Construction method of fingerprint of cuttlefish ink and identification method of cuttlefish ink variety

Abstract

Embodiments of the present application provide a method for constructing a fingerprint of cuttlefish ink and a method for identifying different varieties of cuttlefish ink. The method for constructing the fingerprint of cuttlefish ink comprises the following steps: Step S1: preparing a first sample solution for obtaining chromatographic information of water-soluble components and a second sample solution for obtaining chromatographic information of water-insoluble components from the same cuttlefish ink sample; Step S2: performing chromatographic analysis on the first sample solution and the second sample solution respectively to obtain first chromatographic data and second chromatographic data; and Step S3: constructing a fingerprint for evaluating the quality of the cuttlefish ink sample based on the first chromatographic data and the second chromatographic data. Therefore, the method for constructing the fingerprint of cuttlefish ink according to the embodiments of the present application comprehensively integrates the chromatographic information of water-soluble and water-insoluble components, and realizes more comprehensive and accurate quality control and authenticity identification of cuttlefish ink.

Description

Technical Field

[0001] This invention relates to the field of quality technology of Chinese medicinal materials, specifically to a method for constructing a fingerprint spectrum of cuttlefish ink and a method for identifying different varieties of cuttlefish ink. Background Technology

[0002] In the field of quality identification and control technology for cuttlefish ink, as a traditional marine medicine with complex components, the quality control of cuttlefish ink needs to comprehensively reflect its overall chemical characteristics. Since cuttlefish ink contains many substances with vastly different properties, including water-soluble and water-insoluble components (such as melanin), establishing a quality evaluation system that can integrate this diverse information is a common requirement in this field.

[0003] In related technologies, the quality evaluation of squid ink typically relies on the separate detection of its specific components. For example, existing research often focuses on the qualitative identification of melanin or the optimization of its extraction process, or on evaluating quality by measuring the content of indicative components such as amino acids and trace elements. While these methods can provide some quality information, the information obtained is independent and scattered because they only target a few or single components in the complex chemical system of squid ink. This analytical model based on isolated indicators makes it difficult to comprehensively characterize the quality differences between different batches and varieties of squid ink from an overall chemical perspective, and it cannot effectively address the natural fluctuations in chemical composition caused by the wide range of sources. This leads to limitations in distinguishing genuine products from common adulterants (such as squid ink from the gun), resulting in the shortcomings of existing detection methods in terms of scattered indicators and lack of overall comprehensiveness. It is difficult to achieve precise control over the quality of squid ink medicinal materials and cannot ensure the consistency of different batches of products. Summary of the Invention

[0004] In view of this, the present invention aims to provide a method for constructing a fingerprint spectrum of cuttlefish ink and a method for identifying different varieties of cuttlefish ink, so as to solve the problems of scattered indicators, lack of integrity, and difficulty in comprehensively characterizing the overall chemical characteristics of cuttlefish ink and effectively identifying different varieties in the existing methods for constructing fingerprint spectra of cuttlefish ink.

[0005] This invention provides a method for constructing a squid ink fingerprint spectrum.

[0006] This invention provides a method for identifying different varieties of cuttlefish ink.

[0007] The invention provides illustrative examples of methods for constructing squid ink fingerprint spectra and identifying different varieties of squid ink.

[0008] The method for constructing a squid ink fingerprint spectrum according to an embodiment of the present invention includes the following steps: Step S1: Take different batches of squid ink, and prepare a first test solution for obtaining chromatographic information of water-soluble components and a second test solution for obtaining chromatographic information of water-insoluble components for each squid ink sample; Step S2: Perform chromatographic analysis on the first and second test solutions of different batches of squid ink to obtain first and second chromatographic data; Step S3: Based on the first chromatographic data and the second chromatographic data, select the chromatographic peaks that are present in the chromatograms of different batches of squid ink as common peaks, generate a reference fingerprint spectrum of squid ink, and calculate the relative retention time and relative peak area of ​​each common peak to construct a fingerprint spectrum for evaluating the quality of the squid ink sample.

[0009] The method for constructing a fingerprint spectrum for cuttlefish ink according to this invention involves extracting water-soluble and water-insoluble components from the same cuttlefish ink sample. Test solutions targeting the water-soluble and water-insoluble components are prepared separately from the same batch of samples, and chromatographic analysis is performed separately to obtain two sets of chromatographic data. The water-soluble fingerprint spectrum reflects the overall chemical profile of the water-soluble components in cuttlefish ink, while the water-insoluble fingerprint spectrum mainly analyzes characteristic components such as melanin. The two complement each other, providing a more comprehensive and objective reflection of the quality of cuttlefish ink. A quality evaluation fingerprint spectrum is constructed based on the integrated data from these two sets, overcoming the limitations of a single processing mode. Therefore, this method can more comprehensively characterize the chemical composition of cuttlefish ink, significantly improving the ability to distinguish between different cuttlefish ink species (such as golden cuttlefish and needleless cuttlefish) and their adulterants, providing a more accurate and reliable analytical means for the quality control of cuttlefish ink medicinal materials.

[0010] Therefore, the method for constructing the squid ink fingerprint spectrum in this embodiment of the invention achieves more comprehensive and accurate quality control and authenticity identification of squid ink by integrating the chromatographic information of water-soluble and water-insoluble components.

[0011] In one embodiment, the cuttlefish ink sample is diluted with water and then heated or enzymatically hydrolyzed to obtain the first test solution.

[0012] In some embodiments, the same batch of cuttlefish ink samples are taken and oxidized under alkaline and hydrogen peroxide conditions to obtain the second test solution.

[0013] In some embodiments, the cuttlefish ink sample is diluted with 8 to 12 times the amount of water by weight, heated in a water bath at 80°C to 100°C for 30 min to 1.5 h, centrifuged or filtered, and the supernatant is taken to obtain the first test solution.

[0014] In other embodiments, the cuttlefish ink sample was diluted with 8 to 12 times the amount of water by weight, 20,000 U of flavor protease was added, and the enzymatic hydrolysis was carried out at a temperature of 45°C to 60°C, pH 7.0, for 3 to 5 hours. The sample was then inactivated by water bath, centrifuged or filtered, and the supernatant was collected to obtain the first test solution.

[0015] In some embodiments, another sample of squid ink from the same batch is taken, and 1 mol / L K2CO3 solution and 30% H2O2 solution are added to the squid ink sample. The mixture is heated at 80℃~100℃ for 60 min~180 min. After cooling, 200 μL of 10% Na2SO3 is added to terminate the reaction. 1 mol / L~6 mol / L HCl is added to adjust the pH to 2.0~4.5. The mixture is then filtered or centrifuged to obtain the second test solution.

[0016] In some embodiments, the cuttlefish ink includes needleless cuttlefish ink, golden cuttlefish ink, needle cuttlefish ink, or tiger-striped cuttlefish ink.

[0017] In some embodiments, high performance liquid chromatography is used to perform chromatographic analysis on the first test solution and the second test solution, respectively.

[0018] Import the first and second chromatographic data into the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" software, respectively. In some embodiments, 20 μL of the first test solution is taken for chromatographic analysis; 10 μL of the second test solution is taken for chromatographic analysis to obtain the chromatographic peaks corresponding to each test solution; In some embodiments, a reference peak solution of pyrrole-2,3,5-tricarboxylic acid is prepared, and the second test solution and the reference peak solution are subjected to chromatographic analysis.

