Establishment method of specific chromatogram of angelica sinensis blood-enriching oral liquid as well as specific chromatogram and application of specific chromatogram

Abstract

The invention provides an establishment method of a specific chromatogram of an angelica sinensis blood-enriching oral liquid as well as the specific chromatogram and application of the angelica sinensis blood-enriching oral liquid, and the establishment method is characterized by comprising the following steps: taking ferulic acid, calycosin, calycosin glucoside, formononin and ligustilide reference substances, and adding a methanol solution to prepare a reference substance solution; adding a methanol solution into the angelica sinensis blood-enriching oral liquid, filtering, and taking filtrate to prepare a test solution; a reference substance solution and a test solution are taken respectively, a high performance liquid chromatograph is adopted for detection, a mobile phase A is an acetonitrile solution, a mobile phase B is a phosphoric acid aqueous solution with the volume concentration being 0.05%-0.2%, elution is conducted according to an elution procedure, and the specific chromatogram of the angelica sinensis blood replenishing oral liquid is established through chromatographic analysis. According to the method, the peak corresponding to the calycosin-7-glucoside peak is determined as the S peak, and the specific chromatogram of the angelica sinensis blood-enriching oral liquid is evaluated by adopting the relative retention time, so that the quality of the angelica sinensis blood-enriching oral liquid can be detected and evaluated comprehensively, objectively and accurately.

Description

Technical Field

[0001] The present invention relates to the field of quality inspection, and particularly relates to a method for establishing a characteristic chromatogram of Danggui Buxue Oral Liquid, the characteristic chromatogram thereof, and an application thereof. Background Art

[0002] Danggui Buxue Oral Liquid is a traditional Chinese medicine preparation, which is a brownish-yellow to yellowish-brown liquid with a fragrant smell, a sweet and slightly pungent taste. This product is composed of two traditional Chinese medicines, Angelica sinensis and Astragalus membranaceus, and has the efficacy of nourishing qi and blood, and is applicable to the syndrome of qi and blood deficiency. In the prior art, the content of astragaloside IV in Danggui Buxue Oral Liquid is determined by high performance liquid chromatography, which is used as an index component for evaluating the quality control of this preparation. The evaluation index of this detection method is relatively single, and it cannot fully detect and evaluate the quality and efficacy of Danggui Buxue Oral Liquid comprehensively. In addition, for the medicinal materials from different origins and qualities, there will be batch differences in the detected index components, which will affect the final quality evaluation results. Therefore, in order to objectively reflect the types of chemical components contained in Danggui Buxue Oral Liquid and better control the production quality of this preparation, at present, a detection method for establishing the characteristic chromatogram of Danggui Buxue Oral Liquid is needed, which can detect and evaluate the quality of Danggui Buxue Oral Liquid more comprehensively, objectively and accurately, and ensure the efficacy of the product. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide a method for establishing a characteristic chromatogram of Danggui Buxue Oral Liquid, the characteristic chromatogram thereof, and an application thereof, which can detect and evaluate the quality of Danggui Buxue Oral Liquid more comprehensively, objectively and accurately, and ensure the efficacy of the product.

[0004] The technical solution of the present invention is realized as follows:

[0005] A method for constructing a characteristic chromatogram of Danggui Buxue Oral Liquid includes the following steps:

[0006] (1) Take ferulic acid, calycosin, calycosin-7-O-β-D-glucoside, ononin, and ligustilide reference substances, add them to a methanol solution to prepare a reference substance solution;

[0007] (2) Take Danggui Buxue Oral Liquid, add methanol, filter, and take the filtrate to prepare a test sample solution;

[0008] (3) Take the reference substance solution and the test sample solution respectively, and perform detection using a high performance liquid chromatograph. Among them, mobile phase A is an acetonitrile solution, and mobile phase B is an aqueous phosphoric acid solution with a volume concentration of 0.05% - 0.2%;

[0009] The elution procedure is as follows:

[0010]

[0011] (4) Perform high performance liquid chromatography (HPLC) analysis on the reference solution and the test solution. Select a representative compound peak from the common peaks of the test solution of Danggui Buxue Oral Liquid as the characteristic peak, and establish the characteristic fingerprint of Danggui Buxue Oral Liquid.

