A method for detecting a fingerprint spectrum of a gold-st Johns wort extract (tablet)

Abstract

The application provides a kind of Jinguangdancao extract (tablet) fingerprint detection method, comprising the following steps: A) taking test sample raw material and being pretreated, obtaining the liquid to be measured;The pretreatment mode includes using solvent to dissolve, extraction;B) the liquid to be measured is determined using high performance liquid chromatography-evaporation light scattering method, and the HPLC-ELSD fingerprint of Jinguangdancao extract (tablet) is obtained;High performance liquid chromatography chromatographic conditions are as follows: column is C18 column;Mobile phase A is acetonitrile solution, mobile phase B is 0.1% formic acid aqueous solution, gradient elution;The application uses high performance liquid chromatography-evaporation light scattering method, selects acetonitrile-0.1% formic acid aqueous solution as mobile phase for gradient elution, with ursolic acid, oleanolic acid, β-amyrin, α-amyrin, friedelinone as reference, establishes the fingerprint detection method of Jinguangdancao extract (tablet), and the scientificity and internal quality of Jinguangdancao extract (tablet) are evaluated as a whole.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, and in particular to a fingerprint spectral detection method for Gentiana macrophylla extract (tablets). Background Technology

[0002] Gentian extract is prepared using gentian root as raw material and 70% ethanol as the percolation solvent through a series of processes including percolation extraction, concentration, pH adjustment, alcohol precipitation, and drying. Upon verification, the 2022 re-registration approval document for this product specifies spray drying as the drying method for the extract. This method is poorly compatible with current production equipment, and spray drying yields are unstable, resulting in low utilization of the medicinal material. Therefore, upon resuming production, the drying process must be simultaneously changed from spray drying to vacuum drying.

[0003] Gentiana scabra extract tablets are made from gentiana scabra extract as the active ingredient (API), through processes such as adding excipients, granulation, and tableting. Gentiana scabra extract tablets are commonly used to treat cough and excessive phlegm symptoms caused by bronchitis. Its instructions clearly state that pentacyclic triterpenoid saponins are the main components. During the preparation of this drug extract, a pH adjustment process can selectively remove flavonoid components while retaining saponin components. However, the original drug quality standards lacked specific testing methods for this type of saponin, making it impossible to meet the assessment requirements for product quality consistency and stability after the process change.

[0004] Therefore, it is urgent to establish a fingerprint spectrum detection method for Gentiana scabra extract (tablets) to provide technical support for the overall quality evaluation of its medicinal components. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a fingerprint spectrum detection method for Gentiana scabra extract (tablets). The fingerprint spectrum method constructed by the present invention is stable and reliable, and can control the quality of Gentiana scabra extract (tablets).

[0006] This study used Gentiana macrophylla extract as the main research object and established a corresponding fingerprint chromatographic detection method. The chromatographic conditions for the fingerprint chromatogram of Gentiana macrophylla extract were explored, determining the detection wavelength, elution gradient, mobile phase type, column parameters, and sample pretreatment method. By synthesizing fingerprint chromatograms from multiple batches, common peaks in the fingerprint chromatogram of Gentiana macrophylla extract were preliminarily identified and identified using reference standards. Referring to the analytical method validation guidelines in Part IV (9101) of the 2020 edition of the Chinese Pharmacopoeia, the specificity, precision, repeatability, and intermediate precision of the fingerprint chromatogram method for Gentiana macrophylla extract were validated. Finally, the applicability of the fingerprint chromatogram method for the detection of Gentiana macrophylla extract tablets was evaluated through specificity tests.

[0007] This invention provides a fingerprint spectral detection method for Gentiana macrophylla extract (tablets), comprising the following steps:

[0008] A) Take the test sample raw material and pretreat it to obtain the test solution; the pretreatment method includes solvent dissolution and extraction;

[0009] B) The test solution was analyzed by high performance liquid chromatography-evaporative light scattering to obtain the HPLC-ELSD fingerprint of Gentiana scabra extract (tablets);

[0010] The chromatographic conditions for high performance liquid chromatography were as follows: a C18 column; mobile phase A was acetonitrile solution, mobile phase B was 0.1% formic acid aqueous solution, and gradient elution was used.

[0011] The gradient elution specifically refers to:

[0012] 0~5min, Phase A: 15%, Phase B: 85%;

[0013] 5–10 min, Phase A: 15% → 25%, Phase B: 85% → 75%;

[0014] 10–45 min, Phase A: 25% → 30%, Phase B: 75% → 70%;

[0015] 45~50 min, Phase A: 30%→50%, Phase B: 70%→50%;

[0016] 50~65 min, Phase A: 50%→85%, Phase B: 50%→15%;

[0017] 65~66 min, Phase A: 85%→15%, Phase B: 15%→85%;

[0018] 66~71 min, Phase A: 15%, Phase B: 85%.

[0019] The test sample described in this invention is Gentiana scabra extract (tablets); the source of the extract is not limited, and it can be commercially available or prepared according to the method of this invention. The main active ingredient of Gentiana scabra extract is a pentacyclic triterpenoid.

[0020] This invention provides a fingerprint spectrum detection method for Gentiana macrophylla extract (tablets). First, the raw sample is pretreated to obtain the test solution.

[0021] The concentration of the Gentiana scabra extract (tablets) of the present invention is preferably 2 mg / ml to 25 mg / ml; specifically, it can be 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, or 25 mg / ml; particularly preferably 10 mg / ml.

[0022] The pretreatment method includes solvent dissolution and extraction.

[0023] The solvent used in this invention is 70% ethanol; the extraction is ultrasonic extraction; the ultrasonic time is 30 min; the ratio of the mass (g) of the test sample raw material to the volume (mL) of the solvent is (0.1~0.25):(10~50).

[0024] In some embodiments, the ratio of the mass (g) of the test sample raw material to the volume (mL) of the solvent is 0.1 g: 10 mL;

[0025] In some embodiments, the ratio of the mass (g) of the test sample raw material to the volume (mL) of the solvent is 0.1 g: 50 mL;

[0026] In some embodiments, the ratio of the mass (g) of the test sample raw material to the volume (mL) of the solvent is 0.15 g: 10 mL;

[0027] In some embodiments, the ratio of the mass (g) of the test sample raw material to the volume (mL) of the solvent is 0.2 g: 10 mL;

[0028] In some embodiments, the ratio of the mass (g) of the test sample raw material to the volume (mL) of the solvent is 0.25 g: 10 mL.

[0029] This invention also includes the preparation of a reference solution:

[0030] Preparation of reference solution: Ursolic acid, oleanolic acid, β-amyrin, α-amyrin, and tretinoin were dissolved in a solvent to obtain a reference solution; the solvent mentioned above is preferably 70% ethanol.

[0031] The specific concentrations of the reference solutions are: ursolic acid 0.2~0.25 mg / mL, oleanolic acid 0.2~0.25 mg / mL, β-amyrin 0.15~0.2 mg / mL, α-amyrin 0.2~0.25 mg / mL, and tretinoin 0.2~0.25 mg / mL.

[0032] In some preferred embodiments, ursolic acid 0.2080 mg / mL, oleanolic acid 0.2044 mg / mL, β-amyrin 0.1929 mg / mL, α-amyrin 0.2024 mg / mL, and tretinoin 0.2056 mg / mL are used.

[0033] The present invention also contains rutin 0.1958 mg / mL, β-amyrin 0.1929 mg / mL, chlorogenic acid 0.2121 mg / mL, arbutin 0.2102 mg / mL, luteolin 0.2047 mg / mL, apigenin 0.2051 mg / mL, and caffeic acid 0.2128 mg / mL.

[0034] The reference solution was analyzed by high performance liquid chromatography-evaporative light scattering to obtain the chromatogram of the reference; and the components of the fingerprint spectrum of Gentiana scabra extract (tablets) were qualitatively identified based on the chromatogram of the reference.

