High performance liquid chromatography characteristic chromatogram of Chinese hawthorn seed and determination method and application thereof

Abstract

The invention belongs to the technical field of pharmaceutical analysis. The invention provides a method for determining the high performance liquid chromatography characteristic chromatogram of Chinese hawthorn seed, which comprises the following steps: crushing and sieving a Chinese hawthorn seed sample, adding an ethanol solution for extraction, then carrying out post-treatment, evaporating the supernatant to dryness, dissolving the residue with the ethanol solution, and filtering to obtain a Chinese hawthorn seed sample solution to be determined; dissolving vanillin with methanol to obtain a reference substance solution to be detected; and determining the to-be-detected solution of the hawthorn seed sample and the to-be-detected reference substance solution by adopting high performance liquid chromatography, and obtaining the high performance liquid chromatography characteristic spectrum of the hawthorn seeds according to the chromatogram. According to methodology verification, the method for measuring the hawthorn seed high performance liquid chromatography characteristic chromatogram is good in durability, high in specificity, good in precision, repeatability and stability, simple and convenient to operate and low in cost.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a high performance liquid chromatography characteristic spectrum of hawthorn kernels and a determination method and application thereof. Background Art

[0002] Hawthorn kernel is the dried seed of the plant Shanlihong or Hawthorn of the Rosaceae family. It is mild in nature, bitter in taste, and enters the liver and stomach meridians. It has the effects of digestion, dispersing knots, inducing labor, killing insects, and relieving itching. It is often used for indigestion, hernia, testicular prolapse, dystocia, damp-heat, etc. The main chemical components of hawthorn kernel include lignin, flavonoids, organic acids, triterpenes, carotene, stigmasterol, vitamin C, lycopene, etc. According to the study of pharmacologically active ingredients, hawthorn kernel has antioxidant, lipid-lowering, anti-atherosclerotic, antibacterial, analgesic, and anti-inflammatory effects.

[0003] The hawthorn kernel slices currently in circulation are mostly prepared by washing. The original medicinal materials are taken, the pulp, stalks or other impurities are removed, washed, dried, and the purity of the medicine is guaranteed after preparation. Clinical medication is often taken orally, decocted or ground into powder for swallowing. The research on medicinal hawthorn kernel slices is mostly focused on chemical component analysis and medicinal mechanism research. The current quality standards for hawthorn kernel slices include water content, ash content, thin layer identification, etc., and there is little research on its characteristic spectrum standard, which is not conducive to the quality control of hawthorn kernel slices.

[0004] Chinese herbal medicines have complex ingredients, and most of the effective ingredients have not yet been identified. After proper treatment of Chinese herbal medicines, a certain analytical method is used to obtain a spectrum (characteristic spectrum of Chinese herbal medicines) that can mark the common peaks of the characteristics of various component groups. This is a new quality control mode for Chinese herbal medicines. Compared with the quality method that commonly uses a single component or several components as control indicators, the characteristic spectrum can identify the common peaks of the characteristics of various component groups. It is a comprehensive and quantifiable identification method that can be used to identify the authenticity of Chinese herbal medicines and evaluate the uniformity and stability of the quality of Chinese herbal medicines. Therefore, the establishment of the HPLC characteristic spectrum of hawthorn kernels can not only provide a reference for the comprehensive evaluation and control of the intrinsic quality of hawthorn kernel slices, but also provide a strong basis for improving the quality standards of hawthorn kernels. Summary of the invention

[0005] In order to solve the above problems, the present invention proposes a method for determining the high-performance liquid chromatography characteristic spectrum of hawthorn kernels, which can accurately, simultaneously and quickly present 21 common characteristic peaks in hawthorn kernel pieces, and is a feasible comprehensive and quantifiable identification method for hawthorn kernels. The present invention evaluates the uniformity and stability of the quality of Chinese medicinal materials by applying the high-performance liquid chromatography characteristic spectrum of hawthorn kernels to the quality evaluation and control of hawthorn kernel pieces, and solves the difficult problem of quality evaluation and control in each link of production and circulation of hawthorn kernel pieces.

[0006] In order to achieve the above technical purpose, the present invention provides a method for determining the high performance liquid chromatography characteristic spectrum of hawthorn kernel, comprising the following steps:

[0007] 1) crushing the hawthorn kernel sample, sieving it, then adding an ethanol solution to extract it, and then performing post-treatment, evaporating the supernatant to dryness, dissolving the residue with an ethanol solution, and filtering it to obtain a hawthorn kernel sample test solution;

[0008] 2) dissolving vanillin in methanol to obtain a reference solution;

[0009] 3) The hawthorn kernel sample solution and the reference solution are determined by high performance liquid chromatography, and a high performance liquid chromatography characteristic spectrum of the hawthorn kernel is obtained according to the chromatogram.

