Multi-index quality control method of Naoxinan capsule

Abstract

The invention discloses a multi-index quality control method of Naoxinan capsules, and relates to the technical field of medical drugs, the method adopts a high performance liquid chromatography to establish a wavelength characteristic spectrum for detection, and the method specifically comprises the following steps: detecting a characteristic spectrum 1 under the wavelength of 260nm: taking a puerarin reference substance peak (marked as 1 (S)) as a reference, and detecting the characteristic spectrum 2 under the wavelength of 260nm; five characteristic peaks are required to be presented in the chromatogram of the test sample, and the similarity between the retention time of the five characteristic peaks and the contrast characteristic chromatogram is greater than or equal to 0.90; and / or detecting a characteristic spectrum 2 under the wavelength of 320nm: by taking a puerarin reference substance peak (marked as 1 (S)) as a reference, requiring that five characteristic peaks appear in a chromatogram of the test sample, and the similarity between the retention time of the five characteristic peaks and the contrast characteristic spectrum is greater than or equal to 0.90. According to the invention, five active ingredients such as flavonoids (puerarin), saponins (astragaloside) and phenolic acids (tanshinol) are covered through a wavelength characteristic spectrum (260nm and / or 320nm).

Description

Technical Field

[0001] This invention relates to the field of medical pharmaceutical technology, and in particular to a multi-index quality control method for Naoxin'an capsules. Background Technology

[0002] Cardiovascular and cerebrovascular diseases are prevalent worldwide, and traditional Chinese medicine has unique advantages in their prevention and treatment. Naoxin'an preparation, a commonly used compound traditional Chinese medicine in clinical practice, contains 19 medicinal herbs in its basic formula, including Astragalus membranaceus, Codonopsis pilosula, and saffron. It is prepared through a process of partially pulverizing and partially decocting and concentrating the herbs.

[0003] However, existing technologies have the following limitations:

[0004] Inadequate quality control:

[0005] The standard for Naoxin'an capsules relies solely on routine tests (such as disintegration time and weight variation), lacking a comprehensive quality control method for multiple active ingredients, making it difficult to guarantee batch-to-batch stability and consistency in clinical efficacy.

[0006] Compound traditional Chinese medicines have complex components, and a single content measurement indicator cannot fully characterize the quality of the drug, which poses a risk of fluctuation in efficacy. Summary of the Invention

[0007] The technical solution of this invention to solve the above-mentioned technical problems is to provide a multi-index quality control method for Naoxin'an capsules, including the establishment of wavelength characteristic spectrum detection using high performance liquid chromatography, specifically including the following steps:

[0008] Detection of characteristic chromatogram 1 at 260 nm wavelength: Based on the puerarin reference peak (labeled as 1(S)), the chromatogram of the test sample is required to show 5 characteristic peaks, and the similarity between the retention time and the reference characteristic chromatogram is ≥0.90.

[0009] And / or, detect characteristic chromatogram 2 at a wavelength of 320 nm: based on the puerarin reference peak (labeled as 1(S)), the chromatogram of the test sample is required to show 5 characteristic peaks, and the similarity of their retention time with the reference characteristic chromatogram is ≥0.90.

[0010] Furthermore, the high-performance liquid chromatography conditions include:

[0011] Column: Octadecylsilane-bonded silica gel packed column (C18);

[0012] Mobile phase:

[0013] Phase A: Acetonitrile;

[0014] Phase B: 0.05% phosphoric acid solution;

[0015] Gradient elution procedure:

[0016]

[0017] Flow rate: 1.0 mL / min;

[0018] Column temperature: 30℃.

[0019] Furthermore, the method for preparing the puerarin reference solution is as follows: accurately weigh the puerarin reference standard, dissolve it in methanol, and dilute to 0.1 mg / mL.

[0020] Furthermore, the test solution is directly the solution used in the assay section.

[0021] Furthermore, the theoretical plate number, calculated based on the puerarin peak, is no less than 97,000 at both 260 nm and 320 nm wavelengths.

[0022] Compared with the prior art, the present invention has the following beneficial effects: multi-component overall monitoring, wavelength characteristic spectrum (260nm and / or 320nm) covering 5 classes of active ingredients such as flavonoids (puerarin), saponins (astragaloside A), and phenolic acids (tanshinone), similarity threshold (≥0.90) combined with the fingerprint spectrum guidance principle of the Chinese Pharmacopoeia, batch consistency is improved to 98%; and the corresponding components are also confirmed. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0024] Figure 1 The image shows the results of the repeatability characteristic spectrum test of the Naoxin'an capsule of the present invention (260nm).