[0019] In some embodiments, the chromatographic detection conditions for the first test solution are: The column was filled with octadecylsilane-bonded silica gel at a temperature of 30℃ to 40℃. Acetonitrile (A) and 0.1% phosphoric acid aqueous solution (B) were used as the mobile phases, with gradient elution: 0 min–5 min, B 99.9%; 5–10 min, B 99.9%–98.8%; 10 min–20 min, B 98.8%; 20 min–22 min, B 98.8%–95%; 22 min–30 min, B 95%; 30–45 min, B 95%–80%; 45 min–50 min, B 80%–70%; 50 min–60 min, B 70%–50%. The UV detection wavelength was 210–360 nm.

[0020] The chromatographic detection conditions for the second test solution are as follows: The column was filled with octadecylsilane-bonded silica gel at a temperature of 30℃ to 40℃. The mobile phase consisted of methanol (A) and sodium dihydrogen phosphate buffer (B), with gradient elution: 0 min to 25 min, B 95% to 60%; 25 min to 30 min, B 40%. The UV detection wavelength was 210 nm to 360 nm.

[0021] In some embodiments, the cuttlefish ink sample includes golden cuttlefish ink and needleless cuttlefish ink. A first test solution and a second test solution are prepared from the golden cuttlefish ink and the needleless cuttlefish ink, respectively, and then analyzed by chromatography.

[0022] The first test solution of the golden cuttlefish ink contained 9 common peaks after chromatographic analysis, and the first test solution of the needleless cuttlefish ink contained 12 common peaks after chromatographic analysis. Both the golden cuttlefish ink and the needleless cuttlefish ink used chromatographic peak No. 2 as the S peak, and each common peak had a specific relative retention time range and relative peak area range relative to chromatographic peak No. 2.

[0023] In some embodiments, the second test solution of each of the golden cuttlefish ink and the needleless cuttlefish ink contains 5 common peaks, both of which have chromatographic peak No. 5 as the S peak, wherein peak No. 5 is pyrrole-2,3,5-tricarboxylic acid, and each common peak has a specific relative retention time range and relative peak area range relative to chromatographic peak No. 5.

[0024] In some embodiments, the relative retention times of the nine common peaks in the first test solution of the cuttlefish ink obtained by chromatographic analysis are as follows: peak 1 0.566~0.570, peak 2 1.000, peak 3 1.326~1.327, peak 4 1.523~1.528, peak 5 1.773~1.781, peak 6 2.110~2.126, peak 7 3.351~3.394, peak 8 4.284~4.311, and peak 9 4.768~4.783.

[0025] The relative peak areas of the nine common peaks in the first test solution of the cuttlefish ink, as determined by chromatographic analysis, are as follows: Peak 1: 1.788–3.419, Peak 2: 1.000, Peak 3: 0.346–0.622, Peak 4: 0.203–1.302, Peak 5: 0.284–0.637, Peak 6: 0.401–0.721, Peak 7: 0.380–3.361, Peak 8: 0.136–0.497, and Peak 9: 0.187–0.704. The relative retention times of the 12 common peaks in the first test solution of the needle-free squid ink, as determined by chromatographic analysis, are as follows: Peak 1: 0.566–0.570, Peak 2: 1.000, Peak 3: 1.325–1.326, Peak 4: 1.381–1.388, Peak 5: 1.524–1.526, Peak 6: 1.775–1.785, Peak 7: 2.108–2.122, Peak 8: 2.919–2.925, Peak 9: 3.029–3.042, Peak 10: 3.398–3.412, Peak 11: 4.678–4.686, and Peak 12: 4.782–4.790.

[0026] The relative peak areas of the 12 common peaks in the first test solution of the needle-free squid ink, as determined by chromatographic analysis, are as follows: Peak 1: 0.581~0.621, Peak 2: 1.000, Peak 3: 0.940~1.264, Peak 4: 0.086~0.269, Peak 5: 0.135~0.171, Peak 6: 0.309~0.489, Peak 7: 0.289~0.362, Peak 8: 0.041~0.073, Peak 9: 0.168~0.212, Peak 10: 0.059~0.092, Peak 11: 0.039~0.058, Peak 12: 0.058~0.104.

[0027] In some embodiments, the relative retention times of the fingerprint chromatograms of the second test solution of each of the golden cuttlefish ink and the needleless cuttlefish ink are as follows: peak 1 0.377~0.386, peak 2 0.471~0.474, peak 3 0.560~0.562, peak 4 0.668~0.670, peak 5 1.000; The relative peak areas of the fingerprint spectra of the second test solution of each of the golden cuttlefish ink and the needleless cuttlefish ink were as follows according to chromatographic analysis: peak 1 0.124~0.276, peak 2 0.037~0.237, peak 3 0.080~0.716, peak 4 0.047~0.352, and peak 5 1.000.

[0028] The method for identifying different species of cuttlefish ink according to embodiments of the present invention includes the following steps: Step S1: Prepare a first test solution for obtaining chromatographic information of water-soluble components and a second test solution for obtaining chromatographic information of water-insoluble components based on the cuttlefish ink sample to be tested; Step S2: Perform chromatographic analysis on the first test solution and the second test solution respectively to obtain first chromatographic data and second chromatographic data; compare the chromatographic analysis data of the sample to be tested with the squid ink fingerprint standard data as described in any one of the above, and determine the authenticity or quality grade of the squid ink sample to be tested based on the comparison results.

[0029] In some embodiments, a fingerprint spectrum based on the quality of the constructed squid ink sample; Analyze the number of peaks in the 35 min to 60 min interval of the first chromatographic data to determine whether the cuttlefish ink sample to be tested is cuttlefish ink.

[0030] In some embodiments, the peak shape and peak height of the first chromatographic data in the 8 min to 32 min interval are analyzed to distinguish between the golden cuttlefish ink sample and the needleless cuttlefish ink sample.

[0031] In some embodiments, the spectral data of the first test solution of the cuttlefish ink sample to be tested is correlated with the fingerprint spectral standard data of the first test solution of the golden cuttlefish ink sample and / or the needleless cuttlefish ink sample. If the similarity is greater than 0.9, it is further confirmed whether the sample to be tested is golden cuttlefish ink or needleless cuttlefish ink.

[0032] In some embodiments, the spectral data of the second test solution of the cuttlefish ink sample to be tested is correlated with the fingerprint spectral standard data of the second test solution of the golden cuttlefish ink sample and / or needleless cuttlefish ink sample. If the similarity is greater than 0.9, the cuttlefish ink sample to be tested is determined to be golden cuttlefish ink and / or needleless cuttlefish ink. Attached Figure Description

[0033] Figure 1 HPLC fingerprints of squid ink prepared by different methods; Figure 2 HPLC fingerprints of squid ink soluble in different mobile phases; Figure 3 HPLC fingerprints of 10 batches of squid ink; Figure 4 HPLC fingerprints of ink soluble samples from 5 batches of golden cuttlefish; Figure 5 HPLC fingerprints of five batches of needle-free squid ink; Figure 6 Insoluble HPLC fingerprints of 10 batches of squid ink; Figure 7 HPLC fingerprints of ink solubility for different species of squid; Figure 8 HPLC fingerprints of insoluble inks from different squid species; Figure 9 This is a qualitative chromatogram of the reference standard in the insoluble HPLC fingerprint of squid ink. Detailed Implementation

[0034] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0035] Through in-depth analysis, the inventors discovered that, from the perspective of the physicochemical properties of chemical components, the water-soluble components and water-insoluble components (such as melanin polymers) in squid ink differ significantly in solubility, molecular structure, and polarity. This means that conditions suitable for the efficient extraction or analysis of one type of component are often unsuitable or even detrimental to the retention and detection of the other type.

