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

Abstract

The invention provides a fingerprint detection method for a traditional Chinese medicine preparation for treating heart failure, and a method for determining chemical characteristic index components in medicinal materials including semen plantaginis, radix paeoniae rubra, cassia twig, radix salviae miltiorrhizae, polyporus umbellatus and herba leonuri in Shenfu heart-tonifying granules. Three batches of the Shenfu heart-benefiting granules are determined, and four index components including geniposidic acid, paeoniflorin, cinnamaldehyde and salvianolic acid B. The detection method has the advantages that the detection speed is high, the relative components of qualitative detection indexes are relatively comprehensive, and the inherent quality of the Shenfu heart-benefiting granules can be objectively represented.

Description

Technical Field

[0001] The invention belongs to the field of quality detection of effective ingredients of traditional Chinese medicines, and in particular to a fingerprint spectrum detection method for traditional Chinese medicine preparations for treating heart failure. Background Art

[0002] The Chinese medicine preparation of the present invention is Shenfu Yixin Granule, which is the core variety of Shandong Buchang Pharmaceutical Co., Ltd. It is extracted and processed from Chinese medicinal materials including ginseng, white aconite root, cinnamon twig, salvia miltiorrhiza, red peony root, motherwort, oriental rhizome, umbellate, plantain seed, scutellaria baicalensis, amomum villosum, tangerine peel and jujube. It has the effects of warming yang and invigorating 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 obstruction. Symptoms include palpitations, asthma, cold body and cold limbs, abdominal distension, lower limb edema, oliguria, dark complexion, cyanosis of lips, angulation of neck pulse, lumps under hypochondria, pale tongue or dark blood stasis, white or greasy tongue coating, deep and thin pulse, and irregular or rapid pulse.

[0003] The applicant has previously applied for CN101199812A (detection method) for this product, with the publication date of 2008-06-18. The patent application for the detection method records: the quality control method of the Chinese medicine granules includes: thin layer identification of salvia miltiorrhiza, red peony root, motherwort, scutellaria baicalensis, jujube, amomum villosum, white aconite root, and cassia twig, limit inspection of aconitine, and determination of ginseng content. Take ginsenoside Rg1 reference substance and add methanol to prepare 1 ml of solution containing 1 mg as reference substance solution; according to the thin layer chromatography test in the "Chinese Pharmacopoeia", use the upper layer solution of n-butanol-ethyl acetate-water = 4:1:5 as the developing solvent, develop at below 10°C, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 100°C until the spots are clearly colored, cover the thin layer plate with a glass plate of the same size, fix with adhesive tape, scan by thin layer scanning method, wavelength λS520nm, λR690nm, measure the absorbance integral value of the test sample and the absorbance integral value of the reference substance, the present invention contains ginsenoside Rg1 with a molecular formula of C 42 H 72 O 14 The content of dopamine hydrochloride in Shenfu Qiangxin Granules shall not be less than 0.028%. Zhang Chi, Wu Jianguo, Xu Huiyan, etc., RP-HPLC determination of dopamine hydrochloride in Shenfu Qiangxin Granules, the article adopts acid water dissolution, JADE-PAK ODS-AQ-C18 chromatographic column (4.6mm×250mm, 5μm) analysis, acetonitrile-0.1% phosphoric acid aqueous solution as mobile phase gradient elution, flow rate of 0.6mL / min, column temperature of 35℃, detection wavelength of 280nm. This detection method is used for the content of dopamine hydrochloride in Shenfu Qiangxin Granules. Since the Chinese medicine preparation of the present invention is extracted from 13 Chinese medicines such as ginseng and white aconite, the obtained Chinese medicine extract is a mixture of many effective ingredients. It is difficult to effectively control the overall quality of the Chinese medicine preparation of the present invention by only detecting a single indicator effective ingredient as the basis for evaluating the qualification of the Chinese medicine compound. Summary of the invention

[0004] The present invention provides a fingerprint spectrum detection method for Chinese medicine preparations for treating heart failure. The detection method comprises constructing a characteristic spectrum of the Chinese medicine preparation of the present invention and a method for determining the content of multi-index effective index components. The detection method has the advantages of short detection time, comprehensive detection index components, and simple operation, thereby enabling the intrinsic quality control of the Chinese medicine preparation of the present invention and providing a reference basis.

