A construction method of a polygonum pernyi characteristic atlas

Abstract

This invention belongs to the field of traditional Chinese medicine analysis technology, specifically relating to a method for constructing a characteristic chromatogram of *Phytolacca americana*. The method includes preparing a test solution and employing a specific gradient elution procedure under chromatographic conditions to obtain the characteristic chromatogram of *Phytolacca americana*. The characteristic chromatogram of *Phytolacca americana* obtained by the method provided by this invention exhibits good separation of characteristic peaks, clear material basis, and the ability to distinguish different sources of *Phytolacca americana*. It can separate the main chemical components of *Phytolacca americana*, resulting in more accurate, reliable, reproducible, and sensitive results.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine analysis technology, specifically relating to a method for constructing a characteristic map of vertically arranged Phytolacca acinosa. Background Technology

[0002] Pokeweed, a perennial robust herb belonging to the Phytolaccaceae family and the Phytolacca genus, is widely distributed in the red soil low hilly areas south of the Yangtze River. The main varieties currently distributed in China are *Pokeweed var. chinensis* (wild radish) and *Pokeweed pilosa* (American pokeweed, *Pokeweed pilosa*, and *Pokeweed pilosa* var. chinensis). It is also known by various names based on its morphology, such as large amaranth, mountain radish, flowering pokeweed, and rouge.

[0003] Currently, there are few reports on the characteristic spectra of *Phytolacca americana*. Relevant literature uses evaporative light scattering detectors for analysis, but the detection sensitivity is low, reproducibility is poor, and a systematic comparison of different source materials is lacking. Existing techniques also use isocratic elution procedures, which are difficult to use to separate the main chemical components of *Phytolacca americana*. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the defects of the prior art in obtaining the characteristic spectrum of vertically arranged pokeweed, such as low detection sensitivity, poor reproducibility, and difficulty in separating the main chemical components of vertically arranged pokeweed, thereby providing a method for constructing the characteristic spectrum of vertically arranged pokeweed.

[0005] To this end, the present invention provides the following technical solution.

[0006] This invention provides a method for constructing a vertically ordered feature map of Phytolacca acinosa, comprising the following steps:

[0007] Preparation of test solution: Prepare a test solution from the test sample;

[0008] Determination: The chromatographic conditions for high performance liquid chromatography include: acetonitrile as mobile phase A, phosphoric acid aqueous solution as mobile phase B, and gradient elution;

[0009] The gradient elution program includes: 0-5 min, 1% mobile phase A, 99% mobile phase B; 5-15 min, 1→8% mobile phase A, 99→92% mobile phase B; 15-25 min, 8→20% mobile phase A, 92→80% mobile phase B; 25-32 min, 20→30% mobile phase A, 80→70% mobile phase B; 32-37 min, 30→35% mobile phase A, 70→65% mobile phase B; 37-50 min, 35% mobile phase A, 65% mobile phase B; or...

[0010] 0-8 min, 1% mobile phase A, 99% mobile phase B; 8-18 min, 1→8% mobile phase A, 99→92% mobile phase B; 18-28 min, 8→20% mobile phase A, 92→80% mobile phase B; 28-35 min, 20→30% mobile phase A, 80→70% mobile phase B; 35-40 min, 30→35% mobile phase A, 70→65% mobile phase B; 40-50 min, 35% mobile phase A, 65% mobile phase B; or...

[0011] 0-5 min, 1% mobile phase A, 99% mobile phase B; 5-15 min, 1→8% mobile phase A, 99→92% mobile phase B; 15-25 min, 8→20% mobile phase A, 92→80% mobile phase B; 25-32 min, 20→30% mobile phase A, 80→70% mobile phase B; 32-40 min, 30→35% mobile phase A, 70→65% mobile phase B; 40-50 min, 35% mobile phase A, 65% mobile phase B.

[0012] The chromatographic conditions for the high-performance liquid chromatography (HPLC) method also include: using a Poroshell 120SB-Aq column with the following specifications: inner diameter 4.6 mm, column length 250 mm, and particle size 4 μm; and / or, column temperature 25-35℃; and / or, wavelength 210-254 nm; and / or, injection volume 5-15 μL; and / or, flow rate 0.8-1.2 ml / min; and / or, using 0.03-0.05% phosphoric acid aqueous solution as mobile phase B.

[0013] The gradient elution program includes: 0-5 min, 1% mobile phase A, 99% mobile phase B; 5-15 min, 1→8% mobile phase A, 99→92% mobile phase B; 15-25 min, 8→20% mobile phase A, 92→80% mobile phase B; 25-32 min, 20→30% mobile phase A, 80→70% mobile phase B; 32-37 min, 30→35% mobile phase A, 70→65% mobile phase B; 37-50 min, 35% mobile phase A, 65% mobile phase B.

[0014] The chromatographic conditions for the high-performance liquid chromatography (HPLC) method include: using a Poroshell 120SB-Aq column with an inner diameter of 4.6 mm, a column length of 250 mm, and a particle size of 4 μm; using acetonitrile as mobile phase A and 0.04% phosphoric acid aqueous solution as mobile phase B; a flow rate of 1 ml / min; a column temperature of 30 °C; and a detection wavelength of 210 nm.

[0015] The test samples were Phytolacca americana granules, Phytolacca americana standard decoction freeze-dried powder, Phytolacca americana medicinal material, or Phytolacca americana processed slices.

[0016] The preparation method of the test sample solution includes: taking the test sample, adding extraction solvent, extracting, and filtering;

[0017] Preferably, when preparing the test solution, the extraction solvent is at least one of methanol and water;

[0018] Preferably, the extraction solvent is an aqueous methanol solution; preferably, the volume fraction of methanol in the aqueous methanol solution is not less than 60%.

[0019] Preferably, the extraction method used in preparing the test solution is reflux extraction or ultrasonic extraction.

[0020] Furthermore, when the test sample is lyophilized powder or formulation granules, the mass of the test sample corresponding to each 1 ml of extraction solvent is 0.01 g to 0.08 g;

[0021] When the test sample is a medicinal material or decoction piece, the mass of the water extract corresponding to each 1 ml of extraction solvent is 0.01 g to 0.08 g.

[0022] The construction method also includes the preparation of a reference solution of a control medicinal material;

[0023] The preparation method of the reference solution of the reference medicinal material includes: taking the reference medicinal material, extracting it with water to obtain an aqueous extract, adding an extraction solvent to the aqueous extract, mixing well, and filtering.

[0024] Preferably, when preparing the reference medicinal material solution, the extraction solvent is at least one of methanol and water;

[0025] Preferably, the extraction solvent is an aqueous methanol solution; preferably, the volume fraction of methanol in the aqueous methanol solution is not less than 60%.

[0026] Preferably, the volume of water corresponding to each 1g of *Phytolacca acinosa* reference material is 25-100ml;

[0027] Preferably, the mass of the water extract corresponding to each 1 ml of extraction solvent is 0.01 g - 0.08 g;

[0028] Preferably, when obtaining the aqueous extract, the extraction method is reflux extraction or ultrasonic extraction.

