Construction method of characteristic chromatogram of traditional Chinese medicine compound preparation for Alzheimer's disease

Abstract

The invention belongs to the technical field of traditional Chinese medicine analysis, and particularly relates to a construction method of a characteristic spectrum of a traditional Chinese medicine compound preparation for Alzheimer's disease. According to the method, five characteristic components are scientifically selected as reference substances aiming at the complex chemical composition characteristics of the traditional Chinese medicine compound preparation for the first time, a characteristic spectrum for simultaneously representing various components such as flavone, saponin and oligosaccharide ester is established, the limitation of single index component control is overcome, and the overall quality of a product can be more comprehensively and truly reflected. According to the method, an integrated pretreatment strategy of composite solvent extraction, liquid-liquid distribution low-temperature precipitation and mixed mode solid phase extraction (SPE) purification is innovatively adopted. According to the method, the solvent composition is optimized, so that efficient and synchronous extraction of various target components is realized.

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 a traditional Chinese medicine compound preparation for Alzheimer's disease. Background Technology

[0002] Alzheimer's disease (AD) is a common neurodegenerative disease, clinically characterized by progressive cognitive impairment and memory decline. Currently, the selection of drugs for treating this disease is limited, and most primarily aim to alleviate symptoms, failing to fundamentally slow or reverse disease progression. Traditional Chinese medicine compound preparations, due to their multi-component, multi-target, and multi-pathway holistic regulatory characteristics, are receiving increasing attention in the prevention and treatment of Alzheimer's disease.

[0003] The present invention relates to a compound granule preparation for treating Alzheimer's disease, composed of Epimedium, Polygala tenuifolia, ginseng, and natural borneol. In this preparation, Epimedium tonifies kidney yang and strengthens bones and muscles; its flavonoid glycosides are considered an important material basis for improving cognitive function. Polygala tenuifolia calms the mind and improves intelligence; its saponins and oligosaccharide esters have protective effects on the nervous system. Ginseng greatly replenishes vital energy, improves intelligence, and calms the mind; its ginsenosides have significant effects on improving memory and anti-aging. Natural borneol opens the orifices and refreshes the mind, helping to guide the medicine upwards and promote blood-brain barrier permeability. The combined effects of these herbs are to improve intelligence, strengthen the brain, tonify the kidneys, and calm the mind, making it suitable for the adjunctive treatment of Alzheimer's disease and related cognitive impairments.

[0004] However, this formulation is a typical complex system consisting of multiple medicinal materials and various types of components. Its chemical composition encompasses different categories with a wide range of polarities: including weakly polar icariin flavonoids, moderately polar ginsenosides, and strongly polar polygala saponins and oligosaccharide esters. Under current analytical techniques, a single chromatographic method is insufficient to achieve the simultaneous and effective separation and detection of these diverse active ingredients with significantly different properties, resulting in an incomplete quality evaluation and an inability to truly reflect the overall material basis and intrinsic quality of the formulation.

[0005] Furthermore, the excipients introduced during the preparation of this formulation, as well as the complex matrices such as polysaccharides, pigments, and proteins contained in the medicinal materials themselves, can severely interfere with the detection of target components. Traditional pretreatment methods often fail to effectively remove matrix interference while efficiently extracting multiple target components, easily leading to problems such as unstable baselines, masking of characteristic peaks, and poor method reproducibility in chromatographic analysis. Meanwhile, how to scientifically select a set of multi-component references that can represent the overall chemical characteristics of the formulation and construct a characteristic spectrum that is comprehensive, specific, and transferable is also a key technical bottleneck in achieving stable and controllable quality of this formulation.

[0006] Therefore, for this traditional Chinese medicine compound preparation for treating Alzheimer's disease, there is an urgent need to establish a feature map construction method that can systematically solve the problems of its component complexity, matrix interference, and method robustness, so as to provide a scientific basis and technical guarantee for its production process control, batch-to-batch quality consistency, and stable and reliable clinical efficacy.

