A method for detecting traditional Chinese medicine fingerprint of a traditional Chinese medicine preparation

Abstract

This invention discloses a method for detecting the fingerprint spectrum of traditional Chinese medicine (TCM) preparations, comprising the following steps: S1, preparing a test solution: taking a TCM preparation sample, pulverizing, extracting with a solvent, and filtering to obtain a test solution; S2, preparing a reference herb solution: taking a reference herb, extracting with the same solvent, and filtering to obtain a reference herb solution; S3, preparing a reference standard solution: accurately weighing a reference standard with known components, dissolving it in a solvent to obtain a reference standard solution; S4, determination by high-performance liquid chromatography (HPLC): injecting the test solution, reference herb solution, and reference standard solution into an HPLC instrument, performing determination under set chromatographic conditions, and recording the chromatogram. This invention, by introducing a similarity calculation formula, comprehensively considers peak area similarity and retention time consistency, improving the accuracy and comprehensiveness of TCM preparation quality evaluation, and providing a scientific basis for TCM quality control and standardized production.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine detection technology, and in particular to a method for detecting the fingerprint spectrum of traditional Chinese medicine preparations. Background Technology

[0002] Traditional Chinese medicine (TCM) quality evaluation mainly relies on the determination of single-index components, which is insufficient to comprehensively reflect the overall quality of medicinal materials and preparations. TCM fingerprinting technology, by systematically characterizing the overall features of multiple chemical components in the complex system of TCM and constructing a unique "chemical fingerprint," has become a core technical means for TCM quality control and standardization.

[0003] However, existing fingerprint detection methods often only consider the similarity of peak areas when calculating similarity, while ignoring the consistency of retention time, resulting in incomplete evaluation results. Summary of the Invention

[0004] This invention provides a method for detecting the fingerprint spectrum of traditional Chinese medicine preparations to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A method for detecting the fingerprint spectrum of a traditional Chinese medicine preparation includes the following steps: S1, Preparation of test solution: Take a sample of traditional Chinese medicine preparation, and after crushing, solvent extraction and filtration, prepare the test solution; S2, Preparation of reference medicinal material solution: Take the reference medicinal material, extract it with the same solvent, filter it, and then prepare the reference medicinal material solution; S3, Preparation of reference solution: Accurately weigh a reference standard with known components, dissolve it in a solvent, and prepare a reference solution; S4, Determination by high performance liquid chromatography: Inject the test solution, reference medicinal material solution and reference solution into the high performance liquid chromatograph, and perform the determination under the set chromatographic conditions, and record the chromatogram; S5, Fingerprint similarity calculation: The similarity between the fingerprint spectrum of the test sample solution and the fingerprint spectrum of the reference medicinal material is calculated using similarity software, and the characteristic peaks are identified using the reference solution. S6 introduces the similarity calculation formula and the quality consistency index formula: Similarity calculation formula: ; in, To assess overall similarity, The area of ​​the i-th characteristic peak of the test sample, The area of ​​the i-th characteristic peak of the reference medicinal material. The retention time of the test solution. The retention time of the control herbal solution; Formula for quality consistency index: ; in, It is the peak area of ​​the i-th characteristic peak of the test sample; It is the peak area of ​​the i-th characteristic peak of the reference medicinal material; It is the retention time of the i-th characteristic peak of the test sample; is the retention time of the i-th characteristic peak of the control herb; n is the total number of characteristic peaks; k is an adjustment factor used to control the degree of influence of retention time deviation on the QCI value; S7, Result Judgment: The quality of traditional Chinese medicine preparations is judged based on the comprehensive similarity S and QCI value. If S≥0.90 and QCI value is between 0.9 and 1.1, it is judged as qualified and of highly consistent quality.

[0006] As a further improvement to this technical solution, the chromatographic conditions are as follows: the chromatographic column is a C18 column with a size of 4.6 mm × 250 mm and a packing particle size of 5 μm; the mobile phase is a gradient elution of 0.2 vol% formic acid aqueous solution and acetonitrile; the flow rate is 0.8 ml / min; the detection wavelength is 262 nm; the column temperature is 30 ℃; and the recording time is 65 min.

[0007] As a further improvement to this technical solution: the preparation method of the test solution is as follows: take 1-2g of the traditional Chinese medicine preparation sample, crush it, weigh it accurately, add 20ml of methanol, sonicate for 30min, filter, add 10ml of methanol to the filter residue, sonicate for 15min, filter, combine the filtrates, evaporate to dryness, add 5ml of water to the residue and heat gently to dissolve, add to a C18 column, elute with 20ml of water, discard the water, then elute with 20ml of 10% methanol, discard the eluent, finally elute with 20ml of methanol, collect the eluent, evaporate to dryness, dissolve in methanol and make up to 5ml, filter through a microporous membrane to obtain the test solution.