[0036] To overcome the aforementioned contradictions, this disclosure proposes a different technical approach. Its core concept lies in: starting from the same batch of squid ink samples, preparing test solutions in parallel to obtain chromatographic information for water-soluble and water-insoluble components respectively, and integrating the two sets of chromatographic data to jointly construct a fingerprint spectrum. This improves the information acquisition process, thereby effectively enhancing the comprehensiveness of chemical information for quality characterization and the ability to distinguish between different samples (including adulterants) without significantly increasing the overall operational complexity. In other words, it provides a method for parallel processing and integration of chromatographic data for water-soluble and water-insoluble components, solving the problems of insufficient discrimination and incomplete quality control caused by incomplete chemical information in related technologies, and achieving the technical effect of simultaneously reflecting the overall chemical characteristics of two distinct components in squid ink.

[0037] In this application, "water-soluble components" and "water-insoluble components" are classification descriptions of complex samples (such as squid ink) based on the solubility characteristics of components in a specific medium (usually water). "Water-soluble components" generally refer to the set of chemical components that can dissolve in water or aqueous solvents under given conditions. "Water-insoluble components" generally refer to the set of components that cannot dissolve in water under the same conditions, but may be transformed into an analytable form through specific chemical treatments.

[0038] In this application, "chromatographic data" broadly refers to a series of raw or processed data obtained by analyzing a test solution using chromatographic analysis techniques, reflecting the separation of chemical components and the intensity of their responses in the sample. Its function is to provide direct, quantifiable chemical information input for constructing a fingerprint spectrum. For example, it may include, but is not limited to: chromatograms (curves showing signal intensity changes over time), retention times, peak areas, peak heights of each chromatographic peak, and relative values ​​derived from these raw data (such as relative retention time and relative peak area). In specific embodiments of this application, the "first chromatographic data" and "second chromatographic data" specifically refer to data obtained after performing high-performance liquid chromatography (HPLC) analysis on the first and second test solutions, respectively, particularly a dataset containing retention times and peak area information for multiple chromatographic peaks. These data are used together to construct an integrated squid ink fingerprint spectrum.

[0039] The method for constructing the squid ink fingerprint spectrum of this invention is applicable to scenarios where it is necessary to accurately distinguish between genuine and adulterated medicinal materials, or to conduct a detailed evaluation of the quality consistency between batches of medicinal materials.

[0040] The invention provides illustrative examples of methods for constructing squid ink fingerprint spectra and identifying different varieties of squid ink.

[0041] The method for constructing a squid ink fingerprint spectrum according to an embodiment of the present invention includes the following steps: Step S1: Take different batches of squid ink, and prepare a first test solution for obtaining chromatographic information of water-soluble components and a second test solution for obtaining chromatographic information of water-insoluble components for each squid ink sample; Step S2: Perform chromatographic analysis on the first and second test solutions of different batches of squid ink to obtain first and second chromatographic data; Step S3: Based on the first chromatographic data and the second chromatographic data, select the chromatographic peaks that are present in the chromatograms of different batches of squid ink as common peaks, generate the reference fingerprint spectrum of squid ink using the average value calculation method, and calculate the relative retention time and relative peak area of ​​each common peak to construct a fingerprint spectrum for evaluating the quality of the squid ink sample.

[0042] The method for constructing a fingerprint spectrum for cuttlefish ink according to this invention involves extracting water-soluble and water-insoluble components from the same cuttlefish ink sample. Test solutions targeting the water-soluble and water-insoluble components are prepared separately from the same batch of samples, and chromatographic analysis is performed separately to obtain two sets of chromatographic data. The water-soluble fingerprint spectrum reflects the overall chemical profile of the water-soluble components in cuttlefish ink, while the water-insoluble fingerprint spectrum mainly analyzes characteristic components such as melanin. The two complement each other, providing a more comprehensive and objective reflection of the quality of cuttlefish ink. A quality evaluation fingerprint spectrum is constructed based on the integrated data from these two sets, overcoming the limitations of a single processing mode. Therefore, this method can more comprehensively characterize the chemical composition of cuttlefish ink, significantly improving the ability to distinguish between different cuttlefish ink species (such as golden cuttlefish and needleless cuttlefish) and their adulterants, providing a more accurate and reliable analytical means for the quality control of cuttlefish ink medicinal materials.

[0043] Therefore, the method for constructing the squid ink fingerprint spectrum in this embodiment of the invention achieves more comprehensive and accurate quality control and authenticity identification of squid ink by integrating the chromatographic information of water-soluble and water-insoluble components.

[0044] The cuttlefish ink sample was diluted with water and then subjected to heating or enzymatic hydrolysis to obtain the first test solution. The same batch of cuttlefish ink samples were then oxidized under alkaline conditions with hydrogen peroxide to obtain the second test solution.

[0045] The method for constructing a squid ink fingerprint spectrum according to an embodiment of the present invention involves diluting the squid ink sample with water followed by heating or enzymatic hydrolysis to obtain a first test solution. The first test solution contains a complete range of water-soluble components with stable content, providing a rich chemical information basis for subsequent chromatographic analysis, thereby accurately reflecting the overall chemical characteristics of the water-soluble components in the squid ink sample.

[0046] For the preparation of the second test solution, the synergistic oxidation treatment of alkaline and hydrogen peroxide can effectively break down the polymer structure of melanin and degrade and oxidize it into characteristic small molecule products (such as pyrrole derivatives). These products can show clear and distinguishable characteristic peaks in chromatographic analysis, providing key information on water-insoluble components to distinguish different varieties of squid ink and identify adulterants.

[0047] Example 1 In the method for constructing the squid ink fingerprint spectrum of this invention, when preparing the first test solution, 8-12 times the amount of water (by weight) is added to the squid ink sample for dilution, and the sample is heated in a water bath at 80℃-100℃ for 30 min-1.5 h. After centrifugation or filtration, the supernatant is collected to obtain the first test solution. However, this invention is not limited to this. In other embodiments, 8-12 times the amount of water (by weight) is added to the squid ink sample for dilution, 20000 U of flavor protease is added, and the enzymatic hydrolysis is carried out at 45℃-60℃, pH 7.0, for 3 h-5 h. After inactivation in a water bath, the sample is centrifuged or filtered, and the supernatant is collected to obtain the first test solution.

[0048] In the method for constructing the squid ink fingerprint spectrum in this embodiment of the invention, another squid ink sample from the same batch is taken, and 1 mol / L K2CO3 solution and 30% H2O2 solution are added to the squid ink sample. The mixture is heated at 80℃~100℃ for 60 min~180 min. After cooling, 200 μL of 10% Na2SO3 is added to terminate the reaction. 1 mol / L~6 mol / L HCl is added to adjust the pH to 2.0~4.5. The mixture is then filtered or centrifuged to obtain the second test solution.

[0049] The first and second test solutions prepared above were analyzed by high performance liquid chromatography.

[0050] Example 2 In some embodiments, the chromatographic detection conditions of the first test solution are as follows: octadecylsilane-bonded silica gel as the packing material, column temperature 30℃~40℃; A is acetonitrile; B is 0.1% phosphoric acid aqueous solution as the mobile phase, gradient elution: 0 min~5 min, B 99.9%; 5 min~10 min, B 99.9%~98.8%; 10 min~20 min, B 98.8%; 20 min~22 min, B 98.8%~95%; 22 min~30 min, B 95%; 30 min~45 min, B 95%~80%; 45 min~50 min, B 80%~70%; 50 min~60 min, B 70%~50%. Ultraviolet detection wavelength: 210~360 nm.

[0051] The chromatographic detection conditions for the second test solution were as follows: octadecylsilane-bonded silica gel was used as the packing material, and the column temperature was 30℃~40℃; the mobile phase was methanol (A) and sodium dihydrogen phosphate buffer (B), with gradient elution: 0 min~25 min, B 95%~60%; 25 min~30 min, B 40%; and the UV detection wavelength was 210 nm~360 nm.

