Fingerprint spectrum of Jian'er qianggu granules, method for constructing the same and method for evaluating quality of Jian'er qianggu granules

Abstract

The application relates to the technical field of pharmaceutical analysis, in particular to a Jianer Qianggu granule fingerprint spectrum, a construction method thereof and a quality evaluation method of the Jianer Qianggu granule. The fingerprint spectrum construction method takes 5-hydroxymethyl furfural and 4-coumaric acid as the control samples, the test sample is extracted by water ultrasonic extraction, an HPLC column with octadecylsilane bonded silica gel as the filling agent is used, acetonitrile and phosphoric acid aqueous solution are used as the mobile phase for gradient elution, detection is carried out at a wavelength of 280-320 nm, and the Jianer Qianggu granule fingerprint spectrum containing six characteristic peaks is constructed. The application can simultaneously detect various components in the Jianer Qianggu granule, is simple to operate, has good precision, repeatability and stability, and can be effectively used for evaluating the quality stability of the Jianer Qianggu granule product.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical analysis, in particular to a Jianer Qianggu granule fingerprint, a construction method thereof and a quality evaluation method of the Jianer Qianggu granule. BACKGROUND

[0002] The Jianer Qianggu granule is made of five medicinal materials, namely, jujube, oyster, ophiopogon, atractylodes and codonopsis, and has the effects of invigorating the spleen and tonifying qi, and tonifying the kidney and strengthening the bone, and is suitable for children with rickets and related symptoms caused by calcium deficiency. The Jianer Qianggu granule has complex components, and its efficacy is jointly embodied by multiple active components.

[0003] The current quality standard adopts physicochemical identification, thin layer identification and titration method to determine the content. The above methods can only qualitatively or quantitatively control a single or a few indicators, and cannot comprehensively characterize and monitor the complex chemical substance basis related to the clinical efficacy, and it is difficult to effectively evaluate the quality consistency and stability between batches of the Jianer Qianggu granule.

[0004] Therefore, it is necessary to develop a method for simultaneously detecting multiple components in the Jianer Qianggu granule to control the quality stability of the Jianer Qianggu granule. SUMMARY

[0005] Therefore, the purpose of the present application is to overcome the shortcomings of the prior art, and provide a Jianer Qianggu granule fingerprint and a construction method thereof and a quality evaluation method of the Jianer Qianggu granule. The above Jianer Qianggu granule fingerprint and the construction method and the quality evaluation method can simultaneously detect multiple components in the Jianer Qianggu granule, and can be effectively used to evaluate the quality stability of the Jianer Qianggu granule product.

[0006] To achieve the above purpose, the present application adopts the following technical solutions:

[0007] Firstly, the present application provides a construction method of a Jianer Qianggu granule fingerprint, comprising the following steps:

[0008] S1, preparation of reference solution: 5-hydroxymethylfurfural and 4-coumaric acid are taken, and water is added for quantitative dilution to prepare a mixed reference solution containing 5-hydroxymethylfurfural and 4-coumaric acid;

[0009] S2, preparation of test solution: the Jianer Qianggu granule is ground, water is added for ultrasonic extraction, and the solution is cooled to room temperature, shaken uniformly, filtered, and the filtrate is taken as the test solution;

[0010] S3, the reference solution and the test solution are taken respectively, and injected into a high performance liquid chromatograph, and the chromatogram is recorded to obtain the Jianer Qianggu granule fingerprint;

[0011] The chromatographic conditions of the high performance liquid chromatography include: a chromatographic column with water-resistant octadecylsilane-bonded silica gel as the filler, a column temperature of the chromatographic column of 28-32℃, acetonitrile as the mobile phase A, and phosphoric acid aqueous solution as the mobile phase B, gradient elution, and a detection wavelength of 280-320 nm;

[0012] The gradient elution program is: within 0-30 min, the volume fraction of the mobile phase A is increased from 1%-3% to 3%-6%; within 30-50 min, the volume fraction of the mobile phase A is increased to 10%-20%; within 50-70 min, the volume fraction of the mobile phase A is increased to 25%-35%; within 70-80 min, the volume fraction of the mobile phase A is increased to 35%-45%; within 80-85 min, the volume fraction of the mobile phase A is decreased to 1%-3%; and within 85-95 min, the volume fraction of the mobile phase A is kept at 1%-3%.

