Construction and application of high performance liquid chromatography-mass spectrometry method for content determination of cardamom medicinal materials, decoction pieces, extracts and preparations

Abstract

The present invention provides a method for determining the content of cardamom medicinal materials, decoction pieces, extracts, or preparations using high-performance liquid chromatography (HPLC). By using HPLC for cardamom content determination, two new indicator components are established. This method exhibits excellent stability, high precision, good reproducibility, convenience, and ease of use. This method ensures drug efficacy and enables more standardized quality control of the medicinal materials and their related preparations, thereby enabling comprehensive and effective quality control of cardamom medicinal materials, decoction pieces, and their related preparations.

Description

Technical Field

[0001] The present invention belongs to the technical field of analysis and detection, and in particular relates to a construction method for high-performance liquid phase content determination of cardamom medicinal materials, decoction pieces, extracts and preparations and an application thereof. Background Art

[0002] Cardamom is a plant of the genus Cardamom in the Zingiberaceae family. It is an herbal medicine included in the "Chinese Pharmacopoeia". The herbal medicine comes from the dried mature fruits of the white cardamom or Java white cardamom of the Zingiberaceae family. According to the place of origin, it is divided into original cardamom (white cardamom) and Indonesian white cardamom (Java white cardamom).

[0003] Javanese white cardamom is the dried mature fruit of Amomum compactum Soland ex Maton, a plant of the Zingiberaceae family. It is warm in nature and pungent in taste, entering the lung, spleen, and stomach meridians. It has the effects of dispelling dampness and promoting qi, warming the middle and stopping vomiting, and stimulating the appetite and promoting digestion. It is clinically used to treat dampness and turbidity in the middle of the body, loss of appetite, the initial onset of damp-heat, chest tightness without hunger, vomiting due to cold and dampness, chest and abdominal distension and pain, and indigestion. Modern research shows that the fruit of Javanese white cardamom is primarily used as a medicinal substance, containing volatile oils, essential oils, terpenes, and flavonoids. The volatile oil is generally considered to be its main active ingredient. Studies on flavonoids are limited, with only one report by Li Lihua et al. on the extraction of total flavonoids from white cardamom. Javanese white cardamom formula granules are made from Javanese white cardamom slices through water extraction, vacuum concentration, and spray drying, resulting in granules for clinical formulation. However, research on its extracts and granules is limited. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a construction method for high-performance liquid phase content determination of cardamom medicinal materials, decoction pieces, extracts and preparations and its application. The construction method can effectively separate and determine the protocatechuic acid and vanillic acid components of cardamom extracts and preparations, thereby effectively reflecting the quality level of cardamom and related preparations, and providing guidance for the quality control of cardamom medicinal materials, decoction pieces and related preparations.

[0005] The present invention establishes two new index components by determining the content of cardamom using high-performance liquid chromatography. The method is characterized by good stability, high precision, good reproducibility, convenience, and ease of use. This method ensures the efficacy of the drug and enables more standardized quality control of the medicinal material and its related preparations, thereby enabling comprehensive and effective quality control of cardamom medicinal materials, decoction pieces, and their related preparations.

[0006] The present invention provides a method for constructing a high-performance liquid phase content determination method for cardamom medicinal materials, decoction pieces, extracts or preparations, comprising the following steps:

[0007] S1) taking a reference solution, detecting it by high performance liquid chromatography, and establishing a standard curve based on the concentration of the reference solution and the corresponding peak area;

[0008] S2) taking a test sample raw material, extracting it with a solvent to obtain a test sample solution; and determining the peak area of ​​the test sample solution by high performance liquid chromatography;

[0009] S3) obtaining the content of the target substance in the cardamom medicinal material, decoction piece, extract or preparation according to the standard curve and the peak area of ​​the test solution;

[0010] The reference solution includes a protocatechuic acid reference solution and / or a vanillic acid reference solution;

[0011] The chromatographic conditions of the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; the mobile phase A is acetonitrile, the mobile phase B is a 0.1% phosphoric acid solution, and the elution is performed in a gradient manner.

[0012] In a specific embodiment provided by the present invention, the reference substance solution is prepared according to the following method: mixing the reference substance with a solvent to obtain a reference substance solution; the reference substance is protocatechu or vanillic acid; and the solvent is preferably 70% methanol.

[0013] In a specific embodiment provided by the present invention, reference solutions of different concentrations are provided, and high performance liquid chromatography is used for detection, and a standard curve is established based on the concentration of the reference solution and the corresponding peak area.

[0014] In a specific embodiment provided by the present invention, the test sample raw material is preferably cardamom medicinal materials, decoction pieces, extracts or preparations, more preferably Javanese white cardamom medicinal materials, decoction pieces, extracts or preparations; the preparations are preparations well known to those skilled in the art and are not particularly limited. In the present invention, they include but are not limited to standard decoctions, formula granules, etc.

[0015] In a specific embodiment provided by the present invention, the test sample raw material is taken and extracted with a solvent to obtain a test sample solution; the solvent is preferably 70% methanol; in terms of g / mL, the mass ratio of the test sample to the solvent is preferably 0.5:(10-25), more preferably 0.5:25.

[0016] In a specific embodiment provided by the present invention, the test sample raw material is taken, extracted with a solvent, preferably cooled, weighed, and the lost weight is supplemented with a solvent, shaken, filtered, and the filtrate is obtained to obtain the test sample solution.

[0017] In a specific embodiment provided by the present invention, the extraction is reflux extraction; the extraction time is preferably 15 to 45 minutes, more preferably 30 minutes.

[0018] In a specific embodiment provided by the present invention, the chromatographic column is preferably a C18 column; specifically, the chromatographic column may be Waters C18 4.6mm×250mm, 5μm, Kromasil C18 4.6mm×250mm, 5μm or Shimadzu C18 4.6mm×250mm, 5μm.

[0019] In a specific embodiment provided by the present invention, the theoretical plate number of the chromatographic column is not less than 5000, calculated based on the protocatechuic acid peak.

[0020] In a specific embodiment provided by the present invention, the specifications of the chromatographic column are preferably 5 μm, 4.6×250 mm; the column temperature of the chromatographic column is preferably 30° C. to 40° C., specifically 30° C., 35° C. or 40° C.

