A method for separating chemical components of a pepper or a preparation thereof or a method for constructing a characteristic map

Abstract

The application belongs to the field of traditional Chinese medicine quality detection, and specifically discloses a method for separating chemical components in peppers and preparations thereof or a method for constructing characteristic chromatograms, which comprises the following steps: (1) preparing a test sample solution; (2) detecting the test sample solution by high performance liquid chromatography, using octadecylsilane-bonded silica gel as a filler, using methanol as a mobile phase A, using a water solution containing phosphoric acid as a mobile phase B, and optimizing a gradient elution program; finally, a plurality of common characteristic peaks are obtained, the plurality of common characteristic peaks are effectively separated, the characteristic chromatogram has good characteristic peak shapes and a stable baseline, the detection time is short, and the peak height or peak area is uniform.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine detection technology, specifically relating to a method for separating chemical components or constructing characteristic spectra in chili peppers and their preparations. Background Technology

[0002] Chili pepper is the dried, ripe fruit of *Capsicum annuum* L., a plant in the Solanaceae family, or its cultivated varieties. It has warming and dispersing properties, and can also stimulate appetite and aid digestion. It is used for abdominal pain due to cold stagnation, vomiting, diarrhea, and frostbite.

[0003] The 2020 edition of the Chinese Pharmacopoeia includes quality control measures for chili peppers, covering aspects such as the original plant variety, characteristics, physicochemical identification, and content determination. Literature also describes the chemical components of chili peppers, including capsaicin and organic acids. However, on the one hand, determining or identifying the content of a single component in chili peppers and their preparations (e.g., granules) cannot comprehensively detect and control their quality. On the other hand, combining the determination of a single component with the identification of other components is time-consuming and labor-intensive, making it difficult to widely apply in production practice. Summary of the Invention

[0004] Therefore, the purpose of this invention is to provide a method for separating chemical components or constructing characteristic spectra in chili peppers and their preparations. This method establishes characteristic spectra of the variety based on the characteristics of the chili pepper and its preparations, and achieves effective separation of each characteristic peak. The method is simple and easy to operate, and provides a scientific basis for comprehensively establishing quality control standards for chili peppers and their preparations (e.g., formulation granules).

[0005] Specifically, this invention discloses a method for isolating chemical components or constructing characteristic spectra in chili peppers and their preparations, comprising the following steps:

[0006] (1) Preparation of the test solution;

[0007] (2) The test solution was analyzed by high performance liquid chromatography (HPLC). Octadecylsilane-bonded silica gel was used as the stationary phase, methanol as mobile phase A, and an aqueous solution containing phosphoric acid as mobile phase B. Gradient elution was performed, and the gradient elution procedure included:

[0008] For 0-5 min, the volume ratio of mobile phase A to mobile phase B is 10%:90%.

[0009] Over 5-10 minutes, the volume ratio of mobile phase A to mobile phase B changes from 10% to 15% and from 90% to 85%.

[0010] Over 10-30 minutes, the volume ratio of mobile phase A to mobile phase B changes from 15% to 30% and from 85% to 70%.

[0011] Over 30-55 minutes, the volume ratio of mobile phase A to mobile phase B changes from 30% to 35% and from 70% to 65%.

[0012] During the 55-85 min period, the volume ratio of mobile phase A to mobile phase B was 35% → 50% and 65% → 50%, respectively.

[0013] During the 85-95 min time, the volume ratio of mobile phase A to mobile phase B was 50% → 65% and 50% → 35%.

[0014] 95-110 min, the volume ratio of mobile phase A to mobile phase B is 65%:35%;

[0015] Over 110-120 minutes, the volume ratio of mobile phase A to mobile phase B changed from 65% to 10% and from 35% to 90%.

[0016] In some preferred embodiments, step (2) further satisfies at least one of the following 1)-5):

[0017] 1) The detection wavelength is 206-280nm, preferably 225-235nm; the flow rate is 0.8-1.2mL / min, preferably 1.0mL / min; the column temperature is 25-35℃, preferably 30-35℃;

[0018] 2) The concentration of phosphoric acid in the phosphoric acid-containing aqueous solution is 0.05%-0.2%;

[0019] 3) A chromatographic column with dimensions of 4.6 mm × 250 mm and a diameter of 5 μm was used during the detection process;

[0020] 4) The injection volume is 5-15 μL;

[0021] 5) The gradient elution program also includes 120-125 min, with the volume of mobile phase A and mobile phase B being 10% → 10% : 90% → 90%.

[0022] In some preferred embodiments, step (1) includes weighing the chili pepper sample, adding solvent to extract it, obtaining the extract, separating the solid and liquid, and taking the liquid, which is the sample solution.

[0023] In some preferred embodiments, step (1) further satisfies any one or more of the following AEs:

[0024] A. The ratio of the mass of the chili pepper sample to the volume of the solvent is 0.25-1.0:5-20; the relationship between mass and volume is g / mL.

[0025] B. The extraction method is either reflux extraction or ultrasonic extraction;

[0026] C. The extraction time is ≥10 min, preferably 30-60 min;

[0027] D. The solid-liquid separation is selected from centrifugation or membrane filtration;

[0028] E. The solvent is selected from one or more of methanol, ethanol, and water; methanol is preferred.

[0029] In some preferred embodiments, the method further includes the step of preparing a reference solution by adding at least one of phenylalanine, vanillic acid, apigenin, vanillin nonamide, capsaicin, and dihydrocapsaicin to a solvent, and the step of detecting the reference solution by high performance liquid chromatography according to any of the methods of the present invention to obtain a reference chromatogram.

[0030] Preferably, each 1 mL of the reference solution contains 10-50 μg of each reference standard; and / or, the solvent used in the preparation of the reference solution is selected from methanol or an aqueous methanol solution.

[0031] Furthermore, the chili pepper and its preparations include one or more of the following: chili pepper medicinal materials, chili pepper slices, or chili pepper preparations; and / or, the method further includes the step of determining, or simultaneously determining, the content of at least one chemical component among phenylalanine, vanillic acid, apigenin, vanillin nonamide, capsaicin, and dihydrocapsaicin contained in the chili pepper and its preparations using an external standard method. Chili pepper preparations include, for example, powders, granules, tablets, etc.