[0029] In this invention, approximately 0.2g-0.3g of water extract can be obtained from every 1g of medicinal material or decoction pieces.

[0030] The construction method also includes the preparation of a reference solution;

[0031] The reference standard is at least one of the following: phytolaccoside A, erythrine, adenosine, guanosine, and L-tryptophan.

[0032] The feature map obtained by the construction method includes 9 feature peaks;

[0033] With peak 5 as reference peak 1, the relative retention time of peak 1 is 0.47, the relative retention time of peak 2 is 0.55, the relative retention time of peak 3 is 0.67, the relative retention time of peak 4 is 0.81, the relative retention time of peak 6 is 1.24, and the relative retention time of peak 7 is 1.64.

[0034] With peak 8 as the reference, the relative retention time of peaks 2 and 9 is 1.07.

[0035] The relative retention time should be within ±10% of the specified value.

[0036] The technical solution of this invention has the following advantages:

[0037] 1. The present invention provides a method for constructing a characteristic spectrum of *Phytolacca americana*, comprising the preparation of a test solution and the determination of the characteristic spectrum of *Phytolacca americana* under chromatographic conditions using a specific gradient elution procedure. The characteristic spectrum of *Phytolacca americana* obtained by the method provided by the present invention exhibits good separation of characteristic peaks, clear material basis, and the ability to distinguish different sources of *Phytolacca americana*. It can separate the main chemical components of *Phytolacca americana*, resulting in more accurate, reliable, reproducible, and sensitive results. Attached Figure Description

[0038] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0039] Figure 1 These are the characteristic spectra of the vertically arranged pokeweed formula granules and the vertically arranged pokeweed control medicinal material of Example 1 of the present invention; Figure 2 These are the characteristic chromatograms of three batches of *Phytolacca americana* formulation granules and 16 batches of *Phytolacca americana* standard decoction freeze-dried powder of this invention; Figure 3 This is a comparative feature map of vertically ordered pokeweed according to the present invention; Figure 4 This is a comparison diagram of the characteristic chromatogram of the vertical sequence pokeweed formula granules of this invention with that of the reference standard; Figure 5 This is the chromatogram obtained under chromatographic condition 1 in section 1.2 of Experimental Example 1 of this invention; Figure 6 This is the chromatogram obtained under chromatographic condition 2 in section 1.2 of Experimental Example 1 of this invention; Figure 7 This is the chromatogram obtained under chromatographic condition 3 in section 1.2 of Experimental Example 1 of this invention; Figure 8 This is the chromatogram obtained under chromatographic condition 4 in section 1.2 of Experimental Example 1 of this invention; Figure 9This is the chromatogram obtained under chromatographic condition 5 in section 1.2 of Experimental Example 1 of this invention; Figure 10 This is the chromatogram obtained under chromatographic condition 6 in section 1.2 of Experimental Example 1 of this invention; Figure 11 This is the chromatogram obtained under chromatographic condition 7 in section 1.2 of Experimental Example 1 of this invention; Figure 12 This is the chromatogram obtained under chromatographic condition 8 in section 1.2 of Experimental Example 1 of this invention; Figure 13 This is the chromatogram obtained under chromatographic condition 9 in section 1.2 of Experimental Example 1 of this invention; Figure 14 This is the chromatogram obtained under chromatographic condition 10 in section 1.2 of Experimental Example 1 of this invention; Figure 15 This is the chromatogram obtained under chromatographic condition 11 in section 1.2 of Experimental Example 1 of this invention; Figure 16 This is the chromatogram obtained under chromatographic condition 12 in section 1.2 of Experimental Example 1 of this invention; Figure 17 This is the chromatogram obtained under chromatographic condition 13 in section 1.2 of Experimental Example 1 of this invention; Figure 18 These are the chromatograms obtained at different wavelengths in Section 1.3 of Experimental Example 1 of this invention; Figure 19 This is the chromatogram obtained from the specificity investigation in Section 3.1 of Experimental Example 3 of this invention. Detailed Implementation

[0040] The following embodiments are provided to better understand the present invention and are not intended to limit the preferred embodiments. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention. Where specific experimental steps or conditions are not specified in the embodiments, they can be performed according to the conventional experimental steps or conditions described in the literature in the art. Reagents or instruments used, unless otherwise specified, are all commercially available conventional reagent products.

[0041] instrument

[0042]

[0043] Reagents and reagents:

[0044] reagents and reagents Grade / Specification batch number factory Phytolacca saponin A ≥98％ 14541 Shanghai Shidande Standard Technical Service Co., Ltd. Acetic acid Chromatographic purity P1622914 Thermo Fisher Scientific (China) Co., Ltd. Formic acid mass spectrometry U9260048 Shanghai Anpu Experimental Technology Co., Ltd. methanol Chromatographic purity 0212230404 Shanghai Xingke High Purity Solvent Co., Ltd. methanol Analytical Pure P2304265 Shanghai Titan Technology Co., Ltd. Phosphoric acid Chromatographic purity F6130152 Shanghai Titan Technology Co., Ltd. Acetonitrile Chromatographic purity 0114230403 Shanghai Xingke High Purity Solvent Co., Ltd. Vertical pokeweed formula granules 1g / bag 2208001-2208003 China Resources Sanjiu Modern Chinese Medicine Pharmaceutical Co., Ltd. Vertical order Pokeweed standard decoction freeze-dried powder / 2102001-2102016 China Resources Sanjiu Modern Chinese Medicine Pharmaceutical Co., Ltd.

[0045] Example 1

[0046] This embodiment provides a method for constructing a characteristic map of vertically ordered pokeweed formulation particles, including the following steps:

[0047] Preparation of the test solution: Weigh approximately 0.2 g of the Phytolacca acinosa granules, place it in a stoppered conical flask, add 10 ml of 60% methanol aqueous solution, sonicate (300 W power, 40 kHz frequency) for 30 min, remove, cool, shake well, filter, and collect the filtrate to obtain the test solution.

[0048] Preparation of reference solution for reference medicinal material: Take 1g of Phytolacca acinosa (Phytolacca pilosa) reference medicinal material, place it in a stoppered conical flask, add 50ml of water, heat under reflux for 60min, centrifuge, take the supernatant and concentrate under reduced pressure to dryness to obtain water extract, accurately add 10ml of 60% methanol aqueous solution, sonicate (power 300W, frequency 40kHz) for 30min, cool, shake well, filter, and take the filtrate as reference solution for reference medicinal material.

[0049] Preparation of reference solution: Take appropriate amounts of phytolaccoside A reference standard and erythrine reference standard, accurately weigh them, and add 60% methanol aqueous solution to prepare a mixed solution containing 0.2 mg of phytolaccoside A and 0.1 mg of erythrine per 1 ml.

[0050] Take appropriate amounts of adenosine reference standard, guanosine reference standard, and L-tryptophan reference standard, weigh them accurately, and add them to 60% methanol aqueous solution to prepare a mixed solution containing 0.1 mg of adenosine, 0.1 mg of guanosine, and 0.1 mg of L-tryptophan per ml.

