Compound sanguis draxi pill characteristic spectrum, construction method thereof and quality detection method of compound sanguis draxi pill

Abstract

This invention relates to a characteristic chromatogram of compound ferrous sulfate pills, its construction method, and a quality detection method for compound ferrous sulfate pills. Belonging to the field of traditional Chinese medicine detection technology, the method for constructing the characteristic chromatogram of compound ferrous sulfate pills involves controlling the liquid chromatography conditions, mobile phase composition, and elution procedure to establish a characteristic chromatogram of compound ferrous sulfate pills, obtaining 12 common characteristic peaks. This characteristic chromatogram fully presents the chromatographic peaks, exhibits good homogeneity, and demonstrates good peak resolution, comprehensively reflecting the types and quantities of chemical components contained in the compound ferrous sulfate pills. It achieves a holistic description and evaluation of the quality of the compound ferrous sulfate pills, ensuring their effectiveness and safety. This characteristic chromatogram detection method has advantages such as simple operation, stability and reliability, high precision, good reproducibility, and ease of mastery.

Description

Technical Field

[0001] This invention relates to a characteristic spectrum of compound ferrous sulfate pills and its construction method, as well as a quality detection method for compound ferrous sulfate pills, belonging to the field of traditional Chinese medicine detection technology. Technical Background

[0002] Compound Ferrous Sulfate Pills are a traditional Chinese medicine preparation made from six herbs: ferrous sulfate, American ginseng, cinnamon, seahorse, jujube, and walnut kernels. It has the effects of warming the kidneys and strengthening the marrow, replenishing qi and nourishing yin, promoting blood production and stopping bleeding. It is mainly used for aplastic anemia, leukopenia, thrombocytopenia, myelodysplastic syndrome, and bone marrow damage and leukopenia caused by radiotherapy and chemotherapy, belonging to the syndrome of kidney yang deficiency and qi and blood deficiency. Compound Ferrous Sulfate Pills are listed under the "Compound Ferrous Sulfate Pills" item in the 2020 edition of the Chinese Pharmacopoeia. However, this standard only includes simple characteristics, microscopic identification, physicochemical identification, thin-layer chromatography identification, routine examination, and content determination; it lacks fingerprint and characteristic chromatogram items, making comprehensive quality control of Compound Ferrous Sulfate Pills impossible. Current literature reports on the quality testing of Compound Ferrous Sulfate Pills mainly include "Determination of American Ginseng Content in Compound Ferrous Sulfate Pills by HPLC" and "Colorimetric Determination of Ferrous Sulfate in Compound Ferrous Sulfate Pills." These studies primarily focus on methods for determining the content of Compound Ferrous Sulfate Pills. Currently, there is no research on establishing fingerprint or characteristic spectral methods, leading to incomplete quality testing of Compound Ferrous Sulfate Pills. To better control product quality, we are developing and validating a characteristic spectral method for Compound Ferrous Sulfate Pills to ensure its scientific validity and rationality. Establishing a comprehensive and quantifiable method for monitoring the overall quality of the components plays a crucial role in improving the stability and consistency of the quality of Compound Ferrous Sulfate Pills. Summary of the Invention

[0003] The purpose of this invention is to solve the problems existing in the prior art by providing a characteristic spectrum of compound ferrous sulfate pills and its construction method, as well as a quality detection method for compound ferrous sulfate pills, which can comprehensively evaluate the quality of compound ferrous sulfate pills, thereby ensuring that the product quality is controllable and stable.

[0004] Therefore, this invention discloses a method for constructing a characteristic spectrum of compound ferrous sulfate pills, comprising the following steps:

[0005] (1) Chromatographic conditions and system suitability: Octadecylsilane-bonded silica gel was used as the packing material; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B. Gradient elution was performed according to the specifications in the table below, with a flow rate of 1.0 ml / min, a column temperature of 40℃, and a detection wavelength of 203 nm. The theoretical plate number, calculated based on the ginsenoside Rb1 peak, should not be less than 5000.

[0006]

[0007]

[0008] (2) Preparation of test solution: Take an appropriate amount of this product, cut it into small pieces, take about 3g, weigh it accurately, put it in an Erlenmeyer flask, add 25ml of methanol accurately, seal it tightly, weigh it, sonicate it for 2 hours, cool it, weigh it, make up the weight loss with methanol, shake it well, filter it, and the test solution is obtained.

[0009] (3) Preparation of reference solution: Take appropriate amounts of ginsenoside Re, ginsenoside Rg1 and ginsenoside Rb1 reference standards, add methanol to prepare a solution containing 0.2 mg of each per 1 ml.

[0010] (4) Determination method: Accurately pipette 10 μl each of blank solvent, reference solution and test solution into the liquid chromatograph.

[0011] The chromatograms were measured and recorded to obtain the characteristic chromatogram of Compound Ferrous Sulfate Pills.

[0012] Preferably, it also includes the construction of a reference characteristic chromatogram of compound ferrous sulfate pills. The characteristic chromatograms obtained from the detection of multiple batches of compound ferrous sulfate pill test samples are used to generate a reference characteristic chromatogram of compound ferrous sulfate pills using the 2012 version of the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" of the National Pharmacopoeia Commission.

[0013] Preferably, after generating the reference characteristic chromatogram of Compound Ferrous Sulfate Pill using the 2012 version of the "Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine" of the National Pharmacopoeia Commission, the method further includes the step of marking common characteristic peaks.

[0014] Preferably, the characteristic spectrum of the compound ferrous sulfate pill includes 12 characteristic peaks. The common characteristic peaks are: peak 1 is a common characteristic peak, peak 2 is a common characteristic peak, peak 3 is a common characteristic peak, peak 4 is a common characteristic peak, peak 5 is a common characteristic peak, peak 6 is ginsenoside Rg1, peak 7 is ginsenoside Re, peak 8 is a common characteristic peak, peak 9 is a common characteristic peak, peak 10 (S) is ginsenoside Rb1, peak 11 is a common characteristic peak, and peak 12 is a common characteristic peak.

[0015] Preferably, the characteristic spectrum of the compound ferrous sulfate pill includes 12 common characteristic peaks, of which peak 10 is the reference peak S of ginsenoside Rb1; the relative retention time of each characteristic peak and peak S is within ±5% of the specified value; the specified values ​​are: 0.30 (peak 1), 0.31 (peak 2), 0.48 (peak 3), 0.55 (peak 4), 0.57 (peak 5), 0.80 (peak 6), 0.81 (peak 7), 0.82 (peak 8), 0.94 (peak 9), 1.00 (peak 10, S), 1.03 (peak 11), 1.08 (peak 12);

[0016] Preferably, the chromatographic column used in step (1) of the method for constructing the characteristic chromatogram of the compound ferrous sulfate pill is selected from Shim-pack GISS C 18 Or an equivalent chromatographic column, with a length of 25 cm, an inner diameter of 4.6 mm, and a particle size of 5 μm.

[0017] Preferably, the ultrasonic treatment conditions used in step (2) of the method for constructing the characteristic spectrum of the compound ferrous sulfate pill are: power 500W and frequency 40KHz.

[0018] The present invention also provides a characteristic spectrum of compound ferrous sulfate pills, obtained by any of the above-described construction methods.

[0019] The present invention also provides a comparative characteristic chromatogram of compound ferrous sulfate pills, selected from any one of the following (1)-(3):

[0020] (1) It has 12 common characteristic peaks with retention times of 29.880 min, 31.233 min, 47.823 min, 54.867 min, 56.157 min, 79.547 min, 80.507 min, 81.923 min, 93.180 min, 99.693 min, and 102.317 min, respectively.

[0021] 107.710 min;

[0022] (2) It has 12 common characteristic peaks, of which peak 10 is the reference peak S of ginsenoside Rb1; the relative retention times of each characteristic peak and peak S are within ±5% of the specified values; the specified values ​​are: 0.30 (peak 1), 0.31 (peak 2), 0.48

[0023] (Peak 3), 0.55 (Peak 4), 0.57 (Peak 5), 0.80 (Peak 6), 0.81 (Peak 7), 0.82 (Peak 8), 0.94

[0024] (Peak 9), 1.00 (Peak 10, S), 1.03 (Peak 11), 1.08 (Peak 12);

[0025] (3) Using multiple batches of compound ferrous sulfate pills, the characteristic spectra obtained according to the above construction method were used to make a control spectrum by the average value or median method.

[0026] This invention tested 15 batches of compound ferrous sulfate pills and identified 12 common characteristic peaks. The selection of common characteristic peaks can be carried out using the fingerprint similarity evaluation software of the 2012 version of the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" formulated by the National Pharmacopoeia Commission.

[0027] This invention also discloses a method for constructing the characteristic spectrum of compound ferrous sulfate pills as described above, or the use of the comparative characteristic spectrum of compound ferrous sulfate pills in the quality testing of compound ferrous sulfate pill products.

[0028] This invention also provides a quality testing method for compound ferrous sulfate pills, including the step of comparing the characteristic spectrum of the compound ferrous sulfate pill product to be tested with the characteristic spectrum of a control compound ferrous sulfate pill; the characteristic spectrum of the compound ferrous sulfate pill product to be tested is obtained by using the compound ferrous sulfate pill product to be tested according to any of the above-described construction methods.

[0029] If the similarity between the characteristic chromatogram of the Compound Ferrous Sulfate Pill product to be tested and the characteristic chromatogram of the Compound Ferrous Sulfate Pill control is not less than 0.9, the product is considered qualified; if it is less than 0.9, it is considered unqualified. Specifically, the similarity is obtained by using the fingerprint chromatogram similarity evaluation software of the 2012 version of the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" formulated by the National Pharmacopoeia Commission.

[0030] Preferably, the compound ferrous sulfate pill product and the reference product are tested in the same way as the method described above for constructing the characteristic spectrum of the compound ferrous sulfate pill, and the corresponding characteristic spectrum is obtained. The characteristic spectrum of the compound ferrous sulfate pill product to be tested is compared with the characteristic spectrum of the compound ferrous sulfate pill reference and the characteristic spectrum of the reference product to evaluate its quality.

[0031] The beneficial effects of this invention are as follows:

[0032] 1. Compound ferrous sulfate pills are prepared using traditional honey pill technology. Seahorse and jujubes (pitted) are dried at 75-80℃ and pulverized into fine powder. Walnut kernels are crushed and ground into fine powder along with the remaining American ginseng, ferrous sulfate, and cinnamon. This mixture is then combined with the above powders, sieved, and made into small honey pills with refined honey. Compound ferrous sulfate pills have the functions of warming the kidneys and strengthening the marrow, replenishing qi and nourishing yin, promoting blood production and stopping bleeding. They are mainly used to treat leukopenia, bone marrow abnormalities, and deficiency of both qi and blood. The representative components of each herb in this prescription were analyzed. Ferrous sulfate is a sulfate mineral, specifically the ore of ferrous sulfate (FeSO4·7H2O), and its main component is hydrated ferrous sulfate. The original standard already controlled the content of this component. American ginseng has the effects of enhancing the function of the central nervous system, protecting the cardiovascular system, promoting blood vitality, promoting bone marrow protein synthesis, and improving immunity. Its main components are ginsenosides, with representative components being ginsenosides Rg1, Rb1, and Re. Cinnamon contains volatile components, among which cinnamaldehyde has sedative, analgesic, and anticoagulant effects. Jujube has the effects of tonifying the middle energizer, nourishing blood and calming the mind, protecting the liver, and antibacterial and anti-inflammatory properties. The thin-layer chromatography identification indicators in the 2020 edition of the Chinese Pharmacopoeia, Part I, are oleanolic acid and betulinic acid. The main components of seahorse and walnut kernel are proteins, oils, and trace elements. High-performance liquid chromatography (HPLC) was used to conduct fingerprint analysis on these components, as no specific indicator components were found. In summary, ginsenosides Rg1, Rb1, and Re from American ginseng; cinnamaldehyde from cinnamon; and oleanolic acid and betulinic acid from jujube were selected as indicator components. The same liquid chromatography method was used for localization analysis, and a characteristic chromatographic method was established to strengthen the overall quality control of compound ferrous sulfate pills.

