A construction method for determining the HPLC content of lotus nodes and lotus node related processed products

Abstract

This invention provides a method for determining the content of lotus root nodes and related processed products by HPLC, comprising: A) dissolving and extracting the test sample raw material in a solvent to obtain a test solution; the test sample raw material includes lotus root nodes or related processed products; B) determining the test solution by high-performance liquid chromatography to obtain an HPLC chromatogram of the lotus root nodes or related processed products; the chromatographic conditions of the high-performance liquid chromatography are: a C18 column; mobile phase A is a methanol solution, mobile phase B is a 0.5% phosphoric acid aqueous solution, and gradient elution is performed; the gradient elution is specifically as follows: 0-1 min, phase A: 2%, phase B: 98%; 1-4 min, phase A... 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%; 16–30 min, Phase A: 10%, Phase B: 90%. This invention employs high-performance liquid chromatography (HPLC) with a methanol-0.5% phosphoric acid solution as the mobile phase for gradient elution. Using protocatechuic acid and p-hydroxybenzoic acid as reference standards, an HPLC method for determining the content of lotus root nodes and related processed products was established, providing more scientific technical means for controlling the medicinal quality of lotus root nodes and related processed products.

Description

Technical Field

[0001] This invention relates to the field of analytical testing technology, and in particular to a method for constructing an HPLC method for determining the content of lotus root nodes and related processed products. Background Technology

[0002] Lotus root nodes are the dried rhizome nodes of Nelumbo nucifera Gaertn., a plant in the Nymphaeaceae family. They possess astringent, hemostatic, and blood-stasis-removing properties. They are used for hematemesis, hemoptysis, hemorrhage, hematuria, and metrorrhagia. Literature review reveals that phenolic acids have diverse structures, are widely found in plants, and possess various biological activities, particularly excelling in anti-inflammatory and hemostatic effects. For example, herbs such as Sanguisorba officinalis, Loropetalum chinense, and Rhizoma Cyathulae rubra all exhibit strong hemostatic effects, and their active ingredients are mostly phenolic acids. Experimental studies have found that lotus root nodes contain various phenolic acids, similar to other hemostatic drugs, which resonates with the astringent and hemostatic effects of lotus root nodes.

[0003] Lotus root charcoal-processed products are the dried rhizome nodes of the lotus plant (Nelumbo nucifera), processed according to the main quality indicators of standard decoctions, and made into granules or slices. Distinguishing them from other processed lotus root charcoal products is a major challenge. Specifically, lotus root slices are usually made by removing impurities, washing, drying, and slicing the lotus root nodes. Charcoal-processed lotus root charcoal slices, on the other hand, are made by taking clean lotus root nodes, charring them according to the charcoal-making method (such as the General Processing Rules 0213 of the Chinese Pharmacopoeia) until the surface is dark brown or charred black, and the interior is yellowish-brown or brownish-brown, before slicing. Standard lotus root decoctions are freeze-dried powders made from lotus root charcoal through a fixed preparation process.

[0004] Therefore, the present invention aims to construct a method for determining the content of lotus root nodes and related processed products by HPLC, thereby distinguishing processed products of lotus root nodes from carbonized processed products of lotus root nodes, and thus providing guidance for the quality control of processed products related to lotus root nodes. 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 determining the content of lotus root nodes and related processed products by HPLC. The method of the present invention is accurate and reliable for the detection of protocatechuic acid reference standard and p-hydroxybenzoic acid reference standard, and can also distinguish between processed lotus root node products and carbonized lotus root node products.

[0006] This invention provides a method for constructing an HPLC method for determining the content of lotus root nodes and related processed products, comprising:

[0007] A) The raw material of the test sample is dissolved and extracted with a solvent to obtain the test solution; the raw material of the test sample includes lotus root nodes or related processed products of lotus root nodes;

[0008] B) The test solution was analyzed by high performance liquid chromatography to obtain the HPLC chromatogram of lotus root nodes or related processed products;

[0009] The chromatographic conditions for the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; mobile phase A is a methanol solution, mobile phase B is a 0.5% phosphoric acid aqueous solution, and gradient elution is used.

[0010] The gradient elution specifically refers to:

[0011] 0–1 min, 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] 16–30 min, Phase A: 10%, Phase B: 90%.

[0016] The chromatographic conditions for the high-performance liquid chromatography (HPLC) are as follows: the chromatographic column is a C18 column; preferably a C18 column. 18 A 150×2.1mm chromatographic column with a diameter of 1.8~2.7μm.

[0017] Including but not limited to InfinityLab Poroshell 120HILIC-Z 2.1*150mm, 2.7μm, ZORBAXSB-C18 2.1*150mm, 1.8μm, and InfinityLab Poroshell 120EC-C18 2.1*150mm, 1.9μm.

[0018] In this invention, mobile phase A is a methanol solution, and mobile phase B is a 0.5% phosphoric acid aqueous solution, with gradient elution.

[0019] The gradient elution specifically refers to:

[0020] 0–1 min, 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] 16–30 min, Phase A: 10%, Phase B: 90%.

[0025] The processed products related to lotus root nodes described in this invention include processed lotus root node products and carbonized lotus root node products; wherein the processed lotus root node products include lotus root node slices, standard lotus root node decoctions, and lotus root node formula granules. Carbonized lotus root node products include charred lotus root node slices, charred lotus root node standard decoctions, or charred lotus root node formula granules.

[0026] This invention provides a method for determining the content of lotus root nodes and related processed products by HPLC. First, the raw material of the test sample is dissolved and extracted with a solvent to obtain the test solution.

[0027] Specifically, the sample raw material is dissolved in a solvent, extracted, cooled, shaken, and filtered to obtain the final product. This invention does not limit the specific methods and operations for cooling, shaking, and filtering described above; those familiar with these techniques are acceptable.

[0028] The extraction method described in this invention is either reflux extraction or ultrasonic extraction; preferably, ultrasonic treatment is used. The ultrasonic power is 600W, the frequency is 40kHz, and the ultrasonic time is 20–40 min; preferably 30 min.

[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 used in this invention is 0% methanol to 100% methanol; specifically, it can be water, 30% methanol, 50% methanol, 70% methanol, or methanol; preferably 70% methanol.

[0031] The inventors have discovered that extraction with 70% methanol yields the best results.

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

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

[0034] For the test solution of the medicinal slices, accurately weigh 3.0 g of the powder of this product, place it in a stoppered conical flask, accurately add 50 ml of 70% methanol, weigh it, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the weight loss with 70% methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0035] For the standard decoction test solution, take an appropriate amount of this product, about 1g, accurately weigh it, place it in a stoppered conical flask, accurately add 20ml of 70% methanol, seal tightly, weigh it, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, weigh it again, replenish the lost weight with 70% methanol, shake well, filter, and collect the filtrate to obtain the test solution.

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

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

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

[0039] The reference solution was analyzed by high performance liquid chromatography to obtain a chromatogram of the reference; and the components of lotus root nodes or related processed products were qualitatively identified based on the chromatogram of the reference.

[0040] The preferred concentration of the reference solution in this invention is as follows: the concentration of the reference solution is 0.40677-0.81354 μg / ml for protocatechuic acid reference standard and 0.40650-40.65040 μg / ml for p-hydroxybenzoic acid reference standard.

[0041] Specifically, the concentration range of protocatechuic acid was 0.40677–0.81354 μg / ml, with a linear relationship of y = 35.0807x - 0.2876, R0 2 =1.0000, showing a good linear relationship.

