Qualitative detection method of blood entering components of musk heart-tonifying drop pills

Abstract

The application provides a qualitative detection method of blood components of Moschus Tongxin dripping pills, specifically, 43 blood components of radix et rhizoma Ranae, salvia miltiorrhiza, total ginsenosides of ginseng stem and leaf, artificial ox gall and bear gall powder in Moschus Tongxin dripping pills are synchronously detected by using ultra-high performance liquid chromatography-tandem mass spectrometry. In the liquid phase condition, a carbon eighteen filler chromatographic column is used, the mobile phase A is 0.05%-0.2% formic acid water, the mobile phase B is 0.05%-0.2% formic acid acetonitrile, and gradient elution is adopted; in the mass spectrometry condition, an electrospray ion source is used, positive and negative ion scanning is adopted, the ion source temperature is 550 DEG C-600 DEG C, the atomizing gas pressure is 50 psi, the auxiliary gas pressure is 45-55 psi, the curtain gas pressure is 30-40 psi, the declustering potential is 80-120 V, the collision energy is 10-60 V, and the mass range is m / z 50-1500 Da. The detection method provided by the application can study the compound traditional Chinese medicine as a whole, has many detection indexes, short detection time and simple pretreatment, and is suitable for promotion.

Description

Technical Field

[0001] This technical solution belongs to the field of compound traditional Chinese medicine component detection, specifically involving the qualitative detection of the blood-entering components of Musk Tongxin Dripping Pills. Background Technology

[0002] Musk Heart-Clearing Pills are composed of seven medicinal materials: artificial musk, total saponins from ginseng stems and leaves, toad venom, salvia miltiorrhiza, bear bile powder, artificial bezoar, and borneol. They possess aromatic and orifice-opening, qi-tonifying, and yang-warming, qi-regulating, and pain-relieving effects. They are used for stable exertional angina pectoris in coronary heart disease, diagnosed in Traditional Chinese Medicine as qi deficiency and blood stasis syndrome, characterized by chest pain and tightness, palpitations, shortness of breath, fatigue, and weakness. Among them, artificial musk and toad venom are the principal herbs, acting as the chief herbs to clear the meridians and relieve pain; total saponins from ginseng stems and leaves and salvia miltiorrhiza tonify heart qi and invigorate blood circulation; artificial bezoar and bear bile powder clear the meridians, cool the body, resolve phlegm, and dispel blood stasis; borneol opens the orifices and awakens the mind, working synergistically with musk to guide the other herbs into the heart meridian.

[0003] The theory of "serum pharmacochemistry" was first proposed by German scientist Gerhard Domagk, and later, Chinese scientist Professor Wang Xijun proposed a theory of serum pharmacochemistry of traditional Chinese medicine that aligns with traditional Chinese medicine theory. The chemical composition of traditional Chinese medicine is extremely complex; after oral administration, some components are absorbed into the bloodstream through metabolism by enzymes in the intestines and liver, thereby exerting their effects.

[0004] The components entering the bloodstream are different from the components of medicinal materials and drugs. The study "Analysis of Chemical Constituents of Rhodiola Rosea Granules and Blood-Entering Components After Oral Administration in Rats Based on HPLC-Q-TOF-MS Technology" (Journal of Shanghai University of Traditional Chinese Medicine, 2021, 35(01):61-73) disclosed that 49 chemical components were identified in Rhodiola Rosea Granules, including flavonoids, glycosides, phenolic acids, tannins, proanthocyanidins, and other components. After oral administration of Rhodiola Rosea Granules to rats, 26 blood-entering components were identified in plasma samples, including 4 prototype components and 22 metabolites. The metabolites underwent Phase I metabolism, mainly deglycosylation, dehydrogenation, and hydroxylation reactions, and Phase II metabolism, mainly glucuronidation and sulfation reactions. This shows that after the prototype drug enters the bloodstream, it undergoes biotransformation through different metabolic pathways, resulting in blood-entering components with different structures. Methods used for detecting the prototype drug components are not necessarily applicable to the detection of blood-entering components.

