A method for fingerprint spectrum analysis of seven-flavor radix stephaniae and radix astragali granules

Abstract

This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a fingerprint analysis method for Qiwei Fangji Huangqi granules. The fingerprint analysis method for Qiwei Fangji Huangqi granules includes the following steps: preparation of a test solution; preparation of a reference solution; determination of the fingerprint spectrum of Qiwei Fangji Huangqi granules or its aqueous extract by high-performance liquid chromatography (HPLC). This invention's detection method is highly versatile, low-cost, and can comprehensively reflect the quantitative fingerprint spectrum of the product quality.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a fingerprint analysis method for Qiwei Fangji Huangqi granules. Background Technology

[0002] Fangji Huangqi Decoction, selected from Zhang Zhongjing's *Synopsis of Prescriptions of the Golden Chamber*, is composed of Fangji (Stephania tetrandra), Huangqi (Astragalus membranaceus), Baizhu (Atractylodes macrocephala), and Gancao (Glycyrrhiza uralensis). It is one of the classic ancient formulas, known for its effects of invigorating qi and dispelling wind, strengthening the spleen and promoting diuresis. It is remarkably effective in treating chronic heart failure, nephrotic syndrome, and rheumatoid arthritis. Seven-Ingredient Fangji Huangqi Granules are a modified version of Fangji Huangqi Decoction. While retaining the formula's structure, the proportions of Huangqi and Fangji are changed, and Danshen (Salvia miltiorrhiza) for promoting blood circulation and removing blood stasis, Tinglizi (Lepidium apetalum) for purging the lungs and promoting diuresis, and Xiakucao (Prunella vulgaris) for reducing swelling and dissipating nodules are added. The combined effects of these herbs invigorate qi and blood, promote diuresis and reduce swelling, and are mainly used for qi deficiency and blood stasis, and water retention in chronic heart failure caused by coronary heart disease or pulmonary heart disease. Since no chromatographic quantitative fingerprinting method for intermediates and preparations of Qiwei Fangji Huangqi granules has been found, this study developed a quantitative fingerprinting method based on the analytical quality-origin design concept. This method is highly versatile, has low detection cost, and can comprehensively reflect product quality. It was applied to the detection of intermediates and preparations in the production process of Qiwei Fangji Huangqi granules.

[0003] Relevant patent documents retrieved: For example, Chinese patent CN105181855A, published on December 23, 2015, discloses a method for simultaneously determining the content of 10 chemical components in Fangji Huangqi Decoction using UHPLC-MS / MS technology, belonging to the field of traditional Chinese medicine component analysis. The analytical method established by this invention for simultaneously determining the content of 10 chemical components in Fangji Huangqi Decoction is highly specific, rapid, sensitive, accurate, and reliable. While completing the content determination, it can also accurately provide the mass information of the compounds, achieving the effect of component identification. This method can provide a basis for comprehensive quality control of Fangji Huangqi Decoction.

[0004] Relevant non-patent literature retrieved: Journal or book title: Journal of Shanghai University of Traditional Chinese Medicine; Article title: UPLC-MS / MS quantitative analysis of 18 components in Fangji Huangqi Decoction; Volume number: 2025.01.006. This study established an analytical method for the simultaneous determination of multiple components in Fangji Huangqi Decoction using UPLC-QQQ-MS / MS technology, and characterized the compound for multiple components. A high-sensitivity, reliable, and efficient UPLC-QQQ-MS / MS method was established for the simultaneous determination of multiple components in the Fangji Huangqi Decoction compound. This method was applied to the quantitative analysis of 18 components in Fangji Huangqi Decoction, including alkaloids (magnolinine, tetrandrine, tebufenozide), saponins (astragaloside III, astragaloside A, astragaloside I, glycyrrhetinic acid), flavonoids (verrucoside glucoside, gentianoside, 5-O-methylvisamidolol, verrucoside, gentianoside, glycyrrhizin, isoglycyrrhizin, glycyrrhizin, isoglycyrrhizin, and isoglycyrrhizin), and terpenoids (atractylodes lactone III, atractylodes lactone II). Tebufenozide was the most abundant alkaloid in the Fangji Huangqi Decoction compound. This method is highly specific and can be used for the quantitative detection of multiple components in Fangji Huangqi Decoction.

[0005] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: The aforementioned literature needs improvement in achieving separation between chromatographic peaks.

[0006] In solving the above problems or overcoming the above defects, the present invention encountered the following difficulties and obstacles: (1) Complex compound ingredients and insufficient chromatographic separation efficiency: Qiwei Fangji Huangqi Granules contain two more herbs than Fangji Huangqi Decoction, resulting in a greater number of chemical components and more similar component properties (for example, Danshen and Xiakucao both contain a large amount of phenolic acid components), which increases the difficulty of chromatographic separation. Under conventional chromatographic conditions, the chromatographic peaks of each component are poorly separated, with severe peak overlap and tailing phenomena, making it impossible to obtain clear and distinguishable fingerprint spectra and difficult to achieve accurate quantification of multiple indicator components.