[0007] The challenges in detecting the characteristic chromatograms of this formulation lie in its diverse chemical composition and wide polarity, including weakly polar icariin, moderately polar ginsenosides, and strongly polar polygala saponins and oligosaccharide esters. Under single chromatographic conditions, it is difficult to achieve simultaneous and effective separation and detection of all characteristic components. Furthermore, the presence of numerous coexisting polysaccharides, pigments, and excipients in the compound formulation causes significant matrix interference, making it difficult for traditional pretreatment methods to balance high extraction efficiency with high sample purity. This results in unstable baselines, masking of characteristic peaks, or poor method reproducibility in the characteristic chromatograms. In addition, scientifically selecting representative multi-component references to construct a characteristic chromatogram that can comprehensively characterize product quality and is stably transferable across different instruments and laboratories presents a significant technical challenge. Summary of the Invention

[0008] The purpose of this invention is to provide a method for constructing a characteristic spectrum of a traditional Chinese medicine compound preparation for Alzheimer's disease, and to improve the efficacy and safety of the traditional Chinese medicine compound preparation for treating Alzheimer's disease in clinical applications by establishing quality control standards.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution: A method for constructing a characteristic profile of a traditional Chinese medicine compound preparation for Alzheimer's disease, comprising the following steps: Step 1: Preparation of reference solution: Take icariin, cuscuta C, polygala saponin, 3,6'-disinoyl sucrose and ginsenoside Rg1 reference standards, add 70% (volume fraction) methanol solution, sonicate to fully dissolve, cool, dilute to the mark with 70% methanol, shake well, filter through a 0.22µm microporous membrane to prepare a mixed reference solution containing 10µg of icariin, 10µg of cuscuta C, 8µg of polygala saponin, 8µg of 3,6'-disinoyl sucrose and 12µg of ginsenoside Rg1 per mL.

[0010] Step Two: Preparation of the test solution: Take the powder of this product, add a methanol-acetonitrile-water mixed solvent, weigh it, sonicate it, and cool it. Then, replenish the weight lost after sonication with a methanol-acetonitrile-water mixed solvent, shake well, and filter. Accurately measure the filtrate, place it in a centrifuge tube, add an equal volume of ethyl acetate-n-butanol mixed solvent, vortex, and allow it to stand for separation. Separate the upper organic phase, let it stand overnight at 4°C to precipitate and remove impurities, centrifuge, and obtain the supernatant. Take the supernatant and concentrate it to dryness under reduced pressure in a 40°C water bath to obtain the residue. Dissolve the residue obtained by concentrated to dryness under reduced pressure in a 5% formic acid aqueous solution to obtain the solution. Load the solution onto activated Oasis. The MAX mixed-mode solid-phase extraction column was washed sequentially with 5% formic acid and methanol, and the washings were discarded. Then, 5 mL of methanol solution containing 2% ammonia was used for elution. The eluent was collected and concentrated to dryness under reduced pressure in a 40°C water bath to obtain the residue. The residue obtained by concentrating the eluent to dryness under reduced pressure was dissolved and diluted with 70% methanol. The diluted solution was filtered through a 0.22µm microporous membrane, and the filtrate was collected to obtain the test solution.

[0011] Step 3: Determination method: Accurately pipette 10 µL each of the reference solution and the test solution into the liquid chromatograph and record the chromatogram.

