A method for constructing a characteristic chromatogram of walnut kernel and preparation thereof

Abstract

The application belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a construction method of characteristic chromatogram of walnut kernels and preparations thereof, which comprises the following steps: (1) preparation of a test sample solution; (2) detection of the test sample solution by high performance liquid chromatography, wherein octadecylsilane bonded silica gel is used as a filler, a mobile phase comprises a phosphoric acid-containing aqueous solution and acetonitrile, and a gradient elution procedure comprises: 0→5 min→15 min→20 min→40 min→50 min, and the volume percentage of acetonitrile in the mobile phase is: 2%-3%→3%→8%→8%→22%→40%. The application can realize effective separation of 11 common characteristic peaks, wherein the separation of the common characteristic peaks including the tannic acid peak is realized, the elution procedure is simple, the obtained characteristic chromatogram has a smooth baseline, the characteristic peaks have good peak shapes and high resolution, and the characteristic chromatogram can fully reflect the integrity and characteristics of the walnut kernels and the preparations thereof.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a construction method of a characteristic spectrum of walnut kernels and preparations thereof. BACKGROUND

[0002] Walnut kernels are the dried mature seeds of Juglans regia L. of Juglandaceae. They are collected in autumn when the fruits are mature, the fleshy pericarp is removed, and then dried, and the kernel shell and woody diaphragm are removed. They are sweet in flavor and warm in nature, and are attributed to the kidney, lung and large intestine channels, and have the functions of tonifying the kidney, warming the lung and moistening the intestine, and can be used for kidney yang deficiency, soreness of the waist and knees, impotence and spermatorrhea, cold and asthmatic cough, and dry constipation. Only the properties, microscopic identification, and inspection of moisture, rancidity, carbonyl value and peroxide value are recorded under the entry of walnut kernels in the Chinese Pharmacopoeia 2020 edition.

[0003] In the prior art, there are documents that disclose a TLC (thin layer chromatography) identification method of walnut kernels by taking glansreginins A as a reference substance, and the content is determined by HPLC (high performance liquid chromatography), but only a few peaks are relatively obvious in the obtained HPLC image, the separation degree is poor, the definition is poor, and the chemical component characteristics of walnut kernels cannot be fully displayed. SUMMARY

[0004] Therefore, the purpose of the present application is to provide a construction method of a characteristic spectrum of walnut kernels and preparations thereof, which establishes the characteristic spectrum of the variety according to the characteristics of walnut kernels and preparations thereof, the number of characteristic peaks is large and the separation degree is high, the characteristic is obvious, and the walnut kernels and preparations thereof can be comprehensively controlled in quality.

[0005] To this end, the present application provides the following technical solution:

[0006] The present application provides a construction method of a characteristic spectrum of walnut kernels and preparations thereof, which comprises the following steps: (1) preparation of a test sample solution; (2) detection of the test sample solution by high performance liquid chromatography, taking octadecylsilane bonded silica gel as a filler, and a mobile phase comprising a water solution containing phosphoric acid and acetonitrile, and a gradient elution program comprising: 0→5min→15min→20min→40min→50min, the volume percentage of acetonitrile in the mobile phase is: 2%~3%→3%→8%→8%→22%→40%.

[0007] Optionally, step (2) further satisfies at least one of the following 1) to 4): 1) the detection wavelength is 210 to 300 nm, preferably 210 to 220 nm, and more preferably 220 nm; the flow rate is 0.6 to 0.9 mL / min, preferably 0.8 mL / min; the column temperature is 20 to 30 °C, preferably 20 to 25 °C, and more preferably 20 °C; 2) a chromatographic column with a specification of 4.6 mm x 150 mm and 2.7 μm is used in the detection process, and the chromatographic column is preferably a Poroshell 120 EC-C18 chromatographic column; 3) the injection volume is 1 to 5 μL, and preferably 2 μL; and 4) the volume percentage of phosphoric acid in the phosphoric acid-containing aqueous solution is 0.05% to 0.15%.

[0008] Preferably, the gradient elution program comprises: 0→5 min→15 min→20 min→40 min→50 min, and the volume percentage of acetonitrile in the mobile phase is: 2%→3%→8%→8%→22%→40%.

[0009] Optionally, step (1) comprises weighing the walnut kernel test sample, adding a solvent for extraction to obtain an extract, and performing solid-liquid separation to obtain a liquid, which is the test sample solution.

[0010] Optionally, step (1) further satisfies any one or more of the following A to E: A, the mass-to-volume ratio of the walnut kernel test sample to the solvent is 0.05 to 0.4 g: 10 to 100 mL; B, the extraction method is reflux extraction or ultrasonic extraction; preferably, the extraction method is reflux extraction; C, the extraction time is 20 to 40 min; D, the solid-liquid separation is selected from centrifugation or filtration; and E, the solvent is selected from one or more of methanol and water; preferably, the solvent is a 25% to 75% methanol aqueous solution by volume; and more preferably, the solvent is a 50% methanol aqueous solution by volume.

[0011] Optionally, the construction method further comprises a step of preparing a reference solution of the control by using at least one of gallic acid, gallic acid, casuarine, casuarine, isocasuarine, 4-methylumbelliferone-β-D-glucopyranoside, and ellagic acid-4-O-β-D-xyloside, and a solvent, and a step of obtaining a control atlas by detecting the reference solution of the control according to the high-performance liquid chromatography in the construction method described above; and / or, a step of preparing a reference solution of the control by using walnut kernel reference medicinal material as a reference according to step (1) in the construction method described above, and a step of obtaining a reference atlas of the control by detecting the reference solution of the control according to the high-performance liquid chromatography in the construction method described above.

[0012] Optionally, each 1 mL of the reference solution of the control contains 10 to 80 μg of the control.

[0013] Optionally, the solvent used in the preparation of the reference solution of the control is methanol.

[0014] Optionally, the characteristic pattern of the walnut kernel and its preparation has 11 common characteristic peaks, peak 10 corresponds to the retention time of the reference peak of the reference substance of the gallic acid, the peak corresponding to the reference peak of the gallic acid is the S peak, the relative retention time of peaks 1-9, 11 and the S peak is within ±10% of the specified value; the specified value is: 0.10 (peak 1), 0.15 (peak 2), 0.21 (peak 3), 0.23 (peak 4), 0.26 (peak 5), 0.34 (peak 6), 0.52 (peak 7), 0.86 (peak 8), 0.95 (peak 9), 1.09 (peak 11).