[0007] (2) It is difficult to cover all target components with the detection wavelength: The maximum ultraviolet absorption wavelengths of different active ingredients in the compound are different. Choosing a single detection method is difficult to achieve the ideal detection sensitivity for all components, which may result in weak or even missing signals for some important components.

[0008] (3) Types and amounts of reagents added to the mobile phase: The selection, amount, and ratio of mobile phase additives will significantly affect the number and area of ​​chromatographic peaks in the chromatogram. Even a slight change in the additive content is enough to cause a significant change in the resolution of key chromatographic peaks. This not only affects the initial retention of chromatographic peaks, but also the maintenance of separation stability throughout the gradient elution process. Summary of the Invention

[0009] The purpose of this invention is to provide a fingerprint analysis method for Qiwei Fangji Huangqi granules, and related technologies, to solve technical problems such as low sensitivity and weak specificity of the analysis method, or a combination thereof.

[0010] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0011] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0012] The definition of standard chemical terminology can be found in the reference "Analytical Chemistry, Higher Education Press, Wuhan University (Chief Editor), 2004".

[0013] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0014] In a first aspect, the present invention provides a fingerprint analysis method for Qiwei Fangji Huangqi granules, comprising the following steps: (1) Prepare the test solution; (2) Preparation of reference solution; (3) The fingerprint spectrum of Qiwei Fangji Huangqi granules or Qiwei Fangji Huangqi aqueous extract was determined by high performance liquid chromatography; The linear gradient elution program for the high-performance liquid chromatography (HPLC) method is as follows: 0-10 min, 8%-18% B; 10-40 min, 18%-25% B; 40-55 min, 25%-42% B; 55-60 min, 42%-75% B; 60-65 min, 75%-90% B; and the mobile phase A of the HPLC method is 0.2% formic acid in water, and the mobile phase B is 0.2% formic acid in acetonitrile.

[0015] Preferably, the specific steps for preparing the test solution in step (1) are as follows: Seven-Ingredient Fangji Astragalus Granules Test Sample: Take seven-ingredient fangji astragalus granules, add ethanol to dissolve, sonicate, centrifuge, and take the supernatant to obtain the sample; The test sample of the aqueous extract of Astragalus membranaceus and Stephania tetrandra was prepared by extracting the extract according to the compound ratio of the Astragalus membranaceus and Stephania tetrandra granules, adding water, heating, decocting, discarding the dregs, centrifuging, and collecting the supernatant. The compound ratio of the Astragalus membranaceus and Stephania tetrandra granules was prepared according to patent CN114984087A.

[0016] Preferably, the specific steps for the reference solution in step (2) are as follows: weigh out the following ingredients respectively: tetrandrine, tetrandrine, isoflavone glycoside, gentianin, astragaloside A, atractylodes lactone II, atractylodes lactone III, atractylone, glycyrrhizin, glycyrrhizic acid monoammonium salt, tanshinone sodium, rosmarinic acid, lithospermic acid, salvianolic acid B, isorhomaric acid glycoside, quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside, dissolve and dilute with ethanol, mix well, centrifuge, and take the supernatant to obtain the solution.

[0017] Preferably, in step (1) the preparation of the test sample of Qiwei Fangji Huangqi granules, the material-liquid ratio of Qiwei Fangji Huangqi granules to ethanol is 1g:8-12mL, the volume fraction of ethanol is 50%-55%, the ultrasonic time is 25-35min, after ultrasonication, the sample is cooled, the weight loss is replenished with ethanol, shaken well, and centrifuged at 11000-13000rpm for 10-20min. Other specific point values ​​within the above range can be selected, and all can achieve the technical effect of the present invention. For example, the ultrasonic time includes but is not limited to 25min, 26min, 27min, 28min, 29min, 30min, 31min, 32min, and 35min.

[0018] More preferably, in step (1) the preparation of the test sample of Qiwei Fangji Huangqi granules, the ratio of Qiwei Fangji Huangqi granules to ethanol is 1g:10mL, the volume fraction of ethanol is 50%, the sonication time is 30min, after sonication, the sample is cooled, the weight loss is replenished with ethanol, shaken well, and centrifuged at 12000rpm for 15min.

[0019] Preferably, in step (1) the preparation of the water extract of Astragalus membranaceus, the amount of water added is 6-10 times the total weight of Astragalus membranaceus, and the decoction is carried out for 1-2 hours and centrifuged at 11000-13000 rpm for 10-20 minutes. Other specific values ​​within the above range can be selected, and all can achieve the technical effect of the present invention. For example, the decoction time includes but is not limited to 1 hour, 1.2 hours, 1.4 hours, 1.6 hours, 1.8 hours, and 2.0 hours.

[0020] More preferably, in step (1) the preparation of the water extract of Astragalus membranaceus, the amount of water added is 8 times the total weight of Astragalus membranaceus, and the decoction is 1.5h and centrifuged at 12000rpm for 15min.

[0021] Preferably, the volume fraction of ethanol in step (2) is 50%-55%, and the mixture is centrifuged at 11000-13000 rpm for 10-20 min.

