Fingerprint detection method of traditional Chinese medicine preparation for treating heart failure

Abstract

This invention provides a fingerprinting method for traditional Chinese medicine preparations used to treat heart failure, and determines the chemical characteristic components of Plantago asiatica, Paeonia lactiflora, Cinnamomum cassia, Salvia miltiorrhiza, Polyporus umbellatus, and Leonurus japonicus in Shenfu Yixin Granules. Three batches of Shenfu Yixin Granules were tested, and four indicator components—genipin, paeoniflorin, cinnamaldehyde, and salvianolic acid B—were identified. This detection method features rapid detection speed, comprehensive qualitative analysis of relatively complete components, and can objectively characterize the intrinsic quality of Shenfu Yixin Granules of this invention.

Description

Technical Field

[0001] This invention belongs to the field of quality testing of effective components of traditional Chinese medicine, and in particular, a fingerprint spectrum detection method for traditional Chinese medicine preparations used to treat heart failure. Background Technology

[0002] The traditional Chinese medicine preparation described in this invention is Shenfu Yixin Granules, a core product of Shandong Buchang Pharmaceutical Co., Ltd. It is prepared by extracting and processing Chinese medicinal herbs including ginseng, aconite root, cinnamon twig, salvia miltiorrhiza, red peony root, motherwort, alisma rhizome, polyporus umbellatus, plantain seed, lepidium seed, amomum villosum, areca peel, and jujube. It has the effects of warming yang and tonifying qi, promoting blood circulation and removing blood stasis, and promoting diuresis and reducing swelling. It is used for congestive heart failure caused by yang deficiency and qi deficiency, blood stasis and water retention. Symptoms include palpitations, shortness of breath, cold limbs, abdominal distension, lower limb edema, scanty urine, dark complexion, cyanosis of the lips, distended neck veins, hypochondriac mass, pale or dark tongue, white or greasy tongue coating, deep and thready pulse, intermittent or rapid and scattered pulse, etc.

[0003] The applicant has filed a prior patent application for this product, CN101199812A (Test Method), published on June 18, 2008. The patent application for this test method states that the quality control method for this traditional Chinese medicine granule includes: thin-layer identification of Danshen, Chishao, Yimucao, Tinglizi, Dazao, Sharen, Baifupian, and Guizhi, aconitine limit test, and ginseng content determination. Prepare a 1 mg / mL solution of ginsenoside Rg1 reference standard using methanol. Perform thin-layer chromatography (TLC) according to the Chinese Pharmacopoeia, using the upper layer of n-butanol-ethyl acetate-water (4:1:5) as the developing solvent. Develop at below 10°C. Remove the sample, air dry, spray with 10% sulfuric acid ethanol solution, and heat at 100°C until the spots are clearly visible. Cover the TLC plate with a glass plate of the same size and fix with adhesive tape. Scan using TLC scanning at wavelengths λS 520 nm and λR 690 nm. Measure the absorbance integral values ​​of the test sample and the reference standard. This invention contains ginsenoside Rg1, with the molecular formula C. 42 H 72 O 14 The content shall not be less than 0.028%. Zhang Chi, Wu Jianguo, Xu Huiyan, et al., determined the dopamine hydrochloride content in Shenfu Qiangxin Granules by RP-HPLC. This article used acid-water dissolution, analysis on a JADE-PAK ODS-AQ-C18 column (4.6 mm × 250 mm, 5 μm), gradient elution with acetonitrile-0.1% phosphoric acid aqueous solution as the mobile phase, a flow rate of 0.6 mL / min, a column temperature of 35℃, and a detection wavelength of 280 nm. This detection method was used to determine the dopamine hydrochloride content in Shenfu Qiangxin Granules. Since the traditional Chinese medicine preparation of this invention is extracted from 13 kinds of traditional Chinese medicines such as ginseng and white aconite root, the resulting extract is a mixture of many effective components. Detecting only a single indicative effective component as the basis for evaluating the qualification of the traditional Chinese medicine compound is insufficient to effectively control the overall quality of the traditional Chinese medicine preparation of this invention. Summary of the Invention

[0004] This invention provides a fingerprint spectrum detection method for traditional Chinese medicine preparations used to treat heart failure. The detection method includes constructing a characteristic spectrum of the traditional Chinese medicine preparation and a method for determining the content of multiple effective indicator components. This detection method has the advantages of short detection time, comprehensive detection of indicator components, and simple operation, thereby providing a reference for the internal quality control of the traditional Chinese medicine preparation of this invention.

[0005] This invention employs high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) to establish characteristic spectra and to determine the chemical characteristic components of Plantago asiatica, Paeonia lactiflora, Cinnamomum cassia, Salvia miltiorrhiza, Polyporus umbellatus, and Leonurus japonicus in Shenfu Yixin Granules. Three batches of Shenfu Yixin Granules were analyzed, and four indicator components—genipin, paeoniflorin, cinnamaldehyde, and salvianolic acid B—were identified. This detection method features rapid detection speed (65 minutes), comprehensive qualitative detection indicators, and can objectively characterize the quality of Shenfu Yixin Granules of this invention.