[0015] The application also provides an application of the construction method of the characteristic pattern of the walnut kernel and its preparation in quality detection of walnut kernel pharmaceutical preparations.

[0016] The application provides a quality detection method of walnut kernel and its preparation, which comprises the step of comparing the characteristic pattern of the walnut kernel product to be detected with the reference characteristic pattern of the walnut kernel and its preparation; the characteristic pattern of the walnut kernel product to be detected is obtained by using the walnut kernel product to be detected and the construction method described above, and the reference characteristic pattern of the walnut kernel and its preparation is selected from any one of (1)-(3) as follows: (1) it has 11 common characteristic peaks, peak 10 corresponds to the retention time of the reference peak of the reference substance of the gallic acid, the peak corresponding to the reference peak of the gallic acid is the S peak, the relative retention time of peaks 1-9, 11 and the S peak is within ±10% of the specified value; the specified value is: 0.10 (peak 1), 0.15 (peak 2), 0.21 (peak 3), 0.23 (peak 4), 0.26 (peak 5), 0.34 (peak 6), 0.52 (peak 7), 0.86 (peak 8), 0.95 (peak 9), 1.09 (peak 11); (2) the characteristic pattern of the walnut kernel and / or its preparation obtained by using a single batch or multiple batches of walnut kernel and / or its preparation and the construction method described above; (3) the reference characteristic pattern obtained by using multiple batches of walnut kernel and / or its preparation and the construction method described above through the average value or median number method.

[0017] The technical scheme of the application has the following advantages:

[0018] 1.The method for constructing the characteristic map of walnut kernel and its preparation provided by the present application, using octadecylsilane bonded silica gel as the filler, the mobile phase including a phosphoric acid-containing aqueous solution and acetonitrile, and through a specific gradient elution procedure, 11 common characteristic peaks are obtained, wherein the separation of the common characteristic peaks including the ellagic acid peak is achieved, the elution procedure is simple, the obtained characteristic map has a smooth baseline, the characteristic peaks have good peak shape and high resolution, and the method provides a basis for the quality detection and control of walnut kernel and its preparation. The peak positions of ellagic acid, gallic acid, casuarine, casuarine, isocasuarine, 4-methylumbelliferyl-β-D-glucopyranoside, and ellagic acid-4-O-β-D-xyloside can be accurately located, fully reflecting the integrity and characteristics of walnut kernel and its preparation.

[0019] 2.The method for constructing the characteristic map of walnut kernel and its preparation provided by the present application, through the investigation of the extraction conditions such as the optimization of chromatographic conditions, detection wavelength, extraction solvent, extraction method, extraction time, and extraction solvent dosage, the optimal extraction process and chromatographic conditions are determined, so that the peak area is higher, the separation effect is better, and the quality of walnut kernel and its preparation can be more comprehensively monitored. The test sample processing of the method is simple, the chromatographic conditions are easy to realize, the operation is simple, the result is accurate, and the method has good reproducibility.

[0020] 3.The quality detection method of walnut kernel and its preparation provided by the present application, through the comparison between the characteristic map of the walnut kernel and its preparation product to be detected and the control characteristic map of walnut kernel and its preparation, the quality of walnut kernel and its preparation can be comprehensively, clearly, and effectively detected. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings needed to be used in the specific embodiments or prior art description. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0022] Figure 1 is the chromatogram under condition 1 in the pre-study of chromatographic conditions in experimental example 1;

[0023] Figure 2 is the chromatogram under condition 2 in the pre-study of chromatographic conditions in experimental example 1;

[0024] Figure 3 is the chromatogram under condition 3 in the pre-study of chromatographic conditions in experimental example 1;

[0025] Figure 4 is the chromatogram under condition 4 in the pre-study of chromatographic conditions in experimental example 1;

[0026] Figure 5 is the chromatogram of Experimental Example 1 under the chromatographic conditions of the pre-study, condition 5;

[0027] Figure 6 is the chromatogram of Experimental Example 1 under the chromatographic conditions of the pre-study, condition 6;

[0028] Figure 7 is the chromatogram of Experimental Example 1 under the chromatographic conditions of the pre-study, condition 7;

[0029] Figure 8 is the chromatogram of Experimental Example 1 under the chromatographic conditions of the pre-study, condition 8;

[0030] Figure 9 is the chromatogram of Experimental Example 1 under the chromatographic conditions of the pre-study, condition 9;

[0031] Figure 10 is the chromatogram of Experimental Example 1 under the chromatographic conditions of the pre-study, condition 10;

[0032] Figure 11 is the chromatogram of Experimental Example 1 under the chromatographic conditions of the pre-study, condition 11;

[0033] Figure 12 is the chromatogram of Experimental Example 1 under the chromatographic conditions of the optimization (1), 210 nm;

[0034] Figure 13 is the chromatogram of Experimental Example 1 under the chromatographic conditions of the optimization (1), 220 nm;

[0035] Figure 14 is the chromatogram of Experimental Example 1 under the chromatographic conditions of the optimization (1), 240 nm;

[0036] Figure 15 is the chromatogram of Experimental Example 1 under the chromatographic conditions of the optimization (1), 260 nm;

[0037] Figure 16 is the chromatogram of Experimental Example 1 under the chromatographic conditions of the optimization (1), 280 nm;

[0038] Figure 17 is the chromatogram of Experimental Example 1 under the chromatographic conditions of the optimization (1), 300 nm;

[0039] Figure 18 is the chromatogram of Experimental Example 1 under the chromatographic conditions of the optimization (3), acetonitrile-water system;

[0040] Figure 19 is the chromatogram of Experimental Example 1 under the chromatographic conditions of the optimization (3), acetonitrile-0.1% phosphoric acid solution system;

[0041] Figure 20is the chromatogram of experimental example 1, optimization of chromatographic conditions (6) with column temperature of 20℃;

[0042] Figure 21 is the chromatogram of experimental example 1, optimization of chromatographic conditions (6) with column temperature of 25℃;

[0043] Figure 22 is the chromatogram of experimental example 1, optimization of chromatographic conditions (6) with column temperature of 30℃;

[0044] Figure 23 is the chromatogram of experimental example 1, optimization of chromatographic conditions (7) with elution gradient 1 elution;

[0045] Figure 24 is the chromatogram of experimental example 1, optimization of chromatographic conditions (7) with elution gradient 2 elution;

[0046] Figure 25 is the chromatogram of experimental example 1, optimization of chromatographic conditions (7) with elution gradient 3 elution;