[0022] More preferably, the volume fraction of ethanol in step (2) is 50%, and the mixture is centrifuged at 12,000 rpm for 15 min.

[0023] Preferably, the chromatographic conditions for the high performance liquid chromatography (HPLC) in step (3) are as follows: the column is an Agilent Infinity Lab Poroshell HPH-C18 column (4.6×250mm, 2.7μm), the column temperature is 22℃, the flow rate is 0.70 mL / min, and the injection volume is 5μL; the UV detection wavelength is 280nm, the CAD detector nebulization temperature is 35℃, the sampling frequency is 10 Hz, the filter is 5s, and the power function is 1.0.

[0024] Secondly, the present invention also provides the application of the fingerprint spectrum analysis method of the above-mentioned Qiwei Fangji Huangqi granules in the detection of intermediates and preparations of Qiwei Fangji Huangqi granules in the production process.

[0025] The present invention has the following beneficial effects: Compared with existing technologies, the present invention has better technical effects in terms of versatility and detection cost.

[0026] This invention provides a fingerprint analysis method for Qiwei Fangji Huangqi granules with different concepts. The differences between this invention and existing technologies include, but are not limited to, different reference standards, different mobile phases, different gradient elution programs, and different column temperatures. Attached Figure Description

[0027] Figure 1 The chromatogram corresponding to the optimized CAD detector is shown in blue (Qiwei Fangji Huangqi Granules test sample, black is mixed reference standard). Figure 2 The results of the analysis of variance for the multiple linear regression model; Figure 3 The contour map of Y1 (where Figure 3 (A in the diagram is the contour map between X2 and X4, and B is the contour map between X3 and X4). Figure 4 Y2 contour map (where Figure 4(A is the contour map between X1 and X4, B is the contour map between X2 and X3, C is the contour map between X2 and X4, and D is the contour map between X3 and X4). Figure 5 Y3 contour map (where Figure 5 (A in the diagram is the contour map between X1 and X3, B is the contour map between X1 and X4, and C is the contour map between X2 and X4). Figure 6 Y4 contour map (where Figure 6 (The middle section shows the contour lines between X1 and X3). Figure 7 Y5 contour map (where Figure 7 (A in the diagram is the contour map between X1 and X2, B is the contour map between X2 and X3, and C is the contour map between X4 and X6). Figure 8 The Y6 contour map (where Figure 8 (A in the diagram is the contour map between X2 and X3, and B is the contour map between X4 and X5). Figure 9 For MODR and verification points ( Figure 9 In the model A, X4=18%, X5=40 min, X6=24%; in the model B, X1=0.2%, X2=22℃, X3=0.70 mL / min; in the model C, X2=22℃, X3=0.70 mL / min, X4=18%, X5=40 min (△ represents the MODR internal parameter verification point); and in the model D, X3=0.70 mL / min, X4=20%, X5=38 min, X6=26% (× represents the MODR external parameter verification point). Figure 10 The similarity of fingerprint spectra of 10 batches of Qiwei Fangji Huangqi granules ( Figure 10 (A in the image represents the CAD detector; B represents the UV detector). Figure 11 Clustering results of 10 batches of Qiwei Fangji Huangqi granules ( Figure 11 (A represents the tree-like clustering result, and B represents the principal component analysis result). Figure 12 The chromatogram of Comparative Example 1 under HPLC conditions ( Figure 12 (A is a mixed reference standard, and B is a sample of Qiwei Fangji Huangqi granules extracted with 50% ethanol). Figure 13 The chromatogram of Comparative Example 2 under HPLC conditions ( Figure 13 Sample A is a granule of Astragalus membranaceus extracted with 50% ethanol; Sample B is a granule of Astragalus membranaceus extracted with water; Sample C is an aqueous extract of the medicinal material. Figure 14The chromatogram of Comparative Example 3 under HPLC conditions ( Figure 14 In this formula, A contains 0.1% formic acid, and B contains 0.2% formic acid. Figure 15 The chromatogram of Comparative Example 3 under HPLC conditions ( Figure 15 In the diagram, A represents using acetonitrile as mobile phase B, and B represents using 0.1% formic acid acetonitrile as mobile phase B. Figure 16 The chromatogram of Comparative Example 5 under HPLC conditions ( Figure 16 In the formula, A contains 0.1% formic acid, B contains 0.2% formic acid, and C contains 0.3% formic acid. Figure 17 Comparison of chromatograms at different column temperatures ( Figure 17 (A is 20℃, B is 25℃). Figure 18 Comparison of chromatograms at different flow rates ( Figure 18 (Among them, A is 1.0 mL / min, B is 0.8 mL / min, and C is 1.2 mL / min). Figure 19 A locally magnified chromatogram under certain conditions during the gradient optimization process ( Figure 19 In the middle, A is 0-10 min, 8%-15% B; 10-13 min, 15%-21% B; 13-28 min, 21%-24% B; 28-42 min, 24%-40% B; 42-45 min, 40%-75% B; 45-50 min, 75%-90% B; B is 0-15 min, 8%-15% B; 15-18 min, 15%-21% B; 18-33 min, 21%-24% B; 33-50 min, 24%-40% B; 50-55 min, 40%-75% B; 55-60 min, 75%-90% B; C is 0-15 min, 8%-15% B; 15-18 min, 15%-19% B; 18-22 min, 19%-20% B. B; 22-38 min, 20%-24% B; 38-52min, 24%-40% B; 52-55 min, 40%-75% B; 55-60 min, 75%-90% B); Figure 20 To optimize the obtained chromatogram (corresponding gradients of 0-15 min, 8%-15% B; 15-18 min, 15%-19% B; 18-21 min, 19%-23% B; 21-30 min, 23% B; 30-45 min, 23%-40% B; 45-50 min, 40%-75% B; 50-55 min, 75%-90% B). Detailed Implementation