[0025] Figure 2 The image shows the results of the repeatability characteristic spectrum test (320nm) of the Naoxin'an capsule of the present invention;

[0026] Figure 3 This is a graph (260nm) showing the results of the stability characteristic test of the Naoxin'an capsules of the present invention.

[0027] Figure 4 This is a graph (320nm) showing the results of the stability characteristic test of the Naoxin'an capsules of the present invention.

[0028] Figure 5 The chromatographic column robustness test result (260nm) of the characteristic chromatogram of the Naoxin'an capsule of the present invention is shown.

[0029] Figure 6 The image shows the column robustness test results (320 nm) of the characteristic chromatogram of the Naoxin'an capsule of the present invention.

[0030] Figure 7 This is a comparative characteristic spectrum (260nm) of the Naoxin'an Capsules of the present invention;

[0031] Figure 8 The image shows the characteristic chromatogram results (260nm) of sixteen batches of Naoxin'an capsules of this invention.

[0032] Figure 9 The comparative characteristic spectrum (320nm) of the Brain Heart Care Capsules of this invention;

[0033] Figure 10 The image shows the characteristic chromatogram results (320nm) of sixteen batches of Naoxin'an capsules of this invention.

[0034] Figure 11 This is the spectrum of gastrodin at 260 nm wavelength for 13.091 min according to the present invention;

[0035] Figure 12 The spectrum of puerarin at 260 nm wavelength for 28.772 min is shown in this invention.

[0036] Figure 13 The spectrum of ferulic acid at 260 nm wavelength for 43.755 min is shown in this invention.

[0037] Figure 14 The spectrum of verbascoside at 260 nm wavelength for 48.385 min is shown in the image.

[0038] Figure 15 This is the spectrum of stilbene glycoside at 260 nm wavelength for 51.991 min according to the present invention;

[0039] Figure 16 This is the spectrum of tanshinone IIA at 260 nm wavelength for 85.919 min according to the present invention;

[0040] Figure 17 The spectrum of tanshinone B at 260 nm wavelength is shown at 99.916 min.

[0041] Figure 18 The spectrum of emodin at 260 nm wavelength for 117.293 min is shown in this invention.

[0042] Figure 19 The spectrum of emodin methyl ether at 260 nm wavelength for 137.278 min is shown in the figure.

[0043] Figure 20 This is the full spectrum at 260nm wavelength of this invention;

[0044] Figure 21 This is the full spectrum of the present invention at 320nm wavelength. Detailed Implementation

[0045] This invention proposes a multi-index quality control method for Naoxin'an capsules, aiming to design a Naoxin'an capsule with strong stability.

[0046] The following will describe the Naoxin'an Capsule for cardiovascular and cerebrovascular diseases proposed in this invention through specific embodiments:

[0047] Example 1:

[0048] A multi-index quality control method for Naoxin'an capsules includes the use of high-performance liquid chromatography (HPLC) to establish a wavelength characteristic spectrum for detection, specifically comprising the following steps:

[0049] Detection of characteristic chromatogram 1 at 260 nm wavelength: Based on the puerarin reference peak (labeled as 1(S)), the chromatogram of the test sample is required to show 5 characteristic peaks, and the similarity between the retention time and the reference characteristic chromatogram is ≥0.90.

[0050] And / or, detect characteristic chromatogram 2 at a wavelength of 320 nm: based on the puerarin reference peak (labeled as 1(S)), the chromatogram of the test sample is required to show 5 characteristic peaks, and the similarity of their retention time with the reference characteristic chromatogram is ≥0.90.

[0051] Furthermore, the high-performance liquid chromatography conditions include:

[0052] Column: Octadecylsilane-bonded silica gel packed column (C18);

[0053] Mobile phase:

[0054] Phase A: Acetonitrile;

[0055] Phase B: 0.05% phosphoric acid solution;

[0056] Gradient elution procedure:

[0057]

[0058] Flow rate: 1.0 mL / min;

[0059] Column temperature: 30℃.

[0060] Furthermore, the method for preparing the puerarin reference solution is as follows: accurately weigh the puerarin reference standard, dissolve it in methanol, and dilute to 0.1 mg / mL.

[0061] Furthermore, the test solution is directly the solution used in the assay section.

[0062] Furthermore, the theoretical plate number, calculated based on the puerarin peak, is no less than 97,000 at both 260 nm and 320 nm wavelengths.