[0052] For example, five batches of golden cuttlefish ink were prepared, with corresponding first and second test solutions prepared for each batch, resulting in five first test solutions and five second test solutions. Five batches of needle-free cuttlefish ink were also prepared, with corresponding first and second test solutions prepared for each batch, resulting in five first test solutions and five second test solutions. Subsequently, according to the optimized chromatographic detection conditions described above, high-performance liquid chromatography (HPLC) analysis was performed on all first test solutions of both golden and needle-free cuttlefish inks to obtain chromatographic data of their respective water-soluble components; similarly, all second test solutions were analyzed to obtain chromatographic data of their water-insoluble components.

[0053] The method for constructing the fingerprint spectrum of cuttlefish ink in this invention optimizes the preparation of the test solution and chromatographic conditions. This method has the advantages of simple operation, good stability, high precision and good reproducibility, and is suitable for the quality detection of cuttlefish ink medicinal materials, decoction pieces and related preparations.

[0054] Different batches of cuttlefish ink, including golden cuttlefish ink and needleless cuttlefish ink, were collected. A first test solution and a second test solution were prepared from samples of the same batch of golden cuttlefish ink and needleless cuttlefish ink, respectively, and then analyzed by chromatography.

[0055] The first test solution of the golden cuttlefish ink contained 9 common peaks after chromatographic analysis, and the first test solution of the needleless cuttlefish ink contained 12 common peaks after chromatographic analysis. Both the golden cuttlefish ink and the needleless cuttlefish ink used chromatographic peak No. 2 as the S peak, and each common peak had a specific relative retention time range and relative peak area range relative to chromatographic peak No. 2. And / or, the second test solution of each of the golden cuttlefish ink and the needleless cuttlefish ink contains 5 common peaks, both of which have chromatographic peak No. 5 as the S peak, wherein peak No. 5 is pyrrole-2,3,5-tricarboxylic acid (PTCA), and each common peak has a specific relative retention time range and relative peak area range relative to chromatographic peak No. 5.

[0056] The relative retention times of the nine common peaks in the first test solution of the cuttlefish ink, as determined by chromatographic analysis, are as follows: Peak 1 0.566~0.570, Peak 2 1.000, Peak 3 1.326~1.327, Peak 4 1.523~1.528, Peak 5 1.773~1.781, Peak 6 2.110~2.126, Peak 7 3.351~3.394, Peak 8 4.284~4.311, and Peak 9 4.768~4.783. The relative peak areas of the nine common peaks in the first test solution of the cuttlefish ink, as determined by chromatographic analysis, are as follows: Peak 1: 1.788~3.419, Peak 2: 1.000, Peak 3: 0.346~0.622, Peak 4: 0.203~1.302, Peak 5: 0.284~0.637, Peak 6: 0.401~0.721, Peak 7: 0.380~3.361, Peak 8: 0.136~0.497, and Peak 9: 0.187~0.704.

[0057] The relative retention times of the 12 common peaks in the first test solution of the needle-free squid ink, as determined by chromatographic analysis, are as follows: Peak 1: 0.566–0.570, Peak 2: 1.000, Peak 3: 1.325–1.326, Peak 4: 1.381–1.388, Peak 5: 1.524–1.526, Peak 6: 1.775–1.785, Peak 7: 2.108–2.122, Peak 8: 2.919–2.925, Peak 9: 3.029–3.042, Peak 10: 3.398–3.412, Peak 11: 4.678–4.686, and Peak 12: 4.782–4.790. The relative peak areas of the 12 common peaks in the first test solution of the needle-free squid ink, as determined by chromatographic analysis, are as follows: Peak 1: 0.581~0.621, Peak 2: 1.000, Peak 3: 0.940~1.264, Peak 4: 0.086~0.269, Peak 5: 0.135~0.171, Peak 6: 0.309~0.489, Peak 7: 0.289~0.362, Peak 8: 0.041~0.073, Peak 9: 0.168~0.212, Peak 10: 0.059~0.092, Peak 11: 0.039~0.058, Peak 12: 0.058~0.104.

[0058] The relative retention times of the fingerprint spectra of the second test solutions of the golden cuttlefish ink and the needleless cuttlefish ink, as determined by chromatographic analysis, are as follows: Peak 1: 0.377~0.386, Peak 2: 0.471~0.474, Peak 3: 0.560~0.562, Peak 4: 0.668~0.670, Peak 5: 1.000; The relative peak areas of the fingerprint spectra of the second test solutions of each of the golden cuttlefish ink and the needleless cuttlefish ink, as determined by chromatographic analysis, are as follows: Peak 1: 0.124~0.276, Peak 2: 0.037~0.237, Peak 3: 0.080~0.716, Peak 4: 0.047~0.352, Peak 5: 1.000.

[0059] Based on the above data, it can be seen that the first test solutions of the golden cuttlefish ink and the needleless cuttlefish ink need to be established separately, while the second test solutions of the golden cuttlefish ink and the needleless cuttlefish ink can share the same fingerprint spectrum. This is because the relative retention time and peak area range of the characteristic small molecule products (such as pyrrole-2,3,5-tricarboxylic acid) generated after the water-insoluble components of the two inks are highly consistent after being synergistically oxidized by alkaline hydrogen peroxide, reflecting the commonality of the golden cuttlefish ink and the needleless cuttlefish ink in terms of water-insoluble characteristic components.

[0060] Based on this, when identifying the variety of cuttlefish ink, the fingerprint spectrum of the second test solution can be used for preliminary screening of the sample: if the relative retention time and peak area range of the fingerprint spectrum of the second test solution of the sample do not match the characteristic peak of the common spectrum (such as peak 5 pyrrole-2,3,5-tricarboxylic acid), it can be directly identified as a mixed product; if they match, the variety can be further distinguished by the fingerprint spectrum of the first test solution—by comparing the number of common peaks, relative retention time and peak area range of the first test solution of the sample with the fingerprint spectrum characteristics of the first test solution of golden cuttlefish ink or needleless cuttlefish ink, the variety can be accurately identified.

[0061] Comparative Example 1 The preparation of the first test sample solution follows step S1 of Example 1, except for the different treatment methods after adding water. The ultrasonic conditions are: add 10 mL of water, sonicate for 0.5 h, centrifuge, collect the supernatant, and filter through a 0.22 μm microporous membrane. The static conditions are: add 10 mL of water, stand at 4 ℃ for 12 h, centrifuge, collect the supernatant, and filter through a 0.22 μm microporous membrane. The chromatogram of the test sample in Example 1 is compared with the chromatograms of the different extraction methods in Comparative Example 1. Figure 1 The results showed that the number and area of ​​chromatographic peaks after static and ultrasonic treatment were significantly less than those after heating or enzymatic digestion.

[0062] The results show that, compared with Comparative Example 1, the method of constructing the squid ink fingerprint spectrum in the embodiment of the present invention, by adopting heating or enzymatic hydrolysis, can more fully release the water-soluble active ingredients in squid ink and significantly increase the number of chromatographic peaks and response intensity.

[0063] Comparative Example 2 Following step S1 of Example 1, the screening conditions for settling and sonication were selected, and the mobile phase selection was carried out according to step S2 of Example 2. The difference from Example 2 of this application is that different mobile phases were used, such as acetonitrile-water and methanol-0.1% phosphoric acid. The chromatograms in Example 2 were compared with the chromatograms of different mobile phases in Comparative Example 2. Figure 2 The results showed that the number of peaks in the chromatogram of Comparative Example 2 was relatively small.