[0010] Preferably, the mesh size of the sieving in step 1) is 40-60 meshes; and the volume concentration of the ethanol solution is 40-60%.

[0011] Preferably, in step 1), the mass volume ratio of the hawthorn kernel sample and the ethanol solution is 2 g:40-60 mL.

[0012] Preferably, the extraction in step 1) is ultrasonic extraction or reflux extraction, the extraction time is 30 to 45 minutes, the extraction power is 400 to 600 W, and the extraction frequency is 30 to 50 kHz.

[0013] Preferably, the post-treatment in step 1) is centrifugation or filtration.

[0014] Preferably, the mass concentration of the reference substance solution to be tested in step 2) is 15-25 μg / mL.

[0015] Preferably, the filler determined by high performance liquid chromatography in step 3) is C 18 The column temperature is 30-40°C and the detection wavelength is 300-350nm.

[0016] Preferably, the mobile phase A of the high performance liquid chromatography determination in step 3) is acetonitrile, the mobile phase B is a phosphoric acid aqueous solution, and the volume concentration of the phosphoric acid aqueous solution is 0.05 to 0.15%;

[0017] The elution gradient is 0-30 min, 10-18% A; 30-45 min, 18% A; 45-55 min, 18-26% A; 55-85 min, 26% A; 85-90 min, 26-10% A;

[0018] Flow rate: 0.5-1.5 mL / min;

[0019] The injection volume is 10-20 μL.

[0020] The present invention also provides a high performance liquid chromatography characteristic spectrum of hawthorn kernels obtained by the method for determining the high performance liquid chromatography characteristic spectrum of hawthorn kernels. The high performance liquid chromatography characteristic spectrum of hawthorn kernels has 21 common characteristic peaks.

[0021] The invention also provides application of the high performance liquid chromatography characteristic spectrum of the hawthorn kernel in the quality control of the hawthorn kernel.

[0022] Compared with the prior art, the beneficial effects of the present invention include the following points:

[0023] 1) The present invention takes hawthorn kernel as the research object and establishes a method for determining its high performance liquid chromatography characteristic spectrum. In the established characteristic spectrum, a total of 21 common characteristic peaks are presented, peak No. 13 is taken as the S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time and relative peak area of ​​each characteristic peak and the S peak differ by no more than 5%. The establishment of its characteristic spectrum method can more comprehensively evaluate and control the overall quality of hawthorn kernel, and solve the difficult problem of quality evaluation and control in each link of production and circulation of hawthorn kernel medicinal pieces.

[0024] 2) The method for determining the high performance liquid chromatography characteristic spectrum of hawthorn kernels of the present invention has been verified by methodology, has good durability, strong specificity, good precision, repeatability and stability, and is easy to operate and cost-saving.

[0025] 3) The present invention provides a new method for comprehensive evaluation and overall quality control of hawthorn kernel medicinal materials, which can provide a strong basis for the improvement and improvement of hawthorn kernel inspection standards, and provide an effective method for fully guiding the quality control of hawthorn kernel use and scientifically evaluating product efficacy and quality.

[0026] 4) The method for preparing the hawthorn seed sample test solution described in the technical solution of the present invention is simple and has good repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0028] Figure 1 The fingerprint superposition patterns and reference patterns of 14 batches of hawthorn kernel sample test solutions obtained in 2.5.1 of Example 1;

[0029] Figure 2 This is the spectrum of the vanillin reference solution in 2.5.2 in Example 1;

[0030] Figure 3 The characteristic spectra of the hawthorn kernel sample test solution in Example 2 at different detection wavelengths are shown in Figure 2, wherein A is the characteristic spectrum obtained at a detection wavelength of 210 nm, B is the characteristic spectrum obtained at a detection wavelength of 280 nm, and C is the characteristic spectrum obtained at a detection wavelength of 320 nm;

[0031] Figure 4 The characteristic spectra of the HPLC of the hawthorn kernel sample test solution in different extraction solvents of Example 3, wherein A is the characteristic spectrum obtained by 30% ethanol solution, B is the characteristic spectrum obtained by 50% ethanol solution, and C is the characteristic spectrum obtained by 70% ethanol solution;