[0032] In certain preferred embodiments, the characteristic chromatograms of chili peppers and / or their preparations satisfy at least one of the following (1)-(2):

[0033] (1) It has 10 common characteristic peaks: peak 1 is phenylalanine, peak 2 is vanillic acid, peak 5 is apigenin, peak 8 is vanillin nonamide, peak 9 is capsaicin, and peak 10 is dihydrocapsaicin.

[0034] (2) It has 10 common characteristic peaks, of which peaks 2 and 9 correspond to the retention times of vanillic acid reference standard peak and capsaicin reference standard peak, respectively; the peak corresponding to the retention time of vanillic acid reference standard peak is S1 peak, and the relative retention times of peaks 1, 3-7 with peak S1 are within ±10% of the specified value, and the specified values ​​of peaks 1 and 3-7 are: 0.38, 1.74, 2.00, 2.50, 2.58, and 2.85; the peak corresponding to the retention time of capsaicin reference standard peak is S2 peak, and the relative retention times of peaks 8 and 10 with peak S2 are within ±10% of the specified value, and the specified values ​​of peaks 8 and 10 are: 0.99 and 1.07.

[0035] In some preferred embodiments, the method further includes constructing a reference characteristic chromatogram of chili pepper formula granules. A reference characteristic chromatogram of chili pepper formula granules is generated by using a similarity evaluation system for chromatograms of traditional Chinese medicine chromatograms obtained from multiple batches of chili pepper formula granules. At least two batches of chili pepper formula granules are used, for example, four, seven, eight, fifteen, or eighteen batches.

[0036] In some preferred embodiments, after generating the control characteristic chromatogram of chili pepper formula granules using traditional Chinese medicine chromatographic feature chromatogram similarity evaluation software, the method further includes a step of marking common characteristic peaks. The similarity of the control characteristic chromatogram of the chili pepper formula granules is greater than 0.99.

[0037] The present invention also provides the application of the separation method or characteristic spectrum construction method of chemical components in chili peppers and their preparations as described above in the quality detection or identification of chili peppers and their preparations.

[0038] The present invention also provides a method for quality detection or identification of chili peppers and their preparations, comprising constructing a characteristic spectrum of the product to be tested according to any of the above-described methods for separating chemical components in chili peppers and their preparations or for constructing characteristic spectra.

[0039] The chili product to be tested can be chili peppers and / or their preparations.

[0040] In this invention, the terms "chili pepper and its preparations" or "chili pepper and / or its preparations" refer to one or more of chili peppers and chili pepper preparations. Among them, chili pepper preparations are preparations made from chili peppers using conventional processes, especially powders, granules, tablets, etc., obtained from chili pepper water extracts using conventional processes.

[0041] In this invention, vt% represents volume percentage, vt%methanol or %methanol both refer to the volume percentage of methanol in a methanol aqueous solution, and vt%ethanol or %ethanol refer to the volume percentage of ethanol in an ethanol aqueous solution.

[0042] The technical solution of this invention has the following advantages:

[0043] 1. The method for separating chemical components or constructing characteristic spectra in chili peppers and their preparations according to the present invention uses octadecylsilane-bonded silica gel as a filler, methanol as mobile phase A, and an aqueous solution containing phosphoric acid as mobile phase B, through an optimized gradient elution program; ultimately, multiple common characteristic peaks are obtained, and the effective separation of multiple common characteristic peaks is achieved. Moreover, the obtained characteristic spectra have good peak shapes, stable baselines, short detection times, and uniform peak heights or peak areas. The method is simple and easy to operate, providing a scientific basis for comprehensively establishing quality control standards for chili pepper formulation granules. Furthermore, it can accurately locate the peak positions of phenylalanine, vanillic acid, apigenin, vanillin nonamide, capsaicin, and dihydrocapsaicin, fully reflecting the integrity and characteristics of chili peppers and their preparations (such as formulation granules).

[0044] The detection method provided by this invention has good separation and can simultaneously achieve the effective separation of phenylalanine, vanillic acid, apigenin, vanillin nonamide, capsaicin, and dihydrocapsaicin, so as to simultaneously determine the content of these six active ingredients. Therefore, it can comprehensively and quickly detect the active ingredients and their content in chili powder granules.

[0045] 2. The method for separating chemical components or constructing characteristic chromatograms in chili peppers and their preparations described in this invention, through optimization of chromatographic conditions, extraction solvent type and amount, extraction time, etc., determines the optimal extraction process and chromatographic conditions, resulting in higher peak areas, better separation effects, and more comprehensive quality monitoring of chili pepper formulation particles. Attached Figure Description

[0046] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0047] Figure 1 The characteristic spectrum of the chili pepper formulation granules in Example 1;

[0048] Figure 2 This is the chromatogram of the mixed reference standard in Example 1;

[0049] Figure 3 This is the chromatogram under gradient condition 1 in Experiment Example 1;

[0050] Figure 4 This is the chromatogram under gradient condition 2 in Experiment Example 1;

[0051] Figure 5 This is the chromatogram under gradient condition 3 in Experiment Example 1;

[0052] Figure 6 The chromatogram at a wavelength of 206 nm is shown in Example 1.

[0053] Figure 7 The chromatogram at a wavelength of 230 nm is shown in Experiment Example 1.

[0054] Figure 8 The chromatogram at a wavelength of 245 nm is shown in Experiment Example 1.

[0055] Figure 9 The chromatogram at a wavelength of 280 nm is shown in Example 1.

[0056] Figure 10 The chromatogram of phosphoric acid obtained in Experimental Example 1;

[0057] Figure 11 The chromatogram of formic acid obtained in Experiment Example 1;

[0058] Figure 12 The chromatogram of acetic acid obtained in Experiment Example 1;

[0059] Figure 13 The chromatogram is obtained from 0.05% phosphoric acid in Experimental Example 1;

[0060] Figure 14 The chromatogram is obtained from 0.1% phosphoric acid in Experimental Example 1;

[0061] Figure 15 The chromatogram obtained from 0.2% phosphoric acid in Experimental Example 1;

[0062] Figure 16 The chromatogram obtained at a column temperature of 25℃ in Experiment Example 1;

[0063] Figure 17 The chromatogram obtained at a column temperature of 30℃ in Experiment Example 1;

[0064] Figure 18 The chromatogram obtained at a column temperature of 35℃ in Experiment Example 1;

[0065] Figure 19 The chromatogram obtained at a flow rate of 0.8 / min in Experiment Example 1;

[0066] Figure 20 The chromatogram obtained at a flow rate of 1.0 ml / min in Experiment Example 1;

[0067] Figure 21 The chromatogram obtained at a flow rate of 1.2 ml / min in Experiment Example 1;

[0068] Figure 22 The chromatogram obtained from Kromasil C18 in Experimental Example 1;

[0069] Figure 23 The chromatogram of phenomen C18 obtained in Experiment Example 1;

[0070] Figure 24 The chromatogram obtained from Agilent 5TC-C18(2) in Experimental Example 1;

[0071] Figure 25 The chromatogram of methanol in Experiment Example 1 is shown.