[0013] Preferably, the gradient elution program is: within 0-30 min, the volume fraction of the mobile phase A is increased from 1.5%-2.5% to 3.5%-4.5%; within 30-50 min, the volume fraction of the mobile phase A is increased to 12%-18%; within 50-70 min, the volume fraction of the mobile phase A is increased to 27%-33%; within 70-80 min, the volume fraction of the mobile phase A is increased to 37%-43%; within 80-85 min, the volume fraction of the mobile phase A is decreased to 1.5%-2.5%; and within 85-95 min, the volume fraction of the mobile phase A is kept at 1.5%-2.5%.

[0014] Preferably, the mobile phase B is phosphoric acid aqueous solution with a volume fraction of 0.05%-0.3%. More preferably, the mobile phase B is phosphoric acid aqueous solution with a volume fraction of 0.08%-0.12%. The phosphoric acid aqueous solution can inhibit the dissociation of acidic components, improve the peak shape of phenolic acid components, and has low ultraviolet absorption at the detection wavelength, which is conducive to a smooth baseline.

[0015] Preferably, the chromatographic column is a water-resistant octadecylsilane-bonded silica gel chromatographic column with a column length of 200-300 mm, an inner diameter of 4.0-4.6 mm, and a filler particle size of 3-5 μm. In the initial stage of the gradient elution, the proportion of the water phase in the mobile phase is as high as 95% or more, and the use of the water-resistant octadecylsilane-bonded silica gel chromatographic column can avoid the collapse of the stationary phase of ordinary chromatographic columns under high water phase conditions, thereby ensuring the stability of the retention time. More preferably, the chromatographic column is an Ultimate AQ-C18 chromatographic column with a column length of 250 mm, an inner diameter of 4.6 mm, and a filler particle size of 5 μm.

[0016] Preferably, the chromatographic conditions of the high performance liquid chromatography further include: an injection volume of 8-12 μL; and a flow rate of the mobile phase of 0.9-1.1 mL / min.

[0017] Preferably, the detection wavelength is 290-310 nm, more preferably 295-305 nm. In this wavelength range, there are more and more evenly distributed chromatographic peak information, and the detection of 5-hydroxymethylfurfural and 4-coumaric acid and other components can be considered.

[0018] Preferably, in step S2, the power of ultrasonic extraction is 400-600 W, and the frequency is 35-50 kHz; the ultrasonic extraction time is 20-40 min; the dosage ratio of Jianer Qianggu Granules to water is 1 g: 2-5 mL.

[0019] Preferably, in step S1, the mass concentration of 5-hydroxymethylfurfural in the mixed reference solution is 8-12 μg / mL, and the mass concentration of 4-coumaric acid is 8-12 μg / mL. More preferably, the mass concentration of 5-hydroxymethylfurfural in the mixed reference solution is 9-11 μg / mL, and the mass concentration of 4-coumaric acid is 9-11 μg / mL.

[0020] Preferably, the Jianer Qianggu Granules fingerprint spectrum includes 6 characteristic peaks, with the chromatographic peak of 5-hydroxymethylfurfural as the reference peak, and the relative retention times of the 6 characteristic peaks are as follows: peak 1 is 0.60-0.70, peak 2 is 1.00, peak 3 is 1.22-1.40, peak 4 is 1.78-2.00, peak 5 is 2.95-3.30, and peak 6 is 3.45-3.85; wherein, peak 2 is the 5-hydroxymethylfurfural peak, and peak 6 is the 4-coumaric acid peak.

[0021] Based on one overall inventive concept, the application also provides a Jianer Qianggu Granules fingerprint spectrum, which is obtained according to the construction method of the Jianer Qianggu Granules fingerprint spectrum described above.

[0022] Based on one overall inventive concept, the application also provides a quality evaluation method of Jianer Qianggu Granules, which obtains the fingerprint spectrum of the Jianer Qianggu Granules to be tested by using the construction method described above, imports the fingerprint spectrum into a traditional Chinese medicine chromatographic fingerprint spectrum similarity evaluation system, calculates the similarity, and evaluates the quality of the Jianer Qianggu Granules according to the similarity.