[0051] Determination: The chromatographic conditions for high performance liquid chromatography included: using a Poroshell 120SB-Aq column with an inner diameter of 4.6 mm, a column length of 250 mm, and a particle size of 4 μm; using acetonitrile as mobile phase A and 0.04% phosphoric acid aqueous solution as mobile phase B; a flow rate of 1 ml / min; a column temperature of 30 ℃; and a detection wavelength of 210 nm. The gradient elution program includes: 0-5 min, 1% mobile phase A, 99% mobile phase B; 5-15 min, 1→8% mobile phase A, 99→92% mobile phase B; 15-25 min, 8→20% mobile phase A, 92→80% mobile phase B; 25-32 min, 20→30% mobile phase A, 80→70% mobile phase B; 32-37 min, 30→35% mobile phase A, 70→65% mobile phase B; 37-50 min, 35% mobile phase A, 65% mobile phase B.

[0052] Accurately pipette 10 μL each of the test solution and the reference medicinal material solution into the liquid chromatograph and determine the concentration. The characteristic chromatogram of the *Phytolacca acinosa* formula granules is shown below. Figure 1 See section 1a, the vertical sequence of Phytolacca acinosa reference medicinal material atlas. Figure 1 1b in the middle.

[0053] Example 2

[0054] This embodiment provides a method for constructing a vertically ordered pokeweed feature map, including the following steps:

[0055] Preparation of the test solution: Weigh approximately 0.2 g of the lyophilized powder of the standard decoction of Phytolacca acinosa accurately, place it in a stoppered conical flask, add 10 ml of 60% methanol aqueous solution accurately, sonicate (power 300W, frequency 40kHz) for 30 min, remove, cool, shake well, filter, and collect the filtrate to obtain the test solution.

[0056] Preparation of reference solution for reference medicinal material: Take 1g of Phytolacca acinosa (Phytolacca pilosa) reference medicinal material, place it in a stoppered conical flask, add 50ml of water, heat under reflux for 60min, centrifuge, take the supernatant and concentrate under reduced pressure to dryness to obtain water extract, accurately add 10ml of 60% methanol aqueous solution, sonicate (power 300W, frequency 40kHz) for 30min, cool, shake well, filter, and take the filtrate as reference solution for reference medicinal material.

[0057] Preparation of reference solution: Take appropriate amounts of phytolaccoside A reference standard and erythrine reference standard, accurately weigh them, and add 60% methanol aqueous solution to prepare a mixed solution containing 0.2 mg of phytolaccoside A and 0.1 mg of erythrine per 1 ml.

[0058] Take appropriate amounts of adenosine reference standard, guanosine reference standard, and L-tryptophan reference standard, weigh them accurately, and add them to 60% methanol aqueous solution to prepare a mixed solution containing 0.1 mg of adenosine, 0.1 mg of guanosine, and 0.1 mg of L-tryptophan per ml.

[0059] Determination: The chromatographic conditions for high performance liquid chromatography included: using a Poroshell 120SB-Aq column with an inner diameter of 4.6 mm, a column length of 250 mm, and a particle size of 4 μm; using acetonitrile as mobile phase A and 0.04% phosphoric acid aqueous solution as mobile phase B; a flow rate of 1 ml / min; a column temperature of 30 ℃; and a detection wavelength of 210 nm. The gradient elution program includes: 0-5 min, 1% mobile phase A, 99% mobile phase B; 5-15 min, 1→8% mobile phase A, 99→92% mobile phase B; 15-25 min, 8→20% mobile phase A, 92→80% mobile phase B; 25-32 min, 20→30% mobile phase A, 80→70% mobile phase B; 32-37 min, 30→35% mobile phase A, 70→65% mobile phase B; 37-50 min, 35% mobile phase A, 65% mobile phase B.

[0060] Accurately pipette 10 μL each of the test solution and the reference medicinal material solution into the liquid chromatograph and determine the result.

[0061] Example 3

[0062] This embodiment provides a method for constructing a vertically ordered pokeweed feature map, including the following steps:

[0063] Preparation of the test solution: Take about 1g of Phytolacca acinosa medicinal material and place it in a stoppered conical flask. Add 50ml of water, heat under reflux for 60min, centrifuge, and concentrate the supernatant under reduced pressure to dryness to obtain about 0.2g-0.3g of water extract. Accurately add 10ml of 60% methanol aqueous solution, sonicate (power 300W, frequency 40kHz) for 30min, cool, shake well, filter, and collect the filtrate to obtain the test solution.

[0064] Preparation of reference solution for reference medicinal material: Take 1g of Phytolacca acinosa (Phytolacca pilosa) reference medicinal material, place it in a stoppered conical flask, add 50ml of water, heat under reflux for 60min, centrifuge, take the supernatant and concentrate under reduced pressure to dryness to obtain water extract, accurately add 10ml of 60% methanol aqueous solution, sonicate (power 300W, frequency 40kHz) for 30min, cool, shake well, filter, and take the filtrate as reference solution for reference medicinal material.

[0065] Preparation of reference solution: Take appropriate amounts of phytolaccoside A reference standard and erythrine reference standard, accurately weigh them, and add 60% methanol aqueous solution to prepare a mixed solution containing 0.2 mg of phytolaccoside A and 0.1 mg of erythrine per 1 ml.

[0066] Take appropriate amounts of adenosine reference standard, guanosine reference standard, and L-tryptophan reference standard, weigh them accurately, and add them to 60% methanol aqueous solution to prepare a mixed solution containing 0.1 mg of adenosine, 0.1 mg of guanosine, and 0.1 mg of L-tryptophan per ml.

[0067] Determination: The chromatographic conditions for high performance liquid chromatography included: using a Poroshell 120SB-Aq column with an inner diameter of 4.6 mm, a column length of 250 mm, and a particle size of 4 μm; using acetonitrile as mobile phase A and 0.04% phosphoric acid aqueous solution as mobile phase B; a flow rate of 1 ml / min; a column temperature of 30 ℃; and a detection wavelength of 210 nm. The gradient elution program includes: 0-5 min, 1% mobile phase A, 99% mobile phase B; 5-15 min, 1→8% mobile phase A, 99→92% mobile phase B; 15-25 min, 8→20% mobile phase A, 92→80% mobile phase B; 25-32 min, 20→30% mobile phase A, 80→70% mobile phase B; 32-37 min, 30→35% mobile phase A, 70→65% mobile phase B; 37-50 min, 35% mobile phase A, 65% mobile phase B.

[0068] Accurately pipette 10 μL each of the test solution and the reference medicinal material solution into the liquid chromatograph for determination.