[0042] The concentration range of p-hydroxybenzoic acid was 0.40650–40.65040 μg / ml, and the linear relationship was y = 56.7956x - 0.5472, R0 2 =1.0000, showing a good linear relationship.

[0043] The test solution was analyzed by high performance liquid chromatography to obtain the HPLC chromatogram of lotus root nodes or related processed products.

[0044] The chromatographic conditions for the high-performance liquid chromatography (HPLC) method are as follows: a C18 column with dimensions of 150 × 2.1 mm × 2.7 μm; a column temperature of 20–30 °C, preferably 20 °C. Under the above column temperatures, the chromatographic peaks of this invention are symmetrical, exhibiting good resolution and complete elution.

[0045] In this invention, mobile phase A is a methanol solution and mobile phase B is a 0.5% phosphoric acid aqueous solution, with gradient elution.

[0046] Specifically, the gradient elution is as follows:

[0047] 0–1 min, 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] 16–30 min, Phase A: 10%, Phase B: 90%.

[0052] The theoretical plate number, calculated based on p-hydroxybenzoic acid, should be no less than 3000.

[0053] The present invention exhibits good baseline separation, good peak separation, and stable baseline under the above elution gradient.

[0054] The flow rate of the mobile phase described in this invention is 0.2 to 0.4 mL / min; more preferably 0.3 mL / min.

[0055] The present invention found that the chromatographic peaks were well separated and the resolution was moderate at the above flow rates, which is the optimal solution.

[0056] The injection volume is 2–5 μL; preferably 3 μL.

[0057] The detection wavelength is 260 nm. The inventors have discovered that at 260 nm, the chromatographic peak information content is greater, the chromatographic baseline is more stable, and the peak areas are larger.

[0058] A method for distinguishing between processed lotus root segments and carbonized lotus root segments, employing the method described above, and analyzing the transfer rate of total content of protocatechuic acid and p-hydroxybenzoic acid:

[0059] The measured transfer rate from lotus root nodes to processed lotus root slices ranged from 93.77% to 97.25%; the measured transfer rate from lotus root nodes to charred lotus root slices ranged from 97.21% to 244.90%.

[0060] The measured transfer rate from processed lotus root slices to standard lotus root decoction ranged from 32.7% to 93.4%; the measured transfer rate from charred lotus root slices to standard charred lotus root decoction ranged from 19.1% to 48.5%.

[0061] The total content of the same amount of lotus root medicinal material converted to lotus root formula granules ranges from 0.16 mg / g to 1.02 mg / g; the total content of the same amount of lotus root medicinal material converted to lotus root charcoal formula granules ranges from 0.11 mg / g to 0.76 mg / g.

[0062] (1) This invention establishes a method for distinguishing between processed and carbonized lotus root nodes. Using the total amounts of protocatechuic acid and p-hydroxybenzoic acid reference standards as indicators, it allows for overall and macroscopic control of the intrinsic quality of lotus root nodes, medicinal slices, standard decoctions, their formulations, and related preparations, ensuring the efficacy of the drugs and providing more standardized quality control for the medicinal materials and related preparations. Simultaneously, it provides a scientific basis for distinguishing between processed and carbonized lotus root nodes. The difference in the transfer rate from medicinal materials to medicinal slices, standard decoctions, and their formulations in HPLC analysis can serve as a distinguishing point between lotus root nodes and carbonized lotus root nodes.

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

[0064] This invention provides a method for determining the content of lotus root nodes and related processed products by HPLC, comprising: A) dissolving and extracting the test sample raw material in a solvent to obtain a test solution; the test sample raw material includes lotus root nodes or related processed products; B) determining the test solution by high-performance liquid chromatography to obtain an HPLC chromatogram of the lotus root nodes or related processed products; the HPLC chromatographic conditions are as follows: the chromatographic column is a C18 column; mobile phase A is a methanol solution, mobile phase B is a 0.5% phosphoric acid aqueous solution, and gradient elution is performed; the gradient elution is specifically as follows: 0-1 min, phase A: 2%, phase B: 98%; 1-4 min... n, 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%; 16–30 min, Phase A: 10%, Phase B: 90%. This invention employs high-performance liquid chromatography (HPLC) with methanol-0.5% phosphoric acid solution as the mobile phase for gradient elution. Using protocatechuic acid and p-hydroxybenzoic acid as reference standards, an HPLC method for determining the content of lotus root nodes and related processed products was established, providing more scientific technical means for controlling the medicinal quality of lotus root nodes and related processed products. Attached Figure Description

[0065] Figure 1 The spectrum of protocatechuic acid;

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

[0067] Figure 3 Chromatograms for different flow rates;

[0068] Figure 4 Chromatograms at different column temperatures;

[0069] Figure 5 Chromatograms of different injection volumes;

[0070] Figure 6 Comparison chart of the specificity of lotus root decoctions;

[0071] Figure 7 The standard curve of protocatechuic acid;

[0072] Figure 8 This is a standard curve for p-hydroxybenzoic acid.

[0073] Figure 9 The spectrum of protocatechuic acid;

[0074] Figure 10 This is the spectrum of p-hydroxybenzoic acid;

[0075] Figure 11 Chromatograms at different flow rates;

[0076] Figure 12 Chromatograms at different column temperatures;

[0077] Figure 13 Chromatograms of different injection volumes;

[0078] Figure 14 Comparison chart of the specific medicinal properties of lotus root nodes;

[0079] Figure 15 Standard curve of protocatechuic acid;

[0080] Figure 16 p-Hydroxybenzoic acid standard curve;

[0081] Figure 17 Spectrum of protocatechuic acid;

[0082] Figure 18 p-Hydroxybenzoic acid spectrum;

[0083] Figure 19 Chromatograms at different flow rates;

[0084] Figure 20 Chromatograms at different column temperatures;

[0085] Figure 21 Chromatograms of different injection volumes;

[0086] Figure 22Comparison chart of the specificity of lotus root decoctions;

[0087] Figure 23 Standard curve of protocatechuic acid;

[0088] Figure 24 p-Hydroxybenzoic acid standard curve;

[0089] Figure 25 The chromatogram is for Comparative Example 1;

[0090] Figure 26 The chromatogram for Comparative Example 2 is shown below.

[0091] Figure 27 This is the chromatogram of Comparative Example 3. Detailed Implementation

[0092] This invention provides a method for the HPLC content determination of lotus root medicinal materials, processed slices, standard decoctions, and their formulated granules. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and fall within the scope of protection of this invention. The method and application of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the method and application described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0093] To further illustrate the present invention, the following describes in detail, with reference to embodiments, a method for determining the HPLC content of lotus root nodes and related processed products provided by the present invention.

[0094] Experimental instruments and materials

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

[0096] Electronic balances: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments Ltd.);

[0097] Ultrapure water system: Cellular type 1810A (Shanghai Moler Scientific Instruments Co., Ltd.);

[0098] Ultrasonic cleaner: KQ600DB model (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 all other reagents were of analytical grade.