[0021] In a specific embodiment provided by the present invention, the gradient elution is specifically as follows, in volume percentage:

[0022] 0-18 min, mobile phase A 5%→16%, mobile phase B 95%→84%;

[0023] 18-32 min, mobile phase A 16% → 22%, mobile phase B 84% → 78%;

[0024] 32-37 min, mobile phase A 22% → 28%, mobile phase B 78% → 72%;

[0025] 37-38 min, mobile phase A 28%→5%, mobile phase B 72%→95%.

[0026] In a specific embodiment provided by the present invention, the flow rate of the mobile phase is 0.8-1.2 mL / min, specifically 0.8 mL / min, 1 mL / min or 1.2 mL / min.

[0027] In a specific embodiment provided by the present invention, the detection wavelength of the HPLC is preferably 260 nm.

[0028] In a specific embodiment provided by the present invention, the injection volume of the HPLC is 10 μL.

[0029] In a specific embodiment provided by the present invention, the protocatechuic acid presents a linear relationship in the range of 4.0471 to 202.3562 μg / mL, and the linear relationship is y=37015x-11353, R 2 =0.9999.

[0030] In a specific embodiment provided by the present invention, the vanillic acid presents a linear relationship in the range of 4.0183 to 200.9174 μg / mL, and the linear relationship is y=37102x-6291.7, R 2 =0.9999.

[0031] The content of the target substance in the cardamom medicinal material, decoction piece, extract or preparation can be obtained based on the obtained standard curve of protocatechuic acid, standard curve of vanillic acid and peak area of ​​the test solution.

[0032] The present invention establishes a method for determining the content of cardamom (Java white cardamom) and establishes two new indicator components. The method can control the intrinsic quality of cardamom (Java white cardamom) and related preparations from a holistic and macroscopic perspective, ensure the efficacy of the medicine, and enable the medicinal material and related preparations to be subjected to more formal quality control. In addition, the method provided by the present invention has good stability, high precision, good reproducibility, is convenient, and is easy to master.

[0033] The present invention also provides a method for evaluating the quality of cardamom medicinal materials, decoction pieces, extracts or preparations, wherein a sample to be tested is taken and tested using the above-mentioned constructed method to obtain the contents of protocatechuic acid and vanillic acid in the sample to be tested;

[0034] The sample to be tested is cardamom medicinal material, decoction pieces, extract or preparation.

[0035] In a specific embodiment provided by the present invention, the preparation is any preparation well known to those skilled in the art without any particular limitation, and includes but is not limited to standard decoctions, formula granules, etc. in the present invention.

[0036] In a specific embodiment provided by the present invention, if the total content of protocatechuic acid and vanillic acid in the cardamom medicinal material is not less than 0.010% calculated on a dry basis, it is judged to be qualified; if the total content of protocatechuic acid and vanillic acid in the cardamom medicinal material is less than 0.010%, it is judged to be unqualified.

[0037] In a specific embodiment provided by the present invention, if the total content of protocatechuic acid and vanillic acid in the cardamom slices is not less than 0.010% when calculated on a dry basis, it is judged to be qualified; if the total content of protocatechuic acid and vanillic acid in the cardamom slices is less than 0.010%, it is judged to be unqualified.

[0038] In a specific embodiment provided by the present invention, calculated on a dry basis, the total amount of vanillic acid and protocatechuic acid contained in each 1 g of the cardamom standard decoction is 1.20 mg to 3.50 mg.

[0039] The present invention adopts HPLC method and establishes content determination methodology with protocatechuic acid and vanillic acid as index components, which can effectively control the quality of cardamom (Java white cardamom) formula granules from medicinal materials, decoction pieces, extracts to products. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Chromatogram selected for mobile phase;

[0041] Figure 2 is the UV absorption spectrum of protocatechuic acid;

[0042] Figure 3 is the UV absorption spectrum of vanillic acid;

[0043] Figure 4 This is the column temperature investigation result diagram;

[0044] Figure 5 This is the flow velocity investigation result diagram;

[0045] Figure 6 This is the result diagram of the specificity investigation;

[0046] Figure 7 This is the standard curve of vanillic acid;

[0047] Figure 8 This is the standard curve of protocatechuic acid;

[0048] Figure 9 The following are the results of investigations with different instruments;

[0049] Figure 10 The results of the investigation of different chromatographic columns are shown;

[0050] Figure 11 It is a special survey map;

[0051] Figure 12 This is the standard curve of protocatechuic acid;

[0052] Figure 13 This is the standard curve of vanillic acid;

[0053] Figure 14 Overlay images for different instruments;

[0054] Figure 15 Overlay of different chromatographic columns. DETAILED DESCRIPTION

[0055] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0056] To further illustrate the present invention, the following describes in detail a method for constructing an HPLC characteristic spectrum of a cardamom (Java white cardamom) medicinal material, decoction pieces, standard decoction, intermediate and its formula granules provided by the present invention in combination with examples.

[0057] The reagents used in the following examples are all commercially available.

[0058] Instruments and reagents

[0059] High performance liquid chromatograph: Agilent Inifity1260 high performance liquid chromatograph, Waters e2695 high performance liquid chromatograph;

[0060] Electronic balance: ME204E / 02, MS205DM, XP26 (Mettler-Toledo Instruments Co., Ltd.);

[0061] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0062] Ultrasonic cleaner: KQ600-DB (600 W, 40 kHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0063] Chromatographic columns: Waters C18 4.6 mm × 250 mm, 5 μm, Kromasil C18 4.6 mm × 250 mm, 5 μm, Shimadzu C18 4.6 mm × 250 mm, 5 μm.

[0064] Acetonitrile (Sigma-Aldrich, chromatographic grade); phosphoric acid, glacial acetic acid, formic acid (chromatographic grade); water was ultrapure water; other reagents were of analytical grade.

[0065] Protocatechuic acid reference substance (China Food and Drug Inspection Institute, batch number: 110809-202207, purity: 97.5%);

[0066] Vanillic acid reference substance (China Food and Drug Inspection Institute, batch number: 110776-201503, purity: 99.8%).

[0067] Cardamom (Java white cardamom) standard decoction freeze-dried powder was prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: TQW-01, TQW-02, TQW-03, TQW-04, TQW-05, TQW-06, TQW-07, TQW-08, TQW-09, TQW-10, TQW-11, TQW-12, TQW-13, TQW-14, TQW-15, TQW-16.

