Construction method and application of fingerprint spectrum of Lingze tablet extract

Abstract

This invention provides a method for constructing a fingerprint spectrum of Lingze tablet extract and its application, relating to the field of pharmaceutical testing technology. The method includes analyzing the sample to be tested using high-performance liquid chromatography (HPLC) to obtain a fingerprint spectrum of the Lingze tablet extract. The Lingze tablet extract is composed of Alisma plantago-aquatica, Fritillaria thunbergii, and Curcuma zedoaria residue after volatile oil extraction. The HPLC analytical conditions include using octadecylsilane-bonded silica gel as the packing material. The sample to be tested includes a test solution, which is prepared according to the following method: extracting the Lingze tablet extract using an extraction solvent selected from water, methanol, methanol-water solution, ethanol, or ethanol-water solution. This method can fully reflect the chemical components in the Lingze tablet extract and has the advantages of simple operation, stable results, good reproducibility, high precision, and good robustness.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical testing technology, and in particular to a method for constructing a fingerprint spectrum of Lingze tablet extract and its application. Background Technology

[0002] Currently, the quality standards for traditional Chinese medicine (TCM) are not standardized and need improvement. The existing methods of quality analysis and evaluation cannot accurately reflect the current quality status of TCM. The fingerprinting of prepared TCM products borrows the concept of fingerprint identification from forensic medicine. It involves using appropriate methods to analyze processed medicinal materials or drugs to obtain a common peak spectrum that reflects their characteristics. By analyzing this common peak spectrum, the quality of medicinal materials or drugs can be identified and determined, ensuring stable and controllable quality.

[0003] Lingze tablets are included in the 2020 edition of the Chinese Pharmacopoeia, with a clear prescription and a manufacturing method containing detailed process parameters. The extract of Lingze tablets is made from Alisma plantago-aquatica, Fritillaria thunbergii slices, and Curcuma zedoaria residue after volatile oil extraction, and is an intermediate in the preparation of Lingze tablets.

[0004] The original quality standard for Lingze tablets extract did not include fingerprint spectroscopy. Existing thin-layer chromatography methods can only identify individual herbs and their corresponding components separately, and cannot simultaneously identify multiple components of the extract. Establishing a fingerprint spectroscopy method for identifying Lingze tablets extract will present the main components of Alisma plantago-aquatica, Fritillaria thunbergii, and the turmeric residue after volatile oil extraction all in a single fingerprint spectrum, allowing for a more complete identification of the Lingze tablets extract.

[0005] In view of this, the present invention is hereby introduced. Summary of the Invention

[0006] The purpose of this invention is to provide a method for constructing a fingerprint spectrum of Lingze tablet extract, so as to alleviate the problem of lacking effective means for quality control of Lingze tablet extract.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] In a first aspect, a method for constructing a fingerprint spectrum of Lingze tablet extract is provided, comprising analyzing the sample to be tested using high performance liquid chromatography (HPLC) to obtain a fingerprint spectrum of Lingze tablet extract; the Lingze tablet extract is composed of Alisma plantago-aquatica, Fritillaria thunbergii, and Curcuma zedoaria residue after extraction of volatile oil; the HPLC analytical conditions include using octadecylsilane-bonded silica gel as the column packing; the sample to be tested includes a test solution, which is prepared by the following method: extracting the Lingze tablet extract using an extraction solvent selected from water, methanol, methanol-water solution, ethanol, or ethanol-water solution.

[0009] In an optional implementation, the extraction is performed using ultrasound or reflux extraction.

[0010] In an optional implementation, the extraction time is 20–60 min.

[0011] In an optional embodiment, the sample to be tested further includes a reference solution, which comprises a solution containing known amounts of one or more of the following components: hypoxanthine, uridine, adenine, guanosine, and adenosine.

[0012] In an optional embodiment, the known content is 20–100 μg / mL.

[0013] In an optional embodiment, the solvent of the reference solution is selected from water, methanol, methanol-water solution, ethanol, or ethanol-water solution.

[0014] In an optional embodiment, the mobile phase of the high-performance liquid chromatography includes mobile phase A and mobile phase B; mobile phase A is selected from methanol or acetonitrile; and mobile phase B is selected from sodium acetate aqueous solution, phosphoric acid aqueous solution, or water.

[0015] In an optional embodiment, the elution conditions for the high-performance liquid chromatography are selected from any one of the following groups:

[0016] (a) Mobile phase A is methanol, and mobile phase B is a 0.02 mol / L sodium acetate aqueous solution; the gradient elution program is as follows:

[0017] Mobile phase B: 0–5 min, 100% v / v; 5–30 min, 100% v / v to 45% v / v; 30–52 min, 45% v / v to 10% v / v; 55–60 min, 10% v / v;

[0018] (b) Mobile phase A is methanol, and mobile phase B is 0.1% v / v aqueous phosphoric acid solution; the gradient elution program is as follows:

[0019] Mobile phase B: 0–5 min, 100% v / v; 5–35 min, 100% v / v to 45% v / v; 35–55 min, 45% v / v to 15% v / v; 55–60 min, 15% v / v;

[0020] (c) Mobile phase A is acetonitrile, and mobile phase B is a 0.02 mol / L sodium acetate aqueous solution; the gradient elution program is as follows:

[0021] Mobile phase B: 0~5min, 100% v / v; 5~12min, 100%v / v to 80%v / v; 12~50min, 80%v / v to 32%v / v; 55~60min, 32%v / v~5%v / v; 60~70min, 5%v / v;

[0022] (d) Mobile phase A is acetonitrile, and mobile phase B is a 0.05% v / v aqueous solution of phosphoric acid; the gradient elution program is as follows:

[0023] Mobile phase B: 0–12 min, 100% v / v to 80% v / v; 12–50 min, 80% v / v to 32% v / v; 50–60 min, 32% v / v to 5% v / v; 60–70 min, 5% v / v;

[0024] (e) Mobile phase A is acetonitrile, and mobile phase B is water; the gradient elution program is as follows:

[0025] Mobile phase B: 0–5 min, 100% v / v; 5–18 min, 100% v / v to 80% v / v; 18–55 min, 80% v / v to 35% v / v; 55–60 min, 35% v / v.

[0026] In an optional embodiment, the chromatographic column is packed with octadecylsilane-bonded silica gel, with dimensions of 2.1 × 100 mm and 1.6 μm.

