Content detection method for salvia miltiorrhiza medicinal material, salvia miltiorrhiza clear paste and musk heart-dredging dropping pill

Abstract

The invention provides a content detection method for a salvia miltiorrhiza medicinal material, a salvia miltiorrhiza clear paste and a musk heart-dredging dropping pill. According to the method, a high performance liquid chromatography method is adopted, octadecylsilane chemically bonded silica is used as a chromatographic column filler, acetonitrile is used as a mobile phase A, 0.02%-0.1% trifluoroacetic acid is used as a mobile phase B, gradient elution is carried out, and rosmarinic acid and salvianolic acid B in the salvia miltiorrhiza medicinal material are synchronously and quantitatively detected. And synchronously and quantitatively detecting tanshinol, protocatechuic aldehyde, caffeic acid, salvianolic acid E, rosmarinic acid, alkannic acid and salvianolic acid B in the salvia miltiorrhiza clear paste, and synchronously and quantitatively detecting tanshinol, protocatechuic aldehyde, caffeic acid, salvianolic acid E, rosmarinic acid, alkannic acid, salvianolic acid B, arenobufagin and bufalin in the musk Tongxin dropping pill. The method is used for monitoring the content of specific components, can also judge the transfer rate in the medicine production process, and facilitates traceability in the production link. In addition, the method is relatively low in equipment requirement and suitable for industrial popularization.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of traditional Chinese medicine detection, and particularly relates to a content detection method for Danshen medicinal materials, Qinggao and Mosha Tongxin Dropping Pills. BACKGROUND

[0002] Danshen is the dried root and rhizome of the plant Salviamiltiorrhiza Bge. of the Labiatae family. It has the effects of activating blood and removing stasis, dredging channels and relieving pain, clearing heart and relieving restlessness, and cooling blood and resolving abscess, and is one of traditional Chinese medicinal materials for treating cardiovascular and cerebrovascular diseases in China. Danshen contains various active components, mainly including water-soluble components represented by phenolic acids and fat-soluble components represented by tanshinones. The water-soluble components mainly include salvianolic acid B, caffeic acid, rosmarinic acid and danshensu sodium. Among them, rosmarinic acid has broad prospects in the prevention and treatment of cardiovascular and cerebrovascular diseases through multiple mechanisms such as antioxidant, anti-inflammatory, anti-apoptosis and signal pathway regulation. The 2025 edition of Chinese Pharmacopoeia stipulates the content detection methods of cryptotanshinone, tanshinone I, tanshinone IIA and salvianolic acid B in Danshen medicinal materials, but does not mention the detection methods and content requirements of other water-soluble components.

[0003] Mosha Tongxin Dropping Pills are composed of artificial musk, total ginsenosides of ginseng stem and leaf, toad venom, Danshen, artificial ox gall, bear gall powder and borneol, and have the effects of aromatherapy, tonifying qi and dredging vessels, activating blood and resolving stasis and relieving pain, and are used for stable exertional angina pectoris of coronary heart disease. In the prescription, the artificial musk and toad venom are the monarch drugs, which have the effects of aromatherapy, dredging vessels and relieving pain; the total ginsenosides of ginseng stem and leaf and Danshen are the ministerial drugs, which have the effects of tonifying heart qi and activating blood and resolving stasis; the artificial ox gall and bear gall powder are the auxiliary drugs, which have the effects of clearing vessels, relieving heat, resolving phlegm and removing stasis; and the borneol is the ministerial drug, which has the effects of opening the orifices, refreshing the mind and directly reaching the lesion. Among them, toad venom has the effects of enhancing myocardial contractility, improving coronary circulation and resisting ischemia. Bufadienols and bufalin are two important components of toad venom, which have similar structures and can inhibit Na + ,K + -ATPase, thereby enhancing myocardial contractility. Establishing the component detection method of Danshen medicinal materials and Mosha Tongxin Dropping Pills, including the quality detection of toad venom and Danshen components, is helpful to improve the quality control of Mosha Tongxin Dropping Pills and realize the whole-process supervision from medicinal materials to medicinal products.

[0004] The prior art discloses a synchronous detection method for Danshen and toad venom components in Mosha Tongxin Dropping Pills.

[0005] CN111103369A "A compound traditional Chinese medicine characteristic spectrum construction and content determination method" (publication date: 2022.03.08) discloses high performance liquid chromatography conditions: the chromatographic column is SynergiTM Hydro-RP, 4.6x250mm, 4μm; the mobile phase is 0.005-0.05% phosphoric acid acetonitrile (A) and 0.005-0.05% phosphoric acid water (B); gradient elution; the column temperature is 29-31℃; the detection wavelength is 198-206nm; the flow rate is 1.28-1.32mL / min, and the high performance liquid chromatography characteristic spectrum of musk heart-protecting dropping pills is established. The technical scheme establishes the characteristic spectrum, identifies chromatographic peaks such as sodium danshensu, protocatechuic aldehyde, salvianolic acid D, salvianolic acid B, saucerephrin and bufalin, and simultaneously realizes content detection of ginsenoside Re, ginsenoside Rg1, cinobufagin and lipocidin. The method fails to realize synchronous quantification of Danshen components and ChanSu components.

[0006] CN117030912A "A method for quantitatively detecting blood components of ChanSu or Danshen in musk heart-protecting dropping pills" (publication date: 2023.11.10) discloses ultra-high performance liquid chromatography-tandem mass spectrometry detection of drug-containing plasma or tissue. In the liquid chromatography conditions, 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, gradient elution is used; the electrospray ion source is in positive ion mode, the dry gas temperature is 300-400℃, the dry gas flow rate is 4-8L / min, the sheath gas temperature is 300-400℃, the sheath gas flow rate is 8-12L / min, the atomization gas pressure is 40-50psi; the capillary inlet voltage is 3-4kV; the cone hole voltage is 450-550V, the collision energy is 10-30eV, and the fragmentation voltage is 100-190V. The detection method has high requirements for equipment, and after oral administration or administration of medicinal materials, the components finally enter the blood through absorption, metabolism and other processes, including the original components and metabolites. The detection method is not suitable for component detection and industrialization of medicinal materials and drugs.

[0007] The content detection method of Danshen or ChanSu medicinal materials is also disclosed in the prior art.