[0006] The technical solution of this invention patent application is as follows:

[0007] A fingerprint spectrum detection method for a traditional Chinese medicine preparation for treating heart failure, the detection method comprising the following steps:

[0008] (1) Preparation of reference solution: Weigh genipin, paeoniflorin, cinnamaldehyde and salvianolic acid B reference standards, add 20-50% methanol to prepare the reference solution;

[0009] (2) Preparation of the test solution: Take the traditional Chinese medicine preparation of the present invention, add 20-50% methanol, sonicate to dissolve, dilute to the mark, shake well, filter, and take the filtrate to obtain the test solution;

[0010] (3) Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B. Gradient elution was performed according to the specifications in the table below; the column temperature was 30-40℃; the flow rate was 0.2-0.4 ml per minute; and the detection wavelength was 220-240 nm.

[0011]

[0012] (4) Determination method: Accurately pipette the reference solution and the test solution separately, inject them into the liquid chromatograph, and determine the result.

[0013] Preferably, in step (1) of the detection method, the preparation of the reference solution comprises a mixed reference solution with the following concentrations: genipin acid concentration of 4-8 μg / mL, paeoniflorin concentration of 8-14 μg / mL, cinnamaldehyde concentration of 2-3 μg / mL, and salvianolic acid B concentration of 20-40 μg / mL.

[0014] In a further preferred embodiment, in step (1) of the detection method, the preparation of the reference solution comprises a mixed reference solution with the following concentrations: genipin acid concentration of 6 μg / mL, paeoniflorin concentration of 12 μg / mL, cinnamaldehyde concentration of 2.4 μg / mL, and salvianolic acid B concentration of 30 μg / mL.

[0015] Preferably, in the preparation of the reference solution in step (1) and the test solution in step (2) of the detection method, the concentration of methanol is preferably 30%.

[0016] Preferably, in step (3) of the detection method, C... 18 The chromatographic column has a length of 10 cm, an inner diameter of 2.1 mm, a particle size of 1.7 μm, and a carbon loading of 18%.

[0017] Preferably, in step (3) of the detection method, C... 18 The column model is: ACQUITY UPLC BEH.

[0018] Preferably, in step (3) of the detection method, the chromatographic conditions are: column temperature 35℃, flow rate 0.3 ml / min, and detection wavelength 230 nm. Accurately pipette 2 μl each of the reference solution and the test solution, inject them into the liquid chromatograph, and determine the result.

[0019] In a further preferred embodiment, the fingerprint spectrum of the traditional Chinese medicine preparation of the present invention was determined according to the fingerprint spectrum determination method established in this invention. After comparative analysis, 10 common peaks were obtained. The paeoniflorin peak was selected as the S peak. The relative retention times of each peak were as follows: peak 1: 0.270±0.19%, peak 2: 0.354±0%, peak 3: 0.402±0.06%, peak 4: 0.812±0.09%, peak 5: 1.000%, peak 6: 1.115±0.02%, peak 7: 1.740±0.09%, peak 8: 1.778±0.03%, peak 9: 1.853±0.11%, peak 10: 2.123±0.09%.

[0020] Preferably, four chromatographic peaks were identified and calibrated using standard samples: peak 2: genipin, peak 5: paeoniflorin, peak 8: cinnamaldehyde, and peak 10: salvianolic acid B.

[0021] To further demonstrate the innovativeness of the fingerprinting technology solution of this invention, we have summarized the optimization process of some parameters of the technical solution of this invention as follows:

[0022] 1.1 Confirmation of detection wavelength

[0023] By searching pharmacopoeias and literature, chromatographic collection of most of the indicative components of the 13 medicinal ingredients contained in the preparation can be achieved at a detection wavelength of 203 nm.

[0024] 1.2 Investigation on the preparation of the test solution

[0025] 1.2.1 Method 1: Solid Phase Extraction

[0026] Sample preparation: Weigh 0.40g of Shenfu Yixin Granules-G (T240401), add 10ml of water and shake to dissolve. Let stand for 30min, then centrifuge at 5000r for 5min. Load 5ml onto a solid-phase extraction column (pre-activated with 5ml methanol and 5ml water sequentially). Rinse with 5ml of water and discard the eluent. Elute with 5ml of 70% methanol to a 10ml volumetric flask, dilute to volume with water, shake well, and filter. Column: ACQUITY UPLC BEH C18 1.7um, 2.1×100mm; Chromatographic conditions: column temperature 35°C, sample chamber 25°C, detection wavelength 230nm, injection volume 4ul; gradient elution as shown in the table below; chromatogram as described in the instruction manual. Figure 8 .

[0027]

[0028] Method 2: Extraction with 30% methanol solution

[0029] Weigh 0.40 g of sample powder, add 25 ml of 30% methanol solution, sonicate to aid dissolution, shake well, filter through a membrane, and the product is obtained. Chromatographic column: ACQUITYUPLC BEH C18 1.7 μm, 2.1 × 100 mm;

[0030] Chromatographic conditions: column temperature 35°C, sample chamber temperature 25°C, detection wavelength 230nm, injection volume 4µL, gradient elution as shown in the table below; see the instruction manual for the detection chromatogram. Figure 9 .