[0023] Preferably, when the similarity is not less than 0.90, it is determined that the quality of the Jianer Qianggu Granules is qualified.

[0024] Compared with the prior art, the application has the following beneficial effects:

[0025] (1) This application uses 5-hydroxymethylfurfural and 4-coumaric acid as reference standards. By eluting with an acetonitrile-phosphoric acid aqueous solution gradient, multiple polar to moderately polar components in Jianer Qianggu Granules are separated in a single injection. A fingerprint spectrum containing 6 characteristic peaks is constructed, which can simultaneously detect multiple components in Jianer Qianggu Granules and comprehensively characterize its chemical basis. This makes up for the shortcomings of the current standard, which can only control a few indicators by physicochemical identification, thin-layer identification and titration.

[0026] (2) The method validation results show that the similarity of the precision test of the method in this application is 0.997~1.000, the similarity of the repeatability test is 0.981~1.000, and the similarity of the stability test of the test solution within 44 hours is not less than 0.95. The method is stable and reliable and has good reproducibility.

[0027] (3) The method of this application is simple to operate, the test solution is prepared with water as solvent, the cost is low and the safety is good, and it can be effectively used to evaluate the quality stability of Jianer Qianggu Granules. Attached Figure Description

[0028] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the embodiments of this application will be briefly introduced below. Obviously, the drawings described below are only some implementation methods of this application. For those skilled in the art, other drawings can be obtained based on the drawings without creative effort.

[0029] Figure 1 This is a chromatogram of the test solution under the gradient elution program of methods 1 to 7 in Example 1.

[0030] Figure 2 This is a 3D chromatogram of the test solution at different detection wavelengths in Example 2.

[0031] Figure 3 The chromatograms are of the test solution at different detection wavelengths in Example 2.

[0032] Figure 4 The chromatograms are of the test solutions under different column temperatures in Example 3.

[0033] Figure 5 The chromatograms are of the reference solution and the test solution in Example 4.

[0034] Figure 6 The chromatograms are of the test solutions obtained under different extraction solvent conditions in Example 5.

[0035] Figure 7 The chromatogram is obtained by injecting the same test solution 6 times in Example 6.

[0036] Figure 8 The chromatograms are of the six test sample solutions prepared in Example 6.

[0037] Figure 9 The chromatograms are of the same test solution in Example 6 after storage under different time conditions.

[0038] Figure 10 The fingerprint spectrum and characteristic peak markings of the Jian'er Qianggu Granules determined in Example 6. Detailed Implementation

[0039] The embodiments described in this specification are merely for explaining this application and are not intended to limit this application.

[0040] For simplicity, this specification only explicitly discloses certain numerical ranges. However, any lower limit can be combined with any upper limit to form a range not explicitly stated; and any lower limit can be combined with other lower limits to form a range not explicitly stated, just as any upper limit can be combined with any other upper limit to form a range not explicitly stated. Furthermore, although not explicitly stated, every point or individual value between the endpoints of a range is included within that range. Therefore, each point or individual value can be used as its own lower or upper limit and combined with any other point or individual value, or with other lower or upper limits, to form a range not explicitly stated.

[0041] Those skilled in the art will understand that the order in which the steps are written in the various embodiments or examples does not imply a strict execution order and does not limit the implementation process in any way. The detailed execution order of each step should be determined by its function and possible internal logic. Unless otherwise specified, all steps of the present invention may be performed sequentially or randomly, but sequentially is preferred.

[0042] The present application is further illustrated below with reference to embodiments. It should be understood that these embodiments are merely illustrative, as various modifications and variations will be apparent to those skilled in the art within the scope of the disclosure of this application. Unless otherwise stated, all parts, percentages, and ratios reported in the following embodiments are based on mass, and all reagents used in the embodiments are commercially available or synthesized by conventional methods and can be used directly without further processing, and the instruments used in the embodiments are commercially available.