[0005] The present invention adopts a characteristic spectrum established by high performance liquid chromatography-mass spectrometry, and determines the chemical characteristic index components in plantago, red peony root, cinnamon twig, salvia miltiorrhiza, umbellatum, and motherwort in Shenfu Yixin granules. Three batches of Shenfu Yixin granules are measured, and four index components, namely, geniposide, paeoniflorin, cinnamaldehyde, and salvianolic acid B, are identified. The detection method has fast detection speed (detection time 65 minutes), and the relative components of qualitative detection indicators are relatively comprehensive, and can objectively characterize the quality of the Shenfu Yixin granules of the present invention.

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

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

[0008] ⑴. Preparation of reference solution: weigh geniposide, paeoniflorin, cinnamaldehyde and salvianolic acid B reference substances, add 20-50% methanol to prepare a mixed reference solution;

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

[0010] ⑶、Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler; acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; column temperature was 30-40℃; flow rate was 0.2-0.4ml per minute; detection wavelength was 220-240nm;

[0011]

[0012] (4) Determination method: Accurately aspirate the reference solution and the test solution respectively, inject into the liquid chromatograph, and determine.

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

[0014] Further preferably, in the preparation of the reference solution in step (1) of the detection method, the mixed reference solution comprises: a solution having an acid concentration of 6 μg / mL of geniposide, a concentration of 12 μg / mL of paeoniflorin, a concentration of 2.4 μg / mL of cinnamaldehyde, and a concentration of 30 μg / mL of salvianolic acid B.

[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 the chromatographic conditions of step (3) of the detection method, 18 The column length is 10 cm, the inner diameter is 2.1 mm, the particle size is 1.7 um, and the carbon loading is 18%.

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

[0018] Preferably, in the chromatographic conditions of step (3) of the detection method, the column temperature is 35°C, the flow rate is 0.3 ml per minute, and the detection wavelength is 230 nm. Accurately pipette 2 μl of the reference solution and the test solution, inject them into the liquid chromatograph, and measure to obtain the result.

[0019] Further preference, according to the fingerprint determination method established by the present invention, the fingerprint of the traditional Chinese medicine preparation of the present invention is determined, and 10 common peaks are obtained through comparison and analysis, and the paeoniflorin peak is selected as the S peak. The relative retention times of each peak are, 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 are identified and calibrated using standard substances, namely, chromatographic peak 2: geniposide acid, chromatographic peak 5: paeoniflorin, chromatographic peak 8: cinnamaldehyde, and chromatographic peak 10: salvianolic acid B.

[0021] In order to further reflect the innovativeness of the fingerprint spectrum technical solution of the present invention, we organize the process of optimizing some parameters of the technical solution of the present invention as follows:

[0022] 1.1 Confirmation of detection wavelength

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

[0024] 1.2 Preparation of test solution

[0025] 1.2.1 Method 1: Solid Phase Extraction

[0026] Sample preparation: Weigh 0.40g of Shenfu Yixin Granule-G (T240401), add 10ml of water and shake to dissolve, let stand for 30min, and centrifuge at 5000r for 5min. Take 5ml and load it on a solid phase extraction column (previously activated with 5ml of methanol and 5ml of water). Rinse with 5ml of water and discard the eluent. Elute with 5ml of 70% methanol into a 10ml volumetric flask, make up to volume with water, shake well, filter the membrane, and you're done. Chromatographic column: ACQUITY UPLC BEH C181.7um, 2.1×100mm, chromatographic conditions: column temperature 35 degrees, sample chamber 25 degrees, detection wavelength 230nm, injection volume 4ul, gradient elution as shown in the following table, detection chromatogram see the instruction manual Figure 8 .