[0028] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0029] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0030] The Qiwei Fangji Huangqi Granules described in the embodiments and comparative examples of this invention were prepared according to the formula in Example 1 of patent CN114984087A.

[0031] The compound formula of Qiwei Fangji Huangqi Granules described in the embodiments and comparative examples of the present invention was prepared according to the proportions in Example 1 of patent CN114984087A.

[0032] The instruments used in this invention include: an Ultimate3000 high-performance liquid chromatograph (UV-electrospray detector coupled with a detector, Thermo Scientific, USA); a precision electronic balance (XS105, Mettler-Toledo, Switzerland); a Milli-Q ultrapure water system (Millipore, USA); and an ultrasonic cleaner (LMDTC15, Beijing Lvmian Technology Co., Ltd.).

[0033] Acetonitrile (chromatographic grade, Tianhe Pharmaceuticals, Inc., USA); formic acid (chromatographic grade, 99%, ROE Scientific Inc., USA); ultrapure water for the experiment was prepared by the Milli-Q ultrapure water system (Millipore, USA). The medicinal slices and Qiwei Fangji Huangqi granules were provided by Zhengda Qingchunbao Pharmaceutical Co., Ltd., and batch information is shown in Tables 1 and 2 respectively.

[0034] Table 1

[0035] Table 2

[0036] Reference standards: Tetrandrine (230930, ≥99%), Tetrandrine (230928, ≥98%), Verrucoside (180630, >98%), Aristolochic acid (180929, >98%), Astragaloside A (200307, ​​>98%), Atractylodes lactone II (231103, ≥99%), Atractylodes lactone III (231109, ≥98%), Atractylodes lancea (231006, ≥98%), Glycyrrhizin (231205, ≥99%), Glycyrrhizin ... Ammonium oxalate monosalt (231217, ≥98%), sodium tanshinone (160120, ≥98%), rosmarinic acid (150901, ≥95%), shikonin (190530, >98%), salvianolic acid B (221106, ≥99%), isorhamnetin (240107, ≥98%), and quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside (231023, ≥98%) were all purchased from Shanghai Ronghe Pharmaceutical Technology Development Co., Ltd.

[0037] Example 1 (1) Preparation of test solution Seven-Ingredient Fangji Astragalus Granules Test Sample: Accurately weigh the seven-ingredient Fangji Astragalus Granules and place them in a stoppered conical flask. Add the corresponding volume of 50% ethanol according to the material-to-liquid ratio of 1:10 (g:mL). Seal tightly and weigh. Sonicate for 30 minutes, cool, and weigh again. Make up the lost weight with 50% ethanol, shake well, and centrifuge at 12000 rpm for 15 minutes. Take the supernatant to obtain the test sample.

[0038] The test sample of the aqueous extract of Astragalus membranaceus and Stephania tetrandra was prepared by extracting according to the compound ratio of Astragalus membranaceus and Stephania tetrandra granules. After adding 8 times the amount of water and heating to boiling, the extract was decocted for 1.5 hours. The dregs were discarded, and the extract was centrifuged at 12,000 rpm for 15 minutes. The supernatant was then collected.

[0039] (2) Preparation of reference solution: Preparation of Reference Solutions: Accurately weigh each reference standard into a volumetric flask (specifically: 4.35 mg of tebufenozide, 5.04 mg of tetrandrine, 4.57 mg of isoflavone glycoside, 4.75 mg of gentiopicroside, 2.33 mg of astragaloside A, 7.58 mg of glycyrrhizin, 4.94 mg of glycyrrhizic acid monoammonium salt, 5.36 mg of tanshinone sodium, 9.82 mg of rosmarinic acid, 4.54 mg of shikonin, 1.49 mg of salvianolic acid B1, 4.33 mg of isorhomaric acid glycoside, 1.38 mg of quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside, and 1.29 mg of atractylodes lactone II, 0.90 mg of atractylodes lactone III, and 1.53 mg of atractylone). (Place mg in a 5 mL volumetric flask), add 50% ethanol to dissolve, and dilute to the mark. Mix thoroughly, centrifuge at 12000 rpm for 15 minutes, and collect the supernatant.