[0027] In an optional embodiment, the chromatographic conditions further include a mobile phase flow rate of 0.08–0.12 mL / min.

[0028] In an optional embodiment, the chromatographic conditions further include a column temperature of 23–27°C.

[0029] In an optional embodiment, the detection wavelength of high performance liquid chromatography is 260-280 nm, preferably 260 nm.

[0030] In an optional embodiment, the construction method further includes providing fingerprint spectra from different batches to synthesize a control fingerprint spectra; the control fingerprint spectra selects chromatographic peaks present in fingerprint spectra from different batches of Lingze tablet extract as common peaks.

[0031] In an optional embodiment, the construction method further includes comparing the fingerprint spectrum of the sample to be evaluated with the control fingerprint spectrum and calculating the similarity between the fingerprint spectrum of the sample to be evaluated and the control fingerprint spectrum.

[0032] Secondly, the method for constructing the fingerprint spectrum of Lingze tablet extract from the first aspect is also provided for its application in the quality control of Lingze tablet extract.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] In this invention, an ultra-high performance liquid chromatography (UHPLC) method is used to establish a fingerprint spectrum using an octadecylsilane-bonded silica gel column, which fully reflects the chemical components in the Lingze tablet extract. This method has the advantages of simple operation, stable results, good reproducibility, high precision, and good durability.

[0035] Under liquid chromatography conditions, the material group of Lingze tablet extract is controlled by fingerprint spectrum. In some embodiments, the fingerprint spectrum is located by reference standards of hypoxanthine, uridine, adenine, guanosine and adenosine. This enables the qualitative detection of chemical components in Lingze tablet extract to be identified simultaneously under the same chromatographic conditions and using the same wavelength.

[0036] The method for constructing the fingerprint spectrum of Lingze tablet extract provided by this invention is applied to the quality control of Lingze tablet extract. By evaluating similarity, the quality consistency between batches can be objectively judged, thereby comprehensively monitoring product quality, avoiding the one-sidedness of a few indicators, and has the characteristics of being convenient, fast, stable, precise and reproducible. Attached Figure Description

[0037] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0038] Figure 1 This is a schematic diagram of the fingerprint spectrum of the Lingze tablet extract from Example 1.

[0039] Figure 2 This is a schematic diagram of the overlay of the chromatographic fingerprint of the Lingze tablets extract in Example 1;

[0040] Figure 3 This is a schematic diagram of the superimposed fingerprint spectra of Lingze tablets extract extracted with different solvents in Example 2;

[0041] Figure 4 This is a schematic diagram of the overlay of fingerprint spectra of Lingze tablets extracted by reflux extraction with different solvents in Example 2;

[0042] Figure 5 The image shows the absorption spectrum, including the fingerprint pattern, of the Lingze tablet extract in Example 4.

[0043] Figure 6 This is a schematic diagram of the superimposed fingerprint spectra of different wavelengths of the Lingze tablet extract in Example 4;

[0044] Figure 7 This is a schematic diagram of the superimposed fingerprint spectra of different chromatographic columns for the Lingze tablet extract in Example 5;

[0045] Figure 8 This is a schematic diagram of the superimposed fingerprint spectra of different mobile phases of the Lingze tablet extract in Example 6;

[0046] Figure 9 To verify the superimposed diagram of the extract of Lingze tablets in Example 1 and the control herbs Alisma plantago-aquatica, Fritillaria thunbergii, and Curcuma zedoaria;

[0047] Figure 10 To verify the superposition diagram of Lingze tablet extract, 30% methanol aqueous solution, Alisma plantago-aquatica, Fritillaria thunbergii, Curcuma zedoaria, and reference standard in Example 2. Detailed Implementation

[0048] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0049] Firstly, a method for constructing the fingerprint spectrum of Lingze tablet extract is provided.

[0050] In this invention, "Lingze tablet extract" refers to the extract obtained by extracting, precipitating with alcohol, concentrating and drying Alisma plantago-aquatica, Fritillaria thunbergii and Curcuma zedoaria residue after extracting volatile oil during the preparation process of Lingze tablets.

[0051] Lingze tablets are included in Part I of the 2020 edition of the Chinese Pharmacopoeia, with a clear prescription and a manufacturing method containing detailed process parameters. The specifics are as follows:

[0052] Prescription: 250g of *Ulmus pumila* powder, 1000g of *Curcuma zedoaria*, 667g of *Fritillaria thunbergii*, and 500g of *Alisma plantago-aquatica*.

[0053] Preparation: For the above four ingredients, steam distill the Curcuma zedoaria for 12 hours to extract the volatile oil. Set aside the residue and the distilled aqueous solution. Add the volatile oil to a saturated aqueous solution of betacyclodextrin (the ratio of volatile oil, betacyclodextrin and water is 1:6:24), stir for 3 hours, let stand overnight, discard the supernatant, filter the lower precipitate of inclusion complex until no water droplets are extracted, collect the inclusion complex and set aside. Add water to Fritillaria thunbergii and Alisma plantago-aquatica, combine with the decoction and residue after extracting the volatile oil, and decoct three times, 2 hours each time. Combine the decoctions. Filter, concentrate the filtrate under reduced pressure to a clear extract with a relative density of 1.02–1.06 (50°C), adjust the pH to 7.5, and add ethanol to make the extract contain... With an alcohol content of 70%, let stand overnight, then concentrate the supernatant under reduced pressure to obtain a clear extract with a relative density of 1.10–1.15 (60°C), and spray dry. dry Add *Lingzei* powder and the above-mentioned inclusion complex, then add an appropriate amount of dicalcium phosphate, mix well, use 2% hydroxypropyl methylcellulose solution as a binder, granulate, dry, add 1.7g of magnesium stearate, compress into 1000 tablets, and coat with a film to obtain the final product. The underlined part is about the preparation method of "Lingzei tablet extract".

[0054] To better control the quality of extracts, this invention provides a method for constructing a fingerprint spectrum of Lingze tablet extract. The method includes analyzing the sample to be tested using high-performance liquid chromatography to obtain a fingerprint spectrum of Lingze tablet extract.

[0055] The test sample includes a test solution, which is prepared by extracting the Lingze tablets extract using an extraction solvent selected from water, methanol, methanol-water solution, ethanol, or ethanol-water solution. Exemplary extraction solvents include, but are not limited to, any one of the following: anhydrous methanol, 70% v / v methanol-water solution, 50% v / v methanol-water solution, 30% v / v methanol-water solution, 10% v / v methanol-water solution, water, anhydrous ethanol, 70% v / v ethanol-water solution, 50% v / v ethanol-water solution, 30% v / v ethanol-water solution, or 10% v / v ethanol-water solution.

