A method for constructing a characteristic chromatogram of a radix eupatii throat-clearing mixture and application thereof

Abstract

The present application belongs to the technical field of traditional Chinese medicine analysis, and particularly relates to a construction method of a characteristic spectrum of a Radix et Rhizoma Aconiti Lateralis Praeparata throat clearing mixture and application thereof. The present application realizes a leap from single index control to overall quality evaluation. The present application first establishes a characteristic spectrum method special for the Radix et Rhizoma Aconiti Lateralis Praeparata throat clearing mixture, and breaks through the limitation of the prior art that only the content of a single component is determined. By simultaneously monitoring the consistency of five key characteristic peaks of chlorogenic acid, caffeic acid, scandenoside, gardenia glycoside and catechin and the overall spectrum, the material basis feeding condition and the proportioning stability of the monarch, minister, assistant and guide medicinal ingredients in the preparation can be comprehensively reflected, so that the overall quality and the uniformity of the curative effect of the product are more scientifically ensured.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine analysis technology, and specifically relates to a method for constructing a characteristic spectrum of Achyranthes bidentata throat-clearing compound and its application. Background Technology

[0002] Tu Niu Xi Qing Yan He Ji is a traditional Chinese medicine compound preparation made from four medicinal herbs: Guangdong Tu Niu Xi (Achyranthes bidentata), Shui Yang Mei (Myrica rubra), Gang Mei (Ilex chinensis), and Dan Zhu Ye (Lophatherum gracile). It has the effects of clearing heat and detoxifying, relieving sore throat and reducing swelling. Clinically, it is often used for symptoms such as sore throat and dry mouth caused by excess heat in the lungs and stomach, and is one of the commonly used traditional Chinese medicine preparations for treating upper respiratory tract infections. Ensuring the stable quality and reliable efficacy of this preparation is fundamental to realizing its clinical value.

[0003] Currently, quality control of this preparation relies heavily on traditional appearance and property inspections, as well as quantitative determination of a few single indicators, which is insufficient to comprehensively reflect the overall quality of the multi-component, multi-target compound preparation. High-performance liquid chromatography (HPLC) characteristic chromatographic technology, as a comprehensive identification and evaluation method, can characterize multiple common characteristic peaks and their relative relationships in a sample, thus depicting the overall chemical characteristics of the product like a chemical fingerprint, and has been widely used in the quality evaluation of traditional Chinese medicine. However, to date, no reports have been found in publicly available literature and standards regarding the establishment of an efficient, specific, and practical characteristic chromatographic quality control method for Achyranthes bidentata throat-clearing compound.

[0004] Furthermore, establishing characteristic chromatograms for simultaneously monitoring five components—chlorogenic acid, caffeic acid, bergenin, geniposide, and catechins—is particularly important. These chromatograms can systematically reflect the authenticity of the ingredients, the stability of the proportions, and the consistency of the process, thus achieving a leap in quality control from single-indicator to holistic "chemical fingerprint" evaluation. However, currently, there is a lack of a method that can simultaneously identify and correlate the main characteristic components of the prescription using commercially available and reliable standards. This technological gap means that the production quality control, product authenticity identification, and batch-to-batch consistency evaluation of this preparation lack advanced and comprehensive technical tools.

[0005] The difficulty in testing Achyranthes bidentata throat-clearing compound lies in:

[0006] 1. Complexity of components and interference: The compound is obtained by decocting multiple medicinal materials together, and its chemical composition is extremely complex. In addition to the target active ingredient, there are also a large number of sugars, tannins, pigments and unknown high molecular weight compounds. These coexisting substances seriously interfere with the chromatographic separation and accurate localization of the target component, resulting in the characteristic peaks being difficult to identify clearly, the baseline being unstable or the column efficiency decreasing rapidly.