[0040] A six-factor, three-level deterministic screening experimental design was used for optimization. The studied method parameters included: formic acid addition in the mobile phase (X1), column temperature (X2), flow rate (X3), the proportion of phase B at the end of the first gradient (X4), the end time of the first gradient (X5), and the proportion of phase B at the end of the second gradient (X6). Specific level settings are shown in Table 3. To account for the influence of the analyte type, a dummy element method was used to add the material type parameter Z, with the Qiwei Fangji Huangqi granules sample designated as 0 and the Qiwei Fangji Huangqi aqueous extract sample designated as 1. The gradient elution program for the analytical conditions is shown in Table 4. After each run, the sample was equilibrated with the initial mobile phase for 10 min. Here, -1, 0, and 1 represent the low, medium, and high levels of the experimental design, respectively.

[0041] Table 3 DSD Factors and Corresponding Levels

[0042] Table 4 Analysis of Gradient Elution Procedures

[0043] The DSD experimental design table was generated using Design Expert 12.0.1.0 software. This required 21 experiments and 2 additional center point experiments. Based on this, experiments were conducted on both analytes under 23 parameter combinations, for a total of 46 experiments. Specific experimental conditions are detailed below. The aim of this study was to achieve as many chromatographic peaks as possible for quantitative fingerprint analysis. For chromatographic peaks that were difficult to separate during the study, the retention time differences between them were calculated as CQAs. Specifically, these included the retention time differences between glycyrrhizin and isoflavone glycoside (Y1), glycyrrhizin and tetrandrine (Y2), isorhamnetin and its subsequent retention time difference (Y3), tetrandrine and the large peak (Y4), rosmarinic acid and its preceding retention time difference (Y5), and astragaloside A and glycyrrhizin G2 (Y6), totaling 6 retention time differences. Figure 1 As shown in Table 5, the response values ​​for each experiment are listed below.

[0044] Table 5. DSD Experimental Conditions and Corresponding Response Values

[0045] Table 5 (continued)

[0046] The experimental design results were analyzed using Design Expert 12.0.1.0 software. A quantitative model of method evaluation index, material type parameters, and method parameters was established using formula (1). The model was simplified using stepwise regression. The significance level for adding and deleting terms in the model was set to 0.05. The remaining terms in the model were considered to be MT and CMPs.

[0047]

[0048] Where Y is a key quality attribute, a0 is a constant term, and b i c k Here, f represents the partial regression coefficient, g represents the number of method parameters, and X represents the number of material type parameters. i Z is a method parameter. k This is a parameter for the material type.

[0049] A quantitative model was established using formula (2) to establish the relationship between key method attributes and key material type parameters and key method parameters. The model was simplified using stepwise regression, and the significance level for added and deleted items was set to 0.05.

[0050]

[0051] Where b0 is a constant term, n is the number of critical method parameters, m is the number of critical material type parameters, and b ib ii b ij c k X represents the partial regression coefficients for each term. c,i X c,j Z represents the parameters of each key method. c,k These are key material type parameters.

[0052] The similarity of chromatographic fingerprints was calculated using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)". The experimental design results were analyzed using Design Expert 12.0.1.0 software, and the MODR was calculated using Matlab R2020b software.

[0053] Example 2 Based on the method described in Example 1, stepwise regression was used to simplify the model, and the remaining terms in the model were considered as key method parameters or key material type parameters. Analysis of variance (ANOVA) was used to determine the impact of the parameters on the response variable. Figure 2 The regression coefficients and p-value ranges for the regression model are listed. Based on the p-values ​​of the linear term in the analysis of variance results, all method parameters are critical. The amount of acid added to the mobile phase (X1) and the flow rate (X3) significantly affect all CQAs, while X5 and X6 only affect Y6. The material type parameter Z has no effect on Y1-Y6; therefore, Z is not a critical material type parameter. Material properties have no effect on the response variables under study. Within the quality attribute range of interest in this study, there is no difference in chromatographic separation between the Qiwei Fangji Huangqi granules and the Qiwei Fangji Huangqi aqueous extract.

[0054] To describe the quantitative relationship between key method parameters (CMPs) and key quality attributes (CQAs), a quadratic polynomial regression method was used to establish a mathematical model between the CMPs and the response variables. Design Expert 12.0.1.0 software was used, and a stepwise regression method was employed to establish the polynomial model, with the significance level for added and deleted terms set to 0.05. The regression coefficients and variance analysis results for each quantitative model are shown in Table 6.

[0055] Table 6. Results of Analysis of Variance for the Binomial Regression Model

[0056] Note: * The representative model item has a p-value less than 0.05; ** The representative model item has a P-value of less than 0.01.

[0057] Based on the established regression model, to intuitively express the influence of key method parameters in the UPLC method on the response value, a two-dimensional contour plot is used to represent the relationship between the independent and dependent variables, such as... Figures 3-8As shown. In each contour map, apart from the two parameters under consideration, the levels of other parameters are fixed at the center point level.