[0056] In an optional embodiment, the concentration of methanol in the methanol aqueous solution does not exceed 30% v / v. The concentration of methanol can be, for example, but not limited to, 5, 10, 15, 20, 25 or 30% v / v, or a concentration range between any two of the aforementioned points.

[0057] In an optional embodiment, the concentration of ethanol in the aqueous ethanol solution does not exceed 10% v / v. The concentration of ethanol can be, for example, but not limited to, 1, 5, or 10% v / v, or a concentration range between any two of the aforementioned points.

[0058] In an optional embodiment, the extraction solvent is selected from 30% v / v methanol aqueous solution, 10% v / v methanol aqueous solution, water, or 10% v / v ethanol aqueous solution. The fingerprint chromatograms of the above extraction solvents have slightly more peaks, and the peak patterns are basically consistent. Among them, the 30% methanol solvent is relatively stable, thus resulting in more stable detection results.

[0059] In optional embodiments, the extraction is performed using ultrasound or reflux. The peak patterns of the fingerprint spectra obtained by ultrasound and reflux are basically the same. Ultrasound is relatively simple, so it is preferred to use ultrasound to prepare the test solution, which can make the detection process simpler.

[0060] In an optional embodiment, the extraction is performed using ultrasonic processing with a power of 500W and a frequency of 45KHz.

[0061] In optional embodiments, the extraction time is 20–60 min, for example, but not limited to 20, 30, 40, 45, 50, or 60 min, or any time range between the aforementioned two points. The peak characteristics of the fingerprint spectrum obtained by ultrasound for 20–60 min are basically consistent. Among them, the ultrasound method for 20 min has a relatively shorter extraction time, so ultrasound for 20 min is preferred to make the detection process more time-saving.

[0062] In an optional embodiment, the feed ratio of Lingze tablet extract to extraction solvent is 5 to 20 mg / mL, for example, but not limited to 5, 10, 15 or 20 mg / mL.

[0063] In an optional embodiment, 0.5g of Lingze tablet extract is extracted using 50mL of extraction solvent.

[0064] In an optional embodiment, the sample to be tested further includes a reference solution, which comprises a solution containing known amounts of one or more of the following components: hypoxanthine, uridine, adenine, guanosine, and adenosine.

[0065] In an optional embodiment, the reference solution includes one or a combination of the following solutions: a solution containing hypoxanthine, a solution containing uridine, a solution containing adenine, a solution containing guanosine, and a solution containing adenosine.

[0066] In an optional embodiment, the reference solution comprises a solution containing at least two of the following: hypoxanthine, uridine, adenine, guanosine, and adenosine.

[0067] In an optional embodiment, the known content of hypoxanthine is 20 to 100 μg / mL, for example, but not limited to 20, 30, 40, 50, 60, 70, 80, 90 or 100 μg / mL.

[0068] In an optional embodiment, the known content of uridine is 20 to 100 μg / mL, for example, but not limited to 20, 30, 40, 50, 60, 70, 80, 90 or 100 μg / mL.

[0069] In an optional embodiment, the known content of adenine is 20 to 100 μg / mL, for example, but not limited to 20, 30, 40, 50, 60, 70, 80, 90 or 100 μg / mL.

[0070] In an optional embodiment, the known content of guanosine is 20 to 100 μg / mL, for example, but not limited to 20, 30, 40, 50, 60, 70, 80, 90 or 100 μg / mL.

[0071] In an optional embodiment, the known content of adenosine is 20 to 100 μg / mL, for example, but not limited to 20, 30, 40, 50, 60, 70, 80, 90 or 100 μg / mL.

[0072] In an optional embodiment, the solvent of the reference solution is selected from water, methanol, aqueous methanol solution, ethanol, or aqueous ethanol solution. Exemplary reference solutions may include, but are not limited to, any of the following: anhydrous methanol, 70% v / v aqueous methanol solution, 50% v / v aqueous methanol solution, 30% v / v aqueous methanol solution, 10% v / v aqueous methanol solution, water, anhydrous ethanol, 70% v / v aqueous ethanol solution, 50% v / v aqueous ethanol solution, 30% v / v aqueous ethanol solution, or 10% v / v aqueous ethanol solution.

[0073] In an optional embodiment, the concentration of methanol in the solvent of the reference solution does not exceed 30% v / v. The concentration of methanol can be, for example, but not limited to, 5, 10, 15, 20, 25 or 30% v / v, or a concentration range between any two of the aforementioned points.

[0074] In an optional embodiment, the concentration of ethanol in the solvent of the reference solution does not exceed 10% v / v. The concentration of ethanol can be, for example, but not limited to, 1, 5, or 10% v / v, or a concentration range between any two of the aforementioned points.

[0075] In an optional embodiment, the solvent of the reference solution is selected from 30% v / v methanol aqueous solution, 10% v / v methanol aqueous solution, water, or 10% v / v ethanol aqueous solution.

[0076] In an optional embodiment, the solvent of the reference solution and the extraction reagent of the test solution may be the same or different.

[0077] In the method for constructing the fingerprint spectrum of Lingze tablets provided by the present invention, the chromatographic column of the high performance liquid chromatography uses octadecylsilane-bonded silica gel as the packing material.

[0078] In an optional embodiment, the chromatographic column is packed with octadecylsilane-bonded silica gel, with dimensions of 2.1 × 100 mm and 1.6 μm.

[0079] In an optional embodiment, the chromatographic column is a Waters column. T3, with dimensions of 2.1×100mm and 1.6μm.

[0080] In an optional embodiment, the mobile phase of the high-performance liquid chromatography includes mobile phase A and mobile phase B; mobile phase A is selected from methanol or acetonitrile; and mobile phase B is selected from sodium acetate aqueous solution, phosphoric acid aqueous solution, or water.

[0081] In an optional embodiment, the phosphoric acid concentration in the aqueous phosphoric acid solution is 0.05–0.1% v / v.

[0082] In an optional embodiment, the concentration of sodium acetate in the sodium acetate aqueous solution is 0.01–0.02 mol / L.