[0035] In this invention, mobile phase A is an acetonitrile solution, and mobile phase B is a 0.1% formic acid aqueous solution, with gradient elution.

[0036] The gradient elution specifically refers to:

[0037] 0~5min, Phase A: 15%, Phase B: 85%;

[0038] 5–10 min, Phase A: 15% → 25%, Phase B: 85% → 75%;

[0039] 10–45 min, Phase A: 25% → 30%, Phase B: 75% → 70%;

[0040] 45~50 min, Phase A: 30%→50%, Phase B: 70%→50%;

[0041] 50~65 min, Phase A: 50%→85%, Phase B: 50%→15%;

[0042] 65~66 min, Phase A: 85%→15%, Phase B: 15%→85%;

[0043] 66~71 min, Phase A: 15%, Phase B: 85%.

[0044] Under the above-mentioned elution gradient, compared with other gradient conditions, the present invention has good baseline separation, good peak separation, and stable baseline.

[0045] The chromatographic column of this invention is a C18 column; preferably, the chromatographic column is a Thermo BDS HYPERSIL C18 with dimensions of 250*4.6mm and 5μm; the column temperature is 30℃.

[0046] The present invention preferably uses the above-described chromatographic column for robustness testing, and the column exhibits good robustness. At the above-described column temperature, the chromatographic peaks of the present invention are symmetrical, with good resolution and complete peak elution.

[0047] The mobile phase flow rate was 1.0 ml / min; the injection volume was 10 μL.

[0048] The present invention found that the chromatographic peaks were well separated and the resolution was moderate at a flow rate of 1.0 mL / min, which is the optimal solution.

[0049] The detector of this invention is an evaporative light scattering detector; the drift tube temperature is 109°C and the atomizing gas flow rate is 3.0 L / min.

[0050] This invention uses oleanolic acid as the reference peak S, and calculates the relative retention times of each characteristic peak and the S peak. The relative retention times of each characteristic peak are: 0.476 (peak 1), 0.727 (peak 2), 0.753 (peak 3), 0.788 (peak 4), 0.805 (peak 5), 0.821 (peak 6), 0.870 (peak 7), 1.184 (peak 9), 1.279 (peak 10), 1.371 (peak 11), 1.418 (peak 12), 1.432 (peak 13), 1.801 (peak 14), and 1.942 (peak 15).

[0051] According to the present invention, peak 2 is ursolic acid, peak 8 is oleanolic acid, peak 10 is β-amyrin, peak 11 is α-amyrin, and peak 14 is trefoil ketone.

[0052] This invention provides a fingerprint chromatogram detection method for Gentiana scabra extract (tablets), comprising the following steps: A) pretreatment of the test sample raw material to obtain the test solution; the pretreatment method includes solvent dissolution and extraction; B) determination of the test solution by high performance liquid chromatography-evaporative light scattering (HPLC-ELSD) to obtain the HPLC-ELSD fingerprint chromatogram of Gentiana scabra extract (tablets); the HPLC-ELSD chromatographic conditions are: C18 column; mobile phase A is acetonitrile solution, mobile phase B is 0.1% formic acid aqueous solution, gradient elution; this invention uses HPLC-ELSD, selects acetonitrile-0.1% formic acid aqueous solution as the mobile phase for gradient elution, and uses ursolic acid, oleanolic acid, β-amyrin, α-amyrin, and styraxone as references to establish a fingerprint chromatogram detection method for Gentiana scabra extract (tablets), which is beneficial for the overall evaluation of the scientificity and rationality of the relevant process of Gentiana scabra extract (tablets), and can better assess its intrinsic quality and ensure the clinical efficacy of the drug. Attached Figure Description

[0053] Figure 1 Chromatogram of fingerprinting analysis of Gentiana macrophylla extract in preliminary experimental studies;

[0054] Figure 2 Preliminary experiment to explore full-wavelength scanning chromatogram of fingerprint spectroscopy for Gentiana macrophylla extract;

[0055] Figure 3 3D full-wavelength scanning fingerprint spectrum of Gentiana macrocarpa extract;

[0056] Figure 4 Fingerprint spectrum of Gentiana macrophylla extract (wavelength 210nm);

[0057] Figure 5 Comparison of chromatograms for fingerprint elution gradient of Gentiana macrophylla extract (VWD);

[0058] Figure 6 Fingerprint analysis of mobile phase types and chromatographic comparison of Gentiana macrophylla extract (VWD).

[0059] Figure 7 Fingerprint (VWD) of Gentiana macrocarpa herb, extract, and reference standard;

[0060] Figure 8 Magnified partial fingerprint chromatograms (VWD) of Gentiana scabra herbal material, extract, and reference standard.

[0061] Figure 9 Fingerprint spectrum (ELSD) of Gentiana macrophylla herb and extract;

[0062] Figure 10 Comparison of chromatograms under different elution gradients for fingerprint analysis of Gentiana macrophylla extract (ELSD).

[0063] Figure 11 Comparison of chromatograms of fingerprints of Gentiana scabra extract with different mobile phases (ELSD).

[0064] Figure 12 Comparison of chromatograms of Gentiana macrophylla extract using different chromatographic columns (ELSD).

[0065] Figure 13 The fingerprint chromatogram of Gentiana macrophylla extract;

[0066] Figure 14 Comparison of chromatograms for fingerprint analysis of gentian extract samples at concentration (ELSD);

[0067] Figure 15 Comparison of chromatograms for fingerprint analysis of extraction solvents in Gentiana macrophylla extract;

[0068] Figure 16 Fingerprint spectrum of Gentiana macrophylla extract contains peak matching;

[0069] Figure 17 Comparison of chromatograms of Gentiana macrophylla extract (Z241101) with reference standard;

[0070] Figure 18 Identification of reference standard for Gentiana macrophylla extract (Z241101);

[0071] Figure 19 Fingerprint specificity of Gentiana macrophylla extract;

[0072] Figure 20 Fingerprint spectroscopy precision of Gentiana macrophylla extract;

[0073] Figure 21 Fingerprint repeatability of Gentiana macrophylla extract;

[0074] Figure 22 Intermediate precision of fingerprint spectroscopy for Gentiana scabra extract;

[0075] Figure 23 Fingerprint stability of Gentiana macrophylla extract;

[0076] Figure 24 Fingerprint analysis of Gentiana macrocarpa extract; flow rate durability;

[0077] Figure 25 Fingerprint analysis of Gentiana macrocarpa extract and column temperature durability;

[0078] Figure 26 Application assessment of Gentiana macrophylla extract tablets;

[0079] Figure 27 Fingerprint spectroscopy of samples with long-term stability of Gentiana scabra extract after 3 months;

[0080] Figure 28 Fingerprint spectroscopy of samples with accelerated stability after 3 months of Gentiana macrophylla extract;

[0081] Figure 29 Fingerprint chromatogram of 3-month long-term stability sample of Gentiana scabra extract tablets;

[0082] Figure 30 Fingerprint chromatogram of 3-month accelerated stability sample of Gentiana scabra extract tablets; Detailed Implementation

[0083] This invention provides a fingerprint spectrum detection method for Gentiana macrophylla extract (tablets). Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and fall within the scope of protection of this invention. The method and application of this invention have been described through preferred embodiments. Those skilled in the art can obviously modify or appropriately change and combine the method and application described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0084] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0085] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0086] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural.

[0087] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items.

[0088] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0089] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0090] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0091] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0092] The embodiments and comparative examples of this invention describe some examples, in which the embodiments illustrate certain implementations of the invention. However, this does not mean that the effects of the invention can only be achieved in these examples.

[0093] To further illustrate the present invention, the following describes in detail, with reference to embodiments, a fingerprint spectrum detection method for Gentiana scabra extract (tablets) provided by the present invention.