[0058] The MODR was calculated using the exhaustive Monte Carlo method with Matlab R2020b software. The probability of non-compliance was used to quantitatively describe the risk and measure the reliability of the MODR. To ensure separation between peak pairs of interest, lower limits were set for each CQA (Critical Quality Assurance), but no upper limit was set. The lower limits for Y1-Y6 corresponded to 1.0 min, 1.0 min, 0.2 min, 0.5 min, 0.2 min, and 0.5 min, respectively. The calculation steps for CMPs X1, X2, X3, X4, X5, and X6 were 0.005%, 0.01℃, 0.002 mL / min, 0.05%, 0.05 min, and 0.05%, respectively. The simulation was performed 500 times. The significance level for stepwise regression was set to 0.05, the highest acceptable risk in the design space was set to 0.20, and the corresponding lowest acceptable probability of compliance was 0.80. The results of the multiple linear regression model indicate that material type is not a key parameter of the key method attributes; therefore, the material type parameter Z is ignored when calculating the design space. Consequently, the established MODR is consistent for the two materials studied in this research, as shown in the results below. Figure 9 In the diagram, different colors represent the probability of satisfying the optimization objective within the design space.

[0059] Verify by selecting one parameter combination both inside and outside MODR, such as... Figure 9 As shown in C and D, under the parameter combinations corresponding to the validation points within the MODR, all CQAs met the set standards, and the chromatographic results satisfied the separation and analysis requirements. However, for the parameter validation combinations outside the MODR, Y3, Y4, and Y6 failed to meet the set requirements. Based on the validation results, the established MODR is robust and reliable.

[0060] Example 3 according to Figure 9The parameter combination in C yielded the determined chromatographic conditions, specifically: an Agilent Infinity Lab Poroshell HPH-C18 column (4.6 × 250 mm, 2.7 μm); mobile phase A was 0.2% formic acid in water, and mobile phase B was 0.2% formic acid in acetonitrile. Gradient elution was used, specifically: 0–10 min, 8%–18% B; 10–40 min, 18%–25% B; 40–55 min, 25%–42% B; 55–60 min, 42%–75% B; 60–65 min, 75%–90% B. Column temperature was 22℃, flow rate was 0.70 mL / min, and injection volume was 5 μL. UV detection wavelength was 280 nm, CAD detector nebulization temperature was 35℃, sampling frequency was 10 Hz, filter time was 5 s, and power function was 1.0.

[0061] Methodological validation was performed under the above parameter combination. For the fingerprint spectrum methodology, the B peak of salvianolic acid was selected as the reference peak. Experiments were conducted on instrument precision, method repeatability, and sample 24-hour stability. The final results are shown in Table 7, which shows that the overall methodology requirements are met.

[0062] Table 7. RSD Results of Fingerprint Mapping Methodology

[0063] For the content determination methodology, experiments included instrument precision, method repeatability, 24-hour sample stability, linearity, and recovery. For both analytes, the RSDs for instrument precision and method repeatability were both less than 3.0%, and the RSDs for the 24-hour stability of the Qiwei Fangji Huangqi granules and the Qiwei Fangji Huangqi aqueous extract were less than 4.0% and 3.0%, respectively. The results of the method linearity and recovery experiments are shown in Tables 8 and 9, respectively. Overall, this method has good accuracy and can be used for the quantitative detection of intermediates and preparations in the production process of Qiwei Fangji Huangqi granules.

[0064] Table 8. Results of linear experiments on quantitative components

[0065] Table 9 Results of the recovery experiment of quantitative components

[0066] Example 4 The established analytical method was applied to 10 batches of Qiwei Fangji Huangqi granules, and the results are as follows: Figure 10As shown. Under the CAD detector, the similarity between each batch and the control spectrum ranged from 0.992 to 1.000; under the UV detector, the similarity between each batch and the control spectrum ranged from 0.988 to 1.000. The results obtained by the two detectors are similar, proving that the 10 batches of granular finished products tested have good batch-to-batch consistency.

[0067] Simultaneously, the content of 13 active ingredients in the above-mentioned batches of finished formulations was determined, and the results are shown in Table 10. Further cluster analysis was performed based on the production year, and the results are as follows: Figure 11 As shown, there are differences in the content of active ingredients between different batches. The content is more concentrated between batches produced in 2021 and 2023, and more dispersed in 2022. The 2023 batches show significant differences from other batches, with increased content of Stephania tetrandra alkaloids and decreased content of salvianolic acid compounds.

[0068] Table 1010: Content of active ingredients in batches of Qiwei Fangji Huangqi granules

[0069] Table 10 (continued)

[0070] Comparative Example 1 The difference from the chromatographic conditions in Example 3 is that the mobile phase is water (phase A) and acetonitrile (phase B), and the results are as follows. Figure 12 As shown. Figure 12 The sample actually contains 13 reference standards (excluding isorhamnetin, sodium tanshinone, and shikonin), but the actual number of chromatographic peaks is less than 13. Comparison revealed that alkaloids and phenolic acids did not elute, presumably because the mobile phase consisted of water and acetonitrile without the addition of acid / base, causing these components to ionize. Therefore, the mobile phase system needs improvement to achieve better analytical results.