[0008] Simultaneous determination of 10 water-soluble and 4 fat-soluble components in Salvia miltiorrhiza by high performance liquid chromatography (J Pharm Anal. 2015, 35(06): 991-996) HPLC method was established to simultaneously determine the contents of 10 water-soluble compounds (salvianic acid sodium, protocatechuic aldehyde, caffeic acid, isovanillic acid, salvianolic acid D, rosmarinic acid, shikonin, salvianolic acid B, salvianolic acid A, salvianolic acid C, dihydro-tanshinone I, cryptotanshinone, tanshinone IIA) and 4 fat-soluble compounds in Salvia miltiorrhiza from different sources. The Agilent Zorbax SB-aq column (250 mm x 4.6 mm, 5 μm) was used with acetonitrile-0.03% phosphoric acid solution as the mobile phase, gradient elution (0-60 min, 10% A→68% A; 60-70 min, 68% A→80% A), flow rate 0.8 mL·min-1, column temperature 30 ℃, detection wavelength 280 nm, injection volume 20 μL. The results showed that the 14 components in Salvia miltiorrhiza could be completely separated within 65 min, and the mass concentration in the range of 0.01-1766.67 μg / mL had a good linear relationship with the peak area. Simultaneous determination of seven phenolic acid components in Danshen dispensing granules by one method for multiple evaluations (South China Pharmaceutical Journal. 2023, 21(05): 1352-1357) HPLC method was established to detect the contents of salvianic acid, protocatechuic aldehyde, caffeic acid, salvianolic acid E, rosmarinic acid, shikonin, and salvianolic acid B in Danshen dispensing granules. Danshen dispensing granules are a traditional Chinese medicine product made from Salvia miltiorrhiza decoction pieces that meet the standards of the Pharmacopoeia, through modern industrial water extraction, separation, concentration, drying, and granulation under the guidance of traditional Chinese medicine theory. The detection method is as follows: Waters Xselect HSS T3 column (250 mm x 4.6 mm, 5 μm), with acetonitrile (A)-0.05% phosphoric acid solution (B) as the mobile phase, gradient elution, flow rate 1.2 mL / min; column temperature 25 ℃, detection wavelength 286 nm.

[0009] Simultaneous determination of 11 bufadienolides in Venenum Bufonis by HPLC (J Pharm Anal. 2023, 43(05): 739-747) HPLC method was established to simultaneously determine the contents of 11 bufadienolides (pseudoisobatrachotoxin, dayaotailing, batrachotoxin, bufadienolide, bufalin, cinobufaglycoside, resibufotenol, resibufotenol, cinobufaglycoside, resibufotenol, and bufadienolide) in Venenum Bufonis. The method used an Agilent InfinityLab Poroshell 120EC-C18 column (150 mm x 3.0 mm 2.7 μm) with 0.3% acetic acid solution-acetonitrile as the mobile phase, gradient elution, flow rate 0.5 mL·min-1, column temperature 30 ℃, detection wavelength 296 nm.

[0010] The above method is only used for the quantitative detection of salvia miltiorrhiza or venonatium components, and cannot be directly used for the quantitative detection of salvia miltiorrhiza components and venonatium components. Single herb medicine is different from the components of traditional Chinese medicine compound, and the compatibility of traditional Chinese medicine seven emotions includes single line, mutual need, mutual help, mutual fear, mutual killing, mutual evil, and mutual opposition. Different medicine components influence each other, thereby increasing the difficulty of detecting the components of the compound traditional Chinese medicine; moreover, the effective component content in the medicine is low, the detection sensitivity is high, and the traditional Chinese medicine clear paste concentration process increases the concentration of other non-detected components, thereby interfering with the component quantification. SUMMARY

[0011] To solve the above problems, the present application establishes a content detection method suitable for salvia miltiorrhiza medicine, salvia miltiorrhiza clear paste and musk Tongxin dripping pills, which can better realize the whole process supervision of medicine, production process and medicine, ensure the traceability of the production link and the reliability of the medicine quality. The high performance liquid detection method established in the present application can realize the content detection of rosmarinic acid and salvianolic acid B in salvia miltiorrhiza medicine; can realize the content detection of salvianin, protocatechuic aldehyde, coffee acid, salvianolic acid E, rosmarinic acid, shikonin acid and salvianolic acid B in salvia miltiorrhiza clear paste; and can realize the content detection of salvianin, protocatechuic aldehyde, coffee acid, salvianolic acid E, rosmarinic acid, shikonin acid, salvianolic acid B, saucerephine and bufalin in musk Tongxin dripping pills. By monitoring the content of specific components, the transfer rate of the drug production process can also be judged, which is convenient for the traceability of the production link. In addition, the detection method has relatively low equipment requirements and is suitable for industrialization promotion.

[0012] To achieve the above purpose, the present application realizes the following technical scheme:

[0013] A content detection method of salvia miltiorrhiza medicine, salvia miltiorrhiza clear paste and musk Tongxin dripping pills, characterized in that the detection method comprises the following steps:

[0014] (1) Preparation of test sample includes: taking salvia miltiorrhiza medicine, crushing, adding 30% to 80% methanol 10 to 100 mL, ultrasonic extraction for 10 to 120 min, filtering, and taking the continuous filtrate, namely obtained;

[0015] (2) Liquid phase condition: the chromatographic column uses octadecylsilane bonded silica gel as the filler; acetonitrile is used as the mobile phase A, and 0.02% to 0.1% trifluoroacetic acid is used as the mobile phase B, which is gradient eluted according to the following table; the column temperature is 15 to 25 DEG C; the detection wavelength is 280 to 320 nm; the flow rate is 0.5 to 1.0 mL per minute. The theoretical plate number calculated according to salvianolic acid B should not be less than 20000; the elution gradient of the mobile phase is as follows:

[0016]

[0017] Or

[0018]

[0019] Preferably, the elution gradient of the mobile phase is as follows:

[0020]

[0021] or

[0022]

[0023]

[0024] Further, the mobile phase B is 0.05% trifluoroacetic acid; the detection wavelength is 286 nm, and the flow rate is 0.8 mL per minute.

[0025] Further, the test sample is Danshen clear paste, and the preparation of the test sample comprises: taking Danshen clear paste, adding 30%-80% methanol 10-100 mL, ultrasonic extraction for 10-120 min, filtering, and taking the filtrate, thereby obtaining the test sample. Preferably, the column temperature of the liquid phase condition is 15-20°C, the chromatographic column comprises ZORBAX Eclipse XDB-C18 and ZORBAX SB-C18, the chromatographic column has a specification of 4.6*250 mm, and the filler particle size is 5 μm.

[0026] Further, the test sample is Danshen clear paste, and the preparation of the test sample comprises: taking Danshen clear paste, adding 30%-80% methanol 10-100 mL, ultrasonic extraction for 10-120 min, filtering, and taking the filtrate, thereby obtaining the test sample. Preferably, the column temperature of the liquid phase condition is 15-20°C, the chromatographic column comprises ZORBAX Eclipse XDB-C18 and ZORBAX SB-C18, the chromatographic column has a specification of 4.6*250 mm, and the filler particle size is 5 μm.

[0027] Further, the reference substance for the detection method of Danshen medicinal material comprises one or both of rosmarinic acid and salvianolic acid B.

[0028] Further, the reference substance for the detection method of Danshen clear paste comprises one or more of danshensu, protocatechuic aldehyde, caffeic acid, salvianolic acid E, rosmarinic acid, shikonin, and salvianolic acid B.