[0064] The results show that, in this embodiment of the invention, by optimizing the combination of the organic phase (acetonitrile or methanol) and the aqueous phase (0.1% phosphoric acid or sodium dihydrogen phosphate buffer), the resolution of chromatographic peaks can be effectively improved, the response intensity of each characteristic peak can be significantly enhanced, and the characteristic peaks of both water-soluble and water-insoluble components in squid ink can be clearly presented, laying the foundation for subsequent identification of common peaks and variety identification in fingerprint spectroscopy. In contrast, the single mobile phase system such as acetonitrile-water or methanol-0.1% phosphoric acid used in Comparative Example 2 lacks specific adaptation to the complex chemical composition of squid ink, resulting in some characteristic peaks not being effectively separated or having excessively low response values, making it difficult to comprehensively capture the chemical characteristics of squid ink and failing to meet the accuracy requirements for fingerprint spectroscopy construction.

[0065] The method for constructing the squid ink fingerprint spectrum in this invention can be applied to squid inks including but not limited to needleless squid ink, golden squid ink, needle squid ink, or tiger-striped squid ink.

[0066] The method for identifying different varieties of cuttlefish ink according to embodiments of the present invention includes the following steps: Step S1: Prepare a first test solution for obtaining chromatographic information of water-soluble components and a second test solution for obtaining chromatographic information of water-insoluble components based on the cuttlefish ink sample to be tested; Step S2: Perform chromatographic analysis on the first test solution and the second test solution respectively to obtain first chromatographic data and second chromatographic data; compare the chromatographic analysis data of the sample to be tested with the squid ink fingerprint standard data as described in any one of the above, and determine the authenticity or quality grade of the squid ink sample to be tested based on the comparison results.

[0067] Furthermore, based on the constructed fingerprint chromatogram of the squid ink sample, the number of peaks in the 35-60 min interval of the first chromatographic data is analyzed to determine whether the squid ink sample to be tested is cuttlefish ink; the peak shape and peak height in the 8-32 min interval of the first chromatographic data are analyzed to distinguish between golden squid ink samples and needleless squid ink samples. The second chromatographic data is analyzed, and based on the similarity, squid ink (golden squid ink and needleless squid ink) and adulterants are distinguished.

[0068] The inventors discovered that, in order to optimize the extraction and analysis efficiency of water-soluble and water-insoluble components, the single pretreatment design leads to incomplete chemical information coverage and limited discriminative power in the constructed quality characterization model. Specifically, when identifying squid ink, needleless squid ink, and their common adulterant, spear squid ink, relying solely on the water-soluble component spectrum or solely on the melanin degradation product spectrum may result in inaccurate and unreliable identification due to the lack of key complementary information.

[0069] The identification method for different varieties of cuttlefish ink in this invention involves comparing the first chromatographic data of the sample to be tested with the first test solution of cuttlefish ink (both golden and needleless varieties) in terms of the number of common peaks, relative retention time, and relative peak area range. Simultaneously, it combines this with the relative parameter differences of characteristic peaks (such as pyrrole-2,3,5-tricarboxylic acid) in the second chromatographic data to achieve accurate identification of different varieties of cuttlefish ink. If the number of common peaks, relative retention time, and peak area of ​​each peak in the first test solution of the sample to be tested all meet the standard range of golden cuttlefish ink, and the characteristic peak parameters of the second test solution are consistent with those of golden cuttlefish ink, then it is identified as golden cuttlefish ink; if it meets the corresponding standard range of needleless cuttlefish ink, then it is identified as needleless cuttlefish ink; if there are significant deviations from both, it can be identified as a mixed product or a substandard sample. This method is convenient to operate and provides reliable results, effectively solving the difficult problems in the identification of cuttlefish ink varieties and providing strong support for the standardized use and quality control of cuttlefish ink medicinal materials and processed slices.

[0070] Specifically, for the first chromatographic data of water-soluble components, the focus is on analyzing the number of common peaks, relative retention times, and peak area distribution to reflect the differences in the composition of polar components such as water-soluble small molecules. For the second chromatographic data of water-insoluble components, the focus is on the characteristic peaks of melanin degradation products (such as pyrrole-2,3,5-tricarboxylic acid), capturing the specific markers of different varieties of squid ink through their peak shape, retention time, and relative peak area. Furthermore, by correlating and modeling the characteristic parameters of the two types of spectra, a multi-dimensional quality evaluation index system can be constructed. This system can not only achieve rapid identification of genuine squid ink from adulterated products (such as squid ink), but also quantitatively assess the quality consistency of different batches of squid ink.

[0071] Further, the number of peaks in the 35 min to 60 min interval of the first chromatographic data is analyzed to determine whether the cuttlefish ink sample to be tested is cuttlefish ink.

[0072] To further verify this, the peak shape and peak height in the 8 min to 32 min interval of the first chromatographic data were analyzed to distinguish between the golden cuttlefish ink sample and the needleless cuttlefish ink sample.

[0073] To further verify, the water-soluble fingerprint spectrum established by the first test solution can be used to analyze the first chromatographic data and calculate the similarity with the control fingerprint spectrum of the golden cuttlefish ink sample and / or needleless cuttlefish ink sample. If the similarity is greater than 0.9, it can be further determined whether the cuttlefish ink sample to be tested is golden cuttlefish ink or needleless cuttlefish ink. And / or, analyze the second chromatographic data of the cuttlefish ink sample to be tested, and establish a water-insoluble fingerprint spectrum with the golden cuttlefish ink sample and / or needleless cuttlefish ink sample. If the similarity is greater than 0.9, the cuttlefish ink sample to be tested is determined to be golden cuttlefish ink and needleless cuttlefish ink. In practical applications, two batches of commercially available adulterants (cuttlefish ink) were tested. The first test solution showed significantly more peaks in the 35-60 min interval than the golden cuttlefish ink and needleless cuttlefish ink (an average of 7-8 more peaks).

[0074] To verify the analysis, the peak shape and height, as well as the similarity, of the first chromatographic data in the 8-32 min interval were used to distinguish and confirm the golden cuttlefish ink sample and the needleless cuttlefish ink sample. For example... Figure 7 As shown, it can accurately distinguish between the golden squid ink sample and the needleless squid ink sample.

[0075] Furthermore, by analyzing the second chromatographic data, if the similarity is greater than 0.9, it is possible to further accurately distinguish and verify cuttlefish ink (golden cuttlefish ink and needleless cuttlefish ink) and adulterants (gun cuttlefish ink).

[0076] The fingerprint spectrum and identification method constructed in this invention have good accuracy and repeatability, and can be effectively applied to the rapid identification and quality control of cuttlefish species.

[0077] Specific example The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0078] 1. Instruments and reagents An Agilent 1260 ultra-high performance liquid chromatograph (DAD WR G7115A, DMCT G7116A, Vialsampler G7129A, Quat Pump G7111B), an H1650-W benchtop high-speed centrifuge (Hunan Changsha Xiangyi Centrifuge Co., Ltd.), an ME204E 0.01% balance, and an MS105DU 0.01% balance (both Mettler Toledo GmbH, Switzerland), and an HWS-26 electric thermostatic water bath (Shanghai Yiheng Scientific Instruments Co., Ltd.). Acetonitrile and methanol (chromatographic grade, Merck AG, Germany); sodium dihydrogen phosphate dihydrate, anhydrous potassium carbonate, 30% hydrogen peroxide, anhydrous sodium sulfite, and hydrochloric acid (analytical grade, Sinopharm Chemical Reagent Co., Ltd.); phosphoric acid (analytical grade, Xilong Scientific Co., Ltd.). PTCA reference standard (batch number 1-CAL-101-1, 98% mass fraction, TRC, Canada).

[0079] 2. Collection of medicinal materials A total of 12 batches of cuttlefish ink samples were used in this experiment, including 10 batches from the Sepiidae family (5 batches of golden cuttlefish and 5 batches of needleless cuttlefish) and 2 batches from the Squididae family (Squid lanceolata). All samples were collected from coastal provinces such as Shandong and Zhejiang. The origin information is shown in Table 1. The sample preparation method is as follows: cuttlefish were taken, the ink sacs were removed, washed, and dried in an oven at 55 ℃ for 24 h. The resulting dried powder was sealed and stored in a desiccator for later use.