[0101] Protocatechuic acid (China National Institutes for Food and Drug Control, batch number: 110809-202207, purity: 97.5%)

[0102] p-Hydroxybenzoic acid (Shanghai Shidander Standard Technical 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 herbs (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 charred lotus root slices (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-2416, OJT-YP-2417, OJT-YP-2418, OJT-YP-2419, OJT-YP-2410, OJT-YP-2411, OJT-YP-2412, OJT-YP-2413, OJT-YP-2414, OJT-YP-2415, OJT-YP-2418, OJT-YP-2419 ... 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 granule formula (OJ-KL-2401, OJ-KL-2402, OJ-KL-2403);

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

[0110] Example 1: Drafting Instructions for HPLC Content Determination of Lotus Root Node Standard Decoction

[0111] 1.1 Proposed chromatographic conditions

[0112] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase (column length 150 mm, inner diameter 2.1 mm, particle size 2.7 μm); methanol was used as mobile phase A, and 0.5% phosphoric acid aqueous solution was used as mobile phase B; gradient elution was performed according to the specifications in the table below; the detection wavelength was 260 nm. The flow rate was 0.3 ml / min; the column temperature was 20 °C; the theoretical plate number, calculated based on the p-hydroxybenzoic acid peak, should not be less than 3000.

[0113]

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

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

[0116] The assay involves precisely pipetting 3 μl of both the reference solution and the test solution into a liquid chromatograph and measuring the results.

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

[0118] 1.2.1 Determination of detection wavelength

[0119] Based on the above-specified experimental conditions, a full-band scan of protocatechuic acid and p-hydroxybenzoic acid was performed using a diode array detector, see [link to results]. Figure 1 and Figure 2 . Figure 1 The spectrum of protocatechuic acid; Figure 2 This is the spectrum of p-hydroxybenzoic acid.

[0120] According to the spectral analysis, both of the measured index peaks have large absorption at 260 nm, so the absorption wavelength for future experiments will be set at 260 nm.

[0121] 1.2.2 Flow velocity investigation

[0122] Under the proposed chromatographic conditions, the separation efficiency of protocatechuic acid and p-hydroxybenzoic acid peaks at flow rates of 0.2 ml / min, 0.3 ml / min, and 0.4 ml / min was investigated as an evaluation index. The results are shown in [Figure number missing]. Figure 3 . Figure 3 Chromatograms at different flow rates are shown. The results indicate that at flow rates of 0.2 ml / min, 0.3 ml / min, and 0.4 ml / min, the retention time and peak resolution are best at 0.3 ml / min, therefore the flow rate was set at 0.3 ml / min.

[0123] 1.2.3 Column Temperature Investigation

[0124] Under the proposed chromatographic conditions, the analysis of the test solution at column temperatures of 20℃, 25℃, and 30℃ was investigated. The separation efficiency of the protocatechuic acid and p-hydroxybenzoic acid peaks was used as the evaluation index. The results are shown in [Figure number missing]. Figure 4 . Figure 4 Chromatograms at different column temperatures are shown. The results indicate that the retention time and resolution of the index peaks are better at a column temperature of 20℃, therefore the experimental column temperature is specified as 20℃.

[0125] 1.2.4 Injection Volume Investigation

[0126] Under the proposed chromatographic conditions, 2 μl, 3 μl, and 5 μl of sample were injected, respectively. The separation efficiency of the protocatechuic acid and p-hydroxybenzoic acid peaks was used as the evaluation index. The results are shown in [Figure number missing]. Figure 5 . Figure 5 Chromatograms with different injection volumes; the results show that the peak shape and peak resolution are better when the injection volume is 3 μl, therefore, the injection volume for this method is specified as 3 μl.

[0127] 1.3 Results of chromatographic conditions and system suitability tests

[0128] In summary, the chromatographic conditions and system suitability test results for the determination of lotus root standard decoction content are as follows: Octadecylsilane-bonded silica gel was used as the packing material (column length 150 mm, inner diameter 2.1 mm, particle size 2.7 μm); methanol was used as mobile phase A, and 0.5% phosphoric acid aqueous solution was used as mobile phase B; gradient elution was performed according to the specifications in the table below; the detection wavelength was 260 nm; the flow rate was 0.3 ml / min; the column temperature was 20℃; and the theoretical plate number, calculated based on the p-hydroxybenzoic acid peak, should not be less than 3000.

[0129] 1.4 Investigation on the preparation of the test solution

[0130] 1.4.1 Investigation of Extraction Solvents

[0131] Take an appropriate amount of standard lotus root decoction, approximately 1.0 g, accurately weigh it, and place it in a stoppered conical flask. Accurately add 20 ml each of water, ethanol, methanol, 30% methanol, 50% methanol, and 70% methanol, respectively. Seal the flask tightly, weigh it, and sonicate it (600 W, 40 kHz) for 30 minutes. Remove it, cool it, weigh it again, and replenish the lost weight with 70% methanol. Shake well, filter it, and collect the filtrate. Inject 3 μl of each solution into a chromatograph to analyze and calculate the total content of protocatechuic acid and p-hydroxybenzoic acid under different extraction solvents. The results are shown in Table 1.

[0132] Table 1. Analytical 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 lotus root decoction.

[0136] 1.4.2 Examination of Extraction Methods

[0137] Take an appropriate amount of standard lotus root decoction, approximately 1.0 g, accurately weigh it, place it in a stoppered conical flask, accurately add 20 ml of 70% methanol, seal tightly, weigh, and reflux and sonicate (600 W power, 40 kHz frequency) for 30 minutes respectively. Remove, cool, weigh again, and replenish the lost weight with 70% methanol. Shake well, filter, and collect the filtrate. Inject 3 μl of each into the chromatograph to analyze and calculate the total content of protocatechuic acid and p-hydroxybenzoic acid 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 in efficiency between ultrasonic and reflux extraction. Therefore, ultrasonic extraction was chosen as the extraction method after comprehensive consideration.

[0141] 1.4.3 Examination of extraction time

[0142] Take an appropriate amount of standard lotus root decoction, approximately 1.0 g, accurately weigh it, place it in a stoppered conical flask, accurately add 20 ml of 70% methanol, seal tightly, weigh, and sonicate (600 W, 40 kHz) for 20, 30, and 40 minutes respectively. Remove, cool, weigh again, and replenish the lost weight with 70% methanol. Shake well, filter, and collect the filtrate. Inject 3 μl of each into a chromatograph to analyze and calculate the total content of protocatechuic acid and p-hydroxybenzoic acid 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, therefore the extraction time for the sample was determined to be 30 minutes.

[0146] 1.4.4 Sample Weight Examination

[0147] Take appropriate amounts of standard lotus root decoction, weighing 0.5g, 1.0g, and 1.5g respectively, and place them in stoppered conical flasks. Accurately add 20ml of 70% methanol, seal tightly, and weigh. Sonicate (600W, 40kHz) for 30 minutes, remove, cool, weigh again, and replenish the lost weight with 70% methanol. Shake well, filter, and collect the filtrate. Inject 3μl of each into a chromatograph to analyze and calculate the total content of protocatechuic acid and p-hydroxybenzoic acid under different extraction solvents. The results are shown in Table 4.

[0148] Table 4. Analytical results for different sample sizes

[0149]

[0150] The results showed that the total content of protocatechuic acid and p-hydroxybenzoic acid did not differ significantly with different sample amounts. Taking all factors into consideration, the sample 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 lotus root standard decoction, about 1.0 g, accurately weigh it, place it in a stoppered conical flask, accurately add 20 ml of 70% methanol, seal tightly, weigh it, sonicate (power 600W, frequency 40kHz) for 30 minutes, remove it, let it cool, weigh it again, replenish the lost weight with 70% methanol, shake well, filter, and collect the filtrate to obtain the test sample.

