Establishment method of fingerprint of xueshu sheng granules and fingerprint thereof

Abstract

The present application relates to a kind of blood speed upgrade granule fingerprint's establishment method, comprising: S1, take multiple batches blood speed upgrade granule preparation test sample solution;Take icariin control preparation reference sample solution;S2, using octadecylsilane bonded silica as the chromatographic column of filler, detection wavelength is 270nm, flow rate is 0.8-1.2ml / min, column temperature is 25-35 ℃;Mobile phase A is acetonitrile, mobile phase B is 0.2% formic acid solution, gradient elution is carried out;Injection analysis obtains the liquid chromatogram of multiple batches test sample solution and reference sample solution;S3, the liquid chromatogram of different batches test sample solution is introduced into traditional Chinese medicine chromatographic fingerprint similarity evaluation system, the chromatographic peak of each batch test sample is selected to generate blood speed upgrade granule control fingerprint, with icariin peak as reference peak, the relative peak area and relative retention time of the rest each common peak are calculated.The present application has the advantages of good repeatability, stability and precision, and the established fingerprint can comprehensively control and evaluate the quality of blood speed upgrade product.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of traditional Chinese medicine preparation analysis, and particularly relates to a method for establishing a fingerprint of Xueshusheng granules and the fingerprint. BACKGROUND

[0002] The formula of Xueshusheng granules is: angelica, astragalus, epimedium, caudate vitex, donkey-hide gelatin and hawthorn. In addition to the above main components, Xueshusheng granules also contain sucrose, dextrin and other auxiliary materials, which help the molding of the medicine and improve the taste. The function of Xueshusheng granules is mainly to benefit qi and warm yang, nourish blood and activate blood, and it is suitable for anemia and various blood loss diseases caused by deficiency of qi and blood. It can be used for the treatment of anemia due to malnutrition, anemia due to iron deficiency, rehabilitation after blood loss, induced abortion, postpartum blood supplementation, adjuvant therapy after tumor radiotherapy and chemotherapy, and rehabilitation after various surgeries.

[0003] At present, there is no method for detecting the content of effective components of Xueshusheng granules in various standards, and the quality standard is not recorded in the pharmacopoeia. Only the prescription and quality standard of Xueshusheng granules are recorded in the seventeenth volume of traditional Chinese medicine formula preparation, but the quality standard is low, there is no detection method to control the content, and the medicine of Xueshusheng granules is complex in flavor and composition. The analysis of individual components has certain one-sidedness, and it is not enough to only characterize and control one or two chemical components. It is difficult to fully reflect the information of the medicine, and the use amount of precious medicinal materials cannot be strictly evaluated and controlled, which is not conducive to the quality control of Xueshusheng granules.

[0004] Among the current domestic literature reports on the determination of multiple indicators of Xueshusheng, “Quality Evaluation of Xueshusheng Granules Based on HPLC Multi-index Component Combined with Chemometrics [J]. Modern Medicine and Clinical, 2022, 37(11)” uses high performance liquid chromatography to simultaneously determine the content of 10 components, and establishes a quality evaluation method for Xueshusheng granules. However, the article requires the use of double wavelength for quality control, which has high requirements for instrument facilities. The reference substances include 10 reference substances such as baohuoside I, vitexin, icariin, calycosin-7-glucoside, vitexin glucoside, and vitexin rhamnoside. The purchase cost is high, expensive and not easy to obtain. The cost of detecting the overall quality of the medicine is large. The 10 reference substances need to be accurately weighed and prepared, which is complex and has a large workload. In the detection index of the literature, the content of 8 components is very low (less than 0.1%), which has a great risk for the quality standard control in the actual production process. The content of some components may be lower than the detection limit due to slight fluctuations in the batch of medicinal materials or the production environment, resulting in unqualified product quality, which is not conducive to the quality monitoring in the actual production process and has poor production applicability.

[0005] In order to carry out the blood speed upgrade particle quality standard promotion research, gradually improve its quality control standard, improve the product quality and market competitiveness, it is necessary to build the characteristic fingerprint of blood speed upgrade particle, for comprehensive evaluation of blood speed upgrade particle quality, analysis of the product contains more ingredients, realize the quality control of different batches of blood speed upgrade particle product, ensure the product quality and curative effect. SUMMARY

[0006] (1) The technical problems to be solved

[0007] In view of the above-mentioned defects, deficiencies of the prior art, the present application provides a method for establishing the fingerprint of blood speed upgrade particles and the fingerprint thereof, which solves the problem of lack of overall evaluation standard for the quality control of blood speed upgrade particles, and can effectively ensure the stability, consistency and controllability of the quality of blood speed upgrade particles.

