Construction method for HPLC content determination of rhizoma nelumbinis and rhizoma nelumbinis related processed products

Abstract

The invention provides a construction method for HPLC (High Performance Liquid Chromatography) content determination of rhizoma nelumbinis and rhizoma nelumbinis related processed products, which comprises the following steps: A) dissolving a test raw material with a solvent, and extracting to obtain a to-be-detected solution; the raw material of the test sample comprises rhizoma nelumbinis or a rhizoma nelumbinis related processed product; b) measuring the liquid to be measured by adopting a high performance liquid chromatography to obtain an HPLC spectrum of the rhizoma nelumbinis or the rhizoma nelumbinis related processed products; chromatographic conditions of the high performance liquid chromatography are as follows: a chromatographic column is a C18 column; a mobile phase A is a methanol solution, and a mobile phase B is a 0.5% phosphoric acid aqueous solution; the gradient elution is specifically as follows: 0-1 min, the phase A is 2%, and the phase B is 98%; 1-4 min, the phase A accounts for 2%-8%, and the phase B accounts for 98%-92%; 4-6 min, 8% to 0% of the phase A and 92% to 100% of the phase B; the phase A accounts for 0% and the phase B accounts for 100% in 6-16 min; according to the method, a high performance liquid chromatography method is adopted, a methanol-0. 5% phosphoric acid solution is selected as a mobile phase for gradient elution, a protocatechuic acid reference substance and a p-hydroxybenzoic acid reference substance are taken as reference substances, and the HPLC content determination method for the rhizoma nelumbinis and the rhizoma nelumbinis related processed products is established. And more scientific technical means are provided for controlling the medicinal quality of the rhizoma nelumbinis and the rhizoma nelumbinis related processed products.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection, and in particular to a construction method for HPLC content determination of lotus root nodes and lotus root node related processed products. Background Art

[0002] Lotus root nodes are the dried rhizome nodes of the lotus plant Nelumbo nucifera Gaertn. of the Nymphaeaceae family. It has the effects of astringent hemostasis and blood stasis removal. It is used for hematemesis, hemoptysis, hemostasis, hematuria, and metrorrhagia. According to the literature, phenolic acid compounds have diverse structures and are widely present in plants. They have multiple biological activities, especially in anti-inflammatory and hemostasis. For example, medicinal materials such as Sanguisorba officinalis, Loropetalum leucoderma, and Golden Silk Belt all have strong hemostatic effects, and their active ingredients are mostly phenolic acid components; experimental studies have found that there are a variety of phenolic acid components in lotus root nodes, which are similar to other hemostatic drugs, and echo the astringent and hemostatic effects of lotus root nodes.

[0003] The charcoal processed lotus root node is the dried rhizome node of the lotus plant of the Nymphaeaceae family, which is processed and processed according to the main quality indicators of the standard decoction to make formula granules or pieces. How to distinguish it from the processed lotus root node is a big problem. Specifically, the lotus root node pieces are usually made by removing impurities from the lotus nodes, washing, drying and then slicing them. The charcoal processed lotus root node pieces are made by taking the clean lotus root nodes, frying them according to the charcoal frying method (such as the "Chinese Pharmacopoeia" 0213 General Rules for Processing) until the surface is dark brown or burnt black, and the inside is yellow brown or tan, and then slicing them. The standard decoction of lotus root node is a freeze-dried powder made from the lotus root node medicinal material after processing according to a fixed preparation process.

[0004] Therefore, the present invention aims to construct a method for HPLC content determination of lotus root nodes and lotus root node related processed products, so as to identify lotus root node processed products and lotus root node carbonized processed products, thereby providing guidance for quality control of lotus root node related processed products. Summary of the invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a method for constructing HPLC content determination of lotus root nodes and lotus root node-related processed products. The method of the present invention is accurate and reliable in detecting protocatechuic acid reference substances and para-hydroxybenzoic acid reference substances, and can also identify lotus root node processed products and lotus root node carbonized processed products.

[0006] The present invention provides a method for constructing HPLC content determination of lotus root nodes and lotus root node related processed products, comprising:

[0007] A) dissolving the raw material of the test sample with a solvent, extracting, and obtaining a test solution; the raw material of the test sample includes lotus root nodes or lotus root node-related processed products;

[0008] B) using high performance liquid chromatography to measure the test solution to obtain an HPLC spectrum of the lotus root node or a lotus root node-related processed product;

[0009] 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 a methanol solution, the mobile phase B is a 0.5% phosphoric acid aqueous solution, and the elution is performed in a gradient manner;

[0010] The gradient elution is specifically:

[0011] 0-1min, phase A: 2%, phase B: 98%;

[0012] 1-4 min, phase A: 2%→8%, phase B: 98%→92%;

[0013] 4-6 min, phase A: 8% → 0%, phase B: 92% → 100%;

[0014] 6-16 min, phase A: 0%, phase B: 100%;

[0015] 16-30min, phase A: 10%, phase B: 90%.

[0016] The chromatographic conditions of the high performance liquid chromatography are: the chromatographic column is a C18 column; preferably a C 18 150×2.1mm 1.8~2.7μm chromatographic column.

[0017] Including but not limited to InfinityLab Poroshell 120HILIC-Z 2.1*150mm, 2.7μm, ZORBAXSB-C182.1*150mm, 1.8μm, InfinityLab Poroshell 120EC-C182.1*150mm, 1.9μm.

[0018] The mobile phase A of the present invention is a methanol solution, the mobile phase B is a 0.5% phosphoric acid aqueous solution, and the gradient elution is performed;

[0019] The gradient elution is specifically:

[0020] 0-1min, phase A: 2%, phase B: 98%;

[0021] 1-4 min, phase A: 2%→8%, phase B: 98%→92%;

[0022] 4-6 min, phase A: 8% → 0%, phase B: 92% → 100%;

[0023] 6-16 min, phase A: 0%, phase B: 100%;

[0024] 16-30min, phase A: 10%, phase B: 90%.

[0025] The lotus root node related processed products of the present invention include lotus root node processed products and lotus root node carbonized processed products; wherein the lotus root node processed products include lotus root node decoction pieces, lotus root node standard decoction, and lotus root node formula granules. The lotus root node carbonized processed products include lotus root node charcoal processed pieces, lotus root node charcoal standard decoction, or lotus root node charcoal formula granules.

[0026] The invention provides a construction method for HPLC content determination of lotus root nodes and lotus root node related processed products. Firstly, a test sample raw material is dissolved and extracted by a solvent to obtain a test solution.

[0027] Specifically, the test sample raw material is dissolved in a solvent, extracted, cooled, shaken, and filtered to obtain the product. The present invention does not limit the specific methods and operations of cooling, shaking, and filtering, which are well known to those skilled in the art.

[0028] The extraction of the present invention is heating reflux extraction or ultrasonic extraction, preferably ultrasonic treatment. The power of the ultrasonic treatment is 600W, the frequency is 40kHz, and the ultrasonic treatment time is 20 to 40 minutes, preferably 30 minutes.

[0029] According to the present invention, the ratio of the mass g of the test sample raw material to the volume mL of the solvent is 1 to 4:20.

[0030] The solvent of the present invention is 0% methanol to 100% methanol; specifically, it can be water, 30% methanol, 50% methanol, 70% methanol, methanol; preferably, it is 70% methanol.

[0031] The inventors found that the extraction effect was best with 70% methanol.

[0032] In some specific embodiments of the present invention,

[0033] Medicinal material test solution: Take 3.0 g of the powder of this product, weigh it accurately, put it in a stoppered conical flask, add 50 ml of 70% methanol accurately, weigh it, treat it with ultrasound (power 600 W, frequency 40 kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0034] Decoction piece test solution: Take 3.0g of the powder of this product, weigh it accurately, put it in a stoppered conical flask, add 50ml of 70% methanol accurately, weigh it, treat it with ultrasound (power 600W, frequency 40kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0035] Standard decoction test solution: Take an appropriate amount of the product, take about 1g, accurately weigh, place in a stoppered conical flask, accurately add 20ml of 70% methanol, stopper, weigh, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate.

[0036] Formula granules test solution: Take an appropriate amount of the product, grind it into powder, take about 1g, accurately weigh it, put it in a stoppered conical flask, accurately add 50ml of 70% methanol, stopper it, weigh it, treat it with ultrasound (power 600W, frequency 40kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0037] The above raw materials of the present invention can all be subjected to quality control and qualitative and quantitative detection by the method of the present invention.

[0038] The present invention also includes preparing a reference solution: taking a protocatechuic acid reference substance and a p-hydroxybenzoic acid reference substance respectively, dissolving them in 70% methanol to obtain a reference solution;

[0039] The reference solution is measured by high performance liquid chromatography to obtain a chromatogram of the reference; and the components of the lotus root node or lotus root node related processed products are qualitatively identified according to the chromatogram of the reference.

[0040] The concentration of the reference solution of the present invention is preferably as follows: the concentration of the reference solution is as follows: the protocatechuic acid reference substance is 0.40677-0.81354 μg / ml, and the p-hydroxybenzoic acid reference substance is 0.40650-40.65040 μg / ml.

[0041] Specifically, the concentration of protocatechuic acid ranges from 0.40677 to 0.81354 μg / ml, and the linear relationship is y=35.0807x-0.2876, R 2 =1.0000, showing a good linear relationship.

[0042] The concentration range of p-hydroxybenzoic acid is 0.40650~40.65040μg / ml, and the linear relationship is y=56.7956x-0.5472, R 2 =1.0000, showing a good linear relationship.

[0043] The test liquid is measured by high performance liquid chromatography to obtain the HPLC spectrum of the lotus root node or lotus root node related processed products.

[0044] The HPLC chromatographic conditions are: the chromatographic column is a C18 column, the specification is 150×2.1mm 2.7μm, the column temperature is 20-30°C, preferably 20°C. At the above chromatographic column temperature, the chromatographic peaks of the present invention are symmetrical, the separation is good, and the peaks are more complete.