[0072] Figure 26 The chromatogram of 50% methanol in Experiment Example 1 is shown.

[0073] Figure 27 The chromatogram of ethanol in Experiment Example 1 is shown.

[0074] Figure 28 The chromatogram for 50% ethanol in Experiment Example 1 is shown.

[0075] Figure 29 The chromatogram of water in Experiment Example 1 is shown.

[0076] Figure 30 Characteristic chromatograms of 18 batches of chili pepper formulation granules in Experimental Example 2, S1~S18: 1802002W, 1804002S, 1905001S, 1907001W, 2003001W, 2005002S, 2007003S, 2012001W, 2102001S, 2104001S, 2110002W, 2112001S, 2203002S, 2204001S, 2206002S; R: Control chromatogram;

[0077] Figure 31 The reference characteristic chromatogram of the chili pepper formulation granules in Experiment Example 2; chromatographic column: Agilent 5TC-C18(2), 250mm×4.6mm, 5μm;

[0078] Figure 32 The characteristic spectrum of the chili pepper control material in Experiment Example 2;

[0079] Figure 33 The chromatograms are for different reference standards in Experiment Example 2. S1 is the reference characteristic chromatogram of chili pepper formulation granules; S2 is the chromatogram of a mixed reference standard containing vanillic acid, capsaicin and dihydrocapsaicin; S3 is the chromatogram of phenylalanine reference standard; S4 is the chromatogram of apigenin reference standard; and S5 is the chromatogram of vanillin nonamide reference standard.

[0080] Figure 34 This is the chromatogram of the negative control solution from Experiment Example 3. Detailed Implementation

[0081] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described herein. They do not constitute a limitation on the content or scope of protection of the present invention. Any product identical or similar to the present invention derived by anyone under the guidance of the present invention or by combining the features of the present invention with other prior art falls within the scope of protection of the present invention. Where specific experimental steps or conditions are not specified in the embodiments, they can be performed according to the conventional experimental steps or conditions described in the literature in the art. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products. The chili pepper preparation described in this invention is prepared by the following method:

[0082] Take chili peppers and extract them under reflux at least once, adding 6-12 times their weight of water each time for at least 0.5 hours. Filter the mixture, combine the filtrates, and concentrate them to a relative density of 1.05-1.10 g / mL at 60°C. Add conventional excipients and follow conventional processes to produce clinically acceptable tablets, capsules, pills, granules, honey-processed pills, sustained-release preparations, immediate-release preparations, controlled-release preparations, oral liquid preparations, or injectable preparations. The pharmaceutically acceptable excipients include: fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives, and matrix, etc. Fillers include: starch, pregelatinized starch, lactose, mannitol, chitosan, microcrystalline cellulose, sucrose, etc.; disintegrants include: starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, croscarmellose, low-substituted hydroxypropyl cellulose, croscarmellose sodium, etc.; lubricants include: magnesium stearate, sodium lauryl sulfate, talc, silica, etc.; suspending agents include: polyvinylpyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methylcellulose, etc.; binders include: starch paste, polyvinylpyrrolidone, hydroxypropyl methylcellulose, etc.; sweeteners include: sodium saccharin, aspartame, sucrose, cyclamate, glycyrrhetinic acid, etc.; flavoring agents include: sweeteners and various flavorings; preservatives include: parabens, benzoic acid, sodium benzoate, sorbic acid and its salts, benzalkonium bromide, chlorethidium acetate, eucalyptus oil, etc.; matrix includes: PEG6000, PEG4000, insect wax, etc.

[0083] The test sample used in the following examples and experimental cases is chili pepper formulation granules. The specific preparation method of the chili pepper formulation granules is as follows: take chili peppers, heat and reflux extract twice. For the first extraction, add 10 times the weight of water to the chili peppers and soak for 30 minutes, heat and reflux extract for 0.5 hours, filter, add 8 times the weight of water to the chili peppers and continue to heat and reflux extract for 0.5 hours, filter, combine the filtrates, concentrate the filtrate to a relative density of 1.05 g / mL at 60°C, spray dry, add 0.04 times the weight of chili peppers as excipient maltodextrin to the dry powder, mix evenly, and then dry granulate to make granules.

[0084] The main instruments and reagents involved in this invention are as follows:

[0085] 1. Instruments and Equipment:

[0086] Chromatograph 1: High Performance Liquid Chromatograph 1: Thermo Ultimate 3000, including Pump: LPG-3400SD; Colum Compartment: TCC-3000RS; Autosumpler: WPS-3000SL; Photometer: DAD-3000.

[0087] Other instruments: High-power CNC ultrasonic instrument (KQ-400KDB, Kunshan Ultrasonic Instrument Co., Ltd.), 0.001 g electronic balance (Shimadz μAY120, Shimadzu Corporation, Japan), 0.001 g electronic balance (Sartoriμs SQP SECMRA225D-1CN, Sartorius Scientific Instruments (Beijing) Co., Ltd.), Digital display constant temperature water bath (HH-S6, Jiangsu Jinyi Instrument Technology Co., Ltd.)

[0088] Column: Agilent 5TC-C18(2) (4.6mm×250mm, 5μm);

[0089] Kromasil C18 (4.6mm×250mm, 5μm);

[0090] Pheromon C18 (4.6mm×250mm, 5μm).

[0091] 2. Reagents:

[0092] Methanol and acetonitrile were chromatographic grade (Merck); phosphoric acid (chromatographic grade, 85-90%, Aladdin); glacial acetic acid (HPLC, >99.9%, Aladdin); formic acid (chromatographic grade, ≥98%, Aladdin); water (Milli-Q).

[0093] 3. Drug trials:

[0094] Chili pepper reference material (purchased from Chengdu DST Biotechnology Co., Ltd., batch number DST-L0001).