[0071] Comparative Example 2 The difference in chromatographic conditions compared to Example 3 is that the mobile phase is 0.1% formic acid in water (phase A) and acetonitrile (phase B), and the results are as follows. Figure 13 As shown. Comparison Figure 12 B and Figure 13 As shown in Figure A, the number of chromatographic peaks has increased significantly, but further separation is required. Therefore, formic acid was added to the mobile phase system.

[0072] Comparative Example 3 The difference in chromatographic conditions compared to Example 3 is that the mobile phase is 0.2% formic acid water (phase A) and acetonitrile (phase B), and the results obtained are as follows. Figure 14As shown, the separation effect of 0.1% formic acid solution is not as good as that of 0.2% formic acid solution. Based on the comparison with the retention time of the reference standard, the left circle in the figure represents the area near the peak position of tetrandrine, and the right circle includes verbascoside, tetrandrine, and glycyrrhizin, all of which are indicator components of the medicinal materials and are the focus of this study. Therefore, a 0.2% formic acid concentration will be selected for further investigation.

[0073] When formic acid is added only to phase A and phase B is pure acetonitrile, due to the ultraviolet absorption of formic acid at low wavelengths, the chromatogram baseline shifts downward as the proportion of phase B increases under a 203 nm ultraviolet detector. Figure 15 As shown in A. To solve this problem, the chromatogram obtained after adding an equal amount of formic acid to phase B is shown in Figure A. Figure 15 As shown in Figure B, this method can significantly reduce the baseline drift problem at 203 nm caused by changes in the proportion of the mobile phase.

[0074] Comparative Example 4 The difference from the chromatographic conditions in Example 3 is that the amount of formic acid added is 0.3%, and the results are as follows. Figure 16 As shown, at a formic acid addition of 0.2%, the number of chromatographic peaks was the highest during this time period, and the peak separation effect was better than that at 0.1% and 0.3%. Therefore, in the subsequent optimization of the experimental design, 0.2% was selected as the central level for further optimization of the formic acid addition in the mobile phase. Comparative Example 5 The difference between the chromatographic conditions in Example 3 and those in Example 4 lies in the column temperature. The changes in separation efficiency at 25°C, 30°C, and 35°C were examined, revealing that lower temperatures were more favorable for peak separation in this system. Further reductions in column temperature to 20°C were compared with those at 25°C, and the results are as follows... Figure 17 As shown in the figure, red circles represent relatively poor separation results, while green circles represent relatively good separation results. Compared to 25℃, 20℃ is less favorable for the separation of tetrandrine, but more favorable for the separation of tetrandrine from its nearby peaks and salvianolic acid B from its subsequent peaks. Both 20℃ and 25℃ have their advantages and disadvantages; it is speculated that the optimal conditions are between 20-25℃.

[0075] Comparative Example 6 The difference between the chromatographic conditions in Example 3 and those in Example 4 is the flow rate. The effects of different flow rates on the separation performance were compared, for example... Figure 18As shown in the figure, overall, with the increase of flow rate, the retention time of the chromatogram shifts forward, and the elution rate becomes faster. Compared with 1.0 mL / min, at 0.8 mL / min, the peak shape of tebuconazole deteriorates, and its resolution with subsequent peaks decreases; the resolution of rosmarinic acid with subsequent peaks also decreases; the number of peaks of verrucoside and tetrandrine increases; the resolution of astragaloside A with its preceding small peak increases; and the resolution and peak shape of glycyrrhizic acid improve. At 1.2 mL / min, the resolution of tebuconazole, the resolution between verrucoside, tetrandrine, and glycyrrhizin, the resolution of astragaloside A, and the peak shape of glycyrrhizic acid all deteriorate.

[0076] Comparative Example 7 The difference between the chromatographic conditions and those in Example 3 lies in the gradient. Further gradient optimization was performed, while the flow rate remained at 1.0 mL / min. Chromatograms under different gradients were compared, and some chromatograms obtained under different gradients are shown below. Figure 19 As shown, a partial magnification has been made for easier comparison.

[0077] Through multiple optimizations using gradient spectroscopy, the resulting chromatogram is as follows: Figure 20 As shown, some chromatographic peaks exhibit tailing. The separation of salvianolic acid B from subsequent peaks is still incomplete; the separation of rosmarinic acid and salvianolic acid A from preceding peaks is insufficient; there are numerous small impurity peaks near lithospermic acid; and the separation of glycyrrhizin and isorhamnetin has room for improvement. These all require further optimization through experimental design.