[0007] 2. Significant differences in the physicochemical properties of target components: The characteristic components of the various herbs in the prescription (such as chlorogenic acid, caffeic acid, bergenin, geniposide, and catechins) exhibit significant differences in polarity, acidity / alkalinity, and solubility. Achieving efficient baseline separation of all target components in a single chromatographic system, while simultaneously obtaining good peak shapes, presents extremely high challenges in terms of mobile phase composition, gradient program, and column selection.

[0008] 3. Balance between the integrity and specificity of characteristic chromatograms: Characteristic chromatograms should not only reflect the overall "fingerprint" outline, but also include characteristic peaks that can specifically characterize each medicinal ingredient. There is a lack of existing standards and methods to scientifically screen and identify these characteristic peaks that possess both "commonality" and "representativeness" from a vast number of chromatographic peaks, and to establish their correlation with the feeding of medicinal materials and the stability of the process.

[0009] 4. High difficulty in sample pretreatment: The matrix of compound samples is complex and has high viscosity. Direct injection can easily contaminate the chromatographic system and result in insufficient sensitivity. Conventional dilution, centrifugation, or simple extraction are insufficient to effectively enrich the target components and remove strong interfering substances. Therefore, it is necessary to develop a dedicated pretreatment method that combines high purification efficiency and high recovery rate.

[0010] Therefore, developing a proprietary feature mapping method based on multiple characteristic control standards that can systematically reflect the overall chemical characteristics of Achyranthes bidentata throat-clearing compound and is easy to promote and apply is of urgent need and great significance for improving the quality standards of this product, ensuring the safety and efficacy of clinical medication, and promoting the standardized development of the industry. Summary of the Invention

[0011] The purpose of this invention is to provide a method for constructing a characteristic spectrum of Achyranthes bidentata throat-clearing compound and its application. The characteristic spectrum constructed by this method can be used for quality control, authenticity identification and production process stability evaluation of the preparation, thereby ensuring its clinical efficacy and medication safety.

[0012] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0013] A method for constructing a characteristic spectrum of Achyranthes bidentata throat-clearing compound includes the following steps:

[0014] Step 1:

[0015] Preparation of reference solution: Accurately weigh chlorogenic acid, caffeic acid, bergenin, geniposide, and catechin reference standards, place them in the same volumetric flask, add a 1:1 mixture of methanol and 0.05% phosphoric acid aqueous solution, sonicate at 250W, 40kHz for 15 minutes to ensure complete dissolution, cool, then dilute with the same mixed solvent and filter through a 0.22µm microporous membrane to prepare a mixed reference solution containing 100µg chlorogenic acid, 50µg caffeic acid, 120µg bergenin, 150µg geniposide, and 80µg catechin per mL.

[0016] Step Two:

[0017] Preparation of test solution: Accurately measure the sample of Achyranthes bidentata throat-clearing compound, add a multiphase composite solvent composed of water-methanol-0.1% formic acid aqueous solution, and ultrasonically extract in a 45℃ water bath for 20 minutes. Remove, cool, replenish the lost weight with water-methanol-0.1% formic acid aqueous solution, shake well, and filter through a 0.45μm microporous membrane. Accurately measure 10mL of the subsequent filtrate and pass it through a Waters Oasis HLB solid phase extraction column that has been balanced and activated with 5mL methanol and 5mL water at a flow rate of 2mL / min. Elute successively with water and 5% methanol aqueous solution, discard the eluent, and finally elute with 80% methanol solution containing 0.1% formic acid. Collect the eluent and dry it with nitrogen at 40℃ to obtain the residue. Accurately dissolve the residue in 50% methanol solution containing 0.05% formic acid, vortex mix well, filter through a 0.22μm nylon microporous membrane, and collect the subsequent filtrate to obtain the test solution.