[0005] Qualitative detection of the blood components of Musk Tongxin Dripping Pills has been reported. The paper "Investigation of the Mechanism of Musk Tongxin Dripping Pills in Treating Coronary Heart Disease Based on Serum Pharmaceutical Chemistry and Network Pharmacology" (Traditional Chinese Medicine, 2020, 42(10):2768-2777) discloses the extraction of serum samples in methanol, followed by concentration and detection by HPLC-Q-TOF / MS. The chromatographic column used was a Hydro-RP C18 column (250mm×4.6mm, 4μm); the mobile phase was 0.4% formic acid acetonitrile-0.2% formic acid, with gradient elution; the ion source was ESI, and the scanning mode was ESI+ / ESI-; the drying gas temperature was 320℃, the flow rate was 10L / min, the sheath gas temperature was 350℃, the flow rate was 12L / min, and the fragmentation energy was 175V; the capillary voltage was 3kV; and the positive and negative ion acquisition range was m / z 50~1500. This technical solution can qualitatively detect bufotalin, taurine, far-infrared bufotalin, glycocholic acid, deacetylated bufotalin, bufotalin, ursodeoxycholic acid, and bufotalin ligand. However, this technical solution can only simultaneously detect components of toad venom, artificial bezoar, and bear bile powder, and the number of components that can be detected simultaneously is limited.

[0006] CN114858959A, "A Method for Detecting Bufodiene Lactone Components from Musk-containing Pills in Rat Plasma and Its Use" (Application Date: 2021.01.20; Publication Date: 2022.08.05), discloses a method for qualitative and quantitative detection of bufotoxin, far-infrared bufotoxin, deacetylated bufotoxin, bufotoxin ether, and bufotoxin ligand in rat plasma using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). The plasma is extracted with methanol and then subjected to UPLC detection. The chromatographic column is an AcquityBEH C18 (100mm×2.1mm, 1.7μm). Mobile phase A is 1–3 mmol / L ammonium acetate solution, and mobile phase B is acetonitrile. Gradient elution allows detection to be completed within 13 min. This technical solution can only detect bufotoxin components, and the number of components detected simultaneously is limited.

[0007] In addition, there are also reports on the detection of blood-entering components of single medicinal herbs and similar compound Chinese medicine formulas.

[0008] CN107843671A, "A High-Throughput Liquid Chromatography-Tandem Mass Spectrometry Detection Method" (Application Date: 2017.12.21; Publication Date: 2018.03.27), discloses a technical scheme for the detection of 15 bile acids in human serum by UPLC-MS / MS. The UPLC chromatography uses a C18 column, with mobile phase A being 0.1% formic acid aqueous solution and mobile phase B being 0.1% formic acid-acetonitrile:methanol (3:1) solution. The mass spectrometry detection conditions are as follows: electrospray needle voltage 3.0 kV, desolventizing gas flow rate 800 L / h, desolventizing gas temperature 400℃, cone gas flow rate 50 L / h, detection in negative ion mode, and multiple reaction monitoring. This technical solution can simultaneously detect 15 bile acids, namely cholic acid, lithocholic acid, deoxycholic acid, ursodeoxycholic acid, chenodeoxycholic acid, taurocholic acid, glycocholic acid, taurolithocholic acid, glycocholic acid, taurodeoxycholic acid, tauroursodeoxycholic acid, taurochenodeoxycholic acid, glycocholic acid, glycocholic acid, and glycoursodeoxycholic acid.