[0078] Comparative Example 8 The difference from the chromatographic conditions in Example 3 is that the chromatographic columns are different. In the previous experiment, the DIKMA Diamonsil C18 (2) column (4.6×250mm, 5.0μm), the DIKMA Diamonsil PlusC18-A column (4.6×250mm, 5.0μm), and the Agilent ZORBAX Extend-C18 column (4.6×250mm, 5μm) were compared. Finally, the Agilent ZORBAX Extend-C18 column (4.6×250mm, 5μm) was selected for DoE optimization. However, when verifying MODR, it was found that the previous identification of chromatographic peaks was incorrect, and the analysis effect of the verification point was not ideal. The separation effect of this column was limited. The column with smaller packing particle size was replaced and re-optimized. The selected column was the Agilent Infinity Lab Poroshell HPH-C18 column (4.6×250mm, 2.7μm) for further optimization.

[0079] This study employed experimental design to investigate the parameters affecting CQAs and established a quantitative model between CMPs and CQAs. Subsequently, a Monte Carlo method was used to calculate the operable design region, obtain the suggested MODR, and validate it, evaluating the robustness of the operating space. Optimal chromatographic conditions were selected and the method was validated, with good results. Finally, it was successfully applied to the fingerprint consistency and content determination of 10 batches of Qiwei Fangji Huangqi granules. In summary, this study developed a quantitative fingerprint HPLC method for the determination of the content of 13 active ingredients, applicable to both aqueous extract intermediates and finished formulations, covering six medicinal materials in the formula except for Atractylodes macrocephala, and suitable for quality control in the production process of Qiwei Fangji Huangqi granules.

[0080] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A fingerprint analysis method for Qiwei Fangji Huangqi granules, characterized in that, Includes the following steps: (1) Preparation of the test solution; (2) Preparation of reference solution; (3) The fingerprint spectrum of Qiwei Fangji Huangqi granules or Qiwei Fangji Huangqi aqueous extract was determined by high performance liquid chromatography; The gradient elution program for the high performance liquid chromatography method is as follows: 0-10 min, 8%-18% B; 10-40 min, 18%-25% B; 40-55 min, 25%-42% B; 55-60 min, 42%-75% B; 60-65 min, 75%-90% B; The mobile phase A of the high-performance liquid chromatography (HPLC) is 0.2% formic acid in water, and the mobile phase B is 0.2% formic acid in acetonitrile; The chromatographic column of the HPLC is an Agilent Infinity Lab Poroshell HPH-C18 column, 4.6 × 250 mm, 2.7 μm; The specific steps for preparing the test solution in step (1) are as follows: Seven-Ingredient Fangji Astragalus Granules Test Sample: Take seven-ingredient fangji astragalus granules, add ethanol to dissolve, sonicate, centrifuge, and take the supernatant to obtain the sample; The test sample of the aqueous extract of Seven-Flavor Fangji Astragalus: Extraction was carried out according to the compound ratio of Seven-Flavor Fangji Astragalus Granules, water was added, heated, decocted, the dregs were discarded, centrifuged, and the supernatant was taken. The specific steps for preparing the reference solution in step (2) are as follows: Weigh out the following ingredients separately: tetrandrine, tetrandrine, isoflavone glycoside, gentianin, astragaloside A, atractylodes lactone II, atractylodes lactone III, atractylone, glycyrrhizin, glycyrrhizic acid monoammonium salt, tanshinone sodium, rosmarinic acid, lithospermic acid, salvianolic acid B, isorhomaric acid glycoside, and quercetin-3-O-β-D-glucose-7-O-β-D-gentiopicroside. Dissolve and bring to volume with ethanol, mix well, centrifuge, and collect the supernatant to obtain the final product.

2. The fingerprint spectrum analysis method according to claim 1, characterized in that, In the preparation of the test sample of Qiwei Fangji Huangqi Granules, the material-to-liquid ratio of Qiwei Fangji Huangqi Granules to ethanol is 1g:8-12mL, the volume fraction of ethanol is 50%-55%, the sonication time is 25-35min, after sonication, the sample is cooled, the weight loss is replenished with ethanol, shaken well, and centrifuged at 11000-13000rpm for 10-20min.

3. The fingerprint spectrum analysis method according to claim 1, characterized in that, In the preparation of the water extract of Astragalus membranaceus and Stephania tetrandra, the amount of water added is 6-10 times the total weight of Astragalus membranaceus and Stephania tetrandra. The mixture is decocted for 1-2 hours and centrifuged at 11000-13000 rpm for 10-20 minutes.

4. The fingerprint spectrum analysis method according to claim 1, characterized in that, The volume fraction of ethanol is 50%-55%, and the centrifugation conditions are 11000-13000 rpm for 10-20 min.

5. The fingerprint spectrum analysis method according to claim 1, characterized in that, The column temperature of the high performance liquid chromatography method described in step (3) is 22℃, the flow rate is 0.70 mL / min, and the injection volume is 5μL.

6. The fingerprint spectrum analysis method according to claim 1, characterized in that, The high performance liquid chromatography method described in step (3) has an ultraviolet detection wavelength of 280 nm, a CAD detector nebulization temperature of 35 °C, a sampling frequency of 10 Hz, a filtration constant of 1.0 s, and a power function of 1.

0.

7. The application of the fingerprint analysis method according to any one of claims 1-6 in detecting intermediates and preparations of Qiwei Fangji Huangqi Granules during the production process.

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