[0152] 1.4.5 Methodological Examination

[0153] 1.4.5.1 Specificity Examination

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

[0155] Preparation of the test solution: Take an appropriate amount of lotus root standard decoction, about 1.0 g, accurately weigh it, place it in a stoppered conical flask, accurately add 20 ml of 70% methanol, stopper tightly, weigh it, sonicate (power 600W, frequency 40kHz) for 30 minutes, remove it, let it cool, weigh it again, replenish the lost weight with 70% methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0156] Preparation of negative control solution: Prepare negative control solution of standard decoction without lotus root nodes according to the experimental conditions proposed above.

[0157] Accurately pipette 3 μl each of the reference solution and the test solution into the liquid chromatograph and determine the results. (See attached figure.) Figure 6 . Figure 6 Comparison of the specificity of lotus root standard decoction; as shown in the figure above, the negative solution has no effect on the target peaks of protocatechuic acid and p-hydroxybenzoic acid, indicating that this method has good specificity.

[0158] 1.4.5.2 Precision Examination

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

[0160] Table 5 Precision test results

[0161]

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

[0163] 1.4.5.3 Linear Relationship

[0164] Take 2.086 mg of protocatechuic acid (purity 97.8%), place it in a 50 ml volumetric flask, dilute to the mark with 70% methanol, shake well, and filter to obtain mother liquor 1; separately take 2.074 mg of p-hydroxybenzoic acid (purity 98.0%), place it in a 50 ml volumetric flask, dilute to the mark with 70% methanol, shake well, and filter to obtain mother liquor 2. Prepare reference solutions of different concentrations respectively. Inject different concentrations of protocatechuic acid and p-hydroxybenzoic acid reference standards into the liquid chromatograph, analyze the peak area, and plot the response curve with protocatechuic acid or p-hydroxybenzoic acid (X) as the abscissa and peak area (Y) as the ordinate. The results are shown in Tables 6-7 and 7. Figures 7-8 .

[0165] Table 6. Results of the standard curve analysis of protocatechuic acid

[0166]

[0167] Figure 7 The standard curve for protocatechuic acid was plotted. Results showed that the concentration range of protocatechuic acid was 0.40677–0.81354 μg / ml, with a linear relationship of y = 35.0807x - 0.2876, R0. 2 =1.0000, showing a good linear relationship.

[0168] Table 7. Analytical results of the standard curve of p-hydroxybenzoic acid

[0169]

[0170] Figure 8 The standard curve for p-hydroxybenzoic acid is shown. The results indicate that the linear relationship for p-hydroxybenzoic acid concentrations in the range of 0.40650–40.65040 μg / ml is y = 56.7956x - 0.5472, R0. 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 have the same operator prepare the test sample solution according to the proposed method. Calculate the total content of protocatechuic acid and p-hydroxybenzoic acid in the 6 test samples. The results are shown in Table 8.

[0173] Table 8 Results of Repeatability Experiments

[0174]

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

[0176] 1.4.5.5 Intermediate Precision

[0177] Based on the above-mentioned experimental conditions, six portions of the standard decoction of lotus root were accurately weighed to prepare the test solution. The total contents of protocatechuic acid and p-hydroxybenzoic acid were investigated and calculated using a Waters (Ⅰ) and Agilent 1260 (Ⅱ) high performance liquid chromatograph. The results are shown in Table 9.

[0178] Table 9. Results of the investigation of different personnel and time periods

[0179]

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

[0181] 1.4.5.6 Recovery rate

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

[0183]

[0184] Table 10 Results of the Protocatechuic Acid Recovery Experiment

[0185]

[0186] Table 11 Results of p-hydroxybenzoic acid recovery experiment

[0187]

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

[0189] 1.4.5.7 Durability Assessment

[0190] Based on the above-established experimental conditions, the contents of protocatechuic acid and p-hydroxybenzoic acid were investigated using 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). The results are shown in Table 12.

[0191] Table 12 Durability test results

[0192]

[0193] The results showed that the analytical chromatographic parameters of different columns were good, and the RSD value of the six measurements was 0.7%, indicating that the columns of this method have good robustness.

[0194] 1.4.5.8 Stability Test

[0195] Take the same test solution and measure the peak areas of protocatechuic acid and p-hydroxybenzoic acid at 0, 2, 4, 8, 16 and 24 h 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 of the total content of protocatechuic acid and p-hydroxybenzoic acid was 0.5%, and the test solution had good stability within 24 hours.

[0199] 1.5 Validation of Sample Content Determination

[0200] 1.5.1 Verification of Standard Decoction for Lotus Root Nodes

[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 Results of Content Determination in Standard Lotus Root Decoction

[0203]

[0204]

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

[0206] In summary, the provisional limits for the total content of protocatechuic acid and p-hydroxybenzoic acid in the standard decoction of lotus root nodes are set at -2SD of the average value of the standard decoction as the lower limit and +3SD of the average value as the upper limit, i.e., 0.15-0.75 mg / g.

[0207] The range for determining 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 Standard Decoction for Charred Lotus Root Nodes

[0209] 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 15.

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

[0211]

[0212]

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

[0214] In summary, the total content limits of protocatechuic acid and p-hydroxybenzoic acid in the standard decoction of lotus root charcoal are tentatively set as the lower limit of -2SD of the average value of the standard decoction and the upper limit of +3SD of the average value, i.e., 0.16-0.73 mg / g.

[0215] Example 2: HPLC content determination of lotus root segments and medicinal slices:

[0216] 2.1 Proposed chromatographic conditions

[0217] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase (column length 150 mm, inner diameter 2.1 mm, particle size 2.7 μm); methanol was used as mobile phase A, and 0.5% phosphoric acid aqueous solution was used as mobile phase B; gradient elution was performed according to the specifications in the table below; the detection wavelength was 260 nm. The flow rate was 0.3 ml / min; the column temperature was 20 °C; the theoretical plate number, calculated based on the p-hydroxybenzoic acid peak, should not be less than 3000.

[0218]

[0219]

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

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

[0222] The assay involves precisely pipetting 3 μl of both the reference solution and the test solution into a liquid chromatograph and measuring the results.

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

[0224] 2.2.1 Determination of detection wavelength

[0225] Based on the above-specified experimental conditions, a full-band scan of protocatechuic acid and p-hydroxybenzoic acid was performed using a diode array detector, see [link to results]. Figure 9 and Figure 10 . Figure 9 The spectrum of protocatechuic acid; Figure 10 This is the spectrum of p-hydroxybenzoic acid.

[0226] According to the spectral analysis, both of the measured index peaks have large absorption at 260 nm, so the absorption wavelength for future experiments will be set at 260 nm.

[0227] 2.2.2 Flow velocity investigation

[0228] Under the proposed chromatographic conditions, the separation efficiency of protocatechuic acid and p-hydroxybenzoic acid peaks at flow rates of 0.2 ml / min, 0.3 ml / min, and 0.4 ml / min was investigated as an evaluation index. The results are shown in [Figure number missing]. Figure 11 . Figure 11 Chromatograms at different flow rates; the results showed that when the flow rates were 0.2 ml / min, 0.3 ml / min, and 0.4 ml / min, the retention time and peak resolution were better at 0.3 ml / min, so the flow rate was set at 0.3 ml / min.