[0012] A further solution is that the conditions for high performance liquid chromatography are as follows:

[0013] Chromatographic column: C18 chromatographic column;

[0014] Column temperature: 27 - 33 °C;

[0015] Sample injection volume: 8 - 12 μL;

[0016] Flow rate: 0.8 - 1.2 mL / min.

[0017] A further solution is that in step (1), a methanol solution is added to prepare a solution containing 22 - 28 μg of the reference substance per 1 mL, and the reference solution is prepared.

[0018] A further solution is that in step (2), 4.5 - 5.5 mL of Danggui Buxue Oral Liquid is taken, added with a methanol solution to dissolve and dilute to 10 mL, filtered, and the filtrate is taken to prepare the test solution.

[0019] A further solution is that the characteristic fingerprint of Danggui Buxue Oral Liquid includes 8 characteristic peaks; among them, peak 2 is the peak of calycosin - 7 - O - glucoside, peak 3 is the peak of ferulic acid, peak 6 is the peak of ononin, peak 7 is the peak of calycosin, and peak 8 is the peak of ligustilide.

[0020] An even further solution is that taking peak 2 as the reference S peak, calculate the relative retention time of each peak. The specified values of the relative retention time are: peak 1 is 0.76, peak 2 is 1.00, peak 3 is 1.08, peak 4 is 1.25, peak 5 is 1.40, peak 6 is 1.47, peak 7 is 1.72, and peak 8 is 3.14.

[0021] On the one hand, the present invention provides a quality inspection method for Danggui Buxue Oral Liquid, and the quality inspection method includes the following steps:

[0022] S1: Use the characteristic fingerprint of Danggui Buxue Oral Liquid obtained by the construction method as described above, take the calycosin - 7 - O - glucoside peak of peak 2 as the reference S peak, and calculate the relative retention time of each peak;

[0023] S2: Compare the relative retention time obtained in step S1 with the relative retention time of the characteristic peaks in the characteristic fingerprint of Danggui Buxue Oral Liquid constructed by the construction method as described above;

[0024] S3: Judge whether the quality of the Danggui Buxue Oral Liquid to be detected meets the standard according to the comparison result of step S2.

[0025] On the other hand, the present invention also provides an application of the aforementioned construction method in the identification and quality detection of Danggui Buxue Oral Liquid products.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] The method for establishing the characteristic chromatogram of Danggui Buxue Oral Liquid provided by the present invention obtains the chemical components of 5 characteristic peaks, including ferulic acid, calycosin, calycosin-7-O-β-D-glucoside, ononin, ligustilide, etc. By the separation and retention time of the characteristic peaks, it is determined that the peak corresponding to the calycosin-7-O-β-D-glucoside peak is the S peak. Using the relative retention time to evaluate the characteristic chromatogram of Danggui Buxue Oral Liquid can more comprehensively, objectively and accurately detect and evaluate the quality of Danggui Buxue Oral Liquid.

[0028] The method for establishing the characteristic chromatogram of Danggui Buxue Oral Liquid provided by the present invention has the advantages of high precision, good repeatability, high stability and good durability. The method is simple and can effectively provide a certain reference basis for the quality control of the production process of Danggui Buxue Oral Liquid. Description of the Drawings

[0029] Figure 1 is the characteristic chromatogram of Danggui Buxue Oral Liquid;

[0030] Figure 2 is the chromatogram of the negative reference substance of Danggui;

[0031] Figure 3 is the chromatogram of the negative reference substance of Astragalus membranaceus;

[0032] Figure 4 is the control chromatogram of the reference substance solution;

[0033] Figure 5 is the chromatogram of the test sample under the detection wavelength of 210 nm;

[0034] Figure 6 is the chromatogram of the test sample under the detection wavelength of 230 nm;

[0035] Figure 7 is the chromatogram of the test sample under the detection wavelength of 250 nm;