[0043] In the embodiments, Jian'er Qianggu Granules can be commercially available products or prepared by the following method: The above five ingredients, Codonopsis pilosula, Ophiopogon japonicus, Atractylodes macrocephala, and jujube, are decocted twice with water, the first time for 2 hours and the second time for 1.5 hours. The decoctions are combined, filtered, and the filtrate is concentrated to a relative density of 1.08 (90~95℃). After cooling, ethanol is added to make the alcohol content reach 60%. After standing for 24 hours, the supernatant is taken and concentrated to a clear extract with a relative density of 1.20~1.40 (60℃). Oyster shell is calcined and 150ml of lactic acid is added to make calcium lactate powder. Take 1 part of the clear extract, 1 part of calcium lactate powder, 4 parts of sucrose, and 2.6 parts of dextrin, mix them well, granulate, and dry at 60~70℃ to make 1000g.

[0044] Example 1: Determination of mobile phase system and gradient elution procedure.

[0045] Preparation of the test solution: Take an appropriate amount of Jianer Qianggu Granules, grind them, accurately weigh 15g, place them in an Erlenmeyer flask, add 50mL of water, sonicate for 30 minutes (power 500W, frequency 45kHz), remove the Erlenmeyer flask, cool to room temperature, shake well, filter, and take the filtrate to obtain the test solution.

[0046] Chromatographic conditions: A column (Yuexu Ultimate AQ-C18, 4.6 mm × 250 mm, 5 μm) packed with octadecylsilane-bonded silica gel was used; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid aqueous solution or water was used as mobile phase B, eluting according to the gradient programs of Methods 1 to 8 shown in Tables 1 to 8, respectively; the flow rate was 1 mL / min; the column temperature was 30℃; the detection wavelength was 300 nm; and the injection volume was 10 μL. Accurately pipette the test solution and inject it into the liquid chromatograph, recording the chromatogram. (See table below for chromatogram details.) Figure 1 The x-axis represents time in minutes, and the y-axis represents the light absorption intensity Au.

[0047] Table 1 Gradient elution procedure for Method 1

[0048]

[0049] Table 2 Gradient elution procedure for Method 2

[0050]

[0051] Table 3 Gradient elution procedure for Method 3

[0052]

[0053]

[0054] Table 5 Gradient elution procedure for Method 5

[0055]

[0056] Table 6 Gradient elution procedure for Method 6

[0057]

[0058] Table 7 Gradient elution procedure for Method 7

[0059]

[0060] from Figure 1 It can be seen that under the conditions of Method 5, the chromatogram baseline is relatively flat, the chromatographic peaks are evenly distributed, and the chromatographic parameters are better. Further comparison of mobile phase systems based on Method 5: using acetonitrile-water (i.e., Method 7, with the same gradient elution program as Method 5) as the mobile phase, the results show that the acetonitrile-0.1% phosphoric acid system yields more chromatographic peak information and better chromatographic parameters. Therefore, the mobile phase system is determined to be acetonitrile-0.1% phosphoric acid aqueous solution, and the gradient elution program is Method 5 (see Table 5).

[0061] Example 2: Determination of detection wavelength.

[0062] Preparation of the test solution: Same as in Example 1.

[0063] Chromatographic conditions: An octadecylsilane-bonded silica column (Yuexu Ultimate AQ-C18, 4.6 mm × 250 mm, 5 μm) was used as the packing material; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid aqueous solution was used as mobile phase B, eluting according to the gradient program listed in Table 5; the flow rate was 1 mL / min; the column temperature was 30℃; the injection volume was 10 μL; the test solution was scanned at all wavelengths. The chromatogram was recorded; see [link to chromatogram]. Figure 2 , Figure 3 ;in Figure 2 3D chromatograms under different detection wavelength conditions. Figure 3 Chromatograms under different detection wavelengths are shown.

[0064] from Figure 2 , Figure 3 It can be seen that the chromatographic peaks of Jianer Qianggu Granules are mainly concentrated in the range of 250~340nm. By comparing the chromatograms of 6 different wavelengths, the chromatographic peak information at 300nm is more abundant and evenly distributed. Taking into account the response of each characteristic peak, the detection wavelength was determined to be 300nm.