[0008] As a further improvement to this technical solution: the preparation method of the reference medicinal material solution is as follows: take 1-2g of reference medicinal material, crush it, accurately weigh it, add 20ml of methanol, soak overnight, sonicate for 30min, filter, add 10ml of methanol to the filter residue, sonicate for 15min, filter, combine the filtrates, evaporate to dryness, add 5ml of water to the residue and heat gently to dissolve, add to a C18 column, elute with 20ml of water, discard the water, then elute with 20ml of 10% methanol, discard the eluent, finally elute with 20ml of methanol, collect the eluent, evaporate to dryness, dissolve in methanol and make up to 5ml, filter through a microporous membrane to obtain the solution.

[0009] As a further improvement to this technical solution: the preparation method of the reference solution is as follows: accurately weigh appropriate amounts of ophiopogonin b, ophiopogonin c and ophiopogonin d, add methanol to dissolve them into a mixed reference solution with concentrations of 0.1 mg / ml, 0.2 mg / ml and 0.3 mg / ml, respectively.

[0010] As a further improvement to this technical solution: in the similarity calculation formula, and The value ranges from 0 to 1, representing the relative value of the characteristic peak area; and The value ranges from 0 to 100 min, representing the retention time.

[0011] As a further improvement to this technical solution: the method is applicable to the quality evaluation of Chinese medicinal materials, processed Chinese medicinal materials, and prepared Chinese medicines.

[0012] As a further improvement to this technical solution: the method also includes a stability test on the test solution and the control medicinal material solution, with RSD < 5% within 24 hours.

[0013] As a further improvement to this technical solution: a QCI value between 0.9 and 1.1 indicates that the quality of traditional Chinese medicine preparations is highly consistent; A QCI value between 0.7 and 0.9 or between 1.1 and 1.3 indicates that the quality of traditional Chinese medicine preparations is basically consistent, but attention is still needed. A QCI value less than 0.7 or greater than 1.3 indicates inconsistent quality of traditional Chinese medicine preparations.

[0014] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention innovatively introduces a comprehensive similarity calculation formula that considers both peak area similarity and retention time consistency, building upon traditional fingerprint similarity calculations. This formula calculates the cosine similarity of peak areas and corrects for retention time deviations, thus providing a more comprehensive evaluation of the quality of traditional Chinese medicine preparations. This innovation effectively solves the problem in existing technologies that only consider peak area similarity while neglecting retention time consistency, improving the accuracy and comprehensiveness of quality evaluation.

[0015] 2. This invention employs a multi-stage solvent extraction and purification process in the preparation of the test solution and the reference herbal solution. Specifically, it includes multiple steps such as primary ultrasonic extraction, secondary ultrasonic extraction, filtrate merging, evaporation to dryness, residue dissolution, and C18 column purification. This innovation enables more effective extraction of target components from traditional Chinese medicine preparations while removing impurities, thus improving the sensitivity and accuracy of detection.

[0016] 3. In the preparation of the reference solution, this invention employs a multi-component calibration method, that is, simultaneously and accurately weighing multiple known components of the reference standard and dissolving them in a solvent to prepare a mixed reference standard solution. This allows for the simultaneous calibration of multiple characteristic peaks during fingerprint similarity calculation, further improving the accuracy and reliability of quality evaluation.

[0017] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Specific embodiments of the present invention are given in detail below with reference to the accompanying drawings. Attached Figure Description

[0018] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings: Figure 1 This is a flowchart of the overall process for detecting fingerprint spectra of traditional Chinese medicine proposed in this invention; Figure 2 This is a detailed flowchart of the preparation of the test sample solution proposed in this invention; Figure 3 This is a schematic diagram of the steps in the traditional Chinese medicine fingerprint detection method for traditional Chinese medicine preparations proposed in this invention. Detailed Implementation

[0019] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. The invention is described more specifically in the following paragraphs by way of example with reference to the accompanying drawings. It should be noted that the drawings are in a very simplified form and use non-precise proportions, and are only used to facilitate and clarify the illustration of the embodiments of the present invention.

[0020] It should be noted that when a component is described as "fixed to" another component, it can be directly on the other component or may have a component in between. When a component is considered "connected to" another component, it can be directly connected to the other component or may have a component in between. When a component is considered "set on" another component, it can be directly set on the other component or may have a component in between. The terms "vertical," "horizontal," "left," "right," and similar expressions used in this document are for illustrative purposes only.