[0012] Chromatographic conditions: Column: GL Sciences InertSustain C18; Mobile phase: 10 mmol / L potassium dihydrogen phosphate buffer (pH 3.5)-methanol (95:5 v / v) as mobile phase A, and acetonitrile-isopropanol solution (80:20 v / v) as mobile phase B, with gradient elution as specified; Flow rate: 1.0 mL / min; Column temperature: 30℃; Detection wavelength: 320 nm; The gradient elution procedure is as follows: 0~15min, mobile phase A: phase B, volume ratio 80:20; 15~30min, the volume ratio of mobile phase A: phase B changes from 80:20 to 76:24; For 30-50 minutes, the volume ratio of mobile phase A to phase B changes from 76:24 to 75:25. 50~55 min, the volume ratio of mobile phase A: phase B changes from 75:25 to 73:27; From 55 to 75 minutes, the volume ratio of mobile phase A to phase B changes from 73:27 to 72:28. At 75-76 min, the volume ratio of mobile phase A to phase B changed from 72:28 to 65:35. 76~85min, mobile phase A: phase B, volume ratio 65:35; 85~110min, the volume ratio of mobile phase A: phase B changes from 65:35 to 64:36; 110~120min, the volume ratio of mobile phase A: phase B changes from 64:36 to 60:40; 120~121 min, the volume ratio of mobile phase A: phase B changes from 60:40 to 80:20; Step 4: Generate a reference characteristic chromatogram: Select chromatographic peaks that are present in the chromatograms of different batches of traditional Chinese medicine compound preparations as common peaks, and use the average value calculation method to generate a reference characteristic chromatogram of the traditional Chinese medicine compound preparations.

[0013] Furthermore, in step one, the ultrasonic conditions are: 250 W, 40 kHz ultrasonic treatment for 30 minutes.

[0014] Furthermore, in step two, the volume ratio of the methanol-acetonitrile-water mixed solvent is 4:3:3.

[0015] Furthermore, in step two, the volume ratio of ethyl acetate to n-butanol is 1:1.

[0016] Furthermore, in step two, the Oasis MAX mixed-mode solid-phase extraction column has a specification of 60 mg / 3 mL.

[0017] 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 3,6'-disinoyl sucrose, ginsenoside Rg1, asparagine C, polygala tenuifolia saponin, and icariin, respectively.

[0018] Furthermore, in step four, the relative retention times of each characteristic peak and the S peak are calculated using the icariin reference peak as the S peak. The relative retention times are all within ±10% of the specified values. The specified values ​​are: 0.30 (peak 1), 0.84 (peak 2), 0.89 (peak 3), and 0.95 (peak 4).

[0019] Compared with the prior art, the advantages of the present invention are as follows: 1. This invention constructs a comprehensive and exclusive multi-index feature map: For the first time, based on the complex chemical composition of this traditional Chinese medicine compound preparation, five characteristic components were scientifically selected as references, and a feature map was established to simultaneously characterize multiple components such as flavonoids, saponins, and oligosaccharide esters. This overcomes the limitations of controlling single-index components and can more comprehensively and realistically reflect the overall quality of the product.

[0020] 2. A highly efficient and selective sample pretreatment method was developed: An innovative integrated pretreatment strategy of "compound solvent extraction - liquid-liquid partitioning low-temperature precipitation - mixed-mode solid-phase extraction (SPE) purification" was adopted. This method achieves efficient simultaneous extraction of multiple target components by optimizing solvent composition; through low-temperature precipitation and selective elution using mixed-mode SPE (reverse-phase / anion exchange), matrix interferences such as polysaccharides, pigments, and proteins are significantly removed, greatly improving the purity of the sample solution. This ensures stable baselines and clear peaks in the characteristic chromatograms, significantly enhancing the specificity and signal-to-noise ratio of the method.

[0021] 3. Optimized chromatographic separation conditions suitable for complex systems: A binary gradient elution system composed of composite solvents was designed and adopted. This mobile phase system uses buffer salts to control pH to improve peak shape and introduces isopropanol to adjust solvent selectivity. In particular, it optimizes the separation between structurally similar saponins and flavonoid glycosides, enabling baseline separation of all five characteristic peaks in a single analysis, resulting in rich and highly characteristic chromatograms.