[0032] Figure 5 These are the HPLC characteristic spectra of the hawthorn kernel sample test solution at different extraction times in Example 4, wherein A is the characteristic spectrum obtained at 20 min, B is the characteristic spectrum obtained at 30 min, and C is the characteristic spectrum obtained at 40 min. DETAILED DESCRIPTION

[0033] The present invention provides a method for determining a high performance liquid chromatography characteristic spectrum of a hawthorn kernel, comprising the following steps:

[0034] 1) crushing the hawthorn kernel sample, sieving it, then adding an ethanol solution to extract it, and then performing post-treatment, evaporating the supernatant to dryness, dissolving the residue with an ethanol solution, and filtering it to obtain a hawthorn kernel sample test solution;

[0035] 2) dissolving vanillin in methanol to obtain a reference solution;

[0036] 3) The hawthorn kernel sample solution and the reference solution are determined by high performance liquid chromatography, and a high performance liquid chromatography characteristic spectrum of the hawthorn kernel is obtained according to the chromatogram.

[0037] In the present invention, the mesh size of the sieving in step 1) is preferably 40-60 mesh, more preferably 45-55 mesh, and more preferably 50 mesh; the volume concentration of the ethanol solution is preferably 40-60%, more preferably 45-55%, and more preferably 50%.

[0038] In the present invention, the mass volume ratio of the hawthorn kernel sample and the ethanol solution in step 1) is preferably 2g:40-60mL, more preferably 2g:45-55mL, and more preferably 2g:50mL.

[0039] In the present invention, the extraction in step 1) is preferably ultrasonic extraction or reflux extraction; the extraction time is preferably 30 to 45 min, more preferably 35 to 40 min; the extraction power is preferably 400 to 600 W, more preferably 450 to 550 W, more preferably 500 W; the extraction frequency is preferably 30 to 50 kHz, more preferably 35 to 45 kHz, more preferably 40 kHz.

[0040] In the present invention, the post-treatment in step 1) is preferably centrifugation or filtration.

[0041] In the present invention, the mass concentration of the reference substance solution to be tested in step 2) is preferably 15-25 μg / mL, more preferably 18-22 μg / mL, and more preferably 20 μg / mL.

[0042] In the present invention, the filler measured by high performance liquid chromatography in step 3) is preferably C 18 Chromatographic column; the column temperature is preferably 30 to 40°C, more preferably 35°C; the detection wavelength is preferably 300 to 350nm, more preferably 310 to 340nm, more preferably 320nm.

[0043] In the present invention, the mobile phase A of the high performance liquid chromatography determination in step 3) is preferably acetonitrile, and the mobile phase B is preferably a phosphoric acid aqueous solution;

[0044] The volume concentration of the phosphoric acid aqueous solution is preferably 0.05 to 0.15%, more preferably 0.08 to 0.12%, and more preferably 0.1%;

[0045] The elution gradient is preferably 0-30 min, 10-18% A; 30-45 min, 18% A; 45-55 min, 18-26% A; 55-85 min, 26% A; 85-90 min, 26-10% A;

[0046] The flow rate is preferably 0.5 to 1.5 mL / min, more preferably 0.8 to 1.2 mL / min, and more preferably 1 mL / min;

[0047] The injection volume is preferably 10 to 20 μL, more preferably 14 to 16 μL, and even more preferably 15 μL.

[0048] The present invention also provides a high performance liquid chromatography characteristic spectrum of hawthorn kernels obtained by the method for determining the high performance liquid chromatography characteristic spectrum of hawthorn kernels. The high performance liquid chromatography characteristic spectrum of hawthorn kernels has 21 common characteristic peaks.

[0049] The invention also provides application of the high performance liquid chromatography characteristic spectrum of the hawthorn kernel in the quality control of the hawthorn kernel.

[0050] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0051] Example 1

[0052] Determination of the HPLC Characteristic Spectrum of Hawthorn Seed

[0053] 1. Experimental instruments and materials

[0054] Waters high performance liquid chromatograph (manufacturer: Waters Technologies Inc.); electronic balance XS105 (Mettler Toledo, Switzerland); KS-500XDS ultrasonic cleaner (Kunshan Jielimei Ultrasonic Instrument Co., Ltd.);

[0055] Chromatographic column: Agilent ZORBAX C 18 , CAPCELLPAK C 18 , Ultimate C 18 ,

[0056] (250 mm × 4.6 mm, 5 μm);

[0057] Acetonitrile, chromatographic grade, produced by Thermo Fisher Scientific, 4 L, concentration>99.9%;

[0058] Methanol, chromatographic grade, produced by Thermo Fisher Scientific, 4 L, concentration >99.9%;

[0059] Water was ultrapure water, and other reagents were of analytical grade;

[0060] Vanillin reference substance was analytically pure.