[0069] Confirmation of characteristic peaks

[0070] The spectra of three batches of *Phytolacca acinosa* granules were determined according to Example 1 (see...). Figure 2 In Example 2(b), the chromatograms of 16 batches of *Phytolacca acinosa* standard decoction freeze-dried powder were determined according to Example 2 (see 2b). Figure 2 In section 2a), the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" from the fingerprint similarity evaluation software compiled by the Pharmacopoeia Commission was used. The "multi-point correction, Mark peak matching" mode was employed for fitting and generating the characteristic chromatogram of vertically ordered Phytolacca acinosa. (See section 2a). Figure 3 Comparing the nine common characteristic peaks in the characteristic chromatograms, with peak 5 as reference peak 1, the relative retention times of peak 1 are 0.47, peak 2 is 0.55, peak 3 is 0.67, peak 4 is 0.81, peak 6 is 1.24, and peak 7 is 1.64; with peak 8 as reference peak 2, the relative retention times of peaks 9 are 1.07. The relative retention times should be within ±10% of the specified values. The characteristic chromatograms of the *Phytolacca americana* formula granules, *Phytolacca americana* standard decoction lyophilized powder, and *Phytolacca americana* formula granule extract are basically consistent, indicating good transferability. The preparation method of the extract of Phytolacca acinosa granules includes: taking Phytolacca acinosa medicinal material, removing impurities, and preparing Phytolacca acinosa slices; taking Phytolacca acinosa slices, adding water and decocting twice, filtering with a 200-mesh filter cloth, combining the filtrates, concentrating to a paste, and spray drying to obtain the extract.

[0071] Nine common chromatographic peaks were observed in the HPLC characteristic chromatogram of *Phytolacca americana* granules. The characteristic peaks were identified and assigned using HPLC and LC-MS / MS combined with reference standards. The results are shown in the table below. Peak 1 was identified as adenosine, peak 2 as guanosine, peak 3 as L-tryptophan, peak 5 as erythrine, and peak 8 as phytolaccaside A. Further, through literature review and mass spectrometry analysis, the structures of peaks 4, 6, 7, and 9 were analyzed. It was speculated that peak 4 is 4-aminotryptophan or its isomer, peak 6 is Saccharumoside C, peak 7 is Siberian polygalactosyl sugar A5, and peak 9 is phytolaccaside B. Due to the unknown structures of the above four substances or the difficulty in obtaining reference standards, their positional confirmation could not be achieved using reference standards at this time. The results are shown in the table below. Figure 4 .

[0072] Table 1. LC-MS / MS Analysis Results of Vertically Arranged Pokeweed Granules

[0073]

[0074]

[0075] Note: Because mass spectrometry is incompatible with phosphoric acid solution, 0.1% formic acid solution was used to replace the original 0.04% phosphoric acid solution in the mobile phase during UPLC-Q-TOF-MS analysis. Therefore, the retention time and elution order of each characteristic peak are different from the original spectrum. The compounds corresponding to each characteristic peak were determined by comparison with reference standards and spectral analysis.

[0076] Example 1: Determination of Chromatographic Conditions

[0077] 1.1 Gradient elution procedure

[0078] Take the same batch of vertically arranged pokeweed granules as test samples, prepare 3 test sample solutions according to Example 1, and measure them to obtain characteristic chromatograms. The difference is that the gradient elution program is different. The gradient elution program is shown in the table below, and the results are shown in Table 2.

[0079] (1) Gradient elution procedure 1:

[0080] Time (min) Mobile phase A (%) Mobile phase B (%) 0～5 1 99 5～15 1→8 99→92 15～25 8→20 92→80 25～32 20→30 80→70 32～37 30→35 70→65 37～50 35 65

[0081] (2) Gradient elution procedure 2:

[0082] Time (min) Mobile phase A (%) Mobile phase B (%) 0～8 1 99 8～18 1→8 99→92 18～28 8→20 92→80 28～35 20→30 80→70 35～40 30→35 70→65 40～50 35 65

[0083] (3) Gradient elution procedure 3:

[0084] Time (min) Mobile phase A (%) Mobile phase B (%) 0～5 1 99 5～15 1→8 99→92 15～25 8→20 92→80 25～32 20→30 80→70 32～40 30→35 70→65 40～50 35 65

[0085] Table 2. Test results obtained from different gradient elution programs.

[0086]

[0087]

[0088] The results above show that gradient elution programs 1-3 can all produce characteristic chromatograms that meet the requirements. Furthermore, the chromatograms obtained by gradient elution program 1 show uniform peak distribution and relatively short acquisition time. Therefore, gradient elution program 1 is the preferred gradient elution program for constructing characteristic chromatograms of vertically arranged pokeweed.

[0089] 1.2 Chromatographic conditions

[0090] The same batch of *Phytolacca americana* granules were used to prepare test solutions according to Example 1, and the characteristic chromatograms were obtained. The difference was that the chromatographic conditions were different, and the chromatographic conditions were as follows.

[0091] (1) Chromatographic conditions 1: Waters H-class instrument was used, with acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B. The T3 (2.1×100mm, 1.6μm) column was used for gradient elution according to the table below; the flow rate was 0.35ml / min; the column temperature was 30℃; the detection wavelengths were 210nm and 254nm, and the injection volume was 2μL.

[0092] Time (min) Mobile phase A (%) Mobile phase B (%) 0～10 1→8 99→92 10～20 8→30 92→70 20～30 30→42 70→58

[0093] from Figure 5 The results show that the chromatographic information at 210 nm is richer than that at 254 nm, and the main characteristic component, phytolaccoside A, can also be detected. Therefore, 210 nm was used for subsequent optimization.

[0094] (2) Chromatographic conditions 2: Waters H-class instrument was used, with acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B. The T3 (2.1×100mm, 1.6μm) column was used for gradient elution as specified in the table below; the flow rate was 0.35ml / min; the column temperature was 30℃; the detection wavelength was 210nm; and the injection volume was 2μL.

[0095] Time (min) Mobile phase A (%) Mobile phase B (%) 0～5 1 99 5～13 1→8 99→92 13～23 8→20 92→80 23～28 20→30 80→70 28～40 30→42 70→58

[0096] Figure 6 It can be seen that increasing the aqueous phase elution time did not improve the separation of the chromatographic peaks in the first 5 minutes, and resulted in even worse separation of subsequent chromatographic peaks.

[0097] (3) Chromatographic conditions 3: Waters H-class instrument was used, with acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B. The T3 (2.1×100mm, 1.6μm) column was used for gradient elution as specified in the table below; the flow rate was 0.35ml per minute; the column temperature was 30℃; the detection wavelength was 210nm; and the injection volume was 2μL.

[0098]

[0099]

[0100] from Figure 7 It can be seen that the chromatographic peaks with retention times of 8-12 min need further optimization.

[0101] (4) Chromatographic conditions 4: Waters H-class instrument was used, with acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B. The T3 (2.1×100mm, 1.6μm) column was used for gradient elution as specified in the table below; the flow rate was 0.35ml / min; the column temperature was 30℃; the detection wavelength was 210nm; and the injection volume was 2μL.

[0102] Time (min) Mobile phase A (%) Mobile phase B (%) 0～8 1→6 99→94 8～15 6 94 15～25 6→20 94→80 25～30 20→30 80→70 30～40 30→42 70→58

[0103] from Figure 8 It can be seen that the chromatographic peaks with retention times of 9-12 min need further optimization.

[0104] (5) Chromatographic conditions 5: A Waters e2695 instrument was used, with acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B. The chromatographic column is (4.6×250mm, 5μm). Gradient elution is performed according to the specifications in the table below. The flow rate is 1ml per minute. The column temperature is 30℃. The detection wavelength is 210nm. The injection volume is 10μL.