[0033] 2. The method for constructing the characteristic spectrum of compound ferrous sulfate pills described in this invention overcomes the defect that the complex chemical composition of compound ferrous sulfate pills causes interference, making it impossible to detect the quality of the relevant dosage form from multiple angles and in all aspects. This invention obtains 12 common characteristic peaks and achieves effective separation of the 12 common characteristic peaks. The peak shapes are good and there is no interference, thereby realizing the quality detection of compound ferrous sulfate pills from multiple angles and in all aspects. This further realizes the quality control of compound ferrous sulfate pills. Moreover, this quality detection method is simple to operate. After methodological verification, it has good reproducibility, stability, reliable recovery rate, low detection cost, and high detection efficiency. The application of this method plays a vital role in the quality stability of compound ferrous sulfate pills.

[0034] 3. A set of rapid, comprehensive, and highly specific methods for quality testing of compound ferrous sulfate pills was established.

[0035] 4. The characteristic spectrum can perform quality control of compound ferrous sulfate pills from multiple angles and in all aspects, providing a stronger guarantee for the identification of genuine and counterfeit compound ferrous sulfate pills and large-scale production.

[0036] 5. The characteristic chromatogram / fingerprint chromatogram test results of each batch of Compound Ferrous Sulfate Pills show that the corresponding characteristic peaks were detected in different batches of Compound Ferrous Sulfate Pills, and the fingerprint chromatogram similarity was greater than 0.9, indicating that the product quality is uniform and stable. Attached Figure Description

[0037] The invention will now be further described with reference to the accompanying drawings:

[0038] Figure 1 This invention's experimental example 1, a literature review of compound ferrous sulfate pills, involves method development 1-gradient 1 UV wavelength scanning of a reference solution.

[0039] Figure (190–400 nm);

[0040] Figure 2 This invention's experimental example 1, a literature review of compound ferrous sulfate pills, includes method development 1-gradient 1, test solution 1-DAD full-wavelength analysis.

[0041] Long 3D scan image (190–400 nm);

[0042] Figure 3 This invention's experimental example 1, a literature review of compound ferrous sulfate pills, includes method development 1 - gradient 1 (test solution) 2 - DAD full-wavelength analysis.

[0043] Long 3D scan image (190–400 nm);

[0044] Figure 4 This is the superimposed chromatogram (203nm) of the literature method review of Compound Ferrous Sulfate Pills in Experimental Example 1 of this invention - Method Development 1 - Gradient 1;

[0045] Figure 5 This invention relates to Experiment 1, a literature review of the compound ferrous sulfate pill method, method development 2, gradient 2, and UV wavelength scanning of the reference solution.

[0046] Figure (190–400 nm);

[0047] Figure 6 This invention relates to Experiment 1, a literature review of the compound ferrous sulfate pill method, method development 2, gradient 2, test solution 1, and DAD full-wavelength method.

[0048] Long 3D scan image (190–400 nm);

[0049] Figure 7 This is the experimental example 1 of the present invention, a literature review of the compound ferrous sulfate pill method - method development 2 - gradient 2 test solution 2 - DAD full wavelength 3D scan image (190~400nm);

[0050] Figure 8 This is the superimposed chromatogram (203nm) of the literature method review of Compound Ferrous Sulfate Pills in Experimental Example 1 of this invention - Method Development 2 - Gradient 2.

[0051] Figure 9 This is an investigation into the preparation method of the test solution for compound ferrous sulfate pills in Experiment 1 of this invention - Method Development 3 - Gradient 3 Test Solution

[0052] 1-DAD full-wavelength 3D scan (190–400 nm);

[0053] Figure 10 This is an investigation into the preparation method of the test solution for compound ferrous sulfate pills in Experiment 1 of this invention - Method Development 3 - Gradient 3 Test Solution

[0054] 2-DAD full-wavelength 3D scan (190–400 nm);

[0055] Figure 11 This is an investigation into the preparation method of the test solution for compound ferrous sulfate pills in Experiment 1 of this invention - Method Development 3 - Gradient 3 Test Solution

[0056] 3D-DAD full-wavelength 3D scan (190–400 nm);

[0057] Figure 12 This is an investigation into the preparation method of the test solution for compound ferrous sulfate pills in Experiment 1 of this invention - Method Development 3 - Gradient 3 Test Solution

[0058] 4-DAD full-wavelength 3D scan (190–400 nm);

[0059] Figure 13 This is an example of the preparation method of the compound ferrous sulfate pill test solution in Experiment 1 of the present invention - method development 3 - gradient 3 chromatogram overlay (190-400nm);

[0060] Figure 14 This is an investigation into the preparation method of the test solution for compound ferrous sulfate pills in Experiment 1 of this invention - Method Development 4 - Gradient 3 Test Solution

[0061] 1-DAD full-wavelength 3D scan (190–400 nm);

[0062] Figure 15 This invention relates to Experiment 1, which investigates the preparation method of the compound ferrous sulfate pill test solution – Method Development 4 – Gradient 3 Test Solution.

[0063] 2-DAD full-wavelength 3D scan (190–400 nm);

[0064] Figure 16 This invention relates to Experiment 1, which investigates the preparation method of the compound ferrous sulfate pill test solution – Method Development 4 – Gradient 3 Test Solution.

[0065] 4-DAD full-wavelength 3D scan (190–400 nm);

[0066] Figure 17 This is an example of the preparation method of the compound ferrous sulfate pill test solution in Experiment 1 of this invention - method development 4 - gradient 3 chromatogram overlay; Figure 18 This invention is Example 1, Investigation of Chromatographic Conditions for Compound Ferrous Sulfate Pills - Method Development 5 - Gradient 3 Test Solution 1 - DAD Full Wave

[0067] Long 3D scan image (190–400 nm);

[0068] Figure 19 This is an example of the chromatographic conditions investigation of compound ferrous sulfate pills in Experiment 1 of the present invention - method development 5 - gradient 3 chromatogram overlay (190-400nm);

[0069] Figure 20 This invention is Example 1, Investigation of Chromatographic Conditions for Compound Ferrous Sulfate Pills - Method Development 6 - Gradient 4 Test Solution 1 - DAD Full Wave

[0070] Long 3D scan image (190–400 nm);

[0071] Figure 21 This is an example of the chromatographic conditions investigation of compound ferrous sulfate pills in Experiment 1 of the present invention - method development 6 - gradient 4 chromatogram overlay (190-400nm);

[0072] Figure 22 This invention is Example 1, Investigation of Chromatographic Conditions for Compound Ferrous Sulfate Pills - Method Development 7 - Gradient 5 Test Solution 1 - DAD Full Wave

[0073] Long 3D scan image (190–400 nm);

[0074] Figure 23 This is an example of the chromatographic conditions investigation of compound ferrous sulfate pills in Experiment 1 of the present invention - method development 7 - gradient 5 chromatogram overlay (190-400nm);

[0075] Figure 24 This is the chromatographic condition investigation of Compound Ferrous Sulfate Pills in Experimental Example 1 of the present invention - Method Development 8 - Gradient 5 chromatogram overlay (203nm);

[0076] Figure 25 This is an example of the chromatographic conditions investigation of compound ferrous sulfate pills in Experiment 1 of this invention - method development 9 - gradient 4 chromatogram overlay (203nm);

[0077] Figure 26 This is the chromatogram overlay (203nm) of Compound Ferrous Sulfate Pills, Experimental Example 1 of the present invention, under investigation of chromatographic conditions - method development 9 - gradient 6 chromatogram;

[0078] Figure 27This is the chromatographic condition investigation of Compound Ferrous Sulfate Pills in Experimental Example 1 of the present invention - Method Development 10 - Gradient 7 chromatogram overlay (203nm); Figure 28 This is the chromatogram of Compound Ferrous Sulfate Pills, Experimental Example 1 of the present invention, underwent chromatographic condition investigation - method development 11 - gradient 8 - flow rate 0.9 ml / min.

[0079] Overlay image (203nm);

[0080] Figure 29 This is the chromatogram overlay (203nm) of Compound Ferrous Sulfate Pills, Experimental Example 1 of the present invention, under investigation of chromatographic conditions - method development 12 - gradient 8 - column temperature 35℃;

[0081] Figure 30 This is the chromatogram of Compound Ferrous Sulfate Pills, Experimental Example 1 of the present invention, underwent chromatographic condition investigation - method development 13 - gradient 8 - flow rate 0.9 ml / min.

[0082] Overlay image (203nm);

[0083] Figure 31 This is the chromatographic condition investigation of Compound Ferrous Sulfate Pills in Experimental Example 1 of the present invention - Method Development 14 - Gradient 9 chromatogram overlay (203nm);

[0084] Figure 32 This is the chromatographic condition investigation of Compound Ferrous Sulfate Pills in Experimental Example 1 of the present invention - Method Development 15 - Gradient 8 chromatogram overlay (203nm);

[0085] Figure 33 This is the chromatographic condition investigation of Compound Ferrous Sulfate Pills in Experimental Example 1 of the present invention - Method Development 16 - Gradient 8 chromatogram overlay (203nm);

[0086] Figure 34 This is an example of the chromatographic conditions investigation of compound ferrous sulfate pills in Experiment 1 of this invention - method development 16 - gradient 10 chromatogram overlay (203nm);

[0087] Figure 35 This is the chromatographic condition investigation of Compound Ferrous Sulfate Pills in Experimental Example 1 of the present invention - Method Development 16 - Gradient 11 chromatogram overlay (203nm);

[0088] Figure 36 This is the chromatographic condition investigation of Compound Ferrous Sulfate Pills in Experimental Example 1 of the present invention - Method Development 17 - Gradient 8 chromatogram overlay (203nm);

[0089] Figure 37 This is an example of the chromatographic conditions investigation and method development of Compound Ferrous Sulfate Pills in Experiment 1 of this invention, 18-gradient 9 chromatogram overlay (203nm); Figure 38 This is Example 1 of the present invention, chromatographic conditions investigation of compound ferrous sulfate pills - method development 19 - gradient 12 reference solution UV wavelength.

[0090] Scan image (190–400 nm);

[0091] Figure 39 This is Experimental Example 1 of the present invention, Investigation of Chromatographic Conditions for Compound Ferrous Sulfate Pills - Method Development 19 - Gradient 12 Test Solution - DAD Complete

[0092] 3D scanning pattern of wavelength (190–400 nm);

[0093] Figure 40 This is an example of the chromatographic conditions investigation of compound ferrous sulfate pills in Experiment 1 of the present invention - method development 19 - gradient 12 chromatogram overlay (190-400nm);

[0094] Figure 41 This is the chromatogram of Experimental Example 1 of the present invention, Compound Ferrous Sulfate Pill Chromatography Conditions Investigation - Method Development 21 - Gradient 12;

[0095] Figure 42 This invention is Example 1, Chromatographic Conditions Investigation of Compound Ferrous Sulfate Pills - Method Development 22 - Gradient 13 Test Solution - DAD Complete

[0096] 3D scanning pattern of wavelength (190–400 nm);

[0097] Figure 43 This is an example of the chromatographic conditions investigation of compound ferrous sulfate pills in Experiment 1 of the present invention - method development 22 - gradient 13 chromatogram overlay (190-400nm);

[0098] Figure 44 This is the reproducibility study of the compound ferrous sulfate pill method in Experiment 1 of this invention - method reproducibility - gradient 13 test solution 1 - DAD full wave

[0099] Long 3D scan image (190–400 nm);

[0100] Figure 45 This is the reproducibility study of the compound ferrous sulfate pill method in Experiment Example 1 of the present invention - method reproducibility - gradient 13 test solution 2 - DAD full wavelength 3D scan image (190~400nm);

[0101] Figure 46 This is the reproducibility study of the compound ferrous sulfate pill method in Experimental Example 1 of the present invention - method reproducibility - gradient 13 chromatogram overlay (190-400nm);

[0102] Figure 47 This is the reproducibility study of the compound ferrous sulfate pill method in Experimental Example 1 of the present invention - Method Development 23 - Ultrasonic conditions to determine the UV wavelength scan of the reference solution (190-400nm);

[0103] Figure 48 This is the reproducibility study of the compound ferrous sulfate pill method in Experimental Example 1 of the present invention - Method Development 23 - Determination of test solution under ultrasonic conditions.

[0104] 1-DAD full-wavelength 3D scan (190–400 nm);

[0105] Figure 49 This is the reproducibility study of the compound ferrous sulfate pill method in Experimental Example 1 of the present invention - Method Development 23 - Determination of test solution under ultrasonic conditions.

[0106] 2-DAD full-wavelength 3D scan (190–400 nm);

[0107] Figure 50 This is the reproducibility study of the compound ferrous sulfate pill method in Experimental Example 1 of the present invention - Method Development 23 - Determination of test solution under ultrasonic conditions.