[0083] In an optional embodiment, the elution conditions for the high-performance liquid chromatography are selected from any one of the following groups, wherein in the gradient elution program, the total volume percentage of mobile phase A and mobile phase B, expressed as a volume percentage of mobile phase B, is 100% v / v:

[0084] (a) Mobile phase A is methanol, and mobile phase B is a 0.02 mol / L sodium acetate aqueous solution; gradient elution program:

[0085] Mobile phase B: 0–5 min, 100% v / v; 5–30 min, 100% v / v to 45% v / v; 30–52 min, 45% v / v to 10% v / v; 55–60 min, 10% v / v;

[0086] (b) Mobile phase A is methanol, and mobile phase B is 0.1% v / v aqueous phosphoric acid solution; the gradient elution program is as follows:

[0087] Mobile phase B: 0–5 min, 100% v / v; 5–35 min, 100% v / v to 45% v / v; 35–55 min, 45% v / v to 15% v / v; 55–60 min, 15% v / v;

[0088] (c) Mobile phase A is acetonitrile, and mobile phase B is a 0.02 mol / L sodium acetate aqueous solution; the gradient elution program is as follows:

[0089] Mobile phase B: 0~5min, 100% v / v; 5~12min, 100%v / v to 80%v / v; 12~50min, 80%v / v to 32%v / v; 55~60min, 32%v / v~5%v / v; 60~70min, 5%v / v;

[0090] (d) Mobile phase A is acetonitrile, and mobile phase B is a 0.05% v / v aqueous solution of phosphoric acid; the gradient elution program is as follows:

[0091] Mobile phase B: 0–12 min, 100% v / v to 80% v / v; 12–50 min, 80% v / v to 32% v / v; 50–60 min, 32% v / v to 5% v / v; 60–70 min, 5% v / v;

[0092] (e) Mobile phase A is acetonitrile, and mobile phase B is water; the gradient elution program is as follows:

[0093] Mobile phase B: 0–5 min, 100% v / v; 5–18 min, 100% v / v to 80% v / v; 18–55 min, 80% v / v to 35% v / v; 55–60 min, 35% v / v.

[0094] In an optional embodiment, the elution conditions for the high-performance liquid chromatography (HPLC) are selected as follows: mobile phase A is acetonitrile, and mobile phase B is water; the gradient elution program is as follows: mobile phase B: 0–5 min, 100% v / v; 5–18 min, 100% v / v to 80% v / v; 18–55 min, 80% v / v to 35% v / v; 55–60 min, 35% v / v. Using mobile phase A (acetonitrile) and mobile phase B (water) as the mobile phase gradient elution, the fingerprint chromatogram of Lingze tablets extract shows a large number of chromatographic peaks, with good separation of each peak. In the chromatograms of different batches of Lingze tablets extract samples, hypoxanthine, uridine, adenine, guanosine, adenosine, and other common peaks are well separated from adjacent peaks, showing a significant improvement compared to other elution gradients, greatly improving the accuracy of detection, and yielding the most accurate detection results.

[0095] In an optional embodiment, the chromatographic conditions further include a mobile phase flow rate of 0.08 to 0.12 mL / min, for example, but not limited to 0.08, 0.10, or 0.12 mL / min, or a flow rate range between any two of the aforementioned points.

[0096] In an optional embodiment, the chromatographic conditions further include a column temperature of 23 to 27°C, for example, but not limited to 23, 24, 25, 26 or 27°C, or a temperature range between any two of the aforementioned points.

[0097] In an optional embodiment, the detection wavelength of high performance liquid chromatography is 260-280 nm, more preferably 260 nm.

[0098] In an optional implementation, the injection volume for high-performance liquid chromatography is 1 μL.

[0099] The optimized high-performance liquid chromatography (HPLC) conditions described above enable better retention of chemical components. Column temperature and flow rate ensure the stability and accuracy of the detection results. The most stable and accurate results are obtained at a column temperature of 25℃ and a flow rate of 0.1 ml / min.

[0100] In an optional embodiment, the construction method further includes providing fingerprint spectra from different batches to synthesize a control fingerprint spectra, wherein the control fingerprint spectra select chromatographic peaks present in fingerprint spectra from different batches of Lingze tablet extract as common peaks.

[0101] In an optional embodiment, the fingerprint spectrum includes 15 characteristic peaks, including hypoxanthine characteristic peak, guanosine characteristic peak, adenine characteristic peak, guanosine characteristic peak and adenosine characteristic peak.

[0102] In an optional embodiment, the construction method further includes comparing the fingerprint spectrum of the sample to be evaluated with the control fingerprint spectrum and calculating the similarity between the fingerprint spectrum of the sample to be evaluated and the control fingerprint spectrum.

[0103] In some specific embodiments, the method for constructing the fingerprint spectrum of Lingze tablets extract includes the following steps:

[0104] S1, Preparation of reference solutions: Take hypoxanthine, uridine, adenine, guanosine and adenosine reference standards, add 10-30% v / v methanol to prepare mixed solutions or individual reference solutions with concentrations of 20-100 μg / mL respectively.

[0105] S2, Preparation of the test solution: Provide 0.5g of Lingze tablet extract from different batches, add 50ml of the extraction solvent, extract, cool, shake well, filter, and take the filtrate to obtain the test solution;

[0106] S3, Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase (2.1 × 100 mm, 1.6 μm); acetonitrile was used as mobile phase A, and water as mobile phase B, eluting according to the gradient elution program described above; flow rate was 0.08–0.12 ml / min; column temperature was 23–27 °C; detection wavelength was 260 nm; the test sample and reference solution were injected separately into the ultra-high performance liquid chromatograph, and eluted according to the following program:

[0107]

[0108]

[0109] S4, Establishing the reference fingerprint spectrum: The integrated signals of fingerprint spectra of multiple batches of Lingze tablet extract are imported into the similarity evaluation system of chromatographic fingerprint spectrum of traditional Chinese medicine. Exemplary similarity evaluation systems of chromatographic fingerprint spectrum of traditional Chinese medicine include, but are not limited to, the 2012 version of the "Similarity Evaluation System of Chromatographic Fingerprint Spectra of Traditional Chinese Medicine" software of the Chinese Pharmacopoeia Commission; chromatographic peaks present in the fingerprint spectra of different batches of Lingze tablet extract are selected as common peaks; chromatograms are superimposed using the Mark peak matching method, and a reference fingerprint spectrum of Lingze tablet extract is generated; the similarity between each batch of Lingze tablet extract and the reference fingerprint spectrum of Lingze tablet extract is calculated.