[0094] 1.1 Experimental Materials

[0095] Preparation of Gentiana macrophylla extract: Grind Gentiana macrophylla into coarse powder (24-mesh sieve), soak in 3 times the amount of 70% ethanol overnight, then add 6 times the amount of 70% ethanol for percolation (3-5 ml / kg*min), recover the ethanol from the percolation liquid, let stand, filter, wash the filter residue with 70% ethanol, combine the washing liquid and filtrate, add lime milk to adjust the pH to 12, let stand, filter, adjust the pH of the filtrate to 7 with 4 mol / L sulfuric acid solution, let stand, filter, concentrate the filtrate to a thick paste with a relative density of 1.30-1.35 (50℃), slowly add ethanol to make the ethanol content over 80%, let stand, filter, collect the filtrate for later use; dissolve and dilute the precipitate with a small amount of water to a thick paste with a relative density of 1.30-1.35 (50℃), then add ethanol to make the ethanol content over 85%, let stand, filter, collect the filtrate; combine the above filtrates, recover the ethanol, concentrate to a thick paste, and dry below 80℃ to obtain the final product.

[0096] Preparation of Gentiana macrophylla extract tablets: The excipients starch, microcrystalline cellulose, talc, and magnesium stearate are passed through a 50-mesh sieve. 60g of Gentiana macrophylla extract, 24g of starch, and 108g of microcrystalline cellulose are mixed together and wet-granulated with 70% ethanol. The granules are dried at 60℃~80℃. After passing the granules through a 20-mesh sieve, 4g of talc and 2g of magnesium stearate are added and mixed together. The mixture is then compressed into tablets and coated with a film to obtain the final product.

[0097] Table 1 Experimental Materials

[0098]

[0099] 1.2 Instruments and Equipment

[0100] Table 2 Instruments and Equipment

[0101]

[0102] Example 1

[0103] 1.1.1 Preliminary Experiment Exploration of Fingerprint Spectrum of Gentiana macrophylla Extract

[0104] Preparation of test solution: Weigh 0.1 g of Gentiana scabra extract accurately, place it in a 10 mL volumetric flask, add an appropriate amount of methanol, sonicate for 30 min, cool, dilute to the mark with methanol, shake well, filter, and filter the filtrate through a 0.45 μm microporous membrane to obtain the test solution.

[0105] Chromatographic column: Agilent ZORBAX SB-C18 (250*4.6mm, 5µm); wavelength: 254 nm; column temperature: 30℃; flow rate: 1.0 ml / min; injection volume: 10 μl; mobile phase: acetonitrile as mobile phase A and 0.1% phosphoric acid water as mobile phase B.

[0106] Elution gradients are shown in Table 3

[0107] Table 3

[0108]

[0109] Figure 1 The chromatogram of the fingerprint spectrum of Gentiana macrophylla extract was obtained from preliminary experimental studies. Figure 1 It can be seen that the number of chromatographic peaks is small, and the peak area is much smaller than that of the solvent peaks.

[0110] 1.1.2 Preliminary Experiment Exploration of Fingerprint Spectrum of Gentiana macrophylla Extract

[0111] Preparation of test solution: Weigh approximately 0.3 g of Gentiana scabra extract accurately, place it in a 10 ml volumetric flask, add an appropriate amount of 70% ethanol solution, sonicate for 30 min, cool, dilute to the mark with 70% ethanol solution, shake well, filter using a 0.45 μm microporous membrane, and collect the filtrate.

[0112] Table 4 Screening of fingerprint spectroscopy methods for Gentiana scabra extract

[0113]

[0114] Figure 2 Preliminary experiment to explore full-wavelength scanning chromatogram of fingerprint spectroscopy for Gentiana macrophylla extract; Figure 3 3D full-wavelength scanning fingerprint spectrum of Gentiana macrocarpa extract. Figure 4 Fingerprint pattern of Gentiana macrocarpa extract (wavelength 210 nm). For example... Figures 2-4 As shown, Gentiana macrophylla extract exhibits good UV absorption response at low wavelengths, especially at 210 nm, where the UV absorption response is highest and the chromatographic peaks (substances) are more abundant, making it a suitable wavelength for subsequent detection. However, current chromatographic methods exhibit problems such as poor peak resolution and undesirable peak shapes, indicating that the aforementioned elution gradient is not applicable under current conditions. Therefore, priority should be given to investigating fingerprint chromatographic conditions to screen for a more suitable elution gradient method.

[0115] 1.2 Investigation of chromatographic conditions for fingerprinting of Gentiana scabra extract

[0116] 1.2.1 Velocity gradient investigation (VWD)

[0117] In the preliminary experimental stage, the optimal detection wavelength of Gentiana scabra extract was determined using a DAD (diode array detector). Given that the VWD (variable wavelength detector) offers stable detection at a fixed wavelength, is easy to operate, and facilitates the evaluation of other chromatographic conditions to ensure the scientific rigor and efficiency of the method development, the initial method (Table 4) was used with other chromatographic conditions unchanged. Different gradient methods were then employed (see Tables 5-10) to optimize the elution gradient of the fingerprint chromatogram. The results of the elution gradient evaluation are shown in Tables 5-10. Figure 5 .

[0118] Table 5 Elution gradient (M1) for Method 250210

[0119]

[0120] Table 6 Elution gradient (M2) for Method 250211

[0121]

[0122] Table 7 Elution gradient (M3) for Method 250212-1

[0123]

[0124] Table 8 Elution gradient (M4) for Method 250212-2

[0125]

[0126] Table 9 Elution gradient (M5) for Method 250213

[0127]

[0128] Table 10 Elution gradient of Method 250214 (M6)

[0129]

[0130] Figure 5 Fingerprint analysis of elution gradient of Gentiana macrophylla extract: chromatogram comparison (VWD); such as Figure 5 As shown in the elution gradient study, the chromatogram obtained by method M6 in Table 10 can present the chromatographic peak information of the fingerprint spectrum of Gentiana scabra extract relatively completely. Therefore, this gradient was used for subsequent studies. Given the poor peak resolution during the early retention time of this elution gradient, the type of mobile phase was investigated to improve the separation effect of the fingerprint chromatographic peaks by adjusting the mobile phase composition.

[0131] 1.2.2 Investigation of mobile phase type (VWD)

[0132] Using the M6 ​​elution gradient and keeping other chromatographic conditions unchanged, the composition of three mobile phases—acetonitrile-0.02% phosphoric acid solution, acetonitrile-water, and acetonitrile-0.1% formic acid solution—was investigated. The chromatograms were compared with those obtained using the original method (acetonitrile-0.1% phosphoric acid solution) to screen for the optimal mobile phase composition for the fingerprint chromatogram of Gentiana scabra extract. The results of the mobile phase composition investigation are shown in […]. Figure 6 . Figure 6 Fingerprint analysis of Gentiana macrophylla extract, mobile phase type comparison (VWD); such as Figure 6 As shown, baseline stability was poor when acetonitrile-0.1% formic acid water was used as the mobile phase. Compared with acetonitrile-0.02% phosphoric acid water and acetonitrile-water, acetonitrile-0.1% phosphoric acid water could improve the peak shape of small chromatographic peaks under low organic phase ratio, but it still could not improve the chromatographic peak resolution in the early retention time of the fingerprint spectrum of Gentiana scabra extract.

[0133] Based on the above findings, the peak resolution and overall chromatographic effect of the fingerprint chromatogram of Gentiana scabra extract are still not ideal. Future research plans to utilize existing fingerprint analysis methods for Gentiana scabra medicinal materials to test extract samples and evaluate the applicability of these methods to extract samples.

[0134] 1.2.3 Applicability Study of Fingerprint Spectroscopy Methods for Gentiana macrophylla (VWD, ELSD)

[0135] Table 11 Fingerprint Spectroscopy Methods for Gentiana macrophylla Medicinal Materials

[0136]

[0137] 1.2.3.1 Applicability Study of Fingerprint Spectroscopy Method for Gentiana macrophylla (VWD)

[0138] Preparation of Gentiana macrophylla extract solution: Weigh approximately 0.3g of Gentiana macrophylla extract accurately, place it in a 10ml volumetric flask, add an appropriate amount of 70% ethanol solution, sonicate for 30min, cool, dilute to the mark with 70% ethanol solution, shake well, filter using a 0.45μm microporous membrane, and collect the filtrate.