[0065] Example 3: Determination of column temperature.

[0066] Preparation of the test solution: Same as in Example 1.

[0067] Chromatographic conditions: An octadecylsilane-bonded silica gel column (Yuexu Ultimate AQ-C18, 4.6 mm × 250 mm, 5 μm) was used; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid aqueous solution was used as mobile phase B, eluting according to the gradient program listed in Table 5; the flow rate was 1 mL / min; the detection wavelength was 300 nm; and the injection volume was 10 μL. The chromatographic separation under different column temperatures was investigated. See the chromatograms below. Figure 4 .

[0068] from Figure 4 It can be seen that under different column temperature conditions, the chromatographic parameters of each chromatographic peak are optimal when the column temperature is 30℃, so the column temperature is determined to be 30℃.

[0069] Example 4: Chromatographic peak identification.

[0070] Preparation of reference solutions: Take appropriate amounts of 5-hydroxymethylfurfural reference standard and 4-coumaric acid reference standard, accurately weigh them, add water to prepare reference solution 1 containing 10 μg of 5-hydroxymethylfurfural per 1 mL and reference solution 2 containing 10 μg of 4-coumaric acid per 1 mL, respectively, shake well, and the solution is obtained.

[0071] Preparation of the test solution: Same as in Example 1.

[0072] Chromatographic conditions: A column (Yuexu Ultimate AQ-C18, 4.6 mm × 250 mm, 5 μm) packed with octadecylsilane-bonded silica gel was used; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid aqueous solution was used as mobile phase B, eluting according to the gradient program listed in Table 5; the flow rate was 1 mL / min; the column temperature was 30℃; the detection wavelength was 300 nm; and the injection volume was 10 μL. Accurately pipette 10 μL each of reference solutions 1 and 2 and the test solution were injected into the liquid chromatograph, and the chromatograms were recorded. See the chromatogram below. Figure 5 .

[0073] from Figure 5 It can be seen that the chromatographic peak at approximately 15.7 minutes in the Jian'er Qianggu Granules chromatogram is 5-hydroxymethylfurfural, and the chromatographic peak at approximately 57.2 minutes is 4-coumaric acid. Therefore, the provisional reference solution is a mixed solution containing 10 μg of 5-hydroxymethylfurfural and 10 μg of 4-coumaric acid per 1 mL.

[0074] Example 5: Determination of the extraction solvent for the test sample solution.

[0075] Preparation of the test solution: Take an appropriate amount of Jianer Qianggu Granules, grind them, weigh 15g of each of the 4 groups of Jianer Qianggu Granules powder, place them in an Erlenmeyer flask, and add 50mL of water, methanol, 60% methanol and ethanol respectively. Sonicate for 30 minutes (power 500W, frequency 45kHz), take it out, cool it to room temperature, shake it well, filter it, and take the filtrate to obtain the test solution.

[0076] Chromatographic conditions: A column (Yuexu Ultimate AQ-C18, 4.6 mm × 250 mm, 5 μm) was used as the stationary phase, packed with octadecylsilane-bonded silica gel; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid aqueous solution was used as mobile phase B, eluting according to the gradient program listed in Table 5; the flow rate was 1 mL / min; the column temperature was 30℃; the detection wavelength was 300 nm; and the injection volume was 10 μL. Accurately pipettes of the test solution were injected into the liquid chromatograph, and the chromatograms were recorded. See the chromatograms below. Figure 6 .

[0077] from Figure 6 It can be seen that the chromatogram of ethanol extraction has the flattest baseline, but fewer chromatographic peaks, making it impossible to fully characterize the sample components. The chromatographic peak information completeness of pure methanol and 60% methanol extraction is not as good as that of water extraction, and the stability is also not as good. Water extraction has a relatively flat baseline and more chromatographic peak information, so water was chosen as the extraction solvent for Jian'er Qianggu Granules.

[0078] Example 6: Establishment and methodological verification of fingerprint spectrum of Jian'er Qianggu Granules.