[0110] S5, Calculation of similarity between the test sample and the reference fingerprint spectrum: The fingerprint spectrum integral signal of the test sample is imported into the Chinese medicine chromatographic fingerprint spectrum similarity evaluation system, such as the 2012 version of the "Chinese Medicine Chromatographic Fingerprint Spectrum Similarity Evaluation System" software of the Chinese Pharmacopoeia Commission mentioned above; the common peaks of the test sample and the reference fingerprint spectrum are selected; the chromatograms are superimposed using the Mark peak matching method to calculate the similarity between the test sample and the reference fingerprint spectrum of Lingze tablet extract.

[0111] Secondly, the method for constructing the fingerprint spectrum of Lingze tablet extract from the first aspect is also provided for its application in the quality control of Lingze tablet extract.

[0112] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0113] Example 1

[0114] Instruments and reagents

[0115] Thermo Vanquish-F ultra-high performance liquid chromatograph (Thermo GmbH, Germany).

[0116] Reagents: Acetonitrile and methanol were of chromatographic grade, water was ultrapure water, and all other reagents were of analytical grade.

[0117] Hypoxanthine, uridine, adenine, guanosine, and adenosine were all purchased from the National Institutes for Food and Drug Control.

[0118] Test drug: A total of 25 batches of Lingze tablets extract were tested. Sample information is shown in Table 1.

[0119] Table 1 Sample Information Table

[0120]

[0121] The method for establishing the fingerprint spectrum of Lingze tablet extract includes the following steps:

[0122] S1, Preparation of reference solution: Take appropriate amounts of hypoxanthine, uridine, adenine, guanosine and adenosine reference standards, add 30% methanol to prepare a solution containing 20 μg of each per ml.

[0123] S2, Preparation of the test solution: Weigh approximately 0.5g of Lingze tablet extract accurately, add 50ml of 30% methanol, sonicate for 20min, cool, shake well, filter, and collect the filtrate to obtain the test solution.

[0124] S3, Chromatographic conditions: Octadecylsilane-bonded silica gel (2.1×100mm, 1.6μm) was used as the stationary phase; acetonitrile was used as mobile phase A and water was used as mobile phase B, with gradient elution according to Table 2; flow rate was 0.1 ml / min; column temperature was 25℃; detection wavelength was 260 nm; 1 μl of the test solution and 1 μl of the reference solution were accurately pipetted into the ultra-high performance liquid chromatograph.

[0125] Table 2 Gradient elution program

[0126] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～5 0 100 5～18 0→20 100→80 18～55 20→65 80→35 55～60 65 35

[0127] S4, Establishing the fingerprint chromatogram: The integrated signals of the fingerprint chromatograms of 25 batches of Lingze tablet extracts were imported into the 2012 version of the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" software of the Chinese Pharmacopoeia Commission; chromatographic peaks present in all fingerprint chromatograms of Lingze tablet extracts were selected as common peaks; the chromatograms were superimposed using the Mark peak matching method, and the software superimposed graph is shown below. Figure 2 A fingerprint spectrum of Lingze tablet extract was generated, and a comparison fingerprint was obtained. Figure 1 (Peak 1: hypoxanthine, peak 2: uridine, peak 3: adenine, peak 4: guanosine, peak 5: adenosine); The similarity between the fingerprint chromatograms of each batch of Lingze tablet extract and the control fingerprint chromatogram of Lingze tablet extract is calculated. The results are shown in Table 3. The similarity is greater than 0.99.

[0128] Table 3. Similarity Evaluation Results of Lingze Tablet Extract

[0129]

[0130] Example 2

[0131] Selection of extraction solvent and extraction method:

[0132] The difference from Example 1 is that the test drug was one batch of 25 batches of Lingze tablets extract. In step S2, anhydrous methanol, 70% methanol, 50% methanol, 30% methanol, 10% methanol, water, anhydrous ethanol, 70% ethanol, 50% ethanol, 30% ethanol, and 10% ethanol were used instead of the 30% methanol solvent in Example 1 as new solvents. Ultrasonication or reflux for 30 minutes was used instead of ultrasonication for 20 minutes in Example 1 as new extraction methods. Chromatograms were obtained and compared after extraction. Step S4 involves importing the integrated signals of the chromatograms of the samples extracted with the above solvents into the 2012 version of the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software of the Chinese Pharmacopoeia Commission for superposition, to obtain... Figure 3 , Figure 4 .like Figure 3 As shown, from bottom to top (S1 to S11), the solutions are respectively anhydrous methanol, 70% methanol, 50% methanol, 30% methanol, 10% methanol, water, anhydrous ethanol, 70% ethanol, 50% ethanol, 30% ethanol, and 10% ethanol, which are solutions of Lingze tablets extracted by ultrasound. Figure 4As shown, from bottom to top (S1 to S11), the solutions of Lingze tablet extract refluxed with anhydrous methanol, 70% methanol, 50% methanol, 30% methanol, 10% methanol, water, anhydrous ethanol, 70% ethanol, 50% ethanol, 30% ethanol, and 10% ethanol are respectively. The results show that the chromatograms of 30% methanol, 10% methanol, water, and 10% ethanol after ultrasonic or reflux for 30 min yielded more chromatographic peaks, which were basically consistent. Comparing the results after extraction with these solvents, in step S5, the similarity of the test samples is calculated: the fingerprint spectrum integral signal of the test sample is imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" 2012 version software of the Chinese Pharmacopoeia Commission; the common peaks of the test sample and the reference fingerprint spectrum are selected; the chromatograms are superimposed using the Mark peak matching method, and the similarity between the test sample and the reference fingerprint spectrum of Lingze tablet extract is calculated. The similarity results are shown in Table 4.

[0133] Table 4. Similarity results for different solvents and extraction methods

[0134] Extraction solvent Extraction method Similarity Extraction solvent Extraction method Similarity 30% methanol ultrasound 0.997 30% methanol reflux 0.997 10% methanol ultrasound 0.996 10% methanol reflux 0.997 water ultrasound 0.995 water reflux 0.996 10% ethanol ultrasound 0.997 10% ethanol reflux 0.996

[0135] Results: The chromatograms of Lingze tablet extracts obtained by ultrasonication or reflux for 30 min with 30% methanol, 10% methanol, water, and 10% ethanol all showed a similarity greater than 0.9 with the control fingerprint chromatograms, indicating extremely high similarity. The fingerprint chromatograms of samples extracted with other solvents had fewer peaks than those extracted with the aforementioned four solvents. Therefore, all four solvents can be used as extraction solvents. Among them, 30% methanol is the most stable extraction solvent, hence its optimal selection. Similarly, the similarity between ultrasonication and reflux extraction with the same solvent was also greater than 0.9, indicating extremely high similarity. Therefore, both ultrasonication and reflux can be used as extraction conditions. Ultrasonication is the simplest extraction method, hence its superior performance.