[0029] Further, the reference substance for the detection method of Danshen clear paste comprises one or more of danshensu, protocatechuic aldehyde, caffeic acid, salvianolic acid E, rosmarinic acid, shikonin, and salvianolic acid B.

[0030] Danshen contains rich salvianolic acid components, and salvianolic acid has the effects of improving microcirculation, resisting thrombosis, resisting atherosclerosis, promoting angiogenesis, and protecting nerves; saucisse is one of the important active components in toad venom, has the pharmacological effects of strengthening heart, resisting arrhythmia, and resisting angiogenesis, and the quantitative detection of the above components can better realize the quality control of Moschatos Herba Pill.

[0031] Danshensu is a strong hydrogen bond donor and acceptor; as a steroid toxin, bufalin also contains polar groups such as hydroxyl, carbonyl, and epoxy groups in its structure, which can act as hydrogen bond acceptors or donors. The above-mentioned components in traditional Chinese medicine compound mix with each other, and can form non-covalent complexes through intermolecular hydrogen bonds. The overall polarity and molecular size / shape of such complexes are different from those of the bufalin molecule alone. Moreover, the polyphenolic acid "wraps" around the bufalin molecule, which can shield or expose some groups, forming a "Danshensu B-bufalin" complex, which changes its interaction with the chromatographic stationary phase or solvent. Therefore, when bufalin coexists with shikonin or danshensu, the liquid phase retention time of bufalin can change significantly.

[0032]

[0033] The present application has the following advantages:

[0034] (1) The present application realizes the synchronous detection of danshensu and bufalin in musk heart-protecting dripping pills, and solves the problem of mutual interference between different medicinal materials in the component detection process.

[0035] The present application uses the liquid phase detection conditions of the 2025 edition of Chinese Pharmacopoeia Danshen water extract fingerprint, i.e. elution with acetonitrile (A)-0.1% phosphoric acid (B) and corresponding mobile phase gradient under chromatographic condition 1; the experimental results do not detect the bufalin chromatographic peak; under chromatographic condition 2, the acidity is increased, the mobile phase is modified to acetonitrile (A)-0.05% trifluoroacetic acid (B); and the elution time is extended from 70 min to 75 min for gradient elution, and the liquid phase condition also does not destroy the stability of the "Danshensu B-bufalin" complex. The experimental results show that simply increasing the acidity of the mobile phase and reducing the elution gradient cannot realize the synchronous detection of danshensu B and bufalin.

[0036] The present application uses acetonitrile (A)-0.02%-0.1% trifluoroacetic acid (B), and under chromatographic conditions 3, 4 and 5, the synchronous detection of danshensu, protocatechuic aldehyde, caffeic acid, danshensu E, rosemary acid, shikonin, bufalin, danshensu B and bufalin is realized. Compared with chromatographic condition 2, chromatographic condition 3 keeps the mobile phase B at 78% from 43 to 50 min, which weakens the electrostatic interaction between danshensu B and bufalin, adjusts the hydrogen bond effect, reduces the stability of the "Danshensu B-bufalin" complex, and realizes the complete separation of bufalin and danshensu B. The bufalin chromatographic peak retention time is 49 min, and the danshensu B chromatographic peak retention time is 52 min.

[0037] Compared with chromatographic condition 3, the acidity is reduced in chromatographic condition 4, but the resolution is higher. Increasing the acidity of the mobile phase is usually used to improve the peak shape, but the present technical condition obtains better resolution under the condition of reducing the acidity, and unexpected technical effects are produced. The retention time of the chromatographic peak of bufalin is 43.5 min, and the retention time of the chromatographic peak of salvianolic acid B is 47.5 min, and the detection time is shortened by 15 min compared with chromatographic condition 3.

[0038] In chromatographic condition 5, the mobile phase is 76.4%→72.7% of mobile phase B at 43-50 min, and the acidity continues to decrease, and the resolution of bufalin and salvianolic acid B is relatively reduced, but the quantitative requirements are still met. The retention time of the chromatographic peak of bufalin is 48 min, and the retention time of the chromatographic peak of salvianolic acid B is 49.5 min, and the detection time is shortened by 20 min compared with chromatographic condition 3.

[0039] According to the detection results of chromatographic conditions 1-5, the destruction of the stability of the “salvianolic acid B-bufalin” complex is not simply increasing the acidity of the mobile phase, but under the specific mobile phase and elution gradient, the electrostatic interaction between salvianolic acid B and bufalin is weakened, and the effect of the hydrogen bond is adjusted. The separation requirements of salvianolic acid B and bufalin in the protection scope of the present application are met.

[0040] (2) The present application realizes the synchronous quantitative detection of nine components of salvia and toad venom in musk heart-acting drop pills, and realizes the synchronous detection of organic acid compounds, phenolic compounds and steroidal compounds.

[0041] In the present application, acetonitrile (A)-0.02%-0.05% trifluoroacetic acid (B) is used, and under the conditions of chromatographic conditions 3, 4 and 5, the synchronous detection of salvianic acid, protocatechuic aldehyde, coffee acid, salvianolic acid E, rosmarinic acid, shikonin, bufalin, salvianolic acid B and bufonidine is realized. The regression equation and linear range of the corresponding nine components are shown in Table 1.

[0042] Table 1 Regression equation of toad venom and salvia components in musk heart-acting drop pills

[0043]

[0044]

[0045] In addition, the present application is also applicable to the content detection of salvia decoction pieces and salvia clear paste, and the regression equation and linear range of the salvia components are the same as those in Table 1. When rosmarinic acid and salvianolic acid B are used as detection indexes in the production process, the transfer rate monitoring from medicinal materials to clear paste and musk heart-acting drop pill production process can be realized, and the production link traceability is facilitated.

[0046] (3) The application realizes synchronous detection of the content of Danshen decoction pieces, Danshen clear paste and Moschatos heart-activating dropping pills by high performance liquid chromatography, has low requirement on experimental equipment and is convenient for industrial popularization. BRIEF DESCRIPTION OF DRAWINGS

[0047] Part of the drawings of the specification is used to provide further understanding of the application, the schematic embodiments of the application and the description thereof are used to explain the application and do not constitute improper limitation on the application.

[0048] Figure 1 Liquid chromatogram of Danshen decoction pieces, Danshen clear paste and Moschatos heart-activating dropping pills under chromatographic condition 1.

[0049] Figure 2 Liquid chromatogram of Moschatos heart-activating dropping pills under chromatographic condition 2.

[0050] Figure 3 Liquid chromatogram of Moschatos heart-activating dropping pills under chromatographic condition 3.

[0051] Figure 4 Liquid chromatogram of Moschatos heart-activating dropping pills under chromatographic condition 4.

[0052] Figure 5 Liquid chromatogram of Moschatos heart-activating dropping pills under chromatographic condition 5.

[0053] Figure 6 Liquid chromatogram of Danshen decoction piece detection method specificity test.