[0095] Vanillic acid reference standard (purchased from the National Institutes for Food and Drug Control, batch number 110776-201503, purity 99.8%);

[0096] Apigenin reference standard (purchased from Shanghai Hongyong Biotechnology Co., Ltd., batch number 26544-34-3, purity 98.0%);

[0097] Vanillonamide reference standard (purchased from Shanghai Hongyong Biotechnology Co., Ltd., batch number 2444-46-4, purity 98.0%);

[0098] Capsaicin reference standard (purchased from the National Institutes for Food and Drug Control, batch number 110839-201806, purity 99.0%);

[0099] Dihydrocapsaicin reference standard (purchased from the National Institutes for Food and Drug Control, batch number 111666-201904, purity 97.6%);

[0100] Chili granules (batch numbers: 1802002W, 1804002S, 1905001S, 1907001W, 2003001W, 2005002S, 2007003S, 2012001W, 2102001S, 2104001S, 2110002W, 2112001S, 2203002S, 2204001S, 2206002S).

[0101] Example 1

[0102] This embodiment provides a method for separating chemical components from chili peppers and their preparations, and a method for constructing characteristic spectra, including:

[0103] (1) Preparation of test solution: Using chili granules as test sample, take about 0.5g of test sample powder, accurately weigh it, place it in a stoppered conical flask, accurately add 10mL of methanol, stopper tightly, weigh it, sonicate (power 250W, frequency 40kHz) for 30min, take it out, let it cool, weigh it again, make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain the solution.

[0104] Preparation of mixed reference solution: Accurately weigh appropriate amounts of vanillic acid, capsaicin, and dihydrocapsaicin reference standards, add methanol to prepare a mixed solution containing 30 μg of each reference standard per 1 ml, which is used as the mixed reference solution.

[0105] (2) The test solution and the mixed reference solution were determined by high performance liquid chromatography (HPLC) under the following chromatographic conditions: octadecylsilane-bonded silica gel was used as the packing material (Agilent 5TC-C18(2), column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol was used as mobile phase A, and 0.05% phosphoric acid solution was used as mobile phase B, with gradient elution as specified in the table below; the flow rate was 1.0 ml per minute; the column temperature was 30℃; and the detection wavelength was 230 nm. The theoretical plate number, calculated based on the capsaicin peak, should not be less than 3000.

[0106] Table 1 Gradient Elution Table

[0107]

[0108] The results are shown in the table below. Figure 1-2As shown, the characteristic spectrum of the chili powder granules has 10 characteristic peaks, all of which were effectively separated. Each characteristic peak has a good shape, stable baseline, short detection time, and uniform peak height or area. Peaks 2, 8, and 10 correspond to the retention times of vanillic acid reference standard peaks, capsaicin reference standard peaks, and dihydrocapsaicin reference standard peaks, respectively. The peak corresponding to the retention time of the vanillic acid reference standard peak is peak S1, and the relative retention times of peaks 1, 3-7 with peak S1 are 0.38, 1.74, 2.00, 2.50, 2.57, and 2.84, respectively. The peak corresponding to the retention time of the capsaicin reference standard peak is peak S2, and the relative retention times of peaks 8 and 10 with peak S2 are 0.99 and 1.07, respectively.

[0109] Table 2 System Adaptability Parameters

[0110]

[0111] Experimental Example 1

[0112] I. Investigation of chromatographic conditions

[0113] 1. Investigation of the mobile phase gradient

[0114] The same chili pepper granule test solution (prepared according to the method of Example 1) was analyzed. By comparing the chromatograms of samples measured with different elution programs, the gradient with richer chromatographic information, better resolution of the main chromatographic peaks, more stable baseline, and more reasonable analysis time was selected. The gradient elution conditions are as follows, and the other chromatographic conditions are the same as in Example 1.

[0115] Table 3 Mobile phase gradient 1

[0116]

[0117] Table 4. Mobile phase gradient 2

[0118]

[0119] Table 5. Mobile phase gradient 3

[0120]

[0121] Table 6. System suitability parameters for each gradient chromatographic peak.

[0122]

[0123]

[0124] See results Figure 3-5As shown in the table above, by comparing the chromatograms of the test samples and the system adaptability parameters under different gradients, it can be seen that the peak information and resolution of gradients 1 and 2 are poor, while the separation effect of each chromatographic peak of gradient 3 is better and the chromatographic information is richer. Therefore, gradient 3 is selected for subsequent condition optimization.

[0125] 2. Selection of detection wavelength

[0126] The chili pepper granule (1802002W) test solution prepared according to the method of Example 1 was injected into the liquid chromatograph under the gradient conditions determined above, and a full-wavelength scan was performed. Based on the chromatographic information content, the chromatograms at four different absorption wavelengths (206 nm, 230 nm, 245 nm, and 280 nm) were compared. The remaining chromatographic conditions were the same as in Example 1. The number of chromatographic peaks, response value, and retention time were used as evaluation criteria to screen the detection wavelengths.

[0127] Table 7. System adaptability parameters for chromatographic peaks at different absorption wavelengths.

[0128]

[0129] See results Figure 6-9 As shown in the table above, the chromatogram at a wavelength of 230 nm has more chromatographic peaks, more information, and a stable baseline. Therefore, the absorption wavelength of 230 nm, which has relatively rich chromatographic peak information and relatively better system adaptability parameters, was selected as the detection wavelength for the characteristic chromatogram of chili formulation granules for further investigation.

[0130] 3. Investigation of different types of acids in the mobile phase

[0131] The chili pepper formulation granule test solution prepared according to the method of Example 1 was used to compare the effects of different aqueous phase components (mobile phase B being 0.05% phosphoric acid, 0.05% formic acid, and 0.05% acetic acid) on the separation effect of the fingerprint chromatogram of the chili pepper formulation granules. All other chromatographic conditions were the same as in Example 1. The results are shown in [Figure 1]. Figure 10-12 As shown, the results indicate that the baseline is unstable when using 0.05% formic acid and 0.05% acetic acid as mobile phase B, while the baseline is more stable under the phosphoric acid system. Therefore, the phosphoric acid mobile phase system is tentatively selected for further screening.

[0132] 4. Investigation of acid concentration

[0133] The chili pepper formulation granule test solution prepared according to the method of Example 1 was used to compare the effects of different concentrations of phosphoric acid (0.05% phosphoric acid, 0.1% phosphoric acid, and 0.2% phosphoric acid) on the separation effect of the fingerprint chromatogram of the chili pepper formulation granules. All other chromatographic conditions were the same as in Example 1. The results are shown in the table below.