[0108] 3D-DAD full-wavelength 3D scan (190–400 nm);

[0109] Figure 51 This is the reproducibility study of the compound ferrous sulfate pill method in Experimental Example 1 of the present invention - Method Development 23 - Determination of test solution under ultrasonic conditions.

[0110] 4-DAD full-wavelength 3D scan (190–400 nm);

[0111] Figure 52 This is an example of the reproducibility study of the compound ferrous sulfate pill method in Experiment 1 of this invention - method development 23 - ultrasonic conditions determination of chromatogram overlay;

[0112] Figure 53 This is the HPLC characteristic chromatographic method verification of Compound Ferrous Sulfate Pills in Experimental Example 1 of the present invention - specific chromatographic overlay;

[0113] Figure 54 This is Experiment Example 1 of the present invention, HPLC characteristic chromatogram method verification of Compound Ferrous Sulfate Pills - peak assignment overlay of reference medicinal materials;

[0114] Figure 55 This is the HPLC characteristic chromatogram establishment of Compound Ferrous Sulfate Pills in Experiment 1 of the present invention - HPLC overlay chromatograms of 15 batches of samples;

[0115] Figure 56 This is the HPLC characteristic chromatogram of Compound Ferrous Sulfate Pills in Experiment Example 1 of the present invention - overlapping peaks of cinnamaldehyde in 15 batches;

[0116] Figure 57 This is the HPLC characteristic chromatogram establishment and control characteristic chromatogram of Compound Ferrous Sulfate Pills in Experiment Example 1 of this invention;

[0117] Figure 58 This is the HPLC characteristic chromatogram of the compound ferrous sulfate pill sample (batch number: 2303017) of Experimental Example 1 of the present invention;

[0118] Figure 59This is the peak matching diagram of the HPLC characteristic spectrum of the compound ferrous sulfate pill sample in Experimental Example 1 of this invention;

[0119] Figure 60 The compound ferrous sulfate pill characteristic chromatogram / fingerprint chromatogram and its technical parameters experimental characteristic chromatogram generated by the revision of the characteristic chromatogram in Experiment Example 1 of this invention is the control characteristic chromatogram of compound ferrous sulfate pill (peak 6: ginsenoside Rg1, peak 7: ginsenoside Re, peak 10 (S): ginsenoside Rb1). Detailed Implementation

[0120] The following embodiments are provided to further understand the present invention and are not limited to the preferred embodiments described herein, nor do they constitute a limitation on the content and scope of protection of the present invention.

[0121] The test reference standards, samples, reagents, instruments and equipment used in the embodiments of the present invention are shown in Tables 1-4.

[0122] Table 1. Control standards used in the experiment

[0123]

[0124]

[0125] Table 2 Samples used in the experiment

[0126] Product Name batch number source ferrous sulfate 20230101 Anhui Jiahe Traditional Chinese Medicine Technology Co., Ltd. American ginseng Q2301011 Anhui Jiahe Traditional Chinese Medicine Technology Co., Ltd. Haima 20221002 Guangdong Bengang Seahorse Aquaculture Co., Ltd. Cinnamon 201101 Anhui Jiahe Traditional Chinese Medicine Technology Co., Ltd. Dates (pitted) 20221102 Anhui Jiahe Traditional Chinese Medicine Technology Co., Ltd. Walnuts 220501 Anhui Jiahe Traditional Chinese Medicine Technology Co., Ltd. Compound Ferrous Sulfate Pills 2204027 Shaanxi Haoqijun Pharmaceutical Co., Ltd. Compound Ferrous Sulfate Pills 2204029 Shaanxi Haoqijun Pharmaceutical Co., Ltd. Compound Ferrous Sulfate Pills 2205037 Shaanxi Haoqijun Pharmaceutical Co., Ltd. Compound Ferrous Sulfate Pills 2207051 Shaanxi Haoqijun Pharmaceutical Co., Ltd. Compound Ferrous Sulfate Pills 2207058 Shaanxi Haoqijun Pharmaceutical Co., Ltd. Compound Ferrous Sulfate Pills 2208068 Shaanxi Haoqijun Pharmaceutical Co., Ltd. Compound Ferrous Sulfate Pills 2209076 Shaanxi Haoqijun Pharmaceutical Co., Ltd. Compound Ferrous Sulfate Pills 2209079 Shaanxi Haoqijun Pharmaceutical Co., Ltd. Compound Ferrous Sulfate Pills 2211092 Shaanxi Haoqijun Pharmaceutical Co., Ltd. Compound Ferrous Sulfate Pills 2211098 Shaanxi Haoqijun Pharmaceutical Co., Ltd. Compound Ferrous Sulfate Pills 2212114 Shaanxi Haoqijun Pharmaceutical Co., Ltd. Compound Ferrous Sulfate Pills 2301002 Shaanxi Haoqijun Pharmaceutical Co., Ltd. Compound Ferrous Sulfate Pills 2303015 Shaanxi Haoqijun Pharmaceutical Co., Ltd. Compound Ferrous Sulfate Pills 2303016 Shaanxi Haoqijun Pharmaceutical Co., Ltd. Compound Ferrous Sulfate Pills 2303017 Shaanxi Haoqijun Pharmaceutical Co., Ltd.

[0127] Table 3. Reagents used in the experiment

[0128]

[0129]

[0130] Table 4. Experimental Instruments and Equipment

[0131]

[0132] Example 1

[0133] This embodiment provides a method for constructing the characteristic spectrum of compound ferrous sulfate pills, including the following steps:

[0134] (1) Chromatographic conditions and system suitability: Octadecylsilane-bonded silica gel was used as the packing material (Shim-pack GISSC). 18 Alternatively, an equivalent column (25 cm long, 4.6 mm inner diameter, 5 μm particle size) can be used; acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, with gradient elution as specified in Table 5, at a flow rate of 1.0 mL / min, a column temperature of 40 °C, and a detection wavelength of 203 nm. The theoretical plate number, calculated based on the ginsenoside Rb1 peak, should be no less than 5000.

[0135] Table 5 Gradient elution program table

[0136]

[0137] (2) Preparation of test solution: Take an appropriate amount of this product, cut it into small pieces, take about 3g, weigh it accurately, put it in an Erlenmeyer flask, add 25ml of methanol accurately, seal it tightly, weigh it, sonicate it (power 500W, frequency 40KHz) for 2 hours, cool it, weigh it, make up the lost weight with methanol, shake it well, filter it, and the test solution is obtained.

[0138] (3) Preparation of reference solution: Take appropriate amounts of ginsenoside Re, ginsenoside Rg1 and ginsenoside Rb1 reference standards, add methanol to prepare a solution containing 0.2 mg of each per 1 ml.

[0139] (4) Determination method: Accurately pipette 10 μl of blank solvent, reference solution and test solution into the liquid chromatograph, measure and record the chromatogram to obtain the characteristic chromatogram of compound ferrous sulfate pill.

[0140] This invention also provides a quality testing method for compound ferrous sulfate pills, including obtaining a characteristic spectrum of the compound ferrous sulfate pill to be tested through a method for constructing a characteristic spectrum of compound ferrous sulfate pills, and further including a step of comparing the characteristic spectrum of the compound ferrous sulfate pill to be tested with a control characteristic spectrum of compound ferrous sulfate pills. The characteristic spectrum of the compound ferrous sulfate pill to be tested includes 12 common characteristic peaks with retention times of 29.880 min, 31.233 min, 47.823 min, 54.867 min, 56.157 min, 79.547 min, 80.507 min, 81.923 min, 93.180 min, 99.693 min, 102.317 min, and 107.710 min; it is determined to be of qualified quality.

[0141] Experimental Example 1

[0142] 1. Literature Review

[0143] 1.1 Method Development 1 - Gradient 1

[0144] The method was developed with reference to the chromatographic conditions for the content determination of American ginseng in Part I of the 2020 edition of the Chinese Pharmacopoeia. The chromatographic conditions are shown in Table 6.

[0145] Table 6 HPLC-DAD Determination - Gradient 1

[0146]

[0147] (1) Preparation of reference stock solution

[0148] Accurately weigh 10.33 mg of cinnamaldehyde reference standard into a 50 ml volumetric flask, dissolve and dilute to the mark with methanol, and shake well to obtain the cinnamaldehyde reference standard stock solution.

[0149] Accurately weigh 9.72 mg of oleanolic acid reference standard into a 50 ml volumetric flask, dissolve and dilute to the mark with methanol, and shake well to obtain the oleanolic acid reference standard stock solution.

[0150] Accurately weigh 10.24 mg of betulinic acid reference standard into a 50 ml volumetric flask, dissolve and dilute to the mark with methanol, and shake well to obtain the betulinic acid reference standard stock solution.

[0151] (2) Preparation of reference solution

[0152] Accurately weigh 10.13 mg of ginsenoside Rg1 reference standard, place it in a 50 ml volumetric flask, dissolve and dilute with methanol to the mark, and shake well to obtain the ginsenoside Rg1 reference solution.

[0153] Accurately weigh 10.10 mg of ginsenoside Re reference standard, place it in a 50 ml volumetric flask, dissolve and dilute with methanol to the mark, and shake well to obtain the ginsenoside Re reference standard solution.

[0154] Accurately weigh 10.47 mg of ginsenoside Rb1 reference standard, place it in a 10 ml volumetric flask, dissolve and dilute with methanol to the mark, and shake well to obtain the ginsenoside Rb1 reference solution.

[0155] Accurately measure 1 ml of cinnamaldehyde reference solution into a 20 ml volumetric flask, dilute to the mark with methanol, and shake well to obtain the cinnamaldehyde reference solution.

[0156] Accurately measure 5 ml of oleanolic acid reference standard stock solution, place it in a 20 ml volumetric flask, dilute with methanol to the mark, and shake well to obtain the reference standard solution.

[0157] Accurately measure 5 ml of betulinic acid stock solution and place it in a 20 ml volumetric flask. Dilute to the mark with methanol and shake well to obtain the betulinic acid reference solution.

[0158] (3) Preparation of the test solution

[0159] Test Solution 1: Take an appropriate amount of this product (batch number: 2301002), cut it into small pieces, weigh approximately 3g accurately, place it in an Erlenmeyer flask, accurately add 50ml of water-saturated n-butanol, seal tightly, weigh, reflux in a water bath for 1.5 hours, weigh again, replenish the lost weight with water-saturated n-butanol, shake well, and filter. Accurately measure 25ml of the subsequent filtrate, place it in an evaporating dish, evaporate to dryness, dissolve the residue in an appropriate amount of 50% methanol, transfer to a 10ml volumetric flask, dilute to the mark with 50% methanol, shake well, filter, and collect the subsequent filtrate to obtain the test solution (refer to the preparation method of test solution in the determination of American ginseng medicinal material content in the 2020 edition of the Chinese Pharmacopoeia).

[0160] For test solution 2, take 20 ml of the n-butanol filtrate from test solution 1 and place it in a separatory funnel. Wash twice with 10 ml of ammonia solution each time, discarding the aqueous solution. Extract twice with 10 ml of saturated n-butanol aqueous solution each time, discarding the aqueous solution. Evaporate the n-butanol solution to dryness, dissolve the residue in an appropriate amount of 50% methanol, transfer it to a 5 ml volumetric flask, dilute to the mark with 50% methanol, shake well, filter, and collect the filtrate.

[0161] Accurately pipette 10 μl each of the blank solvent, reference solution, and test solution into the liquid chromatograph. Perform a full-wavelength scan using a DAD detector from 190 to 400 nm and record the chromatograms. The results are shown in the figure. Figure 1 3D absorption scan of the test solution at 190-400 nm, see [link / data]. Figure 2 , 3 .

[0162] According to the method for determining the content of American ginseng in Part I of the Chinese Pharmacopoeia, the results showed that in the chromatogram of the reference standards, the peaks of ginsenoside Rb1, ginsenoside Re, ginsenoside Rg1, and cinnamaldehyde were good, while betulinic acid and oleanolic acid did not show peaks. The reason for this may be that the concentration of organic phase in the mobile phase was insufficient.

[0163] The results showed that both test solutions exhibited maximum absorption in the 190nm–230nm range in their 3D scans. Considering that most known characteristic peaks are ginsenosides, the detection wavelength of 203nm for American ginseng was temporarily selected for method development. The chromatogram at 203nm was extracted and analyzed; the results are shown below. Figure 4 .