[0045] The mobile phase A of the present invention is a methanol solution, the mobile phase B is a 0.5% phosphoric acid aqueous solution, and the elution is performed in a gradient manner.

[0046] Specifically, the gradient elution is specifically:

[0047] 0-1min, phase A: 2%, phase B: 98%;

[0048] 1-4 min, phase A: 2%→8%, phase B: 98%→92%;

[0049] 4-6 min, phase A: 8% → 0%, phase B: 92% → 100%;

[0050] 6-16 min, phase A: 0%, phase B: 100%;

[0051] 16-30min, phase A: 10%, phase B: 90%.

[0052] The theoretical plate number calculated based on p-hydroxybenzoic acid should not be less than 3000.

[0053] The present invention has good baseline separation under the above elution gradient, good separation of each peak and a stable baseline.

[0054] The flow rate of the mobile phase of the present invention is 0.2-0.4 mL / min, and more preferably 0.3 mL / min.

[0055] The present invention found that the chromatographic peaks were well separated and the separation degree was moderate under the above flow rate, which is the most preferred solution.

[0056] The injection volume is 2 to 5 μL, preferably 3 μL.

[0057] The detection wavelength is 260 nm. The inventors found that at 260 nm, the chromatographic peak information volume is larger, the chromatogram baseline is more stable, and the peak area of ​​each peak is larger.

[0058] A method for identifying a processed lotus root node product and a carbonized lotus root node product, using the method described in the above technical solution to perform detection and analyze the transfer rate results of the total content of protocatechuic acid and p-hydroxybenzoic acid:

[0059] The actual measured value range of the transfer rate from lotus root node medicinal materials to lotus root node decoction pieces is 93.77% to 97.25%; the actual measured value range of the transfer rate from lotus root node medicinal materials to lotus root node charcoal decoction pieces is 97.21% to 244.90%;

[0060] The measured values ​​of the transfer rate from lotus root node slices to lotus root node standard decoction ranged from 32.7% to 93.4%; the measured values ​​of the transfer rate from lotus root node charcoal slices to lotus root node charcoal standard decoction ranged from 19.1% to 48.5%.

[0061] The actual measured range of the total content conversion from the same amount of lotus root node medicinal materials to lotus root node formula granules is 0.16mg / g~1.02mg / g; the actual measured range of the total content conversion from the same amount of lotus root node medicinal materials to lotus root node charcoal formula granules is 0.11mg / g~0.76mg / g.

[0062] (1) The present invention establishes a method for distinguishing processed lotus root nodes and processed lotus root node carbonized products. Taking the total amount of protocatechuic acid reference substance and p-hydroxybenzoic acid reference substance as indicators, the intrinsic quality of lotus root node medicinal materials, decoction pieces, standard decoctions and their formula granules and related preparations can be controlled from an overall and macroscopic perspective, thereby ensuring the efficacy of the drugs and subjecting the medicinal materials and their related preparations to more formal quality control. At the same time, a scientific basis is provided for distinguishing processed lotus root nodes and processed lotus root node carbonized products. The difference in the transfer rate from medicinal materials to decoction pieces, standard decoctions and their formula granules in the HPLC content measurement method of lotus root nodes and lotus root node charcoal can become the identification point for distinguishing lotus root nodes and lotus root node charcoal.

[0063] (2) The method of the present invention has good stability, high precision, good reproducibility, is convenient and easy to master.

[0064] The present invention provides a method for constructing HPLC content determination of lotus root nodes and lotus root node related processed products, comprising: A) dissolving a test sample raw material with a solvent, extracting, and obtaining a test solution; the test sample raw material comprises lotus root nodes or lotus root node related processed products; B) determining the test solution with high performance liquid chromatography to obtain an HPLC spectrum of the lotus root node or the lotus root node related processed products; the chromatographic conditions of the high performance liquid chromatography are: the chromatographic column is a C18 column; the mobile phase A is a methanol solution, the mobile phase B is a 0.5% phosphoric acid aqueous solution, and the gradient elution is performed; the gradient elution is specifically: 0-1min, phase A: 2%, phase B: 98%; 1-4mi n, phase A: 2%→8%, phase B: 98%→92%; 4-6min, phase A: 8%→0%, phase B: 92%→100%; 6-16min, phase A: 0%, phase B: 100%; 16-30min, phase A: 10%, phase B: 90%. The present invention adopts high performance liquid chromatography, selects methanol-0.5% phosphoric acid solution as mobile phase for gradient elution, takes protocatechuic acid reference substance and p-hydroxybenzoic acid reference substance as reference substances, and establishes an HPLC content determination method for lotus root nodes and their related processed products, so as to provide more and more scientific technical means for controlling the medicinal quality of lotus root nodes and their related processed products. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 This is the spectrum of protocatechuic acid;

[0066] Figure 2 This is the spectrum of p-hydroxybenzoic acid.

[0067] Figure 3 The chromatograms are at different flow rates;

[0068] Figure 4 The chromatograms are at different column temperatures;

[0069] Figure 5 Chromatograms of different injection volumes;

[0070] Figure 6 Comparison chart of specificity of standard decoction of lotus root nodes;

[0071] Figure 7 This is the standard curve of protocatechuic acid;

[0072] Figure 8 This is the standard curve of p-hydroxybenzoic acid;

[0073] Fig. 9 This is the spectrum of protocatechuic acid;

[0074] Fig.10 is the spectrum of p-hydroxybenzoic acid;

[0075] Fig.11 Chromatograms at different flow rates;

[0076] Fig.12 Chromatograms at different column temperatures;

[0077] Fig.13 Chromatograms of different injection volumes;

[0078] Fig.14 Comparison chart of the specificity of lotus root node medicinal materials;

[0079] Fig.15 Standard curve of protocatechuic acid;

[0080] Fig.16 Standard curve of p-hydroxybenzoic acid;

[0081] Fig.17 Protocatechuic acid spectrum;

[0082] Fig.18 Spectrum of p-hydroxybenzoic acid;

[0083] Fig.19 Chromatograms at different flow rates;

[0084] Fig. 20 Chromatograms at different column temperatures;

[0085] Fig.21 Chromatograms of different injection volumes;

[0086] Fig. 22Comparison chart of specificity of standard decoction of lotus root nodes;

[0087] Fig.23 Standard curve of protocatechuic acid;

[0088] Fig.24 Standard curve of p-hydroxybenzoic acid;

[0089] Fig.25 It is the chromatogram of Comparative Example 1;

[0090] Fig.26 It is the chromatogram of Comparative Example 2;

[0091] Fig. 27 It is the chromatogram of Comparative Example 3. DETAILED DESCRIPTION

[0092] The present invention provides a method for constructing HPLC content determination of lotus root node medicinal materials, decoction pieces, standard decoctions and their formula particles. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they all belong to the scope of protection of the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0093] To further illustrate the present invention, the construction method for HPLC content determination of lotus root nodes and lotus root node-related processed products provided by the present invention is described in detail below in conjunction with the examples.

[0094] Experimental instruments and materials

[0095] High performance liquid chromatograph: Waters 2695 high performance liquid chromatograph, Agilent 1260 high performance liquid chromatograph;

[0096] Electronic balance: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instrument Co., Ltd.);

[0097] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0098] Ultrasonic cleaner: KQ600DB (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0099] Chromatographic columns: Column 1 (InfinityLab Poroshell 120HILIC-Z 2.1*150mm, 2.7μm); Column 2 (ZORBAX SB-C18 2.1*150mm, 1.8μm); Column 3 (InfinityLab Poroshell 120EC-C18 2.1*150mm, 1.9μm)

[0100] Methanol was of chromatographic grade, phosphoric acid was of chromatographic grade, water was ultrapure water, and the other reagents were of analytical grade.

[0101] Protocatechuic acid (China Food and Drug Inspection Institute, batch number: 110809-202207, purity: 97.5%)

[0102] p-Hydroxybenzoic acid (Shanghai Shidande Standard Technology Service Co., Ltd., batch number: 2169, purity: 98%)

[0103] 32 batches of Oujie standard decoction (OJ-BT-2401, OJ-BT-2402, OJ-BT-2403, OJ-BT-2404, OJ-BT-2405, OJ-BT-2406, OJ-BT-2407, OJ- BT-2408, OJ-BT-2409, OJ-BT-2410, OJ-BT-2411, OJ-BT-2412, OJ-BT-2413, OJ-BT-2414, OJ-BT-2415, OJ-BT- 2416, OJ-BT-2417, OJ-BT-2418, OJ-BT-2419, OJ-BT-2420, OJ-BT-2421, OJ-BT-2422, OJ-BT-2423, OJ-BT-24 24. OJ-BT-2425, OJ-BT-2426, OJ-BT-2427, OJ-BT-2428, OJ-BT-2429, OJ-BT-2430, OJ-BT-2431, OJ-BT-2432)

[0104] 32 batches of standard decoctions of carbonized lotus rhizome nodes (OJT-BT-2401, OJT-BT-2402, OJT-BT-2403, OJT-BT-2404, OJT-BT-2405, OJT-BT-2406, OJT-BT-2407, OJT-BT-2408, OJT-BT-2409, OJT-BT-2410, OJT-BT-2411, OJT-BT-2412, OJT-BT-2413, OJT-BT-2414, OJT-BT-2415, OJT-BT-2416, OJT-BT-2417, OJT-BT-2418, OJT-BT-2419, OJT-BT-2420, OJT-BT-2421, OJT-BT-2422, OJT-BT-2423, OJT-BT-2424, OJT-BT-2425, OJT-BT-2426, OJT-BT-2427, OJT-BT-2428, OJT-BT-2429, OJT-BT-2430, OJT-BT-2431, OJT-BT-2432);