[0018] Step 3:

[0019] Determination method: Accurately pipette 10 μL each of the reference solution and the test solution and inject them into the liquid chromatograph, and record the chromatograms;

[0020] Chromatographic conditions: Column: Octadecylsilane-bonded silica gel column (size: 4.6mm × 250mm, 5μm); Mobile phase: Acetonitrile:methanol:tetrahydrofuran (v / v) at a ratio of 65:30:5 as mobile phase A, and 0.05% phosphoric acid (v / v) aqueous solution containing 0.02% (v / v) triethylamine as mobile phase B, with gradient elution as specified; Flow rate: 1.0mL / min; Column temperature: 30℃; Detection wavelength: 330nm; Theoretical plate number calculated based on the chlorogenic acid peak should be no less than 5000.

[0021] The gradient elution procedure is as follows:

[0022] From 0 to 8 minutes, the volume ratio of mobile phase A to phase B changed from 5:95 to 11:89.

[0023] In 8-10 minutes, the volume ratio of mobile phase A to phase B changed from 11:89 to 12:88.

[0024] 10~14 min, the volume ratio of mobile phase A: phase B changes from 12:88 to 13:87;

[0025] 14~25 min, the volume ratio of mobile phase A: phase B changes from 13:87 to 15:85;

[0026] 25~40min, the volume ratio of mobile phase A: phase B changes from 15:85 to 22:78;

[0027] For 40-60 minutes, the volume ratio of mobile phase A to phase B changes from 22:78 to 39:61.

[0028] At 60-61 min, the volume ratio of mobile phase A to phase B changed from 39:61 to 5:95.

[0029] 61~70min, mobile phase A: phase B, volume ratio 5:95;

[0030] Step 4: Generate a control characteristic chromatogram: Select the chromatographic peaks that are present in the chromatograms of different batches of Achyranthes bidentata throat-clearing compound as common peaks, and use the average value calculation method to generate a control characteristic chromatogram of Achyranthes bidentata throat-clearing compound.

[0031] Furthermore, in step two, the volume ratio of water-methanol-0.1% formic acid aqueous solution is 1:3:1.

[0032] Furthermore, in step two, the ultrasonic conditions are 300W power and 40kHz frequency.

[0033] Furthermore, in step two, the Waters Oasis HLB solid-phase extraction column was 200 mg, 6 cc.

[0034] Furthermore, in step three, the chromatographic column dimensions are 4.6 mm × 250 mm and 5 μm.

[0035] Furthermore, the reference characteristic chromatogram generated in step four presents five chromatographic peaks, among which peaks 1, 2, 3, 4, and 5 correspond to the reference peaks of chlorogenic acid, caffeic acid, bergenin, geniposide, and catechin, respectively.

[0036] Furthermore, in step four, the relative retention times of each characteristic peak and the S peak are calculated using the chlorogenic acid reference peak as the S peak. The relative retention times are all within ±10% of the specified values. The specified values ​​are: 1.63 (peak 2), 2.59 (peak 3), 3.03 (peak 4), and 3.52 (peak 5).

[0037] The beneficial effects of this invention are as follows:

[0038] 1. Achieving a leap from single-indicator control to overall quality evaluation: This invention establishes for the first time a characteristic chromatographic method specific to Achyranthes bidentata throat-clearing compound, breaking through the limitations of existing technologies that only measure the content of single components. By simultaneously monitoring the consistency of five key characteristic peaks and their overall chromatograms—chlorogenic acid, caffeic acid, bergenin, geniposide, and catechins—it can comprehensively reflect the material basis and proportion stability of each herb in the preparation, thereby more scientifically ensuring the uniformity of the overall quality and efficacy of the product.

[0039] 2. Significantly improved specificity and discriminative power of the detection method: Through optimized multiphase composite solvent extraction combined with solid-phase extraction purification technology, interference from complex matrices in the compound (such as sugars, pigments, and tannins) was effectively eliminated, enriching the target components. Combined with carefully optimized chromatographic conditions, baseline separation and good peak shapes were achieved for five characteristic components with significantly different physicochemical properties. The constructed characteristic chromatograms exhibit strong specificity, clearly identifying product authenticity and effectively distinguishing samples from different sources or processes.