[0068] Cardamom (Java white cardamom) finished granules were prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: KL-1, KL-2, KL-3;

[0069] Cardamom (Java white cardamom) medicinal materials come from Indonesia and other places; batch numbers are: YC-01, YC-02, YC-03, YC-04, YC-05, YC-06, YC-07, YC-08, YC-09, YC-10, YC-11, YC-12, YC-13, YC-14, YC-15, YC-16;

[0070] Cardamom (Java white cardamom) slices were prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: YP-01, YP-02, YP-03, YP-04, YP-05, YP-06, YP-07, YP-08, YP-09, YP-10, YP-11, YP-12, YP-13, YP-14, YP-15, YP-16.

[0071] Example 1: Establishment of Extract Content Determination Method

[0072] 1.1 Preparation of test solution

[0073] Take about 0.5 g of freeze-dried powder of standard decoction of cardamom (Java white cardamom), accurately weigh it, place it in a stoppered conical flask, accurately add 25 mL of 70% methanol, stopper it, weigh it, heat and reflux it for 30 minutes, cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0074] 1.2 Preparation of reference solution

[0075] Take appropriate amounts of protocatechuic acid reference substance and vanillic acid reference substance, accurately weigh them, and add 70% methanol to prepare a mixed solution containing 10 μg of each per 1 mL.

[0076] 1.3 Chromatographic conditions

[0077] Chromatographic conditions and system suitability testing were performed using octadecylsilane bonded silica gel as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as mobile phase A, and 0.1% phosphoric acid solution as mobile phase B, with gradient elution as specified in the table below; the detection wavelength was 260 nm. The number of theoretical plates, calculated based on the protocatechuic acid peak, should be no less than 5000.

[0078]

[0079] Determination method: Accurately aspirate 10uL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0080] 1.4 Selection of chromatographic conditions and investigation of system suitability

[0081] 1.4.1 Chromatographic conditions

[0082] Based on the experimental conditions proposed above, the separation effects of three different mobile phases were investigated. Except for the mobile phase, the other conditions were proposed, namely: acetonitrile-0.1% acetic acid, acetonitrile-0.1% phosphoric acid, and acetonitrile-0.1% formic acid. Figure 1 . Figure 1 Chromatogram of the selected mobile phase, where Peak 1: protocatechuic acid; Peak 3: vanillic acid.

[0083] 1.4.2 Determination of detection wavelength

[0084] Based on the experimental conditions proposed above, except for the wavelength, all other conditions are proposed. A diode array detector is used to perform full-band scanning of protocatechuic acid, vanillic acid, and 4-coumaric acid. Figure 2 and Figure 3 ,in Figure 2 is the UV absorption spectrum of protocatechuic acid. Figure 3 This is the UV absorption spectrum of vanillic acid.

[0085] The results showed that through analysis of the spectrum and referring to the detection wavelength under the [Content Determination] item of Cardamom (Java White Cardamom) in the 2020 edition of the "Chinese Pharmacopoeia", the optimal inspection wavelength for the determination of protocatechuic acid and vanillic acid in the standard decoction of Cardamom (Java White Cardamom) was determined to be 260nm.

[0086] 1.4.3 Column temperature investigation

[0087] Based on the experimental conditions proposed above, except for the column temperature, all other conditions were proposed. The column temperatures were 30℃, 35℃, and 40℃, respectively. The separation degree of protocatechuic acid and vanillic acid, the number of theoretical plates, etc. were used as evaluation indicators. The results are shown in Figure 4 Compared with Table 1, Figure 4 This is the result of column temperature investigation, where Peak 1: protocatechuic acid; Peak 3: vanillic acid.

[0088] Table 1 Analysis results at different column temperatures

[0089]

[0090]

[0091] The results showed that protocatechuic acid and vanillic acid could be effectively detected at a column temperature of 30℃ to 40℃, and 30℃ was proposed as the detection column temperature.

[0092] 1.4.4 Flow rate investigation

[0093] Based on the experimental conditions proposed above, except for the flow rate, all other conditions were proposed. The flow rates of 0.8mL / min, 1.0mL / min, and 1.2mL / min were investigated, and the separation degree of protocatechuic acid and vanillic acid, the number of theoretical plates, etc. were used as evaluation indicators. The results are shown in Figure 5 and Table 2. Figure 5 This is the flow rate investigation result diagram, where Peak 1: protocatechuic acid; Peak 3: vanillic acid.

[0094] Table 2 Analysis results at different flow rates

[0095]

[0096] Protocatechuic acid and vanillic acid can be effectively detected at flow rates of 0.8 mL / min to 1.0 mL / min, and the final flow rate was determined to be 1.0 mL / min.

[0097] In summary, the chromatographic conditions and system suitability tests for the determination of the content of cardamom (Java white cardamom) standard decoction were determined as follows: octadecylsilane bonded silica gel as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution as specified in the table below; detection wavelength 260 nm. The theoretical plate number calculated based on protocatechuic acid should be no less than 5000.

[0098]

[0099] 1.5 Preparation of test solution

[0100] 1.5.1 Investigation of extraction methods

[0101] Approximately 0.5 g of freeze-dried powder of a standard decoction of cardamom (Java white cardamom) (batch number: TQW-16) was accurately weighed and placed in a stoppered conical flask. 25 mL of 70% methanol was accurately added, the flask was sealed, and the weight was determined. Extraction was then performed using both reflux and ultrasonic methods for 30 minutes. The extract was allowed to cool, then weighed again. The remaining weight was supplemented with 70% methanol, shaken well, filtered, and the filtrate was collected. The protocatechuic acid and vanillic acid contents of the different extraction methods were analyzed and calculated. The results are shown in Table 3.

[0102] Table 3 Analysis results of different extraction methods

[0103]

[0104] The results showed that the protocatechuic acid contents obtained by reflux extraction and ultrasonic extraction were 0.863 mg / g and 0.821 mg / g, respectively, and the vanillic acid contents were 0.812 mg / g and 0.778 mg / g, respectively. Reflux extraction was slightly more efficient than ultrasonic extraction, so reflux extraction was determined to be the preferred extraction method.