[0105] 32 batches of lotus rhizome node medicinal materials (OJ-YC-2401, OJ-YC-2402, OJ-YC-2403, OJ-YC-2404, OJ-YC-2405, OJ-YC-2406, OJ-YC-2407, OJ-YC-2408, OJ-YC-2409, OJ-YC-2410, OJ-YC-2411, OJ-YC-2412, OJ-YC-2413, OJ-YC-2414, OJ-YC-2415, OJ-YC-2416, OJ-YC-2417, OJ-YC-2418, OJ-YC-2419, OJ-YC-2420, OJ-YC-2421, OJ-YC-2422, OJ-YC-2423, OJ-YC-2424, OJ-YC-2425, OJ-YC-2426, OJ-YC-2427, OJ-YC-2428, OJ-YC-2429, OJ-YC-2430, OJ-YC-2431, OJ-YC-2432);

[0106] 32 batches of lotus root slices (OJ-YP-2401, OJ-YP-2402, OJ-YP-2403, OJ-YP-2404, OJ-YP-2405, OJ-YP-2406, OJ-YP-2407, OJ-YP -2408, OJ-YP-2409, OJ-YP-2410, OJ-YP-2411, OJ-YP-2412, OJ-YP-2413, OJ-YP-2414, OJ-YP-2415, OJ-YP-2 416, OJ-YP-2417, OJ-YP-2418, OJ-YP-2419, OJ-YP-2420, OJ-YP-2421, OJ-YP-2422, OJ-YP-2423, OJ-YP-242 4. OJ-YP-2425, OJ-YP-2426, OJ-YP-2427, OJ-YP-2428, OJ-YP-2429, OJ-YP-2430, OJ-YP-2431, OJ-YP-2432);

[0107] 32 batches of charcoal slices of lotus root nodes (OJT-YP-2401, OJT-YP-2402, OJT-YP-2403, OJT-YP-2404, OJT-YP-2405, OJT-YP-2406, OJT-YP-2407, OJT-YP-2408, OJT-YP-2409, OJT-YP-2410, OJT-YP-2411, OJT-YP-2412, OJT-YP-2413, OJT-YP-2414, OJT-YP-2415, OJT-YP-2 416. OJT-YP-2417, OJT-YP-2418, OJT-YP-2419, OJT-YP-2420, OJT-YP-2421, OJT-YP-2422, OJT-YP-2423, OJT-YP-242 4. OJT-YP-2425, OJT-YP-2426, OJT-YP-2427, OJT-YP-2428, OJT-YP-2429, OJT-YP-2430, OJT-YP-2431, OJT-YP-2432);

[0108] Three batches of lotus root node formula granules (OJ-KL-2401, OJ-KL-2402, OJ-KL-2403);

[0109] Three batches of lotus root charcoal formula particles (OJT-KL-2401, OJT-KL-2402, OJT-KL-2403)

[0110] Example 1 HPLC content determination of lotus root node standard decoction Drafting instructions

[0111] 1.1 Proposed chromatographic conditions

[0112] Chromatographic conditions and system suitability test The chromatographic conditions and system suitability test used octadecylsilane bonded silica gel as filler (column length 150mm, inner diameter 2.1mm, particle size 2.7μm); methanol as mobile phase A, 0.5% phosphoric acid aqueous solution as mobile phase B; gradient elution according to the provisions in the table below; detection wavelength 260nm. Flow rate 0.3ml / min; column temperature 20℃; theoretical plate number calculated based on p-hydroxybenzoic acid peak should be no less than 3000.

[0113]

[0114] Preparation of reference solution: Take appropriate amount of protocatechuic acid and p-hydroxybenzoic acid reference substances, weigh accurately, add 70% methanol to prepare a mixed solution containing 20 μg of protocatechuic acid and p-hydroxybenzoic acid per 1 ml.

[0115] Preparation of test solution: Take an appropriate amount of standard lotus root node decoction, take about 1.0 g, accurately weigh, put in a stoppered conical flask, accurately add 20 ml of 70% methanol, stopper, weigh, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate.

[0116] Determination method: Accurately pipette 3μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0117] 1.2 Selection of chromatographic conditions and investigation of system suitability

[0118] 1.2.1 Determination of detection wavelength

[0119] Based on the above experimental conditions, a diode array detector was used to perform full-band scanning of protocatechuic acid and p-hydroxybenzoic acid. Figure 1 and Figure 2 . Figure 1 This is the spectrum of protocatechuic acid; Figure 2 This is the spectrum of p-hydroxybenzoic acid.

[0120] According to the spectral analysis, the two test index peaks have strong absorption at 260nm, so the absorption wavelength of subsequent tests is set at 260nm.

[0121] 1.2.2 Flow rate investigation

[0122] Under the above proposed chromatographic conditions, the separation effect of protocatechuic acid and p-hydroxybenzoic acid chromatographic peaks at flow rates of 0.2 ml / min, 0.3 ml / min, and 0.4 ml / min was used as the evaluation index. The results are shown in Figure 3 . Figure 3 The results show that when the flow rate is 0.2ml / min, 0.3ml / min and 0.4ml / min, the retention time and peak separation are better at 0.3ml / min, so the flow rate is set to 0.3ml / min.

[0123] 1.2.3 Column temperature investigation

[0124] Under the above proposed chromatographic conditions, the analysis of the test solution at column temperatures of 20℃, 25℃, and 30℃ was investigated, and the separation effect of the protocatechuic acid and p-hydroxybenzoic acid chromatographic peaks was used as the evaluation index. The results are shown in Figure 4 . Figure 4 The chromatograms are at different column temperatures; the results show that when the column temperature is 20℃, the retention time and peak separation of the index peaks are better, so the experimental column temperature is specified to be 20℃.

[0125] 1.2.4 Sample volume investigation

[0126] Under the above proposed chromatographic conditions, 2μl, 3μl, and 5μl were injected respectively, and the separation effect of protocatechuic acid and p-hydroxybenzoic acid chromatographic peaks was used as the evaluation index. The results are shown in Figure 5 . Figure 5 Chromatograms of different injection volumes; the results show that when the injection volume is 3μl, the peak shape and peak separation are better, so the injection volume of this method is stipulated to be 3ul.

[0127] 1.3 Chromatographic conditions and system suitability test results

[0128] In summary, the chromatographic conditions and system suitability test results for the determination of the content of the standard decoction of lotus root nodes are as follows: octadecylsilane bonded silica gel is used as the filler (column length is 150 mm, inner diameter is 2.1 mm, particle size is 2.7 μm); methanol is used as mobile phase A, 0.5% phosphoric acid aqueous solution is used as mobile phase B; gradient elution is performed according to the provisions in the table below; the detection wavelength is 260 nm. The flow rate is 0.3 ml / min; the column temperature is 20 ° C; the theoretical plate number calculated according to the p-hydroxybenzoic acid peak should be no less than 3000.

[0129] 1.4 Preparation of test solution

[0130] 1.4.1 Investigation of extraction solvent

[0131] Take an appropriate amount of lotus root node standard decoction, take about 1.0g, accurately weigh, put in a stoppered conical bottle, accurately add water, ethanol, methanol, 30% methanol, 50% methanol, 70% methanol 20ml, plug tightly, weigh, ultrasonic treatment (power 600W, frequency 40kHz) for 30 minutes, take out, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, take the filtrate, and get it. Take 3μl each and inject it into the chromatograph, analyze and calculate the total amount of protocatechuic acid and p-hydroxybenzoic acid content under different extraction solvents, the results are shown in Table 1.

[0132] Table 1 Analysis results of different extraction solvents

[0133]

[0134]

[0135] The results showed that 70% methanol had the best extraction effect, so 70% methanol was selected as the extraction solvent for the standard decoction of lotus root nodes.

[0136] 1.4.2 Investigation of extraction methods

[0137] Take an appropriate amount of lotus root node standard decoction, take about 1.0g, accurately weigh, put in a stoppered conical bottle, accurately add 20ml of 70% methanol, seal, weigh, reflux and ultrasonic treatment (power 600W, frequency 40kHz) for 30 minutes, take out, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, take the filtrate, and get it. Take 3μl each and inject it into the chromatograph, analyze and calculate the total amount of protocatechuic acid and p-hydroxybenzoic acid content under different extraction solvents, the results are shown in Table 2.

[0138] Table 2 Analysis results of different extraction methods

[0139]

[0140] The results showed that there was little difference between the extraction efficiencies of ultrasonic and reflux extraction. After comprehensive consideration, ultrasonic extraction was selected as the extraction method.

[0141] 1.4.3 Extraction time investigation

[0142] Take an appropriate amount of lotus root node standard decoction, take about 1.0g, accurately weigh, put in a stoppered conical bottle, accurately add 20ml of 70% methanol, seal, weigh, ultrasonically treat (power 600W, frequency 40kHz) for 20 minutes, 30 minutes and 40 minutes respectively, take out, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, take the filtrate, and inject 3μl of each into the chromatograph, analyze and calculate the total amount of protocatechuic acid and p-hydroxybenzoic acid content under different extraction solvents, the results are shown in Table 3.

[0143] Table 3 Analysis results of different extraction times

[0144]

[0145] The results showed that the sample was completely extracted after 30 minutes of extraction, so the extraction time of the sample was determined to be 30 minutes.

[0146] 1.4.4 Sample weighing inspection

[0147] Take appropriate amount of lotus root node standard decoction, take 0.5g, 1.0g and 1.5g respectively, weigh accurately, put in a stoppered conical bottle, add 20ml of 70% methanol accurately, stopper tightly, weigh, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, take out, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, take the filtrate, and inject 3μl of each into the chromatograph, analyze and calculate the total amount of protocatechuic acid and p-hydroxybenzoic acid content under different extraction solvents, the results are shown in Table 4.

