A method for constructing fingerprint spectrum of Aguilin nourishing blood preparation

Abstract

The present invention discloses a method for constructing a fingerprint of a gui nourishing blood preparation, and the fingerprint is a method for constructing a HPLC characteristic fingerprint of a gui nourishing blood preparation. The present invention establishes a method for constructing a HPLC characteristic fingerprint of a gui nourishing blood preparation, which overcomes the singleness and one-sidedness of existing quality standards; it can fully reflect the quality information of the gui nourishing blood preparation, thereby achieving the purpose of more comprehensively and effectively controlling the product quality of the gui nourishing blood preparation. For some active ingredients that cannot be identified and the effective ingredients are not clear, the fingerprint method can be used to prove the consistency of product quality. The fingerprint of the gui nourishing blood preparation obtained by the method of the present invention has many peaks, good peak shape, easy identification, high similarity, and is accurate and reliable. The method of the present invention is simple to operate, stable, and repeatable, and the conclusion is objective and accurate.

Description

Technical Field

[0001] The invention belongs to the field of drug quality detection, and particularly relates to a method for constructing a fingerprint spectrum of an Aguilin nourishing blood preparation. Background Art

[0002] Kangpu Pharmaceutical Co., Ltd. prepares the prescription of Agui Yangxue Granules: 386g of Angelica sinensis, 24g of Codonopsis pilosula, 24g of White Peony Root, 12g of Roasted Licorice Root, 24g of Poria, 24g of Astragalus, 24g of Rehmannia glutinosa, 12g of Chuanxiong, and 24g of Ejiao. It is mainly used to nourish qi and blood. It is used for qi and blood deficiency, sallow complexion, dizziness and fatigue, scanty and light menstruation.

[0003] At present, the quality control of Agui Yangxue preparations often selects several traditional Chinese medicine ingredients for identification, and the thin layer chromatography method is mostly used, which makes it impossible to monitor its quality comprehensively.

[0004] Since Chinese medicine and its compound preparations are multi-component complex systems, their quality should be evaluated by detection methods that are suitable for them and can provide rich identification information. However, the current methods such as microscopic identification, physical and chemical identification and content determination are not enough to solve this problem. Chinese medicine fingerprint refers to a chromatogram or spectrum that can indicate the chemical characteristics of certain Chinese medicinal materials or Chinese medicine preparations after appropriate treatment and certain analytical methods. Chinese medicine fingerprint is a comprehensive and quantifiable identification method that can more comprehensively reflect the types and quantities of chemical components contained in Chinese medicine and its preparations, and then provide an overall description and evaluation of the quality of the medicine. Therefore, Chinese medicine fingerprint has become a more advanced quality control method for Chinese medicine in the world. The US Food and Drug Administration (FDA) Industrial Guide for Botanical Drug Products and the WHO Herbal Medicine Evaluation Guide both point out that if the active ingredients of herbal preparations cannot be identified, the fingerprint spectrum can be used to prove the consistency of product quality. The Indian Herbal Codex, the British Herbal Codex, the German Medicinal Plant Association, the Canadian Medicinal and Aromatic Plant Association, etc., also accept the use of fingerprints to ensure the consistency of the quality of herbal products with unknown active ingredients. At present, the State Food and Drug Administration has required Chinese medicine injections to have relevant fingerprint maps, which plays a key role in improving the quality of Chinese medicine injections, but there are no mandatory requirements for other dosage forms of Chinese medicine.

[0005] Agui Yangxue Preparation is composed of Chinese angelica, Codonopsis, White Peony Root, Roasted Licorice, Poria, Astragalus, Rehmannia Glutinosa, Chuanxiong, and donkey-hide gelatin. At present, the quality control of Agui Yangxue Preparation often selects several Chinese medicinal ingredients for identification, and thin layer chromatography is mostly used, which cannot fully monitor its quality. There is no public report on the research of the fingerprint spectrum of Agui Yangxue Preparation, and there is no method for constructing the fingerprint spectrum of Agui Yangxue Preparation. From the aspect of quality control, there is no report on the standard fingerprint spectrum of Agui Yangxue Preparation. Summary of the invention

[0006] The present invention aims to provide a method for constructing a fingerprint spectrum of an Agui nourishing blood preparation.