[0008] (2) Technical scheme

[0009] In the first aspect, the present application provides a method for establishing the fingerprint of blood speed upgrade particles, which comprises the following steps:

[0010] S1, preparing test sample solution and reference material solution:

[0011] The test sample solution is prepared by finely grinding multiple batches of blood speed upgrade particles, precisely quantifying and dissolving in 50% methanol to obtain a clear test sample solution.

[0012] The reference material solution is prepared by precisely quantifying icariin reference substance and dissolving in 50% methanol to obtain icariin reference material solution.

[0013] S2, setting high performance liquid chromatography conditions:

[0014] The chromatographic column adopts octadecylsilane bonded silica gel as the filler, the detection wavelength is 270nm, the flow rate is 0.8-1.2ml / min, the column temperature is 25-35℃, the mobile phase A is acetonitrile, the mobile phase B is 0.2% formic acid solution, gradient elution is carried out, the volume ratio of mobile phase A and B changes as follows: 0-25min, A:B is 2%:98%→5%:95%; 25-35min, A:B is 5%:95%→10%:90%; 35-80min, A:B is 10%:90%→30%:70%; 80-100min, A:B is 30%:70%→35%:65%; 100-125min, A:B is 35%:65%→60%:40%;

[0015] 5-20μl of the test sample solution and the reference material solution of multiple batches of blood speed upgrade particles are precisely sucked respectively, injected into the high performance liquid chromatograph, and determined to obtain the liquid chromatogram of the test sample solution and the reference material solution respectively.

[0016] S3. Establishing the fingerprint spectrum of Xuesusheng Granules: The liquid chromatograms of different batches of test sample solutions were imported into the Chinese medicine chromatographic fingerprint spectrum similarity evaluation system. The chromatographic peaks common to each batch of test samples were selected to generate the reference fingerprint spectrum of Xuesusheng Granules. By comparing with the peaks of the reference solution, the icariin peak on the reference fingerprint spectrum was determined. Using the icariin peak as the reference peak, the relative peak area and relative retention time of the remaining common peaks were calculated.

[0017] According to a preferred embodiment of the present invention, in S1, the test solution is prepared by grinding the granules of Xuesusheng into a fine powder, accurately quantifying them, and then dissolving them in 50% methanol to obtain a clear solution with a concentration of 0.05-1 g / mL.

[0018] Preparation of reference solution: Take icariin reference standard, accurately quantify it, and dissolve it in methanol to obtain 30-80 μg / mL icariin reference solution.

[0019] According to a preferred embodiment of the present invention, in S1, the preparation of the test solution is as follows: Take Xuesusheng granules, grind them finely, weigh them accurately, place them in a stoppered conical flask, dissolve them by adding 12.5 mL of 50% methanol precisely for every 1 g of Xuesusheng granules, sonicate, cool, replenish the lost weight with 50% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0020] For example, take 2g of Xuesusheng granules, grind them into a fine powder, weigh them accurately, place them in a stoppered conical flask, add 25ml of 50% methanol accurately, weigh them, sonicate (power 400W, frequency 50kHz) for 30min, cool them, weigh them again, replenish the lost weight with 50% methanol, shake well, filter them, and take the filtrate to obtain the product.

[0021] According to a preferred embodiment of the present invention, in S1, the preparation of the reference solution is as follows: take an appropriate amount of icariin reference standard, accurately weigh it, and add 50% methanol to prepare a solution containing 50 μg per 1 ml.

[0022] According to a preferred embodiment of the present invention, in S2, the chromatographic column is an Agilent 5TC-C18 column with an inner diameter × length of 4.6 × 250 mm and a packing particle size of 5 μm.

[0023] According to a preferred embodiment of the present invention, in S2, the high-performance liquid chromatography conditions are: detection wavelength of 270 nm; flow rate of 1 ml / min; column temperature of 30 °C; and injection volume of 10 μl.

[0024] According to a preferred embodiment of the present invention, in S3, the fingerprint spectrum of Xuesusheng granules contains 12 common peaks. The peak numbers are marked in order of elution time. By comparing with the peaks of the reference solution, peak No. 10 on the fingerprint spectrum is determined to be the icariin peak. Using peak No. 10 as the reference peak, the relative peak area and relative retention time of the remaining common peaks are calculated.