[0022] 4. A robust and reliable quality control method has been established: Through systematic methodological verification, the characteristic spectral method established in this invention exhibits high precision, good reproducibility, and excellent solution stability. The specified relative retention time in the method ensures good transferability and comparability of the spectral data across different instruments and laboratories, providing a stable and reliable technical means for the production process control, finished product quality evaluation, and batch-to-batch consistency assessment of traditional Chinese medicine compound preparations for the treatment of Alzheimer's disease. Attached Figure Description

[0023] The invention will now be further described with reference to the accompanying drawings; Figure 1 This is a repeatable chromatogram overlay of the present invention; S1-S6 are chromatograms obtained from 6 test solutions prepared according to the test solution preparation method; Figure 2 This is a chromatogram of the stability of the present invention; 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. 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. Figure 4 The chromatogram of the test sample solution is a characteristic chromatogram of the present invention. Detailed Implementation

[0024] 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: I. Instruments and Reagents 1.1 Instruments and Equipment .

[0025] 1.2 Test Materials .

[0026] 1.3 Reference Standard Information .

[0027] 1.4 Sample Information .

[0028] The traditional Chinese medicine compound preparation of this invention is as follows: 37.5g total flavonoids of Epimedium, 150g total saponins of ginseng, 180g total saponins of Polygala tenuifolia, and 25g natural borneol.

[0029] Example 1: Study on the characteristic chromatographic method of the formulation Preparation of reference solution: Accurately weigh appropriate amounts of icariin, astragalin C, polygala tenuifolia saponin, 3,6'-disinoyl sucrose, and ginsenoside Rg1 reference standards, and place them in the same 25 mL volumetric flask. Add an appropriate amount of 70% (v / v) methanol solution, sonicate at 250 W and 40 kHz for 15 minutes to ensure complete dissolution, cool, dilute to the mark with 70% methanol, shake well, and filter through a 0.22 µm microporous membrane to prepare a mixed reference solution containing 10 µg of icariin, 10 µg of astragalin C, 8 µg of polygala tenuifolia saponin, 8 µg of 3,6'-disinoyl sucrose, and 12 µg of ginsenoside Rg1 per mL.

[0030] Preparation of the test solution: Accurately weigh 1.0 g of the powder and place it in a stoppered conical flask. Accurately add 30 mL of a methanol-acetonitrile-water (volume ratio 4:3:3) mixed solvent, weigh the solution, and sonicate (power 250 W, frequency 40 kHz) for 30 minutes. Cool the flask, then replenish the lost weight with the methanol-acetonitrile-water (volume ratio 4:3:3) mixed solvent, shake well, and filter. Accurately measure 15 mL of the filtrate and place it in a centrifuge tube. Add an equal volume of ethyl acetate-n-butanol (volume ratio 1:1) mixed solvent, vortex for 5 minutes, and allow the layers to separate. Separate the upper organic phase, allow it to stand overnight at 4℃ to precipitate and remove impurities, centrifuge, and obtain the supernatant. Concentrate the supernatant to dryness under reduced pressure in a 40℃ water bath to obtain the residue. Dissolve the residue obtained by concentrating the supernatant to dryness under reduced pressure in 2 mL of 5% formic acid aqueous solution and load it onto activated Oasis. The MAX mixed-mode solid-phase extraction column (60 mg / 3 mL) was washed sequentially with 3 mL of 5% (v / v) formic acid solution and 3 mL of methanol. The washings were discarded. The column was then eluted with 5 mL of methanol solution containing 2% (v / v) ammonia solution. The eluent was collected and concentrated to dryness under reduced pressure in a 40°C water bath to obtain a residue. The residue obtained by further concentration of the eluent to dryness under reduced pressure in a 40°C water bath was dissolved in 70% methanol and transferred to a 10 mL volumetric flask. The solution was diluted to the mark with 70% methanol, shaken well, and filtered through a 0.22 µm microporous membrane. The filtrate was collected to obtain the test solution.

[0031] Determination method: Accurately pipette 10 µL each of the reference solution and the test solution into the liquid chromatograph and record the chromatogram.