[0047] Figure 26 is the chromatogram of experimental example 2, the control spectrum of walnut kernel standard decoction freeze-dried powder; wherein, peak 3: gallic acid; peak 9: ellagic acid-4-O-β-D-xyloside; peak 10: ellagic acid;

[0048] Figure 27 is the chromatogram of experimental example 2, (3) with test sample and control sample;

[0049] Figure 28 is the spectrum corresponding to gallic acid in experimental example 2, (3), wherein the left graph is that of the control sample, and the right graph is that of the test sample;

[0050] Figure 29 is the spectrum corresponding to ellagic acid-4-O-β-D-xyloside in experimental example 2, (3), wherein the left graph is that of the control sample, and the right graph is that of the test sample;

[0051] Figure 30 is the spectrum corresponding to ellagic acid in experimental example 2, (3), wherein the left graph is that of the control sample, and the right graph is that of the test sample;

[0052] Figure 31 is the chromatogram of experimental example 3, specificity investigation, negative blank solvent;

[0053] Figure 32 is the chromatogram of the test sample solution in example 1;

[0054] Figure 33 is the chromatogram of the control sample in example 1. DETAILED DESCRIPTION

[0055] The following examples are provided to better enable those skilled in the art to which the application pertains to make and use the application. The examples are not intended to limit the scope of the application and are not intended to be limiting of the scope of the invention. Any product obtained by the practice of the application or by the practice of the application combined with other prior art features is intended to fall within the scope of the application. In the examples, where specific experimental procedures or conditions are not indicated, the procedures or conditions described in the literature are used. Where the manufacturer of reagents or instruments is not indicated, the reagents or instruments are commercially available.

[0056] Investigation of the construction method in experimental example 1

[0057] 1. Instruments, reagents and reagents

[0058] High performance liquid chromatograph: Waters H-class, Waters e 2695, Agilent 1290.

[0059] Chromatographic column: Poroshell 120Aq-C18 (4.6 x 150 mm, 2.7 μm), Poroshell 120EC-C18 (4.6 x 150 mm, 2.7 μm), Poroshell SB-C18 (4.6 x 150 mm, 2.7 μm), Agilent; Waters: T3 (4.6 x 250 mm, 5 μm), ACQUITY HSS T3 (2.1 x 100 mm, 1.8 μm). The size is not specifically mentioned hereinafter.

[0060] Other instruments: Mettler-Toledo International Trade (Shanghai) Co., Ltd.: one-hundredth balance (ME104), one-hundred-thousandth balance (MS105DU / A), one-millionth balance (XPR2); Qun'an Experimental Instrument Co., Ltd.: electric heating constant temperature water bath (WB100-8F); Kunshan Ultrasonic Instrument Co., Ltd.: digital ultrasonic instrument (KQ-300DB).

[0061] Reagents: Shanghai Xingke High Purity Solvent Co., Ltd.: chromatographically pure methanol (0212230404), chromatographically pure acetonitrile (0114230713); Shanghai Titan Technology Co., Ltd.: analytical pure methanol (P2693002); Shanghai Anpu Experimental Science and Technology Co., Ltd.: chromatographically pure phosphoric acid (F6130152); Shanghai Shidan Standard Technology Service Co., Ltd.: 88.8% pure ellagic acid (15285).

[0062] In the reagents, the freeze-dried powder of walnut kernel standard decoction can be prepared by conventional methods in the art, for example, according to the following steps in the present application: according to the provisions of “walnut kernel” in the Chinese Pharmacopoeia 2020 edition, take walnut kernel medicinal materials, remove impurities, and process to obtain walnut kernel decoction pieces that meet the requirements. Take the walnut kernel decoction pieces and crush them. Take about 100 g of the decoction pieces, place them in a sand pot, add 700 ml of purified water, soak for 30 minutes, and decoct for 30 minutes. Filter while hot, and quickly cool the filtrate to room temperature. Add 500 ml of purified water to the residue, decoct for 25 minutes, filter while hot, and quickly cool the filtrate to room temperature. Combine the two filtrates, concentrate the filtrate under reduced pressure, take the concentrated extract, place it in a freeze dryer, freeze-dry it to a dry state, take it out, weigh it, crush it, and package it. Thus, the freeze-dried powder of walnut kernel standard decoction is obtained.

[0063] The freeze-dried powder of walnut kernel standard decoction with batch numbers 2103001Y, 2103002Y, 2103003Y, 2103004Y, 2103005Y, 2103006Y, 2103007Y, 2103008Y, 2103009Y, 2103010Y, 2103011Y, 2103012Y, 2103013Y, 2103014Y, 2103015Y, 2305001Y, 2305002Y, and 2305003Y was prepared using different batches of walnut kernel medicinal materials.

[0064] 2. Pre-study of chromatographic conditions

[0065] Take 0.25 g of the freeze-dried powder of walnut kernel standard decoction, accurately weigh it, place it in a conical flask with a plug, accurately add 10 ml of 50% methanol, weigh the weight, ultrasonically treat it (power 300 W, frequency 40 kHz) for 30 minutes, take it out, cool it down, re-weigh it, make up the weight loss with 50% methanol, shake it well, filter it, and take the filtrate. Thus, the test sample solution is obtained. The test sample solution is detected as follows.

[0066] Condition 1: The above test sample solution is detected by high performance liquid chromatography. The instrument is Waters H-class, acetonitrile is used as mobile phase A, and 0.1% phosphoric acid solution (the concentration of the subsequent phosphoric acid solution refers to the volume percentage) is used as mobile phase B. The column is ACQUITY HSS T3 (2.1 x 100 mm, 1.8 μm), gradient elution is performed according to the provisions in Table 1, the flow rate is 0.3 mL / min, the column temperature is 30°C, the detection wavelength is 220 nm, and the injection volume is 2 μL. The obtained chromatogram is shown in Figure 1 As can be seen from the figure, the separation effect of the chromatographic peaks in the chromatogram is poor, especially the chromatographic peaks in the first 7 minutes and 15-25 minutes.

[0067] Table 1

[0068]

[0069] Condition 2: The instrument Waters e2695 was used, acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, T3 (4.6x250mm, 5um) was used as the chromatographic column, and gradient elution was performed according to the provisions in Table 1; the flow rate was 1 mL / min, the column temperature was 30°C, the detection wavelength was 220 nm, and the injection volume was 5uL. The obtained chromatogram is shown in Figure 2 From the figure, it can be seen that there are many chromatographic peaks in the first 10 minutes, and the separation effect is poor, so the elution gradient needs to be adjusted in the follow-up to improve the separation effect.