[0031]

[0032]

[0033] Experimental Results: Samples were prepared and analyzed using both Method 1 (solid-phase extraction) and Method 2 (30% methanol solution extraction), and the results are shown in the figure. Both methods showed good separation of the formulation components. Method 1 uses a solid-phase extraction column, resulting in higher analytical costs. Method 2 offers simpler sample preparation, more peaks, and larger peak areas. Therefore, Method 2 was chosen as the sample preparation method.

[0034] 2.2 Study on quality transfer and chromatographic peak attribution between processed medicinal materials and finished products

[0035] Chromatographic conditions

[0036] Chromatographic column: ACQUITY UPLC BEH C18 1.7um, 2.1×100mm; Chromatographic conditions: column temperature 35°C, sample chamber 25°C, detection wavelength 230nm, injection volume 2ul, gradient elution as shown in the table below:

[0037]

[0038] 2.3 Optimization of experimental parameters and conditions

[0039] 2.3.1 Optimization of test sample preparation conditions

[0040] This experiment investigated the extraction methods of static extraction, ultrasonic extraction, and solid-phase extraction. Through comparative experiments, it was found that the number of chromatographic peaks in the solutions obtained by the three methods was comparable, with the statically extracted solution showing better peak area and symmetry. Therefore, the static extraction method was selected. The extraction solvents were also investigated: 70% ethanol, 50% methanol, 30% methanol, and water. The results showed that the number of peaks in the extracts was comparable, with 30% methanol exhibiting superior peak shape and good separation. Therefore, 30% methanol was chosen as the extraction solvent.

[0041] 2.3.2 Mobile phase selection and gradient ratio optimization

[0042] This experiment investigated the mobile phase, using acetonitrile-water, acetonitrile-0.05% phosphoric acid solution, acetonitrile-0.1% phosphoric acid solution, and acetonitrile-0.05% formic acid solution as the mobile phase. When the mobile phase was methanol-0.1% phosphoric acid solution, the chromatographic peak shape was good, the resolution between peaks was high, the baseline was relatively stable, and all target compounds were separated with excellent peak shape. To detect more common peaks and achieve high responsiveness, good resolution, and shorter analysis time, the mobile phase ratio was initially high in aqueous phase, as most of the compounds studied in this invention are highly polar. After adjustment and optimization, an initial ratio of 2% A and 98% B resulted in good separation of the chromatographic peaks, and the final ratio was further optimized to 25% A and 75% B. After extensive experimentation and ratio adjustments, the optimal mobile phase ratios were determined to be: 0-5 min, 2%-5% A and 98%-95% B; 5 min-25 min, 5%-18% A and 95%-82% B; 25 min-31 min, 18% A and 82% B; 31 min-38 min, 18%-25% A and 82%-75% B.

[0043] 2.3.3 Basis for Selecting Characteristic Index Components

[0044] This formulation consists of 13 kinds of Chinese medicinal herbs. This method studies the characteristic components of the formulation through quality analysis of the entire prescription. First, using the pharmacopoeia-listed indicators and main active ingredients of each individual herb, and purchasing standard reagents, chromatographic separation conditions were explored to ensure complete chromatographic separation of the main components in the 13 herbs. Based on the proportions of the herbs in the prescription, negative control formulations for each herb, those lacking any herb, and chemical standards were used to determine the chromatographic peak assignments and content composition of each component in the finished product. Genipin, paeoniflorin, cinnamaldehyde, and salvianolic acid B were ultimately identified as characteristic compounds. Subsequent quality analysis of multiple batches of the formulation showed that these four compounds exhibited excellent separation and response values ​​in the characteristic chromatograms, providing an accurate, simple, and reliable analytical method for the quality control of the formulation.

[0045] The beneficial effects of the technical solution of this invention are as follows:

[0046] (1) This invention uses high performance liquid chromatography to establish characteristic chromatograms. Simultaneously, it determines the chemical characteristic components of the herbs *Prunus armeniaca*, *Paeonia lactiflora*, *Cinnamomum cassia*, *Salvia miltiorrhiza*, *Polyporus umbellatus*, and *Leonurus japonicus* in Shenfu Yixin Granules. Three batches of Wenxin Granules were tested. The chromatograms of the three batches showed 10 characteristic peaks, of which 4 peaks should have the same retention time as the corresponding reference peak. The peak corresponding to the paeoniflorin reference peak is the S peak. The relative retention time of each characteristic peak and the S peak should be within ±10% of the specified value. This detection method has the advantages of fast detection speed (detection time less than 60 minutes), comprehensive qualitative detection indicators, and can objectively characterize the intrinsic quality of the traditional Chinese medicine composition of this invention.