[0040] 3. Combining advanced technology with industrial applicability: The five reference standards selected in this invention are all commonly used standards from the National Institutes for Food and Drug Control (NIFDC), ensuring reliable sources and easy access, thus overcoming the technical bottleneck of scarce reference standards for some characteristic components. The established chromatographic conditions are stable and reproducible, and the sample pretreatment steps are efficient and highly standardized, making this method not only advanced and reliable but also easy to promote and apply in manufacturing enterprises and testing institutions, demonstrating good prospects for industrial transformation.

[0041] 4. It provides a powerful tool for whole-process quality control: The standard characteristic spectrum obtained by this method can not only be used for finished product inspection, but also trace back to the quality evaluation of intermediates and raw materials. It provides a scientific chemical basis for optimizing production process parameters, monitoring key process links, and standardizing the feeding of raw materials, realizing whole-process quality traceability from source to finished product. Attached Figure Description

[0042] The invention will now be further described with reference to the accompanying drawings;

[0043] Figure 1 This is a repeatable chromatogram overlay of the present invention; wherein, S1-S6 are chromatograms obtained from 6 test solutions prepared according to the test solution preparation method;

[0044] Figure 2This is a chromatogram of the stability of the present invention; wherein, S1 is the chromatogram obtained by injecting the same test solution at 0 hours, S2 is the chromatogram obtained by injecting the same test solution at 2 hours, S3 is the chromatogram obtained by injecting the same test solution at 4 hours, S4 is the chromatogram obtained by injecting the same test solution at 8 hours, S5 is the chromatogram obtained by injecting the same test solution at 12 hours, and S6 is the chromatogram obtained by injecting the same test solution at 24 hours.

[0045] Figure 3 This is a chromatogram of the reference solution for the characteristic chromatogram of the present invention; wherein, S1 is the test sample and S2 is the reference.

[0046] Figure 4 The chromatogram of the test sample solution is a characteristic chromatogram of the present invention. Detailed Implementation

[0047] The present invention will be further described below with reference to the accompanying drawings. The instruments and equipment used in the embodiments are as follows:

[0048] I. Instruments and Reagents

[0049] 1.1 Instruments and Equipment

[0050] .

[0051] 1.2 Test Materials

[0052] .

[0053] 1.3 Reference Standard Information

[0054] .

[0055] 1.4 Sample Information

[0056] .

[0057] In this embodiment, the Achyranthes bidentata throat-clearing compound is prepared as follows:

[0058] 3.3 parts of Achyranthes bidentata, 2.5 parts of Ilex chinensis, 2.5 parts of Myrica rubra, and 1 part of Lophatherum gracile (by weight). Boil these ingredients twice in water, 1.5 hours each time. Combine the decoctions, filter, and concentrate to 750ml. Add 2.5g of sodium benzoate and an ethanol solution containing 0.5g of ethylparaben, stir to dissolve, let stand for two days, and filter. Add 200g of sucrose, stir to dissolve, boil, add water to 1000ml, and dispense into containers.

[0059] Example 1: Study on the characteristic chromatographic method of Achyranthes bidentata throat-clearing compound

[0060] Preparation of reference solution: Accurately weigh appropriate amounts of chlorogenic acid, caffeic acid, bergenin, geniposide, and catechin reference standards and place them in the same 25 mL volumetric flask. Add an appropriate amount of a 1:1 mixture of methanol and 0.05% phosphoric acid aqueous solution, sonicate at 250 W and 40 kHz for 15 minutes to ensure complete dissolution, cool, dilute to the mark with the same mixed solvent, shake well, and filter through a 0.22 μm microporous membrane to prepare a mixed reference solution containing 100 µg of chlorogenic acid, 50 µg of caffeic acid, 120 µg of bergenin, 150 µg of geniposide, and 80 µg of catechin per mL.