[0079] Chromatographic conditions: A column (Yuxu Ultimate AQ-C18, 4.6 mm × 250 mm, 5 μm) was used as the packing material with octadecylsilane-bonded silica gel; acetonitrile was used as mobile phase A and 0.1% phosphoric acid aqueous solution was used as mobile phase B, eluted according to the gradient program listed in Table 5; the flow rate was 1 mL / min; the column temperature was 30 ℃; the detection wavelength was 300 nm; and the injection volume was 10 μL.

[0080] Preparation of reference solution: Weigh appropriate amounts of 5-hydroxymethylfurfural and 4-coumaric acid reference standards accurately, add water to prepare a mixed solution containing 10 μg of 5-hydroxymethylfurfural and 10 μg of 4-coumaric acid per 1 mL, shake well, and the solution is obtained.

[0081] Preparation of the test solution: Same as in Example 1.

[0082] (1) Instrument precision test: Take the same test solution, inject it 6 times consecutively, and record the chromatogram. See the chromatogram below. Figure 7 The 6-injection chromatograms were imported into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version), and the similarity was calculated. The results are shown in Table 8. The similarity of the 6-injection chromatograms was between 0.997 and 1.000, indicating that the instrument precision was good.

[0083] Table 8 Precision Test Results

[0084]

[0085] In the table, J1~J6 refer to the chromatograms obtained from 1~6 consecutive injections of the same test sample solution.

[0086] (2) Repeatability test: Prepare 6 parallel aliquots of the test solution, accurately pipette 10 μL into each aliquot, inject into the liquid chromatograph, and record the chromatograms. See the chromatograms below. Figure 8 The six chromatograms were imported into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version), and the similarity was calculated. The results are shown in Table 9. The similarity of the six test sample chromatograms was between 0.981 and 1.000, indicating that the method has good repeatability.

[0087] Table 9 Results of Repeatability Tests

[0088]

[0089] In the table, C1 to C6 refer to the chromatograms obtained from the injection of six reproducible test solutions.

[0090] (3) Solution stability test: Take the same test solution and accurately pipette 10 μL at 0, 8, 14, 20, 27, 33, 39, and 44 hours, respectively, and inject it into the liquid chromatograph. Record the chromatograms. See the chromatograms below. Figure 9 The chromatograms at each time point were imported into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version), and the similarity was calculated. The results are shown in Table 10. The similarity of the test solution was not less than 0.95 within 44 hours, indicating that the test solution had good stability within 44 hours.

[0091] Table 10 Results of solution stability test

[0092]

[0093] In the table, T1 to T8 refer to the chromatograms obtained by injecting the same test solution at 0, 8, 14, 20, 27, 33, 39, and 44 hours, respectively.

[0094] (4) Determination of characteristic peaks: Based on the above methodological validation results, six stable characteristic peaks were determined. The 5-hydroxymethylfurfural peak (peak 2) was used as the reference peak S, and the relative retention times of each chromatographic peak were calculated with the chromatographic retention time of the 5-hydroxymethylfurfural peak as 1. The results are shown in Table 11. The fingerprint and characteristic peak markings of Jian'er Qianggu Granules are shown in Table 11. Figure 10 .

[0095] Table 11 Relative retention times of characteristic peaks in the fingerprint spectrum of Jian'er Qiang Gu Granules

[0096]

[0097] Peak 2 is a characteristic peak of 5-hydroxymethylfurfural, and peak 6 is a characteristic peak of 4-coumaric acid.