[0229] 2.2.3 Column Temperature Investigation

[0230] Under the proposed chromatographic conditions, the analysis of the test solution at column temperatures of 20℃, 25℃, and 30℃ was investigated. The separation efficiency of the protocatechuic acid and p-hydroxybenzoic acid peaks was used as the evaluation index. The results are shown in [Figure number missing]. Figure 12 . Figure 12 Chromatograms at different column temperatures; the results showed that the retention time and resolution of the index peaks were better at a column temperature of 20℃, so the experimental column temperature was specified as 20℃.

[0231] 2.2.4 Injection Volume Examination

[0232] Under the proposed chromatographic conditions, 2 μl, 3 μl, and 5 μl of sample were injected, respectively. The separation efficiency of the protocatechuic acid and p-hydroxybenzoic acid peaks was used as the evaluation index. The results are shown in [Figure number missing]. Figure 13 . Figure 13 Chromatograms with different injection volumes; the results show that the peak shape and peak resolution are better when the injection volume is 3 μl, therefore, the injection volume for this method is specified as 3 μl.

[0233] 2.3 Results of chromatographic conditions and system suitability tests

[0234] In summary, the chromatographic conditions and system suitability test results for the determination of lotus root standard decoction content are as follows: Octadecylsilane-bonded silica gel was used as the packing material (column length 150 mm, inner diameter 2.1 mm, particle size 2.7 μm); methanol was used as mobile phase A, and 0.5% phosphoric acid aqueous solution was used as mobile phase B; gradient elution was performed according to the specifications in the table below; the detection wavelength was 260 nm; the flow rate was 0.3 ml / min; the column temperature was 20℃; and the theoretical plate number, calculated based on the p-hydroxybenzoic acid peak, should not be less than 3000.

[0235] 2.4 Investigation on the preparation of test solution

[0236] 2.4.1 Investigation of Extraction Solvents

[0237] Take an appropriate amount of lotus root section (passed through a No. 3 sieve), approximately 3.0g, accurately weigh it, and place it in a stoppered conical flask. Accurately add 20ml each of water, ethanol, methanol, 30% methanol, 50% methanol, and 70% methanol respectively. Seal the flask tightly, weigh it, and sonicate it (600W power, 40kHz frequency) for 30 minutes. Remove it, cool it, weigh it again, and replenish the lost weight with 70% methanol. Shake well, filter it, and collect the filtrate. Inject 3μl of each extract into a chromatograph to analyze and calculate the total content of protocatechuic acid and p-hydroxybenzoic acid under different extraction solvents. The results are shown in Table 16 below.

[0238] Table 16 shows the analytical results for different extraction solvents.

[0239]

[0240] The results showed that water, 50% methanol, and 70% methanol had the best extraction effects. Considering all factors, 70% methanol was selected as the extraction solvent for the standard lotus root decoction.

[0241] 2.4.2 Examination of Extraction Methods

[0242] Take an appropriate amount of lotus root section (passed through a No. 3 sieve), approximately 3.0g, accurately weigh it, place it in a stoppered conical flask, accurately add 20ml of 70% methanol, seal tightly, weigh, and reflux and sonicate (600W power, 40kHz frequency) for 30 minutes respectively. Remove, cool, weigh again, and replenish the lost weight with 70% methanol. Shake well, filter, and collect the filtrate. Inject 3μl of each into a chromatograph to analyze and calculate the total content of protocatechuic acid and p-hydroxybenzoic acid under different extraction solvents. The results are shown in Table 17 below.

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

[0244]

[0245] The results showed that there was little difference in efficiency between ultrasonic and reflux extraction. Therefore, ultrasonic extraction was chosen as the extraction method after comprehensive consideration.

[0246] 2.4.3 Examination of extraction time

[0247] Take an appropriate amount of lotus root section (passed through a No. 3 sieve), approximately 3.0g, accurately weigh it, place it in a stoppered conical flask, accurately add 20ml of 70% methanol, seal tightly, weigh, and sonicate (600W power, 40kHz frequency) for 20 minutes, 30 minutes, and 40 minutes respectively. Remove, cool, weigh again, and replenish the lost weight with 70% methanol. Shake well, filter, and collect the filtrate. Inject 3μl of each into a chromatograph to analyze and calculate the total content of protocatechuic acid and p-hydroxybenzoic acid under different extraction solvents. The results are shown in Table 18 below.

[0248] Table 18 Analysis results for different extraction times

[0249]

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

[0251] 2.4.4 Sample Weight Examination

[0252] Take appropriate amounts of lotus root nodes (passed through a No. 3 sieve), weighing 2.0g, 3.0g, and 4.0g respectively. Place them in stoppered conical flasks, add 20ml of 70% methanol, seal tightly, weigh, and sonicate (600W power, 40kHz frequency) for 30 minutes. Remove, cool, weigh again, and replenish the lost weight with 70% methanol. Shake well, filter, and collect the filtrate. Inject 3μl of each into a chromatograph to analyze and calculate the total content of protocatechuic acid and p-hydroxybenzoic acid under different extraction solvents. The results are shown in Table 19.

[0253] Table 19 Analytical results for different sample sizes

[0254]

[0255] The results showed that the total content of protocatechuic acid and p-hydroxybenzoic acid did not differ significantly with different sample amounts. Taking all factors into consideration, the sample 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 section (passed through a No. 3 sieve), about 3.0g, accurately weigh it, place it in a stoppered conical flask, accurately add 20ml of 70% methanol, seal tightly, weigh, sonicate (power 600W, frequency 40kHz) for 30 minutes, remove, cool, weigh again, replenish the lost weight with 70% methanol, shake well, filter, and collect the filtrate to obtain the test sample.

[0257] 2.5 Methodological Examination

[0258] 2.5.1 Specificity Examination

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

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

[0261] Preparation of negative control solution: Prepare negative control solution of standard decoction without lotus root nodes according to the experimental conditions proposed above.

[0262] Accurately pipette 3 μl each of the reference solution and the test solution into the liquid chromatograph and determine the results. (See attached figure.) Figure 14 . Figure 14 Comparison of the specificity of lotus root nodes as a medicinal material; as shown in the figure above, the negative solution has no effect on the target peaks of protocatechuic acid and p-hydroxybenzoic acid, indicating that this method has good specificity.

[0263] 2.5.2 Precision Examination

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

[0265] Table 20 Precision test results

[0266]

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

[0268] 2.5.3 Linear Relationship

[0269] Take 2.086 mg of protocatechuic acid (purity 97.8%), place it in a 50 ml volumetric flask, dilute to the mark with 70% methanol, shake well, and filter to obtain mother liquor 1; separately take 2.074 mg of p-hydroxybenzoic acid (purity 98.0%), place it in a 50 ml volumetric flask, dilute to the mark with 70% methanol, shake well, and filter to obtain mother liquor 2. Prepare reference solutions of different concentrations respectively. Inject different concentrations of protocatechuic acid and p-hydroxybenzoic acid reference standards into the liquid chromatograph, analyze the peak area, and plot the response curve with protocatechuic acid or p-hydroxybenzoic acid (X) as the abscissa and peak area (Y) as the ordinate. The results are shown in Tables 21-22 and 22. Figures 15-16 .

[0270] Table 21 Results of the standard curve analysis of protocatechuic acid

[0271]

[0272] Figure 15 Standard curve of protocatechuic acid; results showed that the concentration range of protocatechuic acid was 0.40677–0.81354 μg / ml, with a linear relationship of y = 35.0807x - 0.2876, R0. 2 =1.0000, showing a good linear relationship.