[0007] The invention discloses a method for constructing a fingerprint of an Agui nourishing blood preparation, wherein the Agui nourishing blood preparation comprises: 386g of angelica, 24g of codonopsis, 24g of white peony root, 12g of roasted licorice root, 24g of tuckahoe, 24g of astragalus, 24g of prepared rehmannia root, 12g of chuanxiong, and 24g of donkey-hide gelatin. The fingerprint is an HPLC characteristic fingerprint of the Agui nourishing blood preparation, and the method for constructing the fingerprint is as follows:

[0008] (1) Preparation of test solution: Take 1-4 g of the Agui Yangxue preparation or its contents, weigh accurately, place in a conical flask, add 50-100 ml of methanol accurately, weigh the weight, extract by ultrasonic for 0.5-1 hour, cool, weigh again, add methanol to make up the weight, shake well, filter, and take the filtrate to obtain the solution;

[0009] (2) Preparation of reference solution: Take 5-10 mg of paeoniflorin reference substance and place it in a 50 ml volumetric flask. Add methanol to dissolve it and dilute it to the mark. Shake well. Accurately pipette 5 ml and place it in a 25 ml volumetric flask. Add methanol to dilute it to the mark. Shake well.

[0010] (3) Determination: Accurately pipette 5-15 μl of the reference solution and the test solution respectively, inject into the high performance liquid chromatograph, record the chromatogram within 70 minutes, and process the chromatogram with the fingerprint software to obtain the fingerprint of the Agui Yangxue Preparation;

[0011] The chromatographic conditions for the fingerprint high performance liquid chromatography determination are as follows: octadecylsilane bonded silica gel as filler, acetonitrile as mobile phase A, acetonitrile-30mmol / L triethylamine (5:95) as mobile phase B, gradient elution, flow rate 0.5~1.5ml·min-1, column temperature 25~40°C, detection wavelength 185~400nm; the theoretical plate number calculated based on the paeoniflorin peak should be no less than 2500;

[0012] The gradient elution process of the fingerprint HPLC determination is:

[0013] .

[0014] Take 10 batches of Agui nourishing blood samples of the present invention (batch numbers: 180501, 180502, 180503, 180601, 180602, 180603, 180604, 180701, 180702, 180703), prepare the test solution according to the above method, analyze according to the above chromatographic conditions, obtain 10 batches of Agui nourishing blood HPLC fingerprints, and use the average fingerprint of the 10 batches as the control fingerprint. The calculated similarity is 0.98.

[0015] The 9 medicinal materials in the prescription of the Agui Yangxue preparation of the present invention were used to prepare test sample solutions separately according to the above method, and were analyzed separately according to the above chromatographic conditions, and the chromatograms of the Agui Yangxue preparation and each medicinal material were compared to find out the peak attribution. The relative retention time of the common peak was recorded with reference to the paeoniflorin reference substance.

[0016] The method for constructing a fingerprint spectrum of the Agui nourishing blood preparation described in the present invention, there are 14 common peaks in the spectrum, with No. 4 as the reference peak, and the relative retention time and relative peak area of ​​the 14 common peaks are as follows:

[0017] .

[0018] Agui Yangxue preparation contains 9 medicinal materials, but the quality standards of Agui Yangxue preparation are conventional thin layer identification and determination of paeoniflorin on 4 of them, which cannot accurately reflect the quality of Agui Yangxue preparation. At present, there are many components of Agui Yangxue preparation, and its traditional quality control often selects several Chinese medicinal ingredients for identification, and thin layer chromatography is mostly used, which cannot fully monitor its quality. The present invention focuses on developing a new analytical method to improve the quality standard of Agui Yangxue preparation and fully monitor its quality.

[0019] Advantages of the fingerprint spectrum construction method of the Agui nourishing blood preparation of the present invention:

[0020] ① The present invention establishes the HPLC characteristic fingerprint of Agui Yangxue Preparation, which overcomes the singleness and one-sidedness of the existing quality standards; it can fully reflect the quality information of Agui Yangxue Preparation, thereby achieving the purpose of more comprehensive and effective control of the product quality of Agui Yangxue Preparation.

[0021] ② For some active ingredients that cannot be identified or whose effective ingredients are unclear, the fingerprint method can be used to prove the consistency of product quality.

[0022] ③ The fingerprint spectrum of the Agui nourishing blood preparation obtained by the method of the present invention has many peaks, good peak shape, is easy to identify, has high similarity, and is accurate and reliable.

[0023] ④ The method of the present invention is simple to operate, stable, and has good repeatability, and the conclusion is objective and accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 : Fingerprints of ten batches of Agui nourishing blood preparations;

[0025] Figure 2 : Test sample of Agui nourishing blood preparation batch 180501. DETAILED DESCRIPTION

[0026] The following examples are only for further illustrating the present invention and are not intended to limit the scope of the present invention in any form.