[0134] Table 8 Results of the study at different acid concentrations

[0135]

[0136] See results Figure 13-15 As shown in the table above, different formic acid concentrations have little effect on each chromatographic peak, and the chromatograms eluted with 0.05-0.2% phosphoric acid solution show good separation of the main chromatographic peaks.

[0137] 5. Investigation of different column temperatures

[0138] The chili pepper formulation granule test solution prepared according to the method in Example 1 was used to compare the effects of different column temperatures (25℃, 30℃, 35℃) on the separation effect of the chili pepper formulation granule fingerprint chromatogram. Other chromatographic conditions were the same as in Example 1. The results are shown in [Figure 1]. Figure 16-18 and the table below.

[0139] Table 9 Results of investigation at different column temperatures

[0140]

[0141]

[0142] The results showed that different column temperatures had a certain impact on the information content of chromatographic peaks and system adaptability parameters. After comparing and analyzing the above results, the column temperature was tentatively set at 30℃ for subsequent screening.

[0143] 6. Investigation of different flow velocities

[0144] The chili pepper formulation granule test solution prepared according to the method in Example 1 was used to compare the effects of different flow rates (0.8 ml / min, 1.0 ml / min, 1.2 ml / min) on the separation effect of the fingerprint chromatogram of the chili pepper formulation granules. Other chromatographic conditions were the same as in Example 1. The results are shown below. Figure 19-21 and the table below.

[0145] Table 10 Results of investigation at different flow velocities

[0146]

[0147] Different flow rates have a certain impact on the information content of chromatographic peaks and system adaptability parameters. After comparing and analyzing the above results, the number of characteristic peaks was the highest at 1.0-1.2 ml / min, especially the total peak area was the highest at 1.0 ml / min. The flow rate of 1.0 ml / min is tentatively set for subsequent studies.

[0148] 7. Investigation of different brands of chromatographic columns

[0149] Take the chili pepper formula granule test solution prepared according to the method of Example 1, and compare the effects of different chromatographic columns (chromatographic column 1: Kromasil C18; chromatographic column 2: Phyromon C18; chromatographic column 3: Agilent 5TC-C18 (2)) on the separation effect of the fingerprint spectrum of chili pepper formula granules. The other chromatographic conditions are the same as in Example 1.

[0150] Table 11 Results of investigation using different chromatographic columns

[0151]

[0152] See results Figure 22-24 As shown in the table above, different chromatographic columns have a certain impact on the information content of chromatographic peaks and system adaptability parameters. After comparing and analyzing the above results, the Agilent 5TC-C18(2) chromatographic column yielded the most characteristic peaks. The Agilent 5TC-C18(2) chromatographic column will be used for subsequent research.

[0153] II. Optimization of the preparation method of the test sample solution

[0154] 1. Investigation of extraction solvent

[0155] The effects of different extraction solvents (methanol, 50% methanol, ethanol, 50% ethanol, and water) on the characteristic chromatograms of chili pepper formulation granules were investigated. Approximately 0.5 g of the product was accurately weighed and placed in a stoppered conical flask. 10 ml of each different extraction solvent was accurately added, the flask was sealed, and the mixture was sonicated (250 W, 40 kHz) for 30 minutes. After cooling, the mixture was shaken well, filtered, and the filtrate was collected to obtain the test solution. The solution was then analyzed under the chromatographic conditions described in Example 1. The results are shown in the table below.

[0156] Table 12 Parameters for Adaptability of Chromatographic Peak Systems to Different Extraction Solvents

[0157]

[0158]

[0159] See results Figure 25-29 Based on the table above and the chromatographic parameters of each peak, the results show that methanol extraction is more effective than ethanol. The total peak area of ​​the characteristic chromatographic peaks obtained by extraction with different methanol concentrations varies to some extent. The peak area of ​​the chromatographic peaks obtained by methanol extraction is significantly larger than that obtained by extraction with other methanol concentrations. Taking all factors into consideration, methanol is determined to be the preferred extraction solvent.

[0160] 2. Examination of extraction time

[0161] The extraction effects of different extraction times (15, 30, and 45 minutes) on the main characteristic peaks of the chili powder granules were investigated. Approximately 0.5 g of the sample was accurately weighed and placed in a stoppered conical flask. 10 ml of methanol was accurately added, and the flask was sealed tightly. The flasks were then sonicated (250 W, 40 kHz) for 15, 30, and 45 minutes respectively. After cooling, the flasks were shaken well, filtered, and the filtrate was collected to obtain the test solution. The solution was then analyzed using the chromatographic conditions described in Example 1. The results are shown in the table below.

[0162] Table 13 Extraction time parameters for chromatographic peak system adaptability.

[0163]

[0164]

[0165] Results analysis: A comprehensive comparison of the results of the three different extraction times shows that different extraction times do not affect the retention time of each peak; there is no significant difference in peak area between ultrasonic treatment for 30 minutes and 40 minutes, indicating that extraction is complete when ultrasonic treatment is performed for 30 minutes. Considering the need to save sample preparation time, the extraction time is tentatively set at 30 minutes.

[0166] 3. Sampling quantity investigation

[0167] Accurately weigh approximately 0.25 g, 0.5 g, and 1.0 g of the sample, respectively, and place them in stoppered conical flasks. Accurately add 10 ml of methanol, seal tightly, and sonicate (250 W, 40 kHz) for 30 minutes. Cool, shake well, filter, and collect the filtrate to obtain the test solution. Then, perform the chromatographic analysis using the conditions described in Example 1. The results are as follows.

[0168] Table 14 Sampling quantity parameters for chromatographic peak system adaptability assessment

[0169]

[0170]

[0171] Experimental results show that different sample amounts have no effect on the retention time of each characteristic peak, nor on the resolution, symmetry factor, or theoretical plate number. At a sample amount of 0.5g, the peak areas of the characteristic peaks in the chromatogram of the chili pepper formulation granules are relatively moderate. Therefore, 0.5g was chosen as the sample amount for this method.