[0036] Figure 8 is the chromatogram of the test sample under the detection wavelength of 300 nm;

[0037] Figure 9 is the chromatogram of the test sample under the detection wavelength of 320 nm;

[0038] Figure 10 is the chromatogram of the test sample under the detection wavelength of 350 nm;

[0039] Figure 11It is the chromatogram of the test sample at a detection wavelength of 380 nm;

[0040] Figure 12 It is the chromatogram of the test sample under gradient condition 1 in Table 3;

[0041] Figure 13 It is the chromatogram of the test sample under gradient condition 2 in Table 4;

[0042] Figure 14 It is the chromatogram of the test sample under gradient condition 3 in Table 5. Specific Embodiments

[0043] To better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0044] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0045] The materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial channels unless otherwise specified.

[0046] Example 1

[0047] The method for establishing the characteristic fingerprint of Danggui Buxue Oral Liquid is as follows:

[0048] 1. Instruments and Test Drugs

[0049] 1.1 Instruments

[0050] Waters Arc high performance liquid chromatograph (Waters Technology (Shanghai) Co., Ltd., serial number: JY-041); electronic balance (Sartorius Technology Instruments (Beijing) Co., Ltd.); chromatographic column: Agilent SB-C18 (4.6×250 mm, 5 μm) SN: USCL134513.

[0051] 1.2 Reagents

[0052] Acetonitrile (chromatographic grade, Shanghai Xingke High Purity Solvents Co., Ltd.);

[0053] Methanol (analytical grade, Tianjin Fuyu Fine Chemical Co., Ltd.);

[0054] Phosphoric acid (analytical grade, Tianjin Kemiou Fine Chemical Co., Ltd.);

[0055] 1.3 Samples for Research

[0056] Pilot-scale samples of Danggui Buxue Oral Liquid (Fushoutang Pharmaceutical Co., Ltd. in Henan Province, batch numbers: 240401, 240402, 240403, 240404).

[0057] 2. Experimental Part

[0058] 2.1, Chromatographic Conditions

[0059] Using octadecylsilane chemically bonded silica gel as the packing material (Agilent SB-C18, column length 25 cm, inner diameter 4.6 mm, particle size 5 μm); using acetonitrile as mobile phase A and 0.1% phosphoric acid as mobile phase B, perform gradient elution according to the regulations in the following table; flow rate is 1.0 mL / min, detection wavelength is 300 nm, and column temperature is 30 °C. The number of theoretical plates calculated based on the ferulic acid peak should be not less than 3000.

[0060] Table 1 Gradient Elution Table

[0061]

[0062] 2.2, Preparation of Reference Substance Solution

[0063] Accurately weigh appropriate amounts of ferulic acid, calycosin, calycosin-7-O-β-D-glucoside, ononin, and ligustilide reference substances, add methanol, and prepare a solution containing 25 μg per 1 mL, that is obtained.

[0064] 2.3, Preparation of Test Solution

[0065] Take 5 mL of Danggui Buxue Oral Liquid, dilute with methanol and make up the volume to 10 mL, shake well, filter, and take the subsequent filtrate, that is obtained.

[0066] 3, Results and Discussion

[0067] 3.1, Screening of Chromatographic Conditions

[0068] This method optimizes conditions such as detection wavelength (210 nm, 230 nm, 250 nm, 300 nm, 320 nm, 350 nm, 380 nm), mobile phase gradient, etc. Finally, the determined chromatographic conditions are: using octadecylsilane chemically bonded silica gel as the packing material; using acetonitrile as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B, flow rate is 1.0 mL / min, detection wavelength is 300 nm; column temperature is 30 °C.

[0069] 3.1.1 Screening of Detection Wavelength

[0070] Using octadecylsilane chemically bonded silica gel as the packing material; using acetonitrile as mobile phase A and 0.1% phosphoric acid as mobile phase B, perform gradient elution according to the regulations in the following table; flow rate is 1.0 mL / min, column temperature is 30 °C.