[0047] The characteristic peaks constructed in this invention contain 10 chromatographic peaks, of which peaks 1, 7, 9, and 10 are components of Salvia miltiorrhiza, peak 2 is a component of Plantago asiatica, peak 4 is a component of Lepidium apetalum, peak 5 is a component of Paeonia lactiflora, peak 8 is a component of Cinnamomum cassia, peak 3 is a common peak of Salvia miltiorrhiza and Polyporus umbellatus, and peak 6 is a common peak of Paeonia lactiflora and other herbs. Four peaks were identified using standards: peak 2: genipin, peak 5: paeoniflorin, peak 8: cinnamaldehyde, and peak 10: salvianolic acid B. The paeoniflorin peak was selected as the S peak. The relative retention times of each peak were as follows: peak 1: 0.270±0.19%, peak 2: 0.354±0%, peak 3: 0.402±0.06%, peak 4: 0.812±0.09%, peak 5: 1.000%, peak 6: 1.115±0.02%, peak 7: 1.740±0.09%, peak 8: 1.778±0.03%, peak 9: 1.853±0.11%, peak 10: 2.123±0.09%.

[0048] Four chromatographic peaks were identified and calibrated using standard samples: peak 2: genipin, peak 5: paeoniflorin, peak 8: cinnamaldehyde, and peak 10: salvianolic acid B. The present invention selected...

[0049] (2) Experimental results of the fingerprint chromatographic method for Shenfu Yixin Granules constructed in this invention are as follows: ① Precision test results: The RSD of the relative retention time of each characteristic peak is less than 0.27%, and the RSD of the relative peak area of ​​each characteristic peak is less than 2.99%, indicating that the detection method has good precision. ② Reproducibility test results: The RSD of the relative retention time of each characteristic chromatographic peak is less than 0.31%, and the RSD of the relative peak area of ​​each characteristic chromatographic peak is less than 4.08%, indicating that the detection method has good repeatability. ③ Stability test results: The RSD of the relative retention time of each characteristic chromatographic peak is less than 0.46%, and the RSD of the relative peak area of ​​each characteristic chromatographic peak is less than 2.58%, indicating good retention time stability of the analyte within 24 hours. Robustness test results: The maximum RSD% of the relative retention time of each of the 10 characteristic peaks is 1.94%, and the maximum RSD% of the relative peak area is 10.46%. Under chromatographic conditions within the range of 35℃±2℃, the method exhibits better robustness at higher temperatures. The experimental data above show that the method constructed in this invention can be used for internal quality control testing of reference products and is beneficial to improving their intrinsic quality. Attached Figure Description

[0050] Figure 1 -Characteristic chromatogram of Shenfu Yixin Granules (chromatographic peak 2-genipin, chromatographic peak 5-paeoniflorin; chromatographic peak 8-cinnamaldehyde; chromatographic peak 10-tanshinone B);

[0051] Figure 2 -Chromatogram of genipinic acid reference solution;

[0052] Figure 3 -Chromatogram of paeoniflorin reference solution;

[0053] Figure 4 -Chromatogram of cinnamaldehyde reference solution;

[0054] Figure 5 -Chromatogram of salvianolic acid B reference solution;

[0055] Figure 6 -Chromatogram of the reference herb solution of Danshen;

[0056] Figure 7 -Chromatogram of the mixed reference solution;

[0057] Figure 8 -Chromatogram of Shenfu Yixin Granules extracted by solid phase extraction;

[0058] Figure 9-3Spectrum of sample extracted with 0% methanol solution. Detailed Implementation

[0059] Unless otherwise defined, the technical or scientific terms used in the specification and claims of this patent application shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains, the traditional Chinese medicine preparations mentioned in this invention, and SFYX representing Shenfu Yixin Granules.

[0060] Example 1: Detection Method of Characteristic Spectrum of Yixin Granules in this Invention

[0061] 1.1 Instruments and Reagents

[0062] Instruments: Acquity UPLC H-Class ultra-high performance liquid chromatograph (Waters); ML204T / 02 electronic balance (Mettler AG, Switzerland); Reference standards: Genipin, National Institutes for Food and Drug Control (NIFDC), batch number 111828-201805, content calculated as 98.1%; Paeoniflorin, NIFDC, batch number 110736-202246, content calculated as 96.7%; Cinnamaldehyde, NIFDC, batch number 111562-202318, content calculated as 98.8%; Tanshinone B, NIFDC, batch number 111562-202318, content calculated as 97.5%. Samples: Details of the 3 batches of samples are shown in Table 1.

[0063] Table 1. Sample Information Table of Yixin Granules (attached)

[0064]

[0065] 1.2 Method for determining the characteristic spectrum of Yixin Granules in this invention

[0066] 1.2.1 Preparation of the test solution: The test solution is prepared as follows:

[0067] Take an appropriate amount of Shenfu Yixin granules, grind them into a fine powder, accurately weigh 0.40g, place them in a 25ml volumetric flask, add an appropriate amount of 30% methanol solution, sonicate to aid dissolution, then dilute to volume with 30% methanol, and shake to ensure complete dissolution. Accurately measure 2ml of the solution into a 10ml volumetric flask, add 30% methanol solution to dilute to the mark, shake well, and filter through a 0.22μm filter membrane to obtain the final product.

[0068] 1.2.2 Preparation of Specificity Test Solutions

[0069] 1.2.2.1 Preparation of negative sample solution

[0070] Weigh out 0.32g of 12 negative-labeled dry extract powders such as ginseng, and 0.40g of negative-labeled cinnamon twig sample (including excipients), and prepare them in the same way as the test solution.