[0139] Preparation of Gentiana scabra herbal solution: Take about 1.0g of Gentiana scabra powder (passed through a No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 50ml of 70% ethanol, weigh it, reflux in a water bath for 1 hour, cool it, weigh it again, make up the weight loss with 70% ethanol, shake well, and filter it through a 0.22μm filter membrane to obtain the solution.

[0140] Preparation of artemisinin reference solution: Accurately weigh about 10 mg of artemisinin reference standard, place it in a 100 ml volumetric flask, dilute to the mark with methanol, shake well, filter using a 0.45 μm microporous membrane, and collect the filtrate.

[0141] Preparation of the mixed reference solution of Gentiana scabra extract and Artemisia annua: Take 1 ml of Gentiana scabra extract solution and place it in a 10 ml volumetric flask. Add an appropriate amount of Artemisia annua reference solution and dilute to the mark. Filter using a 0.45 μm microporous membrane and collect the filtrate.

[0142] The fingerprinting method for Gentiana scabra (Table 11) was used for sample injection. The results are shown below. Figures 7-8 . Figure 7 Fingerprint chromatograms (VWD) of Gentiana macrocarpa herb, extract, and reference standard. Figure 8 Magnified partial fingerprint spectroscopy (VWD) of Gentiana macrocarpa herb, extract, and reference standard.

[0143] like Figures 7-8As shown, when using the fingerprint chromatogram detection method for Gentiana scabra (VWD detector), the fingerprint chromatogram of the extract showed a stable baseline and good peak resolution and shape. However, there was a difference in the number of chromatographic peaks at the same retention time between the artemisinin reference standard and the mixed reference standard of Gentiana scabra extract + artemisinin, indicating that the main peak component of Gentiana scabra extract is not artemisinin, and the overall number of chromatographic peaks in the chromatogram was relatively small. Considering that saponin components in the extract may not be effectively detected by VWD due to the lack of ultraviolet absorption groups, an evaporative light scattering detector (ELSD) will be used to optimize the experimental conditions for a more comprehensive characterization of the extract components.

[0144] 1.2.3.2 Applicability Study of Fingerprint Spectroscopy Method for Gentiana macrophylla (ELSD)

[0145] The test solution of Gentiana scabra extract was administered using the method described in "1.2.3.1 Applicability Study of Fingerprint Spectroscopy Method for Gentiana scabra (VWD)" and "Table 11 Fingerprint Spectroscopy Method for Gentiana scabra". The results are shown in the table below. Figure 9 . Figure 9 Fingerprint spectroscopy (ELSD) of Gentiana macrophylla extract; such as Figure 9 As shown, when the fingerprint chromatogram of Gentiana scabra extract is analyzed using the same detection conditions combined with an evaporative light scattering detector (ELSD), the number of chromatographic peaks in the extract fingerprint chromatogram increases. However, under the same elution conditions, the extract fingerprint chromatogram exhibits insufficient peak resolution, indicating that this method cannot meet the requirements for fingerprint chromatogram analysis of Gentiana scabra extract. Therefore, it is necessary to re-optimize and investigate the chromatographic conditions for the fingerprint chromatogram of Gentiana scabra extract to establish a specific analytical method suitable for extract samples.

[0146] 1.2.4 Elution gradient investigation (ELSD)

[0147] While maintaining the existing fingerprint chromatogram conditions for Gentiana scabra (except for the elution gradient), the elution gradient was specifically optimized (see Tables 12-21). The drift tube temperature and atomized gas flow rate parameters of the evaporative light scattering detector (ELSD) were scientifically calculated and set based on the ratio of the mobile phase mixed solvent.

[0148] During the elution gradient optimization process, given the actual situation that the fingerprint spectrum of Gentiana scabra extract has overloaded main peak concentration and peak flattening, the sample concentration was investigated first. The relevant research results are detailed in "5.3.1 Sample Concentration Investigation".

[0149] The concentration of the sample was determined according to the results of "1.3.1 Sample Concentration Investigation". The test solution was prepared as follows: Take about 0.1 g of Gentiana scabra extract, accurately weigh it, put it in a 10 ml volumetric flask, add an appropriate amount of 70% ethanol solution, sonicate for 30 min, cool, dilute to the mark with 70% ethanol solution, shake well, filter with a 0.45 μm microporous membrane, and take the filtrate.

[0150] The results of the elution gradient study are shown below. Figure 10 .

[0151] The drift tube temperature of the evaporative light scattering detector is 109℃, and the atomizing gas flow rate is 3.0 L / min.

[0152] Table 12 Elution gradient of Method 250303 (M2-1)

[0153]

[0154] Table 13 Elution gradient of Method 250305 (M2-2)

[0155]

[0156] Table 14 Elution gradient of Method 250305 (M2-3)

[0157]

[0158] Table 15 Elution gradient of Method 250305 (M2-4)

[0159]

[0160] Table 16 Elution gradient of Method 250305 (M2-5)

[0161]

[0162] The drift tube temperature of the evaporative light scattering detector is 111℃, and the atomizing gas flow rate is 3.1 L / min.

[0163] Table 17 Elution gradient of Method 250306 (M2-6)

[0164]

[0165] The drift tube temperature of the evaporative light scattering detector is 87℃, and the atomizing gas flow rate is 2.9 L / min.

[0166] Table 18 Elution gradient of Method 250306 (M2-7)

[0167]

[0168] The drift tube temperature of the evaporative light scattering detector is 109℃, and the atomizing gas flow rate is 3.0 L / min.

[0169] Table 19 Elution gradient of Method 250306 (M2-8)

[0170]

[0171] Table 20 Elution gradient for Method 250306 (M2-9)

[0172]

[0173] Table 21 Elution gradient of Method 250310 (M2-10)

[0174]

[0175] Figure 10 Comparison of chromatograms of different elution gradients for fingerprint analysis of Gentiana macrophylla extract (ELSD); Figure 10 As shown, after systematic investigation and analysis, method M2-10 has a reasonable elution time setting, which can achieve effective separation; the number of chromatographic peaks is moderate, which comprehensively reflects the characteristics of the extract components; and the resolution of each chromatographic peak is good, meeting the requirements of fingerprint analysis. Based on these advantages, method M2-10 was finally selected as the elution gradient program for the fingerprint spectrum of Gentiana scabra extract.

[0176] 1.2.5 Investigation of Mobile Phase Types (ELSD)

[0177] Using the M2-10 elution gradient and keeping other chromatographic conditions unchanged, two mobile phase systems, methanol-water and acetonitrile-0.1% formic acid water, were investigated. The chromatograms obtained by these systems were compared with those obtained by the original method (acetonitrile-water) to screen for the optimal mobile phase system suitable for the fingerprint chromatogram of Gentiana scabra extract. The results of the mobile phase selection are shown in […]. Figure 11 . Figure 11 Comparison of chromatograms of different mobile phases in the fingerprinting of Gentiana macrophylla extract (ELSD); such as Figure 11 As shown, when methanol-water is used as the mobile phase, the elution time of the chromatographic peaks is significantly delayed, and the target components cannot be completely eluted within the predetermined elution time, indicating poor compatibility with existing elution gradient methods. Therefore, this study will not further explore the methanol-0.1% formic acid-water system. In contrast, the overall fingerprint chromatograms of acetonitrile-water and acetonitrile-0.1% formic acid-water mobile phases are similar; however, the acetonitrile-0.1% formic acid-water system can detect more small chromatographic peaks, especially showing better separation for peaks with a retention time of 34 minutes. Considering key indicators such as the completeness of chromatographic peak information and resolution, acetonitrile-0.1% formic acid-water was ultimately determined as the optimal mobile phase system for the fingerprint chromatogram of Gentiana scabra extract.