[0070] Conditions 3-5: The difference from condition 2 is that ① the gradient elution parameters in Table 2 are used, and ② the column temperature is 40°C. The obtained chromatogram is shown in Figures 3-5 From the figure, it can be seen that the separation effect of each peak is still not ideal, so the chromatographic column used is replaced for further experiments.

[0071] Table 2

[0072]

[0073] Condition 6: The above test sample solution was detected by high performance liquid chromatography, the instrument Waters e2695 was used, acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, Poroshell 120Aq-C18 (4.6x150mm, 2.7um) was used as the chromatographic column, and gradient elution was performed according to the provisions in condition 6 of Table 3; the flow rate was 0.6mL / min, the column temperature was 25°C, the detection wavelength was 220nm, and the injection volume was 5uL. Condition 7: The difference from condition 6 is only that the column temperature is 30°C. The obtained chromatogram is shown in Figure 6 7 From the figure, it can be seen that the change of column temperature has no obvious effect on the chromatogram, and the separation of each peak in the first 10 minutes is not ideal, so other parameters need to be changed in the follow-up.

[0074] Table 3

[0075]

[0076] Condition 8: The above test sample solution was detected by high performance liquid chromatography, the instrument Agilent 1290 was used, acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, Poroshell 120EC-C18 (4.6x150mm, 2.7um) was used as the chromatographic column, and gradient elution was performed according to the provisions in condition 8 of Table 3; the flow rate was 0.8mL / min, the column temperature was 20°C, the detection wavelength was 220nm, and the injection volume was 5uL. The obtained chromatogram is shown in Figure 8 ​​The separation of the peaks in the first 10 min is still not ideal, and the other parameters are continuously changed.

[0077] Condition 9: The only difference from Condition 8 is that the flow rate is 0.7 mL / min, and the obtained chromatogram is shown in Figure 9 Condition 10: The only difference from Condition 9 is that the gradient elution is performed according to the provisions of Condition 10 in Table 3, and the obtained chromatogram is shown in Figure 10 It can be seen that Figure 10 the separation of the peaks with retention times of 7-10 min is obviously improved, but the baseline is not flat in the first 5 min, so the next step is to try to reduce the sampling amount of the test sample.

[0078] Condition 11: Preparation of test sample solution: The only difference from the preparation method of the test sample solution described above is that 0.1 g of the freeze-dried powder of walnut kernel standard decoction is taken; the detection parameters are different from those of Condition 24 only in that the flow rate is 0.8 mL / min, and the injection volume is 2 μL. The obtained chromatogram is shown in Figure 11 It can be seen that under this condition, the separation of the peaks with retention times of about 3-10 min is slightly improved, and this method is temporarily used as the initial condition for subsequent system optimization.

[0079] 3. Optimization of chromatographic conditions

[0080] Take 0.1 g of the freeze-dried powder of walnut kernel standard decoction, accurately weigh it, and place it in a conical flask with a stopper. Accurately add 10 ml of 50% methanol, weigh the weight, ultrasonic treat (power 300 W, frequency 40 kHz) for 30 minutes, take it out, cool it down, re-weigh it, make up the weight loss with 50% methanol, shake it well, filter it, and take the filtrate to obtain the test sample solution. The test sample solution is investigated as follows.

[0081] (1) Investigation of absorption wavelength

[0082] The test sample solution is detected by high performance liquid chromatography: Agilent 1290 is used, acetonitrile is used as mobile phase A, 0.1% phosphoric acid solution is used as mobile phase B, Poroshell 120EC-C18 (4.6x150mm, 2.7μm) is used as the chromatographic column, and gradient elution is performed according to the provisions of Condition 10 in Table 3; the flow rate is 0.8 mL / min, the column temperature is 20℃, the injection volume is 2 μL; 210 nm, 220 nm, 240 nm, 260 nm, 280 nm, and 300 nm are used for detection, and the obtained chromatogram is shown in Figures 12-17 .

[0083] From Figures 12-17It can be seen that the UV absorption of the chromatographic peak is obviously reduced at the detection wavelength of 280-300 nm; compared with 240 nm and 260 nm, there is an additional chromatographic peak at a retention time of 18.5 minutes at 210 nm and 220 nm, and the absorption intensity of each chromatographic peak at 210 nm and 220 nm is consistent, so the absorption wavelength for determining the characteristic spectrum is selected as 210-220 nm. Compared with 210 nm, the baseline of 220 nm is more stable, so 220 nm is preferably selected as the detection wavelength of the characteristic spectrum.

[0084] (2) Investigation of different chromatographic columns

[0085] The test solution was detected by high performance liquid chromatography: the instrument Agilent 1290 was used, acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, gradient elution was carried out according to the provisions in Table 3 condition 10; the flow rate was 0.8 mL / min, the column temperature was 20°C, the detection wavelength was 220 nm, and the injection volume was 2 μL; Agilent Poroshell 120EC-C18 (4.6 x 150 mm, 2.7 μm), Agilent Poroshell 120Aq-C18 (4.6 x 150 mm, 2.7 μm), and Agilent Poroshell SB-C18 (4.6 x 150 mm, 2.7 μm) were used for detection, and the system suitability parameters of the obtained chromatographic peaks are shown in Table 4. It can be seen from the table that peak 3 and peak 4 are co-eluted in the chromatogram obtained by the Poroshell 120Aq-C18 chromatographic column, and the peak shape and resolution of the chromatogram obtained by the Poroshell 120EC-C18 chromatographic column are better than those of the Poroshell 120Aq-C18 chromatographic column, which indicates that the chromatographic column has a greater impact on the method. The Agilent Poroshell 120EC-C18 chromatographic column is used as the chromatographic column for the constructed method.

[0086] Table 4

[0087]

[0088]

[0089] (3) Investigation of whether to add phosphoric acid in the mobile phase

[0090] The test sample solution is detected by high performance liquid chromatography: the instrument is Agilent 1290, the mobile phase A is acetonitrile, the mobile phase B is water and 0.1% phosphoric acid solution respectively, the chromatographic column is Poroshell 120EC-C18, gradient elution is carried out according to the provisions in table 3 condition 10; the flow rate is 0.8 mL / min, the column temperature is 20 DEG C, the detection wavelength is 220 nm, the injection volume is 2 mu L; the obtained chromatogram is shown in Figure 18 , 19 It can be seen from the figure that the chromatographic information is rich after adding 0.1% phosphoric acid in the mobile phase, so phosphoric acid is selected as the additive.