[0027]

[0028] Method 2: 30% methanol solution extraction

[0029] Weigh 0.40g of sample powder, add 25ml of 30% methanol solution, dissolve with ultrasonic aid, shake well, filter membrane, and obtain. Chromatographic column: ACQUITYUPLC BEH C181.7um, 2.1×100mm;

[0030] Chromatographic conditions: column temperature 35 degrees, sample chamber 25 degrees, detection wavelength 230nm, injection volume 4ul, gradient elution as shown in the following table: Detection chromatogram see the instructions Fig. 9 .

[0031]

[0032]

[0033] Experimental results: Method 1 solid phase extraction and method 2 30% methanol solution extraction were used for sample preparation and analysis, and the results are shown in the figure. Both methods are good for separating the components of the preparation. Method 1 uses a solid phase extraction column, and the analysis cost is relatively high. Method 2 is simple to prepare samples, with a large number of peaks and a large peak area. Therefore, method 2 is used as the sample preparation method.

[0034] 2.2 Study on mass transfer and chromatographic peak attribution of decoction pieces-preparations

[0035] Chromatographic conditions

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

[0037]

[0038] 2.3 Optimization of experimental parameters

[0039] 2.3.1. Optimization of test sample preparation conditions

[0040] This experiment investigated the methods of static, ultrasonic and solid phase extraction. Through experimental comparison, it was found that the number of chromatographic peaks of the chromatograms of the solutions obtained by the three preparation methods was equivalent, and the peak area and peak symmetry of the static solution were better, so the static extraction method was selected. At the same time, the extraction solvents were investigated, and 70% ethanol, 50% methanol, 30% methanol, and water were investigated respectively. The experimental results showed that the number of peaks of the extract was equivalent, and the peak shape of 30% methanol was better, and the separation was good, so 30% methanol was selected as the extraction solvent.

[0041] 2.3.2 Mobile phase selection and gradient ratio optimization

[0042] This experiment investigated the mobile phase, and selected 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 is methanol-0.1% phosphoric acid water, the chromatographic peak shape is better, the separation between the chromatographic peaks is large, the baseline is relatively stable, and the target compounds are separated, and the peak shape is better. In order to be able to detect more common peaks, and to achieve high responsiveness, good separation and shorten the analysis time. Most of the research objects of the present invention are compounds with high polarity, so the mobile phase ratio is initially a high water phase. After adjustment and optimization, when the initial ratio is 2% A and 98% B, the chromatographic peaks are well separated, and the final optimization is 25% A and 75% B. After a lot of experimental exploration and proportion adjustment, the optimal mobile phase ratio was determined to be 0-5min, 2%-5% A and 98%-95% B; 5min-25min, 5%-18% A and 95%-82% B; 25min-31min, 18% A and 82% B; 31min-38min, 18%-25% A and 82%-75% B.

[0043] 2.3.3. Selection basis of characteristic index components

[0044] The prescription of this preparation is composed of 13 kinds of Chinese medicinal materials. This method studies the characteristic components of the preparation through the quality analysis of the full-flavor prescription. First, the pharmacopoeia-contained index components and main active ingredients of each single medicinal material are used to purchase the inspection standard to explore the chromatographic separation conditions to ensure that the main components of the 13 medicinal materials can be completely separated by chromatography. Combined with the proportion of Chinese medicinal materials in the prescription, the chromatographic peak attribution and content composition of each ingredient in the finished preparation are determined using each single medicinal material, negative preparations lacking each single medicinal material, and chemical standards. Finally, genipine glycine, paeoniflorin, cinnamaldehyde, and salvianolic acid B were determined as characteristic compounds. Later, through the quality analysis of multiple batches of preparations, the separation and response values ​​of the four compounds in the characteristic spectrum were very good, providing an accurate, simple, and reliable analysis method for the quality control of the preparation.

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

[0046] ⑴. The present invention adopts a characteristic spectrum established by high performance liquid chromatography, and simultaneously determines the chemical characteristic index components in the medicinal materials of Qianzi, Red Peony Root, Cinnamon Twig, Salvia miltiorrhiza, Polyporus, and Leonurus japonicus in Shenfu Yixin Granules. The determination was performed on three batches of Wenxin Granules. As a result, 10 characteristic peaks were presented in the chromatograms of the three batches of samples, of which four peaks should have the same retention time as the corresponding reference peaks; the peak corresponding to the paeoniflorin reference peak is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated to be within ±10% of the specified value. The detection method has the advantages of fast detection speed (detection time is less than 60 minutes), relatively comprehensive relative components of qualitative detection indicators, and can objectively characterize the intrinsic quality of the Chinese medicine composition of the present invention.