[0071] 1.2.2.2 Preparation of positive sample solution

[0072] Weigh 10g of each of the 13 medicinal herbs, add 300ml of water, and decoct for 40 minutes (water can be added during decoction). Filter, cool, and bring the volume to 300ml. Transfer 1.0ml of ginseng solution, 1.7ml each of cardamom and aconite root solution, 2.0ml each of cinnamon twig and jujube solution, 5ml each of salvia miltiorrhiza and motherwort solution, and 2.5ml each of the other 6 herbal solutions. Add 30% methanol solution to each and bring the volume to 10ml. Filter through a membrane to obtain the final product.

[0073] Sample injection analysis and experimental results

[0074] Ginseng: The results of the Shenfu Yixin Granules and the negative ginseng sample were consistent, while no chromatographic peaks were observed in the positive ginseng sample.

[0075] Bai Fu Pian: The negative samples of Shen Fu Yi Xin Granules and Bai Fu Pian were consistent, while no chromatographic peaks appeared in the positive samples of Bai Fu Pian.

[0076] Cinnamon twig: There was a difference between the negative samples of ginseng and aconite-containing granules and cinnamon twig, while the positive sample of cinnamon twig had one chromatographic peak (retention time 29.4 min);

[0077] Amomum villosum: The results of the negative sample of Amomum villosum and Ginseng and Aconite granules were consistent (no volatile oil was obtained from cinnamon twig alone, so there was no inclusion compound peak at 29.5 min in the negative chromatogram), while no chromatographic peak appeared in the positive sample of Amomum villosum.

[0078] Danshen: There are differences between Shenfu Yixin Granules, Danshen negative samples and Danshen positive samples. Danshen positive samples have 7 chromatographic peaks (4.5 min, 27.8 min, 28.5 min, 30.2 min, 34.7 min, 35.2 min, 37.8 min);

[0079] Red peony root: There is a difference between the negative results of ginseng and aconite-containing granules and red peony root. The positive results of red peony root have two chromatographic peaks (16.5 min, 18.5 min);

[0080] Plantain seed: There is a difference between the Shenfu Yixin granule preparation and the negative sample of plantain seed. The positive sample of plantain seed has one chromatographic peak (5.9 min).

[0081] Lepidium seed: There is a difference between the Shenfu Yixin granule preparation and the negative sample of Lepidium seed. The positive sample of Lepidium seed has one chromatographic peak (13.3 min);

[0082] Motherwort: There was a difference between the negative sample of Ginseng and Aconite Heart-Nourishing Granules and the negative sample of Motherwort. The positive sample of Motherwort had 3 peaks (11.8 min, 17.2 min, 18.39 min).

[0083] Zhu Ling: The negative sample of Shenfu Yixin Granules was consistent with the negative sample of Zhu Ling, while the positive sample of Zhu Ling showed no chromatographic peak.

[0084] Areca peel: The negative sample of Shenfu Yixin Granules and Areca peel were consistent (Note: The negative sample was prepared by the laboratory and was obtained by direct decoction without the addition of inclusion complex, so there was no inclusion complex peak at 29.5 min in the negative chromatogram). No chromatographic peak appeared in the positive sample of Areca peel.

[0085] Alisma plantago-aquatica: The negative sample of Shenfu Yixin Granules was consistent with that of Alisma plantago-aquatica, while no chromatographic peak appeared in the positive sample of Alisma plantago-aquatica.

[0086] Jujube: The results of the Shenfu Yixin Granules preparation and the negative results were consistent, while no chromatographic peaks appeared in the positive jujube samples.

[0087] 1.2.3 Preparation of reference solution:

[0088] Reference stock solution 1: Accurately weigh 1.084 mg of genipinic acid reference standard, place it in a 10 ml brown volumetric flask, add chromatographic methanol and sonicate to dissolve, then dilute to the mark, shake well, and store frozen.

[0089] Reference stock solution 2: Accurately weigh 2.788 mg of paeoniflorin reference standard, place it in a 10 ml brown volumetric flask, add chromatographic methanol and sonicate to dissolve, then dilute to the mark, shake well, and store frozen.

[0090] Reference stock solution 3: Accurately weigh 4.852 mg of salvianolic acid B reference standard, place it in a 10 ml brown volumetric flask, add chromatographic methanol and sonicate to dissolve, then dilute to the mark, shake well, and store frozen.

[0091] Reference stock solution 4: Accurately weigh 0.0243 g of cinnamaldehyde reference standard, place it in a 10 ml brown volumetric flask, dilute to the mark with methanol, and shake well; measure 1 ml of this solution into a 10 ml brown volumetric flask, dilute to the mark with methanol, shake well, and store frozen.

[0092] Genipin glycoside reference solution: Take 2 ml of reference stock solution 1, dilute to 10 ml with 30% methanol, and shake well.

[0093] Paeoniflorin reference solution: Take 1 ml of reference stock solution 2, dilute it to 10 ml with 30% methanol, and shake well.

[0094] Cinnamaldehyde reference solution: Take 1 ml of the reference stock solution and dilute it to 20 ml with 30% methanol. Shake well to obtain the solution.

[0095] Tanshinone B reference solution: Take 1 ml of reference stock solution 1, dilute to 10 ml with 30% methanol, and shake well.