[0164] The results showed that in the chromatogram of test solution 1, the peak matching degree of ginsenoside Re and ginsenoside Rg1 was both >990, but the resolution was 1.17, indicating poor separation effect; the peak matching degree of ginsenoside Rb1 and cinnamaldehyde was <990, indicating impurity; betulinic acid and oleanolic acid did not elute. In the chromatogram of test solution 2, the response value of water-soluble components decreased, and the peak matching degree of ginsenoside Rb1, ginsenoside Re, and ginsenoside Rg1 was all >990, meeting the requirements; the peak area of ​​cinnamaldehyde was too small, with a peak matching degree of 772 <990, and betulinic acid and oleanolic acid still did not elute. The reason for this is that cinnamaldehyde is a volatile component, and alkaline conditions can cause it to undergo hydrolysis. Additionally, the evaporation of the solution can also lead to its loss. In summary, the preparation method of the test solution and the gradient elution method of the mobile phase need to be further adjusted.

[0165] 1.2 Method Development 2 - Gradient 2

[0166] The method was developed based on the initial elution gradient for the determination of total saponins in ginseng stems and leaves under the "Ginseng and Peony Tablets" section of the 2020 edition of the Chinese Pharmacopoeia. The chromatographic conditions are shown in Table 7.

[0167] Table 7 HPLC-DAD Determination - Gradient 2

[0168]

[0169] Accurately pipette 10 μl each of the blank solvent (methanol), the reference solution from section "1.1 Method Development 1 - Gradient 1", and the test solution, and inject them into the liquid chromatograph. Perform a full-wavelength scan using a DAD detector from 190 to 400 nm, and record the chromatograms. The results are shown in the figure. Figures 5-7 .

[0170] The results showed that the peak matching degree of each reference standard was greater than 990, indicating good peak purity. The maximum absorption wavelength of each reference standard solution (except for cinnamaldehyde) was not significantly different, with significant absorption at the absorption end of 190 nm. The cinnamaldehyde reference standard solution showed maximum absorption at 203 nm, 223 nm, and 291 nm. The 3D scans of the two test solutions showed significant absorption in the wavelength range of 190–230 nm. To reduce interference from the blank solvent, and considering that the detection wavelength for the content determination of American ginseng in the 2020 edition of the Chinese Pharmacopoeia is 203 nm, the absorption wavelength was initially determined to be 203 nm.

[0171] Chromatograms of each solution at a wavelength of 203 nm were obtained and compared for analysis. The results are shown below. Figure 8 .

[0172] The results showed that in the chromatograms of the two test solutions, test solution 1 had significantly more peaks than test solution 2; the elution times of ginsenoside Re and ginsenoside Rg1 were close, resulting in ineffective separation of the two components; the peak matching degree of cinnamaldehyde and ginsenoside Rb1 was greater than 990, indicating good peak purity, but the peak area of ​​cinnamaldehyde in test solution 2 was significantly lower than that in test solution 1; betulinic acid and oleanolic acid were still not effectively detected. In summary, the processing method of test solution 2 was inferior to that of test solution 1, and further adjustments to the test solution preparation method and the gradient elution method of the mobile phase are needed.

[0173] 2. Investigation of the preparation method of the test solution

[0174] 2.1 Method Development 3 - Gradient 3

[0175] Referring to the determination method for the content of American ginseng medicinal materials in Part I of the 2020 edition of the Chinese Pharmacopoeia and the determination method for the total saponin content of ginseng stems and leaves in Part I of the 2020 edition of the Chinese Pharmacopoeia, methanol and n-butanol were selected as solvents, and ultrasonic and reflux extraction methods were used. The chromatographic conditions are shown in Table 8.

[0176] Table 8 HPLC-DA

[0177]

[0178] Preparation of Test Solution 1: Take an appropriate amount of this product (batch number: 2301002), cut it into small pieces, weigh about 3g accurately, place it in an Erlenmeyer flask, accurately add 50ml of methanol, seal tightly, weigh, sonicate (power 500W, frequency 40kHz) for 30 minutes, let stand overnight, sonicate again (power 500W, frequency 40kHz) for 30 minutes, cool, replenish the lost weight with methanol, shake well, filter, and the test solution is obtained.

[0179] Preparation of Test Solution 2: Take an appropriate amount of this product (batch number: 2301002), cut it into small pieces, weigh about 3g accurately, place it in an Erlenmeyer flask, accurately add 50ml of n-butanol, seal tightly, weigh, sonicate (power 500W, frequency 40kHz) for 30 minutes, let stand overnight, sonicate again (power 500W, frequency 40kHz) for 30 minutes, cool, replenish the lost weight with n-butanol, shake well, filter, accurately measure 25ml of the filtrate, place it in a separatory funnel, extract twice with 10ml of saturated aqueous solution of n-butanol each time, discard the aqueous solution, evaporate the n-butanol solution to dryness, dissolve the residue in an appropriate amount of methanol, transfer to a 10ml volumetric flask, dilute to the mark with methanol, shake well, filter, and the test solution is obtained.

[0180] Preparation of test solution 3: Take an appropriate amount of this product (batch number: 2301002), cut it into small pieces, take about 3g, accurately weigh it, accurately add 50ml of methanol, seal tightly, weigh it, reflux in a water bath for 3 hours, cool it, weigh it again, make up the weight loss with methanol, shake well, filter it, and the test solution is obtained.

[0181] Preparation of Test Solution 4: Take an appropriate amount of this product (batch number: 2301002), cut it into small pieces, take about 3g, accurately weigh it, accurately add 50ml of n-butanol, stopper tightly, weigh it, reflux in a water bath for 3 hours, cool, weigh it again, make up the weight loss with n-butanol, shake well, and filter. Accurately measure 25ml of the filtrate, extract it twice with 10ml of n-butanol-saturated aqueous solution each time, discard the aqueous solution, evaporate the n-butanol solution to dryness, dissolve the residue in an appropriate amount of methanol, transfer it to a 10ml volumetric flask, dilute to the mark with methanol, shake well, and filter to obtain the test solution.

[0182] Accurately pipette 10 μl each of the blank solvent (methanol), the reference solution under "1.1 Method Development 1 - Gradient 1", and the above-mentioned test solutions 1-4, and inject them into the liquid chromatograph. Perform a full-wavelength scan using a DAD detector from 190 to 400 nm, and record the chromatograms. The results are shown in [Figure number missing]. Figures 9-12 The chromatogram at 203 nm was extracted and analyzed, see [link to chromatogram]. Figure 13 .

[0183] The results showed that the scan chromatograms of each reference solution at wavelengths of 190–400 nm were basically consistent with those in section “1.2 Method Development 2-Gradient 2”, with peak matching degrees all greater than 990, indicating good peak purity. In the chromatograms of the four test solutions, the elution times of ginsenoside Re and ginsenoside Rg1 were quite close, failing to achieve effective separation. Betulinic acid and oleanolic acid were still not effectively localized. There was no significant difference between methanol ultrasonication and reflux methods, while n-butanol ultrasonication and reflux methods were worse than methanol treatment. Considering all factors, methanol ultrasonication was tentatively chosen as the method for preparing the test solutions, and the grinding method for the diatomaceous earth mixed sample was investigated. Simultaneously, to ensure good separation of ginsenoside Re and ginsenoside Rg1 in the test solutions, the chromatographic column was changed for further investigation.

[0184] 2.2 Method Development 4-Gradient 3

[0185] To enhance the operability of test sample solution preparation, two sampling methods were investigated: directly sampling by cutting the pills into small pieces, or sampling the cut-up sample after grinding it together with diatomaceous earth. The sample processing method was also validated, and the chromatographic conditions are shown in Table 9.

[0186] Table 9 HPLC-DAD Determination - Gradient 3

[0187]

[0188]

[0189] Preparation of test solution 1: Take an appropriate amount of this product (batch number: 2301002), cut it into small pieces, take about 3g, take about 2g of diatomaceous earth, mix and grind them, weigh accurately, add 25ml of methanol accurately, weigh accurately, sonicate (power 500W, frequency 40kHz) for 2 hours, cool, weigh accurately, make up the weight loss with methanol, shake well, filter, and the test solution is obtained.

[0190] Preparation of test solution 2: Take an appropriate amount of this product (batch number: 2301002), cut it into small pieces, take about 3g, weigh it accurately, put it in an Erlenmeyer flask, accurately add 25ml of methanol, seal it tightly, weigh it, sonicate it (power 500W, frequency 40kHz) for 2 hours, cool it, weigh it, make up the weight loss with methanol, shake it well, filter it, and the test solution is obtained.

[0191] Preparation of test solution 3 (diatomaceous earth blank): Weigh approximately 3g of diatomaceous earth accurately, add 25ml of methanol accurately, seal tightly, weigh, sonicate (500W power, 40kHz frequency) for 2 hours, cool, weigh, replenish the lost weight with methanol, shake well, filter, and the test solution is obtained.

[0192] Preparation of test solution 4: Take an appropriate amount of this product (batch number: 2301002), cut it into small pieces, take about 3g, accurately weigh it, accurately add 25ml of methanol, seal tightly, weigh it, sonicate (power 500W, frequency 40kHz) for 30 minutes, let it stand overnight, sonicate again (power 500W, frequency 40kHz) for 2 hours, cool it, weigh it, make up the lost weight with methanol, shake well, filter it, and the test solution is obtained.

[0193] Accurately pipette 10 μl each of the blank solvent (methanol), the reference solution under "1.1 Method Development 1 - Gradient 1", and each of the above-mentioned test solutions, and inject them into the liquid chromatograph. Perform a full-wavelength scan using a DAD detector from 190 to 400 nm, and record the chromatograms. The results are shown in the figure. Figures 14-16 The chromatogram at 203 nm was extracted and analyzed, see [link to chromatogram]. Figure 17 .

[0194] The results showed that the scan chromatograms of each reference solution at wavelengths of 190–400 nm were basically consistent with those under “1.2 Method Development 2-Gradient 2”, with peak matching degrees all greater than 990, indicating good peak purity. There was essentially no significant difference in the number of peaks in the chromatograms of the three test samples. The elution times of ginsenoside Re and ginsenoside Rg1 were quite close, indicating ineffective separation. The peak areas of betulinic acid and oleanolic acid were too small for accurate integration. There was no significant difference in the preparation methods of the three test sample solutions. Therefore, the tentative method for preparing the test sample solutions is to use a simpler method of sample crushing and direct ultrasonication with methanol. Further investigation with a different chromatographic column is needed to ensure effective separation of ginsenoside Re and ginsenoside Rg1.

[0195] 3. Investigation of chromatographic conditions

[0196] 3.1 Method Development 5-Gradient 3

[0197] Methanol was used as the solvent, and direct ultrasonic extraction was employed. GL Inertsustain AQ C was used. 18 The chromatographic column (4.6 mm * 250 mm, 5 μm) and other chromatographic conditions were investigated as shown in Table 9.

[0198] Accurately pipette 10 μL each of the blank solvent (methanol), the reference solution under "1.1 Method Development 1 - Gradient 1", and the test solution under "2.2 Method Development 4 - Gradient 3", and inject them into the liquid chromatograph. Perform a full-wavelength scan using a DAD detector from 190 to 400 nm, and record the chromatograms. The results are shown in the figure. Figure 18 The chromatogram at 203 nm was extracted and analyzed, see [link to chromatogram]. Figure 19 .

[0199] The results showed that the scan chromatograms of each reference solution at wavelengths of 190–400 nm were basically consistent with those under “1.2 Method Development 2-Gradient 2”, with peak matching degrees all greater than 990, indicating good peak purity. In the chromatograms of the test solution, the resolution of ginsenoside Re and ginsenoside Rg1 was 1.32, which was better than the column used in the previous study, but the elution times were still relatively close. The peak areas of betulinic acid and oleanolic acid were too small to be accurately integrated, so the gradient method was adjusted to continue the study.

[0200] 3.2 Method Development 6-Gradient 4

[0201] The test solution and chromatographic column remained unchanged, and other chromatographic conditions are shown in Table 10.

[0202] Table 10 HPLC-DAD Determination - Gradient 4

[0203]

[0204]

[0205] Accurately pipette 10 μL each of the blank solvent (methanol), the reference solution under "1.1 Method Development 1 - Gradient 1", and the test solution under "2.2 Method Development 4 - Gradient 3", and inject them into the liquid chromatograph. Perform a full-wavelength scan using a DAD detector from 190 to 400 nm, and record the chromatograms. The results are shown in the figure. Figure 20 The chromatogram at 203 nm was extracted and analyzed, see [link to chromatogram]. Figure 21 .