[0027] Example 1: Chromatographic Condition Screening

[0028] (1) Screen different chromatographic columns and examine the number of peaks, separation, and peak shape. (Area normalization method, peaks below 0.05% are not counted)

[0029]

[0030] From the results, Agilent TC-C18 (250mm×4.6mm, 5um) detected the most peaks and had the best separation. Agilengt TC-C18 was selected to develop the HPLC characteristic fingerprint detection method for Agui Yangxue preparation.

[0031] (2) Screen different mobile phase salts and examine the number of peaks, separation and peak shape. Use Agilent TC-C18 (250mm×4.6mm, 5um) chromatographic column for the test.

[0032] (Area normalization method, peaks below 0.05% are not counted)

[0033]

[0034] From the results, 30mmol / L triethylamine solution detected the most peaks and had the best separation degree. Therefore, 30mmol / L triethylamine solution was selected as the mobile phase salt for the development of HPLC characteristic fingerprint detection of Agui Yangxue preparation.

[0035] (3) Screen different wavelengths and examine the number of peaks, separation, and peak shape. (Area normalization method, peaks below 0.05% are not counted)

[0036]

[0037] From the results, the wavelength with the most detected peaks and the best separation was 220nm, so 220nm was selected as the wavelength for developing the HPLC characteristic fingerprint detection of Agui Yangxue preparation.

[0038] Example 2: Fingerprint Methodology Validation

[0039] 1. Instrument

[0040] Agilent1260 high performance liquid chromatograph (Agilent, USA); PB-10 pH meter (Sartorius), BP-211D electronic analytical balance (Sartorius).

[0041] 2. Test solution

[0042] Acetonitrile (Tiandi, chromatographic grade), triethylamine (Sinopharm, analytical grade), methanol (Tianjin Best Chemical, analytical grade), and water were Wahaha purified water.

[0043] 3. Reference

[0044] Paeoniflorin (batch number 110736-201842, China Food and Drug Administration, content 97.4%)

[0045] 4. Samples

[0046] Agui Yangxue Preparation (Batch No.: 180501, Kangpu Pharmaceutical Co., Ltd.)

[0047] 5. Chromatographic conditions: Octadecylsilane bonded silica as filler, acetonitrile as mobile phase A, acetonitrile-30mmol / L triethylamine (5:95) as mobile phase B, gradient elution, flow rate 1.0ml·min-1, column temperature 35℃, detection wavelength 220nm; the theoretical plate number calculated based on the paeoniflorin peak should be no less than 2500.

[0048] Gradient elution table:

[0049]

[0050] 6 Sample treatment: Take 3g of the Agui Yangxue preparation or its contents, weigh accurately, place in a conical flask, add 80ml of methanol accurately, weigh the weight, extract by ultrasonic for 50min, cool, weigh again, add methanol to make up the weight, shake well, filter, and take the filtrate to obtain;

[0051] 7. Verification content:

[0052] 1) Precision test:

[0053] Take one sample of Agui Yangxue Preparation (batch number: 180501), prepare it according to the above-mentioned preparation method of the test solution, inject it continuously for 6 times, measure it according to the above-mentioned chromatographic conditions, record the fingerprint spectrum, take the peony inoside chromatographic peak (peak 4 in the fingerprint spectrum) as the internal reference peak, calculate the relative retention time RSD% and relative peak area RSD% of each common peak in the fingerprint spectrum.

[0054] Results: The relative retention time RSD% of the 14 common peaks was less than 2.0; the relative peak area RSD% was less than 2.0, indicating that the precision was good and in compliance with regulations.

[0055] 2) Repeatability test:

[0056] Take one sample of Agui Yangxue Preparation (batch number: 180501), prepare 6 samples in parallel according to the preparation method of the test solution, measure according to the above chromatographic conditions, record the fingerprint, take the peony inoside chromatographic peak (peak 4 in the fingerprint) as the internal reference peak, and calculate the relative retention time RSD% and relative peak area RSD% of each common peak in the fingerprint.

[0057] Results: The relative retention time RSD% of the 14 common peaks was less than 2.0; the relative peak area RSD% was less than 2.0, indicating that the repeatability was good and in compliance with regulations.