[0105] 1.5.2 Extraction solvent investigation

[0106] Approximately 0.5 g of freeze-dried powder of a standard decoction of cardamom (Java white cardamom) (batch number: TQW-16) was accurately weighed and placed in a stoppered conical flask. The sample was extracted with 25 mL each of methanol, 70% methanol, 30% methanol, 50% ethanol, ethanol, and water. The flask was sealed and weighed. The sample was heated under reflux for 30 minutes, cooled, and weighed again. The remaining weight was made up with the appropriate solvent, shaken well, filtered, and the filtrate was collected. The protocatechuic acid and vanillic acid contents were analyzed and calculated using different extraction solvents. The results are shown in Table 4.

[0107] Table 4 Results of investigation on different extraction solvents

[0108]

[0109] The results showed that 70% methanol had a higher extraction efficiency and was selected as the extraction solvent for the standard decoction of cardamom (Java white cardamom).

[0110] 1.5.3 Extraction time investigation

[0111] Accurately weigh approximately 0.5 g of freeze-dried powder of a standard decoction of cardamom (Java white cardamom) (batch number: TQW-16) and place it in a stoppered conical flask. Accurately add 25 mL of 70% methanol, stopper tightly, and weigh the mixture. Heat under reflux for 15, 30, and 45 minutes, cool, and weigh again. Make up the loss with 70% methanol, shake well, filter, and collect the filtrate. Analyze and calculate the protocatechuic acid and vanillic acid contents at different extraction times. The results are shown in Table 5.

[0112] Table 5 Analysis results of different extraction times

[0113]

[0114] The results showed that when the extraction time was 30 minutes, sufficient extraction could be achieved. Therefore, the extraction time of the test sample was determined to be 30 minutes.

[0115] 1.5.4 Investigation of solvent addition amount

[0116] Approximately 0.5 g of freeze-dried powder of a standard decoction of cardamom (Java white cardamom) (batch number: TQW-16) was accurately weighed and placed in a stoppered conical flask. 10 mL, 25 mL, and 50 mL of 70% methanol were accurately added, respectively. The flask was stoppered and weighed. The mixture was heated under reflux for 30 minutes. The mixture was cooled and weighed again. The remaining weight was made up with 70% methanol. The mixture was shaken well, filtered, and the filtrate was collected. The protocatechuic acid and vanillic acid contents were analyzed and calculated at different solvent addition amounts. The results are shown in Table 6.

[0117] Table 6 Investigation results of different solvent addition amounts

[0118]

[0119] The results showed that when the sample size was 0.5 g, the amount of solvent added was 25 mL to ensure complete extraction of the target components, so the amount of solvent added was determined to be 25 mL.

[0120] In summary, the preparation method of the test sample for the content determination of cardamom (Java white cardamom) standard decoction is determined as follows: take about 0.5 g of the product, accurately weigh it, place it in a stoppered conical flask, accurately add 25 mL of 70% methanol, stopper it, weigh it, reflux it for 30 minutes, cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0121] Example 2: Cardamom (Java white cardamom) standard decoction content determination method

[0122] Chromatographic conditions and system suitability testing were performed using octadecylsilane bonded silica gel as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as mobile phase A, and 0.1% phosphoric acid solution as mobile phase B, with gradient elution as specified in the table below; the detection wavelength was 260 nm. The theoretical plate number, calculated based on protocatechuic acid, should be no less than 5000.

[0123]

[0124] Preparation of reference solution: Take appropriate amount of protocatechuic acid reference substance and vanillic acid reference substance, accurately weigh them, and add 70% methanol to make a mixed solution containing 10 μg of each per 1 mL.

[0125] Preparation of test solution: Take about 0.5 g of the product, accurately weigh it, place it in a stoppered conical flask, accurately add 25 mL of 70% methanol, stopper it tightly, weigh it, heat it under reflux for 30 minutes, cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the product.

[0126] Determination method: Accurately aspirate 10 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0127] 2.1 Extract Methodology Investigation

[0128] 2.1.1 Specificity Experiment

[0129] Preparation of test solution: According to the experimental conditions proposed above, prepare the test solution of cardamom (Java white cardamom) standard decoction freeze-dried powder.

[0130] Preparation of reference solution: Take appropriate amount of protocatechuic acid and vanillic acid reference substances, accurately weigh them, and add 70% methanol to make a solution containing 10 μg of each per 1 mL.

[0131] Preparation of negative control solution: According to the experimental conditions proposed above, a negative control solution of the standard decoction lacking cardamom (Java white cardamom) was prepared. Figure 6 . Figure 6 This is the result diagram of the specificity investigation.

[0132] The results showed that the chromatogram of negative solution had no interference with the determination of the peak to be tested, indicating that the method had good specificity.

[0133] 2.1.2 Precision investigation

[0134] The reference solution was injected 6 times continuously, the peak areas of protocatechuic acid and vanillic acid were recorded, and the RSD values ​​were calculated. The results are shown in Table 7.

[0135] Table 7 Precision inspection results

[0136]

[0137] The results showed that the peak area RSD values ​​of protocatechuic acid and vanillic acid were 0.3% and 0.2% respectively. The injection precision of this method was good.

[0138] 2.1.3 Linear Relationship

[0139] Take an appropriate amount of vanillic acid reference substance (purity 99.8%), place it in a 25 mL volumetric flask, dissolve it in 70% methanol to make a solution containing 200.9174 μg per 1 mL, and then dilute it to solutions containing 4.0183 μg, 10.0459 μg, 20.0917 μg, 50.2294 μg, and 100.4587 μg per 1 mL, respectively.

[0140] Take an appropriate amount of protocatechuic acid reference substance (purity 97.5%), place it in a 25 mL volumetric flask, dissolve it in 70% methanol to make a solution containing 202.3562 μg per 1 mL, and then dilute it to concentrations of 4.0471 μg, 10.1178 μg, 20.2356 μg, 50.5891 μg, and 101.1781 μg per 1 mL, respectively.

[0141] 10 μL of each was accurately drawn and injected into the liquid chromatograph to obtain the peak area. The response curve was drawn with the concentration (X, μg / ml) as the abscissa and the peak area (Y) as the ordinate. The results are shown in Tables 8 and 9. Figures 7-8 .in, Figure 7 This is the standard curve of vanillic acid. Figure 8 This is the standard curve of protocatechuic acid.