[0091] (4) Investigation of different phosphoric acid concentrations in the mobile phase

[0092] The test sample solution is detected by high performance liquid chromatography: the instrument is Agilent 1290, the mobile phase A is acetonitrile, the mobile phase B is 0.05%, 0.10%, 0.15% phosphoric acid solution respectively, the chromatographic column is Poroshell 120EC-C18, gradient elution is carried out according to table 3 condition 10; the flow rate is 0.8 mL / min, the column temperature is 20 DEG C, the detection wavelength is 220 nm, the injection volume is 2 mu L. The system suitability parameters of the obtained chromatographic peaks are shown in table 5. It can be known from the analysis table that different phosphoric acid concentrations have no obvious influence on each chromatographic peak, in order to facilitate operation, 0.1% phosphoric acid concentration is selected for subsequent condition screening investigation.

[0093] Table 5

[0094]

[0095] (5) Investigation of different flow rates

[0096] The test sample solution is detected by high performance liquid chromatography: the instrument is Agilent 1290, the mobile phase A is acetonitrile, the mobile phase B is 0.10% phosphoric acid solution, the chromatographic column is Poroshell 120EC-C18, gradient elution is carried out according to the provisions in table 3 condition 10; the flow rate is 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, 0.9 mL / min respectively, the column temperature is 20 DEG C, the detection wavelength is 220 nm, the injection volume is 2 mu L. The system suitability parameters of the obtained chromatographic peaks are shown in table 6. It can be known from the analysis that with the increase of flow rate, each chromatographic peak gradually moves forward, and the system suitability parameters of each chromatographic peak have no obvious influence, considering various factors such as column pressure, 0.8 ml / min flow rate is selected for subsequent condition investigation.

[0097] Table 6

[0098]

[0099]

[0100] (6) Different column temperatures were investigated

[0101] The test sample solution was detected by high performance liquid chromatography: an instrument Agilent 1290 was used, mobile phase A was acetonitrile, mobile phase B was 0.10% phosphoric acid solution, Poroshell 120EC-C18 (4.6*150mm, 2.7um) was used as a chromatographic column, gradient elution was carried out according to the provisions in Table 3 condition 10; the flow rate was 0.8mL / min, the column temperature was 20℃, 25℃ and 30℃ respectively, the detection wavelength was 220nm, and the injection volume was 2uL. The system suitability parameters of the obtained chromatographic peaks are shown in Table 7, and the obtained chromatogram is shown in Figures 20-22 It can be known from the analysis table and image that the separation degree of peak 3 and peak 4 decreases with the increase of column temperature, and the co-flow phenomenon occurs when the column temperature is increased to 30℃. Therefore, the column temperature range of the present application is preferably 20-25℃, and in order to obtain better separation effect, the column temperature is selected as 20℃ for subsequent condition screening investigation.

[0102] Table 7

[0103]

[0104]

[0105] (7) Different elution gradients were investigated

[0106] The test sample solution was detected by high performance liquid chromatography: an instrument Agilent 1290 was used, mobile phase A was acetonitrile, mobile phase B was 0.10% phosphoric acid solution, Poroshell 120EC-C18 (4.6*150mm, 2.7um) was used as a chromatographic column, gradient elution was carried out according to the provisions in Table 8; the flow rate was 0.8mL / min, the column temperature was 20℃, the detection wavelength was 220nm, and the injection volume was 2uL. The system suitability parameters of the obtained chromatographic peaks are shown in Table 9, and the obtained chromatogram is shown in Figures 23-25 It can be known from the analysis table and image that the separation degree of peak 7 and the adjacent interfering peak in elution gradient 2 and elution gradient 3 is obviously better than that in elution gradient 1; the separation degree of peak 1 and peak 2 in elution gradient 3 is better than that in elution gradient 2, so elution gradient 3 is preferably selected as the elution gradient of the present method.

[0107] Table 8

[0108]

[0109] Table 9

[0110]

[0111]

[0112] 4. Investigation of Test Solution

[0113] (1) Investigation of Extraction Solvent

[0114] Take five standard walnut kernel soup freeze-dried powder 0.1 g, accurately weighed, placed in a conical flask with a plug, respectively, accurately add water, 25% methanol, 50% methanol, 75% methanol, methanol 20 ml, tightly sealed, weighed, respectively, ultrasonic treatment (power 300 W, frequency 40 kHz) 30 minutes, take out, cool, reweigh, respectively, with the corresponding solvent to make up the weight loss, shake, filter, accurately suck the filtrate 2 μL, inject high performance liquid chromatograph, according to the chromatographic conditions of elution gradient 3 determination. The chromatogram parameters obtained are shown in Table 10, and the analysis shows that when the extraction solvent is water, peak 7 is missing; when the extraction solvent is methanol, peak 4 is missing, and the peak type of peak 1 and peak 2 is poor; when the extraction solvent is 25% methanol, 50% methanol, 75% methanol, all show 12 characteristic peaks, and the system suitability meets the determination requirements, but when the extraction solvent is 50% methanol, the extraction rate of S peak (ellagic acid) is higher. Comprehensive analysis, select 50% methanol as the extraction solvent for subsequent investigation.

[0115] Table 10

[0116]

[0117]

[0118]

[0119] (2) Investigation of Extraction Method

[0120] Take two standard walnut kernel soup freeze-dried powder 0.1 g, accurately weighed, placed in a conical flask with a plug, respectively, accurately add 50% methanol 20 ml, tightly sealed, weighed, respectively, ultrasonic treatment (power 300 W, frequency 40 kHz), heating reflux treatment 30 minutes, take out, cool, reweigh, with 50% methanol to make up the weight loss, shake, filter, accurately suck the filtrate 2 μL, inject high performance liquid chromatograph, according to the chromatographic conditions of elution gradient 3 determination. The chromatogram parameters obtained are shown in Table 11, and the analysis data shows that the extraction method has no obvious effect on the information amount of chromatographic peak and the system suitability parameters, but the reflux extraction has higher extraction rate of ellagic acid. In order to operate simply and keep consistent with the content determination, select reflux as the extraction method for subsequent investigation.