[0063] The prescription composition of Naoxin'an Capsules is as follows:

[0064] Astragalus membranaceus 50 parts, Codonopsis pilosula 40 parts, Saffron 30 parts, Panax notoginseng 30 parts, Salvia miltiorrhiza 30 parts, Pueraria lobata 30 parts, Ligusticum chuanxiong 30 parts, Curcuma longa 20 parts, processed Polygonum multiflorum 20 parts, Haliotis diversicolor 20 parts, Spatholobus suberectus 20 parts, Gastrodia elata 20 parts, Borneol 5 parts, Prunus persica 20 parts, Arisaema cum bile 10 parts, Buthus martensii 5 parts, Scolopendra subspinipes 5 parts, Artificial musk 0.1 parts, Artificial bezoar 2 parts.

[0065] Specifically, 1000 capsules of Naoxin'an are prepared, with the following components: Astragalus membranaceus 50g, Codonopsis pilosula 40g, Saffron 30g, Panax notoginseng 30g, Salvia miltiorrhiza 30g, Pueraria lobata 30g, Ligusticum chuanxiong 30g, Curcuma longa 20g, processed Polygonum multiflorum 20g, Haliotis diversicolor 20g, Spatholobus suberectus 20g, Gastrodia elata 20g, Borneol 5g, Prunus persica 20g, Arisaema cum bile 10g, Buthus martensii 5g, Scolopendra subspinipes 5g, Artificial musk 0.1g, and Artificial bezoar 2g.

[0066] The preparation method is as follows:

[0067] Step 1: Take 40 parts Astragalus membranaceus, 30 parts Saffron, 30 parts Panax notoginseng, 30 parts Pueraria lobata, 30 parts Ligusticum chuanxiong, 20 parts Gastrodia elata, 5 parts Borneol, 5 parts Scorpion, 5 parts Centipede, 0.1 parts Artificial Musk, and 2 parts Artificial Bezoar and grind them into a fine powder;

[0068] Step 2: Take 10 parts of Astragalus membranaceus, 40 parts of Codonopsis pilosula, 30 parts of Salvia miltiorrhiza, 20 parts of Curcuma longa, 20 parts of processed Polygonum multiflorum, 20 parts of Haliotis diversicolor, 20 parts of Spatholobus suberectus, 20 parts of Prunus persica, and 10 parts of Arisaema cum Bile, add water and decoct three times; the first time for 2 hours, the second and third times for 1.5 hours each, combine the decoctions, filter, and concentrate at 50℃ to a thick paste with a relative density of 1.30;

[0069] Step 3: Add the fine powder obtained in Step 1 to the thick paste in Step 2, mix well, dry, pulverize, and sieve.

[0070] Add 20%-30% starch (20%-30% of the weight of the above components), mix well, fill into capsules, and make into granules.

[0071] Specifically: Quality control is performed using wavelength characteristic maps.

[0072] Feature spectrum 1: Detection wavelength 260nm, with puerarin as the reference peak (marked as S), requiring 5 characteristic peaks and similarity ≥0.90;

[0073] Specifically, octadecylsilane-bonded silica gel was used as the packing material; acetonitrile was used as mobile phase A, and a 0.05% phosphoric acid solution was used as mobile phase B, with gradient elution performed according to the specifications in the table below; the detection wavelength was 260 nm; the column temperature was 30 °C; and the flow rate was 1.0 ml per minute. The theoretical plate number, calculated based on puerarin, should be no less than 97,000 at 260 nm.

[0074]

[0075]

[0076] Preparation of reference solution: Take an appropriate amount of puerarin reference standard, accurately weigh it, and add methanol to prepare a solution containing 0.1 mg per 1 mL.

[0077] Preparation of the test solution: Take the test solution from the [Assay] section to obtain the solution.

[0078] For the determination method, accurately pipette 10 μL each of the reference solution and the test solution, inject them into the liquid chromatograph, measure and record the chromatogram to obtain the result.

[0079] The characteristic chromatogram of the test sample should have a retention time consistent with the peak of the puerarin reference at 260 nm, and should present 5 characteristic peaks, labeled as peaks 1(S), 2, 3, 4, and 5. The similarity between the test sample and the reference characteristic chromatogram should not be less than 0.90, calculated by the Chinese medicine fingerprint chromatogram similarity evaluation system.

[0080] Feature spectrum 2: Detection wavelength 320nm, with puerarin as the reference peak (marked as S), requiring 10 characteristic peaks with a similarity ≥0.90.

[0081] Specifically, octadecylsilane-bonded silica gel was used as the packing material; acetonitrile was used as mobile phase A, and a 0.05% phosphoric acid solution was used as mobile phase B, with gradient elution performed according to the specifications in the table below; the detection wavelength was 320 nm; the column temperature was 30 °C; and the flow rate was 1.0 ml per minute. The theoretical plate number, calculated based on puerarin, should be no less than 97,000 at 320 nm.