[0025] Secondly, the present invention provides a fingerprint spectrum of Xuesusheng granules, comprising: 12 peaks, peak 10 being icariin with a relative retention time of 1.0000 and a relative peak area of ​​1.0000; the remaining peaks are: peak 1 with a relative retention time of 0.1451 and a relative peak area of ​​1.3846, peak 2 with a relative retention time of 0.1634 and a relative peak area of ​​0.4481, peak 3 with a relative retention time of 0.5201 and a relative peak area of ​​0.0600, peak 4 with a relative retention time of 0.5889 and a relative peak area of ​​0.2607, and peak 5 with a relative retention time of 0.7... Peak 5 with a relative peak area of ​​0.4933, peak 6 with a relative retention time of 0.8174 and a relative peak area of ​​0.2565, peak 7 with a relative retention time of 0.9592 and a relative peak area of ​​0.1436, peak 8 with a relative retention time of 0.9707 and a relative peak area of ​​0.1958, peak 9 with a relative retention time of 0.9830 and a relative peak area of ​​1.9460, peak 11 with a relative retention time of 1.3227 and a relative peak area of ​​0.2826, and peak 12 with a relative retention time of 1.3626 and a relative peak area of ​​0.0753.

[0026] Thirdly, the present invention provides a method for evaluating the quality of Xuesusheng granules, comprising: taking a batch of Xuesusheng granules, preparing a test solution according to the preparation method of the test solution, accurately injecting the test solution into a high-performance liquid chromatograph, and measuring to obtain the liquid chromatogram of the test solution; performing a similarity analysis between the liquid chromatogram and the fingerprint spectrum of the Xuesusheng granules; if the similarity is ≥0.90, the batch of products is judged to be of qualified quality, otherwise it is judged to be unqualified.

[0027] As will be understood by those skilled in the art, when evaluating the quality of a batch of Xuesusheng granules, the preparation method of the test solution for that batch of products shall refer to the preparation method of the test solution, and the chromatographic conditions shall refer to the high-performance liquid chromatography conditions used when establishing the fingerprint spectrum.

[0028] (III) Beneficial Effects

[0029] The technical effects of this invention include:

[0030] (1) The method for establishing the blood rapid increase particle fingerprint spectrum established in this invention is simple, reliable, stable, precise and reproducible.

[0031] (2) The present invention established a fingerprint spectrum of Xuesusheng granules and identified 12 common peaks. By comparing with the chromatographic peaks of the reference solution of icariin, one common characteristic peak, namely peak 10, was identified. Using peak 10 as the reference peak, the relative retention time and relative peak area of ​​the remaining 11 common peaks were determined.

[0032] (3) The fingerprint spectrum constructed by the present invention can be used for quality monitoring of Xuesusheng granules, which can control the quality of Xuesusheng granules in a holistic, comprehensive and effective manner, thereby ensuring the stability, reliability and safety of the product. Attached Figure Description

[0033] Figure 1 The HPLC chromatograms are for 10 batches of Xuesusheng Granules.

[0034] Figure 2 The HPLC fingerprint of the Xuesusheng granules established for this invention. Detailed Implementation

[0035] To better explain and facilitate understanding of the present invention, a detailed description of the invention is provided below with reference to the accompanying drawings and specific embodiments. The instruments and reagents used in the following embodiments are as follows:

[0036] 1. Instruments

[0037] Vanquish Core high-performance liquid chromatograph; Agilent 5TC-C18 (4.6×250mm, 5μm) column; JA5003N high-precision analytical balance; XSR-105DU analytical balance; KS-250E ultrasonic cleaner.

[0038] 2. Medicines and reagents

[0039] The drugs and reagents used in the following examples are as follows: Xuesusheng granules were provided by Hebei Yongfeng Pharmaceutical Co., Ltd., with batch numbers: 07823030112, 07823050112, 07823060112, 07823070112, 07823090112, 07823100112, 07824010112, 07824040112, 07824040212, and 07824040312. Icariin (110737-202017) was purchased from the China National Institutes for Food and Drug Control. Acetonitrile was of chromatographic grade; methanol and formic acid were of analytical grade.