[0061] Preparation of the test solution: Accurately measure 5 mL of the Achyranthes bidentata throat-clearing compound sample and place it in a stoppered conical flask. Accurately add 15 mL of a multiphase composite solvent composed of water-methanol-0.1% formic acid aqueous solution (volume ratio 1:3:1), seal tightly, weigh, and extract with ultrasound assistance in a 45℃ water bath (power 300W, frequency 40kHz) for 20 minutes. Remove, cool, and replenish the lost weight with the same composite solvent. Shake well and filter through a 0.45μm microporous membrane. Accurately measure 10 mL of the filtrate and pass it at a flow rate of 2 mL / min through a Waters Oasis HLB solid-phase extraction column (200 mg, 6 cc) that has been activated by equilibrium with 5 mL of methanol and 5 mL of water. Sequentially extract with 5 mL of water and 5 mL of... Elute with 5% methanol aqueous solution, discard the eluent, and finally elute with 8 mL of 80% methanol solution containing 0.1% formic acid. Collect the eluent, dry it under nitrogen at 40°C, accurately dissolve the residue in 50% methanol solution containing 0.05% formic acid and make up to 2 mL, vortex to mix, filter through a 0.22 μm nylon microporous membrane, and collect the filtrate to obtain the test solution.

[0062] Determination method: Accurately pipette 10 μL each of the reference solution and the test solution and inject them into the liquid chromatograph, and record the chromatogram.

[0063] Chromatographic conditions: Column: Octadecylsilane-bonded silica gel column (size: 4.6mm × 250mm, 5μm), such as Phenomenex Luna C18 column or equivalent column; Mobile phase: Acetonitrile:methanol:tetrahydrofuran (v / v) as mobile phase A, and 0.05% phosphoric acid aqueous solution containing 0.02% triethylamine as mobile phase B, gradient elution as specified in the table below; Flow rate: 1.0 mL / min; Column temperature: 30℃; Detection wavelength: 330 nm; The theoretical plate number calculated based on the chlorogenic acid peak should not be less than 5000.

[0064] .

[0065] (1) System applicability

[0066] Accurately pipette 10 µl of the reference solution and repeat the injection 6 times. Calculate the RSD values ​​of the retention times of chlorogenic acid, caffeic acid, bergenin, geniposide, and catechin. The theoretical plate number and retention time are shown in the table below.

[0067] Table 1 System Applicability Results

[0068] .

[0069] Table 2 System applicability retention time (min) results

[0070] .

[0071] The results show that the theoretical plate number of chlorogenic acid is greater than 5000 and the RSD of retention time is less than 2.0%, indicating good system suitability.

[0072] (2) Exclusivity

[0073] Accurately pipette 10 μl each of the negative solution, reference solution, and test solution, and inject them into the liquid chromatograph for determination.

[0074] Summary: The results show that chlorogenic acid, caffeic acid, bergenin, geniposide, and catechin in the test solution and the reference solution have the same chromatographic peaks at the corresponding retention times. The resolution of chlorogenic acid, caffeic acid, bergenin, geniposide, and catechin with adjacent peaks is greater than 1.50, and there is no interference from the negative sample solution, indicating good method specificity.

[0075] (3) Repeatability

[0076] Accurately measure 5 mL of Achyranthes bidentata throat-clearing compound and prepare 6 test solutions according to the test solution preparation method. Inject the solutions, determine the relative retention time and relative peak area of ​​each chromatographic peak, and calculate the relative standard deviation. The results are as follows (see below). Figure 1 .

[0077] Table 3. Repeatability Relative Retention Time Results

[0078] .

[0079] Table 4. Repeatability of relative peak area results

[0080] .

[0081] The results show that the relative retention time RSD of each chromatographic peak is less than 2.0%, and the relative peak area RSD is less than 3.0%, indicating good repeatability.