[0032] Chromatographic conditions: Column: GL Sciences InertSustain C18; Mobile phase: 10 mmol / L potassium dihydrogen phosphate buffer (pH 3.5)-methanol (95:5 v / v) as mobile phase A, and acetonitrile-isopropanol solution (80:20 v / v) as mobile phase B, with gradient elution as specified in the table below; Flow rate: 1.0 mL / min; Column temperature: 30℃; Detection wavelength: 320 nm; .

[0033] (1) System applicability Accurately pipette 10 µL of the reference solution and repeat the injection 6 times. Calculate the retention time RSD values ​​of icariin, astragalin C, polygala tenuifolia saponin, 3,6'-disinanoyl sucrose, and ginsenoside Rg1. The theoretical plate number and retention time are shown in the table below.

[0034] Table 1 System Applicability Results .

[0035] Table 2 System applicability retention time (min) results .

[0036] The results show that the theoretical plate number of icariin is greater than 5000 and the RSD of retention time is less than 2.0%, indicating good system suitability.

[0037] (2) Exclusivity Accurately pipette 10 µL each of the negative solution, reference solution, and test solution, and inject them into the liquid chromatograph for determination.

[0038] Summary: The results show that icariin, cuscuta citrate C, polygala tenuifolia saponin, 3,6'-disinanoyl sucrose, and ginsenoside Rg1 in the test solution and the reference solution have the same chromatographic peaks at the corresponding retention times. The resolution of icariin, cuscuta citrate C, polygala tenuifolia saponin, 3,6'-disinanoyl sucrose, and ginsenoside Rg1 from adjacent peaks is greater than 1.50, and there is no interference from the negative sample solution, indicating good method specificity.

[0039] (3) Repeatability Take an appropriate amount of the traditional Chinese medicine compound preparation, grind it into a fine powder, and accurately weigh about 1.0 g. Prepare 6 test solutions according to the test solution preparation method, inject the samples, and determine the relative retention time and relative peak area of ​​each chromatographic peak. Calculate the relative standard deviation. The results are as follows (see below). Figure 1 .

[0040] Table 3. Repeatability Relative Retention Time Results .

[0041] Table 4. Repeatability of relative peak area results .

[0042] 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 2.0%, indicating good repeatability.

[0043] (4) Intermediate precision At different times, another analyst conducted the following experiments using different chromatographs.

[0044] Take an appropriate amount of traditional Chinese medicine compound preparation, grind it into a fine powder, take about 1.0g, accurately weigh it, 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.

[0045] 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.

[0046] Table 5 Results of intermediate precision relative retention time .

[0047] Table 6 Results of intermediate precision relative peak area .

[0048] The results show that the relative retention time RSD and relative peak area RSD of each characteristic peak are less than 2.0%; when n=12, the relative retention time RSD and relative peak area RSD of each characteristic peak are less than 2.0%, indicating good intermediate precision.

[0049] (5) Solution stability Take an appropriate amount of the traditional Chinese medicine compound preparation, grind it into a fine powder, accurately weigh about 1.0 g, and prepare one test solution according to the test solution preparation method. Inject the sample at 0 hours, 2 hours, 4 hours, 8 hours, 12 hours, 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 .

[0050] Table 7 Results of relative retention time for stability .

[0051] Table 8. Results of relative peak area for stability .

[0052] 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 2.0%, and the solution has good stability.

[0053] (6) Characteristic spectrum determination Chromatographic conditions: Column: GL Sciences InertSustain C18; Mobile phase: 10 mmol / L potassium dihydrogen phosphate buffer (pH 3.5)-methanol (95:5 v / v) as mobile phase A, and acetonitrile-isopropanol solution (80:20 v / v) as mobile phase B, with gradient elution as specified in the table below; Flow rate: 1.0 mL / min; Column temperature: 30℃; Detection wavelength: 320 nm; .