[0040] Example 1

[0041] This embodiment provides a method for establishing the fingerprint spectrum of Xuesusheng granules, the steps of which are as follows:

[0042] (1) Preparation of test solution: Take 10 batches of Xuesusheng granules, grind them into fine powder, accurately weigh 2g of each batch, place them in a stoppered conical flask, accurately add 25ml of 50% methanol, weigh the sample, sonicate (power 400W, frequency 50kHz) for 30min, cool, weigh the sample again, replenish the lost weight with 50% methanol, shake well, filter, and take the filtrate to obtain the test solution of 10 batches of Xuesusheng granules.

[0043] (2) Preparation of reference solution: Take an appropriate amount of icariin reference standard, weigh it accurately, and add 50% methanol to prepare a solution containing 50 μg per ml.

[0044] (3) Determination: Accurately pipette the test solution and reference solution separately and inject them into the high-performance liquid chromatograph for determination. Record the chromatograms. The chromatographic conditions are as follows:

[0045] Chromatographic column: Agilent 5TC-C18 column (4.6×250mm, 5μm); detection wavelength: 270nm; flow rate: 1.0ml / min; column temperature: 30℃; injection volume: 10μl; mobile phase A: acetonitrile, mobile phase B: 0.2% formic acid solution, gradient elution method as follows (all percentages below refer to volume percentage):

[0046] Time (min) Mobile phase A (%) Mobile phase B (%) 0～25 2→5 98→95 25～35 5→10 95→90 35～80 10→30 90→70 80～100 30→35 70→65 100～125 35→60 65→40

[0047] (4) Construction of fingerprint chromatograms for Xuesusheng granules: The chromatograms of 10 batches of Xuesusheng granules were imported into the Chinese herbal medicine chromatographic fingerprint similarity evaluation system. Common chromatographic peaks from each batch of Xuesusheng granules were selected, and the median method was used to generate a reference fingerprint chromatogram (R) (e.g., ...). Figure 1 As shown in the figure, the reference fingerprint spectrum contains 12 common peaks. Peaks 1 to 12 in the reference fingerprint spectrum were labeled according to their elution order. By comparing the peaks of the reference fingerprint spectrum and the reference solution (icariin), peak 10 was determined to be the characteristic peak of icariin. Using peak 10 as the reference peak (the relative retention time and relative peak area of ​​the reference peak are 1.0000 and 1.0000 respectively), the relative peak areas and relative retention times of the remaining 11 common peaks were calculated, as shown in the figure. Figure 2 The HPLC fingerprint of Xuesusheng granules is shown. The relative retention times and peak areas of the 12 peaks in the HPLC fingerprint are as follows:

[0048] Relative retention times of peaks 1-12:

[0049] Peak 1: 0.1451; Peak 2: 0.1634; Peak 3: 0.5201; Peak 4: 0.5889;

[0050] Peak 5: 0.7048; Peak 6: 0.8174; Peak 7: 0.9592; Peak 8: 0.9707;

[0051] Peak 9: 0.9830; Peak 10(S): 1.0000; Peak 11: 1.3227; Peak 12: 1.3626;

[0052] Relative peak areas of peaks 1-12:

[0053] Peak 1: 1.3846; Peak 2: 0.4481; Peak 3: 0.0600; Peak 4: 0.2607;

[0054] Peak 5: 0.4933; Peak 6: 0.2565; Peak 7: 0.1436; Peak 8: 0.1958;

[0055] Peak 9: 1.9460; Peak 10(S): 1.0000; Peak 11: 0.2826; Peak 12: 0.0753.

[0056] (5) The similarity of the fingerprint spectra of 10 batches of Xuesusheng Granules was evaluated using the “Research Version of the Chromatographic Fingerprint Similarity Evaluation System for Traditional Chinese Medicine (2012A)”. The similarity was greater than 0.90. The results are shown in Table 1.

[0057] Table 1: Similarity of 10 batches of Xuesusheng Granules

[0058]

[0059] Example 2

[0060] In this embodiment, the measurement wavelength in step (3) was adjusted to 210nm, 240nm, 270nm, 310nm and 350nm respectively. The remaining conditions were the same as in Example 1. After HPLC detection, the number of chromatographic peaks in the chromatogram of the test solution is shown in Table 2.

[0061] Table 2: Number of chromatographic peaks at different test wavelengths

[0062]

[0063] As shown in Table 2, the number of chromatographic peaks obtained at wavelengths of 210 nm and 270 nm is the highest (10 chromatographic peaks). At the detection wavelength of 270 nm, the chromatographic peak response value is higher and the baseline is more stable. Therefore, 270 nm was selected as the detection wavelength.