[0172] 4. Investigation of different solvent volumes

[0173] Accurately weigh approximately 0.5 g of the sample and place it in a stoppered conical flask. Add 5 ml, 10 ml, and 20 ml of methanol respectively, then seal tightly. Sonicate the flasks (250 W, 40 kHz) for 30 minutes, cool, shake well, and filter. Collect the filtrate to obtain the test solution. Then, perform the chromatographic analysis using the conditions described in Example 1. The results are as follows.

[0174] Table 15 Solvent volume analysis of chromatographic peak system adaptability parameters

[0175]

[0176]

[0177] Based on the information content and separation effect of the chromatographic peaks as the main evaluation indicators, the experimental results show that the main effect of different solvent volumes on the chromatogram is the peak area. When the solvent volume is 20 ml, the solution concentration is low, the peak area is too small, and it is easy to cause integration error. When the solvent volume is 10 ml, the peak area is larger, and the system adaptability parameters are relatively better. Therefore, 10 ml is selected as the extraction solvent volume.

[0178] 5. Sample injection volume analysis

[0179] Accurately weigh approximately 0.5 g of this product and place it in a stoppered conical flask. Accurately add 10 ml of methanol, seal tightly, and sonicate (250 W, 40 kHz) for 30 minutes. Cool, shake well, filter, and collect the filtrate to obtain the test solution. Then, perform the chromatographic analysis using the conditions described in Example 1. The results are as follows.

[0180] Table 16 Injection Volume Assessment Parameters for Chromatographic Peak System Adaptability

[0181]

[0182] Based on the information content and separation effect of the chromatographic peaks as the main evaluation indicators, the experimental results show that different injection volumes mainly affect the peak area of ​​the chromatogram. When the injection volume is 5 μl, the peak response is too small; when the injection volume is 10 μl, the peak area is larger and the system adaptability parameters are relatively better. Therefore, 10 μl was selected as the injection volume.

[0183] Example 2: Establishment of Feature Maps and Their Parameters

[0184] (1) Construction of feature maps

[0185] Fifteen batches of chili pepper granule samples were taken as test samples, and test solution was prepared according to the method in Example 1. 2g of chili pepper reference material was placed in a stoppered conical flask, 20ml of water was added, and the mixture was heated under reflux for 1 hour. The mixture was filtered, the filtrate was evaporated to dryness, and the residue was treated with 10ml of methanol. The mixture was sonicated (250W power, 40kHz frequency) for 30 minutes, cooled, shaken well, and filtered. The filtrate was used as the reference solution for the reference material. Separately, appropriate amounts of vanillic acid, capsaicin, and dihydrocapsaicin reference standards were accurately weighed and dissolved in methanol to prepare a mixed solution containing 30μg of each per ml, which was used as the reference solution for the reference standard.

[0186] The above solution was analyzed using the high-performance liquid chromatography method described in Example 1, and characteristic chromatograms of 18 batches of chili pepper formulation granules and control medicinal materials were obtained. The results are shown in Table 17 and 1888. Figure 30-32 As shown.

[0187] Table 17. Results of Characteristic Spectrum Determination of 18 Batches of Chili Formula Granules

[0188]

[0189] Should Figure 30 In the sample, S1 to S15 correspond to batch numbers S1 to S18: 1802002W, 1804002S, 1905001S, 1907001W, 2003001W, 2005002S, 2007003S, 2012001W, 2102001S, 2104001S, 2110002W, 2112001S, 2203002S, 2204001S, and 2206002S. The fingerprint similarity evaluation software "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Commission was used to generate a reference characteristic chromatogram, as shown below. Figure 31 As shown. According to Figure 31 The reference characteristic chromatograms shown can be used to analyze and compare the detection results of the characteristic chromatograms of formulated granules, and are used for the quality control of formulated granules. The specific method for quality control using reference characteristic chromatograms is as follows:

[0190] The relative retention times of the characteristic chromatograms were determined based on the research results: the characteristic chromatogram of the chili pepper formula granules test sample should show 10 characteristic peaks, which should correspond to the 10 characteristic peaks in the chromatogram of the reference medicinal material, as shown in Table 18-21.

[0191] Table 18. Comparison chart of chili pepper formulation granules and their relative retention times.

[0192]

[0193] Table 19. Comparison of relative peak areas in chili granule formulation chromatograms

[0194]

[0195] Table 20: Relative Retention Time of Common Patterns in Chili Granule Formulation

[0196]

[0197] Table 21 Common Pattern Matching Data for Chili Granule Formulation

[0198]

[0199] The results show that the characteristic chromatogram of the chili pepper formulation granules has 10 characteristic peaks, of which 6 peaks (peaks 1, 2, 5, 8, 9, and 10) are known components. Peaks 2, 8, and 9 should correspond to the retention times of their respective reference standard peaks. The peak corresponding to the retention time of the vanillic acid reference standard peak is peak S1. The relative retention times of peaks 1, 3-7 should be calculated, and their relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.38 (peak 1), 1.74 (peak 3), 2.00 (peak 4), 2.50 (peak 5), 2.58 (peak 6), and 2.85 (peak 7). The peak corresponding to the retention time of the capsaicin reference standard peak is peak S2. The relative retention times of peaks 8 and 10 should be calculated, and their relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.99 (peak 8) and 1.07 (peak 10).

[0200] The detection method described in this embodiment can effectively obtain fingerprint spectra with good separation of various characteristic peaks. It can also simultaneously determine the contents of phenylalanine, vanillic acid, apigenin, vanillin nonamide, capsaicin, and dihydrocapsaicin. Furthermore, by selecting vanillic acid peak S1 and capsaicin peak S2 as internal reference peaks in the fingerprint spectrum, the relative retention times of the common characteristic peaks 1-10 of the chili pepper formulation granules can be determined. Therefore, this method enables comprehensive and rapid detection of chili pepper formulation granules, which is beneficial for comprehensive quality testing and overall quality control of chili pepper formulation granules, thereby contributing to improving the safety and stability of the drug.

[0201] (2) Characteristic peak identification

[0202] Take appropriate amounts of phenylalanine, apigenin, and vanillin nonamide reference standards, accurately weigh them, and add methanol to prepare reference solutions containing 20 μg of phenylalanine, 20 μg of apigenin, and 10 μg of vanillin nonamide per ml.

[0203] Take appropriate amounts of vanillic acid, capsaicin, and dihydrocapsaicin reference standards, accurately weigh them, and add methanol to prepare a mixed solution containing 30 μg of each per 1 ml, which is used as the reference solution.