[0148] Table 4 Analysis results of different sample weights

[0149]

[0150] The results showed that the total amount of protocatechuic acid and p-hydroxybenzoic acid did not differ much with different sample sampling amounts. Taking all factors into consideration, the sampling amount of the test sample was determined to be 1.0 g.

[0151] In summary, the preparation method of the test sample is determined as follows: take an appropriate amount of standard lotus root node decoction, take about 1.0g, accurately weigh, put it in a stoppered conical flask, accurately add 20ml of 70% methanol, stopper it, weigh it, ultrasonically treat it (power 600W, frequency 40kHz) for 30 minutes, take it out, 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.

[0152] 1.4.5 Methodological investigation

[0153] 1.4.5.1 Specificity inspection

[0154] Preparation of reference solution: Take appropriate amount of protocatechuic acid and p-hydroxybenzoic acid reference substances, weigh accurately, add 70% methanol to make a mixed solution containing 20 μg of protocatechuic acid and p-hydroxybenzoic acid per 1 ml.

[0155] Preparation of test solution: Take an appropriate amount of standard lotus root node decoction, take about 1.0g, accurately weigh, put in a stoppered conical flask, accurately add 20ml of 70% methanol, stopper, weigh, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, take out, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate.

[0156] Preparation of negative control solution: According to the experimental conditions proposed above, prepare the negative control solution of the standard decoction lacking lotus root nodes.

[0157] Accurately pipette 3 μl of reference solution and test solution respectively, inject into liquid chromatograph, and measure. Figure 6 . Figure 6 Comparison chart of specificity of standard decoction of lotus root nodes; as can be seen from the above figure, the negative solution has no effect on the target peaks of protocatechuic acid and p-hydroxybenzoic acid, and this method has good specificity.

[0158] 1.4.5.2 Precision investigation

[0159] The reference solution was sampled 6 times continuously, the peak areas of protocatechuic acid and p-hydroxybenzoic acid were recorded, and the RSD values ​​were calculated. The results are shown in Table 6.

[0160] Table 5 Precision investigation results

[0161]

[0162] The results showed that the peak area RSD value of protocatechuic acid and p-hydroxybenzoic acid in the precision study was 0.1%, and the injection precision of this method was good.

[0163] 1.4.5.3 Linear Relationship

[0164] Take 2.086 mg of protocatechuic acid (purity of 97.8%), put it in a 50 ml volumetric flask, dilute it to the mark with 70% methanol, shake well, filter, and obtain mother solution 1; take another 2.074 mg of p-hydroxybenzoic acid (purity of 98.0%), put it in a 50 ml volumetric flask, dilute it to the mark with 70% methanol, shake well, filter, and obtain mother solution 2. Prepare the following reference solutions of different concentrations respectively. Pipette protocatechuic acid and p-hydroxybenzoic acid reference substances of different concentrations and inject them into the liquid chromatograph. Analyze to obtain the peak area, and draw the response curve with protocatechuic acid or p-hydroxybenzoic acid (X) as the horizontal coordinate and the peak area (Y) as the vertical coordinate. The results are shown in Tables 6 to 7 and Figure 7-8 .

[0165] Table 6 Protocatechuic acid standard curve analysis results

[0166]

[0167] Figure 7 The standard curve of protocatechuic acid is shown in Figure 1. The results show that the concentration of protocatechuic acid ranges from 0.40677 to 0.81354 μg / ml, and the linear relationship is y=35.0807x-0.2876, R 2 =1.0000, showing a good linear relationship.

[0168] Table 7 p-Hydroxybenzoic acid standard curve analysis results

[0169]

[0170] Figure 8 This is the standard curve of p-hydroxybenzoic acid. The results show that the concentration of p-hydroxybenzoic acid ranges from 0.40650 to 40.65040 μg / ml, and the linear relationship is y=56.7956x-0.5472, R 2 =1.0000, showing a good linear relationship.

[0171] 1.4.5.4 Repeatability

[0172] Take 6 portions of the same test sample, accurately weigh 1.0 g of each, and prepare the test sample solution by the same operator according to the proposed method. Calculate the total amount of protocatechuic acid and p-hydroxybenzoic acid content of the 6 portions of the test sample. The results are shown in Table 8.

[0173] Table 8 Repeatability test results

[0174]

[0175] The results showed that the RSD value of the total amount of protocatechuic acid and p-hydroxybenzoic acid was 1.5%, and the method had good repeatability.

[0176] 1.4.5.5 Intermediate precision

[0177] On the basis of the experimental conditions proposed above, 6 portions of standard lotus root node decoction were accurately weighed to prepare the test solution, which was examined and calculated on Waters (Ⅰ) and Agilent 1260 (Ⅱ) high performance liquid chromatographs to determine the total contents of protocatechuic acid and p-hydroxybenzoic acid. The results are shown in Table 9.

[0178] Table 9 Results of investigations of different personnel and time

[0179]

[0180] The results showed that the RSD value of the total content of protocatechuic acid and p-hydroxybenzoic acid was 1.5%, and the intermediate precision of this method was good.

[0181] 1.4.5.6 Sample recovery rate

[0182] Take about 0.5g of the test sample with known content (protocatechuic acid content 0.161mg / g, p-hydroxybenzoic acid content 0.157mg / g), a total of 6 portions, accurately weigh, accurately add a certain amount of protocatechuic acid and p-hydroxybenzoic acid reference substances, prepare and measure the test solution according to the proposed method, calculate the recovery rate, and the results are shown in Tables 10 and 11. The calculation formula is as follows:

[0183]

[0184] Table 10 Protocatechuic acid sample recovery test results

[0185]

[0186] Table 11 p-Hydroxybenzoic acid sample recovery test results

[0187]

[0188] The results showed that the average recovery rate of protocatechuic acid was 103.6%, and the RSD value of the result was 1.0%; the average recovery rate of p-hydroxybenzoic acid was 102.8%, and the RSD value of the result was 0.7%; this method had good accuracy.

[0189] 1.4.5.7 Durability test

[0190] Based on the above proposed experimental conditions, the chromatographic columns were investigated as column 1 (InfinityLab Poroshell 120HILIC-Z 2.1*150mm, 2.7μm); column 2 (ZORBAX SB-C18 2.1*150mm, 1.8μm); and column 3 (InfinityLab Poroshell 120EC-C18 2.1*150mm, 1.9μm), and the total contents of protocatechuic acid and p-hydroxybenzoic acid were calculated. The results are shown in Table 12.

[0191] Table 12 Durability test results

[0192]

[0193] The results showed that the analytical chromatographic indicators of different chromatographic columns were good, and the RSD value of 6 measurement results was 0.7%. The chromatographic column of this method had good durability.

[0194] 1.4.5.8 Stability test

[0195] Take the same test solution and determine the peak areas of protocatechuic acid and p-hydroxybenzoic acid at 0, 2, 4, 8, 16 and 24 hours respectively. The results are shown in Table 13.

[0196] Table 13 Stability test results

[0197]

[0198] The results showed that under the experimental conditions, the RSD value of the total amount of protocatechuic acid and p-hydroxybenzoic acid was 0.5%, and the test solution had good stability within 24 hours.

[0199] 1.5 Sample content determination verification

[0200] 1.5.1 Verification of lotus root node standard decoction

[0201] The test solution was prepared and measured according to the proposed method, the peak area was recorded, and the total content of protocatechuic acid and p-hydroxybenzoic acid was calculated. The results are shown in Table 14.

[0202] Table 1432 Batch of Lotus Root Node Standard Decoction Content Determination Results

[0203]

[0204]

[0205] The total content of protocatechuic acid and p-hydroxybenzoic acid in 32 batches of standard decoction of lotus root nodes was measured in the range of 0.25-0.70 mg / g, with an average content of 0.39 mg / g and a SD of 0.119. The range of 70% to 130% of the average value was calculated to be 0.27-0.51 mg / g, the range of the content determination mean plus or minus 3 times SD was 0.03-0.75 mg / g, and the range of the content determination mean plus or minus 2 times SD was 0.15-0.63 mg / g.

[0206] In summary, the provisional limit for the total content of protocatechuic acid and p-hydroxybenzoic acid in the standard decoction of lotus root nodes is -2SD value of the mean of the standard decoction as the lower limit and +3SD value of the mean as the upper limit, that is, 0.15-0.75 mg / g.

[0207] The determination range of the total content of protocatechuic acid and p-hydroxybenzoic acid in the standard decoction of lotus root nodes is tentatively set as "the total content of protocatechuic acid and p-hydroxybenzoic acid per 1g of this product should be 0.15mg-0.75mg.

[0208] 1.5.2 Verification of the standard decoction of lotus root charcoal

[0209] Prepare the test solution and measure it according to the proposed method, record the peak area, and calculate the total content of protocatechuic acid and p-hydroxybenzoic acid. The results are shown in Table 15.

[0210] Table 15 is the content determination results of 32 batches of lotus root charcoal standard decoction

[0211]

[0212]

[0213] The total content of protocatechuic acid and p-hydroxybenzoic acid in 32 batches of standard decoction of lotus root charcoal was measured in the range of 0.25-0.71 mg / g, with an average content of 0.39 mg / g and SD of 0.114. The range of 70% to 130% of the average value was calculated to be 0.27-0.51 mg / g, the range of the content determination mean plus or minus 3 times SD was 0.05-0.73 mg / g, and the range of the content determination mean plus or minus 2 times SD was 0.16-0.62 mg / g.

[0214] In summary, the provisional limit for the total content of protocatechuic acid and p-hydroxybenzoic acid in the standard decoction of lotus node charcoal is -2SD value of the mean of the standard decoction as the lower limit and +3SD value of the mean as the upper limit, that is, 0.16-0.73 mg / g.