[0142] Table 8 Vanillic acid standard curve analysis results

[0143]

[0144] The results showed that when the concentration of vanillic acid ranged from 4.0183 to 200.9174 μg / mL, the linear relationship was y=37102x-6291.7, R 2 =0.9999. This indicates that the concentration range is 4.0183-200.9174 μg / mL with a good linear relationship.

[0145] Table 9 Protocatechuic acid standard curve analysis results

[0146]

[0147] The results showed that when the concentration of protocatechuic acid ranged from 4.0471 to 202.3562 μg / mL, the linear relationship was y=37015x-11353, R 2 =0.9999. This indicates that the concentration range is 4.0471-202.3562 μg / mL with a good linear relationship.

[0148] 2.1.4 Repeatability Experiment

[0149] About 0.5 g of freeze-dried powder of cardamom (Java white cardamom) standard decoction (batch number: TQW-16) was taken and accurately weighed into 6 portions. The same operator prepared the test solution according to the determined method, measured the peak area, and calculated the protocatechuic acid and vanillic acid contents of the 6 test samples. The results are shown in Table 10.

[0150] Table 10 Repeatability test results

[0151]

[0152]

[0153] The RSD values ​​of protocatechuic acid and vanillic acid obtained from 6 repeatability tests were 0.6% and 0.4%, respectively, indicating that this method has good repeatability.

[0154] 2.1.5 Stability test

[0155] The same cardamom (Java white cardamom) standard decoction freeze-dried powder (batch number: TQW-16) test sample (batch number: TQW-16) solution was taken, and the chromatographic peak areas of protocatechuic acid and vanillic acid were determined at 0h, 2h, 4h, 8h, 16h, and 24h, respectively. The results are shown in Table 11.

[0156] Table 11 Stability test results

[0157]

[0158] The results showed that the RSD value of the protocatechuic acid peak area was 0.4%, and the RSD value of the vanillic acid peak area was 0.2%, indicating that the test solution had good stability within 24 hours.

[0159] 2.1.6 Intermediate precision

[0160] Different personnel and time inspections

[0161] The same cardamom (Java white cardamom) standard decoction freeze-dried powder test sample (batch number: TQW-16) was prepared by different personnel (A, B) at different times (Ⅰ, Ⅱ) for analysis. The contents of protocatechuic acid and vanillic acid in the test sample were calculated. The results are shown in Table 12.

[0162] Table 12 Intermediate precision test results

[0163]

[0164] The results showed that the RSD value of protocatechuic acid was 0.6%, and the RSD value of vanillic acid was 1.0%. The intermediate precision of this method was good.

[0165] 2.1.7 Sample recovery rate

[0166] About 0.25 g of a freeze-dried powder of a standard decoction of cardamom (Java white cardamom) with a known content (batch number: TQW-16, protocatechuic acid content 0.90 mg / g, vanillic acid content 0.84 mg / g) was taken as a test sample. Six portions were accurately weighed and 25 mL of a mixed control solution containing protocatechuic acid (purity 97.5%) and vanillic acid (purity: 99.8%) was accurately added to each portion. The test solution was prepared and measured according to the proposed method, and the recovery was calculated. The results are shown in Tables 13 and 14.

[0167] The calculation formula is as follows:

[0168]

[0169] Table 13 Protocatechuic acid sample recovery test results

[0170]

[0171] Table 14 Vanillic acid sample recovery test results

[0172]

[0173] The results showed that the average recovery of protocatechuic acid was 102.4%, and the average recovery of vanillic acid was 99.6%. This method has good accuracy.

[0174] 2.2 Durability inspection

[0175] 2.2.1 Investigation with different instruments

[0176] About 0.1 g of freeze-dried powder of cardamom (Java white cardamom) standard decoction (batch number: TQW-16) was taken and accurately weighed in two portions. The test solution was prepared according to the established method and examined on Waters e2695 and Agilent inifity 1260 high performance liquid chromatographs (chromatographic column: Shimadzu C18 4.6 mm × 250 mm, 5 μm), respectively. The protocatechuic acid and vanillic acid contents were calculated. The results are shown in Figure 9 , Table 15. Figure 9 The following are the results of investigation using different instruments.

[0177] Table 15 Experimental results of different instruments

[0178]

[0179] The results showed that the RSD value of the results measured by Waters e2695 and Agilent inifity 1260 high performance liquid chromatographs was 2.4%, indicating that the instrument durability of this method was good.

[0180] 2.2.2 Investigation of different chromatographic columns

[0181] Different brands of chromatographic columns (Waters C18 4.6mm×250mm, 5μm, Kromasil C18 4.6mm×250mm, 5μm, Shimadzu C18 4.6mm×250mm, 5μm) were used to detect the same cardamom (Java white cardamom) standard decoction freeze-dried powder test sample (batch number: TQW-16). The results are shown in the table. Figure 10 , Table 16. Figure 10 The results of the investigation of different chromatographic columns are shown in Figure 2.

[0182] Table 16 Results of the chromatographic column durability test

[0183]

[0184] The results showed that the analytical chromatographic indicators of different chromatographic columns were good. The theoretical plate number calculated based on the protocatechuic acid peak should not be less than 5000.

[0185] 2.3 Sample content determination verification

[0186] Cardamom (Java white cardamom) standard decoction freeze-dried powder was used as the test sample. The test sample solution was prepared and measured according to the proposed method. The peak area was recorded and the content of protocatechuic acid and vanillic acid was calculated. The results are shown in Table 17.

[0187] Table 17 Protocatechuic acid and vanillic acid contents in 16 batches of standard decoctions of cardamom (Java white cardamom)

[0188]

[0189]

[0190] The results showed that the total content of protocatechuic acid and vanillic acid in the standard decoction of cardamom (Java white cardamom) ranged from 1.65 mg / g to 2.85 mg / g, with an average total content of 2.31 mg / g and an SD value of 0.38. The limit of 70% to 130% of the average value of the total content determination was 1.62 mg / g to 3.00 mg / g, and the limit range of the average value plus or minus 3 times the SD value was 1.17 mg / g to 3.45 mg / g. It was tentatively determined that "the total amount of vanillic acid (C8H8O4) and protocatechuic acid (C7H6O4) per 1g of this product should be 1.20 mg to 3.50 mg."

[0191] 2.4 Final product granule content determination and verification

[0192] Sixteen batches of cardamom (Java white cardamom) finished granules were tested, and the results are shown in Table 18.