[0071] Table 2 Gradient Elution Table

[0072]

[0073] 3.1.2 Screening of Gradient Elution Conditions

[0074] Using octadecylsilane chemically bonded silica as the filler; using acetonitrile as mobile phase A and 0.1% phosphoric acid as mobile phase B, gradient elution was carried out according to the regulations in the following table; the flow rate was 1.0 mL / min, the detection wavelength was 300 nm; the column temperature was 30 °C.

[0075] Table 3 Gradient Condition 1 Elution Table

[0076]

[0077]

[0078] Table 4 Gradient Condition 2 Elution Table

[0079]

[0080] Table 5 Gradient Condition 3 Elution Table

[0081]

[0082] 3.2. Screening of Preparation Conditions for Test Samples

[0083] 3.2.1 Investigation of Extraction Methods

[0084] Method 1: Take 5 mL of Danggui Buxue Oral Liquid, dilute it to 10 mL with methanol, shake well, filter, and take the subsequent filtrate to obtain the sample solution.

[0085] Method 2: Take 5 mL of Danggui Buxue Oral Liquid, evaporate to dryness, dissolve the residue in methanol and dilute to 10 mL, shake well, filter, and take the subsequent filtrate to obtain the sample solution.

[0086] Method 3: Take 5 mL of Danggui Buxue Oral Liquid, extract it 3 times with n-butanol saturated with water, 10 mL each time. Combine the n-butanol layers, add 30 mL of ammonia test solution, shake well, separate the n-butanol layer, evaporate to dryness, dissolve the residue in an appropriate amount of methanol and transfer it to a 10 mL volumetric flask, dilute to the mark with methanol, shake well, filter, and take the subsequent filtrate to obtain the sample solution.

[0087] Table 6 Investigation Table of Extraction Methods

[0088]

[0089] Conclusion: The peak area of direct dilution with methanol is greater than that of dissolution after evaporation to dryness, and the number of peaks is much more than that of n-butanol extraction. Therefore, methanol was selected as the dilution solvent.

[0090] 3.2.2 Investigation of Different Sampling Volumes

[0091] Respectively take 3 mL, 5 mL, and 7 mL of Danggui Buxue Oral Liquid, dilute to 10 mL with methanol, and analyze according to the chromatographic conditions under item "3.1" (see Table 7). The results show that (after converting to the same sampling amount for comparison of peak areas), the peak areas of the labeled peaks are basically the same, and finally a sampling amount of 5 mL is selected.

[0092] Table 7 Investigation Table of Sampling Amount

[0093]

[0094] Conclusion: When the sampling amount is 3 mL, the peak area is relatively small, and when it is 7 mL, it is inconvenient to sample. Therefore, a sampling amount of 5 mL is selected.

[0095] 3.3. Methodological Verification of Characteristic Chromatogram

[0096] 3.3.1 Investigation of Instrument Precision

[0097] Take the reference substance solution and analyze according to the chromatographic conditions under item "2.1" (inject in parallel 6 times). The results show that the RSD of the relative retention times of 9 characteristic peaks ≤ 6.0%, indicating good instrument precision for this method (see Table 8).

[0098] Table 8 Retention Time and Relative Retention Time Table for Investigation of Instrument Precision of Characteristic Chromatogram of Danggui Buxue Oral Liquid

[0099]

[0100] 3.3.2 Investigation of Repeatability

[0101] Take 6 samples of Danggui Buxue Oral Liquid, prepare the test solution according to the method under item "2.3", and analyze according to the chromatographic conditions under item "2.1". The results show that the RSD of the relative retention times of 9 characteristic peaks ≤ 6.0%, indicating good repeatability for this method (see Table 9).

[0102] Table 9 Retention Time and Relative Retention Time Table for Investigation of Repeatability of Characteristic Chromatogram of Danggui Buxue Oral Liquid

[0103]

[0104] 3.3.3 Investigation of Stability

[0105] Take the sample of Danggui Buxue Oral Liquid, prepare the test solution according to the method under item "2.3", and inject for analysis at 0, 6, 12, 24, 48, and 72 hours respectively according to the chromatographic conditions under item "2.1". The results show that the RSD of the relative retention times of 9 characteristic peaks ≤ 6.0%, indicating good stability of the test solution within 72 hours (see Table 10).