[0215] Example 2 HPLC content determination of lotus root node medicinal materials and decoction pieces:

[0216] 2.1 Proposed chromatographic conditions

[0217] Chromatographic conditions and system suitability test The chromatographic conditions and system suitability test used octadecylsilane bonded silica gel as filler (column length 150mm, inner diameter 2.1mm, particle size 2.7μm); methanol as mobile phase A, 0.5% phosphoric acid aqueous solution as mobile phase B; gradient elution according to the provisions in the table below; detection wavelength 260nm. Flow rate 0.3ml / min; column temperature 20℃; theoretical plate number calculated based on p-hydroxybenzoic acid peak should be no less than 3000.

[0218]

[0219]

[0220] Preparation of reference solution: Take appropriate amount of protocatechuic acid and p-hydroxybenzoic acid reference substances, weigh accurately, add 70% methanol to prepare a mixed solution containing 20 μg of protocatechuic acid and p-hydroxybenzoic acid per 1 ml.

[0221] Preparation of test solution: Take an appropriate amount of lotus root node medicinal material (passed through No. 3 sieve), take about 3.0 g, accurately weigh, put in a stoppered conical flask, accurately add 20 ml of 70% methanol, stopper, weigh, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate.

[0222] Determination method: Accurately pipette 3μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0223] 2.2 Selection of chromatographic conditions and investigation of system suitability

[0224] 2.2.1 Determination of detection wavelength

[0225] Based on the above experimental conditions, a diode array detector was used to perform full-band scanning of protocatechuic acid and p-hydroxybenzoic acid. Fig. 9 and Fig.10 . Fig. 9 This is the spectrum of protocatechuic acid; Fig.10 This is the spectrum of p-hydroxybenzoic acid.

[0226] According to the spectral analysis, the two test index peaks have strong absorption at 260nm, so the absorption wavelength of subsequent tests is set at 260nm.

[0227] 2.2.2 Flow rate investigation

[0228] Under the above proposed chromatographic conditions, the separation effect of protocatechuic acid and p-hydroxybenzoic acid chromatographic peaks at flow rates of 0.2 ml / min, 0.3 ml / min, and 0.4 ml / min was used as the evaluation index. The results are shown in Fig.11 . Fig.11 Chromatograms at different flow rates; the results show that when the flow rates are 0.2ml / min, 0.3ml / min and 0.4ml / min, the retention time and peak separation at 0.3ml / min are better, so the flow rate is set to 0.3ml / min.

[0229] 2.2.3 Column temperature investigation

[0230] Under the above proposed chromatographic conditions, the analysis of the test solution at column temperatures of 20℃, 25℃, and 30℃ was investigated, and the separation effect of the protocatechuic acid and p-hydroxybenzoic acid chromatographic peaks was used as the evaluation index. The results are shown in Fig.12 . Fig.12 Chromatograms at different column temperatures; the results show that when the column temperature is 20℃, the retention time and peak separation of the index peaks are better, so the experimental column temperature is specified to be 20℃.

[0231] 2.2.4 Sample injection volume investigation

[0232] Under the above proposed chromatographic conditions, 2μl, 3μl, and 5μl were injected respectively, and the separation effect of protocatechuic acid and p-hydroxybenzoic acid chromatographic peaks was used as the evaluation index. The results are shown in Fig.13 . Fig.13 Chromatograms of different injection volumes; the results show that when the injection volume is 3μl, the peak shape and peak separation are better, so the injection volume of this method is stipulated to be 3ul.

[0233] 2.3 Chromatographic conditions and system suitability test results

[0234] In summary, the chromatographic conditions and system suitability test results for the determination of the content of the standard decoction of lotus root nodes are as follows: octadecylsilane bonded silica gel is used as the filler (column length is 150 mm, inner diameter is 2.1 mm, particle size is 2.7 μm); methanol is used as mobile phase A, 0.5% phosphoric acid aqueous solution is used as mobile phase B; gradient elution is performed according to the provisions in the table below; the detection wavelength is 260 nm. The flow rate is 0.3 ml / min; the column temperature is 20 ° C; the theoretical plate number calculated according to the p-hydroxybenzoic acid peak should be no less than 3000.

[0235] 2.4 Preparation of test solution

[0236] 2.4.1 Investigation of extraction solvent

[0237] Take appropriate amount of lotus root node medicinal material (pass through No. 3 sieve), take about 3.0g, weigh accurately, put in a stoppered conical bottle, add water, ethanol, methanol, 30% methanol, 50% methanol, 70% methanol 20ml respectively, stopper, weigh, ultrasonic treatment (power 600W, frequency 40kHz) for 30 minutes, take out, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, take the filtrate, and get it. Take 3μl each and inject it into the chromatograph, analyze and calculate the total amount of protocatechuic acid and p-hydroxybenzoic acid content under different extraction solvents, the results are shown in Table 16 below.

[0238] Table 16 shows the analysis results of different extraction solvents

[0239]

[0240] The results showed that water, 50% methanol and 70% methanol had the best extraction effects. After comprehensive consideration, 70% methanol was selected as the extraction solvent for the standard decoction of lotus root nodes.

[0241] 2.4.2 Investigation of extraction methods

[0242] Take appropriate amount of lotus root node medicinal material (pass through No. 3 sieve), take about 3.0g, weigh accurately, put in a stoppered conical bottle, add 20ml of 70% methanol accurately, stopper tightly, weigh the weight, reflux and ultrasonic treatment (power 600W, frequency 40kHz) for 30 minutes respectively, take out, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, take the filtrate, and get it. Take 3μl of each and inject it into the chromatograph, analyze and calculate the total amount of protocatechuic acid and p-hydroxybenzoic acid content under different extraction solvents, the results are shown in Table 17 below.

[0243] Table 17 shows the analysis results of different extraction methods

[0244]

[0245] The results showed that there was little difference between the extraction efficiencies of ultrasonic and reflux extraction. After comprehensive consideration, ultrasonic extraction was selected as the extraction method.

[0246] 2.4.3 Extraction time investigation

[0247] Take appropriate amount of lotus root node medicinal material (pass through No. 3 sieve), take about 3.0g, accurately weigh, put in a stoppered conical bottle, accurately add 20ml of 70% methanol, plug tightly, weigh, ultrasonically treat (power 600W, frequency 40kHz) for 20 minutes, 30 minutes and 40 minutes respectively, take out, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, take the filtrate, and inject 3μl of each into the chromatograph, analyze and calculate the total amount of protocatechuic acid and p-hydroxybenzoic acid content under different extraction solvents, the results are shown in Table 18 below.

[0248] Table 18 Analysis results of different extraction times

[0249]

[0250] The results showed that the sample was completely extracted after 30 minutes of extraction, so the extraction time of the sample was determined to be 30 minutes.

[0251] 2.4.4 Sample weighing inspection

[0252] Take appropriate amount of lotus root node medicinal material (pass through No. 3 sieve), take 2.0g, 3.0g and 4.0g respectively, weigh accurately, put in a stoppered conical bottle, add 20ml of 70% methanol accurately, stopper tightly, weigh the weight, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, take out, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, take the filtrate, and inject 3μl of each into the chromatograph, analyze and calculate the total amount of protocatechuic acid and p-hydroxybenzoic acid content under different extraction solvents, the results are shown in Table 19.

[0253] Table 19 Analysis results of different sample weights

[0254]

[0255] The results showed that the total amount of protocatechuic acid and p-hydroxybenzoic acid did not differ much with different sample sampling amounts. Taking all factors into consideration, the sampling amount of the test sample was determined to be 3.0 g.

[0256] In summary, the preparation method of the test sample is determined as follows: take an appropriate amount of lotus root node medicinal material (passed through No. 3 sieve), take about 3.0 g, accurately weigh, put in a stoppered conical flask, accurately add 20 ml of 70% methanol, seal, weigh the weight, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, take out, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate.

[0257] 2.5 Methodological Review

[0258] 2.5.1 Specificity Investigation

[0259] Preparation of reference solution: Take appropriate amount of protocatechuic acid and p-hydroxybenzoic acid reference substances, weigh accurately, add 70% methanol to make a mixed solution containing 20 μg of protocatechuic acid and p-hydroxybenzoic acid per 1 ml.

[0260] Preparation of test solution: Take an appropriate amount of the product, about 1.0g, accurately weigh, place in a stoppered conical flask, accurately add 20ml of 70% methanol, stopper, weigh, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, take out, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate.

[0261] Preparation of negative control solution: According to the experimental conditions proposed above, prepare the negative control solution of the standard decoction lacking lotus root nodes.

[0262] Accurately pipette 3 μl of reference solution and test solution respectively, inject into liquid chromatograph, and measure. Fig.14 . Fig.14 Comparison chart of specificity of lotus root node medicinal materials; as can be seen from the above figure, the negative solution has no effect on the target peaks of protocatechuic acid and p-hydroxybenzoic acid, and this method has good specificity.

[0263] 2.5.2 Precision investigation

[0264] The reference solution was sampled continuously for 6 times, the peak areas of protocatechuic acid and p-hydroxybenzoic acid were recorded, and the RSD values ​​were calculated. The results are shown in Table 20.

[0265] Table 20 Precision inspection results

[0266]

[0267] The results showed that the peak area RSD value of protocatechuic acid and p-hydroxybenzoic acid in the precision study was 0.1%, and the injection precision of this method was good.

[0268] 2.5.3 Linear Relationship

[0269] Take 2.086 mg of protocatechuic acid (purity of 97.8%), put it in a 50 ml volumetric flask, dilute it to the mark with 70% methanol, shake well, filter, and obtain mother solution 1; take another 2.074 mg of p-hydroxybenzoic acid (purity of 98.0%), put it in a 50 ml volumetric flask, dilute it to the mark with 70% methanol, shake well, filter, and obtain mother solution 2. Prepare the following reference solutions of different concentrations respectively. Pipette protocatechuic acid and p-hydroxybenzoic acid reference substances of different concentrations and inject them into the liquid chromatograph. Analyze to obtain the peak area, and draw the response curve with protocatechuic acid or p-hydroxybenzoic acid (X) as the horizontal coordinate and the peak area (Y) as the vertical coordinate. The results are shown in Tables 21 to 22 and Figures 15-16 .