[0061] 2. Experimental Methods

[0062] 2.1 Chromatographic conditions

[0063] Octadecylsilane bonded silica gel was used as filler; acetonitrile was used as mobile phase A, 0.1% phosphoric acid aqueous solution was used as mobile phase B, gradient elution (0-30 min, 10-18% A; 30-45 min, 18% A; 45-55 min, 18-26% A; 55-85 min, 26% A; 85-90 min, 26-10% A); detection wavelength was 320 nm; flow rate was 1.0 mL min -1 ; Column temperature was 35°C; Injection volume was 10 μL.

[0064] 2.2 Preparation of reference solution

[0065] Take an appropriate amount of vanillin reference substance, weigh it accurately, place it in a volumetric flask, add methanol to dissolve and dilute to the scale to make a solution with a mass concentration of 20 μg / mL, filter it, and obtain.

[0066] 2.3 Preparation of hawthorn kernel sample test solution

[0067] The hawthorn kernel sample was crushed, passed through a No. 3 sieve, 2 g was weighed, accurately weighed, placed in a stoppered conical flask, 50 mL of 50% ethanol solution was accurately added, ultrasonic extraction (ultrasonic extraction power was 500 W, ultrasonic extraction frequency was 40 kHz) for 30 min, weighed after cooling, and the lost weight was supplemented with 50% ethanol solution by volume. Filter, accurately measure 25 mL of supernatant, evaporate to dryness, dissolve the residue with 50% ethanol solution by volume and place in a 10 mL volumetric flask, shake well, filter, and obtain the hawthorn kernel sample test solution.

[0068] 2.4 Methodological Review

[0069] According to the above chromatographic conditions and the preparation method of the sample solution to be tested, the sample solution is accurately pipetted and injected into the HPLC, and the chromatogram is recorded. Peak 13 is used as the reference peak (S, the retention time of this peak is relatively centered and has good separation from the other chromatographic peaks), and the relative retention time of each peak (T = tRi / tRs) and the relative area of ​​each characteristic peak are calculated as the relative area of ​​the reference peak (X = Ai / As).

[0070] 2.4.1 Precision test

[0071] Take 2 g of hawthorn kernel sample, follow the preparation method of hawthorn kernel sample test solution in 2.3 and the chromatographic conditions in 2.1, inject the sample 6 times continuously, record the chromatogram, and examine the relative retention time and relative peak area of ​​each common characteristic peak. The RSD results are all less than 3.0%, indicating that the precision of the instrument during the analysis is good.

[0072] 2.4.2 Stability test

[0073] Take 2 g of hawthorn kernel sample, follow the preparation method of hawthorn kernel sample test solution in 2.3 and the chromatographic conditions in 2.1, place at room temperature for 0 h, 2 h, 6 h, 10 h, 14 h, and 24 h before injection, record the total ion current, calculate the relative retention time and relative area of ​​each characteristic peak, and the results show that the RSD of the relative retention time of each characteristic peak is less than 0.6%, and the RSD of the relative peak area is less than 1.9%, indicating that the hawthorn kernel sample test solution has good stability during the analysis.

[0074] 2.4.3 Repeatability test

[0075] Take 2 g of hawthorn kernel sample and prepare 6 hawthorn kernel sample test solutions in parallel according to the preparation method of hawthorn kernel sample test solution in 2.3. According to the chromatographic conditions in 2.1, record the chromatogram and examine the relative retention time and relative peak area of ​​each common characteristic peak. The RSD results are all less than 3.0%, indicating that the method has good repeatability.

[0076] 2.5 Feature Spectrum Analysis

[0077] 2.5.1 Feature map establishment

[0078] Establishment of high performance liquid chromatography characteristic spectrum and similarity calculation of hawthorn kernel: 14 batches of hawthorn kernel sample test solutions were taken for high performance liquid chromatography analysis, and the chromatograms were recorded. The data were analyzed using the software "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2004A)" of the National Pharmacopoeia Committee, and relevant reference spectra were set. After data correction and Mark peak matching, the reference spectrum was established by the average method, and the fingerprint superposition spectra and the reference spectrum of the 14 batches of hawthorn kernel sample test solutions were obtained as shown in the figure. Figure 1 As shown. Taking the peak No. 13 in the control fingerprint spectrum with a relatively central position and good separation as the reference peak, a total of 21 common characteristic peaks were determined. Using the common pattern as a reference, the similarity of the samples was calculated. Among them, the similarity between S1~S14 and the control spectrum was between 0.92 and 0.99.