[0082] (4) Intermediate precision

[0083] At different times, another analyst conducted the following experiments using different chromatographs.

[0084] Accurately measure 5 ml of Achyranthes bidentata throat-clearing compound, take 6 portions, and prepare 6 test solutions according to the test solution preparation method. Inject the samples, determine the relative retention time and relative peak area of ​​each chromatographic peak, and calculate the relative standard deviation.

[0085] Calculate the relative retention time and relative peak area of ​​each chromatographic peak for 12 samples for repeatability and intermediate precision, and calculate the relative standard deviation.

[0086] Table 5 Results of intermediate precision relative retention time

[0087] .

[0088] Table 6 Results of intermediate precision relative peak area

[0089] .

[0090] The results show that the relative retention time RSD of each characteristic peak is less than 2.0% and the relative peak area RSD is less than 3.0%; when n=12, the relative retention time RSD of each characteristic peak is less than 2.0% and the relative peak area RSD is less than 3.0%, indicating good intermediate precision.

[0091] (5) Solution stability

[0092] Accurately measure 5 mL of Achyranthes bidentata throat-clearing compound and prepare one test solution according to the test solution preparation method. Inject the solution at 0, 2, 4, 8, 12, and 24 hours, respectively, and determine the relative retention time and relative peak area of ​​each chromatographic peak. Calculate the relative standard deviation. See [link to relevant documentation]. Figure 2 .

[0093] Table 7 Results of relative retention time for stability

[0094] .

[0095] Table 8. Results of relative peak area for stability

[0096] .

[0097] The results show that the relative retention time RSD of each characteristic peak is less than 2.0%, the relative peak area RSD is less than 3.0%, and the solution has good stability.

[0098] (6) Characteristic spectrum determination

[0099] Chromatographic conditions: Column: Octadecylsilane-bonded silica gel column (size: 4.6mm × 250mm, 5μm), such as Phenomenex Luna C18 column or equivalent column; Mobile phase: Acetonitrile:methanol:tetrahydrofuran (v / v) as mobile phase A, and 0.05% phosphoric acid aqueous solution containing 0.02% triethylamine as mobile phase B, gradient elution as specified in the table below; Flow rate: 1.0 mL / min; Column temperature: 30℃; Detection wavelength: 330 nm; The theoretical plate number calculated based on the chlorogenic acid peak should not be less than 5000.

[0100] .

[0101] Preparation of reference solution: Accurately weigh appropriate amounts of chlorogenic acid, caffeic acid, bergenin, geniposide, and catechin reference standards and place them in the same 25 mL volumetric flask. Add an appropriate amount of a 1:1 mixture of methanol and 0.05% phosphoric acid aqueous solution, sonicate at 250 W and 40 kHz for 15 minutes to ensure complete dissolution, cool, dilute to the mark with the same mixed solvent, shake well, and filter through a 0.22 μm microporous membrane to prepare a mixed reference solution containing 100 µg of chlorogenic acid, 50 µg of caffeic acid, 120 µg of bergenin, 150 µg of geniposide, and 80 µg of catechin per mL.

[0102] Preparation of the test solution: Accurately measure 5 mL of the Achyranthes bidentata throat-clearing compound sample and place it in a stoppered conical flask. Accurately add 15 mL of a multiphase composite solvent composed of water-methanol-0.1% formic acid aqueous solution (volume ratio 1:3:1), seal tightly, weigh, and extract with ultrasound assistance in a 45℃ water bath (power 300W, frequency 40kHz) for 20 minutes. Remove, cool, and replenish the lost weight with the same composite solvent. Shake well and filter through a 0.45μm microporous membrane. Accurately measure 10 mL of the filtrate and pass it at a flow rate of 2 mL / min through a Waters Oasis HLB solid-phase extraction column (200 mg, 6 cc) that has been activated by equilibrium with 5 mL of methanol and 5 mL of water. Sequentially extract with 5 mL of water and 5 mL of... Elute with 5% methanol aqueous solution, discard the eluent, and finally elute with 8 mL of 80% methanol solution containing 0.1% formic acid. Collect the eluent, dry it under nitrogen at 40°C, accurately dissolve the residue in 50% methanol solution containing 0.05% formic acid and make up to 2 mL, vortex to mix, filter through a 0.22 μm nylon microporous membrane, and collect the filtrate to obtain the test solution.