[0270] Table 21 Protocatechuic acid standard curve analysis results

[0271]

[0272] Fig.15 Protocatechuic acid standard curve; the results show that the concentration of protocatechuic acid ranges from 0.40677 to 0.81354 μg / ml, and the linear relationship is y=35.0807x-0.2876, R 2 =1.0000, showing a good linear relationship.

[0273] Table 22 p-Hydroxybenzoic acid standard curve analysis results

[0274]

[0275] Fig.16 Standard curve of p-hydroxybenzoic acid; the results show that the concentration range of p-hydroxybenzoic acid is 0.40650~40.65040μg / ml, and the linear relationship is y=56.7956x-0.5472, R 2 =1.0000, showing a good linear relationship.

[0276] 2.5.4 Repeatability

[0277] Take 6 portions of the same test sample, accurately weigh 3.0 g of each, and prepare the test sample solution by the same operator according to the proposed method. Calculate the total content of protocatechuic acid and p-hydroxybenzoic acid in the 6 portions of the test sample. The results are shown in Table 23.

[0278] Table 23 Repeatability test results

[0279]

[0280] The results showed that the RSD value of the total amount of protocatechuic acid and p-hydroxybenzoic acid was 1.9%, and the method had good repeatability.

[0281] 2.5.5 Intermediate precision

[0282] On the basis of the experimental conditions proposed above, 6 portions of standard lotus root node decoction were accurately weighed to prepare the test solution, which was examined and calculated on Waters (Ⅰ) and Agilent 1260 (Ⅱ) high performance liquid chromatographs to determine the total contents of protocatechuic acid and p-hydroxybenzoic acid. The results are shown in Table 24.

[0283] Table 24 Results of investigations of different personnel and time

[0284]

[0285] The results showed that the RSD value of the total content of protocatechuic acid and p-hydroxybenzoic acid was 1.6%, and the intermediate precision of this method was good.

[0286] 2.5.6 Sample recovery rate

[0287] Take about 0.5g of the test sample with known content (protocatechuic acid content 0.0017%, p-hydroxybenzoic acid content 0.0026%), a total of 6 portions, accurately weigh, accurately add a certain amount of protocatechuic acid and p-hydroxybenzoic acid reference substances, prepare and measure the test solution according to the proposed method, calculate the recovery rate, and the results are shown in Tables 25-26. The calculation formula is as follows:

[0288]

[0289] Table 25 Protocatechuic acid sample recovery test results

[0290]

[0291] Table 26 p-Hydroxybenzoic acid sample recovery test results

[0292]

[0293]

[0294] The results showed that the average recovery rate of protocatechuic acid was 101.1%, and the RSD value of the result was 1.0%; the average recovery rate of p-hydroxybenzoic acid was 104.5%, and the RSD value of the result was 0.3%; this method had good accuracy.

[0295] 2.5.7 Durability test

[0296] Based on the above proposed experimental conditions, the chromatographic columns were investigated as follows: column 1 (InfinityLab Poroshell 120 HILIC-Z 2.1*150 mm, 2.7 μm); column 2 (ZORBAX SB-C18 2.1*150 mm, 1.8 μm); column 3 (InfinityLab Poroshell 120 EC-C18 2.1*150 mm, 1.9 μm), and the total contents of protocatechuic acid and p-hydroxybenzoic acid were calculated. The results are shown in Table 27.

[0297] Table 27 Durability test results

[0298]

[0299] The results showed that the analytical chromatographic indicators of different chromatographic columns were good, and the RSD value of 6 measurement results was 2.4%. The chromatographic column of this method had good durability.

[0300] 2.5.8 Stability test

[0301] Take the same test sample solution and determine the peak areas of protocatechuic acid and p-hydroxybenzoic acid at 0, 2, 4, 8, 16 and 24 hours respectively. The results are shown in Table 28.

[0302] Table 28 Stability test results

[0303]

[0304] The results showed that under the experimental conditions, the RSD value of the total amount of protocatechuic acid and p-hydroxybenzoic acid was 1.5%, and the test solution had good stability within 24 hours.

[0305] 2.6 Sample content determination verification

[0306] 2.6.1 Verification of Lotus Root Node Medicinal Materials

[0307] Prepare the test solution and measure it according to the proposed method, record the peak area, and calculate the total content of protocatechuic acid and p-hydroxybenzoic acid. The results are shown in Table 29.

[0308] Table 29 shows the results of the determination of the medicinal content of 32 batches of lotus root nodes

[0309]

[0310]

[0311] The total content of protocatechuic acid and p-hydroxybenzoic acid in 32 batches of lotus root nodes was measured in the range of 0.0020%-0.0085, with an average content of 0.0049% and a SD of 0.0019. The range of 70%-130% of the average value was calculated to be 0.0034%-0.0064%, the range of the content determination mean plus or minus 3 times SD was -0.0008%-0.0106%, and the range of the content determination mean plus or minus 2 times SD was 0.0011%-0.0087%.

[0312] 2.6.2 Verification of lotus root node slices

[0313] Prepare the test solution and measure it according to the proposed method, record the peak area, and calculate the total content of protocatechuic acid and p-hydroxybenzoic acid. The results are shown in Table 30.

[0314] Table 30 is the content determination results of 32 batches of lotus root node slices

[0315]

[0316]

[0317] The total content of protocatechuic acid and p-hydroxybenzoic acid in 32 batches of lotus root slices was measured in the range of 0.0020%-0.0079, the average content was 0.0047%, and the SD was 0.0017. The calculated range of 70%-130% of the average value was 0.0033%-0.0061%, the range of the content determination mean plus or minus 3 times SD was -0.0004%-0.0098%, and the range of the content determination mean plus or minus 2 times SD was 0.0013%-0.0081%.

[0318] 2.6.3 Verification of charcoal slices of lotus root node

[0319] Prepare the test solution and measure it according to the proposed method, record the peak area, and calculate the total content of protocatechuic acid and p-hydroxybenzoic acid. The results are shown in Table 31.

[0320] Table 31 is the content determination results of 32 batches of lotus root charcoal slices

[0321]

[0322]

[0323] The total content of protocatechuic acid and p-hydroxybenzoic acid in 32 batches of lotus root charcoal slices was measured in the range of 0.0047%-0.0162%, the average content was 0.0095%, and the SD was 0.0027. The calculated range of 70%-130% of the average value was 0.0067%-0.0124%, the range of the content determination mean plus or minus 3 times SD was 0.0014%-0.0176%, and the range of the content determination mean plus or minus 2 times SD was 0.0041%-0.0149%.

[0324] Example 3 HPLC content determination of lotus root recipe granules

[0325] 3.1 Proposed chromatographic conditions

[0326] Chromatographic conditions and system suitability test The chromatographic conditions and system suitability test used octadecylsilane bonded silica gel as filler (column length 150mm, inner diameter 2.1mm, particle size 2.7μm); methanol as mobile phase A, 0.5% phosphoric acid aqueous solution as mobile phase B; gradient elution according to the provisions in the table below; detection wavelength 260nm. Flow rate 0.3ml / min; column temperature 20℃; theoretical plate number calculated based on p-hydroxybenzoic acid peak should be no less than 3000.

[0327]

[0328] Preparation of reference solution: Take appropriate amount of protocatechuic acid and p-hydroxybenzoic acid reference substances, weigh accurately, add 70% methanol to prepare a mixed solution containing 20 μg of protocatechuic acid and p-hydroxybenzoic acid per 1 ml.

[0329] Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take about 1.0 g, weigh it accurately, put it in a stoppered conical flask, add 20 ml of 70% methanol accurately, stopper it tightly, weigh it, treat it with ultrasound (power 600 W, frequency 40 kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the solution.

[0330] Determination method: Accurately pipette 3μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0331] 3.2 Selection of chromatographic conditions and investigation of system suitability

[0332] 3.2.1 Determination of detection wavelength

[0333] Based on the experimental conditions proposed above, a diode array detector was used to perform full-band scanning of protocatechuic acid and p-hydroxybenzoic acid, as shown in the figure. Fig.17 Protocatechuic acid spectrum; Fig.18 Spectrum of p-hydroxybenzoic acid.

[0334] According to the spectral analysis, the two test index peaks have strong absorption at 260nm, so the absorption wavelength of subsequent tests is set at 260nm.

[0335] 3.2.2 Flow rate investigation

[0336] Under the above proposed chromatographic conditions, the separation effect of protocatechuic acid and p-hydroxybenzoic acid chromatographic peaks at flow rates of 0.2 ml / min, 0.3 ml / min, and 0.4 ml / min was used as the evaluation index. The results are shown in Fig.19 . Fig.19 Chromatograms at different flow rates; the results show that when the flow rates are 0.2ml / min, 0.3ml / min and 0.4ml / min, the retention time and peak separation at 0.3ml / min are better, so the flow rate is set to 0.3ml / min.

[0337] 3.2.3 Column temperature investigation

[0338] Under the above proposed chromatographic conditions, the analysis of the test solution at column temperatures of 20℃, 25℃, and 30℃ was investigated, and the separation effect of the protocatechuic acid and p-hydroxybenzoic acid chromatographic peaks was used as the evaluation index. The results are shown in Fig. 20 . Fig. 20 Chromatograms at different column temperatures; the results show that when the column temperature is 20℃, the retention time and peak separation of the index peaks are better, so the experimental column temperature is specified to be 20℃.

[0339] 3.2.4 Sample volume investigation

[0340] Under the above proposed chromatographic conditions, 2μl, 3μl, and 5μl were injected respectively, and the separation effect of protocatechuic acid and p-hydroxybenzoic acid chromatographic peaks was used as the evaluation index. The results are shown in Fig.21 . Fig.21 Chromatograms of different injection volumes; the results show that when the injection volume is 3μl, the peak shape and peak separation are better, so the injection volume of this method is stipulated to be 3ul.