[0136] Example 3

[0137] Selection of extraction time:

[0138] The difference from Example 1 is that the test drug was one batch of 25 batches of Lingze tablet extract. In step S2, 30 min, 45 min, and 60 min were used instead of 20 min in Example 1 as extraction times. Chromatograms were obtained and the results after extraction were compared. Step S5 replaces step S4. Similarity calculation of the test samples: The fingerprint spectrum integral signal of the test sample was imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" 2012 version software of the Chinese Pharmacopoeia Commission; the common peaks of the test sample and the reference fingerprint spectrum were selected; the chromatograms were superimposed using the Mark peak matching method, and the similarity between the test sample and the reference fingerprint spectrum of Lingze tablet extract was calculated. The similarity results are shown in Table 5.

[0139] Table 5. Similarity results at different extraction times

[0140] Extraction time (min) 20 30 45 60 Similarity 0.997 0.997 0.997 0.997

[0141] Results: The chromatograms of Lingze tablet extracts obtained by ultrasonication with 30% methanol for 20, 30, 45, and 60 min all showed a similarity greater than 0.9 with the control fingerprint chromatogram, and the similarity was extremely high. Therefore, 20–60 min can be used as the extraction time, with 20 min being the extraction time, which is shorter and thus the extraction time of 20 min is the best.

[0142] Example 4

[0143] Wavelength selection:

[0144] The difference from Example 1 is that the test drug was one batch of Lingze tablet extract from 25 batches. In step S3, 220nm, 260nm, 280nm, 370nm, and 190-470nm were used instead of 260nm in Example 1 as the detection wavelengths. Chromatograms and isoabsorption spectra were obtained respectively. Figure 5 Step S4 involves importing the integrated chromatogram signals of the above detection wavelengths into the 2012 version of the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software of the Chinese Pharmacopoeia Commission for overlaying, to obtain... Figure 6 ,like Figure 6 As shown, from bottom to top, they are 220nm, 260nm, 280nm, and 370nm, respectively. Figure 5 The isoabsorption spectra shown indicate that the chemical components in the Lingze tablet extract exhibit strong absorption in the 190–280 nm range. Under the same chromatographic conditions, wavelengths with relatively more chromatographic peaks in the 260 nm and 280 nm spectra of the test sample are preferred. Among them, the proportion of each chromatographic peak at 260 nm is relatively appropriate, avoiding maximum peaks, resulting in good separation effect, and it is a non-terminal absorption band. Therefore, the detection wavelength of 260 nm was selected.

[0145] Example 5

[0146] Selection of chromatographic column:

[0147] The difference from Example 1 is that the test drug was one batch out of 25 batches of Lingze tablets extract. In step S3, the chromatographic columns listed in Table 10 below were used for detection to obtain... Figure 7 .like Figure 7 As shown, the chromatographic columns are numbered 1, 2, 3 and 4 from top to bottom.

[0148] Table 6 Chromatographic Column Information

[0149]

[0150] Results: Under the same chromatographic conditions, column 1 exhibited better retention, resolution, and symmetry of the chemical components in the sample; therefore, Waters was selected. The T3 2.1×100mm, 1.6μm column is the best column for fingerprint chromatographic studies.

[0151] Example 6

[0152] Selection of mobile phase:

[0153] The difference from Example 1 is that the test drug was one batch of Lingze tablets extract from 25 batches. In step S3, the gradient elution procedure of mobile phase A-mobile phase B is shown in (1) to (5) for detection, and the results are obtained. Figure 8 .like Figure 8 As shown, the column numbers from top to bottom are (1) to (5).

[0154] (1) Mobile phase A: methanol - Mobile phase B: sodium acetate aqueous solution with a volume mol concentration of 0.02 mol / L; 0-5 min, 100% volume concentration of mobile phase B; 5-30 min, 100-45% volume concentration of mobile phase B; 30-52 min, 45-10% volume concentration of mobile phase B; 52-60 min, 10% volume concentration of mobile phase B.

[0155] (2) Mobile phase A: methanol - Mobile phase B: 0.1% (v / v) phosphoric acid aqueous solution; 0–5 min: 100% (v / v) mobile phase B; 5–35 min: 100% (v / v) mobile phase B; 35–55 min: 45% (v / v) mobile phase B; 55–60 min: 15% (v / v) mobile phase B.

[0156] (3) Mobile phase A acetonitrile - Mobile phase B 0.02 mol / L sodium acetate aqueous solution; 0-5 min, 100% mobile phase B, 5-12 min, 100-80% mobile phase B, 12-50 min, 80-32% mobile phase B, 50-60 min, 32-5% mobile phase B, 60-70 min, 5% mobile phase B;

[0157] (4) Mobile phase A acetonitrile - Mobile phase B 0.05% phosphoric acid aqueous solution (volume percentage); 0-12 min, 100-80% mobile phase B (volume percentage); 12-50 min, 80-32% mobile phase B (volume percentage); 50-60 min, 32-5% mobile phase B (volume percentage); 60-70 min, 5% mobile phase B (volume percentage).

[0158] (5) Mobile phase A acetonitrile-mobile phase B aqueous solution; 0-5 min, 100% volume percentage of mobile phase B, 5-18 min, 100-80% volume percentage of mobile phase B, 18-55 min, 80-35% volume percentage of mobile phase B, 55-60 min, 35% volume percentage of mobile phase B.

[0159] Results: Under different mobile phase conditions, (5) had more peaks in the chromatogram and the separation of each chromatographic peak was good. Therefore, as a gradient elution condition for fingerprint chromatogram detection, it can greatly improve the accuracy of detection.