[0105] Time (min) Mobile phase A (%) Mobile phase B (%) 0～4 1 99 4～13 1→8 99→92 13～23 8→20 92→80 23～33 20→23 80→77 33～50 23→60 77→40

[0106] from Figure 9 It can be seen that the chromatographic information and separation effect of this column are significantly better than those of other columns. T3 (2.1×100mm, 1.6μm), therefore, optimization was carried out based on this chromatographic column.

[0107] (6) Chromatographic conditions 6: A Waters e2695 instrument was used, with acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B. The chromatographic column is (4.6×250mm, 5μm). Gradient elution is performed according to the specifications in the table below. The flow rate is 1ml per minute. The column temperature is 30℃. The detection wavelength is 210nm. The injection volume is 10μL.

[0108] Time (min) Mobile phase A (%) Mobile phase B (%) 0～4 1 99 4～15 1→8 99→92 15～25 8→20 92→80 25～35 20→30 80→70 35～45 30→60 70→40

[0109] from Figure 10 It can be seen that, except for the 7-9 min chromatographic peak, the separation effect of the other chromatographic peaks is relatively better. We will try changing the concentration of phosphoric acid in the mobile phase.

[0110] (7) Chromatographic conditions 7: A Waters e2695 instrument was used, with acetonitrile as mobile phase A and 0.03% phosphoric acid solution as mobile phase B. The chromatographic column is (4.6×250mm, 5μm). Gradient elution is performed according to the specifications in the table below. The flow rate is 1ml per minute. The column temperature is 30℃. The detection wavelength is 210nm. The injection volume is 10μL.

[0111] Time (min) Mobile phase A (%) Mobile phase B (%) 0～4 1 99 4～15 1→8 99→92 15～25 8→20 92→80 25～30 20→30 80→70 30～45 30→60 70→40

[0112] Figure 11 As can be seen, consistent with condition 6, the chromatographic peak separation effect is still poor at 7-9 min, and no chromatographic peak appears at 30-40 min. It is advisable to try shortening the analysis time.

[0113] (8) Chromatographic conditions 8: A Waters e2695 instrument was used, with acetonitrile as mobile phase A and 0.03% phosphoric acid solution as mobile phase B. The chromatographic column is (4.6×250mm, 5μm). Gradient elution is performed according to the specifications in the table below. The flow rate is 1ml per minute. The column temperature is 30℃. The detection wavelength is 210nm. The injection volume is 10μL.

[0114] Time (min) Mobile phase A (%) Mobile phase B (%) 0～4 1 99 4～15 1→8 99→92 15～25 8→20 92→80 25～30 20→30 80→70 30～40 30→60 70→40 40～45 60 40

[0115] from Figure 12 It can be seen that the chromatographic peak separation effect in the 7-9 min period is not good, and the chromatographic peak specificity in the first 10 min period is not good. Therefore, this chromatographic condition should be discarded.

[0116] (9) Chromatographic conditions 9: A Waters e2695 instrument was used, with acetonitrile as mobile phase A and 0.03% phosphoric acid solution as mobile phase B. A chromatographic column (4.6 × 250 mm, 5 μm) was used, and gradient elution was performed according to the specifications in the table below; the flow rate was 1 mL / min; the column temperature was 30 °C; the detection wavelength was 210 nm; and the injection volume was 10 μL. Results are shown below. Figure 13 .

[0117] Time (min) Mobile phase A (%) Mobile phase B (%) 0～4 3 97 4～15 3→8 97→92 15～25 8→20 92→80 25～30 20→30 80→70 30～40 30→60 70→40 40～45 60 40

[0118] (10) Chromatographic conditions 10: Waters e2695 instrument was used, with acetonitrile as mobile phase A and 0.03% phosphoric acid solution as mobile phase B. A chromatographic column (4.6 × 250 mm, 5 μm) was used, and gradient elution was performed according to the specifications in the table below; the flow rate was 1 mL / min; the column temperature was 30 °C; the detection wavelength was 210 nm; and the injection volume was 10 μL. Results are shown below. Figure 14 .

[0119] Time (min) Mobile phase A (%) Mobile phase B (%) 0～8 2 98 8～15 2→8 98→92 15～25 8→20 92→80 25～30 20→30 80→70 30～40 30→60 70→40 40～45 60 40

[0120] As can be seen from chromatographic conditions 9 and 10, the chromatogram in the first 7 minutes is still not ideal. You can try changing the column to Poroshell 120SB-Aq (4.6×250mm, 4μm).

[0121] (11) Chromatographic conditions: A Waters e2695 instrument was used, with acetonitrile as mobile phase A, 0.03% phosphoric acid solution as mobile phase B, and a Poroshell 120SB-Aq (4.6×250mm, 4μm) column. Gradient elution was performed according to the specifications in the table below; the flow rate was 1 ml / min; the column temperature was 30℃; the detection wavelength was 210 nm; and the injection volume was 10 μL. Results are shown below. Figure 15 .

[0122] Time (min) Mobile phase A (%) Mobile phase B (%) 0～4 1 99 4～15 1→8 99→92 15～25 8→20 92→80 25～30 20→30 80→70 30～40 30→60 70→40 40～42 60 40

[0123] The results show that the chromatographic peaks are evenly distributed and the separation effect is good. However, the chromatographic peak at around 32 min (phytolacca saponin A) is close to the adjacent peak and can be further optimized.

[0124] (12) Chromatographic conditions: A Waters e2695 instrument was used, with acetonitrile as mobile phase A, 0.03% phosphoric acid solution as mobile phase B, and a Poroshell 120SB-Aq (4.6×250mm, 4μm) column. Gradient elution was performed according to the specifications in the table below; the flow rate was 1 ml per minute; the column temperature was 30℃; the detection wavelength was 210 nm; and the injection volume was 10 μL. The chromatogram is shown in [reference needed]. Figure 16 .

[0125] Time (min) Mobile phase A (%) Mobile phase B (%) 0～4 1 99 4～15 1→8 99→92 15～25 8→20 92→80 25～35 20→30 80→70 35～50 30→40 70→60

[0126] (13) Chromatographic conditions: A Waters e2695 instrument was used, with acetonitrile as mobile phase A, 0.03% phosphoric acid solution as mobile phase B, and a Poroshell 120SB-Aq (4.6×250mm, 4μm) column. Gradient elution was performed according to the specifications in the table below; the flow rate was 1 ml per minute; the column temperature was 30℃; the detection wavelength was 210 nm; and the injection volume was 10 L. The results are shown in the table below. Figure 17 .

[0127] Time (min) Mobile phase A (%) Mobile phase B (%) 0～5 1 99 5～15 1→8 99→92 15～25 8→20 92→80 25～32 20→30 80→70 32～37 30→35 70→65 37～50 35 65

[0128] The results show that the chromatographic peaks in the chromatogram are evenly distributed and the separation effect is good. Therefore, this method is used as the initial condition for subsequent system optimization.