[0064] In this embodiment, the mobile phase in step (3) was adjusted to methanol-water, acetonitrile-water, acetonitrile-0.1% formic acid solution, methanol-0.2% formic acid solution, and acetonitrile-0.2% formic acid solution, respectively. The remaining conditions were the same as in Example 1. After HPLC detection, the number of chromatographic peaks in the chromatogram of the test sample solution is shown in Table 3.

[0065] Table 3: Number of peaks detected in different mobile phase systems

[0066]

[0067] As shown in Table 3, the number of chromatographic peaks obtained when methanol-0.2% formic acid solution and acetonitrile-0.2% formic acid solution were selected as the mobile phase was the largest (12 chromatographic peaks). When acetonitrile-0.2% formic acid solution was used as the mobile phase, the chromatographic baseline was more stable and the separation between peaks was better. Therefore, acetonitrile-0.2% formic acid solution was selected as the mobile phase.

[0068] In this embodiment, the mobile phase in step (3) is adjusted as follows. The adjustment conditions are as follows. The remaining conditions are the same as those in Example 1. After HPLC detection, a suitable mobile phase ratio is selected.

[0069] Mobile phase condition 1

[0070]

[0071]

[0072] The experiment was conducted according to mobile phase condition 1. The chromatogram showed that the main peaks were mostly concentrated in the first ten minutes, and the peak resolution was poor.

[0073] Mobile phase condition 2

[0074] Time (min) Mobile phase A (%) Mobile phase B (%) 0～5 2→5 98→95 5～15 5→10 95→90 15～30 10→30 90→70 30～50 30→35 70→65 50～60 35→60 65→40

[0075] The experiment was conducted under mobile phase condition 2, but the resolution of each chromatographic peak did not meet the requirements, and the baseline of the chromatogram was uneven.

[0076] Mobile phase condition 3

[0077] Time (min) Mobile phase A (%) Mobile phase B (%) 0～25 2→5 98→95 25～35 5→10 95→90 35～80 10→30 90→70 80～100 30→35 70→65 100～125 35→60 65→40

[0078] Experiments were conducted under mobile phase condition 3. The chromatographic peaks showed good resolution, stable baselines, and well-defined peak shapes. Therefore, mobile phase condition 3 was selected as the chromatographic condition for the fingerprint chromatogram of Xuesusheng particles.

[0079] Example 3

[0080] Take a sample solution of a certain batch of Xuesusheng granules and inject it 6 times consecutively according to the chromatographic conditions of Example 1. The retention time and peak area are measured respectively. Taking the 10th peak of icariin as the reference peak, calculate the relative retention time and relative peak area of ​​each common peak and the reference peak, and calculate the RSD value. The results are shown in Table 4-5.

[0081] Table 4: Relative retention times of each common peak

[0082]

[0083]

[0084] Table 5: Relative Peak Areas of Each Common Peak

[0085]

[0086] As shown in Tables 4 and 5, the relative peak area RSD% and relative retention time RSD% of each common peak are less than 5%, indicating that the chromatographic determination conditions determined in Example 1 have good precision.

[0087] Example 4

[0088] Take the test solution of the same batch of Xuesusheng Granules, prepare the test solution according to the method of Example 1, and determine it at 0, 2, 4, 8, 12, 18 and 24 h according to the chromatographic conditions of Example 1. With the icariin peak of peak 10 as the reference peak, calculate the relative retention time and relative peak area of ​​each common peak and the reference peak, and calculate the RSD value. The results are shown in Table 6-7.

[0089] Table 6: Relative retention times of each common peak

[0090]

[0091] Table 7: Relative Peak Areas of Each Common Peak

[0092]

[0093]

[0094] As shown in Tables 6 and 7, for the test solution of the same batch of Xuesusheng granules, when measured at 0, 2, 4, 8, 12, 18, and 24 hours, the relative peak area RSD% and relative retention time RSD% of each common peak were less than 5%, indicating that the test solution measured at different times within 24 hours according to the fingerprint spectrum establishment conditions determined in Example 1 has stable results.