[0160] Verification Example 1:

[0161] To verify the uniqueness of the detection method in Example 1, the following experiment was conducted:

[0162] The difference from Example 1 is that the test drugs are Lingze tablet extract, Curcuma zedoaria reference material, Alisma plantago-aquatica reference material, and Fritillaria thunbergii reference material. In step S2, approximately 0.5g of Lingze tablet extract is used, along with approximately 1.0g each of Curcuma zedoaria, Alisma plantago-aquatica, and Fritillaria thunbergii reference materials. In step S4, the integrated chromatographic signals of the Lingze tablet extract, Curcuma zedoaria, Alisma plantago-aquatica, and Fritillaria thunbergii reference materials are respectively imported into the 2012 version of the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software of the Chinese Pharmacopoeia Commission for superposition, to obtain... Figure 9 ,like Figure 9 As shown, from bottom to top, are Lingze tablets extract, Zexie control material, Zhebeimu control material, and Ezhu control material.

[0163] As shown in Verification Example 1, the Lingze tablet extract has 18 chromatographic peaks. Among them, Curcuma zedoaria reference material has corresponding peaks 11, 12, 13, 14, 15, 17, and 18; Alisma plantago-aquatica reference material has corresponding peaks 1, 3, 4, 5, and 8; and Fritillaria thunbergii reference material has corresponding peaks 3, 5, 6, 8, and 9. The larger chromatographic peaks in the chromatograms of these three herbs are basically also present in the Lingze tablet extract. Peaks 1 and 4 are unique to Alisma plantago-aquatica. Peaks 6 and 9 are unique to Fritillaria thunbergii, while peaks 11, 12, 13, 14, 15, 17, and 18 are unique to Curcuma zedoaria. Among them, peaks 7, 11, and 16 of Lingze tablets dry extract are relatively small, and these three peaks can be omitted when selecting common peaks in fingerprint spectroscopy. The remaining 15 peaks already include the unique peaks of the three medicinal materials. Only the Lingze tablets extract has 15 peaks, while the other individual medicinal materials do not have all 15 peaks, indicating the uniqueness of this fingerprint spectroscopy method.

[0164] Verification Example 2:

[0165] To verify the specificity of the detection method in Example 1, the following experiment was conducted:

[0166] The difference from Example 1 is that in step S2, the Lingze tablet extract is tested with 30% methanol, Lingze tablet extract solution, Curcuma zedoaria reference material solution, Alisma plantago-aquatica reference material solution, and Fritillaria thunbergii reference material solution. In step S4, the integrated chromatogram signals of 30% methanol, Lingze tablet extract, Curcuma zedoaria reference material, Alisma plantago-aquatica reference material, Fritillaria thunbergii reference material, and reference standards are respectively imported into the 2012 version of the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software of the Chinese Pharmacopoeia Commission to obtain... Figure 10 ,like Figure 10 As shown, from bottom to top, they are blank solution, Lingze tablet extract solution, Zexie reference herb solution, Zhebeimu reference herb solution, Ezhu reference herb solution, and reference solution. The five peaks in the reference solution are, in order, hypoxanthine, uridine, adenine, guanosine, and adenosine.

[0167] As can be seen from Validation Example 2, the chromatographic peak positions of hypoxanthine, uridine, adenine, guanosine, and adenosine, as well as the chromatographic peak positions of the other test samples, are all present. The negative test samples do not interfere with the detection. The reference solutions of Curcuma zedoaria, Alisma plantago-aquatica, and Fritillaria thunbergii all have some corresponding chromatographic peaks with the extract of Lingze tablets, but there are no completely shared peaks, indicating that the method has good specificity.

[0168] Verification Example 3:

[0169] To verify the precision of the detection method in Example 1, the following experiment was conducted:

[0170] The difference from Example 1 is that the test drug is one batch of Lingze tablet extract from 25 batches, and it is injected 6 times consecutively. The chromatogram is recorded, and step S4 is replaced by S5 as follows: Calculation of similarity of test samples: The fingerprint spectrum integral signal of the test sample is imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" 2012 version software of the Chinese Pharmacopoeia Commission; the common peaks of the test sample and the control fingerprint spectrum (constructed in Example 1) are selected; the chromatograms are superimposed using the Mark peak matching method, and the similarity between the test sample and the control fingerprint spectrum of Lingze tablet extract is calculated. The similarity results are shown in Table 7.

[0171] Table 7 Precision Similarity Results

[0172] serial number 1 2 3 4 5 6 RSD% Similarity 0.996 0.996 0.995 0.995 0.995 0.995 0.052％

[0173] Results: The RSD value of the fingerprint spectrum results from 6 consecutive injections was 0.052%, indicating good method precision.

[0174] Verification Example 4

[0175] To verify the repeatability of the detection method in Example 1, the following experiment was conducted.

[0176] The difference from Example 1 is that the test drug is one batch of Lingze tablet extract from 25 batches, and step S2 is to weigh 6 parallel samples and record the chromatograms. Step S4 is replaced by S5 as follows: Calculation of similarity of test samples: The fingerprint spectrum integral signal of the test sample is imported into the 2012 version of the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software of the Chinese Pharmacopoeia Commission; the common peaks of the test sample and the control fingerprint spectrum (constructed in Example 1) are selected; the chromatograms are superimposed using the Mark peak matching method, and the similarity between the test sample and the control fingerprint spectrum of Lingze tablet extract is calculated. The similarity results are shown in Table 8.

[0177] Table 8. Repeatability Similarity Results

[0178] serial number 1 2 3 4 5 6 RSD% Similarity 0.995 0.995 0.995 0.996 0.996 0.996 0.055％

[0179] Results: The RSD value of the fingerprint spectrum results of the 6 parallel samples was 0.055%, indicating good repeatability of the method.

[0180] Verification Example 5:

[0181] To verify the stability of the detection method in Example 1, the following experiment was conducted.

[0182] The difference from Example 1 is that the test drug is one batch of Lingze tablet extract from 25 batches. The test solution was injected at 0, 3, 6, 9, 12, 18, 24 and 36 h, and the chromatograms were recorded. Step S4 was replaced by S5 as follows: Calculation of similarity of test samples: The fingerprint spectrum integral signal of the test sample was imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" 2012 version software of the Chinese Pharmacopoeia Commission; the common peaks of the test sample and the control fingerprint spectrum (constructed in Example 1) were selected; the chromatograms were superimposed using the Mark peak matching method, and the similarity between the test sample and the control fingerprint spectrum of Lingze tablet extract was calculated. The similarity results are shown in Table 9.

[0183] Table 9. Stability Similarity Results

[0184] Time (h) 0 3 6 9 12 18 24 36 RSD% Similarity 0.995 0.995 0.995 0.996 0.996 0.996 0.996 0.995 0.054％

[0185] Results: The RSD value of the fingerprint spectrum results of the test sample after 36 hours was 0.054%, indicating good method stability.