[0178] 1.2.6 Column Investigation (ELSD)

[0179] Given the widespread application of octadecylsilane-bonded silica gel columns in the detection of saponins, this study compared octadecylsilane-bonded silica gel columns of different brands (see Table 22) with the original chromatographic conditions. The columns used were based on a fixed M2-10 elution gradient, an acetonitrile-0.1% formic acid-water mobile phase system, and the sample concentration determined in section "5.3.1 Sample Concentration Investigation." All other chromatographic conditions were kept constant. The aim was to screen for columns suitable for fingerprint analysis of Gentiana macrophylla extract and determine the final chromatographic conditions. The results of the column investigation are shown in Table 22. Figure 12 .

[0180] Table 22 Different Column Parameters

[0181]

[0182] Figure 12 Comparison of chromatograms of Gentiana macrophylla extract fingerprints using different chromatographic columns (ELSD); such as Figure 12 As shown, the Thermo BDS HYPERSIL C18 (16#) column exhibited excellent separation performance in the fingerprint analysis of Gentiana macrophylla extract. This column not only effectively separated most of the chromatographic peaks in the chromatogram, but also ensured that the resolution of each peak met the analytical standards, while maintaining good baseline stability, providing a reliable guarantee for accurate fingerprint analysis. Based on this, the Thermo BDS HYPERSIL C18 was ultimately selected as the optimal column.

[0183] 1.2.7 Determination of Chromatographic Conditions (ELSD)

[0184] The chromatographic column was a Thermo BDS HYPERSIL C18 (250*4.6mm, 5μm). The mobile phase consisted of acetonitrile (A) and 0.1% formic acid (B). The flow rate was 1.0 ml / min, the column temperature was 30℃, the injection volume was 10 μl, the drift tube temperature of the evaporative light scattering detector was 109℃, and the nebulizer gas flow rate was 3.0 L / min. The elution gradient program is detailed in Table 23. The fingerprint chromatogram is shown in [reference needed]. Figure 13 . Figure 13 The image shows the fingerprint chromatogram of Gentiana macrophylla extract.

[0185] Table 23 Fingerprint Spectrum Elution Gradient Program for Gentiana macrophylla Extract

[0186]

[0187] 1.3 Sample Pretreatment Study (ELSD)

[0188] 1.3.1 Sample Concentration Investigation

[0189] Gentiana scabra extract test solutions with concentrations ranging from 2 mg / ml to 25 mg / ml were prepared (see Table 24). Elution gradient 250303 (M2-1) was used under section 1.2.4, with acetonitrile-water as the mobile phase, a flow rate of 1.0 ml / min, a column temperature of 30℃, an injection volume of 10 μl, a drift tube temperature of 109℃ for the evaporative light scattering detector, and a nebulizer gas flow rate of 3.0 L / min. Based on the number, area, and shape of the chromatographic peaks, a suitable sample volume for the Gentiana scabra extract test solution was selected to improve the accuracy of the analytical results. The results of the sample concentration investigation are shown in [Table 24]. Figure 14 .

[0190] Table 24 Preparation of test solutions of Gentiana scabra extract

[0191]

[0192] Figure 14 Gentian extract fingerprint chromatogram sample concentration comparison (ELSD); such as Figure 14 As shown, when the concentration of Gentiana scabra extract is 10 mg / ml, the chromatographic peaks are relatively complete, the main peak area is appropriate, and the peak shape meets the analytical requirements. In contrast, once the sample concentration exceeds 10 mg / ml, the main peak exhibits concentration overload, causing the peak tip to be clipped and deformed, affecting the accuracy and completeness of the fingerprint spectrum. Taking all factors into consideration, 10 mg / ml was ultimately determined to be the optimal detection concentration for Gentiana scabra extract.

[0193] 1.3.2 Investigation of Extraction Solvents

[0194] According to the sample concentration under "1.3.1 Sample Concentration Investigation", approximately 0.1 g of Gentiana scabra extract was placed in a 10 ml volumetric flask, and appropriate amounts of anhydrous ethanol, 70% ethanol, 50% ethanol, anhydrous methanol, 70% methanol, and 50% methanol were added. The mixture was sonicated for 30 min, cooled, and then diluted to the mark with the corresponding solvent. The solution was shaken well and filtered through a 0.45 μm microporous membrane. The filtrate was then collected. The chromatographic conditions under "1.2.7 Chromatographic Condition Determination (ELSD)" were used for injection. Based on the number, peak area, and peak shape of the chromatographic peaks, the best extraction solvent among the pretreatment methods for Gentiana scabra extract was selected. The results of the extraction solvent investigation are shown in [Figure 1]. Figure 15 . Figure 15 Fingerprint analysis of Gentiana macrophylla extract, extraction solvent comparison, and chromatogram comparison; such as... Figure 15As shown, when anhydrous ethanol was used as the extraction solvent, the resulting chromatographic peak areas were generally small, indicating that anhydrous ethanol has limited solubility for the components of Gentiana scabra extract. Under similar sample concentrations, when 70% ethanol and 70% methanol were used as extraction solvents, the total peak area of ​​the fingerprint chromatograms was significantly larger than that of the 50% ethanol and 50% methanol systems, demonstrating that the former two had higher extraction efficiency. Comparison revealed no significant difference in the fingerprint chromatograms obtained from extraction with 70% ethanol and 70% methanol. Considering solvent safety and long-term feasibility, 70% ethanol solution was ultimately determined as the preferred extraction solvent for Gentiana scabra extract.

[0195] 1.4 Confirmation of Common Peak

[0196] Based on the fingerprint chromatograms of multiple batches of Gentiana scabra extract, and by comparing various factors such as chromatographic peak retention time, peak area, and peak shape, common peaks in the fingerprint chromatograms of Gentiana scabra extract were preliminarily identified. Among them, Gentiana scabra extract (Z241101) is the sample before the process change, and Gentiana scabra extract (Z241102~Z241104) are the samples after the process change.

[0197] Preparation of test solution: Weigh approximately 0.1 g each of Gentiana scabra extract (Z241101~Z241104) accurately, place them in 10 ml volumetric flasks, add an appropriate amount of 70% ethanol solution, sonicate for 30 min, cool, dilute to the mark with 70% ethanol solution, shake well, filter using a 0.45 μm microporous membrane, and collect the filtrate.

[0198] See the results of the shared peak confirmation. Figure 16 Tables 25-26. Figure 16 The fingerprint spectrum of Gentiana scabra extract has peak matching (from bottom to top: Z241101, Z241102, Z241103, Z241104, and the reference spectrum).

[0199] Table 25. Determination of common peaks in the fingerprint spectrum of Gentiana scabra extract.

[0200]

[0201] Table 26. Fingerprint Similarity Evaluation of Gentiana macrophylla Extract

[0202]

[0203] like Figure 16As shown in Tables 25 and 26, using peak 8 as a reference peak, 15 common peaks were identified through analysis of the fingerprint spectra of multiple batches of Gentiana macrophylla extract. Comparison revealed that the fingerprint similarity between the Gentiana macrophylla extract sample before the process change (Z241101) and the samples after the process change (Z241102~Z241104) was greater than 0.99. This result preliminarily indicates that the process change had a relatively small impact on the sample composition, and the material basis of the samples before and after the change remained essentially consistent.

[0204] 1.5 Identification of Reference Standards

[0205] Based on the currently reported chemical components of *Gentiana scabra*, reference standards including α-amyrin, rutin, β-amyrin, chlorogenic acid, and oleanolic acid (see Table 27) were prepared to identify the common peaks in the fingerprint chromatogram of *Gentiana scabra* extract. The identification results of the reference standards are shown in [Table 27]. Figures 17-18 .