[0009] CN102486472A, "A Method for Determining Active Pharmaceutical Ingredients in Serum" (Application Date: 2009.04.20; Publication Date: 2015.11.25), discloses the detection of blood-entering components of Musk Heart-Protecting Pill. Musk Heart-Protecting Pill is composed of musk, ginseng extract, artificial bezoar, cinnamon, styrax, toad venom, and borneol. It is used for chest pain caused by qi stagnation and blood stasis, characterized by fixed, unmoving precordial pain; and for angina pectoris and myocardial infarction caused by myocardial ischemia with the above symptoms. This technical solution employs HPLC-ELSD to perform a single-analysis and multiple-evaluation of the blood-borne components of Musk Heart-Protecting Pills. It can simultaneously detect bufotalin (also known as Japanese bufotalin), ginsenoside Re, 1β-hydroxybufotalin, ginsenoside Rb1, ginsenoside Ra1, bufotalinol, ginsenoside Rc, ginsenoside Rb2, ginsenoside Rb3, ginsenoside Rd, bufotalin, cholic acid, deoxycholic acid, bufotalin, chenodeoxycholic acid, and deoxycholic acid. However, the medicinal composition of Musk Heart-Protecting Pills differs from that of Musk Heart-Clearing Pills, as Musk Heart-Protecting Pills do not contain bear bile powder or danshen (Salvia miltiorrhiza).

[0010] The components of compound Chinese medicine that enter the bloodstream are more complex than those of single herbs, and the components entering the bloodstream vary significantly depending on the formulation. On the one hand, changes in the flavor and indications of the herbs during the formulation process lead to changes in the active ingredients; on the other hand, the components of the herbs themselves, after entering the bloodstream, undergo enzymatic catalysis and biotransformation into different derivatives. Furthermore, research on the active ingredients of single herbs deviates from the theory of holistic synergistic effects in traditional Chinese medicine, which is detrimental to the overall evaluation of the efficacy of compound Chinese medicine.

[0011] Therefore, the existing technology has limited capacity to qualitatively detect the number of components of Musk Tongxin Dripping Pills that enter the bloodstream, which cannot fully reflect the drug's entry into the bloodstream and its efficacy. Summary of the Invention

[0012] To address the problems existing in the prior art, this invention provides a qualitative detection method for the blood-entering components of Musk Tongxin Dripping Pills. This method uses ultra-high performance liquid chromatography-tandem mass spectrometry to simultaneously detect 43 blood-entering components of Musk Tongxin Dripping Pills, including toad venom, salvia miltiorrhiza, total saponins from ginseng stems and leaves, artificial bezoar, and bear bile powder, providing a foundation for further research on drug metabolic cycles and pathways.

[0013] The technical solution adopted in this invention is as follows:

[0014] A qualitative detection method for the blood-borne components of musk-containing Tongxin pills, the method comprising ultra-high performance liquid chromatography-tandem mass spectrometry analysis, wherein the liquid chromatography conditions use a C18 packed column, mobile phase A is 0.05%–0.2% formic acid in water, mobile phase B is 0.05%–0.2% formic acid in acetonitrile, and the gradient elution program is as follows:

[0015] Time (min) B(％) 0 4～7 2 9～12 26 90～98 27 4～8

[0016] The flow rate was 0.2–0.4 mL / min, and the column temperature was 35–45 °C.

[0017] Further, the mass spectrometry analysis conditions include: electrospray ionization source, negative ion scan, source voltage of -4.5 kV; positive ion scan, source voltage of 5.5 kV; ion source temperature of 550–600 °C; nebulizing gas pressure of 50 psi, auxiliary gas pressure of 45–55 psi, curtain gas pressure of 30–40 psi, declusivity potential of 80–120 V, collision energy of 10–60 V, and mass range of m / z 50–1500 Da. Preferably, the curtain gas is N2, the ion source temperature is 600 °C for positive ion scan, and 550 °C for negative ion scan. The auxiliary gas pressure is 50 psi, the curtain gas pressure is 35 psi, and the declusivity potential is 100 V.

[0018] Furthermore, the gradient elution program in the mass spectrometry conditions is as follows: 0 min, 5% B; 2 min, 10% B; 26 min, 95% B; 27 min, 5% B; flow rate is 0.3 mL / min, column temperature is 40 °C.

[0019] Furthermore, the detection method also includes pretreatment of plasma samples, which includes: transferring drug-containing plasma, adding methanol to precipitate proteins, vortexing, centrifuging, evaporating the supernatant at below 40°C, redissolving in 90% methanol, vortexing, centrifuging, and taking the supernatant for analysis.