[0206] The results showed that the scan chromatograms of each reference solution at wavelengths of 190–400 nm were basically consistent with those under “1.2 Method Development 2-Gradient 2”, with peak matching degrees all greater than 990, indicating good peak purity. After adjusting elution gradient 4, the resolution of ginsenoside Re and ginsenoside Rg1 in the chromatogram of the test solution was 1.34, which was not significantly different from elution gradient 3, and the elution times were still relatively close. The elution characteristics of other chromatographic peaks did not change much. Therefore, the column was changed again to reduce the rate of organic phase rise in the mobile phase from 25 to 65 minutes, and the column was changed again to continue the study.

[0207] 3.3 Method Development 7-Gradient 5

[0208] The preparation method of the test solution remains unchanged, and other chromatographic conditions are shown in Table 11.

[0209] Table 11 HPLC-DAD Determination - Gradient 5

[0210]

[0211]

[0212] Preparation of the test solution: Take an appropriate amount of this product (batch number: 2301002), cut it into small pieces, weigh about 3g accurately, place it in an Erlenmeyer flask, accurately add 25ml of methanol, seal tightly, weigh, sonicate (power 500W, frequency 40kHz) for 2 hours, cool, weigh, replenish the lost weight with methanol, shake well, filter, and the test solution is obtained.

[0213] Accurately pipette 10 μl each of the blank solvent (methanol), the reference solution from section "1.1 Method Development 1 - Gradient 1", and the above-mentioned test solution into the liquid chromatograph. Perform a full-wavelength scan using a DAD detector from 190 to 400 nm and record the chromatograms. The results are shown in the figure. Figure 22 The chromatogram at 203 nm was extracted and analyzed, see [link to chromatogram]. Figure 23 .

[0214] The results showed that the scan chromatograms of each reference solution at wavelengths of 190–400 nm were basically consistent with those under “1.2 Method Development 2-Gradient 2”, with peak matching degrees all greater than 990, indicating good peak purity. After adjusting elution gradient 5, the resolution of ginsenoside Re and ginsenoside Rg1 in the chromatogram of the test sample was 1.44, and the elution times were still relatively close. The elution of other chromatographic peaks did not change much, so the column was changed for further investigation.

[0215] 3.4 Method Development 8-Gradient 5

[0216] The preparation method of the test solution remains unchanged, but the chromatographic column is changed to Thermo BDS Hypersil. TM C 18 (4.6mm*250mm, 5μm) was investigated using a UV detector. The chromatographic conditions are shown in Table 12.

[0217] Table 12 HPLC determination - gradient 5

[0218]

[0219]

[0220] Preparation of the test solution: Take an appropriate amount of this product (batch number: 2301002), cut it into small pieces, weigh about 3g accurately, place it in an Erlenmeyer flask, accurately add 25ml of methanol, seal tightly, weigh, sonicate (power 500W, frequency 40kHz) for 2 hours, cool, weigh, replenish the lost weight with methanol, shake well, filter, and the test solution is obtained.

[0221] Accurately pipette 10 μl each of the blank solvent (methanol), the reference solution from section "1.1 Method Development 1 - Gradient 1", and the above-mentioned test solution into the liquid chromatograph, record the chromatograms, and see the results shown in the figure. Figure 24 .

[0222] The results showed that the peak shapes of each reference standard were good in the chromatograms of each reference standard; however, no peak of ginsenoside Rg1 was observed in the chromatogram of the test sample, and the resolutions of ginsenoside Re and cinnamaldehyde were 1.27, respectively. Furthermore, the peak shape of ginsenoside Rb1 was poor, with significant peak interference, making the gradient method unsuitable. Therefore, gradient method 4 was used for further investigation.

[0223] 3.5 Method Development 9 - Gradient 4 & Gradient 6

[0224] The preparation method of the test solution and the chromatographic column remain unchanged. Other chromatographic conditions are shown in Table 13.

[0225] Table 13 HPLC determination - gradient 4

[0226]

[0227] Accurately pipette 10 μl each of the blank solvent (methanol), the ginsenoside Rg1 reference solution under "1.1 Method Development 1-Gradient 1", and the test solution under "3.4 Method Development 8-Gradient 5", inject them into the liquid chromatograph, record the chromatograms, and see the results below. Figure 25 .

[0228] The results showed that the resolution of ginsenoside Re and ginsenoside Rg1 in the chromatogram of the test sample was 1.36, with the peak times being earlier and the resolution being poor. Therefore, the organic phase intensification rate was reduced for further investigation. The chromatographic conditions are shown in Table 14.

[0229] Table 14 HPLC determination - gradient 6

[0230]

[0231] Accurately pipette 10 μl each of the blank solvent (methanol), the solution from section "1.1 Method Development 1 - Gradient 1", the reference solution, and the test solution from section "3.4 Method Development 8 - Gradient 5", and inject them into the liquid chromatograph. Record the chromatograms. The results are shown in [Figure number missing]. Figure 26 .

[0232] The results showed that the peak shapes of each reference standard were good in the chromatograms; however, ginsenoside Re had a distinct peak in the chromatogram of the test sample, but the peak shape was poor. Therefore, the elution gradient of the mobile phase was adjusted, and further investigation was conducted.

[0233] 3.6 Method for developing 10-gradient 7

[0234] The method was developed with reference to the chromatographic conditions for the content determination of ginseng medicinal materials in Part I of the 2020 edition of the Chinese Pharmacopoeia. The chromatographic conditions are shown in Table 15.

[0235] Table 15 HPLC determination - gradient 7

[0236]

[0237]

[0238] Preparation of the test solution: Take an appropriate amount of this product (batch number: 2301002), cut it into small pieces, weigh about 3g accurately, place it in an Erlenmeyer flask, accurately add 25ml of methanol, seal tightly, weigh, sonicate (power 500W, frequency 40kHz) for 2 hours, cool, weigh, replenish the lost weight with methanol, shake well, filter, and the test solution is obtained.

[0239] Accurately pipette 10 μl each of the blank solvent (methanol), the reference solution from section "1.1 Method Development 1 - Gradient 1", and the above-mentioned test solution into the liquid chromatograph, record the chromatograms, and see the results shown in the figure. Figure 27 .

[0240] The results showed that oleanolic acid and betulinic acid had poor peak shapes in the chromatograms of the reference standards, while other peak shapes were better. The test solution had fewer peaks and lower response values, which may be due to the low acetonitrile intensification rate, which caused the chromatographic peak retention time to shift later, resulting in a decrease in peak height and detection sensitivity. Therefore, the mobile phase ratio was adjusted and the flow rate was changed for further determination.

[0241] 3.7 Method Development 11-Gradient 8-Flow Rate 0.9 ml / min

[0242] Based on the above results, the separation of ginsenoside Rg1 and ginsenoside Re in elution gradient 4 was relatively good. The elution gradient method of elution gradient 4 was retained before 50 minutes. After 50 minutes, the separation of each chromatographic peak was relatively good. Therefore, the elution rate of the organic phase was increased and the determination was continued. The chromatographic conditions are shown in Table 16.

[0243] Table 16 HPLC Determination - Gradient 8

[0244]

[0245] manual

[0246]

[0247] Accurately pipette 10 μl each of the blank solvent (methanol), the reference solution under "1.1 Method Development 1 - Gradient 1", and the test solution under "3.6 Method Development 10 - Gradient 7", inject them into the liquid chromatograph, record the chromatograms, and see the results below. Figure 28 .

[0248] The results showed that the peak shapes of each reference standard were good in the chromatogram; the number of peaks in the test solution was relatively large. The resolution of ginsenoside Rg1 and ginsenoside Re was 1.33, and the peak area and response value were low. Therefore, the column temperature was adjusted to improve the resolution.

[0249] 3.8 Method Development: 12-gradient 8-column temperature 35℃

[0250] To improve the separation of ginsenoside Rg1 and ginsenoside Re, the column temperature was lowered to 35℃, while other chromatographic conditions remained unchanged, as shown in Table 16.

[0251] Accurately pipette 10 μl each of the blank solvent (methanol), the reference solution under "1.1 Method Development 1 - Gradient 1", and the test solution under "3.6 Method Development 10 - Gradient 7", inject them into the liquid chromatograph, record the chromatograms, and see the results below. Figure 29 .

[0252] The results showed that the peak shapes of the reference standards were good in the chromatograms; in the chromatograms of the test solution, the resolution of ginsenoside Rg1 and ginsenoside Re was 1.50, but the peak shapes of other unknown chromatographic peaks were poor and there was obvious peak engulfment. Based on the above research, the Thermo Acclaim column was used for determination, and the resolution between chromatographic peaks was relatively good. Therefore, this column was used and the determination was continued at an elution flow rate of 0.9 ml / min.

[0253] 3.9 Method Development 13-Gradient 8-Flow Rate 0.9 ml / min

[0254] To screen for better test solution preparation methods, obtain more representative chromatographic peaks, and reduce baseline interference, two methods were used to prepare the test solution: direct ultrasonication with methanol and separation and purification with macroporous resin after ultrasonication. The elution gradient method remained unchanged, the flow rate was 0.9 ml / min, the chromatographic column was Thermo Acclaim™ 120C18 (4.6 mm * 250 mm, 5 μm), and other chromatographic conditions remained unchanged, as shown in Table 16.

[0255] Preparation of test solution 1: Take an appropriate amount of this product (batch number: 2301002), cut it into small pieces, weigh about 3g accurately, place it in an Erlenmeyer flask, accurately add 25ml of methanol, seal tightly, weigh, sonicate (power 500W, frequency 40kHz) for 2 hours, cool, weigh, replenish the lost weight with methanol, shake well, filter, and the test solution is obtained.

[0256] Preparation of test solution 2: Take 10 ml of the filtrate from the above "test solution 1" section, evaporate to dryness in a water bath, place it on a D101 macroporous resin column (inner diameter 1.5 cm, column height 10 cm), elute with 20 ml of water, discard the eluent, then elute with 50 ml of 70% ethanol, collect the eluent, evaporate to dryness in a water bath, transfer to a 5 ml volumetric flask with methanol, shake well, filter, and the test solution is obtained.

[0257] Accurately pipette 10 μl each of the blank solvent (methanol), the reference solution from section "1.1 Method Development 1 - Gradient 1", and the above-mentioned test solution into the liquid chromatograph, record the chromatograms, and see the results shown in the figure. Figure 30 .

[0258] The results showed that the peak shapes of the reference standards were good in the chromatograms. In the chromatograms of the two test solutions, oleanolic acid and betulinic acid did not show peaks, and the separation of ginsenoside Rg1 and ginsenoside Re was poor. Furthermore, after purification using D101 macroporous resin, the peak areas of some test solutions increased because the solution concentration was twice that of test solution 1, but cinnamaldehyde was not detected. This indicates that the D101 macroporous resin purification method is not suitable for preparing the test solutions. Moreover, the results showed little difference at column temperatures of 35℃ and 40℃. Therefore, the column temperature of 40℃ was continued, and the ultrasonic treatment method was used to continue method development for test solution preparation.

[0259] 3.10 Method Development 14-Gradient 9

[0260] The test solution was prepared using the direct ultrasonic method, and the chromatographic column used was an Agilent ZORBAX EclipseXDB-C. 18 To reduce the initial gradient organic phase ratio, the chromatographic conditions are shown in Table 17.

[0261] Table 17 HPLC Determination - Gradient 9

[0262]

[0263]

[0264] Accurately pipette 10 μl each of the blank solvent (methanol), the reference solution under "1.1 Method Development 1 - Gradient 1", and the test solution under "3.9 Method Development 13 - Gradient 8 - Flow Rate 0.9 mL / min", inject them into the liquid chromatograph, record the chromatograms, and see the results below. Figure 31 .

[0265] The results showed that the peak shapes of each reference standard were good in the chromatogram; however, ginsenosides Rb1 and Re were not separated in the chromatogram of the test solution, indicating that the gradient was not suitable. Therefore, gradient 8 was changed and the investigation continued.

[0266] 3.11 Method Development 15-Gradient 8

[0267] The Agilent ZORBAX Eclipse XDB-C18 column (4.6mm*250mm, 5μm) remained unchanged, and other chromatographic conditions are shown in Table 11.