[0106] Table 10 Retention Time and Relative Retention Time Table for Investigation of Stability of Characteristic Chromatogram of Danggui Buxue Oral Liquid

[0107]

[0108] 3.3.4 Intermediate precision investigation

[0109] Six samples of Danggui Buxue Oral Liquid were taken, and the test solution was prepared according to the method under "2.3". Analysis was carried out according to the chromatographic conditions under "2.1". The results showed that the relative retention time RSD of the 9 characteristic peaks ≤ 6.0%, and the relative retention time RSD of the 9 characteristic peaks using different instruments ≤ 6.0. The intermediate precision of this method was good (see Table 11).

[0110] Table 11 Retention time and relative retention time table for intermediate precision investigation of the characteristic chromatogram of Danggui Buxue Oral Liquid

[0111]

[0112] 3.3.5 Robustness investigation

[0113] 3.3.5.1 Different column temperatures

[0114] Samples of Danggui Buxue Oral Liquid were taken, and the test solution was prepared according to the method under "2.3". Analysis was carried out according to the chromatographic conditions under "2.1" with the column temperature set at 27 °C, 30 °C, and 33 °C respectively. The results showed that the relative retention time RSD of the 9 characteristic peaks ≤ 6.0% (see Table 12).

[0115] Table 12 Retention time and relative retention time table for robustness investigation of the characteristic chromatogram of Danggui Buxue Oral Liquid at different column temperatures

[0116]

[0117]

[0118] 3.3.5.2 Different flow rates

[0119] Samples of Danggui Buxue Oral Liquid were taken, and the test solution was prepared according to the method under "2.3". Analysis was carried out according to the chromatographic conditions under "2.1" with the flow rate set at 0.8 mL / min, 1.0 mL / min, and 1.2 mL / min respectively. The results showed that the relative retention time RSD of the 9 characteristic peaks ≤ 6.0% (see Table 13).

[0120] Table 13 Retention time and relative retention time table for robustness investigation of the characteristic chromatogram of Danggui Buxue Oral Liquid at different flow rates

[0121]

[0122] 3.3.5.3 Different acid concentrations

[0123] Take the sample of Danggui Buxue Oral Liquid, prepare the test solution according to the method under "2.3", and analyze it by using 0.05%, 0.10%, and 0.20% phosphoric acid as the aqueous phase respectively under the chromatographic conditions under "2.1". The results show that the RSD of the relative retention times of 9 characteristic peaks ≤ 6.0% (see Table 14).

[0124] Table 14 Retention Times and Relative Retention Time Table for the Durability Investigation of Different Acid Concentrations in the Characteristic Chromatogram of Danggui Buxue Oral Liquid

[0125]

[0126]

[0127] 3.3.5.4 Different Chromatographic Columns

[0128] Take the sample of Danggui Buxue Oral Liquid, prepare the test solution according to the method under "2.3", and analyze it by using different chromatographic columns respectively under the chromatographic conditions under "2.1". The results show that the RSD of the relative retention times of 9 characteristic peaks ≤ 6.0% (see Table 15).

[0129] Table 15 Retention Times and Relative Retention Time Table for the Durability Investigation of Different Chromatographic Columns in the Characteristic Chromatogram of Danggui Buxue Oral Liquid

[0130]

[0131] 3.3.5.5 Different Instruments

[0132] Take the sample of Danggui Buxue Oral Liquid, prepare the test solution according to the method under "2.3", and analyze it by using different instruments respectively under the chromatographic conditions under "2.1". The results show that the RSD of the relative retention times of 9 characteristic peaks ≤ 6.0% (see Table 16).