[0047] The characteristic peaks constructed by the present invention contain 10 chromatographic peaks, wherein peaks 1, 7, 9 and 10 are medicinal components of salvia miltiorrhiza, peak 2 is medicinal components of plantago asiatica, peak 4 is medicinal components of scutellaria baicalensis, peak 5 is medicinal components of red peony root, peak 8 is medicinal components of cassia twig, peak 3 is a common peak of salvia miltiorrhiza and umbellatus, and peak 6 is a common peak of multiple medicinal components such as red peony root. Four peaks were calibrated with standard products, namely peak 2: geniposide acid, peak 5: paeoniflorin, peak 8: cinnamaldehyde, and peak 10: salvianolic acid B. The paeoniflorin peak was selected as the S peak, and the relative retention times of each peak were: 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%.

[0048] Four chromatographic peaks were identified and calibrated using standard products, namely, chromatographic peak 2: geniposide acid, chromatographic peak 5: paeoniflorin, chromatographic peak 8: cinnamaldehyde, and chromatographic peak 10: salvianolic acid B.

[0049] ⑵, the results of the methodological experiment of the fingerprint spectrum of Shenfu Yixin Granule constructed by the present invention show that: ①, precision experiment 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%, which indicates that the detection method has good precision. ②, reproducibility experiment 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%, which indicates that the detection method has good repeatability. ③, stability experiment 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%, and the retention time stability of the measured component within 24 hours is good. Durability experiment results: the maximum value of the relative retention time RSD% of each peak of the 10 characteristic peaks is 1.94%, and the maximum value of the relative peak area RSD% is 10.46%. The chromatographic conditions are within the range of 35℃±2℃, and the temperature is high, and the method has good durability. The above experimental data show that the method constructed by the present invention can be used for internal quality control testing of ginseng and aconite products and is beneficial to improving their intrinsic quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 -Characteristic spectrum of Shenfu Yixin Granule, (chromatographic peak 2-geniposidic acid, chromatographic peak 5-peonidin; chromatographic peak, chromatographic peak 8-cinnamaldehyde; chromatographic peak 10-salvianolic acid B);

[0051] Figure 2 - Chromatogram of geniposide 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 Danshen reference medicinal material solution;

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

[0057] Figure 8 -Solid phase extraction chromatogram of Shenfu Yixin granules;

[0058] Figure 9-3Chromatogram of sample extracted with 0% methanol solution. DETAILED DESCRIPTION

[0059] Unless otherwise defined, the technical terms or scientific terms used in the patent application specification and claims of the present invention shall have the common meanings understood by persons with ordinary skills in the field to which the present invention belongs. The Chinese medicine preparations mentioned in the content of the present invention, and SFYX represent Shenfu Yixin Granules.

[0060] Example 1 Characteristic Spectrum Detection Method of Shenfu Yixin Granules of the Present Invention

[0061] 1.1 Instruments and reagents

[0062] Instrument: Acquity UPLC H-Class ultra-high performance liquid chromatograph (Waters); ML204T / 02 electronic balance (Mettler, Switzerland); Reference substances: Geniposide acid, China National Institute for Food and Drug Control, batch number 111828-201805, content is 98.1%; Paeoniflorin, China National Institute for Food and Drug Control, batch number 110736-202246, content is 96.7%; Cinnamaldehyde, China National Institute for Food and Drug Control, batch number 111562-202318, content is 98.8%; Salvianolic acid B, China National Institute for Food and Drug Control, batch number 111562-202318, content is 97.5%. Samples: The information of the three batches of samples is shown in Table 1.