[0020] Furthermore, the blood-injecting components of Musk Tongxin Dripping Pills are derived from toad venom, salvia miltiorrhiza, total saponins from ginseng stems and leaves, artificial bezoar, and bear bile powder. Preferably, the blood-injecting components of toad venom include bufotalin, bufotalin, bufotalin lipotoxin, bufotalin from South America, bufotalin from sand bufo, deacetylated bufotalin, bufotalin from far-China, bufotalin, bufotalin, bufotalin, bufotalin, bufotalin lipotoxin, bufotalin lipotoxin, bufotalin lipotoxin, and bufotalin lipotoxin.

[0021] Preferably, the blood-entering components of Salvia miltiorrhiza include tanshinone A, neocryptotanshinone, cryptotanshinone, tanshinone, salvianolic acid A, salvianolic acid B, isocryptoshinone, digoxin lactone, tanshinone diol C, and 3,5,7-trimethoxyflavone.

[0022] Preferably, the blood-entering components of total ginseng stem and leaf saponins include ginsenoside Re, ginsenoside Rb1, and ginsenoside Rd.

[0023] Preferably, the blood-inducing components of artificial bezoar and bear bile powder include taurine deoxycholic acid, taurine ursodeoxycholic acid, taurocholic acid, glycocholic acid, glycocholic acid, taurine chenodeoxycholic acid, taurine deoxycholic acid, cholic acid, glycocholic acid, ursodeoxycholic acid, chenodeoxycholic acid, deoxycholic acid, glycolithocholic acid, taurolithocholic acid, and taurine.

[0024] This invention establishes a technical solution for the simultaneous detection of blood-entering components of Musk Tongxin Dripping Pills using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS), including plasma pretreatment and the construction of UHPLC-MS / MS methods. Plasma pretreatment involves precipitating proteins with methanol and concentrating and enriching the blood-entering components with 90% methanol. Compared with existing technologies, this invention can achieve the simultaneous detection of 43 blood-entering components corresponding to 5 medicinal materials, detecting more components and better reflecting the transformation and metabolic pathways of the drug after entering the bloodstream, laying the foundation for the establishment of quantitative detection methods.

[0025] The advantages of this invention are as follows:

[0026] (1) This invention can study compound Chinese medicine as a whole and simultaneously detect 43 blood-entering components in musk Tongxin dripping pills.

[0027] The blood-entering components of this invention encompass the principal drug, toad venom; the assistant drugs, danshen and total saponins from ginseng stems and leaves; and the adjuvant drugs, artificial bezoar and bear bile powder. Specifically, it includes 13 blood-entering components from toad venom, 10 from danshen, 3 from total saponins from ginseng stems and leaves, and 17 from bile acids in artificial bezoar and bear bile powder. On one hand, analyzing these 43 blood-entering components allows for the in vivo metabolic process of the drug prototype; on the other hand, identifying these components clarifies the active ingredients in Musk Tongxin Dripping Pills as a traditional Chinese medicine compound, and allows for the inverse analysis of the drug prototype, serving as a key indicator for optimizing the preparation process and quality standards of Musk Tongxin Dripping Pills. The blood-entering components detected in this invention are shown in Tables 1-4.

[0028] Table 1 Detection Results of Components of Chan Su Entering the Blood

[0029]

[0030] Table 2 Detection Results of Components of Danshen Entering the Blood

[0031]

[0032] Table 3 Detection Results of Components of Total Ginsenosides from Ginseng Stem and Leaf Entering the Blood

[0033]

[0034] Table 4 Detection Results of Components of Artificial Calculus Bovis and Fel Ursi Entering the Blood

[0035]

[0036]

[0037] (2) The detection method established in this invention requires a short time. The ultra-high performance liquid chromatography-tandem mass spectrometry detection can be completed in only 27 minutes, and the pretreatment method is simple, which is suitable for popularization. Specific Embodiments

[0038] To better understand the content of this invention, it is further described in combination with embodiments. It should be understood that the embodiments of this invention should not be limited to the following exemplary embodiments.