[0079] Taking peak No. 13 as the reference peak, the relative retention time (T=tRi / tRs) of the characteristic peaks with relative peak area greater than 10% was calculated. The results are shown in Table 1.

[0080] Table 1 Relative retention time of characteristic peaks

[0081]

[0082]

[0083] In the control spectrum, the relative retention time of each characteristic peak is 0.161 (peak 2), 0.246 (peak 4), 0.517 (peak 6), 0.585 (peak 7), 0.913 (peak 11), 0.950 (peak 12), 1.679 (peak 19), 1.862 (peak 20), 2.054 (peak 21). The relative retention time of each characteristic peak and S peak of the 14 batches of hawthorn kernel sample solutions selected is no more than 10% compared with the control spectrum, which meets the requirements of the characteristic spectrum.

[0084] 2.5.2 Qualitative analysis of characteristic spectrum peaks

[0085] According to the preparation method of hawthorn kernel sample test solution in 2.3, select hawthorn acid, quercetin, vanillin, hyperoside, kaempferol, isoquercetin and other reference substances (reference substance solution is prepared according to the preparation method of reference substance solution to be tested in 2.2), and according to the chromatographic conditions in 2.1, perform qualitative analysis by retention time and UV spectrum. The results show that in the hawthorn kernel chromatogram, the common characteristic peak No. 6 has the same retention time as vanillin. The spectrum of vanillin reference substance solution to be tested is shown in the figure below. Figure 2 As shown, vanillin exists in the unique components of the above hawthorn kernels, which is consistent with relevant literature reports.

[0086] Example 2

[0087] The detection wavelengths in the chromatographic conditions are 210 nm, 280 nm and 320 nm respectively. The rest is carried out according to item "2.1". The hawthorn kernel sample test solution is prepared according to item "2.3". The HPLC characteristic spectrum of the hawthorn kernel sample test solution is as follows: Figure 3 As shown, A is the characteristic spectrum obtained by detecting the wavelength of 210nm, B is the characteristic spectrum obtained by detecting the wavelength of 280nm, and C is the characteristic spectrum obtained by detecting the wavelength of 320nm. Figure 3 It can be seen that the intensities of the common characteristic peaks vary greatly at different detection wavelengths. When using a detection wavelength of 210nm, the baseline interference is strong and the absorption of the common characteristic peaks is not strong. When using a detection wavelength of 320nm, the peak shape and separation of each component peak are good, and the ultraviolet absorption is strong, which meets the requirements for characteristic spectrum determination.

[0088] Example 3

[0089] The extraction solvents in the method for preparing the hawthorn kernel sample test solution are 30% by volume ethanol solution, 50% by volume ethanol solution and 70% by volume ethanol solution respectively. The rest is carried out according to item "2.3". The chromatographic conditions are carried out according to item "2.1". The HPLC characteristic spectrum of the hawthorn kernel sample test solution is as follows: Figure 4 As shown, A is the characteristic spectrum obtained by 30% ethanol solution, B is the characteristic spectrum obtained by 50% ethanol solution, and C is the characteristic spectrum obtained by 70% ethanol solution. Figure 4 It can be seen that when using 50% volume fraction ethanol solution for ultrasonic extraction, the peak area of ​​the characteristic common components of the characteristic spectrum is high, the tailing factor is good, there are few interfering peaks, and it is easy to detect. With the increase of the amount of ethanol, the intensity of most characteristic peaks does not increase significantly, and the number of characteristic peaks no longer increases.

[0090] Example 4

[0091] The extraction times in the preparation method of the hawthorn kernel sample test solution are 20 min, 30 min and 40 min respectively, and the rest is carried out according to item "2.3". The chromatographic conditions are carried out according to item "2.1". The HPLC characteristic spectrum of the hawthorn kernel sample test solution is as follows Figure 5 As shown, A is the characteristic spectrum obtained at 20 minutes, B is the characteristic spectrum obtained at 30 minutes, and C is the characteristic spectrum obtained at 40 minutes. Figure 5 It can be seen that: when the ultrasonic extraction time is ≥30min, the peak area of ​​each component increases slightly, but the change is not large, indicating that the component has been basically extracted completely. After 30min of ultrasonic treatment, the content of each component reaches the highest and tends to be stable.