[0096] Mixed reference solution: Take 1.5 ml each of reference stock solution 1, reference stock solution 2, and reference stock solution 3, and 0.3 ml of reference stock solution 4, and place them in the same volumetric flask. Dilute to 5 ml with water and shake well to obtain the solution.

[0097] Salvia miltiorrhiza reference material solution: Weigh 0.3g of reference material, add 30ml of 30% methanol, sonicate for 40min, take 5ml of supernatant, dilute with 30% methanol solution to 10ml, filter through membrane, and the solution is obtained.

[0098] 1.2.4 Chromatographic conditions and system suitability test

[0099] Octadecylsilane-bonded silica gel was used as the packing material; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the specifications in Table 2; the column temperature was 35℃; the flow rate was 0.3 ml per minute; and the detection wavelength was 230 nm.

[0100] Table 2 Gradient Elution Mobile Phase Ratio

[0101]

[0102] 1.2.5 Sample Solution Injection Analysis

[0103] Genipin glycoside reference solution, paeoniflorin reference solution, cinnamaldehyde reference solution, salvianolic acid B reference solution, mixed reference solution, tanshinone reference medicinal material solution and test solution were injected for analysis.

[0104] 1.2.6 Results Analysis

[0105] The chromatographic results are shown in the figure: For example, genipinic acid reference solution (see instruction manual appendix). Figure 2 ), paeoniflorin reference solution (see instructions attached) Figure 3 Cinnamaldehyde reference solution (see instructions) Figure 4 ), Danshensu B reference solution (see instructions attached) Figure 5 ), Danshen reference herb solution (see instructions attached) Figure 6 ) and mixed reference solution (see instructions attached) Figure 7 The corresponding chromatographic peaks have the same retention time and consistent spectral absorption. Therefore, it can be concluded that the present invention has achieved component separation and standard peak comparison study of genipin, paeoniflorin, cinnamaldehyde and tanshinone B in the characteristic spectrum of Shenfu Yixin Granules by UPLC-UV method, and finally identified 10 characteristic fingerprint peaks in Shenfu Yixin Granules. The specific characteristic peak information is shown in Table 3 below.

[0106] Table 3. Characteristic peak information in the chromatogram of Shenfu Yixin Granules

[0107]

[0108] 1.2.7 Selection of Judgment Method

[0109] The characteristic chromatograms of traditional Chinese medicine can generally be evaluated by retention time, relative retention time / relative peak area, etc. In this method, the four characteristic peaks are known components. Based on the results of the column and instrument robustness test, the characteristic peak retention time method is selected for evaluation. The relevant chromatographic peaks of the test sample characteristic chromatogram should have the same retention time as the corresponding reference peak.

[0110] 1.2.8 Establishment of Feature Map

[0111] According to the optimized chromatographic conditions of this invention, the chromatogram should exhibit four characteristic peaks corresponding to the reference standard chromatogram, and the relative retention times of the remaining six characteristic peaks and the S peak (paeoniflorin) should be within ±10% of the specified value.

[0112] 1.2.9 Determination and Evaluation of Characteristic Peaks

[0113] The relative retention time and relative peak area of ​​multiple batches of samples, such as T240302, T240401, M240601, etc., were calculated to determine the characteristic peak parameters of SFYX particles.

[0114] Table 4. Relative retention times of characteristic peaks in three batches of Shenfu Yixin Granules

[0115]

[0116]

[0117] Table 5. Relative peak areas of characteristic peaks in multiple batches of Shenfu Yixin Granules

[0118]

[0119] As shown in Tables 4 and 5, the fingerprint chromatogram of the Shenfu Yixin Granules of this invention contains 10 chromatographic peaks with relative retention times of: Peak 1: 0.270±0.19%, Peak 2: 0.354±0%, Peak 3: 0.402±0.06%, Peak 4: 0.812±0.09%, Peak 5: 1.000%, Peak 6: 1.115±0.02%, Peak 7: 1.740±0.09%, Peak 8: 1.778±0.03%, Peak 9: 1.853±0.11%, and Peak 10: 2.123±0.09%. The relative peak areas were as follows: peak 1: 1.506±2.54%, peak 2: 0.718±6.69%, peak 3: 0.775±3.81%, peak 4: 0.324±11.88%, peak 5: 1.000%, peak 6: 1.589±0.42%, peak 7: 0.489±4.09%, peak 8: 0.679±1.81%, peak 9: 0.411±1.46%, and peak 10: 3.771±1.63%.

[0120] 1.2.9 Construction of SFYX Particle Feature Map

[0121] Select any SFYX particle separation spectrum as a characteristic spectrum, choose the paeoniflorin peak as the S peak, and calculate the relative retention time and relative peak area for multiple batches of samples, including T240302, T240401, M240601, etc. Construct characteristic spectra using the median of the relative retention time and relative peak area, as shown in the appendix to the instruction manual. Figure 1 .