[0103] Assay: Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph and record the chromatograms. Figure 3 , Figure 4 As shown, Figure 3 This is a chromatogram of the reference solution for the characteristic chromatogram of the present invention. Figure 4The chromatogram of the test sample solution is a characteristic chromatogram of the present invention.

[0104] The chromatogram of the test sample showed five chromatographic peaks. Peaks 1, 2, 3, 4, and 5 corresponded to the reference peaks of chlorogenic acid, caffeic acid, bergenin, geniposide, and catechin, respectively. Using the chlorogenic acid reference peak as the S peak, the relative retention times of each characteristic peak and the S peak were calculated. All relative retention times were within ±10% of the specified values. The specified values ​​were: 1.63 (peak 2), 2.59 (peak 3), 3.03 (peak 4), and 3.52 (peak 5).

Claims

1. A method for constructing a characteristic spectrum of a traditional Chinese medicine formula for clearing the throat, characterized in that, Includes the following steps: Step 1: Preparation of reference solution: Accurately weigh chlorogenic acid, caffeic acid, bergenin, geniposide and catechin reference standards, place them in the same volumetric flask, add a mixed solvent of methanol and 0.05% phosphoric acid aqueous solution, sonicate to dissolve completely, cool, then dilute with a mixed solvent of methanol and 0.05% phosphoric acid aqueous solution, filter through a 0.22μm microporous membrane to prepare a mixed reference solution containing 100µg chlorogenic acid, 50µg caffeic acid, 120µg bergenin, 150µg geniposide and 80µg catechin per mL. Step Two: Preparation of the test solution: Accurately measure the sample of Achyranthes bidentata throat-clearing compound, add a multiphase composite solvent composed of water-methanol-0.1% formic acid aqueous solution, extract ultrasonically in a water bath, then remove and cool, then replenish the lost weight with water-methanol-0.1% formic acid aqueous solution, shake well, and filter first through a 0.45μm microporous membrane; accurately measure the subsequent filtrate, pass it through a Waters Oasis HLB solid phase extraction column activated by methanol and water equilibrium at a flow rate of 2mL / min, wash successively with water and 5% methanol aqueous solution, discard the eluent, and finally elute with 80% methanol solution containing 0.1% formic acid, collect the eluent, blow the eluent dry with nitrogen to obtain the residue, accurately dissolve the residue with 50% methanol solution containing 0.05% formic acid, vortex mix well, filter through a 0.22μm nylon microporous membrane, and collect the subsequent filtrate to obtain the test solution; The preparation of the Achyranthes bidentata throat-clearing compound is as follows: 3.3 parts of Achyranthes bidentata, 2.5 parts of Ilex chinensis, 2.5 parts of Myrica rubra, and 1 part of Lophatherum gracile. All ingredients are decocted twice with water, 1.5 hours each time. The decoctions are combined, filtered, and the filtrate is concentrated to 750 mL. 2.5 g of sodium benzoate and an ethanol solution containing 0.5 g of ethylparaben are added, stirred to dissolve, and allowed to stand for two days. The mixture is then filtered again. 200 g of sucrose is added to the filtrate, stirred to dissolve, boiled, and water is added to 1000 mL. The mixture is then dispensed into containers. Step 3: Determination method: Accurately pipette the reference solution and the test solution separately, inject them into the liquid chromatograph, and record the chromatograms; Chromatographic conditions: Chromatographic column: An octadecylsilane-bonded silica gel column was used as the packing material. Mobile phase: Acetonitrile:methanol:tetrahydrofuran (v / v) was used as mobile phase A, and 0.05% phosphoric acid aqueous solution containing 0.02% triethylamine was used as mobile phase B, with gradient elution performed as specified. Flow rate: 1.0 mL / min; Column temperature: 30℃; Detection wavelength: 330 nm; Theoretical plate number calculated based on the chlorogenic acid peak was not less than 5000. The gradient elution procedure is as follows: From 0 to 8 minutes, the volume ratio of mobile phase A to phase B changed from 5:95 to 11:

89. In 8-10 minutes, the volume ratio of mobile phase A to phase B changed from 11:89 to 12:

88. 10~14 min, the volume ratio of mobile phase A: phase B changes from 12:88 to 13:87; 14~25 min, the volume ratio of mobile phase A: phase B changes from 13:87 to 15:85; 25~40min, the volume ratio of mobile phase A: phase B changes from 15:85 to 22:78; For 40-60 minutes, the volume ratio of mobile phase A to phase B changes from 22:78 to 39:

61. At 60-61 min, the volume ratio of mobile phase A to phase B changed from 39:61 to 5:

95. 61~70min, mobile phase A: phase B, volume ratio 5:95; Step 4: Generate a reference characteristic chromatogram. Select the chromatographic peaks that are present in the chromatograms of different batches of Achyranthes bidentata throat-clearing compound as common peaks, and use the average value calculation method to generate a reference characteristic chromatogram of Achyranthes bidentata throat-clearing compound.

2. The method for constructing the characteristic spectrum of the Achyranthes bidentata throat-clearing compound according to claim 1, characterized in that, In step one, the volume ratio of methanol to 0.05% phosphoric acid aqueous solution in the mixed solvent of methanol and 0.05% phosphoric acid aqueous solution is 1:

1.

3. The method for constructing the characteristic spectrum of the Achyranthes bidentata throat-clearing compound according to claim 1, characterized in that, In step one, the ultrasonic conditions are: 250W, 40kHz ultrasonic treatment for 15 minutes.

4. The method for constructing the characteristic spectrum of the Achyranthes bidentata throat-clearing compound according to claim 1, characterized in that, In step two, the volume ratio of water-methanol-0.1% formic acid aqueous solution is 1:3:

1.

5. The method for constructing the characteristic spectrum of the Achyranthes bidentata throat-clearing compound according to claim 1, characterized in that, In step two, the ultrasonic conditions are 300W power and 40kHz frequency.

6. The method for constructing the characteristic spectrum of the Achyranthes bidentata throat-clearing compound according to claim 1, characterized in that, In step two, the Waters Oasis HLB solid-phase extraction column was 200 mg, 6 cc.

7. The method for constructing the characteristic spectrum of the Achyranthes bidentata throat-clearing compound according to claim 1, characterized in that, In step three, the chromatographic column is 4.6 mm × 250 mm, 5 μm.

8. The method for constructing the characteristic spectrum of the Achyranthes bidentata throat-clearing compound according to claim 1, characterized in that, The reference characteristic chromatogram generated in step four shows five chromatographic peaks, of which peaks 1, 2, 3, 4, and 5 correspond to the reference peaks of chlorogenic acid, caffeic acid, bergenin, geniposide, and catechin, respectively.

9. The method for constructing the characteristic spectrum of the Achyranthes bidentata throat-clearing compound according to claim 1, characterized in that, The reference characteristic spectrum generated in step four uses the chlorogenic acid reference peak as the S peak. The relative retention times of each characteristic peak and the S peak are calculated. The relative retention times are all within ±10% of the specified values. The specified values ​​are: peak 2 is 1.63, peak 3 is 2.59, peak 4 is 3.03, and peak 5 is 3.52.

Images