[0341] 3.3 Chromatographic conditions and system suitability test results

[0342] In summary, the chromatographic conditions and system suitability test results for the determination of the content of the standard decoction of lotus root nodes are as follows: octadecylsilane bonded silica gel is used as the filler (column length is 150 mm, inner diameter is 2.1 mm, particle size is 2.7 μm); methanol is used as mobile phase A, 0.5% phosphoric acid aqueous solution is used as mobile phase B; gradient elution is performed according to the conditions in 3.1; the detection wavelength is 260 nm. The flow rate is 0.3 ml / min; the column temperature is 20 ° C; the theoretical plate number calculated according to the p-hydroxybenzoic acid peak should be no less than 3000.

[0343] 3.4 Preparation of test solution

[0344] 3.4.1 Investigation of extraction solvent

[0345] Take an appropriate amount of lotus root node particles, grind them into powder, take about 1.0g, weigh accurately, put them in a stoppered conical bottle, add 20ml of water, ethanol, methanol, 30% methanol, 50% methanol, and 70% methanol, stopper them, weigh them, treat them with ultrasound (power 600W, frequency 40kHz) for 30 minutes, take them out, cool them, weigh them again, make up the lost weight with 70% methanol, shake them well, filter them, and take the filtrate. Take 3μl of each and inject them into the chromatograph to analyze and calculate the total amount of protocatechuic acid and p-hydroxybenzoic acid content under different extraction solvents. The results are shown in Table 32.

[0346] Table 32 Analysis results of different extraction solvents

[0347]

[0348] The results showed that 70% methanol had the best extraction effect, so 70% methanol was selected as the extraction solvent for the standard decoction of lotus root nodes.

[0349] 3.4.2 Investigation of extraction methods

[0350] Take an appropriate amount of lotus root node particles, grind them into fine powder, take about 1.0g, weigh accurately, put them in a stoppered conical bottle, add 20ml of 70% methanol accurately, stopper tightly, weigh the weight, reflux and ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes respectively, take them out, cool them, weigh them again, make up the lost weight with 70% methanol, shake them well, filter them, and take the filtrate to obtain the product. Take 3μl of each and inject them into the chromatograph to analyze and calculate the total amount of protocatechuic acid and p-hydroxybenzoic acid content under different extraction solvents. The results are shown in Table 33.

[0351] Table 33 Analysis results of different extraction methods

[0352]

[0353] The results showed that there was little difference between the extraction efficiencies of ultrasonic and reflux extraction. After comprehensive consideration, ultrasonic extraction was selected as the extraction method.

[0354] 3.4.3 Extraction time investigation

[0355] Take an appropriate amount of lotus root node particles, grind them into powder, take about 1.0g, weigh accurately, put them in a stoppered conical bottle, add 20ml of 70% methanol accurately, stopper tightly, weigh the weight, ultrasonically treat (power 600W, frequency 40kHz) for 20 minutes, 30 minutes and 40 minutes respectively, take them out, cool them, weigh them again, make up the lost weight with 70% methanol, shake them well, filter them, and take the filtrate to get the result. Inject 3μl of each into the chromatograph, analyze and calculate the total amount of protocatechuic acid and p-hydroxybenzoic acid content under different extraction solvents, and the results are shown in Table 34.

[0356] Table 34 Analysis results of different extraction times

[0357]

[0358] The results showed that the sample was completely extracted after 30 minutes of extraction, so the extraction time of the sample was determined to be 30 minutes.

[0359] 3.4.4 Sample weighing inspection

[0360] Take appropriate amount of lotus root node particles, grind them into fine powder, take 0.5g, 1.0g and 1.5g respectively, weigh accurately, put them in a stoppered conical bottle, add 20ml of 70% methanol accurately, stopper tightly, weigh the weight, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, take out, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, take the filtrate, and inject 3μl of each into the chromatograph, analyze and calculate the total amount of protocatechuic acid and p-hydroxybenzoic acid content under different extraction solvents, the results are shown in Table 35.

[0361] Table 35 Analysis results of different sample weights

[0362]

[0363] The results showed that the total amount of protocatechuic acid and p-hydroxybenzoic acid did not differ much with different sample sampling amounts. Taking all factors into consideration, the sampling amount of the test sample was determined to be 1.0 g.

[0364] In summary, the preparation method of the test sample is determined as follows: take an appropriate amount of lotus root node particles, take about 1.0g, accurately weigh, put in a stoppered conical flask, accurately add 20ml of 70% methanol, stopper, weigh the weight, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, take out, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate.

[0365] 3.5 Methodological Review

[0366] 3.5.1 Specificity inspection

[0367] Preparation of reference solution: Take appropriate amount of protocatechuic acid and p-hydroxybenzoic acid reference substances, weigh accurately, add 70% methanol to make a mixed solution containing 20 μg of protocatechuic acid and p-hydroxybenzoic acid per 1 ml.

[0368] Preparation of test solution: Take appropriate amount of lotus root node particles, grind them into powder, take about 1.0g, weigh accurately, put it in a stoppered conical flask, add 20ml of 70% methanol accurately, stopper it tightly, weigh it, treat it with ultrasound (power 600W, frequency 40kHz) for 30 minutes, take it out, 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.

[0369] Preparation of negative control solution: According to the experimental conditions proposed above, prepare the negative control solution of the standard decoction lacking lotus root nodes.

[0370] Accurately pipette 3 μl of reference solution and test solution respectively, inject into liquid chromatograph, and measure. Fig. 22 . Fig. 22 Comparison chart of specificity of standard decoction of lotus root nodes; as can be seen from the above figure, the negative solution has no effect on the target peaks of protocatechuic acid and p-hydroxybenzoic acid, and this method has good specificity.

[0371] 3.5.2 Precision investigation

[0372] The reference solution was sampled continuously for 6 times, the peak areas of protocatechuic acid and p-hydroxybenzoic acid were recorded, and the RSD values ​​were calculated. The results are shown in Table 36.

[0373] Table 36 Precision inspection results

[0374]

[0375] The results showed that the peak area RSD value of protocatechuic acid and p-hydroxybenzoic acid in the precision study was 0.1%, and the injection precision of this method was good.

[0376] 3.5.3 Linear Relationship

[0377] Take 2.086 mg of protocatechuic acid (purity of 97.8%), put it in a 50 ml volumetric flask, dilute it to the mark with 70% methanol, shake well, filter, and obtain mother solution 1; take another 2.074 mg of p-hydroxybenzoic acid (purity of 98.0%), put it in a 50 ml volumetric flask, dilute it to the mark with 70% methanol, shake well, filter, and obtain mother solution 2. Prepare the following reference solutions of different concentrations respectively. Pipette protocatechuic acid and p-hydroxybenzoic acid reference substances of different concentrations and inject them into the liquid chromatograph. Analyze to obtain the peak area, and draw the response curve with protocatechuic acid or p-hydroxybenzoic acid (X) as the horizontal coordinate and the peak area (Y) as the vertical coordinate. The results are shown in Tables 37 to 38 and Figures 23-24 .

[0378] Table 37 Protocatechuic acid standard curve analysis results

[0379]

[0380] Fig.23 Protocatechuic acid standard curve; the results show that the concentration of protocatechuic acid ranges from 0.40677 to 0.81354 μg / ml, and the linear relationship is y=35.0807x-0.2876, R 2 =1.0000, showing a good linear relationship.

[0381] Table 38 p-Hydroxybenzoic acid standard curve analysis results

[0382]

[0383] Fig.24 Standard curve of p-hydroxybenzoic acid; the results show that the concentration range of p-hydroxybenzoic acid is 0.40650~40.65040μg / ml, and the linear relationship is y=56.7956x-0.5472, R 2 =1.0000, showing a good linear relationship.

[0384] 3.5.4 Repeatability

[0385] Take 6 portions of the same test sample, accurately weigh 1.0 g of each, and have the same operator prepare the test sample solution according to the proposed method. Calculate the total amount of protocatechuic acid and p-hydroxybenzoic acid in the 6 portions of the test sample. The results are shown in Table 39.

[0386] Table 39 Repeatability test results

[0387]

[0388] The results showed that the RSD value of the total amount of protocatechuic acid and p-hydroxybenzoic acid was 1.5%, and the method had good repeatability.

[0389] 3.5.5 Intermediate precision

[0390] Based on the experimental conditions proposed above, 6 portions of lotus root node formula granules were accurately weighed to prepare the test solution, which was examined and calculated on Waters (Ⅰ) and Agilent 1260 (Ⅱ) high performance liquid chromatographs to determine the total contents of protocatechuic acid and p-hydroxybenzoic acid. The results are shown in Table 40.

[0391] Table 40 Results of investigations of different personnel and time

[0392]

[0393] The results showed that the RSD value of the total content of protocatechuic acid and p-hydroxybenzoic acid was 1.1%, and the intermediate precision of this method was good.

[0394] 3.5.6 Sample recovery rate

[0395] Take about 0.5 g of the test sample with known content (protocatechuic acid content 0.136 mg / g, p-hydroxybenzoic acid content 0.128 mg / g), a total of 6 portions, accurately weigh, accurately add a certain amount of protocatechuic acid and p-hydroxybenzoic acid reference substances, prepare and measure the test solution according to the proposed method, calculate the recovery rate, and the results are shown in Tables 41 to 42. The calculation formula is as follows:

[0396]

[0397] Table 41 Protocatechuic acid sample recovery test results

[0398]

[0399] Table 42 p-Hydroxybenzoic acid sample recovery test results

[0400]

[0401] The results showed that the average recovery rate of protocatechuic acid was 99.9%, and the RSD value of the result was 1.3%; the average recovery rate of p-hydroxybenzoic acid was 95.7%, and the RSD value of the result was 0.7%; this method has good accuracy.