[0133] Table 16 Retention Times and Relative Retention Time Table for the Durability Investigation of Different Instruments in the Characteristic Chromatogram of Danggui Buxue Oral Liquid

[0134]

[0135]

[0136] 3.3.5.6 Peak Attribution and Peak Identification

[0137] Take the samples of Danggui Buxue Oral Liquid, prepare the reference substance solutions of ferulic acid, calycosin, calycosin-7-O-β-D-glucoside, ligustilide, and ononin according to the method under "2.2", prepare the test solution and negative test solution according to the method under "2.3" (prepare the negative test solutions lacking Angelica sinensis and Astragalus membranaceus respectively according to the preparation method of Danggui Buxue Oral Liquid), and analyze according to the chromatographic conditions under "3.1". The results show that the compound in peak 1 is unknown and belongs to the Angelica sinensis medicinal material; peak 2 is calycosin-7-O-β-D-glucoside and belongs to the Astragalus membranaceus medicinal material; the compound in peak 3 is ferulic acid and belongs to the Angelica sinensis medicinal material; the compound in peak 4 is unknown and belongs to both Angelica sinensis and Astragalus membranaceus; the compound in peak 5 is unknown and belongs to the Angelica sinensis medicinal material; the compound in peak 6 is ononin and belongs to the Astragalus membranaceus medicinal material; the compound in peak 7 is calycosin and belongs to the Astragalus membranaceus medicinal material; the compound in peak 8 is ligustilide and belongs to the Angelica sinensis medicinal material. See Table 17. Figure 1 。

[0138] Table 17 Results of Peak Attribution of the Characteristic Chromatogram of Danggui Buxue Oral Liquid

[0139]

[0140] 3.4. Sample Determination

[0141] Take the samples of the trial production batches and 3 batches of pilot production samples, prepare the test solutions according to the method under "2.3", and analyze them using different instruments according to the chromatographic conditions under "2.1". The results show that there are 9 characteristic peaks in the characteristic chromatogram. The peak corresponding to the reference peak is the S peak. Calculate the relative retention times of each characteristic peak and the S peak, which should be within ±10% of the specified values. The specified values of the retention times are: 0.76 (peak 1), 1.00 [peak 2 (S)], 1.08 (peak 3), 1.25 (peak 4), 1.40 (peak 5), 1.47 (peak 6), 1.72 (peak 7), 3.14 (peak 8). ( Figure 1 )。

[0142] Table 18 Retention Times and Relative Retention Times of Different Batch Samples of the Characteristic Chromatogram of Danggui Buxue Oral Liquid

[0143]

[0144]

[0145] Method: Determine according to the high performance liquid chromatography method (General Rule 0512).

[0146] 3.4.1 Chromatographic Conditions and System Suitability Test

[0147] Using octadecylsilyl silica gel as the filler (column length: 25 cm, inner diameter: 4.6 mm, particle size: 5 μm, Agilent HC-C18 chromatographic column); using acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, perform gradient elution according to the regulations in the following table; the flow rate is 1.0 mL per minute; the column temperature is 30 °C; the detection wavelength is 300 nm. The number of theoretical plates calculated based on the calycosin-7-O-β-D-glucoside peak should be not less than 6000.

[0148] Table 19 Gradient Elution Table

[0149]

[0150] 3.4.2 Preparation of Reference Substance Solution

[0151] Take appropriate amounts of calycosin-7-O-β-D-glucoside reference substance, ferulic acid reference substance, ononin reference substance, calycosin reference substance, and ligustilide reference substance, weigh accurately, and dissolve them in methanol respectively to prepare solutions containing 20 μg per 1 mL. Record the chromatogram to obtain the solution.

[0152] 3.4.3 Preparation of Test Solution

[0153] Precisely measure 5 mL of Danggui Buxue Oral Liquid, place it in a 10 mL volumetric flask, dilute it to the mark with methanol, and shake well to obtain the solution.

[0154] 3.4.4 Determination Method

[0155] Precisely absorb 10 μL of the reference substance solution and the test solution respectively, inject them into the liquid chromatograph for determination to obtain the results.