[0054] Figure 7 Liquid chromatogram of Danshen clear paste detection method specificity test.

[0055] Figure 8 Liquid chromatogram of Moschatos heart-activating dropping pills and toad venom components.

[0056] Figure 9 Liquid chromatogram of Moschatos heart-activating dropping pill detection method specificity test.

[0057] REFERENCE SIGNS:

[0058] 1-danshensu, 2-protocatechuic aldehyde, 3-caffeic acid, 4-danshensu E, 5-rosemary acid, 6-bistort acid, 7-sand toad venom, 8-danshensu B, 9-bufonidine. DETAILED DESCRIPTION

[0059] The following examples are intended to provide further illustration of the application and are not used to limit the application.

[0060] Example 1: chromatographic condition 1

[0061] Reference to the first edition of 2025 edition of Chinese Pharmacopoeia "Salvia miltiorrhiza water extract" fingerprint liquid detection conditions, test solution and mobile phase elution gradient chromatographic conditions optimization.

[0062] 1, test solution preparation

[0063] Salvia miltiorrhiza decoction sample: take Salvia miltiorrhiza decoction powder 0.15g, precision weighing, precision adding 80% methanol 50mL, weighing, ultrasonic treatment 1 hour, take out, cool, reweigh, with 80% methanol to make up for the weight loss, shake up, filter, take the filter liquid, that is.

[0064] Salvia miltiorrhiza clear paste sample: take Salvia miltiorrhiza clear paste 100mg, precision weighing, add 80% methanol to dissolve and constant volume to 25mL volumetric flask, shake up, filter, take the filter liquid, that is.

[0065] Mushroom heart drop pill sample: take 30 mushroom heart drop pills, break the film coating, precision add water 50mL, weigh, ultrasonic treatment 1 hour, take out, cool, reweigh, make up for the weight loss with water, shake up, filter, take the filter liquid, that is.

[0066] 2, preparation of control solution

[0067] Salvia miltiorrhiza control preparation: take Salvia miltiorrhiza, protocatechuic aldehyde, coffee acid, salvianolic acid E, rosemary acid, lithospermic acid, salvianolic acid B control suitable amount, precision weighing, add 80% methanol to make 1.593mg, 1.005mg, 0.3358mg, 0.6333mg, 1.189mg, 0.998mg, 1.861mg per 1mL control solution.

[0068] Toad venom control preparation: take appropriate amount of bufadienolides and bufalin control, precision weighing, add 80% methanol to make 91.12μg, 102.5μg per 1mL control solution.

[0069] 3, chromatographic conditions

[0070] Chromatographic column: ZORBAX Eclipse XDB-C18Analytical 4.6×250mm, 5μm.

[0071] Chromatographic condition 1: with acetonitrile as mobile phase A, with 0.1% phosphoric acid as mobile phase B, gradient elution according to the following table; the flow rate is 1mL per minute, the column temperature is 30℃, the detection wavelength is 286nm.

[0072] Table 2 mobile phase gradient of chromatographic condition 1

[0073]

[0074] The Danshen decoction sample, Danshen clear paste sample, musk heart-healthy drop pill sample and control product solution were respectively detected under the above liquid phase conditions.

[0075] 4. Test results

[0076] The test results are shown in Table 2. Figure 1 As shown in Table 2, the separation degree of rosmarinic acid and salvianolic acid B in the Danshen decoction sample is good; the chromatographic peaks of salvianolic acid B at the retention time of 33 min in the Danshen clear paste and musk heart-healthy drop pill are not completely separated. After optimization of the liquid phase conditions, it is found that under the conditions, the retention time of the musk heart-healthy drop pill is 28 min to 33 min, i.e. between the lithospermic acid and salvianolic acid B, and no scillarein chrom atographic peak is detected.

[0077] Lithospermic acid and salvianolic acid contain rich phenolic hydroxyl and carboxyl groups, which are strong hydrogen bond donors and acceptors. Scillarein, as a steroidal toxin, also contains hydroxyl, carbonyl, epoxy and other polar groups in its structure, which can act as hydrogen bond acceptors or donors. The above components in traditional Chinese medicine compound are mixed together, which can form non-covalent complexes through intermolecular hydrogen bonds. The overall polarity and molecular size / shape of such complexes are different from those of the scillarein molecule alone. Moreover, the polyphenolic acid "wraps" around the scillarein molecule, which can shield or expose some groups of the scillarein molecule, changing its interaction with the chromatographic stationary phase or solvent. Therefore, when scillarein coexists with lithospermic acid or salvianolic acid, the liquid phase retention time of scillarein can change significantly. Simultaneous detection of scillarein and lithospermic acid or salvianolic acid requires first solving the interference problem between the components. Figure 1 The chromatographic peaks at the retention times of 32 min and 33.5 min may correspond to salvianolic acid B and "salvianolic acid B-scillarein" complex, respectively.

[0078] Since the components of the Danshen decoction sample are relatively simple, and the components of the Danshen clear paste at 286 nm wavelength are the same as those of the Danshen components in the musk heart-healthy drop pill, the musk heart-healthy drop pill is selected as the test product for chromatographic condition optimization.

[0079] Table 3 shows the chromatographic peak separation degree of the Danshen decoction sample, clear paste and musk heart-healthy drop pill under chromatographic condition 1.

[0080] Sample Danshensu Protocatechuic Aldehyde Caffeic Acid Salvianolic Acid E Rosmarinic Acid Shikonin Salvianolic Acid B Bufalin Danshen Decoction / / / / 3.24 / 1.54 / Danshen Qinggao 15.44 6.48 14.48 2.01 3.55 8.78 1.68 / Moshuotongxin Dropping Pills 15.32 6.43 11.39 2.1 3.63 8.51 1.13 6.21

[0081] Example 2: Chromatographic condition 2

[0082] 1. Sample preparation

[0083] Preparation of the test product solution: 30 musk heart-healthy drop pills were taken, the film coating was broken, 50 mL of 30% methanol was precisely added, weighed, ultrasonically treated for 1 hour, taken out, cooled, weighed again, the weight loss was made up with water, shaken, filtered, and the filtrate was taken, i.e. the test product solution was obtained.

[0084] Preparation of the control product solution: prepared in the manner of Example 1.

[0085] 2. Chromatographic conditions

[0086] Chromatographic column: ZORBAX Eclipse XDB-C18 Analytical 4.6 x 250 mm, 5 μm.

[0087] Chromatographic conditions 2: Gradient elution was performed with acetonitrile as mobile phase A and 0.05% trifluoroacetic acid as mobile phase B according to the following table; the flow rate was 0.8 mL per minute, the column temperature was 25 °C, and the detection wavelength was 286 nm.

[0088] Table 4 Mobile phase gradient of chromatographic condition 2

[0089]

[0090] The test sample and control sample solutions were detected respectively under the above liquid chromatographic conditions.