[0122] After identification and authentication, the fingerprint chromatogram of Shenfu Yixin Granules established in this invention contains 10 characteristic peaks. Among them, peaks 1, 7, 9, and 10 are components of Danshen (Salvia miltiorrhiza), peak 2 is a component of Plantago asiatica (Plantago asiatica), peak 4 is a component of Lepidium apetalum (Lepidium apetalum), peak 5 is a component of Paeonia lactiflora (Paeonia veitchii), peak 8 is a component of Cinnamomum cassia (Cinnamomum cassia), peak 3 is a common peak of Danshen and Polyporus umbellatus, and peak 6 is a common peak of Paeonia lactiflora and other components. Four peaks were identified using standards: peak 2: genipinic acid, peak 5: paeoniflorin, peak 8: cinnamaldehyde, and peak 10: salvianolic acid B. The paeoniflorin peak was selected as the S peak. The relative retention times of each peak were as follows: peak 1: 0.270±0.19%, peak 2: 0.354±0%, peak 3: 0.402±0.06%, peak 4: 0.812±0.09%, peak 5: 1.000%, peak 6: 1.115±0.02%, peak 7: 1.740±0.09%, peak 8: 1.778±0.03%, peak 9: 1.853±0.11%, and peak 10: 2.123±0.09%.

[0123] Example 2: Method for detecting the content of Shenfu Yixin Granules in this invention

[0124] 2.1 The specific steps of the method for detecting the content of Shenfu Yixin Granules in this invention are as follows:

[0125] (1) Preparation of reference solution: Weigh genipin, paeoniflorin, cinnamaldehyde and salvianolic acid B reference standards, add 30% methanol to prepare a mixed reference solution, wherein the acid concentration of genipin is 6 μg / mL, the concentration of paeoniflorin is 12 μg / mL, the concentration of cinnamaldehyde is 2.4 μg / mL and the concentration of salvianolic acid B is 30 μg / mL.

[0126] (2) Preparation of the test solution: Take an appropriate amount of Shenfu Yixin granules, grind them finely, accurately weigh 0.40 g, place them in a 25 ml volumetric flask, add an appropriate amount of 30% methanol solution, sonicate to aid dissolution, then dilute to volume with 30% methanol, and shake to ensure complete dissolution. Accurately measure 2 ml of the solution into a 10 ml volumetric flask, add 30% methanol solution to dilute to the mark, shake well, and filter through a 0.22 μm filter membrane to obtain the test solution.

[0127] (3) For chromatographic conditions and system suitability, please refer to section "1.2.4" in Example 1;

[0128] (4) Determination method: Accurately pipette the reference solution and the test solution separately, inject them into the liquid chromatograph, and determine the result.

[0129] 2.2 Methodological Validation

[0130] 2.2.1 Specificity Test

[0131] The specific steps are detailed in "1.2.2" of Example 1. The experiment shows that there is no interference from negative samples and the method has good specificity.

[0132] 2.2.2 System adaptability test

[0133] 2.2.2.1 Preparation of the reference solution:

[0134] Take appropriate amounts of geniposide reference standard, paeoniflorin reference standard, cinnamaldehyde reference standard and salvianolic acid B reference standard, accurately weigh them, and add 30% methanol to prepare solutions containing 6 μg of geniposide, 12 μg of paeoniflorin, 2.4 μg of cinnamaldehyde and 30 μg of salvianolic acid B per ml.

[0135] 2.2.2.2 Preparation of the test solution

[0136] Take an appropriate amount of Shenfu Yixin granules, grind them into a fine powder, accurately weigh 0.40g, place them in a 25ml volumetric flask, add an appropriate amount of 30% methanol solution, sonicate to aid dissolution, then dilute to volume with 30% methanol, and shake to ensure complete dissolution. Accurately measure 2ml of the solution into a 10ml volumetric flask, add 30% methanol solution to dilute to the mark, shake well, and filter through a 0.22μm filter membrane to obtain the final product.

[0137] Determination method: Determine according to high performance liquid chromatography (General Rule 0512). Inject 2 μl of each solution in the injection program into the liquid chromatograph and record the chromatogram.

[0138] Results: No chromatographic peaks were observed in the blank solvent at the retention times of the reference solution and the corresponding retention times of the test solution, indicating that the blank solvent did not interfere with the detection of the reference solution and the test solution. The paeoniflorin plate number in the reference solution was 16495, and the resolution of the cinnamaldehyde peak in the test solution was 1.98, both meeting the requirements.

[0139] 2.2.3 Precision Test

[0140] Prepare the test solution according to the test solution preparation method in section 2.2.2.2.

[0141] Determination method: Inject the same test solution six times consecutively, determine according to the chromatographic method, and record the chromatogram.

[0142] Table 6. Relative Retention Time of Characteristic Peaks - Precision

[0143]

[0144] Table 7 Relative Peak Area of ​​Characteristic Peaks - Precision

[0145]

[0146]

[0147] The experimental results in Tables 6 and 7 show that the RSD of the relative retention time of each characteristic peak is less than 0.27%, and the RSD of the relative peak area of ​​each characteristic peak is less than 2.99%, which indicates that the detection method has good precision.

[0148] 2.2.4 Repeatability Test

[0149] Prepare six test solutions in parallel according to the test solution preparation method described in section 2.2.2.2. Assay: Determine the chromatographic value using the chromatographic method and record the chromatogram.