[0039] Example 1: Detection Method for Plasma Samples

[0040] 1. Administration to Animals and Collection of Plasma Samples

[0041] Healthy SD rats, male, 7 weeks old, weighing 180 - 200 g, were purchased from Shanghai Slake Experimental Animal Co., Ltd. (SPF level, animal license number: SCXK (Shanghai) 2022 - 0004). They were raised according to the requirements of experimental animal operation guidelines, with the environmental temperature at 20℃ - 25℃ and the humidity at 50 - 60%. They were fasted for 12 h before the experiment and had free access to water.

[0042] Weigh an appropriate amount of Musk Tongxin Dripping Pill powder and add it to 0.5% sodium carboxymethyl cellulose solution to prepare a Musk Tongxin Dripping Pill suspension with a mass concentration of 0.4 g / mL. Three SD rats were fasted for 12 hours and then administered the Musk Tongxin Dripping Pill suspension (6.4 g / kg) by gavage. Blood samples were collected from the orbital sinus before administration (0 min) and at 15 min, 30 min, 45 min, 60 min, 90 min, and 120 min after administration (heparin anticoagulation). The blood samples were centrifuged at 5000 r / min for 10 min at 4℃ to separate the plasma. Three blank plasma samples (0 min) and 18 plasma samples from each time point after administration were mixed separately and stored at -80℃ (avoiding repeated freeze-thaw cycles) for later use.

[0043] 2. Plasma sample processing

[0044] Accurately transfer 200 μL of blank plasma and drug-containing plasma into 1.5 mL EP tubes, add 1 mL of methanol to precipitate proteins, vortex for 5 min, centrifuge at 13000 rpm for 10 min at 4 °C, evaporate the supernatant under vacuum in a centrifugal concentration system at 40 °C, add 100 μL of 90% methanol to reconstitute, vortex for 3 min, centrifuge at 13000 rpm for 10 min, and take 80 μL of the supernatant for analysis.

[0045] 3. Liquid chromatography and mass spectrometry conditions

[0046] Liquid chromatography conditions: A Waters UPLC (Waters Corp., Milford, MA, USA) ACQUITY UPLCHSS C18 column (1.8 μm, 2.1 × 100 mm) was used for chromatographic experiments. The mobile phases were 0.1% formic acid-water (A) and 0.1% formic acid-acetonitrile (B). The linear gradient program was as follows: 0 / 5, 2 / 10, 26 / 95, 27 / 5 (min / B%); injection volume 3 μL; column temperature 40 °C; flow rate 0.3 mL / min.

[0047] Mass spectrometry conditions: AB Triple TOF 5600 plus System (AB SCIEX, Framingham, USA). Negative ion mode: source voltage -4.5kV, source temperature 550℃; Positive ion mode: source voltage +5.5kV, source temperature 600℃. Nebulizer gas pressure and auxiliary gas pressure: 50psi; curtain gas (N2) pressure: 35psi. Maximum permissible error: ±5ppm. Declustering potential (DP): 100V; collision energy (CE): 10V. MS / MS acquisition mode parameters are the same: collision energy (CE): 40±20V; ion release delay (IRD): 67; ion release width (IRW): 25. IDA-based automated MS was performed on the eight strongest metabolite ions in one full scan cycle (1s). 2The m / z scan ranges for the precursor and daughter ions were set to 100–1500 Da and 50–1500 Da, respectively. An automated calibration transfer system was used to automatically perform precise quality calibration before each analysis.

[0048] 4. Data Processing

[0049] use (Software version:1.2.0.3(64-bit) (2012) Qualitative analysis of the blood-entering components of Musk Tongxin Dripping Pills was performed. The total ion chromatogram (TIC) and mass spectrometry peaks of blank plasma and drug-containing plasma were compared to obtain the precursor ion peaks with higher abundance in drug-containing plasma at any retention time. These peaks were then compared with the extract peaks, and further secondary mass spectrometry was performed to analyze the fragment ions of the corresponding precursor ions. The mass-to-charge ratio of these difference peaks was preliminarily determined, and compared with corresponding substances that may exist in the literature to screen for components that may enter the bloodstream. Finally, standards were used for verification.