[0268] Accurately pipette 10 μl each of the blank solvent (methanol), the ginsenoside Rg1 reference solution under "1.1 Method Development 1 - Gradient 1", and the test solution under "3.9 Method Development 13 - Gradient 8 - Flow Rate 0.9 ml / min", inject them into the liquid chromatograph, record the chromatograms, and see the results below. Figure 32 .

[0269] The results showed that ginsenosides Rg1 and Re were not effectively separated in the chromatogram of the test solution. Therefore, the Welch Ultimate XB-C18 column, which is relatively better at separating ginsenosides Rg1 and Re, was used for further investigation.

[0270] 3.12 Method Development of 16-Gradient 8, 10, 11

[0271] The chromatographic column used was a Welch Ultimate XB-C18 (4.6mm*250mm, 5μm), and other chromatographic conditions were the same as those in Table 16.

[0272] Accurately pipette 10 μl each of the blank solvent (methanol) and the test solution from section "3.9 Method Development 13-Gradient 8-Flow Rate 0.9 ml / min" into the liquid chromatograph, record the chromatograms, and see the results below. Figure 33 .

[0273] The results showed that the resolution of peaks 4 and 5 in the chromatogram of the test solution was 1.15. Therefore, the acetonitrile intensification rate was reduced by 30–50 minutes to continue the investigation. Other chromatographic conditions are shown in Table 18.

[0274] Table 18 HPLC Determination - Gradient 10

[0275]

[0276] Accurately pipette 10 μl each of the blank solvent (methanol) and the test solution from section "3.9 Method Development 13-Gradient 8-Flow Rate 0.9 ml / min" into the liquid chromatograph, record the chromatograms, and see the results below. Figure 34 .

[0277] The results showed that the resolution of peaks 4 and 5 in the chromatogram of the test solution was 1.18, which was not significantly different from gradient 8. Therefore, the acetonitrile elution rate was further reduced before 37 minutes. Since the peak amount was low between 70 and 90 minutes, the acetonitrile elution rate was increased to continue the investigation. The chromatographic conditions are shown in Table 19.

[0278] Table 19 HPLC Determination - Gradient 11

[0279]

[0280]

[0281] Accurately pipette 10 μl each of the blank solvent (methanol), the reference solution under "1.1 Method Development 1 - Gradient 1", and the test solution under "3.9 Method Development 13 - Gradient 8 - Flow Rate 0.9 mL / min", inject them into the liquid chromatograph, record the chromatograms, and see the results below. Figure 35.

[0282] The results showed that the separation degree of ginsenosides Rg1 and Re in the chromatogram of the test solution was 0.99. Despite repeated adjustments to the mobile phase ratio, they could not be effectively separated. Therefore, the chromatographic column was replaced for further investigation.

[0283] 3.13 Method Development 17-Gradient 8

[0284] The preparation method of the test solution remains unchanged, but the chromatographic column is replaced with a Shim Pack Giss C18 (4.6mm*250mm, 5μm). Other chromatographic conditions are shown in Table 16.

[0285] Preparation of the test solution: Take an appropriate amount of this product (batch number: 2301002), cut it into small pieces, weigh about 3g accurately, place it in an Erlenmeyer flask, accurately add 25ml of methanol, seal tightly, weigh, sonicate (power 500W, frequency 40kHz) for 2 hours, cool, weigh, replenish the lost weight with methanol, shake well, filter, and the test solution is obtained.

[0286] Accurately pipette 10 μl each of the blank solvent (methanol), the reference solution from section "1.1 Method Development 1 - Gradient 1", and the above-mentioned test solution into the liquid chromatograph, record the chromatograms, and see the results shown in the figure. Figure 36 .

[0287] The results showed that the resolution of ginsenosides Rg1 and Re in the chromatogram of the test solution was 1.70, which met the requirements. The chromatographic column provided good separation of ginsenosides Rg1 and Re, so this column was selected for the characteristic chromatographic study of this product. However, the retention times of cinnamaldehyde and ginsenoside Re in the test solution were relatively close, indicating an encapsulation phenomenon. Therefore, the gradient method was further adjusted to optimize the chromatographic conditions.

[0288] 3.14 Method Development 18-Gradient 9

[0289] The test sample was prepared by direct ultrasonication. The chromatographic column used was a Shim Pack Giss C18 (4.6 mm * 250 mm, 5 μm), and other chromatographic conditions were the same as in Table 17.

[0290] Accurately pipette 10 μl each of the blank solvent (methanol), the reference solution under "1.1 Method Development 1 - Gradient 1", and the test solution under "3.13 Method Development 17 - Gradient 8", inject them into the liquid chromatograph, record the chromatograms, and see the results below. Figure 37 .

[0291] The results showed that the resolution of ginsenosides Rg1 and Re in the chromatogram of the test solution was 1.63, which met the requirements; the resolution of cinnamaldehyde was poor; and a gradient peak appeared at the retention time of oleanolic acid, which interfered with its determination. Therefore, the gradient method was adjusted and the investigation continued.

[0292] 3.15 Method Development 19-Gradient 12

[0293] The test sample was prepared by direct ultrasonication, and other chromatographic conditions are shown in Table 20.

[0294] Table 20 HPLC Determination - Gradient 12

[0295]

[0296] Preparation of the test solution: Take an appropriate amount of this product (batch number: 2301002), cut it into small pieces, weigh about 3g accurately, place it in an Erlenmeyer flask, accurately add 25ml of methanol, seal tightly, weigh, sonicate (power 500W, frequency 40kHz) for 2 hours, cool, weigh, replenish the lost weight with methanol, shake well, filter, and the test solution is obtained.

[0297] Accurately pipette 10 μl each of the blank solvent (methanol) and the reference solution from section "1.1 Method Development 1-Gradient 1" and the above-mentioned test solution into the liquid chromatograph. Perform a full-wavelength scan using a DAD detector from 190 to 400 nm and record the chromatograms. The results are shown in the figure. Figure 38 , 39 The chromatogram at 203 nm was extracted and analyzed, see [link to chromatogram]. Figure 40 .

[0298] The results showed that the peak matching degree in the chromatograms of each reference solution was greater than 990, indicating good peak purity. In the chromatogram of the test solution, the number of peaks increased significantly, and the separation degree of ginsenosides Rg1 and Re was 2.61, indicating good separation effect. The peak matching degree of cinnamaldehyde was 997 > 990, which met the requirements. Oleanolic acid and betulinic acid had late elution times and small peak areas, and were interfered with by other peaks. The baseline was poor in the 90-120 minute range, so the elution rate was reduced in the 90-120 minute range for determination.

[0299] 3.16 Method Development 20-Gradient 12

[0300] To screen for a more suitable mobile phase, the mobile phase was changed to acetonitrile-0.2 mol / L potassium dihydrogen phosphate solution, with other chromatographic conditions remaining the same as in Table 20. During the determination with a blank solvent (methanol), the HPLC pump pressure exceeded the limit, indicating that the mobile phase was unsuitable; therefore, the mobile phase was changed for the determination.

[0301] 3.17 Method Development 21-Gradient 12

[0302] Replace the mobile phase with acetonitrile-0.2% formic acid solution, and use the same chromatographic conditions as in Table 20.

[0303] Preparation of the test solution: Take an appropriate amount of this product (batch number: 2301002), cut it into small pieces, weigh about 3g accurately, place it in an Erlenmeyer flask, accurately add 25ml of methanol, seal tightly, weigh, sonicate (power 500W, frequency 40kHz) for 2 hours, cool, weigh, replenish the lost weight with methanol, shake well, filter, and the test solution is obtained.

[0304] Accurately pipette 10 μl each of the blank solvent (methanol), the reference solution from section "1.1 Method Development 1 - Gradient 1", and the above-mentioned test solution, and inject them into the liquid chromatograph. Record the chromatograms. (See figure) Figure 41 .

[0305] The results showed a large baseline in the chromatogram, and abnormal peaks in the test solution. The sequence was terminated, and oleanolic acid and betulinic acid were not measured. Therefore, the mobile phase was changed to acetonitrile-0.1% phosphoric acid solution for further determination.

[0306] 3.18 Method Development 22-Gradient 13

[0307] The mobile phase was continued to be acetonitrile-0.1% phosphoric acid solution, and the elution gradient was further optimized. The chromatographic conditions are shown in Table 21.

[0308] Table 21 HPLC Determination - Gradient 13

[0309]

[0310]

[0311] Accurately pipette 10 μl each of the blank solvent (methanol), the reference solution under "1.1 Method Development 1 - Gradient 1", and the test solution under "3.17 Method Development 21 - Gradient 12", and inject them into the liquid chromatograph. Perform a full-wavelength scan using a DAD detector from 190 to 400 nm, and record the chromatograms. The results are shown in the figure. Figure 42 The chromatogram at 203 nm was extracted and analyzed, see [link to chromatogram]. Figure 43 .

[0312] The results showed that the chromatograms of each reference solution at wavelengths of 190–400 nm were basically consistent with those under “3.15 Method Development 19-Gradient 12”, with a peak matching degree of 1000, indicating good peak purity. In the chromatogram of the test solution, the number of peaks increased significantly, and the separation degree of ginsenosides Rg1 and Re was 2.60, indicating good separation effect. The peak matching degree of cinnamaldehyde was 999 > 990, which met the requirements. Therefore, the method was reproduced using elution gradient 13, and the method was confirmed.

[0313] 4. Method reproducibility test

[0314] 4.1 Method Reproduction - Gradient 13

[0315] To further investigate the feasibility of the method, the test solution was prepared in two parallel batches using the direct ultrasonic method with methanol, and the method was reproduced. Other chromatographic conditions were the same as those in Table 21.

[0316] Preparation of the test solution: Take an appropriate amount of this product (batch number: 2301002), cut it into small pieces, weigh approximately 3g accurately, place it in an Erlenmeyer flask, accurately add 25ml of methanol, seal tightly, weigh, sonicate (power 500W, frequency 40kHz) for 2 hours, cool, weigh again, replenish the lost weight with methanol, shake well, filter, and the test solution is ready. Prepare two parallel solutions.

[0317] Accurately pipette 10 μl each of the blank solvent (methanol), the reference solution from section "1.1 Method Development 1 - Gradient 1", and the above-mentioned test solution into the liquid chromatograph. Perform a full-wavelength scan using a DAD detector from 190 to 400 nm and record the chromatograms. The results are shown in the figure. Figure 44 , 45 The chromatogram at 203 nm was extracted and analyzed, see [link to chromatogram]. Figure 46 .

[0318] The results showed that the scan chromatograms of each reference solution at wavelengths of 190–400 nm were basically consistent with those under “3.15 Method Development 19-Gradient 12”, with a peak matching degree of 1000, indicating good peak purity. In the chromatograms of the two test solutions, the resolutions of ginsenoside Rg1 and Re were 2.68 and 2.66, respectively, indicating good separation. The peak matching degree of cinnamaldehyde was 999 > 990, meeting the requirements, indicating good reproducibility of Gradient 13. Oleanolic acid and betulinic acid had late elution times and poor baselines nearby, causing interference and small peak areas. The reason for this may be related to the natural forms of oleanolic acid and betulinic acid, which are generally mostly in bound form with relatively few free forms and low polarity. Combined with the fact that the extraction method of this product is water extraction, and the complex composition of this product, it is impossible to effectively locate and analyze these components. Generally, acid hydrolysis is used to treat the test solution for the determination of these two components, but this treatment method is not suitable for the determination of other components in this product. Therefore, oleanolic acid and betulinic acid were not considered as characteristic peaks for investigation.

[0319] 4.2 Method Development 23 - Determination of Ultrasound Conditions

[0320] The ultrasonic conditions of the test solution were investigated to screen the preparation method of the test solution. The chromatographic conditions were the same as those in Table 21.

[0321] Preparation of test solution 1: Take an appropriate amount of this product (batch number: 2301002), cut it into small pieces, take about 3g, weigh it accurately, place it in an Erlenmeyer flask, accurately add 25ml of methanol, seal tightly, weigh it, sonicate (power 500W, frequency 40kHz) for 1 hour, cool it, weigh it, make up the weight loss with methanol, shake well, filter it, and the test solution is obtained.

[0322] Preparation of test solution 2: Take an appropriate amount of this product (batch number: 2301002), cut it into small pieces, take about 3g, weigh it accurately, place it in an Erlenmeyer flask, accurately add 25ml of methanol, seal tightly, weigh it, sonicate (power 500W, frequency 40kHz) for 2 hours, cool it, weigh it, make up the weight loss with methanol, shake well, filter it, and the test solution is obtained.