[0150] Table 8. Relative retention time of characteristic peaks - repeatability

[0151]

[0152] Table 9. Relative Peak Area of ​​Characteristic Peaks - Repeatability

[0153]

[0154]

[0155] The experimental results in Tables 8 and 9 show that the RSD of the relative retention time of each characteristic chromatographic peak is less than 0.31%, and the RSD of the relative peak area of ​​each characteristic chromatographic peak is less than 4.08%, indicating that the detection method has good repeatability.

[0156] 2.2.5 Stability Test

[0157] Take the test solution under the repeatability test and inject it at 0, 2, 4, 6, 8, 10, 12 and 24 hours respectively. Use the relative retention time and relative peak area RSD of each peak as indicators to observe the stability of the test component in the test solution during the detection process.

[0158] Table 10 Relative retention time of characteristic peaks - solution stability

[0159]

[0160]

[0161] Table 11 Relative Peak Area of ​​Characteristic Peaks - Solution Stability

[0162]

[0163] The experimental results in Tables 10 and 11 show that the RSD of the relative retention time of each characteristic chromatographic peak is less than 0.46%, and the RSD of the relative peak area of ​​each characteristic chromatographic peak is less than 2.58%. The retention time stability of the analyte is good within 24 hours, which can meet the determination requirements.

[0164] 2.2.6 Durability Test

[0165] Keeping the basic chromatographic conditions unchanged, change the column temperature and compare the relative retention times and relative peak areas of the 10 chromatographic peaks of the test solution.

[0166] Table 12 Relative Retention Time of Characteristic Peaks - Column Temperature Study

[0167]

[0168] Table 13. Characteristic Peak Relative Peak Area - Column Temperature Study

[0169]

[0170]

[0171] As shown in Tables 12 and 13, the maximum RSD% of the relative retention time of the 10 characteristic peaks is 1.94%, and the maximum RSD% of the relative peak area is 10.46%. The method has better robustness when the chromatographic conditions are within the range of 35℃±2℃ and the temperature is higher.

[0172] 3. Summary

[0173] Characteristic chromatograms were established using high performance liquid chromatography. The chemical components of Plantago asiatica, Paeonia lactiflora, Cinnamomum cassia, Salvia miltiorrhiza, Polyporus umbellatus, and Leonurus japonicus in Shenfu Yixin Granules were determined. Three batches of Shenfu Yixin Granules were tested, and the results showed that the chromatograms of the three batches of samples all showed characteristic peaks corresponding to the chromatograms of the reference standard.

Claims

1. A method for detecting the fingerprint of a traditional Chinese medicine preparation for treating heart failure, characterized in that, The aforementioned traditional Chinese medicine preparation for heart failure is Shenfu Yixin Granules, which is prepared by extracting ginseng, white aconite root, cinnamon twig, salvia miltiorrhiza, red peony root, motherwort, alisma rhizome, polyporus umbellatus, plantain seed, lepidium seed, amomum villosum, areca peel, and jujube. The detection method includes the following steps: (1) Preparation of reference solution: Weigh genipin, paeoniflorin, cinnamaldehyde and salvianolic acid B reference standards, add 20-50% methanol to prepare the reference solution; (2) Preparation of test solution: Take Shenfu Yixin granules, add 20-50% methanol, sonicate to dissolve, dilute to the mark, shake well, filter, and take the filtrate to obtain the test solution; (3) Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B. Gradient elution was performed according to the specifications in the table below; the column temperature was 30-40℃; the flow rate was 0.2-0.4 ml per minute; and the detection wavelength was 220-240 nm. (4) Determination method: Accurately pipette the reference solution and the test solution separately, inject them into the liquid chromatograph, and determine the result.

2. The method of claim 1, wherein the fingerprinting comprises: In step (1) of the detection method, the preparation of the reference solution comprises a mixed reference solution with the following concentrations: geniposide 4-8 μg / mL, paeoniflorin 8-14 μg / mL, cinnamaldehyde 2-3 μg / mL, and salvianolic acid B 20-40 μg / mL.

3. The method of claim 1, wherein the fingerprinting comprises: In step (1) of the detection method, the preparation of the reference solution comprises a mixed reference solution with the following concentrations: geniposide 6 μg / mL, paeoniflorin 12 μg / mL, cinnamaldehyde 2.4 μg / mL, and salvianolic acid B 30 μg / mL.

4. The method of claim 1, wherein the fingerprinting comprises: In the preparation of the reference solution (1) and the test solution (2) of the detection method, the concentration of methanol is 30%.

5. The method of claim 1, wherein the fingerprinting comprises: The detection method step 6 chromatographic conditions C 18 The column length of the chromatographic column is 10 cm, the inner diameter is 2.1 mm, the particle size is 1.7 um, and the carbon loading is 18%.

6. The method of claim 1, wherein the fingerprinting comprises: The detection method step 6 chromatographic conditions C 18 The model of the chromatographic column is: ACQUITY UPLC BEH.

7. The fingerprint pattern detection method as described in claim 1, characterized in that, In step (3) of the detection method, the chromatographic conditions are: column temperature 35℃, flow rate 0.3 ml / min, and detection wavelength 230 nm.

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