[0206] Table 27 Preparation of Reference Solution

[0207]

[0208] Figure 17 Comparison of chromatograms of Gentiana macrophylla extract (Z241101) with reference standard; Figure 18 Gentiana macrophylla extract (Z241101) reference standard identification; such as Figures 17-18 As shown, the reference standards for the Gentiana scabra extract (Z241101) sample were identified using peak 8 as the reference peak. The relative retention times were 21.715, 29.941, 38.214, 40.941, and 54.126, respectively, for ursolic acid (peak 2), oleanolic acid (peak 8), β-amyrin (peak 10), α-amyrin (peak 11), and styrax ketone (peak 14). All identified reference standards were pentacyclic triterpenoid saponins, consistent with the pharmacodynamic components of Gentiana scabra extract.

[0209] Example 2 Methodological Validation

[0210] The optimal processing method under "1.3 Sample Pretreatment Investigation" and the chromatographic method under "1.2.7 Determination of Chromatographic Conditions" of Gentiana scabra extract were adopted. The specificity, precision, repeatability, intermediate precision and robustness of the fingerprint chromatogram of Gentiana scabra extract were validated with reference to the analytical method validation guidelines of Part IV (9101) of the 2020 edition of the Chinese Pharmacopoeia.

[0211] 2.6.1 Specificity

[0212] Preparation of blank solvent: Take an appropriate amount of 70% ethanol solution, filter it using a 0.45μm microporous membrane, and collect the filtrate.

[0213] Preparation of the test solution: Weigh approximately 0.1 g of Gentiana scabra extract accurately, place it in a 10 ml volumetric flask, add an appropriate amount of 70% ethanol solution, sonicate for 30 min, cool, dilute to the mark with 70% ethanol solution, shake well, filter using a 0.45 μm microporous membrane, and collect the filtrate.

[0214] Both the blank solvent and the test solution were injected and detected using the established fingerprint spectra. Specificity assessment results are shown below. Figure 19 .

[0215] Figure 19 The fingerprint specificity of Gentiana macrophylla extract; such as Figure 19 As shown in the figure, the spectrum of the blank solvent and the sample of Gentiana scabra extract were compared, and no interfering peaks were found, indicating that the method has good specificity.

[0216] 2.6.2 Precision

[0217] Accurately weigh approximately 0.1 g of Gentiana scabra extract (batch number Z241101), place it in a 10 ml volumetric flask, add an appropriate amount of 70% ethanol solution, sonicate for 30 min, cool, and dilute to the mark with 70% ethanol solution. Shake well, filter using a 0.45 μm microporous membrane, and use the filtrate to prepare one test solution. Inject the solution six times consecutively according to the established fingerprint chromatographic conditions, and record the chromatogram. The "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" (2012 version) was used to evaluate sample similarity. The specific method is as follows: using the S1 chromatogram as the reference chromatogram, the average method was used to generate a control chromatogram; the time window width was set to 0.5, and similarity analysis was carried out through multi-point correction and Mark peak matching. Simultaneously, the relative retention time and peak area of ​​each common chromatographic peak were calculated. The results of the precision test are shown in […]. Figure 20 And Tables 28-30.

[0218] Figure 20 Fingerprint precision of Gentiana macrophylla extract; (S1: precision 1, S2: precision 2, S3: precision 3, S4: precision 4, S5: precision 5, S6: precision 6).

[0219] Table 28. Precision and similarity evaluation of fingerprint chromatograms of Gentiana scabra extract.

[0220]

[0221] Table 29 Relative retention time (precision) of fingerprint chromatograms of Gentiana macrophylla extract

[0222]

[0223] Table 30 Relative peak areas (precision) of fingerprint chromatograms of Gentiana macrophylla extract.

[0224]

[0225] As shown in Table 28, the fingerprint spectra obtained from the gentian extract sample were injected six times consecutively, and the similarity of the fingerprint spectra was greater than 0.99, indicating that the method has good injection precision and the system stability meets the requirements.

[0226] 2.6.3 Repeatability

[0227] Accurately weigh approximately 0.1 g of Gentiana scabra extract (batch number Z241101), place it in a 10 ml volumetric flask, add an appropriate amount of 70% ethanol solution, sonicate for 30 min, cool, and dilute to the mark with 70% ethanol solution. Shake well, filter using a 0.45 μm microporous membrane, and collect the filtrate. Prepare 6 test solutions, and continuously inject and detect them according to the determined fingerprint chromatographic conditions, recording the chromatograms. The "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" (2012 version) was used to evaluate sample similarity. The specific method is as follows: using the S1 chromatogram as the reference chromatogram, the average method was used to generate the control chromatogram; the time window width was set to 0.5, and similarity analysis was carried out through multi-point correction and Mark peak matching. Simultaneously, the relative retention time and peak area of ​​each common chromatographic peak were calculated. The repeatability test results are shown in […]. Figure 21 And Tables 31-33.

[0228] Figure 21 Fingerprint repeatability of Gentiana macrophylla extract; (S1: repeatability 1, S2: repeatability 2, S3: repeatability 3, S4: repeatability 4, S5: repeatability 5, S6: repeatability 6).

[0229] Table 31 Evaluation of the repeatability similarity of fingerprint chromatograms of Gentiana scabra extract

[0230]

[0231] Table 32 Relative retention times (reproducibility) of fingerprint chromatograms of Gentiana macrophylla extract.

[0232]

[0233] Table 33 Relative peak areas (reproducibility) of fingerprint chromatograms of Gentiana macrophylla extract.

[0234]

[0235] As shown in Table 31, the fingerprint similarity of the six test solutions was all greater than 0.99, indicating that the method has good repeatability and the sample preparation and determination process is stable and reliable.

[0236] 2.6.4 Intermediate Precision

[0237] On different dates, different analysts accurately weighed approximately 0.1 g of Gentiana scabra extract (batch number Z241101), placed it in a 10 ml volumetric flask, added an appropriate amount of 70% ethanol solution, sonicated for 30 min, cooled, and diluted to the mark with 70% ethanol solution. The solution was then filtered through a 0.45 μm microporous membrane, and the filtrate was collected. Six test solutions were prepared and continuously injected according to the established fingerprint chromatographic conditions, and the chromatograms were recorded. The "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" (2012 version) was used to evaluate sample similarity. The specific method is as follows: using the S1 chromatogram as the reference chromatogram, the average method was used to generate the control chromatogram; the time window width was set to 0.5, and similarity analysis was conducted through multi-point correction and Mark peak matching. Simultaneously, the relative retention time and peak area of ​​each common chromatographic peak were calculated. The intermediate precision test results are shown in […]. Figure 22 And Tables 34-36. Figure 22 Intermediate precision of fingerprint chromatogram of Gentiana scabra extract (S1: repeatability 1, S2: repeatability 2, S3: repeatability 3, S4: repeatability 4, S5: repeatability 5, S6: repeatability 6, S7: repeatability 1-1, S8: repeatability 2-1, S9: repeatability 3-1, S10: repeatability 4-1, S11: repeatability 5-1, S12: repeatability 6-1).

[0238] Table 34. Evaluation of intermediate precision similarity of fingerprint chromatograms of Gentiana scabra extract.

[0239]

[0240] Table 35 Relative retention times (intermediate precision) of fingerprint chromatograms of Gentiana macrophylla extract.

[0241]

[0242] Table 36 Relative peak areas (intermediate precision) of fingerprint chromatograms of Gentiana macrophylla extract

[0243]

[0244] As shown in Table 34, in the intermediate precision verification, the fingerprint similarity of Gentiana scabra extract was greater than 0.99, indicating that the method has extremely high reproducibility and stability under different operators and time conditions, which meets the strict requirements of fingerprint analysis for precision.

[0245] 2.6.5 Stability

[0246] Take the test solution from section "1.6.2 Precision" and inject it at 0 h, 3 h, 6 h, 14 h, 18 h, and 24 h according to the determined fingerprint chromatographic conditions, and record the chromatograms. The "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" (2012 version) was used to evaluate sample similarity. The specific method is as follows: using the S1 chromatogram as the reference chromatogram, a control chromatogram was generated using the averaging method; the time window width was set to 0.5, and similarity analysis was conducted through multi-point correction and Mark peak matching. Simultaneously, the relative retention time and peak area of ​​each common chromatographic peak were calculated. The stability test results are shown in […]. Figure 23 And Tables 37-39.