[0193] Table 18 Cardamom (Java white cardamom) finished product granule content determination results

[0194]

[0195] The results showed that the content determination method of cardamom (Java white cardamom) extract can effectively detect cardamom (Java white cardamom) finished granules.

[0196] Example 3: Establishment of a method for determining the content of medicinal materials

[0197] 3.1 Preparation of test solution

[0198] Take an appropriate amount of cardamom (Java white cardamom) medicinal material powder (pass through No. 3 sieve), take about 1.0g, accurately weigh, place in a stoppered conical flask, accurately add 25mL of 70% methanol, stopper, weigh the weight, heat and reflux for 30 minutes, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain.

[0199] 3.2 Preparation of reference solution

[0200] Take appropriate amounts of vanillic acid and protocatechuic acid reference substances, accurately weigh them, and add 70% methanol to prepare a mixed solution containing 10 μg of each per 1 mL.

[0201] 3.3 Chromatographic conditions 1.4 Selection of extract chromatographic conditions and investigation of system suitability.

[0202] 3.4 Preparation of test solution

[0203] 3.4.1 Investigation of extraction methods

[0204] Take approximately 1.0 g of cardamom (Java white cardamom) powder (passed through a No. 3 sieve, batch number: YC-16), accurately weigh it, and place it in a stoppered conical flask. Accurately add 25 mL of 70% methanol, stopper tightly, and weigh it. Extraction was then performed using both reflux and ultrasonic methods for 30 minutes. Allow to cool, weigh it again, and make up the loss with 70% methanol. Shake well, filter, and collect the filtrate. The total contents of vanillic acid and protocatechuic acid were analyzed and calculated using different extraction methods. The results are shown in Table 19.

[0205] Table 19 Analysis results of different extraction methods

[0206]

[0207] The results showed that the efficiency of reflux extraction was better than that of ultrasonic extraction, and reflux extraction was determined to be the extraction method.

[0208] 3.4.2 Extraction solvent investigation

[0209] Take approximately 1.0 g of cardamom (Java white cardamom) powder (passed through a No. 3 sieve, batch number: YC-16), accurately weigh it, and place it in a stoppered conical flask. Add 25 mL of each of the extraction solvents, ethanol, methanol, 70% methanol, 50% ethanol, 30% methanol, and water, and seal it tightly. Reflux for 30 minutes, cool, and weigh again. Add the appropriate solvent to compensate for the loss, shake well, filter, and collect the filtrate. Accurately pipette 10 μL of each test solution into a liquid chromatograph. Calculate the total content of vanillic acid and protocatechuic acid using different extraction solvents. The results are shown in Table 20.

[0210] Table 20 Results of investigation on different extraction solvents

[0211]

[0212] The results showed that 70% methanol had the best extraction efficiency and 70% methanol solution was used as the extraction solvent.

[0213] 3.4.3 Extraction time investigation

[0214] Take approximately 1.0 g of cardamom (Java white cardamom) powder (passed through a No. 3 sieve, batch number: YC-16), accurately weigh it, and place it in a stoppered conical flask. Accurately add 25 mL of 70% methanol solution, stopper tightly, and weigh the weight. Reflux extraction was performed for 15 minutes, 30 minutes, and 45 minutes, respectively. Allow to cool, weigh the weight again, and make up the loss with 70% methanol. Shake well, filter, and collect the filtrate. Analyze and calculate the vanillic acid and protocatechuic acid contents at different extraction times. The results are shown in Table 21.

[0215] Table 21 Analysis results of different extraction times

[0216]

[0217] The results showed that when the extraction time was 30 minutes, sufficient extraction could be achieved. Therefore, the extraction time of the test sample was determined to be 30 minutes.

[0218] 3.4.4 Investigation of solvent addition amount

[0219] Take approximately 1.0 g of cardamom (Java white cardamom) powder (passed through a No. 3 sieve, batch number: YC-16), accurately weigh it, and place it in a stoppered conical flask. Accurately add 10 mL, 25 mL, and 50 mL of 70% methanol, respectively, stopper the flask, weigh it, and reflux for 30 minutes. Cool, weigh it again, and make up the loss with 70% methanol. Shake well, filter, and collect the filtrate. Calculate the total vanillic acid and protocatechuic acid content. The results are shown in Table 22.

[0220] Table 22 Investigation results of different solvent addition amounts

[0221]

[0222] The results showed that when the sample size was 1.0 g, the amount of solvent added was 25 mL to ensure complete extraction of the target component, so the amount of solvent added was determined to be 25 mL.

[0223] In summary, the preparation method of the test sample for the determination of the medicinal material content of cardamom (Java white cardamom) is determined as follows: take the powder of this product (passed through No. 3 sieve), take about 1.0 g, accurately weigh, place in a stoppered conical flask, accurately add 25 mL of 70% methanol, stopper, weigh the weight, heat and reflux for 30 minutes, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain.

[0224] Example 4: Cardamom (Java white cardamom) medicinal material content determination method

[0225] Chromatographic Conditions and System Suitability: Use octadecylsilane bonded silica gel as the filler; acetonitrile as mobile phase A, and 0.1% phosphoric acid solution as mobile phase B, using gradient elution as specified in the table below; the detection wavelength is 260 nm. The theoretical plate number, calculated based on the protocatechuic acid peak, should be no less than 5000.

[0226]

[0227] Preparation of reference solution: Take appropriate amount of vanillic acid and protocatechuic acid reference substances, weigh accurately, add 70% methanol to make a mixed solution containing 10 μg of each per 1 mL.

[0228] Preparation of test solution: Take the powder of the product (passed through No. 3 sieve), take about 1.0 g, accurately weigh, place in a stoppered conical flask, accurately add 25 mL of 70% methanol, stopper, weigh the weight, heat under reflux for 30 minutes, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain.

[0229] Determination method: Accurately aspirate 10 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0230] 4.1 Methodological Investigation of Medicinal Materials

[0231] 4.1.1 Specificity Experiment

[0232] Preparation of test solution: According to the experimental conditions proposed above, prepare the test solution of cardamom (Java white cardamom).

[0233] Take appropriate amounts of vanillic acid and protocatechuic acid reference substances, accurately weigh them, and add 70% methanol to make solutions containing 10 μg per 1 mL.