[0082]

[0083]

[0084] Preparation of the reference solution: Take an appropriate amount of puerarin reference standard, accurately weigh it, and add methanol to prepare a solution containing 0.1 mg per 1 mL.

[0085] Preparation of the test solution: Take the test solution from the [Assay] section to obtain the solution.

[0086] For the determination method, accurately pipette 10 μL each of the reference solution and the test solution, inject them into the liquid chromatograph, measure and record the chromatogram to obtain the result.

[0087] The characteristic chromatogram of the test sample should have a retention time consistent with the peak of the puerarin reference at 320 nm, and should present 5 characteristic peaks, labeled as peaks 1, 2(S), 3, 4, and 5. The similarity between the test sample and the reference characteristic chromatogram should not be less than 0.90, calculated by the Chinese medicine fingerprint chromatogram similarity evaluation system.

[0088] Quality Inspection:

[0089] Determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020 Edition).

[0090] Chromatographic conditions and system suitability tests were conducted using octadecylsilane-bonded silica gel as the stationary phase; acetonitrile as mobile phase A and 0.05% phosphoric acid solution as mobile phase B, with gradient elution performed according to the specifications in the table below; detection wavelengths were 260 nm and 320 nm; column temperature was 30 °C; and flow rate was 1.0 mL per minute. The theoretical plate number, calculated based on puerarin, should be no less than 97,000 at both 260 nm and 320 nm.

[0091]

[0092]

[0093] Preparation of reference solution: Take an appropriate amount of puerarin reference standard, accurately weigh it, and add methanol to prepare a solution containing 0.1 mg per 1 mL.

[0094] Preparation of the test solution: Take the test solution from the [Assay] section to obtain the solution.

[0095] For the assay, accurately pipette 10 μL each of the reference solution and the test solution, inject them into the liquid chromatograph, measure and record the chromatograms to obtain the results.

[0096]

Characteristic Spectrum 1

[0097]

Characteristic Spectrum 2

[0098] Characteristic chromatograms are distinctive, reproducible, and practical, possessing sufficient representativeness to reflect multi-component information and characterize the quality features of traditional Chinese medicine. A systematic study of the characteristic chromatogram method was conducted for this product. Using puerarin peak as a reference peak, high-performance liquid chromatography (HPLC) was employed for characteristic chromatogram determination. Systematic experimental investigations were carried out, demonstrating that this method is simple, rapid, stable, reproducible, and highly precise, facilitating the monitoring of this product's quality.

[0099] Reagents and instruments:

[0100] Instruments: Waters 2695 / 2996 high performance liquid chromatograph, Empower3 workstation.

[0101] Column: Agilent 5TC-C18(2) 250×4.6mm;

[0102] Reagents: Acetonitrile was of chromatographic grade, water was purified water, and all other reagents were of analytical grade.

[0103] CN-20 Digital Ultrasonic Cleaner (480W power, 40kHz frequency, Beijing Desay Zhiyuan Trading Co., Ltd.)

[0104] Reference solution: Puerarin was provided by the National Institutes for Food and Drug Control (batch number: 110752-200410) for content determination.

[0105] Sample preparation:

[0106] Preparation of reference solution:

[0107] Take an appropriate amount of puerarin reference standard, accurately weigh it, and add methanol to prepare a solution containing 0.1 mg per 1 mL.

[0108] Preparation of the test solution:

[0109] Take the test solution from the [Assay] section to obtain the solution.

[0110] Selection of measurement conditions:

[0111] Selection of detection wavelength: The instrument used for the characteristic chromatogram test of this product is a Waters 2695 / 2996 high performance liquid chromatograph and an Empower3 chromatography workstation. The test was conducted according to the high performance liquid chromatography method (General Rule 0512). After preliminary testing, 260 nm and 320 nm were selected as the detection wavelengths of the characteristic chromatogram of this product.

[0112] Based on the preliminary test, the mobile phase ratio was optimized. Acetonitrile was selected as mobile phase A and 0.05% phosphoric acid solution as mobile phase B for gradient elution of the product. The chromatographic column Agilent 5TC-C18 (2) 250×4.6mm obtained the best resolution and the relative retention time value. Therefore, the mobile phase selected in this experiment was acetonitrile as mobile phase A and 0.05% phosphoric acid solution as mobile phase B for gradient elution. The chromatograms tested under these conditions were recorded. The theoretical plate number was calculated with reference component puerarin, and the resolution between reference component puerarin and adjacent peaks was calculated. The results are as follows:

[0113] 260nm:

[0114] n = 5.54(t) R / W h / 2 ) 2 =5.54 (7.98 / 0.06) 2 =97997.06;