[0121] Table 11

[0122]

[0123] (3) Preliminary investigation of solvent addition amount

[0124] Take two walnut kernel standard soup freeze-dried powder 0.1 g, precision weighing, placed in a stoppered conical flask, respectively, precision 50% methanol 10 ml, 25 ml, tight, weighed, respectively, heated reflux treatment 30 minutes, removed, put cold, weighed again, with 50% methanol to make up for the weight loss, shake, filter, precision suction continue filtrate 2 μL, injected into high performance liquid chromatograph, according to the elution gradient 3 chromatographic conditions were determined. The chromatogram parameters obtained are shown in Table 12, the analysis data show that with the increase of solvent addition, the peak area of chromatographic peak corresponding multiple reduction, shows that the sample size in the range of 10 ~ 25 ml, walnut kernel standard soup freeze-dried powder can be completely extracted. In order to facilitate operation, choose 25 ml for subsequent conditions of investigation.

[0125] Table 12

[0126]

[0127] (4) extraction time investigation

[0128] Take three walnut kernel standard soup freeze-dried powder 0.1 g, precision weighing, placed in a stoppered conical flask, respectively, precision 50% methanol 25 ml, tight, weighed, respectively, heated reflux treatment 20, 30, 40 minutes, removed, put cold, weighed again, with 50% methanol to make up for the weight loss, shake, filter, precision suction continue filtrate 2 μL, injected into high performance liquid chromatograph, according to the elution gradient 3 chromatographic conditions were determined. The chromatogram parameters obtained are shown in Table 13, the analysis data show that the extraction time has no significant effect on the information content of chromatographic peak and system suitability parameters, in order to extract completely, choose reflux 30 minutes as the extraction time for subsequent investigation.

[0129] Table 13

[0130]

[0131]

[0132] (5) investigation of sample size

[0133] Take four quality of 0.05g, 0.1g, 0.2g, 0.4g walnut kernel standard decoction of freeze-dried powder, precision weighing, placed in a conical flask with plug, respectively, precision 50% methanol 25ml, tight plug, weighed, respectively, heated reflux treatment 30 minutes, remove, put cold, weighed again, with 50% methanol to make up for the weight loss, shake, filter, precision suction continue filtrate 2μL, inject high performance liquid chromatograph, according to elution gradient 3 chromatographic conditions were determined. The chromatogram parameters obtained in table 14, analysis data, with the increase of sampling amount, the peak area of chromatographic peak corresponding multiple increase, showed that the sampling amount in 0.05-0.4g range, walnut kernel standard decoction of freeze-dried powder components can be completely extracted. Based on the overall response value of characteristic peak, comprehensive consideration, therefore, select 0.2g as the sampling amount of this test.

[0134] Table 14

[0135]

[0136]

[0137] (6) solvent addition amount investigation

[0138] Take three walnut kernel standard decoction of freeze-dried powder 0.2g, precision weighing, placed in a conical flask with plug, respectively, precision 50% methanol 25ml, 50ml, 100ml, tight plug, weighed, respectively, heated reflux treatment 30 minutes, remove, put cold, weighed again, with 50% methanol to make up for the weight loss, shake, filter, precision suction continue filtrate 2μL, inject high performance liquid chromatograph, according to elution gradient 3 chromatographic conditions were determined. The chromatogram parameters obtained in table 15, analysis data, with the increase of solvent addition amount, the peak area of chromatographic peak corresponding multiple decrease, showed that the sampling amount in 25-100ml range, walnut kernel standard decoction of freeze-dried powder components can be completely extracted. Based on the overall response value of characteristic peak, comprehensive consideration, therefore, select 50ml as the sampling amount of this test.

[0139] Table 15

[0140]

[0141] (7) different injection volume investigation

[0142] Take four walnut kernel standard soup freeze-dried powder 0.2 g, precision weighing, placed in a plug conical flask, respectively, precision 50% methanol 50 ml, tightly sealed, weighed, respectively, heated reflux treatment 30 minutes, removed, put cold, weighed again, with 50% methanol to make up for the weight loss, shake up, filter, respectively, precision suction filter liquid 1 μL, 2 μL, 3 μL, 5 μL, inject high performance liquid chromatograph, according to the elution gradient 3 chromatographic conditions were determined. The chromatogram parameters obtained in table 16, analysis data can know, with the increase of sample size, the peak area of chromatographic peak in test sample relatively increased, the interference of non characteristic peak is more obvious, thus leading to part of the chromatographic peak in chromatogram stacking. 2 μl of peak type is better, peak height and peak width is relatively moderate, the separation degree of chromatographic peak is relatively good, so the sample volume is selected 2 μl.

[0143] Table 16

[0144]

[0145]

[0146] Experimental example 2 determination of characteristic peak and establishment of control atlas

[0147] (1) construction method

[0148] Take 18 batches of walnut kernel standard soup freeze-dried powder (batch number: 2103001Y, 2103002Y, 2103003Y, 2103004Y, 2103005Y, 2103006Y, 2103007Y, 2103008Y, 2103009Y, 2103010Y, 2103011Y, 2103012Y, 2103013Y, 2103014Y, 2103015Y, 2305001Y, 2305002Y, 2305003Y) as test sample, according to the method of example 1, the test sample solution was prepared. Take appropriate amount of gallic acid reference substance, precision weighing, add methanol to prepare a solution containing 20 μg per 1 ml as reference solution. The above solution was detected by high performance liquid chromatography in example 1, and table 17 was the characteristic atlas determination results of 18 batches of walnut kernel standard soup freeze-dried powder.

[0149] According to the research results, it is determined that 11 characteristic peaks should be present in the freeze-dried powder chromatogram of walnut kernel standard decoction, and should correspond to the retention time of the 11 characteristic peaks in the reference chromatogram of the control medicinal material. Peak 10 should correspond to the retention time of the reference peak of the control sample of ellagic acid. The peak corresponding to the reference peak of ellagic acid is the S peak. The relative retention times of the other characteristic peaks to the S peak are calculated, and the relative retention times should be within ±10% of the specified values, which are: 0.10 (peak 1), 0.15 (peak 2), 0.21 (peak 3), 0.23 (peak 4), 0.26 (peak 5), 0.34 (peak 6), 0.52 (peak 7), 0.86 (peak 8), 0.95 (peak 9), and 1.09 (peak 11).

[0150] Table 17

[0151]

[0152]

[0153] (2) The characteristic chromatogram of the freeze-dried powder of 18 batches of walnut kernel standard decoction was synthesized by using Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version), and the control chromatogram of the freeze-dried powder of walnut kernel standard decoction was established, as shown in Figure 26

[0154] (3) Identification of characteristic peaks

[0155] Gallic acid, ellagic acid-4-O-β-D-xyloside, and ellagic acid were precisely weighed and added to methanol to prepare a control sample solution containing 20 μg of each control sample per 1 ml.