[0234] Preparation of negative control solution: According to the experimental conditions proposed above, a negative control solution lacking cardamom (Java white cardamom) was prepared. Figure 11 . Figure 11 This is a specific inspection map.

[0235] The results showed that the chromatogram of negative solution had no interference with the determination of the peak to be tested, indicating that the method had good specificity.

[0236] 4.1.2 Precision investigation

[0237] The reference solution was sampled six times continuously, the peak areas of protocatechuic acid and vanillic acid were recorded, and the RSD values ​​were calculated. The results are shown in Table 23.

[0238] Table 23 Precision inspection results

[0239]

[0240]

[0241] The results showed that the RSD values ​​of the peak areas of vanillic acid and protocatechuic acid were both 1.0%, indicating good instrument precision.

[0242] 4.1.3 Linear Relationship

[0243] Take an appropriate amount of protocatechuic acid reference substance (purity 97.5%), place it in a 25 mL volumetric flask, dissolve it in 70% methanol to make a solution containing 202.3562 μg per 1 mL, and then dilute it to solutions containing 4.0471 μg, 10.1178 μg, 20.2356 μg, 50.5891 μg, and 101.1781 μg per 1 mL, respectively.

[0244] 10 μL of each solution was accurately drawn and injected into the liquid chromatograph to obtain the peak area. The response curve was drawn with the concentration (X, μg / mL) as the abscissa and the peak area (Y) as the ordinate. The results are shown in Table 24. Figure 12 . Figure 12 This is the standard curve of protocatechuic acid.

[0245] Table 24 Protocatechuic acid standard curve analysis results

[0246]

[0247] The results showed that when the concentration of protocatechuic acid ranged from 4.0471 to 202.3562 μg / ml, the linear relationship was y=37015x-11353, R 2 =0.9999. This indicates that the concentration range is 4.0471-202.3562 μg / ml, with a good linear relationship.

[0248] Take an appropriate amount of vanillic acid reference substance (purity 99.8%), place it in a 25 mL volumetric flask, dissolve it in 70% methanol to make a solution containing 200.9174 μg per 1 mL, and then dilute it to solutions containing 4.0183 μg, 10.0459 μg, 20.0917 μg, 50.2294 μg, and 100.4587 μg per 1 mL, respectively.

[0249] 10 μL of each solution was accurately drawn and injected into the liquid chromatograph to obtain the peak area. The response curve was drawn with the concentration (X, μg / ml) as the abscissa and the peak area (Y) as the ordinate. The results are shown in Table 25. Figure 13 . Figure 13 This is the standard curve of vanillic acid.

[0250] Table 25 Vanillic acid standard curve analysis results

[0251]

[0252] The results showed that when the concentration of vanillic acid ranged from 4.0183 to 200.9174 μg / mL, the linear relationship was y=37102x-6291.7, R 2 =0.9999. This indicates that the concentration range is 4.0183-200.9174 μg / mL with a good linear relationship.

[0253] 4.1.4 Repeatability Experiment

[0254] Take an appropriate amount of the powder of this product (passed through No. 3 sieve, batch number: YC-16), take about 1.0 g, accurately weigh 6 portions, and have the same operator prepare the test solution according to the determined method. Calculate the vanillic acid and protocatechuic acid contents of the 6 test samples. The results are shown in Table 26.

[0255] Table 26 Repeatability test results

[0256]

[0257]

[0258] The RSD value of the total content of protocatechuic acid and vanillic acid in 6 repeatability tests was 0.0%, and this method had good repeatability.

[0259] 4.1.5 Stability test

[0260] A solution of the same cardamom (Java white cardamom) medicinal material test sample (batch number: YC-16) was taken, and the chromatographic peak areas of vanillic acid and protocatechuic acid were determined at 0h, 2h, 4h, 8h, 16h, and 24h, respectively. The results are shown in Table 27.

[0261] Table 27 Stability test results

[0262]

[0263] The results showed that the RSD values ​​of the peak areas of protocatechuic acid and vanillic acid were both 1.0%, and the test solution had good stability within 24 hours.

[0264] 4.1.6 Intermediate precision

[0265] Investigation by different personnel and time: The same cardamom (Java white cardamom) medicinal material test sample (batch number: YC-16) was prepared by different personnel (A, B) at different times (Ⅰ, Ⅱ) for analysis, and the contents of vanillic acid and protocatechuic acid in the test sample were calculated. The results are shown in Table 28.

[0266] Table 28 Intermediate precision test results

[0267]

[0268] The results showed that the RSD value of the total content of vanillic acid and protocatechuic acid was 0.0%, and the intermediate precision of this method was good.

[0269] 4.1.7 Sample recovery

[0270] Six portions of approximately 0.25 g of a cardamom (Java white cardamom) herbal sample (batch number: YC-16, protocatechuic acid content: 0.70%, vanillic acid content: 0.60%) with known contents were accurately weighed and 25 mL of a mixed control solution containing vanillic acid (purity: 99.8%) and protocatechuic acid (purity: 97.9%) was accurately added to each portion. The test solution was prepared and measured according to the proposed method, and the recovery was calculated. The results are shown in Tables 29 and 30. The calculation formula is as follows:

[0271] Recovery rate % = (measured amount - content in sample) / added amount × 100%

[0272] Table 29 Protocatechuic acid sample recovery test results

[0273]

[0274] Table 30 Vanillic acid sample recovery experimental results

[0275]

[0276] The results showed that the recoveries of protocatechuic acid and vanillic acid were both RSD 1.2%, indicating that the method had good accuracy.

[0277] 4.1.8 Durability inspection

[0278] 4.1.8.1 Inspection of different instruments

[0279] According to the determined method, 1.0 g of cardamom (Java white cardamom) medicinal material (batch number: YC-16) was taken and accurately weighed. The chromatographic analysis was performed on Waters e2695 and Agilent inifity 1260 high performance liquid chromatographs (both columns were Shimadzu C18 4.6 mm × 250 mm, 5 μm). The total content of protocatechuic acid and vanillic acid was calculated. The results are shown in Figure 14 , Table 31. Figure 14 Overlay images for different instruments.

[0280] Table 31 Experimental results of different instruments

[0281]

[0282]

[0283] The results showed that the RSD value of the results measured on Waters e2695 and Agilent inifity 1260 high performance liquid chromatographs was 3.8%, indicating that the instrument durability of this method was good.