[0058] 3) Stability test

[0059] Take one sample of Agui Yangxue Preparation (batch number: 180501), prepare it according to the above-mentioned method for preparing the test solution, and measure it according to the above-mentioned chromatographic conditions at 0, 3, 6, 9, 12, 24h, and 36h, record the fingerprint, and use the peony indole glycoside chromatographic peak (peak 4 in the fingerprint) as the internal reference peak to calculate the relative retention time RSD% and relative peak area RSD% of each common peak in the fingerprint.

[0060] Results: The relative retention time RSD% of the 14 common peaks was less than 2.0; the relative peak area RSD% was less than 2.0, indicating that the sample solution was stable within 36 hours.

[0061] Example 3 Common Peaks and Peak Attribution

[0062] The 9 medicinal materials in the prescription of Agui Yangxue Preparation were separately prepared into test sample solutions according to the method of Example 2 above, and analyzed separately according to the above chromatographic conditions. The chromatograms of the Agui Yangxue Preparation sample (180501) and each medicinal material were compared to find out the peak attribution.

[0063] Using the paeoniflorin reference substance as a reference, record the relative retention time of the common peaks. There are 14 common peaks in the fingerprint. See Table 1 below. Figure 2 .

[0064] Table 1 Fingerprint peak attribution

[0065]

[0066] Figure 2 Agui nourishing blood preparation batch 180501 was provided as test sample.

[0067] Example 6: Fingerprint Similarity Evaluation

[0068] Prepare 10 batches of Agui Yangxue preparations (batch numbers: 180501, 180502, 180503, 180601, 180602, 180603, 180604, 180701, 180702, 180703) respectively, prepare the test solution separately according to the above method, and record the chromatogram (see Figure 2 ), the HPLC chromatographic data of 10 batches of Agui Yangxue preparation were imported using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System Version A" software issued by the State Pharmacopoeia Committee, and the common pattern of the control fingerprint was generated using the average method.

[0069] Table 2 Similarity evaluation of fingerprint spectra of 10 batches of Agui Yangxue preparations

[0070]

[0071] The similarity of the spectra of 10 batches of Agui Yangxue preparation was compared with that of the reference spectrum of Example 5. According to the angle cosine method and the correlation coefficient method, the similarity of the relative retention time between the spectra of different batches was above 0.98.

Claims

1. A method for constructing a fingerprint spectrum of a quince blood-nourishing preparation, the quince blood-nourishing preparation comprising: Angelica 386g, Codonopsis pilosula 24g, White Peony Root 24g, Radix Glycyrrhizae Preparata 12g, Poria 24g, Astragalus 24g, Rehmannia glutinosa 24g, Ligusticum chuanxiong 12g, Donkey-hide Gelatin 24g, characterized in that the fingerprint is the HPLC characteristic fingerprint of Donkey-hide Gelatin. The construction method of the fingerprint is: (1) Preparation of test solution: Take 1-4 g of the Agui Yangxue preparation or its contents, weigh accurately, place in a conical flask, add 50-100 ml of methanol accurately, weigh the weight, extract by ultrasonic for 0.5-1 hour, cool, weigh again, add methanol to make up the weight, shake well, filter, and take the filtrate to obtain the solution; (2) Preparation of reference solution: Take 5-10 mg of paeoniflorin reference substance and place it in a 50 ml volumetric flask. Add methanol to dissolve it and dilute it to the mark. Shake well. Accurately pipette 5 ml and place it in a 25 ml volumetric flask. Add methanol to dilute it to the mark. Shake well. (3) Determination: Accurately pipette 5-15 μl of the reference solution and the test solution respectively, inject into the high performance liquid chromatograph, record the chromatogram within 70 minutes, and process the chromatogram with the fingerprint software to obtain the fingerprint of the Agui Yangxue Preparation; The chromatographic conditions for the fingerprint high performance liquid chromatography determination are as follows: octadecylsilane bonded silica gel as filler, acetonitrile as mobile phase A, acetonitrile-30mmol / L triethylamine (5:95) as mobile phase B, gradient elution, flow rate 0.5~1.5ml·min-1, column temperature 25~40°C, detection wavelength 185~400nm; the theoretical plate number calculated based on the paeoniflorin peak should be no less than 2500; the gradient elution process for the fingerprint high performance liquid chromatography determination is as follows: 。 2. The method for constructing the fingerprint spectrum of the Agui nourishing blood preparation according to claim 1, characterized in that: There are 14 common peaks in the spectrum, with No. 4 as the reference peak. The relative retention times and relative peak areas of the 14 common peaks are shown in the following table: 。

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