[0402] 3.5.7 Durability test

[0403] Based on the above proposed experimental conditions, the chromatographic column 1 (InfinityLab Poroshell120HILIC-Z 2.1*150mm, 2.7μm); chromatographic column 2 (ZORBAX SB-C182.1*150mm, 1.8μm); chromatographic column 3 (InfinityLab Poroshell 120EC-C182.1*150mm, 1.9μm) were investigated respectively, and the total content of protocatechuic acid and p-hydroxybenzoic acid was calculated. The results are shown in Table 43.

[0404] Table 43 Durability test results

[0405]

[0406]

[0407] The results showed that the analytical chromatographic indicators of different chromatographic columns were good, and the RSD value of 6 measurement results was 1.0%. The chromatographic column of this method had good durability.

[0408] 3.5.8 Stability test

[0409] Take the same test sample solution and determine the peak areas of protocatechuic acid and p-hydroxybenzoic acid at 0, 2, 4, 8, 16 and 24 hours respectively. The results are shown in Table 44.

[0410] Table 44 Stability test results

[0411]

[0412] The results showed that under the experimental conditions, the RSD value of the total amount of protocatechuic acid and p-hydroxybenzoic acid was 0.5%, and the test solution had good stability within 24 hours.

[0413] 3.6 Verification of content determination of lotus root node samples

[0414] Prepare the test solution and measure it according to the proposed method, record the peak area, and calculate the total content of protocatechuic acid and p-hydroxybenzoic acid. The results are shown in Table 45.

[0415] Table 45 Results of content of three batches of lotus root recipe particles

[0416]

[0417] It can be seen from the above table that the average value of the three batches is 0.28 mg / g, and the range of the average value ± 30% is 0.20-0.36 mg / g.

[0418] 3.7 Verification of content determination of lotus root node charcoal samples

[0419] Prepare the test solution and measure it according to the proposed method, record the peak area, and calculate the total content of protocatechuic acid and p-hydroxybenzoic acid. The results are shown in Table 46.

[0420] Table 46 Results of the particle content of three batches of lotus root charcoal formula

[0421]

[0422] It can be seen from the above table that the average value of the three batches is 0.28 mg / g, and the range of the average value ± 30% is 0.19-0.35 mg / g.

[0423] Example 4 Comparison of transfer rates between lotus root nodes and lotus root node charcoal by HPLC content determination method

[0424] The method of Example 1 was used to determine the content of lotus root nodes and lotus root node related processed products, and the total content transfer rate of protocatechuic acid and p-hydroxybenzoic acid was determined.

[0425] Table 47

[0426]

[0427] From the content of lotus root nodes from medicinal materials to lotus root node decoction pieces and lotus root node charcoal decoction pieces, it can be seen that the contents of protocatechuic acid and p-hydroxybenzoic acid in lotus root node charcoal increase significantly during the processing process, while they decrease in the standard decoction; the measured values ​​of the transfer rate from lotus root node decoction pieces to standard decoction range from 32.7% to 93.4%; the measured values ​​of the transfer rate from lotus root node charcoal decoction pieces to standard decoction range from 19.1% to 48.5%, with significant differences. Therefore, this content measurement method can be used as a distinguishing point between lotus root nodes and lotus root node charcoal.

[0428] Comparative Example 1

[0429] The pretreatment method of Example 1 of the present invention was used, except that the detection method was different. The chromatographic column was a Phenomenex Luna C18 column (150 mm × 2.0 mm), the mobile phase A was acetonitrile, the mobile phase B was 0.4% glacial acetic acid, the flow rate was 0.2 mL / min, the injection volume was 3 μL, and the wavelength of the diode array detector was 280 nm. Linear gradient elution: 0-40 min, 10%-25% mobile phase A; 40-45 min, 25-35% mobile phase A; 45-70 min, 35%-50% mobile phase A. The results are shown in FIG. Fig.25 As stated,

[0430] The lotus root node particle sample 1, lotus root node particle sample 2 and the reference substance were respectively measured using the above detection method.

[0431] according to Fig.25 It can be seen that Fig.25 The chromatogram is shown in Comparative Example 1. Although the method in Comparative Example 1 can produce two components, it cannot identify and separate the two index components in the lotus root node.

[0432] Comparative Example 2

[0433] The pretreatment method of implementation 1 of the present invention is adopted, except that the detection method is different, chromatographic column: YWG-C18 (200×4.6mm10μm). Mobile phase: (1) methanol: water = 8:92 (V / V) pH = 3; (2) methanol: water = 22:78 (V / V) pH = 3; detection wavelength: 280nm; flow rate: 1mL / min column temperature: room temperature, injection volume is 10μL.

[0434] The lotus root node particle samples and the reference substance were respectively measured using the (1) mobile phase ratio and (2) mobile phase ratio detection methods.

[0435] The results are as follows Fig.26 As stated, Fig.26 Be the chromatogram of comparative example 2; According to Fig.26 It can be seen that Fig.26 (1) mobile phase ratio and Fig.26 The following (2) mobile phase ratios cannot identify and separate the two index components in the lotus node patent.

[0436] Comparative Example 3

[0437] The pretreatment method of Example 1 of the present invention was adopted, except that the detection method was different. The chromatographic column was filled with octadecylsilane bonded silica gel (Eclipse Plus C18, 4.6×150 mm, 3.5 μm); the detection wavelength was 270 nm; the mobile phase was 0.2% phosphoric acid solution as mobile phase A and methanol as mobile phase B; the gradient elution program was 0-5 min, 10%-15% B; 5-10 min, 15%-20% B; 10-30 min, 20%-40% B; 30-40 min, 40%-60% B; 40-50 min, 60%-70% B; 50-60 min, 70%-80% B. The column temperature was 30° C., the injection volume was 10 μL, and the flow rate was 1.0 mL / min.

[0438] The lotus root node particle samples and the reference substance were respectively measured using the above detection method.

[0439] The results are as follows Fig. 27 As stated, Fig. 27 This is the chromatogram of Comparative Example 3. The method of Comparative Example 3 can identify one component, but cannot identify and separate both components in the lotus root node of the present invention.

[0440] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for constructing HPLC content determination of lotus root nodes and lotus root node related processed products, comprising: A) dissolving the raw material of the test sample with a solvent, extracting, and obtaining a test solution; the raw material of the test sample includes lotus root nodes or lotus root node-related processed products; B) using high performance liquid chromatography to measure the test solution to obtain an HPLC spectrum of the lotus root node or a lotus root node-related processed product; 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 a methanol solution, the mobile phase B is a 0.5% phosphoric acid aqueous solution, and the elution is performed in a gradient manner; The gradient elution is specifically: 0-1min, phase A: 2%, phase B: 98%; 1-4 min, phase A: 2%→8%, phase B: 98%→92%; 4-6 min, phase A: 8% → 0%, phase B: 92% → 100%; 6-16 min, phase A: 0%, phase B: 100%; 16-30min, phase A: 10%, phase B: 90%.

2. The method according to claim 1, characterized in that: The invention also includes preparing a reference solution: taking a protocatechuic acid reference substance and a p-hydroxybenzoic acid reference substance respectively, dissolving them in 70% methanol to obtain a reference solution; The reference solution is measured by high performance liquid chromatography to obtain a chromatogram of the reference; and the components of the lotus root node or lotus root node related processed products are qualitatively identified according to the chromatogram of the reference.

3. The method according to claim 2, characterized in that The concentrations of the reference solutions are specifically as follows: the protocatechuic acid reference substance is 0.40677-0.81354 μg / ml, and the p-hydroxybenzoic acid reference substance is 0.40650-40.65040 μg / ml.

4. The method according to claim 1, characterized in that: The chromatographic column is C 18 150×2.1mm 1.8~2.7μm; column temperature is 20℃~30℃; the theoretical plate number calculated based on p-hydroxybenzoic acid should be no less than 3000.

5. The method according to claim 4, characterized in that The flow rate of the mobile phase is 0.2-0.4 mL / min; the injection volume is 2-5 μL.

6. The method according to claim 4, characterized in that The detection wavelength is 260 nm.

7. The method according to claim 5, characterized in that The column temperature was 20° C.; the mobile phase flow rate was 0.3 mL / min; and the injection volume was 3 μL.

8. The method according to claim 1, characterized in that In step A), the solvent is 0% to 100% methanol; the extraction is ultrasonic extraction; the ultrasonic power is 600W, the frequency is 40kHz; and the ultrasonic time is 20 to 40 minutes.

9. The method according to claim 8, characterized in that The ratio of the mass g of the test material to the volume mL of the solvent is 1 to 4:20; The raw materials of the test samples are lotus root node medicinal materials, lotus root node decoction pieces, lotus root node standard decoction, lotus root node formula granules, lotus root node charcoal decoction pieces, lotus root node charcoal standard decoction or lotus root node charcoal formula granules.

10. A method for identifying a processed lotus root node and a carbonized lotus root node, characterized in that: The method described in any one of claims 1 to 9 is used for detection, and the transfer rate of the total content of protocatechuic acid and p-hydroxybenzoic acid is analyzed: The actual measured value range of the transfer rate from lotus root node medicinal materials to lotus root node decoction pieces is 93.77% to 97.25%; the actual measured value range of the transfer rate from lotus root node medicinal materials to lotus root node charcoal decoction pieces is 97.21% to 244.90%; The measured values ​​of the transfer rate from lotus root node slices to lotus root node standard decoction ranged from 32.7% to 93.4%; the measured values ​​of the transfer rate from lotus root node charcoal slices to lotus root node charcoal standard decoction ranged from 19.1% to 48.5%. The total content range of the same amount of lotus root node medicinal materials to lotus root node formula granules is 0.16mg / g~1.02mg / g; the actual measured total content range of the same amount of lotus root node medicinal materials to lotus root node charcoal formula granules is 0.11mg / g~0.76mg / g.

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