[0247] Figure 23 Stability of fingerprint chromatogram of Gentiana macrophylla extract; (S1: stability 0 h, S2: stability 3 h, S3: stability 6 h, S4: stability 14 h, S5: stability 18 h, S6: stability 24 h)

[0248] Table 37 Stability and Similarity Evaluation of Fingerprint Spectra of Gentiana macrophylla Extract

[0249]

[0250] Table 38 Relative retention times (stability) of fingerprint chromatograms of Gentiana macrophylla extract.

[0251]

[0252] Table 39 Relative peak areas (stability) of fingerprint chromatograms of Gentiana macrophylla extract.

[0253]

[0254] As shown in Table 37, the fingerprint similarity of the Gentiana scabra extract sample at each time point (0, 3, 6, 14, 18, 24 h) within 24 hours was greater than 0.99. The results indicate that the chemical composition of the test solution is stable for at least 24 hours, which meets the time requirements for fingerprint analysis.

[0255] 2.6.6 Durability

[0256] Robustness refers to the degree to which the measurement results remain unaffected by minor changes in measurement conditions. To ensure the method can be used for routine testing, this experiment investigated the robustness of the chromatographic conditions, including flow rate (0.8 ml / min, 1.0 ml / min, 1.2 ml / min) and column temperature (28℃, 30℃, 32℃), to examine the impact of small changes in measurement conditions on the fingerprint chromatogram of Gentiana scabra extract. The results of the flow rate robustness investigation are shown below. Figure 24 And Table 40.

[0257] Accurately weigh approximately 0.1 g of Gentiana scabra extract (batch number Z241101), place it in a 10 ml volumetric flask, add an appropriate amount of 70% ethanol solution, sonicate for 30 min, cool, and dilute to the mark with 70% ethanol solution. Shake well and filter using a 0.45 μm microporous membrane. Prepare one test solution from the filtrate. Following the chromatographic method under "5.2.7 Determination of Chromatographic Conditions," set different flow rates (0.8 ml / min, 1.0 ml / min, 1.2 ml / min) and column temperatures (28℃, 30℃, 32℃) for injection and detection, and record the chromatograms. The "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" (2012 version) was used to evaluate sample similarity. The specific method is as follows: using the S2 chromatogram as the reference chromatogram, a control chromatogram was generated using the mean method; the time window width was set to 0.5, and similarity analysis was conducted through multi-point correction and Mark peak matching. Simultaneously, the relative retention times and peak areas of each common chromatographic peak were calculated. The results of the flow rate robustness study are shown below. Figure 24 And Table 41.

[0258] 2.6.6.1 Flow rate durability

[0259] Figure 24 Fingerprint robustness of Gentiana macrophylla extract at different flow rates (S1: flow rate 0.8 ml / ml, S2: flow rate 1.0 ml / ml, S3: flow rate 1.2 ml / ml, with S2 as the reference spectrum).

[0260] Table 40. Fingerprint spectrum similarity evaluation of Gentiana scabra extract flow rate durability.

[0261]

[0262] As shown in Table 40, within a certain range (flow rate 0.8~1.0 ml / min), the fingerprint similarity of Gentiana scabra extract is less affected by the flow rate, showing good flow rate durability. However, when the flow rate reaches 1.2 ml / min, the fingerprint similarity decreases significantly, suggesting that excessively high flow rates may have a significant impact on the fingerprint spectrum of Gentiana scabra extract, exceeding the drug's flow rate durability range.

[0263] 2.6.6.2 Column Temperature Durability

[0264] Figure 25 Fingerprint spectroscopy of Gentiana macrophylla extract for column temperature durability; (S1: column temperature 28℃, S2: column temperature 30℃, S3: column temperature 32℃, with S2 as the reference spectrum).

[0265] Table 41. Evaluation of Column Temperature Durability Similarity of Gentiana macrophylla Extract Fingerprint Spectra

[0266]

[0267] As shown in Table 41, the similarity between the fingerprints obtained at column temperatures of 28℃, 30℃, and 32℃, as well as with the control fingerprints, was extremely high, all ranging from 0.997 to 1.000. This indicates that within the temperature range of 28℃ to 32℃, changes in column temperature have minimal impact on the similarity of the fingerprint chromatograms of Gentiana scabra extract. The drug exhibits good column temperature durability within this range, and changes in column temperature do not significantly interfere with the fingerprint chromatogram characteristics of Gentiana scabra extract.

[0268] 2.7 Applicability Study of Fingerprint Spectroscopy Method for Gentiana macrophylla Extract in the Detection of Gentiana macrophylla Extract Tablets

[0269] Gentiana macrophylla extract tablets are made from gentiana macrophylla extract as the API (active ingredient), through processes such as adding excipients, granulation, and tableting. The applicability of the fingerprinting method for gentiana macrophylla extract tablets can be investigated through specificity testing. Specifically, the influence of blank excipients (microcrystalline cellulose, corn starch, magnesium stearate, talc, and gastric-soluble film-coating premix) on the API of gentiana macrophylla extract tablets will be examined to evaluate the applicability of the fingerprinting method for gentiana macrophylla extract tablets.

[0270] Preparation of blank excipient solution: Take an appropriate amount of blank excipient (0.1 g of Gentiana scabra extract can be converted to 0.33 g of Gentiana scabra extract tablets; see Table 42 for specific excipient dosage), accurately weigh it, place it in a 100 ml volumetric flask, add an appropriate amount of 70% ethanol, sonicate for 30 min, cool, dilute to the mark with 70% ethanol, shake well, filter through a 0.45 μm microporous membrane, and take the filtrate to obtain the blank excipient solution.

[0271] Table 42 Excipients for Gentiana macrophylla Extract Tablets

[0272]

[0273] Preparation of the test solution: Weigh approximately 0.33 g of Gentiana scabra extract tablets (batch number C241201) accurately, place them in a 10 ml volumetric flask, add an appropriate amount of 70% ethanol solution, sonicate for 30 min, cool, dilute to the mark with 70% ethanol solution, shake well, filter using a 0.45 μm microporous membrane, and collect the filtrate to obtain the test solution.

[0274] Take blank excipient solution and test sample solution separately, and perform chromatographic analysis using the method described in section "5.2.7 Chromatographic Conditions Determination". Specificity test results are shown below. Figure 26 .

[0275] Figure 26 Application assessment of Gentiana macrophylla extract tablets; such as Figure 26As shown, the chromatogram of the blank excipient solution did not show any interfering peaks at the common peaks corresponding to the Gentiana scabra extract tablets, indicating that the fingerprint analysis method for Gentiana scabra extract is not affected by excipients and can be effectively and accurately used for the detection and analysis of Gentiana scabra extract tablets.

[0276] In addition, the results of other methodological validation projects previously carried out on the fingerprint spectrum of Gentiana scabra extract, including the validation results of precision, repeatability, intermediate precision, stability and robustness, can also be directly applied to the methodological validation of Gentiana scabra extract tablets, providing comprehensive and reliable support for the methodological system of quality control of Gentiana scabra extract tablets.

[0277] Example 3: Process of Gentiana scabra extract (tablets) and sample testing before and after modification

[0278] Using the fingerprint spectroscopy detection method for Gentiana scabra extract (tablets) established in Example 1, the samples of Gentiana scabra extract (tablets) before and after process changes were tested to evaluate the consistency of their material basis.

