 ℃ at 10000 r/min.
4. The method according to claim 1, wherein the column temperature in the liquid chromatography conditions is 35 ℃; the sample injection amount is 5 mu L; the flow rate is 0.3mL/min; mobile phase A was 2mmol/L ammonium acetate solution, B was acetonitrile, gradient elution procedure was as follows:
。
5. The method of claim 1, wherein the ion source interface temperature is 300 ℃; the temperature of the desolventizing pipe is 250 ℃; the temperature of the heating module is 400 ℃; heating air flow rate is 10L/min; the flow rate of the atomized gas is 3L/min; the flow rate of the drying gas is 10L/min.
6. The method according to claim 1, wherein the Q1 Pre Bias, the collision energy, and the Q3 Pre Bias in the MRM monitoring scan are as follows:
。
7. Use of the detection method according to any one of claims 1 to 6 in clinical trials of musk heart-penetrating drops, characterized in that said clinical trials comprise pharmacokinetic trials.
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