[0323] Preparation of test solution 3: Take an appropriate amount of this product (batch number: 2301002), cut it into small pieces, take about 3g, weigh it accurately, put it in an Erlenmeyer flask, accurately add 25ml of methanol, seal it tightly, weigh it, sonicate it (power 500W, frequency 40kHz) for 3 hours, cool it, weigh it, make up the weight loss with methanol, shake it well, filter it, and the test solution is obtained.

[0324] Preparation of test solution 4: Take an appropriate amount of this product (batch number: 2301002), cut it into small pieces, take about 3g, accurately weigh it, place it in an Erlenmeyer flask, accurately add 25ml of methanol, seal tightly, weigh it, sonicate (power 500W, frequency 40kHz) for 10 minutes, soak overnight, sonicate again (power 500W, frequency 40kHz) for 2 hours, cool, weigh it, make up the lost weight with methanol, shake well, filter, and the test solution is obtained.

[0325] Accurately pipette 10 μl each of the blank solvent (methanol), the reference solution under "1.1 Method Development 1 - Gradient 1", and the above-mentioned test solutions 1-4, and inject them into the liquid chromatograph. Perform a full-wavelength scan using a DAD detector from 190 to 400 nm, and record the chromatograms. The results are shown in [Figure number missing]. Figures 47-51 The chromatogram at 203 nm was extracted and analyzed, see [link to chromatogram]. Figure 52 .

[0326] The results showed that the peak matching degree in the chromatograms of each reference solution was 1000, indicating good peak purity. There were no significant differences in the number of peaks, response values, peak areas, and resolution in the chromatograms of the four test solutions. To ensure the applicability of the test solution processing method, the intermediate condition (ultrasonic treatment for 2 hours) was selected as the test solution preparation method, and the method and chromatographic conditions were verified.

[0327] 5. Validation of HPLC characteristic chromatographic method

[0328] 5.1 Exclusivity

[0329] Chromatographic conditions and system suitability tests were conducted using octadecylsilane-bonded silica gel as the packing material. {Shim Pack GissC} 18 (4.6mm*250mm, 5μm)}; using acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, gradient elution was performed according to the specifications in Table 22, with a flow rate of 1.0 ml / min, a column temperature of 40℃, and a detection wavelength of 203 nm. The theoretical plate number, calculated based on the ginsenoside Rb1 peak, should not be less than 5000.

[0330] Table 22 Elution gradient

[0331]

[0332] Preparation of cinnamaldehyde reference standard stock solution: Accurately weigh 10.13 mg of cinnamaldehyde reference standard, place it in a 100 ml brown volumetric flask, add an appropriate amount of methanol to dissolve it, dilute to the mark, mix well, and the solution is ready.

[0333] Preparation of Ginsenoside Re Reference Stock Solution: Accurately weigh 10.90 mg of ginsenoside Re reference standard, place it in a 10 ml brown volumetric flask, add an appropriate amount of methanol to dissolve it, dilute to the mark, mix well, and the solution is ready.

[0334] Preparation of Ginsenoside Rb1 Reference Stock Solution: Accurately weigh 11.18 mg of ginsenoside Rb1 reference standard, place it in a 10 ml volumetric flask, add an appropriate amount of methanol to dissolve it, dilute to the mark, mix well, and the stock solution is obtained.

[0335] Preparation of Ginsenoside Rg1 Reference Stock Solution: Accurately weigh 10.91 mg of ginsenoside Rg1 reference standard, place it in a 10 ml volumetric flask, add an appropriate amount of methanol to dissolve it, dilute to the mark, mix well, and the solution is ready.

[0336] Preparation of cinnamaldehyde reference solution: Accurately measure 1 ml of cinnamaldehyde reference solution into a 10 ml volumetric flask, dilute to the mark with methanol, and shake well to obtain the solution.

[0337] Preparation of Ginsenoside Re Reference Solution: Accurately measure 2 ml of ginsenoside Re reference solution into a 10 ml volumetric flask, dilute to the mark with methanol, and shake well to obtain the solution.

[0338] Preparation of Ginsenoside Rb1 Reference Solution: Accurately measure 2 ml of ginsenoside Rb1 reference solution into a 10 ml volumetric flask, dilute to the mark with methanol, and shake well.

[0339] Preparation of Ginsenoside Rg1 Reference Solution: Accurately measure 2 ml of ginsenoside Rg1 reference solution into a 10 ml volumetric flask, dilute to the mark with methanol, and shake well.

[0340] Preparation of the mixed control solution: Accurately measure 1 ml of cinnamaldehyde reference standard stock solution, 2 ml each of ginsenoside Re, ginsenoside Rb1, and ginsenoside Rb1 reference standard stock solutions, place them in a 10 ml brown volumetric flask, dilute to the mark with methanol, and shake well to obtain the mixed control solution.

[0341] Preparation of the test solution: Take an appropriate amount of this product (batch number: 2301002), cut it into small pieces, weigh about 3g accurately, place it in an Erlenmeyer flask, accurately add 25ml of methanol, seal tightly, weigh, sonicate (power 500W, frequency 40kHz) for 2 hours, cool, weigh, replenish the lost weight with methanol, shake well, filter, and the test solution is obtained.

[0342] The assay method involves precisely injecting 10 μl each of the blank solvent, mixed control solution, and test solution into the liquid chromatograph and measuring the results. The results are shown in the figure. Figure 53 .

[0343] The standard stipulates that the blank solvent should not interfere with the determination of the characteristic spectrum.

[0344] The results showed that a total of 13 characteristic peaks were detected, and the blank solvent did not interfere with the determination of the characteristic spectrum of compound ferrous sulfate pills.

[0345] 5.2 Instrument Precision Assessment

[0346] Accurately pipette 10 μl of the mixed control solution under “5.1 Specificity” and inject it into the liquid chromatograph. Measure it 5 times consecutively. The results are shown in Tables 23 and 24.

[0347] The standard stipulates that the retention time RSD of each control peak should be ≤1.0% (n=5); and the peak area RSD should be ≤2.0% (n=5).

[0348] Table 23 Instrument precision test results - retention time

[0349] Serial Number Cinnamaldehyde <![CDATA[Ginsenoside Rg1]]> Ginsenoside Re <![CDATA[Ginsenoside Rb1]]> Mixed reference solution 1 55.603 80.603 81.413 100.173 Mixed reference solution 2 55.600 80.617 81.423 100.183 Mixed reference solution 3 55.587 80.603 81.413 100.173 Mixed reference solution 4 55.587 80.600 81.413 100.170 Mixed reference solution 5 55.580 80.607 81.413 100.170 RSD (%) 0.02 0.01 0.01 0.01

[0350] Table 24 Instrument Precision Test Results - Peak Area

[0351] Serial Number Cinnamaldehyde <![CDATA[Ginsenoside Rg1]]> Ginsenoside Re <![CDATA[Ginsenoside Rb1]]> Mixed reference solution 1 7.1221 11.5663 9.1783 8.2590 Mixed reference solution 2 7.1238 11.5797 9.1842 8.2379 Mixed reference solution 3 7.0866 11.5733 9.1660 8.2688 Mixed reference solution 4 7.1182 11.5684 9.1766 8.2553 Mixed reference solution 5 7.0777 11.5661 9.1723 8.2269 RSD (%) 0.31 0.05 0.07 0.20

[0352] The results showed that after five consecutive injections of the mixed reference solution, the retention time RSD of each reference peak was less than 1.0%, and the peak area RSD was less than 2.0%, indicating that the instrument precision of this method was good.

[0353] 5.3 Repeatability

[0354] Following the method for preparing the test solution under "5.1 Specificity", prepare 6 parallel aliquots. Accurately pipette 10 μl of each test solution and inject it into the liquid chromatograph for determination. Using ginsenoside Rb1 as the S peak, calculate the relative retention time and relative peak area of ​​each major chromatographic peak and the S peak. The results are shown in Tables 25 and 26.

[0355] Note: Repeatability 1 is the same as the chromatogram of the test solution under "5.1 Specificity".

[0356] The standard stipulates that the relative retention time (RSD) of each major chromatographic peak in the six test solutions should be ≤2.0%, and the relative peak area (RSD) should be ≤10.0% (n=6).

[0357] Table 25 Repeatability Test Results - Relative Retention Time

[0358]

[0359] Table 26 Repeatability Test Results - Relative Peak Area

[0360]

[0361] The results showed that the relative retention time RSD of each major chromatographic peak in the six samples was less than 2.0%, and the relative peak area RSD was less than 10.0%, indicating that the method had good repeatability.

[0362] 5.4 Peak Attribution

[0363] To assign peaks to the 13 chromatographic peaks identified in the test solution, six medicinal materials—ferrous sulfate, walnut kernel, seahorse, cinnamon, American ginseng, and jujube—were used for determination. The preparation methods were the same as those for the test solution under "5.1 Specificity".

[0364] Preparation of ferrous sulfate medicinal material solution: Take an appropriate amount of ferrous sulfate medicinal material, grind it into a fine powder, take 0.2106g, weigh it accurately, put it in an Erlenmeyer flask, add 25ml of methanol accurately, stopper tightly, weigh it, sonicate (power 500W, frequency 40kHz) for 2 hours, cool it, weigh it again, make up the weight loss with methanol, shake well, filter it, and the solution is obtained.

[0365] Preparation of Walnut Kernel Medicinal Material Solution: Take an appropriate amount of walnut kernel medicinal material, grind it into a fine powder, take 0.5172g, weigh it accurately, place it in an Erlenmeyer flask, add 25ml of methanol accurately, seal the flask tightly, weigh it, sonicate it (power 500W, frequency 40kHz) for 2 hours, cool it, weigh it again, replenish the lost weight with methanol, shake it well, filter it, and the solution is obtained.

[0366] Preparation of Seahorse Herbal Solution: Take an appropriate amount of seahorse herbal material, grind it into a fine powder, take 0.5129g, weigh it accurately, place it in an Erlenmeyer flask, add 25ml of methanol accurately, seal the flask tightly, weigh it, sonicate it (power 500W, frequency 40kHz) for 2 hours, cool it, weigh it again, replenish the lost weight with methanol, shake it well, filter it, and the solution is obtained.

[0367] Preparation of Cinnamon Herbal Solution: Take an appropriate amount of cinnamon herbal material, grind it into a fine powder, take 0.5152g, weigh it accurately, place it in an Erlenmeyer flask, add 25ml of methanol accurately, seal the flask tightly, weigh it, sonicate it (power 500W, frequency 40kHz) for 2 hours, cool it, weigh it again, replenish the lost weight with methanol, shake it well, filter it, and the solution is ready.

[0368] Preparation of American ginseng medicinal material solution: Take an appropriate amount of American ginseng medicinal material, grind it into a fine powder, take 0.5-120g, weigh it accurately, put it in an Erlenmeyer flask, add 25ml of methanol accurately, seal it tightly, weigh it, sonicate it (power 500W, frequency 40kHz) for 2 hours, cool it, weigh it again, make up the weight loss with methanol, shake it well, filter it, and the solution is obtained.

[0369] Preparation of Jujube Reference Solution: Take an appropriate amount of jujube medicinal material, grind it into a fine powder, take 0.5190g, weigh it accurately, place it in an Erlenmeyer flask, add 25ml of methanol accurately, seal tightly, weigh it, sonicate (power 500W, frequency 40kHz) for 2 hours, cool it, weigh it again, replenish the lost weight with methanol, shake well, filter it, and the solution is obtained.

[0370] Accurately pipette 10 μl of each of the above-mentioned medicinal material solutions and inject them into the liquid chromatograph. Record the chromatograms and compare them with the "Repeatability 1 Chromatogram" under "5.3 Repeatability". The results are shown in the figure below. Figure 54 .