[0063] Table 1 Sample information of Shenfu Yixin Granules

[0064]

[0065] 1.2 Method for determining the characteristic spectrum of Shenfu Yixin granules of the present invention

[0066] 1.2.1 Preparation of test solution:

[0067] Take an appropriate amount of Shenfu Yixin granules, grind them into powder, accurately weigh 0.40g, put them into a 25ml volumetric flask, add an appropriate amount of 30% methanol solution, sonicate, then dilute with 30% methanol, shake to dissolve fully. 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 solution.

[0068] 1.2.2 Preparation of specific experimental solutions

[0069] 1.2.2.1 Preparation of negative sample solution

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

[0071] 1.2.2.2 Preparation of positive sample solution

[0072] Weigh 10g of each of the 13 medicinal pieces, add 300ml of water, and boil for 40min (water can be added during the boiling process). Filter, cool and dilute to 300ml. Transfer 1.0ml of ginseng medicinal material solution, 1.7ml of Amomum villosum and White Atractylodes rhizome medicinal material solution, 2.0ml of cinnamon twig and jujube medicinal material solution, 5ml of Salvia miltiorrhiza and Leonurus japonicus medicinal material solution, and 2.5ml of the other 6 medicinal material solutions, add 30% methanol solution to dilute to 10ml, filter through a membrane, and you have it.

[0073] Sampling analysis and experimental results

[0074] Ginseng: Shenfu Yixin Granules and ginseng negative samples were consistent, and no chromatographic peak appeared in ginseng positive samples;

[0075] Baifu Pian: Shenfu Yixin Granules and Baifu Pian negative samples were consistent, and no chromatographic peak appeared in Baifu Pian positive samples;

[0076] Cinnamon twig: There are differences between Shenfu Yixin granules and negative samples of Cinnamon twig, and the positive sample of Cinnamon twig has one chromatographic peak (retention time 29.4min);

[0077] Amomum villosum: The negative samples of Shenfu Yixin Granule and Amomum villosum were consistent (volatile oil could not be obtained by extracting cinnamon twig alone, so there was no inclusion complex peak at 29.5min in the negative spectrum), and no chromatographic peak appeared in the positive sample of Amomum villosum;

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

[0079] Red peony root: There is a difference between Shenfu Yixin granules and red peony root negative, and the red peony root positive sample has 2 chromatographic peaks (16.5min, 18.5min);

[0080] Plantago: There is a difference between the Shenfu Yixin granule preparation and the Plantago negative sample, and the Plantago positive sample has one chromatographic peak (5.9min);

[0081] Tinglizi: There is a difference between Shenfu Yixin granule preparation and Tinglizi negative sample, and Tinglizi positive sample has 1 chromatographic peak (13.3min);

[0082] Motherwort: There is a difference between Shenfu Yixin Granule preparation and negative samples of Motherwort, and positive samples of Motherwort have 3 peaks (11.8min, 17.2min, 18.39);

[0083] Poria: The Shenfu Yixin Granule preparation was consistent with the Poria negative sample, and no chromatographic peak appeared in the Poria positive sample.

[0084] Cortex zedoariae: The Cortex zedoariae negative sample was consistent with the Shenfu Yixin granules preparation (Note: the self-made negative sample was obtained by direct water decoction in the laboratory without the addition of inclusion complex, so there was no inclusion complex peak at 29.5min in the negative spectrum), and no chromatographic peak appeared in the Cortex zedoariae positive sample.

[0085] Alisma: The results of Shenfu Yixin Granule preparation and Alisma negative sample were consistent, and no chromatographic peak appeared in the Alisma positive sample.

[0086] Jujube: The Shenfu Yixin Granule preparation was consistent with the negative result, and no chromatographic peak appeared in the jujube positive sample.

[0087] 1.2.3 Preparation of reference solution:

[0088] Reference substance stock solution 1: Accurately weigh 1.084 mg of geniposide reference substance, place in a 10 ml brown volumetric flask, add chromatographic methanol and ultrasonic dissolution, then dilute to the mark, shake well, and store in the freezer.

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

[0090] Reference substance stock solution 3: Accurately weigh 4.852 mg of salvianolic acid B reference substance, place in a 10 ml brown volumetric flask, add chromatographic methanol and ultrasonic dissolution, dilute to the mark, shake well, and store in the freezer.