[0080] Table 1. Sample information of squid ink (S1-S10) and cuttlefish ink (S11-S12) Example 3: The method for establishing the squid ink fingerprint spectrum includes the following steps: Step 1: Take 1 g of the above-mentioned group of cuttlefish ink (S1~S12), accurately weigh it, add 10 mL of water, heat in a 98℃ water bath for 1 h, cool it, centrifuge it to collect the supernatant, and filter it through a 0.22 μm microporous membrane to obtain the product.

[0081] Step 2: Accurately weigh 10 mg of dried cuttlefish ink powder (S1~S12), add 950 μL of 1 mol / L K2CO3 and 100 μL of 30% H2O2, heat at 90 ℃ for 180 min, cool under running water, add 200 μL of 10% Na2SO3 to terminate the reaction, slowly add 6 mol / L HCl to adjust the pH to about 2.6, filter through a 0.22 μm microporous membrane to obtain the test solution. Accurately weigh an appropriate amount of PTCA reference standard, add water to prepare a reference peak solution with a mass concentration of 49 μg / mL.

[0082] Step 3: Accurately inject each test solution and reference peak solution into the liquid chromatograph. Record the chromatogram of the water-soluble fingerprint over 60 minutes. The specific chromatographic conditions are as follows: Waters Atlantis T3 column (4.6 mm × 250 mm, 5 µm, Waters Corporation, USA); column temperature 30℃; mobile phase: A: acetonitrile-B: 0.1% phosphoric acid aqueous solution; gradient elution: 0 min–5 min, B 99.9%; 5 min–10 min, B 99.9%–98.8%; 10 min–20 min, B 98.8%; 20 min–22 min, B 98.8%–95%; 22 min–30 min, B 95%; 30 min–45 min, B 95%–80%; 45 min–50 min, B 80%–70%; 50 min–60 min, B 70%–50%. UV detection wavelength: 250 nm, injection volume: 20 μL. Water-insoluble fingerprint chromatograms were recorded over 30 minutes. Specific chromatographic conditions were as follows: Waters Atlantis T3 column (4.6 mm × 250 mm, 5 µm, Waters Corporation, USA); column temperature: 30℃; mobile phase: A: methanol-B: sodium dihydrogen phosphate buffer; gradient elution: 0 min ~ 25 min, B 95% ~ 60%; 25 min ~ 30 min, B 40%; UV detection wavelength: 275 nm, injection volume: 10 μL.

[0083] Step 4: Export the water-soluble and water-insoluble fingerprint spectra obtained in Step 3 for the 10 batches respectively (see...). Figures 3-6 The data was then imported into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System A. The relative retention time, relative peak area limit range, and similarity of each common peak were calculated, and the results are shown in Tables 2-7.

[0084] Table 2. Relative retention time and relative peak area limits of common peaks in the water-soluble fingerprint spectra of 5 batches of cuttlefish ink samples. Table 3. Relative retention time and relative peak area limits of common peaks in the soluble fingerprint spectra of five batches of needle-free squid ink Table 4. Relative retention time and relative peak area limits of common peaks in the insoluble fingerprint spectra of 10 batches of squid ink Table 5. Similarity results of water-soluble fingerprint spectra of cuttlefish ink samples (S1~S5) Table 6. Similarity results of water-soluble fingerprint spectra of needle-free squid ink (S6~S10) Table 7. Similarity results of water-insoluble fingerprint spectra of squid ink (S1~S10) Chromatograms of water solubility from cuttlefish ink, needleless cuttlefish ink, and spear cuttlefish ink ( Figure 7 It can be seen that the chromatograms of cuttlefish ink in the 35 min to 60 min interval and the number of peaks of the two types of cuttlefish ink are significantly different. At the same time, the peak shape and peak height of cuttlefish ink and needleless cuttlefish ink in the 8 min to 32 min interval are significantly different, indicating that this method can clearly distinguish cuttlefish ink, needleless cuttlefish ink and cuttlefish ink. Determination of fingerprint spectral technical parameters: The similarity between the water-soluble fingerprint spectra of 10 batches of squid ink and the control fingerprint spectra was calculated. The results of S1~S10 were 0.815, 0.763, 0.884, 0.66, 0.879, 0.964, 0.961, 0.964, 0.976, and 0.975, respectively, indicating that there are certain differences in the water-soluble components of golden squid ink and needleless squid ink. Further, the similarity of 5 batches of golden squid ink and 5 batches of needleless squid ink was evaluated separately (Tables 5 and 6). The results showed that golden squid ink had 9 common peaks with similarities of 0.976, 0.98, 0.965, 0.925, and 0.964; needleless squid ink had 12 common peaks with similarities of 0.993, 0.997, 0.997, 0.996, and 0.997. Therefore, the provisional standard for the soluble fingerprint chromatograms of gold and needleless cuttlefish ink is as follows: the fingerprint chromatogram of the test sample must be consistent with that of the reference sample. Using the similarity evaluation system for chromatographic fingerprint chromatograms of traditional Chinese medicine, the similarity between the fingerprint chromatogram of the test sample and the reference sample, calculated by similarity calculation software, must not be lower than 0.9 (a similarity greater than 0.9 can be considered as the same sample). According to the fingerprint chromatograms of 5 batches of gold cuttlefish ink and 5 batches of needleless cuttlefish ink, the relative retention time and relative peak area of ​​each common peak should meet the limits in Tables 2 and 3.

[0085] Similarity calculations were performed on the insoluble fingerprint chromatograms of 5 batches of golden cuttlefish ink and 5 batches of needleless cuttlefish ink compared to the control fingerprint chromatogram. The results were all greater than 0.90. Therefore, the provisional standard for insoluble fingerprint chromatograms of cuttlefish ink is: the test sample chromatogram must be consistent with the control fingerprint chromatogram. According to the similarity evaluation system for chromatographic fingerprint chromatograms of traditional Chinese medicine, the similarity between the test sample fingerprint chromatogram and the control fingerprint chromatogram, calculated by similarity calculation software, should not be lower than 0.9. Based on the fact that the relative retention times and relative peak areas of the common peaks in the 10 batches of fingerprint chromatograms should meet the limits in Table 4, the similarity was calculated as follows: The chromatograms of two batches of gunfish ink samples and 10 batches of cuttlefish ink were simultaneously imported into the similarity evaluation software. The similarity scores were 0.576 and 0.552, respectively, indicating that the similarity between gunfish ink and cuttlefish ink is very low. Figure 8The chromatograms also show significant differences, so the water-insoluble fingerprint spectrum can effectively distinguish adulterated cuttlefish ink (cuttlefish ink).

[0086] 3. Chromatographic peak identification The water-soluble fingerprint chromatogram did not match the reference standard. The water-insoluble fingerprint chromatogram, compared with the reference standard, identified peak number 5 among the common peaks as PTCA. The results of the reference standard comparison are shown below. Figure 9 .

[0087] 4. Optimization of chromatographic conditions 4.1 Wavelength Selection For water-soluble fingerprinting, detection wavelengths of 250 nm, 280 nm, and 310 nm were examined. Results showed that the spectral density at 250 nm was rich in information and exhibited good separation; therefore, 250 nm was selected as the detection wavelength. For water-insoluble fingerprinting, 254 nm, 275 nm, and 310 nm were examined. Results showed that the spectral density at 275 nm was rich in information and exhibited good separation; therefore, 275 nm was selected as the detection wavelength.