[0273] Table 22 Analytical results of the p-hydroxybenzoic acid standard curve

[0274]

[0275] Figure 16 Standard curve of p-hydroxybenzoic acid; results show that the linear relationship of p-hydroxybenzoic acid concentration range is y = 56.7956x - 0.5472, R0 2 =1.0000, showing a good linear relationship.

[0276] 2.5.4 Repeatability

[0277] Take 6 portions of the same test sample, each weighed accurately to 3.0 g, and have them prepared into test solutions by the same operator according to the prescribed method. Calculate the total content of protocatechuic acid and p-hydroxybenzoic acid in the 6 test samples. The results are shown in Table 23.

[0278] Table 23 Results of Repeatability Experiments

[0279]

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

[0281] 2.5.5 Intermediate Precision

[0282] Based on the above-mentioned experimental conditions, six portions of the standard decoction of lotus root were accurately weighed to prepare the test solution. The total contents of protocatechuic acid and p-hydroxybenzoic acid were investigated and calculated using a Waters (Ⅰ) and Agilent 1260 (Ⅱ) high performance liquid chromatograph. The results are shown in Table 24.

[0283] Table 24 Results of the survey on different personnel and time periods

[0284]

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

[0286] 2.5.6 Recovery rate

[0287] Take approximately 0.5 g of the test sample with known content (0.0017% protocatechuic acid and 0.0026% p-hydroxybenzoic acid), make 6 portions, accurately weigh them, and accurately add a certain amount of protocatechuic acid and p-hydroxybenzoic acid reference standards to each. Prepare and determine the test sample solutions according to the prescribed method, calculate the recovery rate, and the results are shown in Tables 25-26. The calculation formula is as follows:

[0288]

[0289] Table 25 Results of Protocatechuic Acid Recovery Experiment

[0290]

[0291] Table 26 Results of p-hydroxybenzoic acid recovery experiment

[0292]

[0293]

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

[0295] 2.5.7 Durability Assessment

[0296] Based on the above-established experimental conditions, the chromatographic columns were investigated for 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 27.

[0297] Table 27 Durability Test Results

[0298]

[0299] The results showed that the analytical chromatographic parameters of different columns were good, and the RSD value of the six measurements was 2.4%, indicating that the columns of this method have good robustness.

[0300] 2.5.8 Stability Test

[0301] 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 h 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 of the total content of protocatechuic acid and p-hydroxybenzoic acid was 1.5%, and the test solution had good stability within 24 hours.

[0305] 2.6 Validation of Sample Content Determination

[0306] 2.6.1 Verification of medicinal properties of lotus root nodes

[0307] 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 29.

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

[0309]

[0310]

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

[0312] 2.6.2 Verification of Lotus Root Slices

[0313] 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 30.

[0314] Table 30 shows the results of content determination in 32 batches of lotus root slices.

[0315]

[0316]

[0317] The measured total content of protocatechuic acid and p-hydroxybenzoic acid in 32 batches of lotus root slices ranged from 0.0020% to 0.0079%, with an average content of 0.0047% and a standard deviation (SD) of 0.0017. The calculated 70%–130% range of the average content was 0.0033%–0.0061%, the range of the average content plus or minus three times the SD was -0.0004%–0.0098%, and the range of the average content plus or minus two times the SD was 0.0013%–0.0081%.

[0318] 2.6.3 Verification of charred lotus root slices

[0319] 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 31.

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

[0321]

[0322]

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

[0324] Example 3: Drafting Instructions for HPLC Content Determination of Lotus Root Node Granules

[0325] 3.1 Proposed chromatographic conditions

[0326] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase (column length 150 mm, inner diameter 2.1 mm, particle size 2.7 μm); methanol was used as mobile phase A, and 0.5% phosphoric acid aqueous solution was used as mobile phase B; gradient elution was performed according to the specifications in the table below; the detection wavelength was 260 nm. The flow rate was 0.3 ml / min; the column temperature was 20 °C; the theoretical plate number, calculated based on the p-hydroxybenzoic acid peak, should not be less than 3000.

[0327]

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

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

[0330] The assay involves precisely pipetting 3 μl of both the reference solution and the test solution into a liquid chromatograph and measuring the results.

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

[0332] 3.2.1 Determination of detection wavelength

[0333] Based on the above-specified experimental conditions, a full-band scan of protocatechuic acid and p-hydroxybenzoic acid was performed using a diode array detector, as shown in the figure. Figure 17 Spectrum of protocatechuic acid; Figure 18 Spectrum of p-hydroxybenzoic acid.

[0334] According to the spectral analysis, both of the measured index peaks have large absorption at 260 nm, so the absorption wavelength for future experiments will be set at 260 nm.

[0335] 3.2.2 Flow velocity investigation

[0336] Under the proposed chromatographic conditions, the separation efficiency of protocatechuic acid and p-hydroxybenzoic acid peaks at flow rates of 0.2 ml / min, 0.3 ml / min, and 0.4 ml / min was investigated as an evaluation index. The results are shown in [Figure number missing]. Figure 19 . Figure 19 Chromatograms at different flow rates; the results showed that when the flow rates were 0.2 ml / min, 0.3 ml / min, and 0.4 ml / min, the retention time and peak resolution were better at 0.3 ml / min, so the flow rate was set at 0.3 ml / min.

[0337] 3.2.3 Column Temperature Investigation

[0338] Under the proposed chromatographic conditions, the analysis of the test solution at column temperatures of 20℃, 25℃, and 30℃ was investigated. The separation efficiency of the protocatechuic acid and p-hydroxybenzoic acid peaks was used as the evaluation index. The results are shown in [Figure number missing]. Figure 20 . Figure 20 Chromatograms at different column temperatures; the results showed that the retention time and resolution of the index peaks were better at a column temperature of 20℃, so the experimental column temperature was specified as 20℃.

[0339] 3.2.4 Sample Injection Volume Examination

[0340] Under the proposed chromatographic conditions, 2 μl, 3 μl, and 5 μl of sample were injected, respectively. The separation efficiency of the protocatechuic acid and p-hydroxybenzoic acid peaks was used as the evaluation index. The results are shown in [Figure number missing]. Figure 21 . Figure 21 Chromatograms with different injection volumes; the results show that the peak shape and peak resolution are better when the injection volume is 3 μl, therefore, the injection volume for this method is specified as 3 μl.

[0341] 3.3 Results of chromatographic conditions and system suitability tests

[0342] In summary, the chromatographic conditions and system suitability test results for the determination of lotus root standard decoction content are as follows: Octadecylsilane-bonded silica gel was used as the packing material (column length 150 mm, inner diameter 2.1 mm, particle size 2.7 μm); methanol was used as mobile phase A, and 0.5% phosphoric acid aqueous solution was used as mobile phase B; gradient elution was performed according to the conditions in 3.1; the detection wavelength was 260 nm; the flow rate was 0.3 ml / min; the column temperature was 20℃; and the theoretical plate number, calculated based on the p-hydroxybenzoic acid peak, should not be less than 3000.

[0343] 3.4 Investigation on the preparation of test solution

[0344] 3.4.1 Investigation of Extraction Solvents

[0345] Take an appropriate amount of lotus root granules, grind them finely, and accurately weigh about 1.0g. Place the weighed flask in a stoppered conical flask, and accurately add 20ml each of water, ethanol, methanol, 30% methanol, 50% methanol, and 70% methanol. Seal the flask tightly, weigh it, and sonicate it (600W power, 40kHz frequency) for 30 minutes. Remove the flask, cool it, weigh it again, and replenish the lost weight with 70% methanol. Shake well, filter it, and collect the filtrate. Inject 3μl of each flask into a chromatograph to analyze and calculate the total content of protocatechuic acid and p-hydroxybenzoic acid under different extraction solvents. The results are shown in Table 32.