[0115] R = 2(t) R2 -t R1 ) / (W1+W2)=2(8.66-7.98) / (0.17+0.21)=3.58;

[0116] 320nm:

[0117] n = 5.54(t) R / W h / 2 ) 2 =5.54 (7.98 / 0.06) 2 =97997.06;

[0118] R = 2(t) R2 -t R1 ) / (W1+W2)=2(8.41-7.98) / (0.17+0.18)=2.46;

[0119] Based on the experimental results, and considering the influence of system factors such as instrument, chromatographic column, mobile phase preparation and temperature, the theoretical plate number is specified to be no less than 97,000 at both 260 nm and 320 nm, calculated based on puerarin.

[0120] Based on literature and experimental results, the selected chromatographic conditions are as follows:

[0121] Column: Agilent 5TC-C18(2) 250×4.6mm;

[0122] Mobile phase: Gradient elution was performed using acetonitrile as mobile phase A and 0.05% phosphoric acid solution as mobile phase B;

[0123]

[0124]

[0125] Flow rate: 1.0 mL / min;

[0126] Detection wavelengths: 260nm; 320nm.

[0127] Column temperature: 30℃.

[0128] Repeatability testing:

[0129] Take the test solution and perform the procedure described in the "Characteristic Chromatography" section above. Perform seven consecutive determinations using the same method. The test results are shown in the table below. Figure 1 and Figure 2 .

[0130] Stability testing:

[0131] Take the same test solution (batch number: 20230917), and operate according to the method under the [Characteristic Chromatography] section above. Measure the characteristic chromatogram of the test sample at time intervals of 0, 4, 8, 12, 16, 20, and 24 hours. The test results are shown in [link to test results]. Figure 3 and Figure 4 The experimental results show that when the test solution is measured within 24 hours after preparation, the reference peak shows no significant change, and the chemical properties of the characteristic spectrum of the test solution are stable within this time period.

[0132] Column durability test:

[0133] Take the same sample solution and use chromatographic columns of different brands to investigate the effect of different column brands on the detection of characteristic chromatograms. The experimental results are shown below. Figure 5 and Figure 6 Note: Column 1: Agilent 5TC-C18(2) 250×4.6mm;

[0134] Column 2: ZORBAX SB-C18 Stable Bond Analytical 4.6×250mm 5-Micron;

[0135] Column 3: μm 4.6×250mm Column.

[0136] The experimental results show that the three different brands of chromatographic columns had no significant impact on the determination of characteristic chromatograms.

[0137] Characteristic spectrum analysis of sixteen batches of Naoxin'an capsules:

[0138] Following the aforementioned method for [characteristic chromatograms], the characteristic chromatograms of the control group and sixteen batches of Naoxin'an capsules were determined, and the test results were verified. Figure 7 , Figure 8 , Figure 9 , Figure 10 Other active ingredients in Naoxin'an capsules were determined in fingerprint spectroscopy as follows: Figures 11 to 21 .

[0139] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A multi-index quality control method for Naoxin'an capsules, characterized in that, This includes establishing wavelength characteristic spectra for detection using high-performance liquid chromatography, specifically comprising the following steps: Detection of characteristic chromatogram 1 at 260 nm wavelength: Based on the puerarin reference peak, the chromatogram of the test sample is required to show 5 characteristic peaks, and the similarity between the retention time and the reference characteristic chromatogram is ≥0.

90. And / or, detect characteristic chromatogram 2 at a wavelength of 320 nm: using the puerarin reference peak as a benchmark, the chromatogram of the test sample is required to show 5 characteristic peaks, and the similarity between the retention time and the reference characteristic chromatogram is ≥0.

90. The high-performance liquid chromatography conditions include: Column: Octadecylsilane-bonded silica gel packed column; Mobile phase: Phase A: Acetonitrile; Phase B: 0.05% phosphoric acid solution; Gradient elution procedure: Flow rate: 1.0 mL / min; Column temperature: 30℃.

2. The multi-index quality control method for Naoxin'an capsules according to claim 1, characterized in that, The method for preparing the puerarin reference solution is as follows: accurately weigh the puerarin reference standard, dissolve it in methanol, and dilute to 0.1 mg / mL.

3. The multi-index quality control method for Naoxin'an capsules according to any one of claims 1-2, characterized in that, The test solution is the same as the solution used in the assay section.

4. The multi-index quality control method for Naoxin'an capsules according to any one of claims 1-3, characterized in that, The theoretical plate number, calculated based on the puerarin peak, is no less than 97,000 at both 260 nm and 320 nm wavelengths.

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