[0156] HPLC and LC / MS / MS were used to analyze peaks 1-11, and it was determined that peak 3 was gallic acid, peak 9 was ellagic acid-4-O-β-D-xyloside, and peak 10 was ellagic acid. Through literature research and analysis, the structures and components of peaks 1, 2, 4, 5, 6, 7, 8, and 11 were speculated, and the LC / MS / MS analysis results are shown in Table 18.

[0157] Table 18

[0158]

[0159]

[0160] Since mass spectrometry is not compatible with phosphoric acid solution, when performing UPLC-Q-TOF-MS analysis, 0.1% formic acid solution was used to replace the original mobile phase 0.1% phosphoric acid solution for analysis, and the obtained chromatogram is shown in Figure 27 , and the obtained spectrum is shown in Figures 28-30 ​, the retention time and peak sequence of each characteristic peak have certain differences with the original spectrum, and the compounds corresponding to each characteristic peak are determined by comparison with reference substances and spectral analysis.

[0161] Methodology verification of experimental example 3

[0162] 1. Precision experiment: the freeze-dried powder of walnut kernel standard decoction prepared by the same method in Example 1 was taken as the test sample solution, and was continuously injected for 6 times according to the chromatographic conditions in Example 1, and the relative retention time and relative peak area of each characteristic peak were calculated. The results showed that the RSD of the relative retention time of each characteristic peak was less than 2%, indicating that the precision of the instrument was good.

[0163] 2. Method repeatability test: the freeze-dried powder of the same batch of walnut kernel standard decoction was taken, and 6 test sample solutions were prepared by repeating the preparation method in Example 1, and were determined according to the chromatographic conditions in Example 1, and the relative retention time and relative peak area of each characteristic peak were calculated. The results showed that the RSD of the relative retention time of each characteristic peak was less than 2%, indicating that the repeatability of the method was good.

[0164] 3. Intermediate precision between different personnel: the freeze-dried powder of the same batch of walnut kernel standard decoction was taken, and 2 test sample solutions were prepared by 3 persons according to the preparation method in Example 1, and were determined according to the chromatographic conditions in Example 1 on the same instrument, and the relative retention time and relative peak area of each characteristic peak were calculated. The results showed that the RSD of the relative retention time of each characteristic peak was less than 2%, indicating that the intermediate precision of the method between different personnel was good.

[0165] 4. Intermediate precision between different instruments: the freeze-dried powder of the same batch of walnut kernel standard decoction was taken, and 6 test sample solutions were prepared by 2 persons according to the preparation method in Example 1, and were determined according to the chromatographic conditions in Example 1 on different instruments, and the relative retention time and relative peak area of each characteristic peak were calculated. The results showed that the RSD of the relative retention time of each characteristic peak was less than 10%, indicating that the intermediate precision between different instruments was good.

[0166] 5. Stability: the freeze-dried powder of walnut kernel standard decoction prepared by the same method in Example 1 was taken as the test sample solution, and was determined according to the method in Example 1 at 0, 2, 4, 8, 12, 18, 24, 36 and 48 hours, and the relative retention time and relative peak area of each characteristic peak were calculated. The RSD of the relative retention time of each characteristic peak was less than 2%, and the test sample solution was stable within 48 hours, meeting the determination requirements.

[0167] 6. Different flow rate durability investigation: the same walnut kernel standard decoction freeze-dried powder test sample solution prepared by the method of Example 1 was taken, and the flow rate was 0.75 ml / min, 0.80 ml / min, and 0.85 ml / min, respectively, and the rest of the determination conditions were the same as in Example 1. The relative retention time and relative peak area of each characteristic peak were calculated. The results showed that the RSD values of the relative retention time of each characteristic peak to the reference S peak were all less than 5%, indicating that the slight change of the flow rate had no significant effect on the relative retention time of the characteristic peak. The method had certain durability to the flow rate.

[0168] 7. Different column temperature durability investigation: the same walnut kernel standard decoction freeze-dried powder test sample solution prepared by the method of Example 1 was taken, and the column temperature was 18°C, 20°C, and 22°C, respectively, and the rest of the determination conditions were the same as in Example 1. The relative retention time and relative peak area of each characteristic peak were calculated. The results showed that the RSD values of the relative retention time of each characteristic peak were all less than 5%, indicating that the slight change of the column temperature had no significant effect on the relative retention time of the characteristic peak. The method had certain durability to the column temperature.

[0169] 8. Different acid concentration of mobile phase durability investigation: the same walnut kernel standard decoction freeze-dried powder test sample solution prepared by the method of Example 1 was taken, and the mobile phase was mobile phase I (acetonitrile-0.08% phosphoric acid solution), mobile phase II (acetonitrile-0.10% phosphoric acid solution), and mobile phase III (acetonitrile-0.12% phosphoric acid solution), respectively, and the rest of the determination conditions were the same as in Example 1. The relative retention time and relative peak area of each characteristic peak were calculated. The results showed that the relative retention time of each characteristic peak to the reference S peak was less than 5%, indicating that the slight change of the acid concentration of the mobile phase had no significant effect on the characteristic peak. The method had certain durability to the acid concentration of the mobile phase.

[0170] 9. Specificity investigation: the test sample solution obtained in Example 1 and an equal amount of negative blank solvent were precisely taken and injected into a high performance liquid chromatograph, respectively, and tested according to the chromatographic conditions of Example 1. As shown in Figure 32 and 31 , the results showed that the negative had no interference.

[0171] Example 1

[0172] The present example provides a method for constructing a characteristic spectrum of a walnut kernel standard decoction freeze-dried powder, comprising the following steps:

[0173] Preparation of the test sample solution: about 0.2 g of the walnut kernel standard decoction freeze-dried powder was precisely weighed and placed in a conical flask with a plug, 50 ml of 50% methanol was precisely added, heated for reflux for 30 min, cooled, and weighed again. The lost weight was supplemented with 50% methanol, shaken well, filtered, and the filtrate was taken, thereby obtaining the test sample solution.

[0174] Preparation of reference solution of control: accurately weigh appropriate amount of ellagic acid control, and add methanol to prepare a solution containing 20 μg per 1 ml, namely the reference solution of control is obtained.