[0129] 1.3 wavelength

[0130] Following chromatographic conditions 13 in Example 1, the *Phytolacca acinosa* granule test solution was injected into the chromatograph, and a full wavelength scan of 210-400 nm was performed. (See...) Figure 18 Based on the amount of chromatographic information, chromatograms at different absorption wavelengths (210 nm, 230 nm, 254 nm, 280 nm, 300 nm, and 330 nm) were compared, with the number of chromatographic peaks, absorption intensity corresponding to retention time, and baseline stability used as evaluation criteria. Experimental results show that the number of chromatographic peaks significantly decreases at detection wavelengths between 280 nm and 330 nm, while there is no significant difference in the number of peaks at 210 nm, 230 nm, and 254 nm. Therefore, 210 nm is the preferred detection wavelength for the characteristic chromatograms of *Phytolacca americana*.

[0131] 1.4 flow rate

[0132] Take the same batch of vertically arranged pokeweed granules and prepare 3 test solutions according to Example 1. Using flow rate as a variable, measure according to Example 1 to obtain the corresponding spectra for different flow rates. The results are as follows: the flow rates are 0.8 ml / min, 1.0 ml / min, and 1.2 ml / min, respectively.

[0133] Table 3 Applicability parameters of the system for different flow velocities

[0134]

[0135]

[0136] The results above show that the effect is optimal when the flow rate is 1.0 ml / min.

[0137] 1.5 column temperature

[0138] Take the same batch of vertically arranged pokeweed granules and prepare 3 test solutions according to Example 1. Using column temperature as a variable, determine the corresponding spectra at different column temperatures according to Example 1. The results are as follows; the column temperatures are 25℃, 30℃ and 35℃.

[0139] Table 4. Applicability parameters of the system for different column temperatures.

[0140]

[0141] The results above show that the separation of each characteristic peak is better when the column temperature is 30℃, and 30℃ is the preferred column temperature.

[0142] 1.6 chromatographic column

[0143] Take the same batch of vertically arranged pokeweed formula granules, and prepare 3 test solutions according to Example 1. Using chromatographic columns of different batches as variables, determine according to the method of Example 1 to obtain the corresponding chromatograms for different batches, namely batches 1-3. The results are as follows;

[0144] Table 5 System suitability parameters for chromatographic peaks obtained from different chromatographic columns

[0145]

[0146] The above results indicate that there are no significant differences in the chromatograms obtained from different batches of chromatographic columns, and the system suitability of each characteristic peak meets the determination requirements.

[0147] 1.7 Mobile phase B concentration

[0148] Four test solutions were prepared according to Example 1 using the same batch of *Phytolacca acinosa* granules. Different mobile phase B concentrations were used as variables, and the results were obtained by measuring the concentrations according to the method in Example 1. The concentrations of mobile phase B were 0.02%, 0.03%, 0.04%, and 0.05%, respectively.

[0149] Table 6. Applicability parameters of the system for different phosphoric acid concentrations.

[0150]

[0151]

[0152] The results above show that when the concentration of mobile phase B is 0.03-0.05%, the system suitability parameters for each chromatographic peak are good, with no significant differences. To improve the robustness of the method, a phosphoric acid concentration of 0.04% was selected for subsequent studies.

[0153] In summary, the preferred chromatographic conditions and system suitability test scheme is as follows: using a Poroshell 120SB-Aq column with the following specifications: inner diameter 4.6 mm, column length 250 mm, particle size 4 μm; acetonitrile as mobile phase A; 0.04% phosphoric acid aqueous solution as mobile phase B; flow rate 1 ml / min; column temperature 30℃; and detection wavelength 210 nm. The gradient elution program includes: 0-5 min, 1% mobile phase A, 99% mobile phase B; 5-15 min, 1→8% mobile phase A, 99→92% mobile phase B; 15-25 min, 8→20% mobile phase A, 92→80% mobile phase B; 25-32 min, 20→30% mobile phase A, 80→70% mobile phase B; 32-37 min, 30→35% mobile phase A, 70→65% mobile phase B; 37-50 min, 35% mobile phase A, 65% mobile phase B.

[0154] Experiment Example 2: Investigation of the preparation method of the test solution

[0155] 2.1 Extraction Solvent

[0156] Take the same batch of vertically arranged pokeweed granules, and prepare the test solution according to Example 1, using the extraction solvent as a variable. The solution is then measured to obtain the chromatogram. The extraction solvents are water, 30% methanol, 60% methanol, 80% methanol, and methanol.

[0157] Table 7. Chromatographic parameters of *Phytolacca acinosa* formulation granules under different extraction solvents.

[0158]

[0159]

[0160] The above results indicate that when the extraction solvent is 60% methanol or 80% methanol, there is no significant change in the peak area and system suitability. To save on methanol usage, 60% methanol is preferred as the extraction solvent.

[0161] 2.2 Extraction Method

[0162] The same batch of *Phytolacca acinosa* granules were used. The sample solution was prepared according to Example 1, with the extraction method as the variable, and the chromatogram was obtained. The extraction method was either ultrasonic treatment or reflux heating. Ultrasonic treatment: power 300W, frequency 40kHz, time 30min; reflux heating: time 30min.

[0163] Table 8. Chromatographic parameters of *Phytolacca acinosa* formulation granules under different extraction methods.

[0164]

[0165] The information content of chromatographic peaks and system suitability parameters were used as the main evaluation indicators. The results show that the extraction method has no significant impact on the information content of chromatographic fractions and system suitability parameters. For ease of operation, ultrasonic extraction is the preferred method.

[0166] 2.3 Examination of extraction time

[0167] Take the same batch of vertically arranged pokeweed formula granules, use extraction time as a variable, prepare test solution according to Example 1, and measure to obtain spectrum; wherein, the extraction time is 20 min, 30 min, and 40 min.

[0168] Table 9. Chromatographic parameters of *Phytolacca acinosa* formulation granules under different extraction times.

[0169]

[0170]

[0171] The main evaluation indicators were chromatographic peak information content and system suitability parameters. The results show that extraction time has no significant impact on chromatographic peak information content and system suitability parameters. Therefore, for complete extraction, ultrasonication for 30 minutes is the preferred extraction time.

[0172] 2.4 Sampling quantity

[0173] Using the sample amount as a variable, the sample amounts were 0.1g, 0.2g, 0.4g, and 0.8g, respectively. The sample solutions were prepared according to Example 1 and measured to obtain the chromatograms corresponding to different sample amounts.

[0174] Table 10 Chromatogram parameters of *Phytolacca acinosa* formulation granules under different sampling amounts

[0175]

[0176]

[0177] The information content of chromatographic peaks and system suitability parameters were used as the main evaluation indicators. The results show that the peak area of ​​the chromatographic peaks increases proportionally with the increase in sample size, indicating that the components in the *Phytolacca acinosa* formulation granules can be completely extracted within the sample size range of 0.1-0.8 g. Considering the response value and baseline stability, 0.2 g is the preferred sample size.

[0178] 2.5 injection volume

[0179] Using the injection volume as a variable, with injection volumes of 5 μL, 10 μL and 15 μL, the test solution was prepared according to Example 1 and measured to obtain the chromatograms corresponding to different injection volumes.