[0050] 5. Results of blood component analysis

[0051] By comparing the components of blank plasma and drug-containing plasma, it was determined that the components entering the bloodstream were not endogenous. The experimental results showed that 43 components entering the bloodstream were identified in the plasma of Musk Tongxin Dripping Pills, including 13 toad venom-like components, 10 tanshinone-like components, 3 ginsenoside-like components, and 17 bile acid-like components, as shown in Tables 1-4.

[0052] Table 1. Results of blood-entry component detection of toad venom

[0053]

[0054]

[0055] Table 2. Results of blood component detection of Salvia miltiorrhiza.

[0056]

[0057] Table 3. Results of Blood-Entering Component Detection of Total Saponins from Ginseng Stems and Leaves

[0058]

[0059] Table 4. Results of Blood Component Detection for Artificial Bezoar and Bear Bile Powder

[0060]

Claims

1. A qualitative detection method for the blood-borne components of musk-containing heart-clearing pills, characterized in that, The detection method includes ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS), wherein the liquid chromatography conditions use a C18 packed column, mobile phase A is 0.05%~0.2% formic acid in water, mobile phase B is 0.05%~0.2% formic acid in acetonitrile, and the gradient elution program is as follows: The flow rate was 0.2–0.4 mL / min, and the column temperature was 35–45 °C. The mass spectrometry analysis conditions include: electrospray ionization source, negative ion scan, source voltage of -4.5 kV; positive ion scan, source voltage of 5.5 kV; ion source temperature of 550~600℃, nebulizer gas pressure of 50 psi, auxiliary gas pressure of 45~55 psi, curtain gas pressure of 30~40 psi, declustering potential of 80~120 V, collision energy of 10~60 V, and mass range of m / z 50~1500 Da; The detection method also includes pretreatment of plasma samples, which includes: transferring drug-containing plasma, adding methanol to precipitate proteins, vortexing, centrifuging, evaporating the supernatant at below 40°C, redissolving in 90% methanol, vortexing, centrifuging, and taking the supernatant for analysis. The blood-inducing components of the Musk Tongxin Dripping Pills are derived from toad venom, salvia miltiorrhiza, total saponins from ginseng stems and leaves, artificial bezoar, and bear bile powder. The blood-inducing components of the toad venom include bufotalin, bufotalin, bufotalin, bufotalin, bufotalin, deacetylated bufotalin, bufotalin, bufotalin, bufotalin, bufotalin, bufotalin, bufotalin, bufotalin, and bufotalin. The blood-entering components of Danshen include tanshinone A, neocryptotanshinone, cryptotanshinone, tanshinone, salvianolic acid A, salvianolic acid B, isocryptoshinone, digeprolactin, tanshinone diol C, and 3,5,7-trimethoxyflavone. The total saponins from ginseng stems and leaves that enter the bloodstream include ginsenoside Re, ginsenoside Rb1, and ginsenoside Rd; The artificial bezoar and bear bile powder contained the following blood-inducing components: taurourdeoxycholic acid, tauroursodeoxycholic acid, taurocholic acid, glycocholic acid, glycochorenocholic acid, taurourchedeoxycholic acid, taurourdeoxycholic acid, cholic acid, glycochorenocholic acid, ursodeoxycholic acid, porcine deoxycholic acid, glycochorenocholic acid, deoxycholic acid, glycolithocholic acid, taurolithocholic acid, and taurine.

2. The detection method according to claim 1, characterized in that, The mass spectrometry analysis conditions include: curtain gas is N2, ion source temperature is 600℃ for positive ion scanning and 550℃ for negative ion scanning; auxiliary gas pressure is 50 psi, curtain gas pressure is 35 psi, and declustering potential is 100 V.

3. The detection method according to claim 1, characterized in that, The mobile phase A is 0.1% formic acid in water, and the mobile phase B is 0.1% formic acid in acetonitrile. The gradient elution program is as follows: 0 min, 5% B; 2 min, 10% B; 26 min, 95% B; 27 min, 5% B; the flow rate is 0.3 mL / min, and the column temperature is 40℃.

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