[0204] The above solution was analyzed using the high-performance liquid chromatography method described in Example 1, and the chromatogram of the reference standard was obtained. The chromatogram was compared with the characteristic chromatogram of the chili pepper formulation granules reference standard. The results are shown in the figure below. Figure 33 As shown, the 10 characteristic peaks were located and identified using reference standards. The retention times of peaks 1, 2, 5, 8, 9, and 10 should correspond to the retention times of the reference peaks of phenylalanine, vanillic acid, apigenin, vanillin nonamide, capsaicin, and dihydrocapsaicin, respectively. Peak 1 was identified as phenylalanine, peak 2 as vanillic acid, peak 5 as apigenin, peak 8 as vanillin nonamide, peak 9 as capsaicin, and peak 10 as dihydrocapsaicin.

[0205] (3) Similarity

[0206] The similarity of characteristic chromatograms of 18 batches of chili pepper formula granules was calculated using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" 2012 version, a fingerprint similarity evaluation software compiled by the Pharmacopoeia Commission. The results are shown in the table below. The results show that the similarity of characteristic chromatograms of all 18 batches of chili pepper formula granules is greater than 0.99.

[0207] Table 22 Similarity results of characteristic maps of 18 batches of chili powder formulation granules

[0208]

[0209] (4) Content determination

[0210] Eighteen batches of chili granule test solutions were used to construct characteristic chromatograms according to the method in Example 1, and the contents of capsaicin and dihydrocapsaicin were simultaneously determined by external standard method. The results are shown in the table below.

[0211] Table 23 Results of capsaicin and dihydrocapsaicin content in 18 batches of chili formulation granules

[0212]

[0213]

[0214] The detection method described in Example 1 effectively obtains fingerprint spectra with good separation of various characteristic peaks. It also allows for the simultaneous determination of capsaicin and dihydrocapsaicin content. Furthermore, by selecting vanillic acid peak S1 and capsaicin peak S2 as internal reference peaks in the fingerprint spectrum, the relative retention times of the common characteristic peaks 1-10 of the chili pepper formulation granules can be determined. Therefore, this method enables comprehensive and rapid detection of chili pepper formulation granules, which is beneficial for comprehensive quality testing and overall quality control, thereby contributing to improved safety and stability of the drug.

[0215] Example 3: Analytical Method Verification

[0216] (1) Instrument precision

[0217] The same chili pepper formulation granules (1802002W) prepared according to Example 1 were used as a test solution, and the injection was repeated 6 times according to the method in Example 1. The RSD of the relative retention time of the characteristic peak should be less than 2.0%. The results show that the instrument precision is good.

[0218] Table 24 Relative retention time results from precision tests

[0219]

[0220] (2) Repeatability test

[0221] Six test solutions were prepared in parallel according to the method in Example 1, and the RSD of the relative retention time of the characteristic peak was calculated to be less than 2.0%. The results showed that the method had good repeatability.

[0222] Table 25 Results of Relative Retention Times in Repeatability Tests

[0223]

[0224] (3) Stability test

[0225] The chili pepper formulation granules (1802002W) prepared by the same method in Example 1 were used as a test solution. The results were measured at 0h, 2h, 4h, 8h, 12h and 24h according to the method in Example 1. The RSD of the relative retention time of the characteristic peak was calculated to be less than 2.0%. The results showed that the test solution was stable within 24h and met the requirements for the determination.

[0226] Table 26 Results of relative retention times in stability tests

[0227]

[0228]

[0229] (4) Exclusivity

[0230] Maltodextrin was granulated by dry method to obtain negative control granules. 2.0 g of negative control granules were taken and a negative control solution was prepared by the same method as the test solution in Example 1.

[0231] Accurately pipette 10 μl each of the test solution prepared in Example 1 and the above negative control solution, and inject them separately into the high-performance liquid chromatograph. Perform the test according to the chromatographic conditions of Example 1. See [example details]. Figure 1 and 34 As shown, the result was negative and there was no interference.

[0232] (5) Intermediate precision (personnel)

[0233] Three researchers prepared a chili pepper formulation granule (1802002W) test solution according to the method in Example 1 and measured it. The RSD of the relative retention time of the characteristic peak should be less than 2.0%. The results showed that the intermediate precision was good.

[0234] Table 27 Intermediate Precision (Personnel) Relative Retention Time

[0235]

[0236] The examples provided are not intended to limit the implementation of the invention. Those skilled in the art will recognize that various variations and modifications can be made based on the foregoing description. It is neither necessary nor possible to exhaustively list all possible implementations. However, obvious variations and modifications derived therefrom remain within the scope of this invention.

Claims

1. A method for isolating chemical components in a Capsicum and preparation thereof, characterized by, The method comprises the following steps: (1) Preparation of the sample solution: a sample of the pepper is weighed and extracted with a solvent to obtain an extract, and then the liquid is separated from the solid to obtain the sample solution, wherein the solvent is methanol, 50% methanol or 50% ethanol; the sample of the pepper is a water extract of the pepper; the preparation of the control solution also comprises the step of preparing a control solution by dissolving phenylalanine, vanillic acid, apioin, vanillyl nitrile, capsaicin and dihydrocapsaicin in a solvent; (2) The sample solution and the control solution are detected by high performance liquid chromatography, and an Agilent 5 TC-C18(2) column is used, methanol is used as the mobile phase A, and a water solution containing phosphoric acid is used as the mobile phase B, and gradient elution is performed, and the gradient elution program comprises: 0-5 min, the volume ratio of the mobile phase A to the mobile phase B is 10%:90%; 5-10 min, the volume ratio of the mobile phase A to the mobile phase B is 10%→15%:90%→85%; 10-30 min, the volume ratio of the mobile phase A to the mobile phase B is 15%→30%:85%→70%; 30-55 min, the volume ratio of the mobile phase A to the mobile phase B is 30%→35%:70%→65%; 55-85 min, the volume ratio of the mobile phase A to the mobile phase B is 35%→50%:65%→50%; 85-95 min, the volume ratio of the mobile phase A to the mobile phase B is 50%→65%:50%→35%; 95-110 min, the volume ratio of the mobile phase A to the mobile phase B is 65%:35%; 110-120 min, the volume ratio of the mobile phase A to the mobile phase B is 65%→10%:35%→90%; the detection wavelength is 206-280 nm; the flow rate is 1.0-1.2 mL / min; and the column temperature is 30-35 ℃.