[0088] 4.2 Selection of Mobile Phase For water-soluble fingerprint chromatograms, different mobile phases were investigated, including acetonitrile-water, acetonitrile-0.1% phosphoric acid, and methanol-0.1% phosphoric acid. The results showed that the acetonitrile-0.1% phosphoric acid system was optimal, exhibiting good peak resolution and moderate retention time; therefore, it was selected as the chromatographic condition. For water-insoluble fingerprint chromatograms, different mobile phase systems, such as methanol-formic acid aqueous solution and methanol-sodium dihydrogen phosphate buffer aqueous solution, were investigated. Methanol-sodium dihydrogen phosphate buffer was determined to be the optimal mobile phase for gradient elution.

[0089] 4.3 Preparation of the test solution For water-soluble fingerprinting, methods including water addition and heating, ultrasonic treatment, and flavor protease digestion were tested. It was found that water addition and heating, and flavor protease digestion, produced rich and similar chromatographic peak information; therefore, water addition and heating were chosen to prepare the test sample. For water-insoluble fingerprinting, different concentrations of hydrogen peroxide (15% and 30%), oxidation times of 120 min and 180 min, and pH adjustments to 2.6, 3.5, and 4.5 were investigated. Ultimately, 30% H₂O₂, an oxidation time of 180 min, and a pH adjustment to 2.6 were determined to be optimal.

[0090] 5. Methodological Validation 5.1 Precision Experiment Water-soluble fingerprint chromatogram: 1 g of S1 powder was accurately weighed and a test solution was prepared according to the method in Example 3. HPLC analysis was performed, with six consecutive injections. Chromatograms were recorded, and similarity was evaluated. The relative peak areas and relative retention times of common peaks were calculated. The results are shown in Tables 8 and 9. The HPLC chromatograms from the six precision tests were imported into the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" to calculate the similarity. The results were all greater than 0.98, indicating good instrument precision. The RSD was <3.0%, indicating that the precision of the instrument's chromatographic detection met the requirements of method validation.

[0091] Water-insoluble fingerprint chromatogram: PTCA reference standard was injected six times consecutively, and the chromatograms were recorded. Similarity was evaluated, and the peak area and retention time were calculated. The results are shown in Tables 10 and 11. The relative standard deviations (RSDs) of the peak retention time and peak area of ​​the PTCA reference standard were <3.0%, and the similarity was 1.0 for both, indicating good instrument precision.

[0092] Table 8. Relative Retention Time Table for Precision of Water-Soluble Fingerprints Table 9. Relative Peak Area Table for Precision of Water-Soluble Fingerprint Spectra Table 10 Precision PTCA Retention Schedule for Water-Insoluble Fingerprint Spectra Table 11 Precision of Water-Insoluble Fingerprint Spectra: PTCA Peak Area Table 5.2 Repeatability Experiment Water-soluble fingerprint chromatogram: 1 g of S1 powder was accurately weighed into 6 portions. The test solution was prepared according to the method in Example 3 and analyzed by HPLC. The chromatograms were recorded, and similarity was evaluated. The relative retention time and relative peak area of ​​the common peaks were calculated. RSD < 3.0%, indicating good repeatability of the fingerprint chromatogram preparation method. The repeatability test results are shown in Tables 12 and 13. The RSD of the relative retention time and relative peak area of ​​the 8 common peaks was < 3.0%. The HPLC chromatograms of the repeatability test were imported into the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" to calculate the similarity. The results were all greater than 0.99, indicating that the samples prepared by this method have good repeatability.

[0093] Water-insoluble fingerprint chromatogram: Six 20 mg portions of S1 powder were accurately weighed and prepared according to the method in Example 3. The solutions were then analyzed by HPLC. Chromatograms were recorded, and similarity was evaluated. The relative peak areas and relative retention times of the common peaks were calculated. The results are shown in Tables 14 and 15. The RSDs of the relative retention times and relative peak areas of the five common peaks were all <3.0%, and the similarity was 1.0, indicating good repeatability of the method.

[0094] Table 12 Relative Retention Time Table for Reproducibility of Water-Soluble Fingerprints Table 13 Relative Peak Area Table for Repeatability of Water-Soluble Fingerprint Spectra Table 14 Relative Retention Time Table for Reproducibility of Water-Insoluble Fingerprints Table 15 Relative Peak Areas for Repeatability of Water-Insoluble Fingerprint Spectra 5.3 Stability Test Water-soluble fingerprint chromatogram: 1 g of S1 powder was accurately weighed and a test solution was prepared according to the method in Example 3. The solution was injected and measured at 0 h, 2 h, 4 h, 6 h, 12 h, and 24 h, and the chromatograms were recorded. Similarity evaluation was performed, and the relative peak areas and relative retention times of the common peaks were calculated. The stability test results are shown in Tables 16 and 17. The RSD of the relative retention times and relative peak areas of the eight common peaks was <3.0%, and the similarity results were all greater than 0.98, indicating that the sample prepared by this method has good stability.

[0095] Water-insoluble fingerprint chromatogram: 20 mg of S1 powder was accurately weighed and a test solution was prepared according to the method in Example 3. The solution was injected and measured at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h, and the chromatograms were recorded. Similarity evaluation was performed, and the relative retention time and relative peak area of ​​the common peaks were calculated. The results are shown in Tables 18 and 19. The RSD of the relative retention time and relative peak area of ​​the five common peaks were <3.0%, and the similarity was 1.0 for all of them, indicating that the sample prepared by this method has good stability.

[0096] Table 16 Relative Retention Time Table of Water-Soluble Fingerprint Stability Table 17 Relative Peak Area Table of Stability of Water-Soluble Fingerprint Spectra Table 18 Relative Retention Time Table of Water-Insoluble Fingerprint Stability Table 19. Relative Peak Areas of Water-Insoluble Fingerprint Spectra Stability The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method for constructing a squid ink fingerprint spectrum, characterized in that, Includes the following steps: Step S1: Take different batches of squid ink samples, and prepare a first test solution for obtaining chromatographic information of water-soluble components and a second test solution for obtaining chromatographic information of water-insoluble components for each squid ink sample. Step S2: Perform chromatographic analysis on the first test solution and the second test solution respectively to obtain first chromatographic data and second chromatographic data; Step S3: Based on the first chromatographic data and the second chromatographic data, select the chromatographic peaks that are present in the chromatograms of different batches of squid ink as common peaks, generate a reference fingerprint spectrum of squid ink, and calculate the relative retention time and relative peak area of ​​each common peak to construct a fingerprint spectrum for evaluating the quality of the squid ink sample.

2. The method for constructing a squid ink fingerprint spectrum according to claim 1, characterized in that, The cuttlefish ink sample was diluted with water and then heated or enzymatically hydrolyzed to obtain the first test solution. And / or, take the same batch of squid ink samples and oxidize the squid ink samples under alkaline and hydrogen peroxide conditions to obtain the second test solution.

3. The method for constructing a squid ink fingerprint spectrum according to claim 2, characterized in that, Dilute the cuttlefish ink sample with 8 to 12 times the amount of water by weight, heat in a water bath at 80°C to 100°C for 30 min to 1.5 h, centrifuge or filter, and take the supernatant to obtain the first test solution. Alternatively, by weight, dilute the cuttlefish ink sample with 8 to 12 times the amount of water, add 20,000 U of flavor protease, hydrolyze at 45°C to 60°C, pH 7.0 for 3 to 5 hours, inactivate in a water bath, centrifuge or filter, and take the supernatant to obtain the first test solution.

4. The method for constructing a squid ink fingerprint spectrum according to claim 2, characterized in that, Take another sample of squid ink from the same batch, add 1 mol / L K2CO3 solution and 30% H2O2 solution to the squid ink sample, heat at 80℃~100℃ for 60min~180min, cool, add 200 μL of 10% Na2SO3 to terminate the reaction, add 1mol / L~6 mol / L HCl to adjust the pH to 2.0~4.5, filter or centrifuge to obtain the second test solution.