[0091] 3. Test results

[0092] The test results are shown in Table 5. Figure 2 After changing the mobile phase to the stronger acid trifluoroacetic acid, the separation degree of the chromatographic peak of salvianolic acid B with a retention time of about 35 min was poor, and the chromatographic peak of shichuan-toxin did not appear after the chromatographic peak of bauhinic acid with a retention time of 32 min. By changing the mobile phase to the stronger acid trifluoroacetic acid and reducing the elution gradient, and by adjusting the mobile phase proportion for the "salvianolic acid B-shichuan-toxin" complex, the percentage of mobile phase B was reduced from 78.75% to 70% at 20-45 min in chromatographic condition 1 to 77% to 73% at 20-45 min in chromatographic condition 2, which did not destroy the stability of the "salvianolic acid B-shichuan-toxin" complex. Simply increasing the acidity of the mobile phase and reducing the elution gradient cannot achieve the simultaneous detection of salvianolic acid B and shichuan-toxin.

[0093] Table 5 Separation degree of chromatographic peaks of musk heart drop pills under chromatographic condition 2

[0094] Danshensu Protocatechuic Aldehyde Caffeic Acid Salvianolic Acid E Rosmarinic Acid Shikonin Salvianolic Acid B Bufalin 2.19 2.41 10.37 2.12 2.25 6.99 1.22 9.49

[0095] Example 3: Chromatographic condition 3

[0096] 1. Sample preparation

[0097] Preparation of test sample solution: 50 musk heart drop pills were crushed, 100 mL of 80% methanol was added, and the weight was recorded. The sample was treated by ultrasonic for 2 hours, and then taken out and cooled. The weight was recorded again, and the weight loss was made up with water. After shaking and filtering, the filtrate was obtained.

[0098] Preparation of control sample solution: prepared according to the method of Example 1.

[0099] 2. Chromatographic conditions

[0100] Chromatographic column: ZORBAX Eclipse XDB-C18 Analytical 4.6×250 mm, 5 μm.

[0101] Chromatographic conditions 3: acetonitrile as mobile phase A, 0.05% trifluoroacetic acid as mobile phase B, gradient elution as specified in the table below; flow rate 0.8 mL / min, column temperature 20°C, detection wavelength 286 nm.

[0102] Table 6 Chromatographic Conditions 3 Mobile Phase Gradient

[0103]

[0104] The test sample and reference sample solutions were tested under the above liquid phase conditions.

[0105] 3. Test results

[0106] The test results are as follows Figure 3 As shown, under this elution gradient, the chromatographic peaks of Danshen (Salvia miltiorrhiza) were well resolved and had normal peak shapes. A peak for neribufotoxin appeared at retention time around 49 minutes, and a peak for salvianolic acid B appeared at retention time around 52 minutes. Compared with chromatographic conditions 1 and 2, this chromatographic condition maintained a 78% mobile phase B percentage from 20 to 50 minutes. Under this elution gradient, the electrostatic interaction between salvianolic acid B and neribufotoxin was weakened, hydrogen bonding was modulated, and the stability of the "salvianolic acid B-neribufotoxin" complex was reduced, achieving complete separation of neribufotoxin from salvianolic acid B. Furthermore, this elution gradient also accommodated the separation requirements of caffeic acid, salvianolic acid E, and lithospermic acid, enabling the simultaneous detection of steroidal, organic acid, and phenolic compounds in the compound traditional Chinese medicine preparation. However, this condition takes a relatively long time to detect, so further optimization of the mobile phase gradient is required.

[0107] Table 7 Chromatographic peak separation of Musk Tongxin Dropping Pills under chromatographic condition 3

[0108] Danshensu Protocatechuic Aldehyde Caffeic Acid Salvianolic Acid E Rosmarinic Acid Shikonin Salvianolic Acid B Bufalin Figure 4 5.73 3.7 20.71 2.61 4.74 10.17 2.06 2.43 6.27

[0109] Example 4: Chromatographic Conditions 4

[0110] 1. Sample preparation

[0111] Preparation of test solution: Take 20 Musk Tongxin Dropping Pills, crush the film coating, accurately add 10 mL of 50% methanol, weigh, ultrasonically treat for 90 minutes, take out, cool, weigh again, make up the weight loss with water, shake well, filter, and take the filtrate.

[0112] Preparation of reference solution: Prepare according to the method of Example 1.

[0113] 2. Chromatographic conditions

[0114] Chromatographic column: ZORBAX Eclipse XDB-C18 Analytical 4.6×250mm, 5μm.

[0115] Chromatographic conditions 4: acetonitrile as mobile phase A, 0.05% trifluoroacetic acid as mobile phase B, gradient elution as specified in the table below; flow rate 0.5 mL / min, column temperature 15°C, detection wavelength 286 nm.

[0116] Table 8 Chromatographic Conditions 4 Mobile Phase Gradient

[0117]

[0118] The test sample and reference sample solutions were tested under the above liquid phase conditions.

[0119] 3. Test results

[0120] The test results are as follows Danshensu As shown, this elution gradient achieved better separation between salvianolic acid B and bufotoxin, with the bufotoxin peak retention time at 43.5 minutes and the salvianolic acid B peak retention time at 47.5 minutes. Compared to chromatographic condition 3, where the mobile phase was 78% mobile phase B from 43 to 50 minutes, this chromatographic condition, where the mobile phase was 78% to 75.2% mobile phase B from 43 to 50 minutes, exhibited even higher separation, demonstrating a decrease in acidity. Increasing mobile phase acidity is often used to improve peak shape, but this technique achieved even better separation while reducing acidity, yielding an unexpected technical benefit. These experimental results also demonstrate that the destabilization of the salvianolic acid B-bufotoxin complex is not achieved by simply increasing mobile phase acidity, but rather by weakening the electrostatic interaction between salvianolic acid B and bufotoxin, modulating hydrogen bonding, under specific mobile phases and elution gradients.

[0121] In addition, the separation degree of Danshen components, bufotoxin and bufalin was good, and the peak shape was normal, meeting the requirements of simultaneous quantitative detection; at the same time, the detection time was shortened to 75 minutes compared with chromatographic condition 3.

[0122] Table 9 Chromatographic peak separation of Musk Tongxin Dropping Pills under chromatographic condition 4

[0123] Protocatechuic Aldehyde Caffeic Acid Salvianolic Acid E Rosmarinic Acid Shikonin Salvianolic Acid B Bufalin Figure 5 Danshensu 51.63 12.57 11.48 1.51 2.93 4.68 7.98 4.95 7.30

[0124] Example 5: Chromatographic Conditions 5

[0125] 1. Sample preparation

[0126] Preparation of test solution: Take 10 Musk Tongxin Dropping Pills, crush the film coating, accurately add 50 mL of 30% methanol, weigh, ultrasonically treat for 10 minutes, take out, cool, weigh again, make up the weight loss with water, shake well, filter, and take the filtrate.

[0127] Preparation of reference solution: Prepare according to the method of Example 1.