2. The method of claim 1, wherein the chemical components are isolated from the pepper and its preparation. Step (2) also satisfies at least one of the following 1)-5): 1) the detection wavelength is 225-235 nm; and / or, the flow rate is 1.0 mL / min; 2) the concentration of phosphoric acid in the water solution containing phosphoric acid is 0.05%-0.2%; 3) a chromatographic column with a specification of 4.6 mm×250 mm and 5 μm is used in the detection process; 4) the injection volume is 5-15 μL; 5) the gradient elution program further comprises 120-125 min, the volume ratio of the mobile phase A to the mobile phase B is 10%→10%:90%→90%.

3. The method of claim 1, wherein the chemical components are isolated from the pepper and its preparation. The step (1) also satisfies any one or more of the following A-D: A, the mass of the sample of the pepper to the volume of the solvent is 0.25-1.0:5-20; the mass to volume relationship is g / mL; B, the extraction method is reflux extraction or ultrasonic extraction; C, the extraction time is ≥10 min; D, the solid-liquid separation is selected from centrifugation or membrane filtration.

4. The method for separating chemical components from chili peppers and their preparations according to any one of claims 1-3, characterized in that, The extraction time is 30-60 min.

5. The method of claim 1, wherein the chemical components are isolated from the pepper and its preparation. Each 1 mL of the control solution contains 10-50 μg of each control; and / or, the solvent used in the preparation of the control solution is selected from methanol or a methanol aqueous solution.

6. The method of claim 1, wherein the chemical components are isolated from the pepper and its preparation. The method further comprises the step of determining or simultaneously determining the content of at least one of phenylalanine, vanillic acid, apioside, vanillylmandelamide, capsaicin, dihydrocapsaicin in the pepper and its preparation by external standard method.

7. A method for constructing a characteristic map of a pepper and its preparation, characterized by, The method comprises the following steps, (1) Preparation of test sample solution: a test sample of pepper is weighed, extracted with a solvent to obtain an extract, and then subjected to solid-liquid separation, and the liquid is taken as the test sample solution, wherein the solvent is methanol, 50% methanol or 50% ethanol, and the test sample of pepper is a water extract of pepper; the method further comprises the step of preparing a control solution by adding phenylalanine, vanillic acid, apioside, vanillylmandelamide, capsaicin and dihydrocapsaicin to a solvent; (2) The test sample solution and the control solution are detected by high performance liquid chromatography, and an Agilent 5 TC-C18(2) chromatographic column is used, methanol is used as the mobile phase A, and a water solution containing phosphoric acid is used as the mobile phase B, gradient elution is performed, and the gradient elution program comprises: 0-5 min, the volume ratio of the mobile phase A to the mobile phase B is 10%:90%; 5-10 min, the volume ratio of the mobile phase A to the mobile phase B is 10%→15%:90%→85%; 10-30 min, the volume ratio of the mobile phase A to the mobile phase B is 15%→30%:85%→70%; 30-55 min, the volume ratio of the mobile phase A to the mobile phase B is 30%→35%:70%→65%; 55-85 min, the volume ratio of the mobile phase A to the mobile phase B is 35%→50%:65%→50%; 85-95 min, the volume ratio of the mobile phase A to the mobile phase B is 50%→65%:50%→35%; 95-110 min, the volume ratio of the mobile phase A to the mobile phase B is 65%:35%; 110-120 min, the volume ratio of the mobile phase A to the mobile phase B is 65%→10%:35%→90%; the detection wavelength is 206-280 nm; the flow rate is 1.0-1.2 mL / min; and the column temperature is 30-35 ℃.

8. The method of claim 7, wherein the method is characterized by, The step (2) further satisfies at least one of the following 1)-5): 1) the detection wavelength is 225-235 nm; and / or, the flow rate is 1.0 mL / min; 2) the concentration of phosphoric acid in the water solution containing phosphoric acid is 0.05%-0.2%; 3) a chromatographic column with a specification of 4.6 mm×250 mm and 5 μm is used in the detection process; 4) the injection volume is 5-15 μL; 5) the gradient elution program further comprises 120-125 min, the volume ratio of the mobile phase A to the mobile phase B is 10%→10%:90%→90%.

9. The method of claim 7, wherein the method is characterized by, The step (1) further satisfies any one or more of the following A-D: A, the mass of the test sample of pepper to the volume of the solvent is 0.25-1.0:5-20, and the mass to volume relationship is g / mL; B, the extraction method is reflux extraction or ultrasonic extraction; C, the extraction time is ≥10 min; D, the solid-liquid separation is selected from centrifugation or membrane filtration.

10. The method of claim 7-9, wherein the method is characterized by, The extraction time is 30-60 min.

11. The method of claim 7, wherein the method is characterized by, Each 1 mL of the control solution contains 10-50 µg of each control; and / or the solvent used in the preparation of the control solution is selected from methanol or aqueous methanol.

12. The method of claim 7-9, wherein the method is characterized by, The characteristic pattern of the pepper and / or its preparation satisfies at least one of the following (1)-(2): (1) has 10 common characteristic peaks, peak 1 is phenylalanine, peak 2 is vanillic acid, peak 5 is apioin, peak 8 is vanillyl nonamide, peak 9 is capsaicin, and peak 10 is dihydrocapsaicin; (2) has 10 common characteristic peaks, wherein peak 2 and peak 9 correspond to the retention time of the reference peak of the vanillic acid control and the reference peak of the capsaicin control, respectively; the peak corresponding to the retention time of the reference peak of the vanillic acid control is S1 peak, peak 1, peaks 3-7 have a relative retention time within ±10% of the specified value of S1 peak, and the specified value of peak 1, 3-7 is: 0.38, 1.74, 2.00, 2.50, 2.58, 2.85; the peak corresponding to the retention time of the reference peak of the capsaicin control is S2 peak, and peaks 8 and 10 have a relative retention time within ±10% of the specified value of S2 peak; the specified value of peak 8 and peak 10 is: 0.99、1.07。 13. The use of the method for separating chemical components in the pepper and its preparation according to any one of claims 1-6 or the method for constructing the characteristic pattern according to any one of claims 7-12 in the quality detection or identification of the pepper and its preparation.

14. A method for detecting or identifying the quality of a pepper and a preparation thereof, characterized by, The characteristic pattern of the product to be tested is constructed according to the method for constructing the characteristic pattern according to any one of claims 7-12.

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