[0346] Table 32 Analytical 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 lotus root decoction.

[0349] 3.4.2 Examination of Extraction Methods

[0350] Take an appropriate amount of lotus root granules, grind them finely, and accurately weigh about 1.0g. Place the weighing result in a stoppered conical flask, add 20ml of 70% methanol, seal tightly, and weigh. Reflux and sonicate (600W power, 40kHz frequency) for 30 minutes each. Remove, cool, weigh again, and replenish the lost weight with 70% methanol. Shake well, filter, and collect the filtrate. Inject 3μl of each solution into a chromatograph to analyze and calculate the total content of protocatechuic acid and p-hydroxybenzoic acid 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 in efficiency between ultrasonic and reflux extraction. Therefore, ultrasonic extraction was chosen as the extraction method after comprehensive consideration.

[0354] 3.4.3 Examination of extraction time

[0355] Take an appropriate amount of lotus root granules, grind them finely, and accurately weigh about 1.0g. Place the weighed flask in a stoppered conical flask, accurately add 20ml of 70% methanol, seal tightly, and weigh. Sonicate the flask (600W power, 40kHz frequency) for 20 minutes, 30 minutes, and 40 minutes respectively. Remove the flask, cool it, weigh it again, and replenish the lost weight with 70% methanol. Shake well, filter, and collect the filtrate. Inject 3μl of each flask into a chromatograph to analyze and calculate the total content of protocatechuic acid and p-hydroxybenzoic acid under different extraction solvents. The results are shown in Table 34.

[0356] Table 34 Analysis results for different extraction times

[0357]

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

[0359] 3.4.4 Sample Weight Examination

[0360] Take an appropriate amount of lotus root granules, grind them finely, and accurately weigh 0.5g, 1.0g, and 1.5g respectively. Place them in a stoppered conical flask, accurately add 20ml of 70% methanol, seal tightly, weigh, and sonicate (600W power, 40kHz frequency) for 30 minutes. Remove, cool, weigh again, and replenish the lost weight with 70% methanol. Shake well, filter, and collect the filtrate. Inject 3μl of each into a chromatograph to analyze and calculate the total content of protocatechuic acid and p-hydroxybenzoic acid under different extraction solvents. The results are shown in Table 35.

[0361] Table 35 Analytical results for different sample sizes

[0362]

[0363] The results showed that the total content of protocatechuic acid and p-hydroxybenzoic acid did not differ significantly with different sample amounts. Taking all factors into consideration, the sample 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 granules, about 1.0 g, accurately weigh it, place it in a stoppered conical flask, accurately add 20 ml of 70% methanol, seal tightly, weigh, sonicate (power 600W, frequency 40kHz) for 30 minutes, remove, cool, weigh again, replenish the lost weight with 70% methanol, shake well, filter, and collect the filtrate to obtain the test sample.

[0365] 3.5 Methodological Examination

[0366] 3.5.1 Specificity Examination

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

[0368] Preparation of the test solution: Take an appropriate amount of lotus root granules, grind them into a fine powder, weigh about 1.0g accurately, place them in a stoppered conical flask, add 20ml of 70% methanol accurately, stopper tightly, weigh, sonicate (power 600W, frequency 40kHz) for 30 minutes, remove, cool, weigh again, replenish the lost weight with 70% methanol, shake well, filter, and collect the filtrate to obtain the test solution.

[0369] Preparation of negative control solution: Prepare negative control solution of standard decoction without lotus root nodes according to the experimental conditions proposed above.

[0370] Accurately pipette 3 μl each of the reference solution and the test solution into the liquid chromatograph and determine the results. (See attached figure.) Figure 22 . Figure 22 Comparison of the specificity of lotus root standard decoction; as shown in the figure above, the negative solution has no effect on the target peaks of protocatechuic acid and p-hydroxybenzoic acid, indicating that this method has good specificity.

[0371] 3.5.2 Precision Examination

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

[0373] Table 36 Precision test results

[0374]

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

[0376] 3.5.3 Linear Relationship

[0377] Take 2.086 mg of protocatechuic acid (purity 97.8%), place it in a 50 ml volumetric flask, dilute to the mark with 70% methanol, shake well, and filter to obtain mother liquor 1; separately take 2.074 mg of p-hydroxybenzoic acid (purity 98.0%), place it in a 50 ml volumetric flask, dilute to the mark with 70% methanol, shake well, and filter to obtain mother liquor 2. Prepare reference solutions of different concentrations respectively. Inject different concentrations of protocatechuic acid and p-hydroxybenzoic acid reference standards into the liquid chromatograph, analyze the peak area, and plot the response curve with protocatechuic acid or p-hydroxybenzoic acid (X) as the abscissa and peak area (Y) as the ordinate. The results are shown in Tables 37-38 and 38. Figures 23-24 .

[0378] Table 37 Results of the standard curve analysis of protocatechuic acid

[0379]

[0380] Figure 23 Standard curve of protocatechuic acid; results showed that the concentration range of protocatechuic acid was 0.40677–0.81354 μg / ml, with a linear relationship of y = 35.0807x - 0.2876, R0. 2 =1.0000, showing a good linear relationship.

[0381] Table 38. Analytical results of the standard curve for p-hydroxybenzoic acid.

[0382]

[0383] Figure 24 Standard curve of p-hydroxybenzoic acid; results show that the linear relationship of p-hydroxybenzoic acid concentration range is y = 56.7956x - 0.5472, R0 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 content of protocatechuic acid and p-hydroxybenzoic acid in the 6 test samples. The results are shown in Table 39.

[0386] Table 39 Results of Repeatability Experiments

[0387]

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

[0389] 3.5.5 Intermediate Precision

[0390] Based on the above-mentioned experimental conditions, six portions of lotus root formula granules were accurately weighed to prepare a test solution. The total content of protocatechuic acid and p-hydroxybenzoic acid was investigated and calculated using a Waters (Ⅰ) or Agilent 1260 (Ⅱ) high performance liquid chromatograph. The results are shown in Table 40.

[0391] Table 40 Results of the surveys on different personnel and time periods

[0392]

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

[0394] 3.5.6 Recovery rate

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

[0396]

[0397] Table 41 Results of Protocatechuic Acid Recovery Experiment

[0398]

[0399] Table 42 Results of p-hydroxybenzoic acid recovery experiment

[0400]

[0401] The results showed that the average recovery rate of protocatechuic acid was 99.9%, with an RSD of 1.3%; the average recovery rate of p-hydroxybenzoic acid was 95.7%, with an RSD of 0.7%; the method had good accuracy.

[0402] 3.5.7 Durability Assessment

[0403] Based on the above-established experimental conditions, the contents of protocatechuic acid and p-hydroxybenzoic acid were investigated using chromatographic columns 1 (InfinityLab Poroshell 120 HILIC-Z 2.1*150 mm, 2.7 μm), 2 (ZORBAX SB-C18 2.1*150 mm, 1.8 μm), and 3 (InfinityLab Poroshell 120 EC-C18 2.1*150 mm, 1.9 μm). The results are shown in Table 43.