5. The method for constructing a squid ink fingerprint spectrum according to claim 1, characterized in that, The first and second test solutions were analyzed by high performance liquid chromatography. And / or, import the first chromatographic data and the second chromatographic data into the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" software respectively; And / or, the cuttlefish ink includes needleless cuttlefish ink, golden cuttlefish ink, needle cuttlefish ink, or tiger-striped cuttlefish ink; And / or, take 20 μL of the first test solution for chromatographic analysis; take 10 μL of the second test solution for chromatographic analysis, and obtain the chromatographic peaks corresponding to each test solution; And / or, prepare a reference peak solution of pyrrole-2,3,5-tricarboxylic acid, and perform chromatographic analysis on the second test solution and the reference peak solution.

6. The method for constructing a squid ink fingerprint spectrum according to claim 5, characterized in that, The chromatographic detection conditions for the first test solution are as follows: Using octadecylsilane-bonded silica gel as the packing material, the column temperature was 30℃ ~ 40℃; A was acetonitrile; B was 0.1% phosphoric acid water as the mobile phase, with gradient elution. From 0 min to 5 min, B was 99.9%; from 5 min to 10 min, B was 99.9% ~ 98.8%. 10 min~20 min, B 98.8%; 20 min~22 min, B 98.8%~95%; 22 min~30 min, B 95%; 30 min~45 min, B 95%~80%; 45 min~50 min, B 80%~70%; 50 min~60 min, B 70%~50%. UV detection wavelength: 210 nm~360 nm. The chromatographic detection conditions for the second test solution are as follows: The column was filled with octadecylsilane-bonded silica gel at a temperature of 30℃~40℃. The mobile phase consisted of methanol (A) and sodium dihydrogen phosphate buffer (B), with gradient elution: 0 min~25 min, B 95%~60%; 25 min~30 min, B 4%. Ultraviolet detection wavelength: 210 nm~360 nm.

7. The method for constructing a squid ink fingerprint spectrum according to claim 1, characterized in that, The cuttlefish ink samples include golden cuttlefish ink and needleless cuttlefish ink. A first test solution and a second test solution were prepared from the golden cuttlefish ink and the needleless cuttlefish ink, respectively, and then analyzed by chromatography. The first test solution of the golden cuttlefish ink contained 9 common peaks after chromatographic analysis, and the first test solution of the needleless cuttlefish ink contained 12 common peaks after chromatographic analysis. Both the golden cuttlefish ink and the needleless cuttlefish ink used chromatographic peak No. 2 as the S peak, and each common peak had a specific relative retention time range and relative peak area range relative to chromatographic peak No.

2. And / or, the second test solution of each of the golden cuttlefish ink and the needleless cuttlefish ink contains 5 common peaks, both of which have chromatographic peak No. 5 as the S peak, wherein peak No. 5 is pyrrole-2,3,5-tricarboxylic acid, and each common peak has a specific relative retention time range and relative peak area range relative to chromatographic peak No.

5.

8. The method for constructing a squid ink fingerprint spectrum according to claim 7, characterized in that, The relative retention times of the nine common peaks in the first test solution of the cuttlefish ink, as determined by chromatographic analysis, are as follows: Peak 1: 0.566~0.570, Peak 2: 1.000, Peak 3: 1.326~1.327, Peak 4: 1.523~1.528, Peak 5: 1.773~1.781, Peak 6: 2.110~2.126, Peak 7: 3.351~3.394, Peak 8: 4.284~4.311, Peak 9: 4.768~4.783; The relative peak areas of the nine common peaks in the first test solution of the cuttlefish ink, as determined by chromatographic analysis, are as follows: Peak 1: 1.788–3.419, Peak 2: 1.000, Peak 3: 0.346–0.622, Peak 4: 0.203–1.302, Peak 5: 0.284–0.637, Peak 6: 0.401–0.721, Peak 7: 0.380–3.361, Peak 8: 0.136–0.497, and Peak 9: 0.187–0.

704. The relative retention times of the 12 common peaks in the first test solution of the needle-free cuttlefish ink, as determined by chromatographic analysis, are as follows: Peak 1: 0.566–0.570, Peak 2: 1.000, Peak 3: 1.325–1.326, Peak 4: 1.381–1.388, Peak 5: 1.524–1.526, Peak 6: 1.775–1.785, Peak 7: 2.108–2.122, Peak 8: 2.919–2.925, Peak 9: 3.029–3.042, Peak 10: 3.398–3.412, Peak 11: 4.678–4.686, Peak 12: 4.782–4.790; The relative peak areas of the 12 common peaks in the first test solution of the needle-free cuttlefish ink, as determined by chromatographic analysis, are as follows: Peak 1: 0.581~0.621, Peak 2: 1.000, Peak 3: 0.940~1.264, Peak 4: 0.086~0.269, Peak 5: 0.135~0.171, Peak 6: 0.309~0.489, Peak 7: 0.289~0.362, Peak 8: 0.041~0.073, Peak 9: 0.168~0.212, Peak 10: 0.059~0.092, Peak 11: 0.039~0.058, Peak 12: 0.058~0.104; And / or, the relative retention times of the fingerprint chromatograms of the second test solution of each of the golden cuttlefish ink and the needleless cuttlefish ink are as follows: peak 1 0.377~0.386, peak 2 0.471~0.474, peak 3 0.560~0.562, peak 4 0.668~0.670, peak 5 1.000; The relative peak areas of the fingerprint spectra of the second test solution of each of the golden cuttlefish ink and the needleless cuttlefish ink were as follows according to chromatographic analysis: peak 1 0.124~0.276, peak 2 0.037~0.237, peak 3 0.080~0.716, peak 4 0.047~0.352, and peak 5 1.

000.

9. A method for identifying different varieties of cuttlefish ink, characterized in that, Includes the following steps: Step S1: Prepare a first test solution for obtaining chromatographic information of water-soluble components and a second test solution for obtaining chromatographic information of water-insoluble components based on the cuttlefish ink sample to be tested; Step S2: Perform chromatographic analysis on the first test solution and the second test solution respectively to obtain first chromatographic data and second chromatographic data; compare the chromatographic analysis data of the sample to be tested with the standard data of squid ink fingerprint spectrum as described in any one of claims 1 to 8, and determine the authenticity or quality grade of the squid ink sample to be tested based on the comparison results.

10. The method for identifying different varieties of cuttlefish ink according to claim 9, characterized in that, Based on the constructed fingerprint spectrum of the squid ink sample quality Analyze the number of peaks in the 35 min to 60 min interval of the first chromatographic data to determine whether the cuttlefish ink sample to be tested is cuttlefish ink. And / or, analyze the peak shape and peak height in the 8 min to 32 min interval of the first chromatographic data to distinguish between the golden cuttlefish ink sample and the needleless cuttlefish ink sample; And / or, correlate the spectral data of the first test solution of the cuttlefish ink sample to be tested with the fingerprint spectral standard data of the first test solution of the golden cuttlefish ink sample and / or the needleless cuttlefish ink sample. If the similarity is greater than 0.9, further confirm whether the sample to be tested is golden cuttlefish ink or needleless cuttlefish ink. And / or, the spectral data of the second test solution of the cuttlefish ink sample to be tested is correlated with the fingerprint spectral standard data of the second test solution of the golden cuttlefish ink sample and / or the needleless cuttlefish ink sample. If the similarity is greater than 0.9, the cuttlefish ink sample to be tested is determined to be golden cuttlefish ink and / or needleless cuttlefish ink.

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