[0284] 4.1.8.2 Investigation of different chromatographic columns

[0285] The same sample (batch number: YC-16) was tested using different brands of C18 columns (Waters C18 4.6mm×250mm, 5μm, Kromasil C18 4.6mm×250mm, 5μm, Shimadzu C18 4.6mm×250mm, 5μm). The results are shown in Figure 15 , Table 32. Figure 15 Overlay of different chromatographic columns.

[0286] Table 32 Results of the chromatographic column durability test

[0287]

[0288] The results showed that the RSD value of the results measured by different chromatographic columns was 3.0%, indicating that the chromatographic column of this method had good durability.

[0289] 4.2 Verification of Cardamom (Java White Cardamom) Medicinal Material Content

[0290] Cardamom (Java white cardamom) was used as the test sample, and 16 batches of medicinal materials were tested using the established method. The results are shown in Table 33.

[0291] Table 33 Test results of medicinal material content of 16 batches of cardamom (Java white cardamom)

[0292]

[0293]

[0294] The content of cardamom (Java white cardamom) in 16 batches ranged from 0.026% to 0.044%, with an average of 0.038% and an SD of 0.008. The range of ±30% of the mean was 0.027% to 0.049%, and the range of ±3SD of the mean was 0.014% to 0.062%. The content limit for cardamom (Java white cardamom) was temporarily set at 0.010%, rounded to 0.010%.

[0295] To sum up, the limit of the total content of protocatechuic acid and vanillic acid in the medicinal material Cardamom (Java white cardamom) is "The total amount of protocatechuic acid (C7H6O4) and vanillic acid (C8H8O4) calculated on the basis of dry product shall not be less than 0.010%."

[0296] 4.3 Validation of Decoction Pieces Content Determination

[0297] 16 batches of cardamom (Java white cardamom) slices were tested, and the results are shown in Table 34.

[0298] Table 34 Cardamom (Java white cardamom) slice content determination results

[0299]

[0300]

[0301] The results showed that the content of 16 batches of cardamom (Java white cardamom) slices ranged from 0.026% to 0.045%, with an average of 0.038% and an SD of 0.008. The range of ±30% of the average was 0.027% to 0.049%, and the range of ±3SD of the average was 0.014% to 0.062%. The content limit for cardamom (Java white cardamom) slices was temporarily set at 0.010%, with a rounded value of 0.010%.

[0302] To sum up, the limit of the total content of protocatechuic acid and vanillic acid in cardamom (Java white cardamom) slices is "the total amount of protocatechuic acid (C7H6O4) and vanillic acid (C8H8O4) calculated on the basis of dry product shall not be less than 0.010%."

[0303] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for constructing a high-performance liquid phase content determination method for cardamom medicinal materials, decoction pieces, extracts or preparations, characterized in that: The following steps are involved: S1) taking a reference solution, detecting it by high performance liquid chromatography, and establishing a standard curve based on the concentration of the reference solution and the corresponding peak area; S2) taking a test sample raw material, extracting it with a solvent to obtain a test sample solution; determining the peak area of ​​the test sample solution by high performance liquid chromatography; the solvent is 70% methanol; S3) obtaining the content of the target substance in the cardamom medicinal material, decoction piece, extract or preparation based on the standard curve and the peak area of ​​the test solution; The cardamom medicinal material, decoction piece, extract or preparation is Java white cardamom medicinal material, decoction piece, extract or preparation; The reference solution includes a protocatechuic acid reference solution and a vanillic acid reference solution; The chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column is a C18 column; the mobile phase A is acetonitrile, the mobile phase B is a 0.1% phosphoric acid solution, and the gradient elution is performed; In terms of volume percentage, the gradient elution is specifically as follows: 0-18 min, mobile phase A 5%→16%, mobile phase B 95%→84%; 18-32 min, mobile phase A 16% → 22%, mobile phase B 84% → 78%; 32-37 min, mobile phase A 22%→28%, mobile phase B 78%→72%; 37-38 min, mobile phase A 28%→5%, mobile phase B 72%→95%; The detection wavelength was 260 nm.

2. The construction method according to claim 1, characterized in that The extraction is reflux extraction; the extraction time is 15 to 45 minutes.

3. The construction method according to claim 1, characterized in that In terms of g / mL, the mass ratio of the test sample to the solvent is 0.5:(10~25).

4. The construction method according to claim 1, characterized in that The chromatographic column is a C18 column with a specification of 5 μm and 4.6×250 mm; the column temperature is 30°C~40°C.

5. The construction method according to claim 1, characterized in that The flow rate of the mobile phase was 0.8-1.2 mL / min, and the injection volume was 10 μL.

6. The construction method according to claim 1, characterized in that The theoretical plate number of the chromatographic column is not less than 5000, calculated based on the protocatechuic acid peak.

7. The construction method according to claim 1, characterized in that The protocatechuic acid showed a linear relationship in the range of 4.0471-202.3562 μg / mL, with a linear relationship of y = 37015x - 11353, R² = 0.9999; The vanillic acid showed a linear relationship in the range of 4.0183~200.9174 μg / mL, with a linear relationship of y = 37102x- 6291.7 and R² = 0.9999.

8. A method for evaluating the quality of cardamom medicinal materials, decoction pieces, extracts or preparations, characterized in that: Taking a sample to be tested, and detecting it using the construction method described in any one of claims 1 to 7 to obtain the content of protocatechuic acid and vanillic acid in the sample to be tested; The sample to be tested is Javanese white cardamom medicinal material, decoction pieces, extract or preparation.

9. The quality evaluation method according to claim 8, characterized in that: Calculated on the dry product, the total content of protocatechuic acid and vanillic acid in the cardamom medicinal material is not less than 0.010%, which is judged to be qualified; the total content of protocatechuic acid and vanillic acid in the cardamom medicinal material is less than 0.010%, which is judged to be unqualified; Calculated on the dry product, the total content of protocatechuic acid and vanillic acid in the cardamom slices is not less than 0.010%, which is considered qualified; the total content of protocatechuic acid and vanillic acid in the cardamom slices is less than 0.010%, which is considered unqualified; Calculated on a dry basis, the total amount of vanillic acid and protocatechuic acid contained in each 1 g of the standard cardamom decoction is 1.20 mg to 3.50 mg.

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