[0180] Table 11. Applicability parameters of the chromatographic peak system under different injection volumes.

[0181]

[0182] The information content of chromatographic peaks and system suitability parameters were used as the main evaluation indicators. The results show that the peak area of ​​the chromatographic peaks increases proportionally with the increase of the injection volume. Considering the peak shape, resolution, and baseline stability, 10 μL is the preferred injection volume.

[0183] In summary, the preferred method for preparing the test solution is as follows: take an appropriate amount of the test sample, grind it finely, take about 0.2g, accurately weigh it, place it in a stoppered conical flask, accurately add 10ml of 60% methanol, sonicate (power 300W, frequency 40kHz) for 30 minutes, remove it, cool it, shake it well, filter it, and collect the filtrate.

[0184] Methodological Investigation of Experiment Example 3

[0185] 3.1 Specificity Examination

[0186] According to Example 1, test solutions, reference solutions, and standard solutions of *Phytolacca acinosa* granules were prepared. Chromatographic conditions and system suitability of the *Phytolacca acinosa* granules were determined according to Example 1, and the interference of the blank excipient (maltodextrin) was investigated. The blank excipient chromatogram is shown in [reference needed]. Figure 19 The results are as follows:

[0187] Table 12 System Suitability Parameters for Reference Standards

[0188] chromatographic peaks name Retention time Peak area Peak height Resolution Tail Factor Theoretical number of plates 1 Erythrine 15.464 4844944 214748 11613 1.10 / 2 Phytolacca saponin A 40.387 536585 581164 444328 1.24 /

[0189] Table 13. Systemic Suitability Parameters for Comparative Medicinal Materials

[0190] chromatographic peaks Retention time Peak area Peak height Resolution Tail Factor Theoretical number of plates 1 7.256 734760 54516 \ 1.60 6422 2 8.457 228720 14380 3.06 1.22 6503 3 10.422 291258 14468 4.18 1.45 7388 4 12.424 339912 20617 4.28 0.87 13516 5 15.503 581058 31390 6.71 1.32 15525 6 19.160 94516 8172 9.57 1.60 75678 7 25.426 90823 12434 2.57 1.54 278058 8 40.372 403796 35645 58.19 1.56 318912 9 43.143 98830 6502 7.56 1.12 215704

[0191] Table 14 System Suitability Parameters for Test Samples

[0192] Serial Number Retention time Peak area Peak Resolution Tail Factor Theoretical number of plates 1 7.155 335069 43301 3.29 1.28 19735 2 8.489 201479 15816 4.69 1.14 10198 3 10.720 335642 28276 6.64 1.18 20200 4 12.649 211725 22721 6.93 1.31 41660 5 15.624 617965 51474 10.58 1.45 40593 6 19.328 70167 8814 1.88 0.82 164439 7 25.573 60013 11889 3.92 1.04 555281 8 40.516 151246 22284 21.1 1.15 832408 9 43.344 31896 3738 8.90 0.93 566417

[0193] The above results indicate that the test solution, reference material solution, and reference solution of the *Phytolacca acinosa* formula granules are suitable for use. Figure 19 This indicates that the blank excipients did not interfere with the characteristic chromatogram of the test sample, demonstrating that the chromatographic method system has good applicability and specificity.

[0194] 3.2 Precision

[0195] A sample solution of vertically arranged pokeweed granules was prepared according to Example 1 and injected 6 times consecutively. The relative retention time and relative peak area of ​​each characteristic peak were calculated. The results showed that the RSD of the relative retention time of each characteristic peak was less than 2% and the RSD of the relative peak area was less than 5%, indicating that the instrument precision was good.

[0196] Table 15 Results of Instrument Precision and Relative Retention Time Tests

[0197]

[0198]

[0199] Table 16 Results of Instrument Precision Relative Peak Area Test

[0200]

[0201] 3.3 Method repeatability

[0202] Six test solutions were prepared according to Example 1 using the same batch of vertically ordered pokeweed granules. The solutions were then analyzed to obtain chromatograms, and the relative retention times and relative peak areas of each characteristic peak were calculated. The results showed that the RSD of the relative retention time of each characteristic peak was less than 2%, and the RSD of the relative peak area was less than 5%, indicating good repeatability of the method.

[0203] Table 17 Results of Relative Retention Time in Repeatability Tests

[0204]

[0205]

[0206] Table 18 Results of Repeatability Relative Peak Area Test

[0207]

[0208] 3.4 Intermediate Precision

[0209] (1) Intermediate precision among different personnel

[0210] Two test solutions were prepared by three different staff members (A, B, and C) according to Example 1. The solutions were measured on the same instrument to obtain characteristic chromatograms of different vertically ordered pokeweed formulation particles. The relative retention time and relative peak area of ​​each characteristic peak were calculated. The results showed that the RSD of the relative retention time of each characteristic peak was less than 2%, and the RSD of the relative peak area was less than 5%, indicating that the method has good precision when operated by different personnel.

[0211] Table 19 Results of Intermediate Precision Relative Retention Time Test for Different Personnel

[0212]

[0213] Table 20 Results of Intermediate Precision Relative Peak Area Test for Different Personnel

[0214]

[0215]

[0216] (2) Intermediate precision of different instruments

[0217] Six solutions of vertically arranged pokeweed granules were prepared according to Example 1. These solutions were injected at different times and measured using different instruments to obtain spectra. The relative retention times and relative peak areas of each characteristic peak were calculated. The results showed that the RSD of the relative retention time of each characteristic peak was less than 5%, and the RSD of the relative peak area was less than 10%, indicating that the method has good intermediate precision across different instruments.

[0218] Table 21 Test results of relative retention time for intermediate precision of different instruments

[0219]

[0220]

[0221] Table 22 Results of Intermediate Precision Relative Peak Area Tests for Different Instruments

[0222]

[0223] 3.5 Durability

[0224] The test solution of *Phytolacca acinosa* granules was prepared according to Example 1. The sample was injected at 0h, 2h, 4h, 8h, 12h, 18h, 24h, 36h, and 48h after preparation, and the relative retention time and relative peak area of ​​each characteristic peak were measured. The results showed that the RSD values ​​of the relative retention times of each characteristic peak were all less than 2%, and the RSD values ​​of the relative peak areas of each characteristic peak were all less than 5%, indicating that the test solution was stable within 48h and met the measurement requirements.

[0225] Table 23 Results of the relative retention time test for stability assessment

[0226]

[0227]

[0228] Table 24 Results of relative peak area test for stability assessment

[0229]

[0230] 3.6 Durability Test at Different Flow Rates

[0231] Three test solutions were obtained from the same batch of vertically arranged pokeweed granules according to Example 1. The flow rate was used as a variable to measure the relative retention time and relative peak area of ​​each characteristic peak. The flow rates were 0.9 ml / min, 1.0 ml / min, and 1.1 ml / min, respectively. The results showed that the RSD values ​​of the relative retention times of each characteristic peak were all less than 5%, and the RSD values ​​of the relative peak areas of each characteristic peak were all less than 10%. This method has a certain robustness to different flow rates.