[0175] Determination: inject 2 μl of the above-mentioned freeze-dried powder sample solution of walnut kernel standard decoction and the reference solution of control into the ultra-high performance liquid chromatograph, and the chromatographic conditions are as follows: Poroshell 120EC-C18 (4.6 mm x 150 mm, 2.7 μm) is used as the chromatographic column; acetonitrile is used as mobile phase A, and 0.1% phosphoric acid solution is used as mobile phase B, and gradient elution is carried out according to the provisions in Table 19; the flow rate is 0.8 ml / min; the column temperature is 20°C; the detection wavelength is 220 nm; and the theoretical plate number should not be less than 5000 calculated according to the peak of ellagic acid. Table 20 is the system suitability parameter of the sample, and Table 21 is the system suitability parameter of the control. The chromatogram is shown in Figure 32 、 Figure 33 .

[0176] Table 19

[0177]

[0178] Table 20

[0179]

[0180] Table 21

[0181]

[0182] It can be seen that the sample solution shows 11 peaks, and the retention time of peak 10 corresponds to the peak of the reference solution of ellagic acid control. In the chromatogram of the sample solution, the characteristic peak has good peak shape, high resolution, and short detection and analysis time.

[0183] Obviously, the above examples are only examples for the purpose of clear illustration, and are not limitations to the embodiments. Based on the above description, other different forms of changes or variations can also be made by those skilled in the art. Here, it is not necessary and impossible to exhaust all the embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A method for constructing a characteristic spectrum of walnut kernels and their preparations, characterized in that, Includes the following steps: (1) Preparation of the test solution includes weighing the walnut kernels as test sample, adding solvent to extract, obtaining the extract, separating the solid and liquid, and taking the liquid, which is the test solution; It also includes the use of ellagic acid, gallic acid, and 4-methylumbelliferyl- β -D-glucopyranoside, ellagic acid-4-O- β -D-xyloside was added to a solvent to prepare a reference solution; (2) The test solution and reference solution were analyzed by ultra-high performance liquid chromatography. Acetonitrile was used as mobile phase A and an aqueous solution containing phosphoric acid was used as mobile phase B. The gradient elution program included: 0→5min→15min→20min→40min→50min. The volume percentage of acetonitrile in the mobile phase was: 2%~3%→3%→8%→8%→22%→40%. The detection process uses an Agilent Poroshell 120 EC-C18 column with dimensions of 4.6 mm × 150 mm and 2.7 μm, or an Agilent Poroshell SB-C18 column with dimensions of 4.6 mm × 150 mm and 2.7 μm. The detection wavelength is 210~220nm; In the preparation of the test solution, the solvent is an aqueous solution of methanol with a volume percentage of 25% to 75%.

2. The construction method according to claim 1, characterized in that, Step (2) also satisfies at least one of the following: 1) to 4) 1) Detection wavelength: 220 nm; flow rate: 0.6~0.9 mL / min; column temperature: 20~30℃; 2) A Poroshell 120 EC-C18 column was used during the detection process; 3) The injection volume is 1~5μL; 4) The volume percentage of phosphoric acid in the phosphoric acid-containing aqueous solution is 0.05%~0.15%.

3. The construction method according to claim 2, characterized in that, Step (2) also requires a flow rate of 0.8 mL / min and a column temperature of 20~25℃. And / or, the injection volume is 2 μL.

4. The construction method according to claim 3, characterized in that, Step (2) also requires the column temperature to be 20°C.

5. The construction method according to claim 1, characterized in that, The gradient elution program includes: 0→5min→15min→20min→40min→50min, and the volume percentage of acetonitrile in the mobile phase is: 2%→3%→8%→8%→22%→40%.

6. The construction method according to claim 1, characterized in that, Step (1) also satisfies any one or more of the following A~E: A. The mass-to-volume ratio of walnut kernel sample to solvent is 0.05~0.4g:10~100mL; B. The extraction method is either reflux extraction or ultrasonic extraction; C. Extraction time is 20~40 minutes; D. The solid-liquid separation is selected from centrifugation or filtration; E. The solvent is a 50% (v / v) aqueous methanol solution.

7. The construction method according to claim 6, characterized in that, Step (1) also satisfies that the extraction method is reflux extraction.

8. The construction method according to any one of claims 1 to 7, characterized in that, The construction method further includes the step of preparing a reference solution by using walnut kernel as a reference material according to step (1) of any of the construction methods described in claims 1 to 7, and detecting the reference solution by ultra-high performance liquid chromatography according to any of the construction methods described in claims 1 to 7 to obtain a reference chromatogram.

9. The construction method according to claim 1, characterized in that, Each 1 mL of the reference solution contains 10–80 μg of reference standard; and / or, the solvent used in the preparation of the reference solution is methanol.

10. The construction method according to claim 1, characterized in that, The characteristic chromatogram of the walnut kernel and its preparation has 11 common characteristic peaks. Peak 10 corresponds to the retention time of the ellagic acid reference peak, and the peak corresponding to the ellagic acid reference peak is peak S. The relative retention times of peaks 1 to 9, peak 11 and peak S are within ±10% of the specified values. The specified values ​​are as follows: 0.10, 0.15, 0.21, 0.23, 0.26, 0.34, 0.52, 0.86, 0.95 and 1.

09.

11. The application of the method for constructing the characteristic spectrum of walnut kernels and their preparations according to any one of claims 1 to 10 in the quality detection of walnut kernel pharmaceutical preparations.

12. A method for quality testing of walnut kernels and their preparations, characterized in that, The process includes comparing the characteristic chromatogram of the walnut kernel product to be tested with the control characteristic chromatogram of walnut kernel and its preparation; the characteristic chromatogram of the walnut kernel product to be tested is constructed using the walnut kernel product to be tested according to any one of the construction methods described in claims 1 to 10, and the control characteristic chromatogram of walnut kernel and its preparation is selected from any one of the following (1) to (3): (1) It has 11 common characteristic peaks. Peak 10 corresponds to the retention time of the ellagic acid reference peak. The peak corresponding to the ellagic acid reference peak is peak S. The relative retention times of peaks 1 to 9, peak 11 and peak S are within ±10% of the specified values. The specified values ​​are: 0.10, 0.15, 0.21, 0.23, 0.26, 0.34, 0.52, 0.86, 0.95, 1.

09. (2) Characteristic chromatograms of walnut kernels and / or their preparations obtained by using a single batch or multiple batches of walnut kernels and / or their preparations according to the construction method described in any one of claims 1 to 10; (3) Using multiple batches of walnut kernels and / or their preparations, the characteristic chromatograms obtained according to the construction method of any one of claims 1 to 10 are used to prepare a control characteristic chromatogram by means of average or median.

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