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

[0092] Geniposide acid reference solution: Take reference stock solution 1, measure 2 ml, dilute to 10 ml with 30% methanol, and shake well.

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

[0094] Cinnamaldehyde reference solution: Take reference stock solution 3, measure 1 ml and dilute to 20 ml with 30% methanol, shake well.

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

[0096] Mixed reference solution: measure 1.5 ml each of reference stock solution 1, reference stock solution 2, reference stock solution 3 and 0.3 ml of reference stock solution 4 into the same volumetric flask, make up to 5 ml with water, shake well, and obtain.

[0097] Salvia miltiorrhiza control medicinal material solution: weigh 0.3 g of the control medicinal material, add 30 ml of 30% methanol, ultrasonicate for 40 min, take 5 ml of the supernatant, dilute to 10 ml with 30% methanol solution, filter through a membrane, and obtain.

[0098] 1.2.4 Chromatographic conditions and system suitability test

[0099] Octadecylsilane bonded silica gel was used as filler; acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed as specified in Table 2 below; column temperature was 35°C; flow rate was 0.3 ml per minute; detection wavelength was 230 nm;

[0100] Table 2 Gradient elution mobile phase ratio

[0101]

[0102] 1.2.5 Sample solution injection analysis

[0103] Geniposide reference solution, paeoniflorin reference solution, cinnamaldehyde reference solution, salvianolic acid B reference solution, mixed reference solution, salvia miltiorrhiza reference medicinal material solution and test sample solution were respectively taken for sampling and analysis.

[0104] 1.2.6 Result Analysis

[0105] The chromatographic results are shown in the figure: Geniposide acid reference solution (see the attached manual Figure 2 ), Paeoniflorin reference solution (see the attached manual Figure 3 ), cinnamaldehyde reference solution (see the instructions for Figure 4 ) Salvianolic acid B reference solution (see the instructions for Figure 5 ), Danshen control medicinal material solution (see the attached manual Figure 6 ) and mixed reference solution (see Appendix Figure 7 ), the retention time of the corresponding chromatographic peaks is the same, and the spectral absorption is consistent. It can be considered that the present invention realizes the component separation and standard peak comparison study of geniposide, paeoniflorin, cinnamaldehyde and salvianolic acid B in the characteristic spectrum of Shenfu Yixin Granules by UPLC-UV method, and finally identifies 10 characteristic peaks of the characteristic fingerprint spectrum in Shenfu Yixin Granules. For specific characteristic peak information, see Table 3 below.

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

[0107]

[0108] 1.2.7 Selection of determination method

[0109] The characteristic spectrum of traditional Chinese medicine can generally be used to evaluate the retention time, relative retention time / relative peak area, etc. In this method, the four characteristic peaks are known components. Combined with the results of the chromatographic column and instrument durability inspection, the characteristic peak retention time method is selected for evaluation. The relevant chromatographic peaks of the characteristic spectrum of the test sample should have the same retention time as the corresponding chromatographic peaks of the reference substance.

[0110] 1.2.8 Establishment of feature maps

[0111] According to the chromatographic conditions optimized by the present invention, four characteristic peaks corresponding to the chromatogram of the reference substance should be present in the specified chromatogram, and the relative retention time of the remaining six characteristic peaks and the S peak (peony root glucoside) should be within ±10% of the specified value.

[0112] 1.2.9 Determination and evaluation of characteristic peaks

[0113] The relative retention times and relative peak areas of multiple batches of samples such as T240302, T240401, M240601, etc. were calculated respectively and used to formulate the characteristic peak parameters of SFYX particles.

[0114] Table 4 Relative retention time of characteristic peaks of three batches of Shenfu Yixin granules

[0115]

[0116]

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

[0118]

[0119] It can be seen from the experimental results in Table 4 and Table 5 that the characteristic peaks of the fingerprint spectrum of Shenfu Yixin Granule of the present invention contain 10 chromatographic peaks, and the relative retention times are 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%. The relative peak areas are 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 characteristic map

[0121] Take any SFYX particle separation spectrum as the characteristic spectrum, select the paeoniflorin peak as the S peak, and calculate the relative retention time and relative peak area of ​​multiple batches of samples such as T240302, T240401, M240601, etc. Take the median of the relative retention time and relative peak area to construct the characteristic spectrum, as shown in the attached manual Figure 1 .