[0232] Table 25 Comparison of relative retention times for different flow velocities

[0233]

[0234] Table 26 Comparison of relative peak area results for different flow velocities

[0235]

[0236] 3.7 Durability test at different column temperatures

[0237] Three test solutions were prepared from the same batch of *Phytolacca acinosa* granules according to Example 1. The relative retention time and relative peak area of ​​each characteristic peak were measured and calculated, using column temperature as a variable. The column temperatures were 28℃, 30℃, and 32℃. The results showed that the RSD values ​​of the relative retention times of each characteristic peak were all less than 5%, and the RSD values ​​of the relative peak areas of each characteristic peak were all less than 10%, indicating a certain degree of robustness to column temperature.

[0238] Table 27 Comparison of relative retention times at different column temperatures

[0239]

[0240] Table 28 Comparison of relative peak areas at different column temperatures

[0241]

[0242] 3.8 Durability Study of Different Chromatographic Columns

[0243] Two test solutions were prepared using the same batch of *Phytolacca acinosa* granules as described in Example 1. The effect of different batches of chromatographic columns on the characteristic peaks was investigated. The results showed that the RSD values ​​of the relative retention times of each characteristic peak were all less than 5%, and the RSD values ​​of the relative peak areas of each characteristic peak were all less than 10%, indicating that the method has good robustness to the chromatographic column. The batch numbers of the chromatographic columns were B21336 and B21193; Poroshell 120SB-Aq, 4.6 × 250 mm, 4 μm.

[0244] Table 29 Comparison of relative retention times for different chromatographic columns

[0245]

[0246] Table 30 Comparison of relative peak areas for different chromatographic columns

[0247]

[0248] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for constructing a feature map of vertically ordered pokeweed, characterized in that, Includes the following steps: Preparation of test solution: Prepare a test solution from the test sample; When the test sample is Phytolacca acuminata medicinal material or Phytolacca acuminata slices, the preparation method includes: taking the test sample, adding water, heating under reflux, centrifuging, concentrating under reduced pressure to dryness to obtain water extract, adding extraction solvent, extracting, and filtering. When the test sample is Phytolacca americana granules or Phytolacca americana standard decoction freeze-dried powder, the preparation method includes: taking the test sample, adding extraction solvent, extracting, and filtering. When preparing the test solution, the extraction solvent is an aqueous methanol solution, and the volume fraction of methanol in the aqueous methanol solution is not less than 60%. Preparation of reference solutions: The reference standards were phytolaccoside A, erythrine, adenosine, guanosine, and L-tryptophan; Determination: The chromatographic conditions for high-performance liquid chromatography (HPLC) included: acetonitrile as mobile phase A, 0.03-0.05% phosphoric acid aqueous solution as mobile phase B, gradient elution; a Poroshell 120 SB-Aq column with the following specifications: inner diameter 4.6 mm, column length 250 mm, particle size 4 μm, and wavelength 210 nm. The gradient elution program includes: 0-5 min, 1% mobile phase A, 99% mobile phase B; 5-15 min, 1→8% mobile phase A, 99→92% mobile phase B; 15-25 min, 8→20% mobile phase A, 92→80% mobile phase B; 25-32 min, 20→30% mobile phase A, 80→70% mobile phase B; 32-37 min, 30→35% mobile phase A, 70→65% mobile phase B; 37-50 min, 35% mobile phase A, 65% mobile phase B; or... 0-8 min, 1% mobile phase A, 99% mobile phase B; 8-18 min, 1→8% mobile phase A, 99→92% mobile phase B; 18-28 min, 8→20% mobile phase A, 92→80% mobile phase B; 28-35 min, 20→30% mobile phase A, 80→70% mobile phase B; 35-40 min, 30→35% mobile phase A, 70→65% mobile phase B; 40-50 min, 35% mobile phase A, 65% mobile phase B; or 0-5 min, 1% mobile phase A, 99% mobile phase B; 5-15 min, 1→8% mobile phase A, 99→92% mobile phase B; 15-25 min, 8→20% mobile phase A, 92→80% mobile phase B; 25-32 min, 20→30% mobile phase A, 80→70% mobile phase B; 32-40 min, 30→35% mobile phase A, 70→65% mobile phase B; 40-50 min, 35% mobile phase A, 65% mobile phase B.

2. The construction method according to claim 1, characterized in that, The chromatographic conditions for the high-performance liquid chromatography method also include: Column temperature is 25-35℃; and / or, The injection volume is 5-15 μL; and / or, The flow rate is 0.8-1.2 ml / min.

3. The construction method according to claim 1 or 2, characterized in that, The chromatographic conditions for the high-performance liquid chromatography (HPLC) method include: using a Poroshell 120 SB-Aq column with an inner diameter of 4.6 mm, a column length of 250 mm, and a particle size of 4 μm; using acetonitrile as mobile phase A and 0.04% phosphoric acid aqueous solution as mobile phase B; a flow rate of 1 ml / min; a column temperature of 30 °C; and a detection wavelength of 210 nm.

4. The construction method according to claim 1, characterized in that, When preparing the test solution, the extraction method is either reflux extraction or ultrasonic extraction.

5. The construction method according to claim 1, characterized in that, When the test sample is lyophilized powder or formulation granules, the mass of the test sample corresponding to each 1 ml of extraction solvent is 0.01 g to 0.08 g; When the test sample is a medicinal material or decoction piece, the mass of the water extract corresponding to each 1 ml of extraction solvent is 0.01 g to 0.08 g.

6. The construction method according to claim 1 or 2, characterized in that, It also includes the preparation of reference solutions for control medicinal materials; The preparation method of the reference solution of the reference medicinal material includes: taking the reference medicinal material, extracting it with water to obtain an aqueous extract, adding an extraction solvent to the aqueous extract, mixing well, and filtering.

7. The construction method according to claim 6, characterized in that, When preparing the reference solution of the control medicinal material, the extraction solvent is at least one of methanol and water.

8. The construction method according to claim 7, characterized in that, The extraction solvent is an aqueous methanol solution.

9. The construction method according to claim 8, characterized in that, The volume fraction of methanol in the methanol-water solution is not less than 60%.

10. The construction method according to claim 7, characterized in that, Each 1g of *Phytolacca americana* reference material corresponds to a water volume of 25-100ml.

11. The construction method according to claim 7, characterized in that, The mass of the water extract corresponding to each 1 ml of extraction solvent is 0.01 g - 0.08 g.

12. The construction method according to claim 7, characterized in that, When obtaining the aqueous extract, the extraction method is either reflux extraction or ultrasonic extraction.

13. The construction method according to any one of claims 1, 2, 4, 5, 7-12, characterized in that, The feature map obtained by the construction method includes 9 feature peaks; With peak 5 as reference peak 1, the relative retention time of peak 1 is 0.47, the relative retention time of peak 2 is 0.55, the relative retention time of peak 3 is 0.67, the relative retention time of peak 4 is 0.81, the relative retention time of peak 6 is 1.24, and the relative retention time of peak 7 is 1.

64. With peak 8 as the reference, the relative retention time of peaks 2 and 9 is 1.

07. The relative retention time should be within ±10% of the specified value.

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