[0092] Example 5

[0093] The column temperatures in the chromatographic conditions are 30°C, 35°C and 40°C respectively, and the rest are carried out according to item "2.1". The hawthorn kernel sample test solution is prepared according to item "2.3". The high performance liquid chromatography characteristic spectrum of the hawthorn kernel sample test solution obtained shows that the peak time, separation, peak shape, peak area and other parameters of the main components of hawthorn kernel all meet the requirements.

[0094] Example 6

[0095] The octadecylsilane bonded silica gel used in the chromatographic conditions was Agilent ZORBAX C 18 ,CAPCELLPAKC 18 and Ultimate C 18 The rest was carried out according to item "2.1". The hawthorn kernel sample test solution was prepared according to item "2.3". The HPLC characteristic spectrum of the hawthorn kernel sample test solution obtained showed that under the conditions of chromatographic columns of three different brands, the common peaks in the characteristic spectrum of the hawthorn kernel sample test solution were well separated. The relative retention time and relative peak area of ​​each characteristic peak and S peak did not differ by more than 3%, indicating that the detection method was applicable to C of different brands. 18 Chromatographic column.

[0096] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for determining the high performance liquid chromatography characteristic spectrum of hawthorn kernels, characterized in that: The following steps are included: 1) crushing the hawthorn kernel sample, sieving it, then adding an ethanol solution to extract it, and then performing post-treatment, evaporating the supernatant to dryness, dissolving the residue with an ethanol solution, and filtering it to obtain a hawthorn kernel sample test solution; 2) dissolving vanillin in methanol to obtain a reference solution; 3) The hawthorn kernel sample solution and the reference solution are determined by high performance liquid chromatography, and a high performance liquid chromatography characteristic spectrum of the hawthorn kernel is obtained according to the chromatogram.

2. The method for determining the high performance liquid chromatography characteristic spectrum of hawthorn kernel according to claim 1, characterized in that: Step 1) The mesh size of the sieve is 40 to 60 meshes; the volume concentration of the ethanol solution is 40 to 60%.

3. The method for determining the high performance liquid chromatography characteristic spectrum of hawthorn kernel according to claim 1 or 2, characterized in that: Step 1) The mass volume ratio of the hawthorn kernel sample and the ethanol solution is 2g:40-60mL.

4. The method for determining the high performance liquid chromatography characteristic spectrum of hawthorn kernel according to claim 3, characterized in that: The extraction in step 1) is ultrasonic extraction or reflux extraction, the extraction time is 30 to 45 minutes, the extraction power is 400 to 600 W, and the extraction frequency is 30 to 50 kHz.

5. The method for determining the high performance liquid chromatography characteristic spectrum of hawthorn kernel according to claim 4, characterized in that: The post-treatment in step 1) is centrifugation or filtration.

6. The method for determining the high performance liquid chromatography characteristic spectrum of hawthorn kernel according to claim 5, characterized in that: Step 2) The mass concentration of the reference substance solution to be tested is 15-25 μg / mL.

7. The method for determining the high performance liquid chromatography characteristic spectrum of hawthorn kernel according to claim 5 or 6, characterized in that: Step 3) The filler determined by high performance liquid chromatography is C 18 The column temperature is 30-40°C and the detection wavelength is 300-350nm.

8. The method for determining the high performance liquid chromatography characteristic spectrum of hawthorn kernel according to claim 7, characterized in that: Step 3) The mobile phase A of the HPLC determination is acetonitrile, the mobile phase B is a phosphoric acid aqueous solution, and the volume concentration of the phosphoric acid aqueous solution is 0.05-0.15%; The elution gradient is 0-30 min, 10-18% A; 30-45 min, 18% A; 45-55 min, 18-26% A; 55-85 min, 26% A; 85-90 min, 26-10% A; Flow rate: 0.5-1.5 mL / min; The injection volume is 10-20 μL.

9. The high performance liquid chromatography characteristic spectrum of hawthorn kernel obtained by the method for determining the high performance liquid chromatography characteristic spectrum of hawthorn kernel according to any one of claims 1 to 8, characterized in that: The HPLC characteristic spectrum of hawthorn kernel has 21 common characteristic peaks.

10. Use of the high performance liquid chromatography characteristic spectrum of hawthorn kernel according to claim 9 in the quality control of hawthorn kernel.

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