[0054] Preparation of reference solution: Accurately weigh appropriate amounts of icariin, astragalin C, polygala saponin, 3,6'-disinoyl sucrose, and ginsenoside Rg1 reference standards, and place them in the same 25 mL volumetric flask. Add an appropriate amount of 70% methanol solution, sonicate at 250 W and 40 kHz for 15 minutes to ensure complete dissolution, cool, dilute to the mark with 70% methanol, shake well, and filter through a 0.22 µm microporous membrane to prepare a mixed reference solution containing 10 µg of icariin, 10 µg of astragalin C, 8 µg of polygala saponin, 8 µg of 3,6'-disinoyl sucrose, and 12 µg of ginsenoside Rg1 per mL.

[0055] Preparation of test solution: Weigh approximately 1.0 g of the powder accurately, add 30 mL of methanol-acetonitrile-water (4:3:3) mixed solvent for ultrasonic extraction, filter, and then take the filtrate for liquid-liquid partitioning with ethyl acetate-n-butanol (1:1) and precipitation at 4°C to remove impurities; after concentration of the supernatant, dissolve the residue in 5% formic acid aqueous solution, and load the sample onto an activated Oasis MAX mixed mode solid phase extraction column (60 mg / 3 mL), wash successively with 5% formic acid aqueous solution and methanol, and finally elute the target component with methanol solution containing 2% ammonia; collect the eluent, concentrate to remove ammonia, dissolve the residue in 70% methanol and make up to 10 mL, filter through a 0.22 µm microporous membrane, and take the filtrate as the test solution.

[0056] Assay: Accurately pipette 10 µL each of the reference solution and the test solution into the liquid chromatograph and record the chromatograms. See the chromatogram for the reference solution below. Figure 3 The chromatogram of the test solution is shown in [reference needed]. Figure 4 .

[0057] The chromatogram of the test sample showed five chromatographic peaks. Peaks 1, 2, 3, 4, and 5 correspond to the reference peaks of 3,6'-disinoyl sucrose, ginsenoside Rg1, asarumin C, polygala tenuifolia saponin, and icariin, respectively. Using the icariin 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: 0.30 (peak 1), 0.84 (peak 2), 0.89 (peak 3), and 0.95 (peak 4).

Claims

1. A method for constructing a characteristic spectrum of a traditional Chinese medicine compound preparation for Alzheimer's disease, characterized in that, Includes the following steps: Step 1: Preparation of reference solution: Take the reference standards of icariin, citrofurantoin C10, polygala tenuifolia saponin, 3,6'-disinoyl sucrose and ginsenoside Rg1, add 70% methanol solution, sonicate to fully dissolve, cool, dilute with 70% methanol and shake well, filter through a microporous membrane to prepare a mixed reference solution containing icariin, citrofurantoin C10, polygala tenuifolia saponin, 3,6'-disinoyl sucrose and ginsenoside Rg1 per mL. Step Two: Preparation of test solution: Take the powder of traditional Chinese medicine compound preparation for Alzheimer's disease, add methanol-acetonitrile-water mixed solvent, weigh, sonicate and cool, then replenish the lost weight with methanol-acetonitrile-water mixed solvent, shake well, filter to obtain the filtrate; accurately measure the filtrate, place it in a centrifuge tube, then add an equal volume of ethyl acetate-n-butanol mixed solvent, vortex, let stand for layering, take the upper organic phase and let stand overnight at low temperature to precipitate and remove impurities, centrifuge to obtain the supernatant; take the supernatant and concentrate it to dryness in a water bath to obtain the residue, concentrate the supernatant to dryness in a water bath to obtain the residue, dissolve the residue in 5% formic acid aqueous solution to obtain the solution, and load the solution onto activated Oasis. The MAX mixed-mode solid-phase extraction column was washed sequentially with 5% formic acid and methanol, and the washings were discarded. Then, it was eluted with 5 mL of methanol solution containing 2% ammonia. The eluent was collected and concentrated to dryness under reduced pressure in a 40°C water bath to obtain the residue. The residue obtained by concentrating the eluent to dryness under reduced pressure in a water bath was dissolved and diluted with 70% methanol. The diluted solution was filtered through a microporous membrane, and the filtrate was collected to obtain the test solution. Step 3: Determination method: Accurately pipette 10 µL each of the reference solution and the test solution into the liquid chromatograph and record the chromatogram; Chromatographic conditions: Column: GL Sciences InertSustain C18; Mobile phase: 10 mmol / L potassium dihydrogen phosphate buffer (pH 3.5) at a volume ratio of 95:5 (methanol) as mobile phase A, and acetonitrile-isopropanol solution at a volume ratio of 80:20 (methanol) as mobile phase B, with gradient elution performed as specified; Flow rate: 1.0 mL / min; Column temperature: 30℃; Detection wavelength: 320 nm; The gradient elution procedure is as follows: 0~15min, mobile phase A: phase B, volume ratio 80:20; 15~30min, the volume ratio of mobile phase A: phase B changes from 80:20 to 76:24; For 30-50 minutes, the volume ratio of mobile phase A to phase B changes from 76:24 to 75:

25. 50~55 min, the volume ratio of mobile phase A: phase B changes from 75:25 to 73:27; From 55 to 75 minutes, the volume ratio of mobile phase A to phase B changes from 73:27 to 72:

28. At 75-76 min, the volume ratio of mobile phase A to phase B changed from 72:28 to 65:

35. 76~85min, mobile phase A: phase B, volume ratio 65:35; 85~110min, the volume ratio of mobile phase A: phase B changes from 65:35 to 64:36; 110~120min, the volume ratio of mobile phase A: phase B changes from 64:36 to 60:40; 120~121 min, the volume ratio of mobile phase A: phase B changes from 60:40 to 80:20; Step 4: Generate a reference characteristic chromatogram: Select chromatographic peaks that are present in the chromatograms of different batches of traditional Chinese medicine compound preparations as common peaks, and use the average value calculation method to generate a reference characteristic chromatogram of the traditional Chinese medicine compound preparations.

2. The method for constructing a characteristic spectrum of a traditional Chinese medicine compound preparation for Alzheimer's disease according to claim 1, characterized in that, In step one, the ultrasound conditions are: 250 W, 40 kHz ultrasound treatment for 30 minutes.

3. The method for constructing a characteristic spectrum of a traditional Chinese medicine compound preparation for Alzheimer's disease according to claim 1, characterized in that, In steps one and two, the pore size of the microporous membrane is 0.22µm.

4. The method for constructing a characteristic spectrum of a traditional Chinese medicine compound preparation for Alzheimer's disease according to claim 1, characterized in that, In step two, the volume ratio of the methanol-acetonitrile-water mixed solvent is 4:3:

3.

5. The method for constructing a characteristic spectrum of a traditional Chinese medicine compound preparation for Alzheimer's disease according to claim 1, characterized in that, In step two, the volume ratio of ethyl acetate to n-butanol is 1:

1.

6. The method for constructing a characteristic spectrum of a traditional Chinese medicine compound preparation for Alzheimer's disease according to claim 1, characterized in that, In step two, the Oasis MAX mixed-mode solid-phase extraction column has a specification of 60 mg / 3 mL.

7. The method for constructing a characteristic spectrum of a traditional Chinese medicine compound preparation for Alzheimer's disease according to claim 1, characterized in that, The reference characteristic chromatogram generated in step four shows five chromatographic peaks, among which peaks 1, 2, 3, 4, and 5 correspond to the reference peaks of 3,6'-disinoyl sucrose, ginsenoside Rg1, asparagine C, polygala tenuifolia saponin, and icariin, respectively.

8. The method for constructing a characteristic spectrum of a traditional Chinese medicine compound preparation for Alzheimer's disease according to claim 7, characterized in that, The control characteristic chromatograms generated in step four are used with the icariin 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: 0.30 for peak 1, 0.84 for peak 2, 0.89 for peak 3, and 0.95 for peak 4.

Images