[0279] Take approximately 0.1 g of each of the long-term 3-month stability samples (Z241101~Z241104) and accelerated 3-month stability samples (Z241101~Z241104) of Gentiana scabra extract, accurately weigh them, and place them in 10 ml volumetric flasks. Add an appropriate amount of 70% ethanol solution, sonicate for 30 min, cool, and dilute to the mark with 70% ethanol solution. Shake well, filter using a 0.45 μm microporous membrane, and collect the filtrate to obtain the Gentiana scabra extract test solution (Gentiana scabra extract with batch number Z241101 is the sample before the process change, and Gentiana scabra extract with batch numbers Z241102~Z241104 is the sample after the process change).

[0280] Take approximately 0.33 g of each of the following samples: long-term stability samples (C241201~C241204) and accelerated stability samples (C241201~C241204) of Gentiana scabra extract tablets after 3 months of processing. Accurately weigh each sample and place them in a 10 ml volumetric flask. Add an appropriate amount of 70% ethanol solution, sonicate for 30 min, cool, and dilute to the mark with 70% ethanol solution. Shake well and filter using a 0.45 μm microporous membrane. Collect the filtrate to obtain the test solution of Gentiana scabra extract tablets (of which, Gentiana scabra extract tablets with batch number C241201 are samples before the process change, and Gentiana scabra extract tablets with batch numbers C241202~C241204 are samples after the process change).

[0281] The test solutions of *Gentiana scabra* extract and *Gentiana scabra* extract tablets were injected and analyzed according to the chromatographic method described in section "1.2.7 Determination of Chromatographic Conditions," and the chromatograms were recorded. The *Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine* (2012 version) was used to evaluate sample similarity. The specific method is as follows: using the S1 chromatogram as the reference chromatogram, a control chromatogram was generated using the average method; the time window width was set to 0.5, and similarity analysis was conducted through multi-point correction and Mark peak matching. The sample test results are shown in […]. Figures 27-30 And Tables 43-26. Figure 27 Fingerprint chromatograms of gentian extract after 3 months of long-term stability; (S1: Gentian extract Z241101 before modification, S2: Gentian extract Z241102 after modification, S3: Gentian extract Z241103 after modification, S4: Gentian extract Z241104 after modification).

[0282] Table 43. Sample Similarity Evaluation of Long-Term Stability of Gentiana macrophylla Extract After 3 Months

[0283]

[0284] Figure 28 Fingerprint chromatograms of 3-month accelerated stability samples of Gentiana scabra extract; (S1: Gentiana scabra extract Z241101 before modification, S2: Gentiana scabra extract Z241102 after modification, S3: Gentiana scabra extract Z241103 after modification, S4: Gentiana scabra extract Z241104 after modification).

[0285] Table 44. Accelerated Stability Evaluation of Gentiana macrophylla Extract after 3 Months: Sample Similarity

[0286]

[0287] Figure 29 Fingerprint chromatograms of Gentiana scabra extract tablets after 3 months of long-term stability; (S1: Gentiana scabra extract tablet C241201 before change, S2: Gentiana scabra extract tablet C241202 after change, S3: Gentiana scabra extract tablet C241203 after change, S4: Gentiana scabra extract tablet C241204 after change).

[0288] Table 45. Calculation of sample similarity for the long-term stability of Gentiana scabra extract tablets over 3 months.

[0289]

[0290] Figure 30Fingerprint chromatograms of accelerated stability samples of Gentiana scabra extract tablets after 3 months; (S1: Gentiana scabra extract tablet C241201 before modification, S2: Gentiana scabra extract tablet C241202 after modification, S3: Gentiana scabra extract tablet C241203 after modification, S4: Gentiana scabra extract tablet C241204 after modification).

[0291] Table 46. Calculation of sample similarity for accelerated stability of Gentiana scabra extract tablets after 3 months.

[0292]

[0293] As shown in Tables 43-44, the long-term (3 months) and accelerated (3 months) stability samples of Gentiana scabra extract before the process change showed a high level of similarity with the corresponding samples after the process change, with similarity values ​​all greater than 0.9. This result indicates that the material basis of Gentiana scabra extract is quite consistent before and after the process change.

[0294] Meanwhile, as shown in Tables 45-46, a comparative analysis of the long-term (3 months) and accelerated (3 months) stability samples of Gentiana scabra extract tablets before the process change with the samples after the change revealed extremely high similarity, with similarity values ​​all exceeding 0.99. This fully demonstrates that the material basis of Gentiana scabra extract tablets remained highly consistent before and after the process change.

[0295] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A fingerprint spectral detection method for Gentiana macrophylla extract and Gentiana macrophylla extract tablets, characterized in that, Includes the following steps: A) The sample raw material is pretreated to obtain the test solution; the pretreatment method includes solvent dissolution and extraction; the solvent is 70% ethanol; Preparation of reference solutions: Ursolic acid, oleanolic acid, β-amyrin, α-amyrin, and trefoil ketone were dissolved in solvents to obtain reference solutions. The reference solution was analyzed by high performance liquid chromatography-evaporative light scattering to obtain the chromatogram of the reference; and the components of the fingerprint chromatograms of Gentiana scabra extract and Gentiana scabra extract tablets were qualitatively identified based on the chromatogram of the reference. B) The test solution was analyzed by high performance liquid chromatography-evaporative light scattering to obtain the HPLC-ELSD fingerprint of Gentiana scabra extract and Gentiana scabra extract tablets; The chromatographic conditions for high performance liquid chromatography-evaporative light scattering were as follows: C18 column; mobile phase A was acetonitrile solution, mobile phase B was 0.1% formic acid aqueous solution, and gradient elution was used. The gradient elution specifically refers to: 0~5min, Phase A: 15%, Phase B: 85%; 5–10 min, Phase A: 15% → 25%, Phase B: 85% → 75%; 10–45 min, Phase A: 25% → 30%, Phase B: 75% → 70%; 45~50 min, Phase A: 30%→50%, Phase B: 70%→50%; 50~65 min, Phase A: 50%→85%, Phase B: 50%→15%; 65~66 min, Phase A: 85%→15%, Phase B: 15%→85%; 66~71 min, Phase A: 15%, Phase B: 85%.

2. The method according to claim 1, characterized in that, The specific concentrations of the reference solutions are: ursolic acid 0.2~0.25 mg / mL, oleanolic acid 0.2~0.25 mg / mL, β-amyrin 0.15~0.2 mg / mL, α-amyrin 0.2~0.25 mg / mL, and tretinoin 0.2~0.25 mg / mL.

3. The method according to claim 1, characterized in that, Detector: Evaporative light scattering detector; drift tube temperature is 109℃, atomizing gas flow rate is 3.0 L / min.

4. The method according to claim 3, characterized in that, The chromatographic column was a Thermo BDS HYPERSIL C18, with dimensions of 250*4.6mm and a diameter of 5μm; the column temperature was 30℃.

5. The method according to claim 1, characterized in that, The flow rate of the mobile phase was 1.0 ml / min; the injection volume was 10 μL.

6. The method according to claim 1, characterized in that, The concentration of the gentian extract and gentian extract tablets is 2 mg / ml to 25 mg / ml.

7. The method according to claim 1, characterized in that, Using oleanolic acid as the reference peak S, the relative retention times of each characteristic peak and peak S were calculated. The relative retention times of each characteristic peak are as follows: 0.476 - Peak 1, 0.727 - Peak 2, 0.753 - Peak 3, 0.788 - Peak 4, 0.805 - Peak 5, 0.821 - Peak 6, 0.870 - Peak 7, 1.000 - Peak 8, 1.184 - Peak 9, 1.279 - Peak 10, 1.371 - Peak 11, 1.418 - Peak 12, 1.432 - Peak 13, 1.801 - Peak 14, 1.942 - Peak 15.

8. The method according to claim 7, characterized in that, Peak 2 is ursolic acid, peak 8 is oleanolic acid, peak 10 is β-amyrin, peak 11 is α-amyrin, and peak 14 is trefoil ketone.

9. The method according to claim 1, characterized in that, Step A) The extraction is ultrasonic extraction; the ultrasonic time is 30 min; the ratio of the mass g of the test sample raw material to the volume mL of the solvent is (0.1~0.25):(10~50).

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