[0098] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in this application, and these modifications or substitutions should all be covered within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A method for constructing a fingerprint spectrum of Jian'er Qianggu Granules, characterized in that, Includes the following steps: S1. Preparation of reference solution: Take 5-hydroxymethylfurfural and 4-coumaric acid, add water to dilute quantitatively, and prepare a mixed reference solution containing 5-hydroxymethylfurfural and 4-coumaric acid; S2. Preparation of test solution: Take Jianer Qianggu Granules, grind them, add water and extract by ultrasonication, cool to room temperature, shake well, filter, and take the filtrate to obtain the test solution. S3. Take the reference solution and the test solution respectively, inject them into the high performance liquid chromatograph, record the chromatogram, and obtain the fingerprint spectrum of Jianer Qianggu Granules; The chromatographic conditions for the high performance liquid chromatography include: a column packed with water-resistant octadecylsilane-bonded silica gel, a column temperature of 28-32°C, acetonitrile as mobile phase A, and phosphoric acid aqueous solution as mobile phase B, with gradient elution and a detection wavelength of 280-320 nm. The gradient elution program is as follows: within 0-30 min, the volume fraction of mobile phase A increases from 1%-3% to 3%-6%; within 30-50 min, the volume fraction of mobile phase A increases to 10%-20%; within 50-70 min, the volume fraction of mobile phase A increases to 25%-35%; within 70-80 min, the volume fraction of mobile phase A increases to 35%-45%; within 80-85 min, the volume fraction of mobile phase A decreases to 1%-3%; and within 85-95 min, the volume fraction of mobile phase A remains at 1%-3%.

2. The method for constructing the fingerprint spectrum of Jian'er Qianggu Granules according to claim 1, characterized in that, The gradient elution program is as follows: within 0–30 min, the volume fraction of mobile phase A increases from 1.5%–2.5% to 3.5%–4.5%; within 30–50 min, the volume fraction of mobile phase A increases to 12%–18%; within 50–70 min, the volume fraction of mobile phase A increases to 27%–33%; within 70–80 min, the volume fraction of mobile phase A increases to 37%–43%; within 80–85 min, the volume fraction of mobile phase A decreases to 1.5%–2.5%; and within 85–95 min, the volume fraction of mobile phase A remains at 1.5%–2.5%.

3. The method for constructing the fingerprint spectrum of Jian'er Qianggu Granules according to claim 1, characterized in that, The mobile phase B is an aqueous solution of phosphoric acid with a volume fraction of 0.05% to 0.3%.

4. The method for constructing the fingerprint spectrum of Jian'er Qianggu Granules according to claim 1, characterized in that, The chromatographic column is a water-resistant octadecylsilane-bonded silica gel column with a length of 200-300 mm, an inner diameter of 4.0-4.6 mm, and a packing particle size of 3-5 μm.

5. The method for constructing the fingerprint spectrum of Jian'er Qianggu Granules according to claim 1, characterized in that, The chromatographic conditions for the high performance liquid chromatography include: an injection volume of 8-12 μL and a mobile phase flow rate of 0.9-1.1 mL / min.

6. The method for constructing the fingerprint spectrum of Jian'er Qianggu Granules according to claim 1, characterized in that, In step S2, the ultrasonic extraction power is 400~600W and the frequency is 35~50kHz; the ultrasonic extraction time is 20~40min; and the ratio of Jianer Qianggu Granules to water is 1g:2~5mL.

7. The method for constructing the fingerprint spectrum of Jian'er Qianggu Granules according to claim 1, characterized in that, In step S1, the mass concentration of 5-hydroxymethylfurfural in the mixed reference solution is 8~12 μg / mL, and the mass concentration of 4-coumaric acid is 8~12 μg / mL.

8. The method for constructing the fingerprint spectrum of Jian'er Qianggu Granules according to claim 1, characterized in that, The fingerprint spectrum of the Jian'er Qianggu Granules includes six characteristic peaks. Taking the chromatographic peak of 5-hydroxymethylfurfural as a reference peak, the relative retention times of the six characteristic peaks are as follows: peak 1 is 0.60~0.70, peak 2 is 1.00, peak 3 is 1.22~1.40, peak 4 is 1.78~2.00, peak 5 is 2.95~3.30, and peak 6 is 3.45~3.

85. Among them, peak 2 is the 5-hydroxymethylfurfural peak, and peak 6 is the 4-coumaric acid peak.

9. A fingerprint spectrum of Jian'er Qianggu Granules, characterized in that, The method described in any one of claims 1 to 8 is used to obtain the product.

10. A method for evaluating the quality of Jian'er Qianggu Granules, characterized in that, The fingerprint spectrum of Jian'er Qianggu Granules to be tested is obtained by the construction method described in any one of claims 1 to 8. The fingerprint spectrum is then imported into a similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine to calculate the similarity and evaluate the quality of Jian'er Qianggu Granules based on the similarity.

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