[0095] Example 5

[0096] Six test solutions were prepared from the same batch of Xuesusheng granules according to the test solution preparation method in Example 1. The solutions were injected and analyzed under the chromatographic conditions of Example 1 to obtain high-performance liquid chromatography (HPLC). Using the icariin peak (peak 10) as the reference peak, the relative retention times and relative peak areas of each common peak and the reference peak were calculated, and the RSD values ​​were calculated. The results are shown in Tables 8-9.

[0097] Table 8: Relative retention times of each common peak

[0098]

[0099] Table 9: Relative Peak Areas of Each Common Peak

[0100]

[0101]

[0102] As shown in Tables 8 and 9, when six test solutions were prepared from the same batch of Xuesusheng granules according to the test solution preparation method in Example 1, and measured under the chromatographic conditions of Example 1, the relative peak area RSD% and relative retention time RSD% of the common peaks of the six test solutions were less than 5%. This indicates that the measurement results have very good repeatability when measured under the chromatographic conditions determined in Example 1.

[0103] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for establishing a fingerprint spectrum of blood-boosting particles, characterized in that, Includes the following steps: S1. Preparation of the test solution and reference solution: Test solution: Take multiple batches of Xuesusheng granules, grind them into a fine powder, accurately quantify each batch, and dissolve them in 50% methanol to obtain a clear test solution; Reference solution: Take icariin reference standard, accurately quantify it, and dissolve it in 50% methanol to prepare the reference solution; S2. Set the high-performance liquid chromatography conditions: The chromatographic column used octadecylsilane-bonded silica gel as the packing material, with a detection wavelength of 270 nm; the flow rate was 0.8-1.2 ml / min; the column temperature was 25-35℃; mobile phase A was acetonitrile, and mobile phase B was 0.2% formic acid solution, with gradient elution. The volume ratio of mobile phases A and B changed as follows: 0-25 min, A:B 2%:98% → 5%:95%; 25-35 min, A:B 5%:95% → 10%:90%; 35-80 min, A:B 10%:90% → 30%:70%; 80-100 min, A:B 30%:70% → 35%:65%; 100-125 min, A:B 35%:65% → 60%:40%. Accurately pipette 5-20 μl of the test solution and reference solution of multiple batches of Xuesusheng granules, inject them into a high-performance liquid chromatograph, and determine the liquid chromatograms of the test solution and reference solution, respectively. S3. Establishing the fingerprint spectrum of Xuesusheng granules: The liquid chromatography of different batches of test solutions is imported into the Chinese medicine chromatographic fingerprint spectrum similarity evaluation system. The common chromatographic peaks of each batch of test samples are selected to generate the Xuesusheng granules reference fingerprint spectrum. By comparing with the peaks of the reference solution, the icariin peak on the reference fingerprint spectrum is determined. Using the icariin peak as the reference peak, the relative peak area and relative retention time of the remaining common peaks are calculated. The Xuesusheng granules reference fingerprint spectrum contains 12 common peaks. Preparation of reference solution: Take icariin reference standard, accurately quantify it, and dissolve it in methanol to obtain 30-80 μg / mL icariin reference solution.

2. The method for establishing according to claim 1, characterized in that, In S1, the preparation of the test solution is as follows: Take the Xuesusheng granules, grind them into a fine powder, weigh them accurately, place them in a stoppered conical flask, dissolve them by adding 12.5 mL of 50% methanol for every 1 g of Xuesusheng granules, sonicate them, cool them, replenish the lost weight with 50% methanol, shake them well, filter them, and take the filtrate to obtain the test solution.

3. The method for establishing according to claim 1, characterized in that, In S1, the preparation of the reference solution is as follows: Take an appropriate amount of icariin reference standard, accurately weigh it, and add 50% methanol to prepare a solution containing 50 μg per ml.

4. The method for establishing according to claim 1, characterized in that, In S2, the chromatographic column was an Agilent 5TC-C18 column with an inner diameter × length of 4.6 × 250 mm and a packing particle size of 5 μm.

5. The method for establishing according to claim 1, characterized in that, In S2, the high-performance liquid chromatography (HPLC) conditions were: detection wavelength 270 nm; flow rate 1 ml / min; column temperature 30 ℃; and injection volume 10 μl.

6. The method for establishing according to claim 1, characterized in that, In S3, peak numbers are marked according to the elution time. By comparing with the peaks in the reference solution, peak 10 on the reference fingerprint spectrum is identified as the icariin peak. Using peak 10 as the reference peak, the relative peak area and relative retention time of the remaining common peaks are calculated.

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