[0122] After identification and appraisal, the characteristic peaks of the fingerprint of Shenfu Yixin Granule established by the present invention contain 10 chromatographic peaks, of which peaks 1, 7, 9 and 10 are the medicinal ingredients of Danshen, peak 2 is the medicinal ingredient of Plantago, peak 4 is the medicinal ingredient of Lemongrass, peak 5 is the medicinal ingredient of Red Peony, peak 8 is the medicinal ingredient of Cinnamomum cassia, peak 3 is the common peak of Danshen and Polyporus, and peak 6 is the common peak of Red Peony and other medicinal ingredients. Four peaks were calibrated with standard products, namely peak 2: geniposide acid, peak 5: paeoniflorin, peak 8: cinnamaldehyde, and peak 10: salvianolic acid B. The paeoniflorin peak was selected as the S peak, and the relative retention times of each peak were: 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%.

[0123] Example 2 Method for detecting the content of Shenfu Yixin granules of the present invention

[0124] 2.1 The specific steps of the method for detecting the content of Shenfu Yixin granules of the present invention are as follows

[0125] (1) Preparation of reference solution: Weigh geniposide acid, paeoniflorin, cinnamaldehyde and salvianolic acid B reference substances, add 30% methanol to prepare a mixed reference solution, wherein the acid concentration of geniposide 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] ⑵、Preparation of test solution: Take an appropriate amount of Shenfu Yixin granules, grind them into powder, accurately weigh 0.40g, put them into a 25ml volumetric flask, add an appropriate amount of 30% methanol solution, sonicate, then dilute with 30% methanol, shake to dissolve fully. Accurately measure 2ml of the solution into a 10ml volumetric flask, add 30% methanol solution to dilute to the mark, shake well, filter through a 0.22μm filter membrane, and you are done;

[0127] (3) Chromatographic conditions and system suitability, see section "1.2.4" in Example 1;

[0128] (4) Determination method: Accurately aspirate the reference solution and the test solution respectively, inject into the liquid chromatograph, and determine.

[0129] 2.2 Methodological validation

[0130] 2.2.1 Specificity test

[0131] The specific steps can be referred to in detail in "1.2.2" of Example 1. The experiment showed that there was no interference with the negative samples and the method had good specificity.

[0132] 2.2.2 System suitability test

[0133] 2.2.2.1 Preparation of reference solution:

[0134] Take appropriate amounts of geniposide reference substance, paeoniflorin reference substance, cinnamaldehyde reference substance and salvianolic acid B reference substance, weigh them accurately, and add 30% methanol to make solutions containing 6 μg of geniposide, 12 μg of paeoniflorin, 2.4 μg of cinnamaldehyde and 30 μg of salvianolic acid B per 1 ml.

[0135] 2.2.2.2 Preparation of test solution

[0136] Take an appropriate amount of Shenfu Yixin granules, grind them into powder, accurately weigh 0.40g, put them into a 25ml volumetric flask, add an appropriate amount of 30% methanol solution, sonicate, then dilute with 30% methanol, shake to dissolve fully. 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 solution.

[0137] Determination method: Determine according to the high performance liquid chromatography method (General Rule 0512). In the injection procedure, 2 μl of each solution is injected into the liquid chromatograph and the chromatogram is recorded.

[0138] Results: The blank solvent had no chromatographic peak at the retention time of the chromatographic peak of the reference solution and the corresponding retention time of the test solution, that is, the blank solvent did not interfere with the detection of the reference solution and the test solution; the plate number of paeoniflorin in the reference solution was 16495; the separation degree of cinnamaldehyde peak in the test solution was 1.98, both of which met the requirements.

[0139] 2.2.3 Precision test

[0140] Prepare the test sample solution according to the test sample solution preparation method under "2.2.2.2".

[0141] Determination method: Inject the same test solution 6 times continuously, determine it according to the chromatographic method, and record the chromatogram.