[0128] 2. Chromatographic conditions

[0129] Chromatographic column: ZORBAX Eclipse XDB-C18 Analytical 4.6×250 mm, 5 μm.

[0130] Chromatographic conditions 5: Flow rate 1.0 mL / min, column temperature 25°C, detection wavelength 286 nm, 0.05% trifluoroacetic acid as mobile phase B, gradient elution as specified in the following table:

[0131] Table 10 Chromatographic Conditions 5 Mobile Phase Gradient

[0132]

[0133] The test sample and reference sample solutions were tested under the above liquid phase conditions.

[0134] 3. Test results

[0135] The test results are as follows Protocatechuic Aldehyde As shown in the figure, the elution gradient conditions for Danshen chromatographic peaks were well resolved and had normal peak shapes. The retention time for the chromatographic peak of neritobuflin was 48 minutes, and that for salvianolic acid B was 49.5 minutes. Compared to chromatographic condition 4, where the mobile phase was 78% to 75.2% mobile phase B from 43 to 50 minutes, the mobile phase for this chromatographic condition was 76.4% to 72.7% mobile phase B from 43 to 50 minutes. The decreased acidity reduced the resolution of neritobuflin and salvianolic acid B, but still met the quantitative requirements. This indicates that only when a specific acidity combined with a mobile phase gradient is neritobuflin and salvianolic acid B present can the simultaneous detection of seven Danshen components, including danshensu, protocatechuic aldehyde, caffeic acid, salvianolic acid E, rosmarinic acid, lithospermic acid, and salvianolic acid B, along with neritobuflin and bufalin, be achieved.

[0136] Under chromatographic condition 5, the separation degree between the chromatographic peak of bufalin and the chromatographic peak of salvianolic acid B meets the quantitative requirements. At the same time, the detection time is shortened to 70 min compared with chromatographic condition 3, which is more suitable for industrial promotion.

[0137] Table 11 Chromatographic peak separation of Musk Tongxin Dropping Pills under chromatographic condition 5

[0138] Caffeic Acid Salvianolic Acid E Rosmarinic Acid Shikonin Salvianolic Acid B Bufalin Figure 6 Ingredient Regression equation 4.95 3.7 12.78 2.55 4.79 11.29 1.52 2.17 5.32

[0139] Example 6: Specificity test of Danshen decoction piece detection method

[0140] 1. Sample preparation

[0141] Negative test sample solution: Take 80% methanol as the negative test sample solution, inject for detection, and record the chromatogram.

[0142] Control solution: Add 80% methanol to prepare a solution of 5 μg of rosmarinic acid and 90 μg of salvianolic acid B per 1 mL.

[0143] Test sample solution: Take 0.15 g of Danshen decoction piece powder, accurately weigh, and accurately add 50 mL of 80% methanol. Weigh, ultrasonically treat for 0.5 hours, take out, cool, weigh again, make up the weight loss with 80% methanol, shake well, filter, and take the filtrate, which is obtained.

[0144] 2. Liquid phase conditions

[0145] According to the liquid phase conditions of Example 4 for detection.

[0146] 3. Test results

[0147] The results are shown in Table 1. Linear range (μg / mL) The test sample has the same spectral absorption as the peaks corresponding to the rosmarinic acid and salvianolic acid B control peaks, and the negative test sample does not interfere with the above peaks, and the method has good specificity.

[0148] Example 7: Linearity test of Danshen decoction piece detection method

[0149] 1. Sample preparation

[0150] Control stock solution: Take appropriate amounts of rosmarinic acid and salvianolic acid B controls, accurately weigh, and add 80% methanol to prepare a control solution containing 1.189 mg and 1.861 mg per 1 mL, respectively, as the respective control stock solutions.

[0151] Rosmarinic acid control linear solution: Take the rosmarinic acid control stock solution to prepare linear solutions with concentrations of 29.73, 14.87, 5.948, 2.379, 1.190, and 0.5950 μg / mL, respectively.

[0152] Salvianolic acid B control linear solution: Take the salvianolic acid B control stock solution to prepare linear solutions with concentrations of 465.3, 232.7, 93.08, 37.23, 18.62, and 9.310 μg / mL, respectively.

[0153] 2. Liquid phase conditions

[0154] According to the liquid phase conditions of Example 4 for detection.

[0155] 3. Test results

[0156] The linear range and regression mode of rosmarinic acid and salvianolic acid B in Danshen medicinal materials are shown in Table 12.

[0157] Table 12 Regression equation of Danshen medicinal material components

[0158] Rosmarinic Acid Salvianolic Acid B Figure 7 Figure 8 [y = 26.9845x - 0.2430, R 2 = 1.0000]] 0.5950～14.87 Figure 9 [y = 16.8527x + 6.3512, R 2 = 1.0000]] 9.310～465.3

[0159] Example 8: Durability test of chromatographic column for Danshen decoction piece detection method

[0160] 1. Sample preparation

[0161] Danshen decoction piece sample: Danshen decoction piece samples were prepared according to the manner of Example 6. The same batch of Danshen decoction pieces was taken to prepare 5 test sample solutions, which were injected into different chromatographic columns for detection, chromatograms were recorded, and contents were calculated.

[0162] 2. Liquid phase conditions

[0163] According to the experimental conditions of Example 4. Among them, the following 5 chromatographic columns were used:

[0164] Chromatographic column 1: ZORBAX SB-C18 StableBond Analytical 4.6x250mm, 5μm;

[0165] Chromatographic column 2: Chrom Core C18 4.6x250mm, 5μm;

[0166] Chromatographic column 3: Ultimate Plus C18 HPLC Column 4.6x250mm, 5μm;

[0167] Chromatographic column 4: ZORBAX Eclipse XDB-C18 Analytical 4.6x250mm, 5μm;

[0168] Chromatographic column 5: Shim-pack C18 250x4.6mm, 5μm.

[0169] 3. Test results

[0170] The test results show that the content detection results of different chromatographic columns under the same mobile phase and elution gradient are similar, and the detection method has certain durability to the chromatographic column.

[0171] Table 13 Chromatographic column durability test results

[0172]

[0173] Example 9: Specificity test of Danshen clear paste detection method

[0174] 1. Sample preparation

[0175] Negative sample solution: 80% methanol was taken as the negative sample solution, and the sample was injected for detection, and the chromatogram was recorded.

[0176] Control solution: Salviol sodium, protocatechuic aldehyde, coffee acid, salvianolic acid E, rosmarinic acid, shikonin acid, and salvianolic acid B controls were taken and 80% methanol was added to prepare a solution containing 40 μg of salviol sodium, 10 μg of protocatechuic aldehyde, 3 μg of coffee acid, 10 μg of salvianolic acid E, 5 μg of rosmarinic acid, 10 μg of shikonin acid, and 90 μg of salvianolic acid B per 1 mL.