[0404] Table 43 Durability Test Results

[0405]

[0406]

[0407] The results showed that the analytical chromatographic parameters of different columns were good, and the RSD value of the six measurements was 1.0%, indicating that the columns of this method have good robustness.

[0408] 3.5.8 Stability Test

[0409] Take the same test solution and measure the peak areas of protocatechuic acid and p-hydroxybenzoic acid at 0, 2, 4, 8, 16 and 24 h 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 of the total content 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 Lotus Root Node Content Determination

[0414] 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 45.

[0415] Table 45 Results of Granule Content in Three Batches of Lotus Root Formula

[0416]

[0417] As shown in the table above, the average value of the three batches was 0.28 mg / g, and the average value ±30% ranged from 0.20 to 0.36 mg / g.

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

[0419] 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 46.

[0420] Table 46 Results of Particle Content in Three Batches of Lotus Root Charcoal Formulation

[0421]

[0422] As shown in the table above, the average value of the three batches was 0.28 mg / g, and the average value ±30% ranged from 0.19 to 0.35 mg / g.

[0423] Example 4: Comparison of transfer rates of HPLC methods for determining the content of lotus root nodes and lotus root node carbon

[0424] The content of lotus root nodes and related processed products was determined using the method described in Example 1, and the total content transfer rate of protocatechuic acid and p-hydroxybenzoic acid was measured.

[0425] Table 47

[0426]

[0427] From the analysis of lotus root nodes from raw medicinal materials to processed lotus root nodes and charred lotus root nodes, it can be seen that the content of protocatechuic acid and p-hydroxybenzoic acid in charred lotus root nodes increases significantly during the processing, while it decreases in the standard decoction. The measured transfer rate from processed lotus root nodes to standard decoction ranges from 32.7% to 93.4%; while the measured transfer rate from charred lotus root nodes to standard decoction ranges from 19.1% to 48.5%, showing a significant difference. Therefore, this content determination method can be used as a distinguishing feature between lotus root nodes and charred lotus root nodes.

[0428] Comparative Example 1

[0429] The pretreatment method of Example 1 of this invention was used, the difference being the detection method. The chromatographic column was a Phenomenex Luna C18 column (150 mm × 2.0 mm), mobile phase A was acetonitrile, 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 diode array detector wavelength was 280 nm. Linear gradient elution was performed: 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 as follows: Figure 25 The above,

[0430] The above detection method was used to determine the results of lotus root granule sample 1, lotus root granule sample 2, and the control standard, respectively.

[0431] according to Figure 25 It can be seen that, Figure 25 This is the chromatogram of Comparative Example 1. Although the method in Comparative Example 1 can extract two components, it cannot identify and separate the two indicator components in the lotus root node.

[0432] Comparative Example 2

[0433] The pretreatment method of Embodiment 1 of this invention is used, the difference being the detection method. 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: 10μL.

[0434] The determination was performed using lotus root granule samples and control samples respectively using the mobile phase ratio detection methods (1) and (2).

[0435] The results are as follows Figure 26 The above, Figure 26 The chromatogram of Comparative Example 2; according to Figure 26 It can be seen that, Figure 26 (1) The proportion of the mobile phase and Figure 26 The ratio of the mobile phase (2) cannot identify and separate the two index components in the lotus root section of this patent.

[0436] Comparative Example 3

[0437] The pretreatment method of Example 1 of this invention is used, the difference being the detection method. The chromatographic column is packed with octadecylsilane-bonded silica gel (Eclipse Plus C18, 4.6 × 150 mm, 3.5 μm); the detection wavelength is 270 nm; the mobile phase is 0.2% phosphoric acid solution as mobile phase A and methanol as mobile phase B; the gradient elution program is: 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 is 30 °C, the injection volume is 10 μL, and the flow rate is 1.0 mL / min.

[0438] The above detection method was used to determine the results of lotus root granule samples and control standards, respectively.

[0439] The results are as follows Figure 27 The above, Figure 27 The chromatogram is for Comparative Example 3. The method in Comparative Example 3 can identify one component, but it cannot identify and separate both components in the lotus root section of this invention.

[0440] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for determining the HPLC content of lotus root nodes and related processed products, comprising: A) The test sample raw material is dissolved and extracted using a solvent to obtain the test solution; the test sample raw material includes lotus root nodes or related processed products; The solvent is 0%~100% methanol; B) The test solution was analyzed by high performance liquid chromatography to obtain the HPLC chromatogram of lotus root nodes or related processed products; It also includes the preparation of reference solutions: taking protocatechuic acid reference standard and p-hydroxybenzoic acid reference standard respectively, dissolving them in 70% methanol to obtain reference solutions; The reference solution was analyzed by high performance liquid chromatography to obtain a chromatogram of the reference; and the components of lotus root nodes or related processed products were qualitatively identified based on the chromatogram of the reference. The chromatographic conditions for the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; mobile phase A is a methanol solution, mobile phase B is a 0.5% phosphoric acid aqueous solution, and gradient elution is used. The gradient elution specifically refers to: 0–1 min, 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%; 16–30 min, Phase A: 10%, Phase B: 90%.

2. The method according to claim 1, characterized in that, The specific concentrations of the reference solutions are as follows: protocatechuic acid reference standard is 0.40677~0.81354 μg / ml, and p-hydroxybenzoic acid reference standard is 0.40650~40.65040 μg / ml.

3. The method according to claim 1, characterized in that, The chromatographic column is C. 18 150×2.1mm, 1.8~2.7μm; column temperature 20℃~30℃; theoretical plate number calculated based on p-hydroxybenzoic acid should not be less than 3000.

4. The method according to claim 3, characterized in that, The flow rate of the mobile phase is 0.2~0.4 mL / min; the injection volume is 2~5 μL.

5. The method according to claim 3, characterized in that, The detection wavelength is 260nm.

6. The method according to claim 4, characterized in that, The column temperature was 20℃; the mobile phase flow rate was 0.3 mL / min; and the injection volume was 3 μL.

7. The method according to claim 1, characterized in that, The extraction in step A) is ultrasonic extraction; the ultrasonic power is 600W, the frequency is 40kHz, and the ultrasonic time is 20~40min.

8. The method according to claim 7, characterized in that, The ratio of the mass (g) of the test sample raw material to the volume (mL) of the solvent is 1~4:20; The raw materials for the test samples are lotus root medicinal materials, lotus root slices, lotus root standard decoction, lotus root formula granules, lotus root charcoal slices, lotus root charcoal standard decoction, or lotus root charcoal formula granules.

9. A method for distinguishing between processed lotus root segments and carbonized lotus root segments, characterized in that, The transfer rate of protocatechuic acid and total p-hydroxybenzoic acid was analyzed by using the method described in any one of claims 1 to 8. The measured transfer rate from lotus root nodes to processed lotus root slices ranged from 93.77% to 97.25%; the measured transfer rate from lotus root nodes to charred lotus root slices ranged from 97.21% to 244.90%. The measured transfer rate from processed lotus root slices to standard lotus root decoction ranged from 32.7% to 93.4%; the measured transfer rate from charred lotus root slices to standard charred lotus root decoction ranged from 19.1% to 48.5%. The total content of the same amount of lotus root medicinal material to lotus root formula granules ranges from 0.16 mg / g to 1.02 mg / g; the measured total content of the same amount of lotus root medicinal material to lotus root charcoal formula granules ranges from 0.11 mg / g to 0.76 mg / g.

Images