[0142] Table 6 Characteristic peak relative retention time-precision

[0143]

[0144] Table 7 Characteristic peak relative peak area-precision

[0145]

[0146]

[0147] The test 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 6 test solutions in parallel according to the test solution preparation method under "2.2.2.2". Determination method: Determine according to the chromatographic method and record the chromatogram.

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

[0151]

[0152] Table 9 Characteristic peak relative peak area-repeatability

[0153]

[0154]

[0155] The test 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%, which indicates that the detection method has good repeatability.

[0156] 2.2.5 Stability test

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

[0158] Table 10 Characteristic peak relative retention time-solution stability

[0159]

[0160]

[0161] Table 11 Characteristic peak relative peak area-solution stability

[0162]

[0163] The test 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 components to be measured within 24 hours is good and can meet the measurement needs.

[0164] 2.2.6 Durability test

[0165] Keep the basic chromatographic conditions unchanged, change the column temperature, and compare the relative retention time and relative peak area of ​​the 10 chromatographic peaks of the test solution.

[0166] Table 12 Characteristic peak relative retention time-column temperature investigation

[0167]

[0168] Table 13 Characteristic peak relative peak area-column temperature investigation

[0169]

[0170]

[0171] From the results in Tables 12 and 13, it can be seen that 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 chromatographic conditions are within the range of 35°C±2°C, and the method is more durable when the temperature is higher.

[0172] 3. Summary

[0173] A characteristic spectrum was established using high performance liquid chromatography, and the chemical components of Plantago seed, red peony root, cinnamon twig, salvia miltiorrhiza, Poria cocos, and Leonurus japonicus in Shenfu Yixin Granules were determined at the same time. Three batches of Shenfu Yixin Granules were determined, and the results showed that the chromatograms of the three batches of samples all showed characteristic peaks corresponding to the chromatograms of the reference substance.

Claims

1. A fingerprint detection method for traditional Chinese medicine preparations for treating heart failure, characterized in that: The detection method comprises the following steps: ⑴. Preparation of reference solution: weigh geniposide, paeoniflorin, cinnamaldehyde and salvianolic acid B reference substances, add 20-50% methanol to prepare a mixed reference solution; (2) Preparation of the test solution: Take the Chinese medicine preparation of the present invention, add 20-50% methanol, dissolve it by ultrasonication, dilute it to the scale, shake it well, filter it, and take the filtrate to obtain the solution; ⑶、Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler; acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed as specified in the following table; column temperature was 30-40℃; flow rate was 0.2-0.4ml per minute; detection wavelength was 220-240nm; (4) Determination method: Accurately aspirate the reference solution and the test solution respectively, inject into the liquid chromatograph, and determine.

2. The fingerprint detection method according to claim 1, characterized in that: In the preparation of the reference solution in step (1) of the detection method, the mixed reference solution comprises: a solution having an acid concentration of 4-8 μg / mL of geniposide, a concentration of 8-14 μg / mL of paeoniflorin, a concentration of 2-3 μg / mL of cinnamaldehyde, and a concentration of 20-40 μg / mL of salvianolic acid B.

3. The fingerprint detection method according to claim 2, characterized in that: In the preparation of the reference solution in step (1) of the detection method, the mixed reference solution comprises: a solution having an acid concentration of 6 μg / mL of geniposide, a concentration of 12 μg / mL of paeoniflorin, a concentration of 2.4 μg / mL of cinnamaldehyde, and a concentration of 30 μg / mL of salvianolic acid B.

4. The fingerprint detection method according to claim 1, characterized in that: 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%.

5. The fingerprint detection method according to claim 1, characterized in that: In the chromatographic conditions of step (3) of the detection method, 18 The column length 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 fingerprint detection method according to claim 1, characterized in that: In the chromatographic conditions of step (3) of the detection method, 18 The model of the chromatographic column is: ACQUITYUPLC BEH.

7. The fingerprint detection method according to claim 1, characterized in that: In the chromatographic conditions of step (3) of the detection method, the column temperature is 35° C., the flow rate is 0.3 ml per minute, and the detection wavelength is 230 nm.

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