[0021] Please see Figure 1 In this embodiment of the invention, a method for detecting the fingerprint spectrum of a traditional Chinese medicine preparation includes the following steps: S1, Preparation of the test solution: Take 1-2g of the traditional Chinese medicine preparation sample, pulverize it, weigh it accurately, add 20ml of methanol, sonicate for 30min, filter, add 10ml of methanol to the residue, sonicate for 15min, filter, combine the filtrates, evaporate to dryness, add 5ml of water to the residue and heat gently to dissolve, add to a C18 column, elute with 20ml of water, discard the water, then elute with 20ml of 10% methanol, discard the eluent, finally elute with 20ml of methanol, collect the eluent, evaporate to dryness, dissolve in methanol and make up to 5ml, filter through a microporous membrane to obtain the test solution.

[0022] S2, Preparation of reference herb solution: Take 1-2g of reference herb, pulverize it, weigh it accurately, add 20ml of methanol, soak overnight, sonicate for 30min, filter, add 10ml of methanol to the residue, sonicate for 15min, filter, combine the filtrates, evaporate to dryness, add 5ml of water to the residue and heat gently to dissolve, add to a C18 column, elute with 20ml of water, discard the water, then elute with 20ml of 10% methanol, discard the eluent, finally elute with 20ml of methanol, collect the eluent, evaporate to dryness, dissolve in methanol and make up to 5ml, filter through a microporous membrane to obtain the solution.

[0023] S3, Preparation of reference solution: Accurately weigh appropriate amounts of ophiopogonin b, ophiopogonin c and ophiopogonin d, dissolve them in methanol to prepare a mixed reference solution with concentrations of 0.1 mg / ml, 0.2 mg / ml and 0.3 mg / ml, respectively.

[0024] S4, Determination by High Performance Liquid Chromatography (HPLC): The test solution, reference medicinal material solution, and reference standard solution were injected separately into the HPLC instrument, and the determination was performed under the set chromatographic conditions. The chromatographic conditions were as follows: C18 column, 4.6 mm × 250 mm, packing particle size 5 μm; mobile phase: 0.2 vol% formic acid aqueous solution and acetonitrile gradient elution; flow rate: 0.8 ml / min; detection wavelength: 262 nm; column temperature: 30 ℃; recording time: 65 min.

[0025] S5, Fingerprint Similarity Calculation: The similarity between the fingerprint spectrum of the test sample solution and the fingerprint spectrum of the reference medicinal material is calculated using similarity software, and the characteristic peaks are identified using the reference solution.

[0026] S6 introduces the similarity calculation formula and the quality consistency index formula: Similarity calculation formula: ; in, To assess overall similarity, The area of ​​the i-th characteristic peak of the test sample, The area of ​​the i-th characteristic peak of the reference medicinal material. The retention time of the test solution. The retention time of the control herbal solution; Formula for quality consistency index: ; in, It is the peak area of ​​the i-th characteristic peak of the test sample; It is the peak area of ​​the i-th characteristic peak of the reference medicinal material; It is the retention time of the i-th characteristic peak of the test sample; is the retention time of the i-th characteristic peak of the control herb; n is the total number of characteristic peaks; k is an adjustment factor used to control the degree of influence of retention time deviation on the QCI value; QCI values ​​between 0.9 and 1.1 indicate highly consistent quality. QCI values ​​between 0.7 and 0.9 or between 1.1 and 1.3: quality is generally consistent, but attention is still needed; QCI value less than 0.7 or greater than 1.3: quality inconsistency.

[0027] S7, Result Judgment: The quality of traditional Chinese medicine preparations is judged based on the comprehensive similarity S and QCI value. If S≥0.90 and QCI value is between 0.9 and 1.1, it is judged as qualified and of highly consistent quality.

[0028] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Those skilled in the art can readily implement the present invention based on the accompanying drawings and the above description. However, any modifications, alterations, or variations made by those skilled in the art without departing from the scope of the present invention, utilizing the disclosed technical content, are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, or variations made to the above embodiments based on the essential technology of the present invention are still within the protection scope of the present invention.