[0186] Verification Example 6:

[0187] To verify the robustness of the detection method in Example 1, the following experiment was conducted.

[0188] The difference from Example 1 is that the test drug is one batch of Lingze tablet extract from 25 batches. The robustness of the column temperature (±2℃), flow rate (±0.02ml / min), and different brands of ultra-high performance liquid chromatographs of the same column in step S3 were tested and the chromatograms were recorded. Step S4 is replaced by step S5 as follows: Calculation of similarity of test samples: The fingerprint spectrum integral signal of the test sample is imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" 2012 version software of the Chinese Pharmacopoeia Commission; the common peaks of the test sample and the control fingerprint spectrum (constructed in Example 1) are selected; the chromatograms are superimposed using the Mark peak matching method, and the similarity between the test sample and the control fingerprint spectrum of Lingze tablet extract is calculated. The similarity results are shown in Table 10.

[0189] Table 10 Durability Similarity Results

[0190]

[0191] Results: The RSD values ​​of fingerprint chromatograms obtained by different column temperatures, flow rates, and brands of ultra-high performance chromatographs were 0.209%, 0.101%, and 0.356%, respectively, indicating good method robustness.

[0192] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting the fingerprint spectrum of Lingze tablet extract, characterized in that, The method includes using ultra-high performance liquid chromatography to analyze the test solution and obtain the fingerprint spectrum of Lingze tablet extract; the Lingze tablet extract is obtained by water extraction, alcohol precipitation, concentration and drying of Alisma plantago-aquatica, Fritillaria thunbergii and Curcuma zedoaria residue after volatile oil extraction; The analytical conditions for ultra-high performance liquid chromatography included: a Waters CORTECS column. ® UPLC ® T3, with dimensions of 2.1×100mm and 1.6μm; the mobile phase includes mobile phase A (acetonitrile) and mobile phase B (water); the gradient elution program is as follows: mobile phase B: 0–5 min, 100% v / v; 5–18 min, 100% v / v to 80% v / v; 18–55 min, 80% v / v to 35% v / v; 55–60 min, 35% v / v; mobile phase flow rate: 0.08–0.12 mL / min; detection wavelength: 260–280 nm. The test solution includes a sample solution, which is prepared by extracting Lingze tablets using an extraction solvent selected from water, methanol aqueous solution, or ethanol aqueous solution; the concentration of methanol in the methanol aqueous solution does not exceed 30% v / v; and the concentration of ethanol in the ethanol aqueous solution does not exceed 10% v / v. The test solution also includes a reference solution, which is a solution containing known amounts of the following components: hypoxanthine, uridine, adenine, guanosine, and adenosine.

2. The detection method according to claim 1, characterized in that, The extraction is performed using ultrasonic or reflux extraction methods; The extraction solvent is selected from 30% v / v methanol aqueous solution, 10% v / v methanol aqueous solution, water or 10% v / v ethanol aqueous solution; Extraction time is 20-60 minutes; The ratio of Lingze tablet extract to extraction solvent is 5~20 mg / mL.

3. The detection method according to claim 1, characterized in that, The known content is 20~100μg / mL; The solvent for the reference solution is selected from water, methanol, methanol-water solution, ethanol, or ethanol-water solution.

4. The detection method according to claim 1, characterized in that, Chromatographic conditions also include a column temperature of 23-27℃.

5. The detection method according to claim 1, characterized in that, The detection wavelength of ultra-high performance liquid chromatography is 260 nm.

6. The detection method according to claim 1, characterized in that, The detection method further includes providing a synthetic control fingerprint spectrum of fingerprint spectra from different batches; the control fingerprint spectrum selects chromatographic peaks that are present in the fingerprint spectra of different batches of Lingze tablet extract as common peaks.

7. The detection method according to claim 6, characterized in that, The fingerprint spectrum includes 15 characteristic peaks, including hypoxanthine characteristic peak, guanosine characteristic peak, adenine characteristic peak, guanosine characteristic peak and adenosine characteristic peak.

8. The detection method according to claim 6, characterized in that, The detection method further includes comparing the fingerprint spectrum of the sample to be evaluated with the control fingerprint spectrum and calculating the similarity between the fingerprint spectrum of the sample to be evaluated and the control fingerprint spectrum.

9. The detection method according to any one of claims 1 to 8, characterized in that, Includes the following steps: S1, Preparation of reference solutions: Take hypoxanthine, uridine, adenine, guanosine and adenosine reference standards, add 10% v / v~30% v / v methanol to prepare mixed solutions with concentrations of 20~100 μg / mL respectively. S2, Preparation of the test solution: Provide extracts of different batches of Lingze tablets, extract with the extraction solvent, cool, shake well, filter, and take the filtrate to obtain the test solution; S3, Chromatographic conditions: Waters CORTECS column. ® UPLC ® T3, with dimensions of 2.1 × 100 mm and 1.6 μm; using acetonitrile as mobile phase A and water as mobile phase B, eluted according to the following gradient program: Mobile phase B: 0–5 min, 100% v / v; 5–18 min, 100% v / v to 80% v / v; 18–55 min, 80% v / v to 35% v / v; 55–60 min, 35% v / v; flow rate 0.08–0.12 ml / min; column temperature 23–27 °C; detection wavelength 260 nm; inject the test solution and reference solution separately into the ultra-high performance liquid chromatograph. S4, Establishing the fingerprint spectrum: The integrated signals of fingerprint spectra of multiple batches of Lingze tablet extract are imported into the fingerprint spectrum similarity evaluation system; the chromatographic peaks present in the fingerprint spectra of different batches of Lingze tablet extract are selected as common peaks; the chromatograms are superimposed using the Mark peak matching method, and a reference fingerprint spectrum of Lingze tablet extract is generated; the similarity between each batch of Lingze tablet extract and the reference fingerprint spectrum of Lingze tablet extract is calculated. S5, Calculation of similarity between the test solution and the fingerprint spectrum of the test solution: The fingerprint spectrum integral signal of the test solution is imported into the fingerprint spectrum similarity evaluation system, and the chromatogram is superimposed using the Mark peak matching method to calculate the similarity between the test solution and the fingerprint spectrum of the Lingze tablet extract.

10. The application of the detection method of fingerprint spectrum of Lingze tablet extract according to any one of claims 1 to 9 in the quality control of Lingze tablet extract.

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