[0177] Test sample solution: 100 mg of Danshen clear paste was precisely weighed and determined, dissolved with 80% methanol and diluted to a 25 mL volumetric flask, shaken, filtered, and the filtrate was taken to obtain the test sample solution.

[0178] 2. Liquid phase conditions

[0179] According to the experimental conditions of Example 4.

[0180] 3. Test results

[0181] The results are shown in Table 1. In the test sample chromatogram, there should be spectral absorption peaks corresponding to the positions of the salviol, protocatechuic aldehyde, coffee acid, salvianolic acid E, rosmarinic acid, shikonin acid, and salvianolic acid B control peaks. The negative sample had no interference with the above chromatographic peaks, and the method had good specificity. Acid concentration

[0182] Example 10: Linear test of Danshen clear paste detection method

[0183] 1. Sample preparation

[0184] Control stock solution preparation: An appropriate amount of salviol, protocatechuic aldehyde, coffee acid, salvianolic acid E, rosmarinic acid, shikonin acid, and salvianolic acid B controls were precisely weighed and determined, and 80% methanol was added to prepare a control solution containing 1.593 mg, 1.005 mg, 0.3358 mg, 0.6333 mg, 1.189 mg, 0.998 mg, and 1.861 mg per 1 mL, respectively, as the respective control stock solutions.

[0185] ​Linear solution preparation: The linear solutions with concentrations of 199.1, 99.55, 39.82, 15.93, 7.965, 3.983 μg / mL were prepared from the salvianic acid A reference substance stock solution; the linear solutions with concentrations of 50.25, 25.13, 10.05, 4.021, 2.011, 1.006 μg / mL were prepared from the protocatechuic aldehyde reference substance stock solution; the linear solutions with concentrations of 16.79, 8.395, 3.358, 1.343, 0.6715, 0.3358 μg / mL were prepared from the caffeic acid reference substance stock solution; the linear solutions with concentrations of 47.50, 23.75, 9.500, 3.800, 1.900, 0.9500 μg / mL were prepared from the lithospermic acid E reference substance stock solution; the linear solutions with concentrations of 29.73, 14.87, 5.948, 2.379, 1.190, 0.5950 μg / mL were prepared from the rosmarinic acid reference substance stock solution; the linear solutions with concentrations of 49.90, 24.95, 9.980, 3.992, 1.996, 0.9980 μg / mL were prepared from the zhebeinosic acid reference substance stock solution; and the linear solutions with concentrations of 465.3, 232.7, 93.08, 37.23, 18.62, 9.310 μg / mL were prepared from the lithospermic acid B reference substance stock solution.

[0186] 2. Liquid phase conditions

[0187] According to the experimental conditions of Example 4.

[0188] 3. Test results

[0189] The regression equations and linear ranges of the seven components in the Danshen Qinggao are shown in Table 14.

[0190] Table 14 Regression equations of components in Danshen Qinggao

[0191]

[0192]

[0193] Example 11: Chromatographic durability of the Danshen Qinggao detection method

[0194] 1. Sample preparation

[0195] Danshen Qinggao sample: The Danshen Qinggao sample was prepared according to the method of Example 9. Five test sample solutions were prepared and injected into different chromatographic columns for detection, and the chromatograms were recorded and the contents were calculated.

[0196] 2. Liquid phase conditions

[0197] According to the liquid phase conditions of Example 4.

[0198] 3. Test results

[0199] The results show that the chromatographic column 2, 3 has poor separation degree for salvianolic acid E, and the chromatographic column 5 has serious peak tailing phenomenon, the RSD of the content of danshensu, salvianolic acid B component is not more than 4%, the RSD of the content of caffeic acid is not more than 8%, and the RSD of the content of the remaining six components is not more than 6%, and the method has certain durability to the chromatographic column.

[0200] Table 15: Content detection results of different chromatographic columns of drop pills (%)

[0201]

[0202] Example 12: Detection method of musk heart drop pills to confirm the bufonid component

[0203] 1. Sample preparation

[0204] Control solution: Take appropriate bufalin and cinobufagin control samples, accurately weigh and add 80% methanol to prepare a control solution of 91.12 μg / mL and 102.5 μg / mL respectively.

[0205] Test solution: Take 60 musk heart drop pills, crush the film coating, accurately add 80% methanol 50 mL, weigh, ultrasonic treat for 1 hour, take out, cool down, weigh again, add 80% methanol to make up the lost weight, shake well, filter, and take the filtrate.

[0206] 2. Liquid phase conditions

[0207] According to the liquid phase conditions of Example 4.

[0208] 3. Test results

[0209] The test results are shown in Table 15. Danshensu The chromatographic peak with a retention time of 47 min, i.e. between the chromatographic peaks of lithospermic acid and salvianolic acid B, is bufalin; the chromatographic peak with a retention time of 69 min is cinobufagin.

[0210] Example 13: Specificity test of musk heart drop pills detection method

[0211] 1. Sample preparation

[0212] Negative test solution: The blank sample prepared according to the prescription proportion without Danshen is the negative test solution, which is injected for detection and the chromatogram is recorded.

[0213] Test solution: Prepared according to the method of Example 12.

[0214] Reference solution: Take sodium danshensu, protocatechuic aldehyde, caffeic acid, salvianolic acid E, rosmarinic acid, shikimic acid, and salvianolic acid B reference substance respectively, and add 80% methanol to prepare a solution containing 40 μg of sodium danshensu, 10 μg of protocatechuic aldehyde, 3 μg of caffeic acid, 10 μg of salvianolic acid E, 5 μg of rosmarinic acid, 10 μg of shikimic acid, and 90 μg of salvianolic acid B per 1 mL.

[0215] 2. Liquid phase condition

[0216] The liquid phase condition is the same as that in Example 4.

[0217] 3. Test result

[0218] The result is shown in Table 3. Protocatechuic Aldehyde As shown in Table 3, in the test sample chromatogram, there should be spectral absorption peaks corresponding to the peaks of sodium danshensu, protocatechuic aldehyde, caffeic acid, salvianolic acid E, rosmarinic acid, shikimic acid, and salvianolic acid B. The negative test sample does not interfere with the detection of the above peaks.

[0219] In addition, according to the detection results of bufadienolides in Example 12, the negative test sample has a bufadienolide peak at a retention time of 43.5 min and a bufalin peak at a retention time of 67 min. This shows that other medicinal ingredients in musk heart-protecting pills, including bufadienolides, do not affect the detection of danshen ingredients, and the method has good specificity.

[0220] Example 14: Acid test of the detection method of musk heart-protecting pills

[0221] 1. Sample preparation

[0222] Test sample preparation: The test sample was prepared according to the method of Example 12.