[0156] The test solution characteristic chromatogram should show 8 characteristic peaks, among which 5 peaks should have the same retention time as the corresponding reference substance peaks; the peak corresponding to the calycosin-7-O-β-D-glucoside reference substance is the S peak. Calculate the relative retention time of each characteristic peak with respect to the S peak, and the relative retention time should be within ±5% of the specified value; the specified values are: 0.76 (peak 1), 1.00 [peak 2 (S)], 1.08 (peak 3), 1.25 (peak 4), 1.40 (peak 5), 1.47 (peak 6), 1.72 (peak 7), 3.14 (peak 8). ( Figure 1 )

[0157] The above is the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications are also considered within the protection scope of the present invention.

Claims

1. A method for constructing a characteristic spectrum of Danggui Buxue Oral Liquid, characterized in that: The steps include: (1) Take ferulic acid, calycosin, calycosin glucoside, formononetin, and ligustilide reference substances, add methanol solution, and prepare reference substance solution; (2) Take Danggui Buxue Oral Liquid, add methanol, filter, and take the filtrate to prepare the test solution; (3) taking the reference solution and the test solution respectively, and detecting them by high performance liquid chromatography, wherein the mobile phase A is an acetonitrile solution, and the mobile phase B is a 0.05% to 0.2% phosphoric acid aqueous solution by volume concentration; The elution procedure is as follows: (4) The reference solution and the test solution were subjected to HPLC analysis, and representative compound peaks were selected from the common peaks of the Danggui Buxue Oral Liquid test solution as characteristic peaks to establish a characteristic spectrum of the Danggui Buxue Oral Liquid.

2. The method for constructing a characteristic spectrum of Danggui Buxue Oral Liquid according to claim 1, characterized in that: The conditions for HPLC were as follows: Chromatographic column: C18 column; Column temperature: 27-33°C; Injection volume: 8-12 μL; Flow rate: 0.8~1.2mL / min.

3. The method for constructing a characteristic spectrum of Danggui Buxue Oral Liquid according to claim 1, characterized in that: In step (1), methanol solution is added to prepare a solution containing 22 to 28 μg of the reference substance per 1 mL to prepare a reference substance solution.

4. The method for constructing a characteristic spectrum of Danggui Buxue Oral Liquid according to claim 1, characterized in that: In step (2), 4.5-5.5 mL of Angelica Blood-Replenishing Oral Liquid is taken, dissolved in methanol solution and diluted to 10 mL, filtered, and the filtrate is taken to prepare a test solution.

5. The method for constructing a characteristic spectrum of Danggui Buxue Oral Liquid according to claim 1, characterized in that: The characteristic spectrum of the Angelica Blood-Replenishing Oral Liquid includes 8 characteristic peaks, wherein peak 2 is the peak of calycosin glucoside, peak 3 is the peak of ferulic acid, peak 6 is the peak of formononetin, peak 7 is the peak of calycosin, and peak 8 is the peak of ligustilide.

6. The method for constructing a characteristic spectrum of Danggui Buxue Oral Liquid according to claim 5, characterized in that: Taking Peak 2 as the reference S peak, the relative retention time of each peak was calculated. The specified values ​​of relative retention time were: Peak 1 was 0.76, Peak 2 was 1.00, Peak 3 was 1.08, Peak 4 was 1.25, Peak 5 was 1.40, Peak 6 was 1.47, Peak 7 was 1.72, and Peak 8 was 3.

14.

7. A quality detection method for Danggui Buxue Oral Liquid, characterized in that: The quality detection method comprises the following steps: S1: using the characteristic spectrum of Danggui Buxue Oral Liquid obtained by the construction method according to any one of claims 1 to 6, taking the peak 2 calycosin glucoside as the reference S peak, calculating the relative retention time of each peak; S2: comparing the relative retention time obtained in step S1 with the relative retention time of the characteristic peak in the characteristic spectrum of Danggui Buxue Oral Liquid constructed by the construction method according to any one of claims 1 to 7; S3: judging whether the quality of the Angelica Blood-Replenishing Oral Liquid to be tested meets the standard according to the comparison result of step S2.

8. Application of the construction method according to any one of claims 1 to 6 in the identification and quality inspection of Danggui Buxue Oral Liquid products.

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