[0371] The results showed that of the 13 characteristic peaks identified in the chromatogram of the test sample, peaks 2 and 3 belonged to walnut kernels; peaks 4 and 5 belonged to cinnamon; and peaks 1, 7, 8, 11, 12, and 13 belonged to American ginseng. Most chromatographic peaks were confirmed to belong to their respective medicinal components. No corresponding chromatographic peaks were detected in ferrous sulfate, jujube, and seahorse. This is related to the main components of these medicinal materials. The main component of ferrous sulfate is ferrous sulfate heptahydrate, which is unsuitable for this method, while the current quality standard specifies a content determination method. Jujube contains a large amount of polysaccharides, which are not suitable for detection by this method. During the development of this method, the representative components oleanolic acid and betulinic acid were analyzed for localization, and the results are shown in the conclusion under "4. Method Reproducibility". Due to the inherent characteristics of these components, they are not suitable for this method and will not be controlled for now. Seahorse is an animal-derived medicinal material, with main components including proteins, oils, and trace elements. This method is not applicable, and the current quality standard provides for microscopic identification. Peaks 6, 9, and 10 could not be effectively assigned. The reason for this is that the product has a complex composition, and the interaction between substances and its own physicochemical properties are also related to this. This is also a characteristic of traditional Chinese medicine compound preparations. Therefore, the establishment of characteristic spectral methods can effectively locate complex components, thereby highlighting the advantages of overall product quality control.

[0372] 5.5 Intermediate Precision

[0373] The effects of different dates, different analysts, and different instruments on precision were investigated.

[0374] Following the method for preparing the test solution under "5.1 Specificity", prepare 6 parallel aliquots. Accurately pipette 10 μl of each test solution and inject it into the liquid chromatograph for determination. Using ginsenoside Rb1 as the S peak, calculate the relative retention time and relative peak area of ​​each major chromatographic peak and the S peak. The results are shown in Tables 27 and 28.

[0375] The standard stipulates that the relative retention time RSD of 6 test solutions should be ≤2.0%, and the relative peak area RSD should be ≤10.0% (n=6). For the repeatability test, the relative retention time RSD of 12 test solutions should be ≤3.0%, and the relative peak area RSD should be ≤12.0% (n=12).

[0376] Table 27 Intermediate Precision Test Results - Relative Retention Time

[0377]

[0378] Table 28 Intermediate Precision Test Results - Relative Peak Area

[0379]

[0380] The results showed that the relative retention time RSD of each major chromatographic peak in the 12 samples was less than 3.0%, and the relative peak area RSD was less than 12.0%, indicating that the intermediate precision of the method was good.

[0381] 5.6 Durability

[0382] 5.6.1 Stability test of the test sample solution

[0383] Take the test solution from section "5.1 Specificity", let it stand at room temperature, and accurately inject 10 μl into the liquid chromatograph at 0 h, 14 h, 20 h, 24 h, and 36 h respectively. Measure the relative retention time and relative peak area of ​​each major chromatographic peak relative to the S peak, using ginsenoside Rb1 as the S peak. The results are shown in Tables 29 and 30.

[0384] The standard stipulates that the relative retention time (RD) of each major chromatographic peak of the test solution at room temperature should be less than 2.0% and the relative peak area (RD) should be less than 5.0% when compared with the result obtained at 0 h.

[0385] Table 29 Stability Test Results - Relative Retention Time

[0386]

[0387]

[0388] Table 30 Stability Test Results - Relative Peak Area

[0389]

[0390]

[0391] The results showed that after the test solution was placed at room temperature for 36 hours, the relative retention time (RD) of each major chromatographic peak was less than 2.0% and the relative peak area (RD) was less than 5.0% compared with the detection result at 0 h, indicating that the test solution had good stability after being placed at room temperature for 36 hours.

[0392] 5.6.2 Chromatographic condition robustness

[0393] The effects of slight changes in chromatographic conditions on the proposed method were investigated. The experimental conditions are shown in Table 31.

[0394] Table 31 Durability Test Conditions

[0395] Factors to be considered Assessment level Column temperature 38℃、40℃、42℃ Flow rate 0.95ml / min, 1.0ml / min, 1.05ml / min

[0396] The standard stipulates that, using the average relative retention time under item "5.3 Repeatability" as the standard condition, the test solution should detect 13 corresponding chromatographic peaks under each condition, and the relative retention time of each chromatographic peak should be within ±5% of the standard condition. The results are shown in Tables 32 and 33.

[0397] Table 32 Durability Test Results - Relative Retention Time

[0398]

[0399]

[0400] Table 33 Durability Test Results - Relative Peak Area

[0401]

[0402] The results showed that the relative retention times of each major chromatographic peak were within ±5% of those measured under standard conditions. However, the relative peak areas of each major chromatographic peak fluctuated significantly compared to those measured under standard conditions, indicating that this method is sensitive to column temperature and mobile phase flow rate, but does not affect the determination of characteristic chromatograms. To ensure more accurate detection of this product, the column temperature was fixed at 40℃ and the mobile phase flow rate at 1.0 ml / min.

[0403] 6. Establishment of HPLC characteristic chromatograms

[0404] Fifteen batches of Compound Ferrous Sulfate Pills were tested, and the chromatograms were recorded. The results are shown in the figure. Figures 55-56 A reference chromatogram was generated using the Chinese herbal chromatographic fingerprint similarity evaluation system (2012 version). Results are shown below. Figure 57 .

[0405] Table 34 Peak area of ​​cinnamaldehyde in 15 batches

[0406] batch number 2204027 2204029 2205037 2207051 2207058 Peak area 0.5896 1.4345 0.6979 2.4123 1.6543 batch number 2208068 2209076 2209079 2211092 2211098 Peak area 1.9115 3.4509 1.9023 5.5342 1.9594 batch number 2212114 2301002 2303015 2303016 2303017 Peak area 6.2254 3.5708 6.3639 5.1773 4.1915

[0407] The results showed that among the 15 batches of samples, those with earlier production dates exhibited a significantly reduced peak area for cinnamaldehyde, indicating a degradation trend. The degradation peak partially overlapped with the cinnamaldehyde peak, resulting in poor separation and interference with cinnamaldehyde determination. This may be attributed to the unstable nature of cinnamaldehyde in acidic or alkaline environments and its susceptibility to oxidation in air. This product is sealed in packaging to prevent oxidation from air. The formula consists of ferrous sulfate, American ginseng, jujube, and other medicinal ingredients. Ferrous sulfate, primarily composed of ferrous sulfate heptahydrate, is acidic; therefore, cinnamaldehyde degradation is closely related to the acidity of this product. Furthermore, the complex composition of this product also affects the stability of cinnamaldehyde. Therefore, to ensure the applicability of the method and to identify the common peaks of this product, this component is not included in the common peaks of the characteristic chromatogram.

[0408] The results showed that the chromatographic peaks with good stability and suitable response values ​​among the characteristic chromatograms of 15 batches of samples were selected as common peaks. A total of 12 common peaks were identified. By comparing the retention times of each peak in the chromatograms of the test sample and the mixed reference sample, three of them could be identified: ginsenoside Rg1 (peak 6), ginsenoside Re (peak 7), and ginsenoside Rb1 (peak 10). Among them, peak 10 had a moderate elution time, a large peak area, a good peak shape, and good separation. Therefore, peak 10 was selected as the reference peak (S peak). The average relative retention time of 15 samples was used as the specified value, which was: 0.30 (peak 1), 0.31 (peak 2), 0.48 (peak 3), 0.55 (peak 4), 0.57 (peak 5), 0.80 (peak 6), 0.81 (peak 7), 0.82 (peak 8), 0.94 (peak 9), 1.00 (peak 10, S), 1.03 (peak 11), and 1.08 (peak 12). Based on the results of previous methodological validation, the relative retention time was tentatively set to be within ±5% of the specified value.

[0409] 7. Analysis of the test results of 15 batches of compound ferrous sulfate pills

[0410] Fifteen batches of Compound Ferrous Sulfate Pills from different batches were analyzed. The S peak was defined as the peak corresponding to the reference peak of ginsenoside Rb1 in the chromatogram. The relative retention times of each characteristic peak and the S peak were calculated, and the results are shown in Tables 35-37. The retention times of the 12 common peaks in sample number 2303017 were: 29.880 min, 31.233 min, 47.823 min, 54.867 min, 56.157 min, 79.547 min, 80.507 min, 81.923 min, 93.180 min, 99.693 min, 102.317 min, and 107.710 min. (See Table 35-37 for details). Figure 58 .

[0411] Table 35 Test Results of 15 Batches of Samples - 1

[0412]

[0413] Table 36 Test Results of 15 Batches of Samples - 2

[0414]

[0415] Table 37 Test Results of 15 Batches of Samples - 3

[0416]

[0417] The results showed that 12 characteristic peaks corresponding to the reference chromatograms were observed in the chromatograms of 15 batches of samples; among them, 3 peaks had retention times consistent with the corresponding reference standard chromatogram peaks, and the relative retention times of the remaining characteristic peaks were within ±5% of the specified values. Matching with the reference chromatograms was performed in the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version). Figures 59-60 The similarity between the characteristic spectra of each sample and the generated control spectra was calculated, and the results are shown in Table 38.

[0418] Table 38 Similarity Comparison Results for 15 Batches

[0419] batch number 2204027 2204029 2205037 2207051 2207058 Similarity 0.965 0.982 0.970 0.990 0.987 batch number 2208068 2209076 2209079 2211092 2211098 Similarity 0.994 0.995 0.991 0.980 0.995 batch number 2212114 2301002 2303015 2303016 2303017 Similarity 0.983 0.987 0.951 0.966 0.994

[0420] The results showed that the similarity between the 15 batches of samples and the control spectrum was greater than 0.90. Therefore, based on the overall spectral information, it can be seen that the types and quantities of chemical components contained in each test sample are basically the same.

[0421] in conclusion

[0422] The characteristic chromatogram / fingerprint chromatogram of compound ferrous sulfate pills developed by this method has certain specificity. The method has good robustness and reproducibility and can be used as a basis for quality control of compound ferrous sulfate pills. It can be used to evaluate the uniformity, quality and stability of the preparation quality and to compare the consistency of product quality before and after the change.

[0423] The characteristic chromatogram / fingerprint chromatogram test results of each batch of Compound Ferrous Sulfate Pills showed that the corresponding characteristic peaks were detected in different batches of Compound Ferrous Sulfate Pills, and the fingerprint chromatogram similarity was greater than 0.9, indicating that the product quality is uniform and stable.

[0424] A characteristic chromatographic method for compound ferrous sulfate pills was developed and validated. The established method is scientific, feasible, and advanced. The validation results are scientific, reasonable, and accurate, and can meet the requirements for multi-component and overall quality control of the product.

Claims

1. A method for constructing a characteristic spectrum of compound ferrous sulfate pills, characterized in that, Includes the following steps: (1) Chromatographic conditions and system suitability: The chromatographic column used was selected from Shim-pack GISS C 18 Or an equivalent chromatographic column; use acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, perform gradient elution as specified in the table below, with a flow rate of 1.0 ml / min, a column temperature of 40℃, a detection wavelength of 203 nm, and a theoretical plate number calculated based on the ginsenoside Rb1 peak that should not be less than 5000; (2) Preparation of the test solution: Take an appropriate amount of this product, cut it into small pieces, weigh approximately 3g accurately, place it in an Erlenmeyer flask, and accurately... Add 25ml of methanol, seal tightly, weigh, sonicate for 2 hours, cool, weigh again, replenish the lost weight with methanol, shake well, filter, and the product is obtained. (3) Preparation of reference solution: Take appropriate amounts of ginsenoside Re, ginsenoside Rg1 and ginsenoside Rb1 reference standards, add methanol to prepare a solution containing 0.2 mg of each per 1 ml. (4) Determination method: Accurately pipette 10 μl of blank solvent, reference solution and test solution into the liquid chromatograph, determine and record the chromatogram to obtain the characteristic chromatogram of compound ferrous sulfate pill.

2. The method for constructing the characteristic spectrum of compound ferrous sulfate pills according to claim 1, characterized in that, The chromatographic column used in step (1) is selected from Shim-pack GISS C 18 Or an equivalent chromatographic column, with a length of 25 cm, an inner diameter of 4.6 mm, and a particle size of 5 μm.

3. The method for constructing the characteristic spectrum of compound ferrous sulfate pills according to claim 1, characterized in that, The conditions for ultrasonic treatment used in step (2) are: power 500W, frequency 40KHz.

4. The use of the method for constructing the characteristic spectrum of compound ferrous sulfate pills according to any one of claims 1-3 in the quality inspection of compound ferrous sulfate pill products.

5. A quality testing method for compound ferrous sulfate pills, characterized in that, The method includes the step of comparing the characteristic spectrum of the compound ferrous sulfate pill product to be tested with the characteristic spectrum of the compound ferrous sulfate pill control; the characteristic spectrum of the compound ferrous sulfate pill product to be tested is obtained by using the compound ferrous sulfate pill product to be tested according to any one of the construction methods described in claims 1-3.

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