[0223] 2. Liquid phase condition

[0224] Chromatographic column: ZORBAX SB-C18 StableBond Analytical 4.6×250 mm, 5 μm;

[0225] Chromatographic condition: The flow rate was 0.5 mL per minute, the column temperature was 15°C, the detection wavelength was 286 nm, 0.01%, 0.02%, 0.05%, and 0.1% trifluoroacetic acid was used as the mobile phase B, and gradient elution was performed according to the following Table 8:

[0226] Table 8 Elution gradient of chromatographic condition 4

[0227]

[0228] 3. Test result

[0229] The separation degree results of 7 Danshen components, bufalin and cinobufagin are shown in Table 16. When the concentration of trifluoroacetic acid is 0.01%, Danshensan B and bufalin are not completely separated, and the separation degree is less than 1.5, which does not meet the quantitative requirements. With the increase of the concentration of trifluoroacetic acid, the separation degree of Danshensan B and bufalin increases, and when the acidity is 0.02%, the separation degree of Danshensan B is 3.54, which meets the quantitative requirements. It can be seen that the increase of acidity can adjust the peak shape of the chromatogram, but for complex component samples, especially for the synchronous detection of multiple components in traditional Chinese medicine compound preparations, the requirements are higher.

[0230] Table 16 Chromatographic peak separation degree under different concentrations of trifluoroacetic acid

[0231] Caffeic Acid Salvianolic Acid E Rosmarinic Acid Shikonin Salvianolic Acid B Bufalin ​ ​ ​ ​ 0.01％ 14.14 15.94 10.11 2.23 3.25 3.28 11.43 0.95 6.55 0.02％ 14.95 13.65 10.37 5.47 3.79 4.00 6.26 3.54 6.49 0.05％ 42.03 10.36 10.31 1.53 2.83 4.48 6.59 5.97 6.48 0.10％ 15.61 8.59 10.16 1.67 2.54 4.57 8.11 6.69 6.43

[0232] Example 15: Linear test of musk heart-healthy drop pill detection method

[0233] 1. Sample preparation

[0234] Preparation of reference substance stock solution: Take appropriate amounts of bufalin and cinobufagin reference substances, accurately weigh, and add 80% methanol to prepare a reference substance solution containing 91.12 mg and 102.5 mg per 1 mL, respectively, as the respective reference substance stock solution.

[0235] Preparation of linear solution: Take the bufalin reference substance stock solution to prepare linear solutions with concentrations of 91.12, 45.56, 18.22, 7.822, 3.644, and 1.822 μg / mL, respectively; take the cinobufagin reference substance stock solution to prepare linear solutions with concentrations of 102.5, 51.25, 20.50, 8.200, 4.100, and 2.050 μg / mL, respectively.

[0236] Preparation of Danshen component stock solution and linear solution according to the method of Example 10.

[0237] 2. Liquid phase conditions

[0238] According to the experimental conditions of Example 4.

[0239] 3. Test results

[0240] The regression equation and linear range of 9 components in musk heart-healthy drop pills are shown in Table 1.

[0241] Table 1 Regression equation of cinobufagin and Danshen components in musk heart-healthy drop pills

[0242]

[0243]

[0244] Example 16: Chromatographic robustness of musk heart-healthy drop pill detection method

[0245] 1. Sample preparation

[0246] Mushroom heart drop pill sample: Mushroom heart drop pill sample was prepared according to Example 12. Five test sample solutions were prepared and injected into different chromatographic columns for detection, chromatograms were recorded, and contents were calculated.

[0247] 2. Liquid phase conditions

[0248] Liquid phase conditions according to Example 8.

[0249] 3. Test results

[0250] The results show that chromatographic columns 2 and 3 have poor separation degree for Dan- shen acid E, and chromatographic column 5 has serious tailing phenomenon. The content RAD of protocatechuic aldehyde and rosmarinic acid in the remaining chromatographic columns is not more than 11%, and the content RAD of the remaining 7 components is not more than 8%. This method has certain durability to chromatographic columns.

[0251] Table 17. Results of content detection for chromatographic column durability (μg / pill)

[0252]

Claims

1. A method for detecting the content of Danshen medicinal materials, Danshen clear paste and Musk Tongxin dripping pills, characterized in that: The detection method comprises the following steps: (1) The preparation of the test sample includes: taking Danshen medicinal material, crushing it, adding 10-100 mL of 30%-80% methanol, ultrasonically extracting it for 10-120 min, filtering it, and taking the filtrate; (2) Liquid Phase Conditions: The chromatographic column is filled with octadecylsilane bonded silica gel; acetonitrile is used as mobile phase A, and 0.02% to 0.1% trifluoroacetic acid is used as mobile phase B, with gradient elution as specified in the table below; the column temperature is 15°C to 25°C; the detection wavelength is 280 to 320 nm; the flow rate is 0.5 to 1.0 mL / min. The theoretical plate number calculated based on salvianolic acid B should be no less than 20,000; the mobile phase elution gradient is as follows: or 2. The detection method according to claim 1, wherein The mobile phase elution gradient is as follows: or 3. The detection method according to claim 1, wherein The mobile phase B was 0.05% trifluoroacetic acid, the detection wavelength was 286 nm, and the flow rate was 0.8 mL per minute.

4. The detection method according to any one of claims 1 to 3, characterized in that: The test sample is Danshen clear paste, and the preparation of the test sample includes: taking Danshen clear paste, adding 10-100 mL of 30%-80% methanol, ultrasonic extraction for 10-120 minutes, filtering, and taking the filtrate to obtain the product.

5. The detection method according to claim 4, characterized in that The test sample is Danshen Qing Gao, the liquid phase column temperature is 15°C to 20°C, the chromatographic columns include ZORBAX Eclipse XDB-C18 and ZORBAX SB-C18, the chromatographic column specifications are 4.6×250mm, and the filler particle size is 5μm.

6. The detection method according to any one of claims 1 to 3, characterized in that: The test sample is Musk Tongxin Dropping Pills, and the preparation of the test sample includes: taking Musk Tongxin Dropping Pills, removing the film coating, adding 10 to 100 mL of 30% to 80% methanol, ultrasonically extracting for 10 to 120 minutes, filtering, and taking the filtrate to obtain the product.

7. The detection method according to claim 6, characterized in that The test sample is Musk Tongxin Dropping Pills, the liquid phase conditions are column temperature of 15°C to 20°C, ZORBAX Eclipse XDB-C18, ZORBAX SB-C18, the chromatographic column specifications are 4.6×250 mm, and the filler particle size is 5 μm.

8. The detection method according to claim 1, wherein The detection method reference substance includes one or both of rosmarinic acid and salvianolic acid B.

9. The detection method according to claim 4, characterized in that The detection method reference substances include one or more of danshensu, protocatechuic aldehyde, caffeic acid, salvianolic acid D, salvianolic acid E, rosmarinic acid, lithospermic acid, and salvianolic acid B.

10. The detection method according to claim 6, characterized in that: The detection method reference substances include one or more of danshensu, protocatechuic aldehyde, caffeic acid, salvianolic acid D, salvianolic acid E, rosmarinic acid, lithospermic acid, salvianolic acid B, bufotoxin and bufalin.

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