Claims

1. A method for detecting the fingerprint spectrum of a traditional Chinese medicine preparation, characterized in that, Includes the following steps: S1, Preparation of test solution: Take a sample of traditional Chinese medicine preparation, and after crushing, solvent extraction and filtration, prepare the test solution; S2, Preparation of reference medicinal material solution: Take the reference medicinal material, extract it with the same solvent, filter it, and then prepare the reference medicinal material solution; S3, Preparation of reference solution: Accurately weigh a reference standard with known components, dissolve it in a solvent, and prepare a reference solution; S4, Determination by high performance liquid chromatography: Inject the test solution, reference medicinal material solution and reference solution into the high performance liquid chromatograph, and perform the determination under the set chromatographic conditions, and record the chromatogram; S5, Fingerprint similarity calculation: The similarity between the fingerprint spectrum of the test sample solution and the fingerprint spectrum of the reference medicinal material is calculated using similarity software, and the characteristic peaks are identified using the reference solution. S6 introduces the similarity calculation formula and the quality consistency index formula: Similarity calculation formula: ; in, To assess overall similarity, The area of ​​the i-th characteristic peak of the test sample, The area of ​​the i-th characteristic peak of the reference medicinal material. The retention time of the test solution. The retention time of the control herbal solution; Formula for quality consistency index: ; in, It is the peak area of ​​the i-th characteristic peak of the test sample; It is the peak area of ​​the i-th characteristic peak of the reference medicinal material; It is the retention time of the i-th characteristic peak of the test sample; is the retention time of the i-th characteristic peak of the control herb; n is the total number of characteristic peaks; k is an adjustment factor used to control the degree of influence of retention time deviation on the QCI value; S7, Result Judgment: The quality of traditional Chinese medicine preparations is judged based on the comprehensive similarity S and QCI value. If S≥0.90 and QCI value is between 0.9 and 1.1, it is judged as qualified and of highly consistent quality.

2. The method for detecting the fingerprint spectrum of a traditional Chinese medicine preparation according to claim 1, characterized in that, The chromatographic conditions were as follows: the column was a C18 column with dimensions of 4.6 mm × 250 mm and a packing particle size of 5 μm; the mobile phase was a gradient elution of 0.2 vol% formic acid aqueous solution and acetonitrile; the flow rate was 0.8 ml / min; the detection wavelength was 262 nm; the column temperature was 30 ℃; and the recording time was 65 min.

3. The method for detecting the fingerprint spectrum of a traditional Chinese medicine preparation according to claim 2, characterized in that, The preparation method of the test solution is as follows: Take 1-2g of the traditional Chinese medicine preparation sample, crush it, weigh it accurately, add 20ml of methanol, sonicate for 30min, filter, add 10ml of methanol to the filter residue, sonicate for 15min, filter, combine the filtrates, evaporate to dryness, add 5ml of water to the residue and heat gently to dissolve, add to a C18 column, elute with 20ml of water, discard the water, then elute with 20ml of 10% methanol, discard the eluent, finally elute with 20ml of methanol, collect the eluent, evaporate to dryness, dissolve in methanol and make up to 5ml, filter through a microporous membrane to obtain the test solution.

4. The method for detecting the fingerprint spectrum of a traditional Chinese medicine preparation according to claim 3, characterized in that, The preparation method of the reference medicinal material solution is as follows: Take 1-2g of reference medicinal material, crush it, accurately weigh it, add 20ml of methanol, soak overnight, sonicate for 30min, filter, add 10ml of methanol to the filter residue, sonicate for 15min, filter, combine the filtrates, evaporate to dryness, add 5ml of water to the residue and heat gently to dissolve, add to a C18 column, elute with 20ml of water, discard the water, then elute with 20ml of 10% methanol, discard the eluent, finally elute with 20ml of methanol, collect the eluent, evaporate to dryness, dissolve in methanol and make up to 5ml, filter through a microporous membrane to obtain the solution.

5. The method for detecting the fingerprint spectrum of a traditional Chinese medicine preparation according to claim 4, characterized in that, The preparation method of the reference solution is as follows: accurately weigh appropriate amounts of ophiopogonin b, ophiopogonin c and ophiopogonin d, dissolve them in methanol to form a mixed reference solution with concentrations of 0.1 mg / ml, 0.2 mg / ml and 0.3 mg / ml, respectively.

6. The method for detecting the fingerprint spectrum of a traditional Chinese medicine preparation according to claim 5, characterized in that, In the similarity calculation formula and The value ranges from 0 to 1, representing the relative value of the characteristic peak area; and The value ranges from 0 to 100 min, representing the retention time.

7. The method for detecting the fingerprint spectrum of a traditional Chinese medicine preparation according to claim 6, characterized in that, The method is applicable to the quality evaluation of Chinese medicinal materials, processed Chinese medicinal herbs, and prepared Chinese medicines.

8. The method for detecting the fingerprint spectrum of a traditional Chinese medicine preparation according to claim 7, characterized in that, The method also includes stability testing of the test solution and the control herbal solution, with RSD < 5% within 24 hours.

9. The method for detecting the fingerprint spectrum of a traditional Chinese medicine preparation according to claim 7, characterized in that, A QCI value between 0.9 and 1.1 indicates that the quality of traditional Chinese medicine preparations is highly consistent. A QCI value between 0.7 and 0.9 or between 1.1 and 1.3 indicates that the quality of traditional Chinese medicine preparations is basically consistent, but attention is still needed. A QCI value less than 0.7 or greater than 1.3 indicates inconsistent quality of traditional Chinese medicine preparations.

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